HSP90 COMBINATION THERAPY

The inventions described herein were made, at least in part, with support from Grant No. ROI CA 155226 from the National Cancer Institute, Department of Health and Human Services; and the U.S. Government has rights in any such subject invention.

Throughout this application numerous public documents including issued and pending patent applications, publications, and the like are identified. These documents in their entireties are hereby incorporated by reference into this application to help define the state of the art as known to persons skilled therein.

BACKGROUND OF THE INVENTION

There is a great need to understand the molecular aberrations that maintain the malignant phenotype of cancer cells. Such an understanding would enable more selective targeting of tumor-promoting molecules and aid in the development of more effective and less toxic anticancer treatments. Most cancers arise from multiple molecular lesions, and likely the resulting redundancy limits the activity of specific inhibitors of signaling molecules. While combined inhibition of active pathways promises a better clinical outcome, comprehensive identification of oncogenic pathways is currently beyond reach.

Application of genomics technologies, including large-scale genome sequencing, has led to the identification of many gene mutations in various cancers, emphasizing the complexity of this disease (Ley et al, 2008; Parsons et al, 2008). However, whereas these genetic analyses are useful in providing information on the genetic make-up of tumors, they intrinsically lack the ability to elucidate the functional complexity of signaling networks aberrantly activated as a consequence of the genetic defect(s). Development of complementary proteomic methodologies to identify molecular lesions intrinsic to tumors in a patient- and disease stage-specific manner must thus follow. Most proteomic strategies are limited to measuring protein expression in a particular tumor, permitting the identification of new proteins associated with pathological states, but are unable to provide information on the functional significance of such findings (Hanash & Taguchi, 2010). Some functional information can be obtained using antibodies directed at specific proteins or post-translational modifications and by activity-based protein profiling using small molecules directed to the active site of certain enzymes (Kolch & Pitt, 2010; Nomura et al., 2010; Brehme et al., 2009; Ashman & Villar, 2009). Whereas these methods have proven useful to query a specific pathway or post-translational modification, they are not as well suited to capture more global information regarding the malignant state (Hanash & Taguchi, 2010). Moreover, current proteomic methodologies are costly and time consuming. For instance, proteomic assays often require expensive SILAC labeling or two- dimensional gel separation of samples. Accordingly, there exists a need to develop simpler, more cost effective proteomic methodologies that capture important information regarding the malignant state. As it is recognized that the molecular chaperone protein heat shock protein (Hsp90) maintains many oncoproteins in a pseudo-stable state (Zuehlke & Johnson, 2010; Workman et al, 2007), Hsp90 may be an important protein in the development of new proteomic methods.

In support of this hypothesis, heat shock protein (Hsp90), a chaperone protein that functions to properly fold numerous proteins to their active conformation, is recognized to play important roles in maintaining the transformed phenotype (Zuehlke & Johnson, 2010; Workman et al, 2007). Hsp90 and its associated co-chaperones assist in the correct conformational folding of cellular proteins, collectively referred to as "client proteins", many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to deregulated proteins (i.e. through mutations, aberrant expression, improper cellular translocation etc) can thus become critically dependent on Hsp90 (Workman et al, 2007). While Hsp90 is expressed in most cell types and tissues, work by Kamal et al demonstrated an important distinction between normal and cancer cell Hsp90 (Kamal et al, 2003). Specifically, they showed that tumors are characterized by a multi-chaperone complexed Hsp90 with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 with low affinity for these inhibitors.

Many of the client proteins of Hsp90 also play a prominent role in disease onset and progression in several pathologies, including cancer. (Whitesell and Lindquist, Nat Rev Cancer 2005, 5, 761; Workman et al, Ann NY Acad Sci 2007, 1113, 202; Luo et al, Mol Neurodegener 2010, 5, 24.) As a result there is also significant interest in the application of Hsp90 inhibitors in the treatment of cancer. (Taldone et al., Opin Pharmacol 2008, 8, 370; Janin, Drug Discov Today 2010, 15, 342.)

Based on the body of evidence set forth above, we hypothesize that proteomic approaches that can identify key oncoproteins associated with Hsp90 can provide global insights into the biology of individual tumor and can have widespread application towards the development of new cancer therapies. Accordingly, the present disclosure provides tools and methods for identifying oncoproteins that associate with Hsp90. Moreover, the present disclosure provides methods for identifying treatment regimens for cancer patient.

SUMMARY OF THE INVENTION

The present disclosure relates to the discovery that small molecules able to target tumor- enriched Hsp90 complexes (e.g., Hsp90 inhibitors) can be used to affinity-capture Hsp90- dependent oncogenic client proteins. The subsequent identification combined with bioinformatic analysis enables the creation of a detailed molecular map of transformation- specific lesions. This map can guide the development of combination therapies that are optimally effective for a specific patient. Such a molecular map has certain advantages over the more common genetic signature approach because most anti-cancer agents are small molecules that target proteins and not genes, and many small molecules targeting specific molecular alterations are currently in pharmaceutical development.

Accordingly, the present disclosure relates to Hsp90 inhibitor-based chemical biology/proteomics approach that is integrated with bioinformatic analyses to discover oncogenic proteins and pathways. We show that the method can provide a tumor-by-tumor global overview of the Hsp90-dependent proteome in malignant cells which comprises many key signaling networks and is considered to represent a significant fraction of the functional malignant proteome.

The disclosure provides small-molecule probes that can affinity-capture Hsp90-dependent oncogenic client proteins. Additionally, the disclosure provides methods of harnessing the ability of the molecular probes to affinity-capture Hsp90-dependent oncogenic client proteins to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in different types of cancer. In one aspect, the disclosure provides small-molecule probes derived from Hsp90 inhibitors based on purine and purine-like (e.g., PU-H71 , MPC-3100, Debio 0932), isooxazole (e.g., NVP-AUY922) and indazol-4-one (e.g., SNX-21 12) chemical classes (see Figure 3). In one embodiment, the Hsp90 inhibitor is PU-H71 8-(6-Iodo-benzo[l ,3]dioxol-5-ylsulfanyl)-9-(3- isopropylamino-propyl)-9H-purin-6-ylamine, (see Figure 3). The PU-H71 molecules may be linked to a solid support (e.g., bead) through a tether or a linker. The site of attachment and the length of the tether were chosen to ensure that the molecules maintain a high affinity for Hsp90. In a particular embodiment, the PU-H71 -based molecular probe has the structure shown in Figure 30. Other embodiments of Hsp90 inhibitors attached to solid support are shown in Figures 32-35 and 38. It will be appreciated by those skilled in the art that the molecule maintains higher affinity for the oncogenic Hsp90 complex species than the housekeeping Hsp90 complex. The two Hsp90 species are as defined in Moulick et al, Nature chemical biology (201 1). When bound to Hsp90, the Hsp90 inhibitor traps Hsp90 in a client- protein bound conformation.

In another aspect, the disclosure provides methods of identifying specific oncoproteins associated with Hsp90 that are implicated in the development and progression of a cancer. Such methods involve contacting a sample containing cancer cells from a subject suffering from cancer with an inhibitor of Hsp90, and detecting the oncoproteins that are bound to the inhibitor of Hsp90. In particular embodiments, the inhibitor of Hsp90 is linked to a solid support, such as a bead. In these embodiments, oncoproteins that are harbored by the Hsp90 protein bound to the solid support can be eluted in a buffer and submitted to standard SDS- PAGE, and the eluted proteins can be separated and analyzed by traditional means. In some embodiments of the method the detection of oncoproteins comprises the use of mass spectroscopy. Advantageously, the methods of the disclosure do not require expensive SILAC labeling or two-dimensional separation of samples.

In certain embodiments of the invention the analysis of the pathway components comprises use of a bioinformatics computer program, for example, to define components of a network of such components.

The methods of the disclosure can be used to determining oncoproteins associated with various types of cancer, including but not limited to a breast cancer, a lung cancer including a small cell lung cancer and a non-small cell lung cancer, a cervical cancer, a colon cancer, a choriocarcinoma, a bladder cancer, a cervical cancer, a basal cell carcinomachoriocarcinoma, a colon cancer, a colorectal cancer, an endometrial cancer esophageal cancer, a gastric cancer, a head and neck cancer, a acute lymphocytic cancer (ACL), a myelogenous leukemia including an acute myeloid leukemia (AML) and a chronic myeloid chronic myeloid leukemia (CML), a multiple myeloma, a T-cell leukemia lymphoma, a liver cancer, lymphomas including Hodgkin's disease, lymphocytic lymphomas neuroblastomas follicular lymphoma and a diffuse large B-cell lymphoma, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a rectal cancer, sarcomas, skin cancers such as melanoma, a testicular cancer, a thyroid cancer, a renal cancer, myeloproliferative disorders, gastrointestinal cancers including gastrointestinal stromal tumors, an esophageal cancer, a stomach cancer, a gallbladder cancer, an anal cancer, brain tumors including gliomas, lymphomas including a follicular lymphoma and a diffuse large B-cell lymphoma. Additionally, the disclosure provides proteomic methods to identify dysregulated signaling networks associated with a particular cancer. In addition, the approach can be used to identify new oncoproteins and mechanisms.

In another aspect, the methods of the disclosure can be used to provide a rational basis for designing personalized therapy for cancer patients. A personalized therapeutic approach for cancer is based on the premise that individual cancer patients will have different factors that contribute to the development and progression of the disease. For instance, different oncogenic proteins and/or cancer- implicated pathways can be responsible for the onset and subsequent progression of the disease, even when considering patients with identical types at cancer and at identical stages of progression, as determined by currently available methods. Moreover, the oncoproteins and cancer-implicated pathways are often altered in an individual cancer patient as the disease progresses. Accordingly, a cancer treatment regimen should ideally be targeted to treat patients on an individualized basis. Therapeutic regimens determined from using such an individualized approach will allow for enhanced anti-tumor activity with less toxicity and with less chemotherapy or radiation.

Hence, in one aspect, the disclosure provides methods of identifying therapeutic regimens for cancer patients on an individualized basis. Such methods involve contacting a sample containing cancer cells from a subject suffering from cancer with an inhibitor of Hsp90, detecting the oncoproteins that are bound to the inhibitor of Hsp90, and selecting a cancer therapy that targets at least one of the oncoproteins bound to the inhibitor of Hsp90. In certain aspects, a combination of drugs can be selected following identification of oncoproteins bound to the Hsp90. The methods of the disclosure can be used to identify a treatment regimen for a variety of different cancers, including, but not limited to a breast cancer, a lung cancer, a brain cancer, a cervical cancer, a colon cancer, a choriocarcinoma, a bladder cancer, a cervical cancer, a choriocarcinoma, a colon cancer, an endometrial cancer an esophageal cancer, a gastric cancer, a head and neck cancer, an acute lymphocytic cancer (ACL), a myelogenous leukemia, a multiple myeloma, a T-cell leukemia lymphoma, a liver cancer, lymphomas including Hodgkin's disease and lymphocytic lymphomas neuroblastomas, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a rectal cancer, sarcomas, a skin cancer, a testicular cancer, a thyroid cancer and a renal cancer.

In another aspect, the methods involve contacting a sample containing cancer cells from a subject suffering from cancer with an inhibitor of Hsp90, detecting the oncoproteins that are bound to the inhibitor of Hsp90, determining the protein network(s) associated with these oncoproteins and selecting a cancer therapy that targets at least one of the molecules from the networks of the oncoproteins bound to the inhibitor of Hsp90.

In certain aspects, a combination of drugs can be selected following identification of oncoproteins bound to the Hsp90. In other aspects, a combination of drugs can be selected following identification of networks associated with the oncoproteins bound to the Hsp90. The methods of the disclosure can be used to identify a treatment regimen for a variety of different cancers, including, but not limited to a breast cancer, a lung cancer, a brain cancer, a cervical cancer, a colon cancer, a choriocarcinoma, a bladder cancer, a cervical cancer, a choriocarcinoma, a colon cancer, an endometrial cancer an esophageal cancer, a gastric cancer, a head and neck cancer, an acute lymphocytic cancer (ACL), a myelogenous leukemia, a multiple myeloma, a T-cell leukemia lymphoma, a liver cancer, lymphomas including Hodgkin's disease and lymphocytic lymphomas neuroblastomas, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a rectal cancer, sarcomas, a skin cancer, a testicular cancer, a thyroid cancer and a renal cancer.

In one embodiment of the present invention, after a personalized treatment regimen for a cancer patient is identified using the methods described above, the selected drugs or combination of drugs is administered to the patient. After a sufficient amount of time taking the selected drug or drug combination, another sample can be taken from the patient and the an assay of the present can be run again to determine if the oncogenic profile of the patient changed. If necessary, the dosage of the drug(s) can be changed or a new treatment regimen can be identified. Accordingly, the disclosure provides methods of monitoring the progress of a cancer patient over time and changing the treatment regimen as needed.

In another aspect, the methods of the disclosure can be used to provide a rational basis for designing personalized combinatorial therapy for cancer patients built around the Hsp90 inhibitors. Such therapeutic regimens may allow for enhanced anti-tumor activity with less toxicity and with less chemotherapy. Targeting Hsp90 and a complementary tumor-driving pathway may provide a better anti-tumor strategy since several lines of data suggest that the completeness with which an oncogenic target is inhibited could be critical for therapeutic activity, while at the same time limiting the ability of the tumor to adapt and evolve drug resistance.

Accordingly this invention provides a method for selecting an inhibitor of a cancer- implicated pathway, or of a component of a cancer-implicated pathway, for coadministration with an inhibitor of Hsp90, to a subject suffering from a cancer which comprises the following steps:

(a) contacting a sample containing cancer cells from the subject with (i) an inhibitor of Hsp90 which binds to Hsp90 when such Hsp90 is bound to cancer pathway components present in the sample; or (ii) an analog, homolog, or derivative of such Hsp90 inhibitor which binds to Hsp90 when such Hsp90 is bound to such cancer pathway components in the sample;

(b) detecting pathway components bound to Hsp90;

(c) analyzing the pathway components detected in step (b) so as to identify a pathway which includes the components detected in step (b) and additional components of such pathway; and

(d) selecting an inhibitor of the pathway or of a pathway component identified in step (c). In connection with the invention a cancer-implicated pathway is a pathway involved in metabolism, genetic information processing, environmental information processing, cellular processes, or organismal systems including any pathway listed in Table 1.

In the practice of this invention the cancer-implicated pathway or the component of the cancer-implicated pathway is involved with a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, thyroid cancer, a leukemia including acute myeloid leukemia and chronic myeloid leukemia, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, myeloproliferative disorders, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. For example the component of the cancer-implicated pathway and/or the pathway may be any component identified in Figure 1.

In a preferred embodiment involving personalized medicine in step (a) the subject is the same subject to whom the inhibitor of the cancer-implicated pathway or the component of the cancer-implicated pathway is to be administered although the invention in step (a) also contemplates the subject is a cancer reference subject.

In the practice of this invention in step (a) the sample comprises any tumor tissue or any biological fluid, for example, blood.

Suitable samples for use in the invention include, but are not limited to, disrupted cancer cells, lysed cancer cells, and sonicated cancer cells.

In connection with the practice of the invention the inhibitor of Hsp90 to be administered to the subject may be the same as or different from the (a) inhibitor of Hsp90 used, or (b) the inhibitor of Hsp90, the analog, homolog or derivative of the inhibitor of Hsp90 used, in step (a). In one embodiment, wherein the inhibitor of Hsp90 to be administered to the subject is PU- H71 or an analog, homolog or derivative of PU-H71 having the biological activity of PU- H71.

In another embodiment PU-H71 is the inhibitor of Hsp90 used, or is the inhibitor of Hsp90, the analog, homolog or derivative of which is used, in step (a). Alternatively, the inhibitor of Hsp90 may be selected from the group consisting of the compounds shown in Figure 3.

In one embodiment in step (a) the inhibitor of Hsp90 or the analog, homolog or derivative of the inhibitor of Hsp90 is preferred immobilized on a solid support, such as a bead.

In certain embodiments in step (b) the detection of pathway components comprises the use of mass spectroscopy, and in step (c) the analysis of the pathway components comprises use of a bioinformatics computer program.

In one example of the invention the cancer is a lymphoma, and in step (c) the pathway component identified is Syk. In another example, the cancer is a chronic myelogenous leukemia (CML) and in step (c) the pathway or the pathway component identified is a pathway or component shown in any of the Networks shown in Figure 15, for example one of the following pathway components identified in Figure 15, i.e. mTOR, IKK, MEK, NFKB, STAT3, STAT5A, STAT5B, Raf-1, bcr-abl, Btk, CARM1, or c-MYC. In one such example in step (c) the pathway component identified is mTOR and in step (d) the inhibitor selected is PP242. In another such example in step (c) the pathway identified is a pathway selected from the following pathways: PI3K/mTOR-, NFKB-, MAPK-, STAT-, FAK-, MYC and TGF-β mediated signaling pathways. In yet another example the cancer is a lymphoma, and in step (c) the pathway component identified is Btk. In a still further example the cancer is a pancreatic cancer, and in step (c) the pathway or pathway component identified is a pathway or pathway component shown in any of Networks 1-10 of Figure 16 and in those of Figure 24. In another example, in step (c) the pathway and pathway component identified is mTOR and in an example thereof in step (d) the inhibitor of mTOR selected is PP242. This invention further provides a method of treating a subject suffering from a cancer comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of a component of a cancer-implicated pathway which in (B) need not be but may be selected by the method described herein. Thus this invention provides a treatment method wherein coadministering comprises administering the inhibitor in (A) and the inhibitor in (B) simultaneously, concomitantly, sequentially, or adjunctive ly. One example of the method of treating a subject suffering from a cancer comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of Btk. Another example of the method of treating a subject suffering from a cancer which comprises coadministering to the subject (A) an inhibitor of Hsp90 and

(B) an inhibitor of Syk. In such methods the cancer may be a lymphoma. Another example of the method of treating a subject suffering from a chronic myelogenous leukemia (CML) comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of any of mTOR, IKK, MEK, NFKB, STAT3, STAT5A, STAT5B, Raf-1, bcr-abl, CARM1,

CAMKIl, or c-MYC. In an embodiment of the invention the inhibitor in (B) is an inhibitor of mTOR. In a further embodiment of the method described above in (a) binding of the inhibitor of Hsp90 or the analog, homolog, or derivative of such Hsp90 inhibitor traps Hsp90 in a cancer pathway components-bound state. Still further the invention provides a method of treating a subject suffering from a pancreatic cancer which comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of the pathway or of a pathway component shown in any of the Networks shown in Figure 16 and 24. This invention also provides a method of treating a subject suffering from a breast cancer which comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of the pathway or of a pathway component shown in any of the Networks shown in Figures 22. Still further this invention provides a method of treating a subject suffering from a lymphoma which comprises coadministering to the subject (A) an inhibitor of Hsp90 and (B) an inhibitor of the pathway or of a pathway component shown in any of the Networks shown in Figures 23. In the immediately preceeding methods the inhibitor in (B) may be an inhibitor of mTOR, e.g.

PP242. Still further this invention provides a method of treating a subject suffering from a chronic myelogenous leukemia (CML) which comprises administering to the subject an inhibitor of CARMl . In another embodiment this invention provides a method for identifying a cancer-implicated pathway or one or more components of a cancer-implicated pathway in a subject suffering from cancer which comprises:

(a) contacting a sample containing cancer cells from the subject with (i) an inhibitor of Hsp90 which binds to Hsp90 when such Hsp90 is bound to cancer pathway components present in the sample; or (ii) an analog, homolog, or derivative of such Hsp90 inhibitor which binds to Hsp90 when such Hsp90 is bound to such cancer pathway components in the sample; (b) detecting pathway components bound to Hsp90,

so as to thereby identify the cancer-implicated pathway or said one or more pathway components. In this embodiment the cancer-implicated pathway or the component of the cancer-implicated pathway may be involved with any cancer selected from the group consisting of colorectal cancer, pancreatic cancer, thyroid cancer, a leukemia including acute myeloid leukemia and chronic myeloid leukemia, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, myeloproliferative disorders, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. Further in step (a) the sample may comprise a tumor tissue or a biological fluid, e.g., blood. In certain embodiments in step (a) the sample may comprise disrupted cancer cells, lysed cancer cells, or sonicated cancer cells. However, cells in other forms may be used.

In the practice of this method the inhibitor of Hsp90 may be PU-H71 or an analog, homo log or derivative of PU-H71 although PU-H71 is currently a preferred inhibitor. In the practice of the invention, however the inhibitor of Hsp90 may be selected from the group consisting of the compounds shown in Figure 3. In an embodiment in step (a) the inhibitor of Hsp90 or the analog, homolog or derivative of the inhibitor of Hsp90 is immobilized on a solid support, such as a bead; and/or in step (b) the detection of pathway components comprises use of mass spectroscopy; and/or in step (c) the analysis of the pathway components comprises use of a bioinformatics computer program.

In one desirable embodiment of the invention in (a) binding of the inhibitor of Hsp90 or the analog, homolog, or derivative of such Hsp90 inhibitor traps Hsp90 in a cancer pathway components-bound state.

This invention further provides a kit for carrying out the method which comprises an inhibitor of Hsp90 immobilized on a solid support such as a bead. Typically, such a kit will further comprise control beads, buffer solution, and instructions for use. This invention further provides an inhibitor of Hsp90 immobilized on a solid support wherein the inhibitor is useful in the method described herein. One example is where the inhibitor is PU-H71. In another aspect this invention provides a compound having the structure:

Still further the invention provides a method for selecting an inhibitor of a cancer-implicated pathway or a component of a cancer-implicated pathway which comprises identifying the cancer-implicated pathway or one or more components of such pathway according to the method described and then selecting an inhibitor of such pathway or such component. In addition, the invention provides a method of treating a subject comprising selecting an inhibitor according to the method described and administering the inhibitor to the subject alone or in addition to administering the inhibitor of the pathway component. More typically said administering will be effected repeatedly. Still further the methods described for identifying pathway components or selecting inhibitors may be performed at least twice for the same subject. In yet another embodiment this invention provides a method for monitoring the efficacy of treatment of a cancer with an Hsp90 inhibitor which comprises measuring changes in a biomarker which is a component of a pathway implicated in such cancer. For example, the biomarker used may be a component identified by the method described herein. In addition, this invention provides a method for monitoring the efficacy of a treatment of a cancer with both an Hsp90 inhibitor and a second inhibitor of a component of the pathway implicated in such cancer which Hsp90 inhibits which comprises monitoring changes in a biomarker which is a component of such pathway. For example, the biomarker used may be the component of the pathway being inhibited by the second inhibitor. Finally, this invention provides a method for identifying a new target for therapy of a cancer which comprises identifying a component of a pathway implicated in such cancer by the method described herein, wherein the component so identified has not previously been implicated in such cancer. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts exemplary cancer-implicated pathways in humans and components thereof. Figure 2 shows several examples of protein kinase inhibitors.

Figure 3 shows the structure of PU-H71 and several other known Hsp90 inhibitors.

Figure 4. PU-H71 interacts with a restricted fraction of Hsp90 that is more abundant in cancer cells, (a) Sequential immuno-purification steps with H9010, an anti-Hsp90 antibody, deplete Hsp90 in the MDA-MB-468 cell extract. Lysate = control cell extract, (b) Hsp90 from MDA-MB-468 extracts was isolated through sequential chemical- and immuno- purification steps. The amount of Hsp90 in each pool was quantified by densitometry and values were normalized to an internal standard, (c) Saturation studies were performed with ¹³¹ I-PU-H71 in the indicated cells. All the isolated cell samples were counted and the specific uptake of ¹³¹ I-PU-H71 determined. These data were plotted against the concentration of ¹³ I- PU-H71 to give a saturation binding curve. Representative data of four separate repeats is presented {lower). Expression of Hsp90 in the indicated cells was analyzed by Western blot (upper). Figure 5. PU-H71 is selective for and isolates Hsp90 in complex with onco-proteins and co- chaperones. (a) Hsp90 complexes in K562 extracts were isolated by precipitation with H9010, a non-specific IgG, or by PU-H71- or Control-beads. Control beads contain ethanolamine, an Hsp90-inert molecule. Proteins in pull-downs were analyzed by Western blot. (b,c) Single or sequential immuno- and chemical-precipitations, as indicated, were conducted in K562 extracts with H9010 and PU-beads at the indicated frequency and in the shown sequence. Proteins in the pull-downs and in the remaining supernatant were analyzed by WB. NS = non-specific, (d) K562 cell were treated for 24h with vehicle (-) or PU-H71 (+), and proteins analyzed by Western blot, (e) Expression of proteins in Hsp70-knocked- down cells was analyzed by Western blot {left) and changes in protein levels presented in relative luminescence units (RLU) {right). Control = scramble siRNA. (f) Sequential chemical-precipitations, as indicated, were conducted in K562 extracts with GM-, SNX- and

NVP -beads at the indicated frequency and in the shown sequence. Proteins in the pull-downs and in the remaining supernatant were analyzed by Western blot, (g) Hsp90 in K562 cells exists in complex with both aberrant, Bcr-Abl, and normal, c-Abl, proteins. PU-H71 , but not H9010, selects for the Hsp90 population that is Bcr-Abl onco-protein bound.

Figure 6. PU-H71 identifies the aberrant signalosome in CML cells, (a) Protein complexes were isolated through chemical precipitation by incubating a K562 extract with PU-beads, and the identity of proteins was probed by MS. Connectivity among these proteins was analyzed in IP A, and protein networks generated. The protein networks identified by the PU- beads (Networks 1 through 13) overlap well with the known canonical myeloid leukemia signaling (provided by IP A). A detailed list of identified protein networks and component proteins is shown in Table 5f and Figure 15. (b) Pathway diagram highlighting the PU-beads identified CML signalosome with focus on Networks 1 (Raf-MAPK and PI3K-AKT pathway), 2 (NF-κΒ pathway) and 8 (STAT5-pathway). Key nodal proteins in the identified networks are depicted in yellow, (c) MS findings were validated by Western blot, (left) Protein complexes were isolated through chemical precipitation by incubating a K562 extract with PU- or control-beads, and proteins analyzed by Western blot. No proteins were detected in the Control-bead pull-downs and those data are omitted for simplicity of presentation. (right) K562 cell were treated for 24h with vehicle (-) or PU-H71 (+), and proteins were analyzed by WB. (d) Single chemical-precipitations were conducted in primary CML cell extracts with PU- and Control-beads. Proteins in the pull-downs were analyzed by WB.

Figure 7. PU-H71 identified proteins and networks are those important for the malignant phenotype. (a) K562 cells were treated for 72 h with the indicated inhibitors and cell growth analyzed by the Alamar Blue assay. Data are presented as means ± SD (n = 3). (b) Sequential chemical-precipitations, as indicated, were conducted in K562 extracts with the PU-beads at the indicated frequency. Proteins in the pull-downs and in the remaining supernatant were analyzed by WB. (c) The effect of CARM1 knock-down on cell viability using Tryptan blue (left) or Acridine orange/Ethidium bromide (right) stainings was evaluated in K562 cells, (d) The expression of select potential Hsp90-interacting proteins was analyzed by WB in K562 leukemia and Mia-PaCa-2 pancreatic cancer cells, (e) Select proteins isolated on PU-beads from K562 and Mia-PaCa-2 cell extracts, respectively, and subsequently identified by MS were tabulated. +++, very high; ++, high; +, moderate and -, no identifying peptides were found in MS analyses, (f) Single chemical-precipitations were conducted in Mia-PaCa-2 cell extracts with PU- and Control-beads. Proteins in the pull-downs were analyzed by WB. (g)

The effect of select inhibitors on Mia-PaCa-2 cell growth was analyzed as in panel (a). Figure 8. Hsp90 facilitates an enhanced STAT5 activity in CML. (a) K562 cells were treated for the indicated times with PU-H71 (5 μΜ), Gleevec (0.5 μΜ) or DMSO (vehicle) and proteins analyzed by WB. (b) Sequential chemical-precipitations were conducted in K562 cells with PU- and Control-beads, as indicated. Proteins in the pull-downs and in the remaining supernatant were analyzed by WB. (c) STAT5 immuno-complexes from cells pre- treated with vehicle or PU-H71 were treated for the indicated times with trypsin and proteins analyzed by WB. (d) K562 cells were treated for the indicated times with vanadate (1 mM) in the presence and absence of PU-H71 (5 μΜ). Proteins were analyzed by WB {upper), quantified by densitometry and graphed against treatment time {lower). Data are presented as means ± SD (n = 3). (e) The DNA-binding capacity of STAT5a and STAT5b was assayed by an ELISA-based assay in K562 cells treated for 24h with indicated concentrations of PU- H71. (f) Quantitative chromatin immunoprecipitation assays (QChIP) performed with STAT5 or Hsp90 antibodies vs. IgG control for two known STAT5 target genes {CCND2 and MYC). A primer that amplifies an intergenic region was used as negative control. Results are expressed as percentage of the input for the specific antibody (STAT5 or Hsp90) over the respective IgG control, (g) The transcript abundance of CCND2 and MYC was measured by QPCR in K562 cells exposed to 1 μΜ of PU-H71. Results are expressed as fold change compared to baseline (time 0 h) and were normalized to RPL13A. HPRT was used as negative control. Experiments were carried out in biological quintuplicates with experimental duplicates. Data are presented as means ± SEM. (h) Proposed mechanism for and Hsp90- facilitated increased STAT5 signaling in CML. Hsp90 binds to and influences the conformation of STAT5 and maintains STAT5 in an active conformation directly within STAT5 -containing transcriptional complexes.

Figure 9. Schematic representation of the chemical-proteomics method for surveying tumor oncoproteins. Hsp90 forms biochemically distinct complexes in cancer cells. A major fraction of cancer cell Hsp90 retains "house keeping" chaperone functions similar to normal cells (green), whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival (yellow). PU-H71 specifically interacts with Hsp90 and preferentially selects for oncoprotein (yellow)/Hsp90 species but not WT protein (green)/Hsp90 species, and traps Hsp90 in a client binding conformation. The PU-H71 beads therefore can be used to isolate the onco-protein/Hsp90 species. In an initial step, the cancer cell extract is incubated with the PU-H71 beads (1). This initial chemical precipitation step purifies and enriches the aberrant protein population as part of PU-bead bound Hsp90 complexes (2). Protein cargo from PU- bead pull-downs is then eluted in SDS buffer, submitted to standard SDS-PAGE (3), and then the separated proteins are extracted and trypsinized for LC/MS/MS analyses (4). Initial protein identification is performed using the Mascot search engine, and is further evaluated using Scaffold Proteome Software (5). Ingenuity Pathway Analysis (IP A) is then used to build biological networks from the identified proteins (6,7). The created protein network map provides an invaluable template to develop personalized therapies that are optimally effective for a specific tumor. The method may (a) establish a map of molecular alterations in a tumor- by-tumor manner, (b) identify new oncoproteins and cancer mechanisms (c) identify therapeutic targets complementary to Hsp90 and develop rationally combinatorial targeted therapies and (d) identify tumor-specific biomarkers for selection of patients likely to benefit from Hsp90 therapy and for pharmacodynamics monitoring of Hsp90 inhibitor efficacy during clinical trials

Figure 10. (a,b) Hsp90 from breast cancer and CML cell extracts (120 μg) was isolated through serial chemical- and immuno-purification steps, as indicated. The supernatant was isolated to analyze the left-over Hsp90. Hsp90 in each fraction was analyzed by Western blot. Lysate = endogenous protein content; PU-, GM- and Control-beads indicate proteins isolated on the particular beads. H9010 and IgG indicate protein isolated by the particular Ab. Control beads contain an Hsp90 inert molecule. The data are consistent with those obtained from multiple repeat experiments (n > 2). (c) Sequential chemical- and immuno-purification steps were performed in peripheral blood leukocyte (PBL) extracts (250 μg) to isolate PU-H71 and H9010-specific Hsp90 species. All samples were analyzed by Western blot, {upper). Binding to Hsp90 in PBL was evaluated by flow cytometry using an Hsp90-PE antibody and PU-H71- FITC. FITC-TEG = control for non-specific binding {lower).

Figure 11. (a) Within normal cells, constitutive expression of Hsp90 is required for its evolutionarily conserved housekeeping function of folding and translocating cellular proteins to their proper cellular compartment ("housekeeping complex"). Upon malignant transformation, cellular proteins are perturbed through mutations, hyperactivity, retention in incorrect cellular compartments or other means. The presence of these functionally altered proteins is required to initiate and maintain the malignant phenotype, and it is these oncogenic proteins that are specifically maintained by a subset of stress modified Hsp90 ("oncogenic complex"). PU-H71 specifically binds to the fraction of Hsp90 that chaperones oncogenic proteins ("oncogenic complex"), (b) Hsp90 and its interacting co-chaperones were isolated in K562 cell extracts using PU- and Control-beads, and H9010 and IgG-immobilized Abs. Control beads contain an Hsp90 inert molecule, (c) Hsp90 from K562 cell extracts was isolated through three serial immuno-purification steps with the H9010 Hsp90 specific antibody. The remaining supernatant was isolated to analyze the left-over proteins. Proteins in each fraction were analyzed by Western blot. Lysate = endogenous protein content. The data are consistent with those obtained from multiple repeat experiments (n > 2).

Figure 12. GM and PU-H71 are selective for aberrant protein/Hsp90 species, (a) Bcr-Abl and Abl bound Hsp90 species were monitored in experiments where a constant volume of PU-H71 beads (80 μί) was probed with indicated amounts of K562 cell lysate (left), or where a constant amount of lysate (1 mg) was probed with the indicated volumes of PU-H71 beads (right), (b) (left) PU- and GM-beads (80 uL) recognize the Hsp90-mutant B-Raf complex in the SKMel28 melanoma cell extract (300 μg), but fail to interact with the Hsp90- WT B-Raf complex found in the normal colon fibroblast CCDI 8C0 extracts (300 μg). H9010 Hsp90 Ab recognizes both Hsp90 species, (c) In MDA-MB-468 cell extracts (300 μg), PU- and GM-beads (80 μΐ) interact with HER3 and Raf-1 kinase but not with the non-oncogenic tyrosine-protein kinase CSK, a c-Src related tyrosine kinase, and p38. (d) (right) PU-beads (80 μΐ,) interact with v-Src/Hsp90 but not c-Src/Hsp90 species. To facilitate c-Src detection, a protein in lower abundance than v-Src, higher amounts of c-Src expressing 3T3 cell lysate (1 ,000 μg) were used when compared to the v-Src transformed 3T3 cell (250 μg), providing explanation for the higher Hsp90 levels detected in the 3T3 cells (Lysate, 3T3 fibroblasts vs v-Src 3T3 fibroblasts). Lysate = endogenous protein content; PU-, GM- and Control-beads indicate proteins isolated on the particular beads. Hsp90 Ab and IgG indicate protein isolated by the particular Ab. Control beads contain an Hsp90 inert molecule. The data are consistent with those obtained from multiple repeat experiments (n > 2). Figure 13. Single chemical-precipitations were conducted in Bcr-Abl-expressing CML cell lines (a) and in primary CML cell extracts (b) with PU- and Control-beads. Proteins in the pull-downs were analyzed by Western blot. Several Bcr-Abl cleavage products are noted in the primary CML samples as reported (Dierov et al., 2004). N/A = not available. Figure 14. PU-H71 is selective for Hsp90. (a) Coomassie stained gel of several Hsp90 inhibitor bead-pulldowns. K562 lysates (60 μg) were incubated with 25 μΙ_, of the indicated beads. Following washing with the indicated buffer, proteins in the pull-downs were applied to an SDS-PAGE gel. (b) PU-H71 (10 μΜ) was tested in the scanMAX screen (Ambit) against 359 kinases. The TKEEspot™ Interaction Map for PU-H71 is presented. Only SNAR (NUAK family SNFl-like kinase 2) (red dot on the kinase tree) appears as a potential low affinity kinase hit of the small molecule.

Figure 15. Top scoring networks enriched on the PU-beads and as generated by bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IP A) software. Analysis was performed in the K562 chronic myeloid leukemia cells, (a) Network 1; Score = 38; mTOR/PI3K and MAPK pathways, (b) Network 2; Score = 36; NFKB pathway, (c) Network 8; Score = 14; STAT pathway, (d) Network 12; Score = 13; Focal adhesion network, (e) Network 7; Score = 22; c-MYC oncogene driven pathway, (f) Network 10; Score = 18; TGF pathway. Scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone.

Gene expression, cell cycle and cellular assembly Individual proteins are displayed as nodes, utilizing gray to represent that the protein was identified in this study. Proteins identified by IPA only are represented as white nodes. Different shapes are used to represent the functional class of the gene product. Proteins are depicted in networks as two circles when the entity is part of a complex; as a single circle when only one unit is present; a triangle pointing up or down to describe a phosphatase or a kinase, respectively; by a horizontal oval to describe a transcription factor; and by circle to depict "other" functions. The edges describe the nature of the relationship between the nodes: an edge with arrow-head means that protein A acts on protein B, whereas an edge without an arrow-head represents binding only between two proteins. Direct interactions appear in the network diagram as a solid line, whereas indirect interactions as a dashed line. In some cases a relationship may exist as a circular arrow or line originating from one molecule and pointing back at that same molecule. Such relationships are termed "self-referential" and arise from the ability of a molecule to act upon itself. Figure 16. Top scoring networks enriched on the PU-beads and as generated by bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IP A) software. Analysis was performed in the MiaPaCa2 pancreatic cancer cells. Figure 17. The mTOR inhibitor PP242 synergizes with the Hsp90 inhibitor PU-H71 in Mia- PaCa-2 cells. Pancreatic cells (Mia-PaCa-2) were treated for 72h with single agent or combinations of PP242 and PU-H71 and cytotoxicity determined by the Alamar blue assay. Computerized simulation of synergism and/or antagonism in the drug combination studies was analyzed using the Chou-Talalay method, (a) In the median-effect equation, fa is the fraction of affected cells, e.g. fractional inhibition; fu=(l-fa) which is the fraction of unaffected cells; D is the dose required to produce fa. (b) Based on the actual experimental data, serial CI values were calculated for an entire range of effect levels (Fa), to generate Fa- CI plots. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively, (c) Normalized isobologram showing the normalized dose of Drug 1 (PU-H71) and Drug2 (PP242). PU = PU-H71, PP = PP242.

Quantitative analysis of synergy between mTOR and Hsp90 inhibitors: To determine the drug interaction between pp242 (mTOR inhibitor) and PU-H71 (Hsp90 inhibitor), the combination index (CI) isobologram method of Chou-Talalay was used as previously described. This method, based on the median-effect principle of the law of mass action, quantifies synergism or antagonism for two or more drug combinations, regardless of the mechanisms of each drug, by computerized simulation. Based on algorithms, the computer software displays median-effect plots, combination index plots and normalized isobolograms (where non constant ratio combinations of 2 drugs are used). PU-H71 (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0125 μΜ) and pp242 (0.5, 0.125, 0.03125, 0.0008, 0.002, 0.001 μΜ) were used as single agents in the concentrations mentioned or combined in a non constant ratio (PU-H71 : pp242; 1 : 1, 1 :2, 1 :4, 1 :7.8, 1 : 15.6, 1 : 12.5). The Fa (fraction killed cells) was calculated using the formulae Fa=l-Fu; Fu is the fraction of unaffected cells and was used for a dose effect analysis using the computer software (CompuSyn, Paramus,New Jersey, USA).

Figure 18. Bcl-6 is a client of Hsp90 in Bcl-6 dependent DLBCL cells and the combination of an Hsp90 inhibitor with a Bcl-6 inhibitor is more efficacious than each inhibitor alone, a)

Cells were treated for 24h with the indicated concentration of PU-H71 and proteins were analyzed by Western blot, b) PU-H71 beads indicate that Hsp90 interacts with Bcl-6 in the nucleus, c) the the combination of the Hsp90 inhibitor PU-H71 with the Bcl-6 inhibitor RI- BPI is more efficacious in Bcl-6 dependent DLBCL cells than each inhibitor alone

Figure 19. Several repeats of the method of the invention identify the B cell receptor network as a major pathway in the OCI-Lyl cells to demonstrate and validate the robustmenss and accuracy of the method

Figure 20. Validation of the B cell receptor network as an Hsp90 dependent network in OCI- LY1 and OCI-LY7 DLBCL cells, a) cells were treated with the Hsp90 inhibitor PU-H71 and proteins analyzed by Western blot, b) PU-H71 beads indicate that Hsp90 interacts with BTK and SYK in the OCI-LY1 and OCI-LY7 DLBCL cells, c) the the combination of the Hsp90 inhibitor PU-H71 with the SYK inhibitor R406 is more efficacious in the Bcl-6 dependent OCI-LY1, OCI-LY7, Farage and SUDHL6 DLBCL cells than each inhibitor alone Figure 21. The CAMKII inhibitor KN93 and the mTOR inhibitor PP242 synergize with the Hsp90 inhibitor PU-H71 in K562 CML cells.

Figure 22. Top scoring networks enriched on the PU-beads and as generated by

bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IP A) software. Analysis was performed in the MDA-MB-468 triple-negative breast cancer cells. Major signaling networks identified by the method were the PI3K/AKT, IGF-IR, NRF2- mediated oxidative stress response, MYC, PKA and the IL-6 signaling pathways, (a)

Simplified representation of networks identified in the MDA-MB-468 breast cancer cells by the PU-beads proteomics and bioinformatic method, (b) IL-6 pathway. Key network components identified by the PU-beads method in M DA-MB-468 breast cancer cells are depicted in grey.

Figiire 23. Top scoring networks enriched on the PU-beads and as generated by

bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IP A) software. Analysis was performed in the OCI-Lyl diffuse large B cell lymphoma (DLBCL) cells. In the Diffuse large B-cel! lymphoma (DLBCL) eel! line OCI-LYL major signaling networks identified by the method were the B receptor, PKCteta, PBK/AKT, CD40, CD28 and the ERK/MAPK signaling pathways, (a) B cell receptor pathway. Key network components identified by the PU-beads method are depicted in grey, (b) CD40 signaling pathway. Key network components identified by the PU-beads method are depicted in grey. (c) CD28 signaling pathway. Key network components identified by the PU-heads method are depicted in grey.

Figure 24, Top scoring networks enriched on the PU-beads and as generated by bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IP A) software. Analysis was performed in the Mia-PaCa-2 pancreatic cancer cells, (a) PU-beads identify the aberrant signalosome in Mia-PaCa-2 cancer cells. Among the protein pathways identified by the PU-beads are those of the PBK-Akt-mTOR-NFkB-pathway, TGF-beta pathway, Wnt-beta-catenin pathway, PKA-pathway, STAT3 -pathway, JNK-pathway and the Rac-cdc42-ras-ERK pathway, (b) Cell cycle-G2/M DNA damage checkpoint regulation. Key network components identified by the PU-beads method are depicted in grey.

Figure 25. PU-H71 synergizes with the PARP inhibitor olaparib in inhibiting the clonogenic survival of MDA-MB-468 (upper panels) and the HCC1937 (lower panel) breast cancer cells. Figure 26. Structures of Hsp90 inhibitors.

Figure 27. A) Interactions of Hsp90a (PDB ID: 2FWZ) with PU-H71 (ball and stick model) and compound 5 (tube model). B) Interactions of Hsp90a (PDB ID: 2VCI) with NVP- AUY922 (ball and stick model) and compound 10 (tube model). C) Interactions of Hsp90a (PDB ID: 3D0B) with compound 27 (ball and stick model) and compound 20 (tube model). Hydrogen bonds are shown as dotted yellow lines and important active site amino acid residues and water molecules are represented as sticks.

Figure 28. A) Hsp90 in K562 extracts (250 μg) was isolated by precipitation with PU-, SNX- and NVP -beads or Control-beads (80 μΐ _^ ). Control beads contain 2-methoxyethylamine, an Hsp90-inert molecule. Proteins in pull-downs were analyzed by Western blot. B) In MDA- MB-468 cell extracts (300 μg), PU-beads isolate Hsp90 in complex with its onco-client proteins, c-Kit and IGF-IR. To evaluate the effect of PU-H71 on the steady-state levels of Hsp90 onco-client proteins, cells were treated for 24 h with PU-H71 (5 μΜ). C) In K562 cell extracts, PU-beads (40 μί) isolate Hsp90 in complex with the Raf-1 and Bcr-Abl oncoproteins. Lysate = endogenous protein content; PU- and Control-beads indicate proteins isolated on the particular beads. The data are consistent with those obtained from multiple repeat experiments (n > 2). Figure 29. A) Hsp90-containing protein complexes from the brains of JNPL3 mice, an Alzheimer's disease transgenic mouse model, isolated through chemical precipitation with beads containing a streptavidin-immobilized PU-H71-biotin construct or control streptavidin- immobilized D-biotin. Aberrant tau species are indicated by arrow, cl, c2 and si, s2, cortical and subcortical brain homogenates, respectively, extracted from 6-month-old female JNPL3 mice {Right). Western blot analysis of brain lysate protein content {Left). B) Cell surface Hsp90 in MV4-11 leukemia cells as detected by PU-H71-biotin. The data are consistent with those obtained from multiple repeat experiments (n > 2).

Figure 30. Synthesis of PU-H71 beads (6). Figure 31. Synthesis of PU-H71-biotin (7). Figure 32. Synthesis of NVP-AUY922 beads (11). Figure 33. Synthesis of SNX-2112 beads (21). Figure 34. Synthesis of SNX-2112.

Figure 35. Synthesis of purine and purine-like Hsp90 inhibitor beads. Both the pyrimidine and imidazopyridine (i.e X= N or CH) type inhibitors are described. Reagents and conditions: (a) CS ₂ CO ₃ , 1 ,2-dibromoethane or 1,3-dibromopropane, DMF, rt; (b) NH ₂ (CH ₂ )6NHBoc, DMF, rt, 24 h; (c) TFA, CH ₂ C1 ₂ , rt, 1 h; (d) Affigel-10, DIEA, DMAP, DMF.

9-(2-Bromoethyl)-8-(6-(dimethylamino)benzo[d] [l,3]dioxol-5-ylthio)-9H-purin-6-amine (2a). la (29 mg, 0.0878 mmol), Cs ₂ C0 ₃ (42.9 mg, 0.1317 mmol), 1 ,2-dibromoethane (82.5 mg, 37.8 μί, 0.439 mmol) in DMF (0.6 mL) was stirred for 1.5 h at rt. Then additional Cs ₂ C0 ₃ (14 mg, 0.043 mmol) was added and the mixture stirred for an additional 20 min. The mixture was dried under reduced pressure and the residue purified by preparatory TLC (CH ₂ Cl ₂ :MeOH:AcOH, 15: 1 :0.5) to give 2a (24 mg, 63%). 1H NMR (500 MHz,

CDCl ₃ /MeOH-<¾) δ 8.24 (s, 1H), 6.81 (s, 1H), 6.68 (s, 1H), 5.96 (s, 2H), 4.62 (t, J= 6.9 Hz, 2H), 3.68 (t, J= 6.9 Hz, 2H), 2.70 (s, 6H); MS (ESI) m/z 437.2/439.1 [M+H] ⁺ . tei-i-Butyl (6-((2-(6-amino-8-((6-(dimethylamino)benzo[d] [l,3]dioxol-5-yl)thio)-9H- purin-9-yl)ethyl)amino)hexyl)carbamate (3a). 2a (0.185 g, 0.423 mmol) and tert-butyl 6- aminohexylcarbamate (0.915 g, 4.23 mmol) in DMF (7 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH:MeOH- NH ₃ (7N), 100:7:3] to give 0.206 g (85%) of 3a; MS (ESI) m/z 573.3 [M+H] ⁺ .

(4a). 3a (0.258 g, 0.45 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue dried under high vacuum overnight. This was dissolved in DMF (12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed, 3 x 50 mL DMF) in a solid phase peptide synthesis vessel. 225 of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (0.085 g, 97 μΐ, 1.13 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ Cl ₂ :Et ₃ N (9: 1, 4 x 50 mL), DMF (3 x 50 mL), Felts buffer (3 x 50 mL) and z ^' -PrOH (3 x 50 mL). The beads 4a were stored in z ^' -PrOH (beads: z-PrOH (1 :2), v/v) at -80°C.

9-(3-Bromopropyl)-8-(6-(dimethylamino)benzo[d] [l,3]dioxol-5-ylthio)-9H-purin-6- amine (2b). la (60 mg, 0.1818 mmol), Cs ₂ C0 ₃ (88.8 mg, 0.2727 mmol), 1,3- dibromopropane (184 mg, 93 uL, 0.909 mmol) in DMF (2 mL) was stirred for 40 min. at rt. The mixture was dried under reduced pressure and the residue purified by preparatory TLC (CH ₂ Cl ₂ :MeOH:AcOH, 15: 1 :0.5) to give 2b (60 mg, 73%). 1H NMR (500 MHz, CDC1 ₃ ) δ 8.26 (s, 1H), 6.84 (br s, 2H), 6.77 (s, 1H), 6.50 (s, 1H), 5.92 (s, 2H), 4.35 (t, J= 7.0 Hz, 2H), 3.37 (t, J= 6.6 Hz, 2H), 2.68 (s, 6H), 2.34 (m, 2H); MS (ESI) m/z 451.1/453.1 [M+H] ⁺ . ieri-Buty\ (6-((3-(6-amino-8-((6-(dimethylamino)benzo[d] [l,3]dioxol-5-yl)thio)-9H- purin-9-yl)propyl)amino)hexyl)carbamate (3b). 2b (0.190 g, 0.423 mmol) and tert-butyl 6- aminohexylcarbamate (0.915 g, 4.23 mmol) in DMF (7 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH:MeOH- NH ₃ (7N), 100:7:3] to give 0.218 g (88%) of 3b; MS (ESI) m/z 587.3 [M+H] ⁺ .

(4b). 3b (0.264 g, 0.45 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue dried under high vacuum overnight. This was dissolved in DMF (12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed, 3 x 50 mL DMF) in a solid phase peptide synthesis vessel. 225 μΐ, of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (0.085 g, 97 μΐ, 1.13 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ Cl ₂ :Et ₃ N (9: 1, 4 x 50 mL), DMF (3 x 50 mL), Felts buffer (3 x 50 mL) and z ^' -PrOH (3 x 50 mL). The beads 4b were stored in z ^' -PrOH (beads: z-PrOH (1 :2), v/v) at -80°C. l-(2-Bromoethyl)-2-((6-(dimethylamino)benzo[d] [l,3]dioxol-5-yl)thio)-lH-imidazo[4,5- c]pyridin-4-amine (5a). lb (252 mg, 0.764 mmol), Cs ₂ C0 ₃ (373 mg, 1.15 mmol), 1,2- dibromoethane (718 mg, 329 μί, 3.82 mmol) in DMF (2 mL) was stirred for 1.5 h at rt. Then additional Cs ₂ C0 ₃ (124 mg, 0.38 mmol) was added and the mixture stirred for an additional 20 min. The mixture was dried under reduced pressure and the residue purified by

preparatory TLC (CH ₂ Cl ₂ :MeOH, 10: 1) to give 5a (211 mg, 63%); MS (ESI) m/z 436.0/438.0 [M+H] ⁺ . tert-Butyl (6-((2-(4-amino-2-((6-(dimethylamino)benzo[d] [l,3]dioxol-5-yl)thio)-lH- imidazo[4,5-c]pyridin-l-yl)ethyl)amino)hexyl)carbamate (6a). 5a (0.184 g, 0.423 mmol) and tert-butyl 6-aminohexylcarbamate (0.915 g, 4.23 mmol) in DMF (7 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed

[CHCl ₃ :MeOH:MeOH-NH ₃ (7N), 100:7:3] to give 0.109 g (45%) of 6a; MS (ESI) m/z 572.3 [M+H] ⁺ .

(7a). 6a (0.257 g, 0.45 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue dried under high vacuum overnight. This was dissolved in DMF (12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed, 3 x 50 mL DMF) in a solid phase peptide synthesis vessel. 225 μΐ, of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (0.085 g, 97 μΐ, 1.13 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ Cl ₂ :Et ₃ N (9: 1, 4 x 50 mL), DMF (3 x 50 mL), Felts buffer (3 x 50 mL) and z ^' -PrOH (3 x 50 mL). The beads 7a were stored in z ^' -PrOH (beads: z-PrOH (1 :2), v/v) at -80°C. The beads 7b were prepared in a similar manner as described above for 7a.

Figure 36. Synthesis of biotinylated purine and purine-like Hsp90 inhibitors. Reagents and conditions: (a) EZ-Link ^® Amine-PE0 ₃ -Biotin, DMF, rt.

(8a). 2a (3.8 mg, 0.0086 mmol) and EZ-Link ^® Amine-PE0 ₃ -Biotin (5.4 mg, 0.0129 mmol) in DMF (0.2 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH-NH ₃ (7N), 10: 1] to give 2.3 mg (35%) of 8a. MS (ESI): m/z 775.2 [M+H] ⁺ .

(9a). 5a (3.7 mg, 0.0086 mmol) and EZ-Link ^® Amine-PE0 ₃ -Biotin (5.4 mg, 0.0129 mmol) in DMF (0.2 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH-NH ₃ (7N), 10: 1] to give 1.8 mg (27%) of 9a. MS (ESI): m/z 774.2 [M+H] ⁺ .

Biotinylated compounds 8b and 9b were prepared in a similar manner from 2b and 5b, respectively. Figure 37. Synthesis of biotinylated purine and purine-like Hsp90 inhibitors. Reagents and conditions: (a) N-(2-bromoethyl)-phthalimide or N-(3-bromopropyl)-phthalimide, Cs ₂ C0 ₃ , DMF, rt; (b) hydrazine hydrate, MeOH, CH ₂ C1 ₂ , rt; (c) EZ-Link ^® NHS-LC-LC-Biotin, DIEA, DMF, rt; (d) EZ-Link ^® NHS-PEG ₄ -Biotin, DIEA, DMF, rt. 2-(3-(6-Amino-8-(6-(dimethylamino)benzo[d] [l,3]dioxol-5-ylthio)-9H-purin-9- yl)propyl)isoindoline-l,3-dione. la (0.720 g, 2.18 mmol), Cs ₂ C0 ₃ (0.851 g, 2.62 mmol), 2- (3-bromopropyl)isoindoline-l,3-dione (2.05 g, 7.64 mmol) in DMF (15 mL) was stirred for 2 h at rt. The mixture was dried under reduced pressure and the residue purified by column chromatography (CH ₂ Cl ₂ :MeOH:AcOH, 15: 1 :0.5) to give 0.72 g (63%) of the titled compound. 1H NMR (500 MHz, CDCl ₃ /MeOH-d ₄ ): δ 8.16 (s, 1H), 7.85-7.87 (m, 2H), 7.74- 7.75 (m, 2H), 6.87 (s, 1H), 6.71 (s, 1H), 5.88 (s, 2H), 4.37 (t, J= 6.4 Hz, 2H), 3.73 (t, J= 6.1 Hz, 2H), 2.69 (s, 6H), 2.37-2.42 (m, 2H); HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₂₅ H ₂₄ N ₇ 0 ₄ S, 518.1610; found 518.1601. 9-(3-Aminopropyl)-8-(6-(dimethylamino)benzo[d] [l,3]dioxol-5-ylthio)-9H-purin-6- amine (10b). 2-(3-(6-Amino-8-(6-(dimethylamino)benzo[d][l,3]dioxol-5-ylth io)-9H-purin-9- yl)propyl)isoindoline-l,3-dione (0.72 g, 1.38 mmol), hydrazine hydrate (2.86 g, 2.78 mL, 20.75 mmol), in CH ₂ Cl ₂ :MeOH (4 mL:28 mL) was stirred for 2 h at rt. The mixture was dried under reduced pressure and the residue purified by column chromatography (CH ₂ Cl ₂ :MeOH- NH ₃ (7N), 20: 1) to give 430 mg (80%) of 10b. 1H NMR (500 MHz, CDC1 ₃ ): δ 8.33 (s, 1H), 6.77 (s, 1H), 6.49 (s, 1H), 5.91 (s, 2H), 5.85 (br s, 2H), 4.30 (t, J= 6.9 Hz, 2H), 2.69 (s, 6H), 2.65 (t, J= 6.5 Hz, 2H), 1.89-1.95 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 154.5, 153.1, 151.7, 148.1, 147.2, 146.4, 144.8, 120.2, 120.1, 109.3, 109.2, 101.7, 45.3, 45.2, 40.9, 38.6, 33.3; HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₁₇ H ₂₂ N ₇ O ₂ S, 388.1556; found 388.1544.

(12b). 10b (13.6 mg, 0.0352 mmol), EZ-Link ^® NHS-LC-LC-Biotin (22.0 mg, 0.0387 mmol) and DIEA (9.1 mg, 12.3 μί, 0.0704 mmol) in DMF (0.5 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1) to give 22.7 mg (77%) of 12b. MS (ESI): m/z 840.2 [M+H] ⁺ .

(14b). 10b (14.5 mg, 0.0374 mmol), EZ-Link ^® NHS-PEG ₄ -Biotin (24.2 mg, 0.0411 mmol) and DIEA (9.7 mg, 13 μΐ,, 0.0704 mmol) in DMF (0.5 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1) to give 24.1 mg (75%) of 14b. MS (ESI): m/z 861.3 [M+H] ⁺ . Biotinylated compounds 12a, 13a, 13b, 14a, 15a and 15b were prepared in a similar manner as described for 12b and 14b.

Figure 38. Synthesis of Debio 0932 type beads. Reagents and conditions: (a) Cs ₂ C0 ₃ , DMF, rt; (b) TFA, CH ₂ C1 ₂ , rt; (c) 6-(BOC-amino)caproic acid, EDCI, DMAP, rt, 2 h; (d) Affigel- 10, DIEA, DMAP, DMF.

8-((6-Bromobenzo[d] [l,3]dioxol-5-yl)thio)-9-(2-(piperidin-4-yl)ethyl)-9H-purin- 6-amine

(18). 16 (300 mg, 0.819 mmol), Cs ₂ C0 ₃ (534 mg, 1.64 mmol), 17 (718 mg, 2.45 mmol) in

DMF (10 mL) was stirred for 1.5 h at rt. The reaction mixture was filtered and dried under reduced pressure and chromatographed (CH ₂ Cl ₂ :MeOH, 10: 1) to give a mixture of Boc- protected N9/N3 isomers. 20 mL of TFA:CH ₂ C1 ₂ (1 : 1) was added at rt and stirred for 6 h. The reaction mixture was dried under reduced pressure and purified by preparatory HPLC to give 18 (87 mg, 22%); MS (ESI) m/z 477.0 [M+H] ⁺ .

6-Amino-l-(4-(2-(6-amino-8-((6-bromobenzo[d] [l,3]dioxol-5-yl)thio)-9H-purin-9- yl)ethyl)piperidin-l-yl)hexan-l-one (19). To a mixture of 18 (150 mg, 0.314 mmol) in CH ₂ C1 ₂ (5 ml) was added 6-(Boc-amino)caproic acid (145 mg, 0.628 mmol), EDCI (120 mg, 0.628 mmol) and DMAP (1.9 mg, 0.0157 mmol). The reaction mixture was stirred at rt for 2 h then concentrated under reduced pressure and the residue purified by preparatory TLC

[CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 15: 1] to give 161 mg (74%) of 19; MS (ESI) m/z 690.1 [M+H] ⁺ .

(20) . 19 (0.264 g, 0.45 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue dried under high vacuum overnight. This was dissolved in DMF (12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed, 3 x 50 mL DMF) in a solid phase peptide synthesis vessel. 225 of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (0.085 g, 97 μΐ, 1.13 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ Cl ₂ :Et ₃ N (9: 1, 4 x 50 mL), DMF (3 x 50 mL), Felts buffer (3 x 50 mL) and z ^' -PrOH (3 x 50 mL). The beads 20 were stored in z ^' -PrOH (beads: z-PrOH (1 :2), v/v) at -80°C.

Figure 39. Synthesis of Debio 0932 linked to biotin. Reagents and conditions: (a) EZ-Link ^® NHS-LC-LC-Biotin, DIEA, DMF, 35°C; (b) EZ-Link ^® NHS-PEG ₄ -Biotin, DIEA, DMF, 35°C.

(21) . 18 (13.9 mg, 0.0292 mmol), EZ-Link ^® NHS-LC-LC-Biotin (18.2 mg, 0.0321 mmol) and DIEA (7.5 mg, 10.2 μί, 0.0584 mmol) in DMF (0.5 mL) was heated at 35°C for 6 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1) to give 7.0 mg (26%) of 21. MS (ESI): m/z 929.3 [M+H] ⁺ . (22). 18 (13.9 mg, 0.0292 mmol), EZ-Link ^® NHS-PEG ₄ -Biotin (18.9 mg, 0.0321 mmol) and DIEA (7.5 mg, 10.2 μί, 0.0584 mmol) in DMF (0.5 mL) was heated at 35°C for 6 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1) to give 8.4 mg (30%) of 22; MS (ESI): m/z 950.2 [M+H] ⁺ .

Figure 40. Synthesis of the SNX 2112type Hsp90 inhibitor linked to biotin. Reagents and conditions: (a) EZ-Link ^® NHS-LC-LC-Biotin, DIEA, DMF, rt; (b) EZ-Link ^® NHS-PEG ₄ - Biotin, DIEA, DMF, rt.

(24) . 23 (16.3 mg, 0.0352 mmol), EZ-Link ^® NHS-LC-LC-Biotin (22.0 mg, 0.0387 mmol) and DIEA (9.1 mg, 12.3 μί, 0.0704 mmol) in DMF (0.5 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH, 10: 1) to give 26.5 mg (82%) of 24; MS (ESI): m/z 916.4 [M+H] ⁺ .

(25) . 23 (17.3 mg, 0.0374 mmol), EZ-Link ^® NHS-PEG ₄ -Biotin (24.2 mg, 0.0411 mmol) and DIEA (9.7 mg, 13 μΐ,, 0.0704 mmol) in DMF (0.5 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH ₂ Cl ₂ :MeOH 10: 1) to give 30.1 mg (78%) of 25; MS (ESI): m/z 937.3 [M+H] ⁺ .

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods of identifying cancer-implicated pathways and specific components of cancer-implicated pathways (e.g., oncoproteins) associated with Hsp90 that are implicated in the development and progression of a cancer. Such methods involve contacting a sample containing cancer cells from a subject suffering from cancer with an inhibitor of Hsp90, and detecting the components of the cancer-implicated pathway that are bound to the inhibitor of Hsp90.

As used herein, certain terms have the meanings set forth after each such term as follows:

"Cancer-Implicated Pathway" means any molecular pathway, a variation in which is involved in the transformation of a cell from a normal to a cancer phenotype. Cancer-implicated pathways may include pathways involved in metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A list of many such pathways is set forth in Table 1 and more detailed information may be found about such pathways online in the KEGG PATHWAY database; and the National Cancer Institute's Nature Pathway Interaction Database. See also the websites of Cell Signaling Technology, Beverly, Mass.; BioCarta, San Diego, Calif; and Invitrogen/Life Technologies Corporation, Clarsbad, Calif. In addition, Figure 1 depicts pathways which are recognized to be involved in cancer.

Table 1. Examples of Potential Cancer-Implicated Pathways.

1. Metabolism 1.1 Carbohydrate Metabolism

Glycolysis / Gluconeogenesis

Citrate cycle (TCA cycle)

Pentose phosphate pathway

Pentose and glucuronate interconversions

Fructose and mannose metabolism

Galactose metabolism

Ascorbate and aldarate metabolism

Starch and sucrose metabolism

Amino sugar and nucleotide sugar metabolism

Pyruvate metabolism

Glyoxylate and dicarboxylate metabolism

Propanoate metabolism

Butanoate metabolism

C5-Branched dibasic acid metabolism

Inositol phosphate metabolism

1.2 Energy Metabolism Oxidative phosphorylation

Photosynthesis

Photosynthesis - antenna proteins

Carbon fixation in photosynthetic organisms

Carbon fixation pathways in prokaryotes

Methane metabolism

Nitrogen metabolism

Sulfur metabolism

1.3 Lipid Metabolism

Fatty acid biosynthesis

Fatty acid elongation in mitochondria

Fatty acid metabolism

Synthesis and degradation of ketone bodies

Steroid biosynthesis

Primary bile acid biosynthesis

Secondary bile acid biosynthesis

Steroid hormone biosynthesis

Glycerolipid metabolism

Glycerophospholipid metabolism

Ether lipid metabolism

Sphingolipid metabolism

Arachidonic acid metabolism

Linoleic acid metabolism

alpha-Linolenic acid metabolism

Biosynthesis of unsaturated fatty acids

1.4 Nucleotide Metabolism

Purine metabolism

Pyrimidine metabolism

1.5 Amino Acid Metabolism

Alanine, aspartate and glutamate metabolism

Glycine, serine and threonine metabolism

Cysteine and methionine metabolism

Valine, leucine and isoleucine degradation

Valine, leucine and isoleucine biosynthesis

Lysine biosynthesis

Lysine degradation

Arginine and proline metabolism

Histidine metabolism

Tyrosine metabolism

Phenylalanine metabolism

Tryptophan metabolism

Phenylalanine, tyrosine and tryptophan biosynthesis

1.6 Metabolism of Other Amino Acids

beta- Alanine metabolism

Taurine and hypotaurine metabolism

Phosphonate and phosphinate metabolism

Selenoamino acid metabolism

Cyanoamino acid metabolism

D-Glutamine and D-glutamate metabolism

D-Arginine and D-ornithine metabolism D-Alanine metabolism

Glutathione metabolism

1.7 Glycan Biosynthesis and Metabolism

N-Glycan biosynthesis

Various types of N-glycan biosynthesis

Mucin type O-Glycan biosynthesis

Other types of O-glycan biosynthesis

Glycosaminoglycan biosynthesis - chondroitin sulfate

Glycosaminoglycan biosynthesis - heparan sulfate

Glycosaminoglycan biosynthesis - keratan sulfate

Glycosaminoglycan degradation

Glycosylphosphatidylinositol(GPI)-anchor biosynthesis

Glycosphingolipid biosynthesis - lacto and neolacto series

Glycosphingolipid biosynthesis - globo series

Glycosphingolipid biosynthesis - ganglio series

Lipopolysaccharide biosynthesis

Peptidoglycan biosynthesis

Other glycan degradation

1.8 Metabolism of Cofactors and Vitamins

Thiamine metabolism

Riboflavin metabolism

Vitamin B6 metabolism

Nicotinate and nicotinamide metabolism

Pantothenate and CoA biosynthesis

Biotin metabolism

Lipoic acid metabolism

Folate biosynthesis

One carbon pool by folate

Retinol metabolism

Porphyrin and chlorophyll metabolism

Ubiquinone and other terpenoid-quinone biosynthesis

1.9 Metabolism of Terpenoids and Polyketides

Terpenoid backbone biosynthesis

Monoterpenoid biosynthesis

Sesquiterpenoid biosynthesis

Diterpenoid biosynthesis

Carotenoid biosynthesis

Brassinosteroid biosynthesis

Insect hormone biosynthesis

Zeatin biosynthesis

Limonene and pinene degradation

Geraniol degradation

Type I polyketide structures

Biosynthesis of 12-, 14- and 16-membered macro lides

Biosynthesis of ansamycins

Biosynthesis of type II polyketide backbone

Biosynthesis of type II polyketide products

Tetracycline biosynthesis

Polyketide sugar unit biosynthesis

Nonribosomal peptide structures Biosynthesis of siderophore group nonribosomal peptides

Biosynthesis of vancomycin group antibiotics

1.10 Biosynthesis of Other Secondary Metabolites

Phenylpropanoid biosynthesis

Stilbenoid, diarylheptanoid and gingerol biosynthesis

Flavonoid biosynthesis

Flavone and flavonol biosynthesis

Anthocyanin biosynthesis

Isoflavonoid biosynthesis

Indole alkaloid biosynthesis

Isoquinoline alkaloid biosynthesis

Tropane, piperidine and pyridine alkaloid biosynthesis

Acridone alkaloid biosynthesis

Caffeine metabolism

Betalain biosynthesis

Glucosinolate biosynthesis

Benzoxazinoid biosynthesis

Penicillin and cephalosporin biosynthesis

beta-Lactam resistance

Streptomycin biosynthesis

Butirosin and neomycin biosynthesis

Clavulanic acid biosynthesis

Puromycin biosynthesis

Novobiocin biosynthesis

1.11 Xenobiotics Biodegradation and Metabolism

Benzoate degradation

Aminobenzoate degradation

Fluorobenzoate degradation

Chloroalkane and chloroalkene degradation

Chlorocyclohexane and chlorobenzene degradation

Toluene degradation

Xylene degradation

Nitrotoluene degradation

Ethylbenzene degradation

Styrene degradation

Atrazine degradation

Caprolactam degradation

DDT degradation

Bisphenol degradation

Dioxin degradation

Naphthalene degradation

Polycyclic aromatic hydrocarbon degradation

Metabolism of xenobiotics by cytochrome P450

Drug metabolism - cytochrome P450

Drug metabolism - other enzymes

1.12 Overview

Overview of biosynthetic pathways

Biosynthesis of plant secondary metabolites

Biosynthesis of phenylpropanoids

Biosynthesis of terpenoids and steroids Biosynthesis of alkaloids derived from shikimate pathway

Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid

Biosynthesis of alkaloids derived from histidine and purine

Biosynthesis of alkaloids derived from terpenoid and polyketide

Biosynthesis of plant hormones enetic 2.1 Transcription

Information R A polymerase

Processing Basal transcription factors

Spliceosome

2.2 Translation

Ribosome

Aminoacyl-tRNA biosynthesis

RNA transport

mRNA surveillance pathway

Ribosome biogenesis in eukaryotes

2.3 Folding, Sorting and Degradation

Protein export

Protein processing in endoplasmic reticulum

SNARE interactions in vesicular transport

Ubiquitin mediated proteolysis

Sulfur relay system

Proteasome

RNA degradation

2.4 Replication and Repair

DNA replication

Base excision repair

Nucleotide excision repair

Mismatch repair

Homologous recombination

Non-homologous end-joining

Environmental 3.1 Membrane Transport

Information ABC transporters

Processing Phosphotransferase system (PTS)

Bacterial secretion system

3.2 Signal Transduction

Two-component system

MAPK signaling pathway

MAPK signaling pathway - fly

MAPK signaling pathway - yeast

ErbB signaling pathway

Wnt signaling pathway

Notch signaling pathway

Hedgehog signaling pathway

TGF-beta signaling pathway

VEGF signaling pathway

Jak-STAT signaling pathway Calcium signaling pathway

Phosphatidylinositol signaling system mTOR signaling pathway

Plant hormone signal transduction

3.3 Signaling Molecules and Interaction

Neuroactive ligand-receptor interaction

Cytokine-cytokine receptor interaction

ECM-receptor interaction

Cell adhesion molecules (CAMs)

ellular Processes 4.1 Transport and Catabolism

Endocytosis

Phagosome

Lysosome

Peroxisome

Regulation of autophagy

4.2 Cell Motility

Bacterial chemotaxis

Flagellar assembly

Regulation of actin cytoskeleton

4.3 Cell Growth and Death

Cell cycle

Cell cycle - yeast

Cell cycle - Caulobacter

Meiosis - yeast

Oocyte meiosis

Apoptosis

p53 signaling pathway

4.4 Cell Communication

Focal adhesion

Adherens junction

Tight junction

Gap junction

rganismal 5.1 Immune System

Systems Hematopoietic cell lineage

Complement and coagulation cascades

Toll-like receptor signaling pathway

NOD-like receptor signaling pathway

RIG-I-like receptor signaling pathway

Cytosolic DNA-sensing pathway

Natural killer cell mediated cytotoxicity

Antigen processing and presentation

T cell receptor signaling pathway

B cell receptor signaling pathway

Fc epsilon RI signaling pathway

Fc gamma R-mediated phagocytosis

Leukocyte transendothelial migration

Intestinal immune network for IgA production

Chemokine signaling pathway 5.2 Endocrine System

Insulin signaling pathway

Adipocytokine signaling pathway

PPAR signaling pathway

GnRH signaling pathway

Progesterone-mediated oocyte maturation

Melanogenesis

"Component of a Cancer-Implicated Pathway" means a molecular entity located in a Cancer- Implicated Pathway which can be targeted in order to effect inhibition of the pathway and a change in a cancer phenotype which is associated with the pathway and which has resulted from activity in the pathway. Examples of such components include components listed in Figure 1.

"Inhibitor of a Component of a Cancer-Implicated Pathway" means a compound (other than an inhibitor of Hsp90) which interacts with a Cancer-Implicated Pathway or a Component of a Cancer-Implicated Pathway so as to effect inhibition of the pathway and a change in a cancer phenotype which has resulted from activity in the pathway. Examples of inhibitors of specific Components are widely known. Merely by way of example, the following U.S. patents and U.S. patent application publications describe examples of inhibitors of pathway components as listed follows:

SYK: U.S. Patent Application Publications US 2009/0298823 Al, US

2010/0152159 Al, US 2010/0316649 Al

BTK: U.S. Patent 6,160,010; U.S. Patent Application Publications US

2006/0167090 Al, US 2011/0008257 Al

EGFR: U.S. Patents 5,760,041; US 7,488,823 B2; US 7,547,781 B2 mTOR: U.S. Patent US 7,504,397 B2; U.S. Patent Application

Publication US 2011/0015197 Al

MET: U.S. Patent US 7,037,909 B2; U.S. Patent Application

Publications US 2005/0107391 Al, US 2006/0009493 Al

MEK: U.S. Patent US 6,703,420 Bl; U.S. Patent Application

Publication US 2007/0287737 Al

U.S. Patent US 7,790,729 B2; U.S. Patent Application Publications US 2005/0234115 Al, US 2006/0074056 Al PTEN: U.S. Patent Application Publications US 2007/0203098 Al, US

2010/0113515 Al

PKC: U.S. Patents 5,552,396; US 7,648,989 B2

Bcr-Abl: U.S. Patent US 7,625,894 B2; U.S. Patent Application

Publication US 2006/0235006 Al

Still further a few examples of inhibitors of protein kinases are shown in Figure 2.

"Inhibitor of Hsp90" means a compound which interacts with, and inhibits the activity of, the chaperone, heat shock protein 90 (Hsp90). The structures of several known Hsp90 inhibitors, including PU-H71, are shown in Figure 3. Many additional Hsp90 inhibitors have been described. See, for example, U.S. Patents US 7,820,658 B2; US 7,834,181 B2; and US 7,906,657 B2. See also the following:

Hardik J Patel, Shanu Modi, Gabriela Chiosis, Tony Taldone. Advances in the discovery and development of heat-shock protein 90 inhibitors for cancer treatment. Expert Opinion on Drug Discovery May 2011, Vol. 6, No. 5, Pages 559-587: 559- 587;

Porter JR, Fritz CC, Depew KM. Discovery and development of Hsp90 inhibitors: a promising pathway for cancer therapy. Curr Opin Chem Biol. 2010 Jun; 14(3): 412- 20;

Janin YL. ATPase inhibitors of heat-shock protein 90, second season. Drug Discov Today. 2010 May; 15(9-10): 342-53;

Taldone T, Chiosis G. Purine-scaffold Hsp90 inhibitors. Curr Top Med Chem. 2009; 9(15): 1436-46; and

Taldone T, Sun W, Chiosis G. Discovery and Development of heat shock protein 90 inhibitors. Bioorg Med Chem. 2009 Mar 15; 17(6): 2225-35. Small molecule Hsp90 Probes

The attachment of small molecules to a solid support is a very useful method to probe their target and the target's interacting partners. Indeed, geldanamycin attached to solid support enabled for the identification of Hsp90 as its target. Perhaps the most crucial aspects in designing such chemical probes are determining the appropriate site for attachment of the small molecule ligand, and designing an appropriate linker between the molecule and the solid support. Our strategy to design Hsp90 chemical probes entails several steps. First, in order to validate the optimal linker length and its site of attachment to the Hsp90 ligand, the linker-modified ligand was docked onto an appropriate X-ray crystal structure of Hsp90a. Second, the linker-modified ligand was evaluated in a fluorescent polarization (FP) assay that measures competitive binding to Hsp90 derived from a cancer cell extract. This assay uses Cy3b-labeled geldanamycin as the FP-optimized Hsp90 ligand (Du et al, 2007). These steps are important to ensure that the solid-support immobilized molecules maintain a strong affinity for Hsp90. Finally, the linker-modified small molecule was attached to the solid support, and its interaction with Hsp90 was validated by incubation with an Hsp90- containing cell extract.

When a probe is needed to identify Hsp90 in complex with its onco-client proteins, further important requirements are (1.) that the probe retains selectivity for the "oncogenic Hsp90 species" and (2.) that upon binding to Hsp90, the probe locks Hsp90 in a client-protein bound conformation. The concept of "oncogenic Hsp90" is further defined in this application as well as in Figure 11.

When a probe is needed to identify Hsp90 in complex with its onco-client proteins by mass spectrometry techniques, further important requirements are (1.) that the probe isolates sufficient protein material and (2.) that the signal to ratio as defined by the amount of Hsp90 onco-clients and unspecifically resin-bound proteins, respectively, be sufficiently large as to be identifiable by mass spectrometry. This application provides examples of the production of such probes.

We chose Affi-Gel ^® 10 (BioRad) for ligand attachment. These agarose beads have an N- hydroxysuccinimide ester at the end of a IOC spacer arm, and in consequence, each linker was designed to contain a distal amine functionality. The site of linker attachment to PU-H71 was aided by the co-crystal structure of it bound to the N-terminal domain of human Hsp90a (PDB ID: 2FWZ). This structure shows that the purine's N9 amine makes no direct contact with the protein and is directed towards solvent (Figure 27A) (Immormino et al., 2006). As well, a previous SAR indicated that this is an attractive site since it was previously used for the introduction of water solubilizing groups (He et al, 2006). Compound 5 (PU-H71-C ₆ linker) was designed and docked onto the Hsp90 active site (Figure 27A). All the interactions of PU-H71 were preserved, and the computer model clearly showed that the linker oriented towards the solvent exposed region. Therefore, compound 5 was synthesized as the immediate precursor for attachment to solid support (see Chemistry, Figure 30). In the FP assay, 5 retained affinity for Hsp90 (IC ₅₀ = 19.8 nM compared to 22.4 nM for PU-H71, Table 8) which then enabled us to move forward with confidence towards the synthesis of solid support immobilized PU-H71 probe (6) by attachment to Affi-Gel ^® 10 (Figure 30).

We also designed a biotinylated derivative of PU-H71. One advantage of the biotinylated agent over the solid supported agents is that they can be used to probe binding directly in cells or in vivo systems. The Iigand-Hsp90 complexes can then be captured on biotin-binding avidin or streptavidin containing beads. Typically this process reduces the unspecific binding associated with chemical precipitation from cellular extracts. Alternatively, for in vivo experiments, the presence of active sites (in this case Hsp90), can be detected in specific tissues (i.e. tumor mass in cancer) by the use of a labeled-streptavidin conjugate (i.e. FITC- streptavidin). Biotinylated PU-H71 (7) was obtained by reaction of 2 with biotinyl-3,6,9- trioxaundecanediamine (EZ-Link ^® Amine -PE0 ₃ -Biotin) (Figure 31). 7 retained affinity for Hsp90 (IC ₅₀ = 67.1 nM) and contains an exposed biotin capable of interacting with streptavidin for affinity purification. From the available co-crystal structure of NVP-AUY922 with Hsp90a (PDB ID: 2VCI, Figure 27B) and co-crystal structures of related 3,4-diarylpyrazoles with Hsp90a, as well as from SAR, it was evident that there was a considerable degree of tolerance for substituents at the /?ara-position of the 4-aryl ring (Brough et al, 2008; Cheung et al, 2005; Dymock et al, 2005; Barril et al, 2006). Because the 4-aryl substituent is largely directed towards solvent and substitution at the /?ara-position seems to have little impact on binding affinity, we decided to attach the molecule to solid support at this position. In order to enable attachment, the morpholine group was changed to the 1,6-diaminohexyl group to give 10 as the immediate precursor for attachment to solid support. Docking 10 onto the active site (Figure

27B) shows that it maintains all of the interactions of NVP-AUY922 and that the linker orients towards the solvent exposed region. When 10 was tested in the binding assay it also retained affinity (IC ₅₀ = 7.0 nM compared to 4.1 nM for NVP-AUY922, Table 8) and was subsequently used for attachment to solid support (see Chemistry, Figure 32). Although a co-crystal structure of SNX-2112 with Hsp90 is not publicly available, that of a related tetrahydro-4H-carbazol-4-one (27) bound to Hsp90a (PDB ID: 3D0B, Figure 27C) is (Barta et al., 2008). This, along with the reported SAR for 27 suggests linker attachment to the hydroxyl of the trans-4-aminocylohexanol substituent. Direct attachment of 6-amino- caproic acid via an ester linkage was not considered desirable because of the potential instability of such bonds in lysate mixtures due to omnipresent esterases. Therefore, the hydroxyl was substituted with amino to give the trans- 1 ,4-diaminocylohexane derivative 18 (Figure 33). Such a change resulted in nearly a 14-fold loss in potency as compared to SNX- 2112 (Table 8). 6-(Boc-amino)caproic acid was attached to 18 and following deprotection, 20 was obtained as the immediate precursor for attachment to beads (see Chemistry, Figure 33). Docking suggested that 20 interacts similarly to 27 (Figure 27C) and that the linker orients towards the solvent exposed region. 20 was determined to have good affinity for Hsp90 (ICso = 24.7 nM compared to 15.1 nM for SNX-2112 and 210.1 nM for 18, Table 8) and to have regained almost all of the affinity lost by 18. The difference in activity between 18 and both 20 and SNX-2112 is well explained by our binding model, as compounds 20 (- C=0, Figure 27C) and SNX-2112 (-OH, Figure not shown) form a hydrogen bond with the side-chain amino of Lys 58. 18 contains a strongly basic amino group and is incapable of forming a hydrogen bond with Lys 58 side chain (NH ₂ , Figure not shown). This is in good agreement with the observation of Huang et al. that basic amines at this position are disfavored. The amide bond of 20 converts the basic amino of 18 into a non-basic amide group capable of acting as an H-bond acceptor to Lys 58, similarly to the hydroxyl of SNX- 2112.

Synthesis of PU-H71 beads (6) is shown in Figure 30 and commences with the 9-alkylation of 8-arylsulfanylpurine (1) (He et al, 2006) with 1,3-dibromopropane to afford 2 in 35% yield. The low yield obtained in the formation of 2 can be primarily attributed to unavoidable competing 3-alkylation. Five equivalents of 1,3-dibromopropane were used to ensure complete reaction of 1 and to limit other undesirable side-reactions, such as dimerization, which may also contribute to the low yield. 2 was reacted with tert-butyl 6- aminohexylcarbamate (3) to give the Boc-protected amino purine 4 in 90% yield. Deprotection with TFA followed by reaction with Affi-Gel 10 resulted in 6. Biotinylated PU-H71 (7) was also synthesized by reacting 2 with EZ-Link ^® Amine -PE0 ₃ -Biotin (Figure 31). Synthesis of NVP-AUY922 beads (11) from aldehyde 8 (Brough et al, 2008) is shown in Figure 32. 9 was obtained from the reductive amination of 8 with 3 in 75% yield with no detectable loss of the Boc group. In a single step, both the Boc and benzyl protecting groups were removed with BC1 ₃ to give isoxazole 10 in 78% yield, which was then reacted with Affi-Gel ^® 10 to give 11.

Synthesis of SNX-2112 beads (21) is shown in Figure 33, and while compounds 17 and 18 are referred to in the patent literature (Serenex et al., 2008, WO-2008130879A2; Serenex et al, 2008, US-20080269193A1), neither is adequately characterized, nor are their syntheses fully described. Therefore, we feel that it is worth describing the synthesis in detail. Tosylhydrazone 14 was obtained in 89% yield from the condensation of tosyl hydrazide (12) with dimedone (13). The one -pot conversion of 14 to tetrahydroindazolone 15 occurs following base promoted cyclocondensation of the intermediate trifluoroacyl derivative generated by treatment with trifluroacetic anhydride in 55% yield. 15 was reacted with 2- bromo-4-fluorobenzonitrile in DMF to give 16 in 91% yield. It is interesting to note the regioselectivity of this reaction as arylation occurs selectively at Nl . In computational studies of indazol-4-ones similar to 15, both \H and 2H-tautomers are known to exist in equilibrium, however, because of its higher dipole moment the 1H tautomer is favored in polar solvents (Claramunt et al., 2006). The amination of 16 with trans- 1 ,4-diaminocyclohexane was accomplished under Buchwald conditions (Old et al, 1998) using tris(dibenzylideneacetone)dipalladium [Pd ₂ (dba) ₃ ] and 2-dicyclohexylphosphino-2'-(N,N- dimethylamino)biphenyl (DavePhos) to give nitrile 17 (24%) along with amide 18 (17%) for a combined yield of 41%. Following complete hydrolysis of 17, 18 was coupled to 6-(Boc- amino)caproic acid with EDCI/DMAP to give 19 in 91% yield. Following deprotection, 20 was obtained which was then reacted with Affi-Gel ^® 10 to give 21.

Several methods were employed to measure the progress of the reactions for the synthesis of the final probes. UV monitoring of the liquid was used by measuring a decrease in for each compound. In general, it was observed that that there was no further decrease in the after 1.5 h, indicating completion of the reaction. TLC was employed as a crude measure of the progress of the reaction whereas LC-MS monitoring of the liquid was used to confirm complete reaction. While on TLC the spot would not disappear since excess compound was used (1.2 eq.), a clear decrease in intensity indicated progress of the reaction. The synthesis and full characterization of the Hsp90 inhibitors PU-H71 (He et al., 2006) and NVP-AUY922 (Brough et al, 2008) have been reported elsewhere. SNX-2112 had previously been mentioned in the patent literature (Serenex et al, 2008, WO-2008130879A2; Serenex et al., 2008, US-20080269193A1), and only recently has it been fully characterized and its synthesis adequately described (Huang et al, 2009). At the time this research project began specific details on its synthesis were lacking. Additionally, we had difficulty reproducing the amination of 16 with trans-4-aminocyclohexanol under conditions reported for similar compounds [Pd(OAc) ₂ , DPPF, NaOtBu, toluene, 120°C, microwave]. In our hands, only trace amounts of product were detected at best. Changing catalyst to PdCl ₂ , Pd(PPh ₃ ) ₄ or Pd ₂ (dba) ₃ or solvent to DMF or 1 ,2-dimethoxyethane (DME) or base to K ₃ P0 ₄ did not result in any improvement. Therefore, we modified this step and were able to couple 16 to trans-4-aminocyclohexanol tetrahydropyranyl ether (24) under Buchwald conditions (Old et al, 1998) using Pd ₂ (dba) ₃ and DavePhos in DME to give nitrile 25 (28%) along with amide 26 (17%) for a combined yield of 45% (Figure 34). These were the conditions used to couple 16 to trans- 1 ,4-diaminocyclohexane, and similarly some of 25 was hydrolysed to 26 during the course of the reaction. Because for our purpose it was unnecessary, we did not optimize this reaction for 25. We surmised that a major hindrance to the reaction was the low solubility of trans-4-aminocyclohexanol in toluene and that using the THP protected alcohol 24 at the very least increased solubility. SNX-2112 was obtained and fully characterized (1H, ¹³ C-NMR, MS) following removal of the THP group from 26.

Next, we investigated whether the synthesized beads retained interaction with Hsp90 in cancer cells. Agarose beads covalently attached to either of PU-H71, NVP-AUY922, SNX- 2112 or 2-methoxyethylamine (PU-, NVP-, SNX-, control-beads, respectively), were incubated with K562 chronic myeloid leukemia (CML) or MDA-MB-468 breast cancer cell extracts. As seen in Figure 28A, the Hsp90 inhibitor, but not the control-beads, efficiently isolated Hsp90 in the cancer cell lysates. Control beads contain an Hsp90 inactive chemical (2-methoxyethylamine) conjugated to Affi-Gel ^® 10 (see Experimental) providing an experimental control for potential unspecific binding of the solid-support to proteins in cell extracts. Further, to probe the ability of these chemical tools to isolate genuine Hsp90 client proteins in tumor cells, we incubated PU-H71 attached to solid support (6) with cancer cell extracts. We were able to demonstrate dose-dependent isolation of Hsp90/c-Kit and Hsp90/IGF-IR complexes in MDA-MB-468 cells (Figure 28B) and of Hsp90/Bcr-Abl and Hsp90/Raf-1 complexes in K562 cells (Figure 28C). These are Hsp90-dependent onco-proteins with important roles in driving the transformed phenotype in triple-negative breast cancers and CML, respectively (Whitesell & Lindquist, 2005; Hurvitz & Finn, 2009; Law et al, 2008). In accord with an Hsp90 mediated regulation of c-Kit and IGF-IR, treatment of MDA-MB-468 cells with PU-H71 led to a reduction in the steady-state levels of these proteins (Figure 28B, compare Lysate, - and + PU-H71). Using the PU-beads (6), we were recently able to isolate and identify novel Hsp90 clients, such as the transcriptional repressor BCL-6 in diffuse large B-cell lymphoma (Cerchietti et al., 2009) and JAK2 in mutant JAK2 driven myeloproliferative disorders (Marubayashi et al., 2010). We were also able to identify Hsp90 onco-clients specific to a triple-negative breast cancer (Caldas-Lopes et al., 2009). In addition to shedding light on the mechanisms of action of Hsp90 in these tumors, the identified proteins are important tumor-specific onco-clients and will be introduced as biomarkers in monitoring the clinical efficacy of PU-H71 and Hsp90 inhibitors in these cancers during clinical studies.

Similar experiments were possible with PU-H71-biotin (7) (Figure 29A), although the PU- H71 -beads were superior to the PU-H71-biotin beads at isolating Hsp90 in complex with a client protein. It is important to note that previous attempts to isolate Hsp90/client protein complexes using a solid-support immobilized GM were of little success (Tsaytler et al, 2009). In that case, the proteins bound to Hsp90 were washed away during the preparative steps. To prevent the loss of Hsp90-interacting proteins, the authors had to subject the cancer cell extracts to cross- linking with DSP, a homobifunctional amino-reactive DTT-reversible cross-linker, suggesting that unlike PU-H71, GM is unable to stabilize Hsp90/client protein interactions.

We observed a similar profile when using beads with GM directly covalently attached to the

Affi-Gel ^® 10 resin. Crystallographic and biochemical investigations suggest that GM preferentially interacts with Hsp90 in an apo, open-conformation, that is unfavorable for certain client protein binding (Roe et al, 1999; Stebbins et al, 1997; Nishiya et al, 2009) providing a potential explanation for the limited ability of GM-beads to capture Hsp90/client protein complexes. It is currently unknown what Hsp90 conformations are preferred by the other Hsp90 chemotypes, but with the NVP- and SNX-beads also available, as reported here, similar evaluations are now possible, leading to a better understanding of the interaction of these agents with Hsp90, and of the biological significance of these interactions.

In another application of the chemical tools designed here, we show that PU-H71-biotin (7) can also be used to specifically detect Hsp90 when expressed on the cell surface (Figure 29B). Hsp90, which is mainly a cytosolic protein, has been reported in certain cases to translocate to the cell surface. In a breast cancer for example, membrane Hsp90 is involved in aiding cancer cell invasion (Sidera & Patsavoudi, 2008). Specific detection of the membrane Hsp90 in live cells is possible by the use of PU-H71-biotin (7) because, while the biotin conjugated Hsp90 inhibitor may potentially enter the cell, the streptavidin conjugate used to detect the biotin, is cell impermeable. Figure 29B shows that PU-H71 -biotin but not D-biotin can detect Hsp90 expression on the surface of leukemia cells.

In summary, we have prepared useful chemical tools based on three different Hsp90 inhibitors, each of a different chemotype. These were prepared either by attachment onto solid support, such as PU-H71 (purine), NVP-AUY922 (isoxazole) and SNX-21 12 (indazol- 4-one)-beads, or by biotinylation (PU-H71 -biotin). The utility of these probes was demonstrated by their ability to efficiently isolate Hsp90 and, in the case of PU-H71 beads (6), isolate Hsp90 onco-protein. containing complexes from cancer cell extracts. Available co- crystal structures and SAR were utilized in their design, and docking to the appropriate X-ray crystal structure of Hsp90a used to validate the site of attachment of the linker. These are important chemical tools in efforts towards better understanding Hsp90 biology and towards designing Hsp90 inhibitors with most favorable clinical profile.

Identification of Oncoproteins and Pathways Using Hsp90 Probes

The disclosure provides methods of identifying components of cancer-implicated pathway (e.g., oncoproteins) using the Hsp90 probes described above. In one embodiment of the invention the cancer-implicated pathway is a pathway involved in metabolism, genetic information processing, environmental information processing, cellular processes, or organismal systems. For example, the cancer-implicated pathway may be a pathway listed in Table 1.

More particularly, the cancer-implicated pathway or the component of the cancer-implicated pathway is involved with a cancer such as a cancer selected from the group consisting of a colorectal cancer, a pancreatic cancer, a thyroid cancer, a leukemia including an acute myeloid leukemia and a chronic myeloid leukemia, a basal cell carcinoma, a melanoma, a renal cell carcinoma, a bladder cancer, a prostate cancer, a lung cancer including a small cell lung cancer and a non-small cell lung cancer, a breast cancer, a neuroblastoma, myeloproliferative disorders, gastrointestinal cancers including gastrointestinal stromal tumors, an esophageal cancer, a stomach cancer, a liver cancer, a gallbladder cancer, an anal cancer, brain tumors including gliomas, lymphomas including a follicular lymphoma and a diffuse large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers.

The following subsections describe use of the Hsp90 probes of the present disclosure to determine properties of Hsp90 in cancer cells and to identifty oncoproteins and cancer- implicated pathways.

Heterogeneous Hsp90 presentation in cancer cells

To investigate the interaction of small molecule Hsp90 inhibitors with tumor Hsp90 complexes, we made use of agarose beads covalently attached to either geldanamycin (GM) or PU-H71 (GM- and PU-beads, respectively) (Figures 4, 5). Both GM and PU-H71, chemically distinct agents, interact with and inhibit Hsp90 by binding to its N-terminal domain regulatory pocket (Janin, 2010). For comparison, we also generated G protein agarose-beads coupled to an anti-Hsp90 antibody (H9010).

First we evaluated the binding of these agents to Hsp90 in a breast cancer and in chronic myeloid leukemia (CML) cell lysates. Four consecutive immunoprecipitation (IP) steps with

H9010, but not with a non-specific IgG, efficiently depleted Hsp90 from these extracts (Figure 4a, 4xH9010 and not shown). In contrast, sequential pull-downs with PU- or GM- beads removed only a fraction of the total cellular Hsp90 (Figures 4b, 10a, 10b).

Specifically, in MDA-MB-468 breast cancer cells, the combined PU-bead fractions represented approximately 20-30% of the total cellular Hsp90 pool, and further addition of fresh PU-bead aliquots failed to precipitate the remaining Hsp90 in the lysate (Figure 4b, PU-beads). This PU-depleted, remaining Hsp90 fraction, while inaccessible to the small molecule, maintained affinity for H9010 (Figure 4b, H9010). From this we conclude that a significant fraction of Hsp90 in the MDA-MB-468 cell extracts was still in a native conformation but not reactive with PU-H71.

To exclude the possibility that changes in Hsp90 configuration in cell lysates make it unavailable for binding to immobilized PU-H71 but not to the antibody, we analyzed binding of radiolabeled ¹³¹ I-PU-H71 to Hsp90 in intact cancer cells (Figure 4c, lower). The chemical structures of ¹³¹ I-PU-H71 and PU-H71 are identical: PU-H71 contains a stable iodine atom ( ¹²⁷ I) and ¹³¹ I-PU-H71 contains radioactive iodine; thus, isotopically labeled ¹³¹ I-PU-H71 has identical chemical and biological properties to the unlabeled PU-H71. Binding of ¹³¹ I-PU- H71 to Hsp90 in several cancer cell lines became saturated at a well-defined, although distinct, number of sites per cell (Figure 4c, lower). We quantified the fraction of cellular Hsp90 that was bound by PU-H71 in MDA-MB-468 cells. First, we determined that Hsp90 represented 2.66-3.33% of the total cellular protein in these cells, a value in close agreement with the reported abundance of Hsp90 in other tumor cells (Workman et al., 2007). Approximately 41.65xl0 ⁶ MDA-MB-468 cells were lysed to yield 3875 μg of protein, of which 103.07-129.04 μg was Hsp90. One cell, therefore, contained (2.47-3.09)xl0 ^~6 μg, (2.74-3.43)xl0 ^"u μιηοΐβ or (1.64-2.06)xl0 ⁷ molecules of Hsp90. In MDA-MB-468 cells, ¹³¹ I- PU-H71 bound at most to 5.5xl0 ⁶ of the available cellular binding sites (Figure 4c, lower), which amounts to 26.6-33.5% of the total cellular Hsp90 (calculated as 5.5xl0 ⁶ /(1.64- 2.06)xl0 ⁷ * 100). This value is remarkably similar to the one obtained with PU-bead pull- downs in cell extracts (Figure 4b), confirming that PU-H71 binds to a fraction of Hsp90 in MDA-MB-468 cells that represents approximately 30% of the total Hsp90 pool and validating the use of PU-beads to efficiently isolate this pool. In K562 and other established t(9;22)+ CML cell lines, PU-H71 bound 10.3-23% of the total cellular Hsp90 (Figures 4c, 10b, 10c).

Collectively, these data suggest that certain Hsp90 inhibitors, such as PU-H71, preferentially bind to a subset of Hsp90 species that is more abundant in cancer cells than in normal cells (Figure 11a).

Onco- and WT-protein bound Hsp90 species co-exist in cancer cells, but PU-H71 selects for the onco-protein/Hsp90 species To explore the biochemical functions associated with these Hsp90 species, we performed immunoprecipitations (IPs) and chemical precipitations (CPs) with antibody- and Hsp90- inhibitor beads, respectively, and we analysed the ability of Hsp90 bound in these contexts to co-precipitate with a chosen subset of known clients. K562 CML cells were first investigated because this cell line co-expresses the aberrant Bcr-Abl protein, a constitutively active kinase, and its normal counterpart c-Abl. These two Abl species are clearly separable by molecular weight and thus easily distinguishable by Western blot (Figure 5a, Lysate), facilitating the analysis of Hsp90 onco- and wild type (WT)-clients in the same cellular context. We observed that H9010, but not a non-specific IgG, isolated Hsp90 in complex with both Bcr- Abl and Abl (Figures 5a and 11, H9010). Comparison of immunoprecipitated Bcr-Abl and Abl (Figures 5a and 5b, left, H9010) with the fraction of each protein remaining in the supernatant (Figure 5b, left, Remaining supernatant), indicated that the antibody did not preferentially enrich for Hsp90 bound to either mutant or WT forms of Abl in K562 cells. In contrast, PU-bound Hsp90 preferentially isolated the Bcr-Abl protein (Figures 5a and 5b, right, PU-beads). Following PU-bead depletion of the Hsp90/Bcr-Abl species (Figure 5b, right, PU-beads), H9010 precipitated the remaining Hsp90/Abl species (Figure 5b, right, H9010). PU-beads retained selectivity for Hsp90/Bcr-Abl species at substantially saturating conditions (i.e. excess of lysate, Figure 12a, left, and beads, Figure 12a, right). As further confirmation of the biochemical selectivity of PU-H71 for the Bcr-Abl/Hsp90 species, Bcr- Abl was much more susceptible to degradation by PU-H71 than was Abl (Figure 5d). The selectivity of PU-H71 for the aberrant Abl species extended to other established t(9;22)+ CML cell lines (Figure 13a), as well as to primary CML samples (Figure 13b). The onco- but not WT-protein bound Hsp90 species are most dependent on co-chaperone recruitment for client protein regulation by Hsp90

To further differentiate between the PU-H71- and antibody-associated Hsp90 fractions, we performed sequential depletion experiments and evaluated the co-chaperone constituency of the two species (Zuehlke & Johnson, 2010). The fraction of Hsp90 containing the Hsp90/Bcr- Abl complexes bound several co-chaperones, including Hsp70, Hsp40, HOP and HIP (Figure 5c, PU-beads). PU-bead pull-downs were also enriched for several additional Hsp90 co- chaperone species (Tables 5a-d). These findings strongly suggest that PU-H71 recognizes co-chaperone-bound Hsp90. The PU-beads-depleted, remaining Hsp90 pool, shown to include Hsp90/Abl species, was not associated with co-chaperones (Figure 5c, H9010), although their abundant expression was detected in the lysate (Figure 5c, Remaining supernatant). Co-chaperones are however isolated by H9010 in the total cellular extract (Figures lib, 11c). These findings suggest the existence of distinct pools of Hsp90 preferentially bound to either Bcr-Abl or Abl in CML cells (Figure 5g). H9010 binds to both the Bcr-Abl and the Abl containing Hsp90 species, whereas PU-H71 is selective for the Bcr-Abl/Hsp90 species. Our data also suggest that Hsp90 may utilize and require more acutely the classical co-chaperones Hsp70, Hsp40 and HOP when it modulates the activity of aberrant (i.e. Bcr-Abl) but not normal (i.e. Abl) proteins (Figure 11a). In accord with this hypothesis, we find that Bcr-Abl is more sensitive than Abl to knock-down of Hsp70, an Hsp90 co-chaperone, in K562 cells (Figure 5e).

The onco-protein/Hsp90 species selectivity and the complex trapping ability of PU-H71 are not shared by all Hsp90 inhibitors

We next evaluated whether other inhibitors that interact with the N-terminal regulatory pocket of Hsp90 in a manner similar to PU-H71, including the synthetic inhibitors SNX-2112 and NVP-AUY922, and the natural product GM (Janin, 2010), could selectively isolate similar Hsp90 species (Figure 5f). SNX-beads demonstrated selectivity for Bcr-Abl/Hsp90, whereas NVP -beads behaved similarly to H9010 and did not discriminate between Bcr- Abl/Hsp90 and Abl/Hsp90 species (see SNX- versus NVP -beads, respectively; Figure 5f). While GM-beads also recognized a subpopulation of Hsp90 in cell lysates (Figure 10a), they were much less efficient than were PU-beads in co-precipitating Bcr-Abl (Figure 5f, GM- beads). Similar ineffectiveness for GM in trapping Hsp90/client protein complexes was previously reported (Tsaytler et al, 2009).

The onco-protein/Hsp90 species selectivity and the complex trapping ability of PU-H71 is not restricted to Bcr-Abl/Hsp90 species

To determine whether selectivity towards onco-proteins was not restricted to Bcr-Abl, we tested several additional well-defined Hsp90 client proteins in other tumor cell lines (Figures

12b-d) (da Rocha Dias et al, 2005; Grbovic et al, 2006). In agreement with our results in

K562 cells, H9010 precipitated Hsp90 complexed with both mutant B-Raf expressed in

SKMel28 melanoma cells and WT B-Raf expressed in CCDI8C0 normal colon fibroblasts

(Figure 12b, H9010). PU- and GM-beads however, selectively recognized Hsp90/mutant B- Raf, showing little recognition of Hsp90/WT B-Raf (Figure 12b, PU-beads and GM-beads). However, as was the case in K562 cells, GM-beads were significantly less efficient than PU- beads in co-precipitating the mutant client protein. Similar results were obtained for other Hsp90 clients (Figures 12c, 12d; Tsaytler et al, 2009).

PU-H71-beads identify the aberrant signalosome in CML

The data presented above suggest that PU-H71, which specifically interacts with Hsp90 (Figure 14; Taldone & Chiosis, 2009), preferentially selects for onco-protein/Hsp90 species and traps Hsp90 in a client binding conformation (Figure 5). Therefore, we examined whether PU-H71 beads could be used as a tool to investigate the cellular complement of oncogenic Hsp90 client proteins. Because the aberrant Hsp90 clientele is hypothesized to comprise the various proteins most crucial for the maintenance of the tumor phenotype (Zuehlke & Johnson, 2010; Workman et al., 2007; Dezwaan & Freeman, 2008), this approach could potentially identify critical signaling pathways in a tumor-specific manner. To test this hypothesis, we performed an unbiased analysis of the protein cargo isolated by PU-H71 beads in K562 cells, where at least some of the key functional lesions are known (Ren, 2005; Burke & Carroll, 2010).

Protein cargo isolated from cell lysate with PU-beads or control-beads was subjected to proteomic analysis by nano liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Initial protein identification was performed using the Mascot search engine, and was further evaluated using Scaffold Proteome Software (Tables 5a-d). Among the PU-bead-interacting proteins, Bcr-Abl was identified (see Bcr and Abll, Table 5a and Figure 6), confirming previous data (Figure 5).

Ingenuity Pathway Analysis (IP A) was then used to build biological networks from the identified proteins (Figures 6a, 6b, 15; Tables 5e, 5f). IPA assigned PU-H71 -isolated proteins to thirteen networks associated with cell death, cell cycle, cellular growth and proliferation. These networks overlap well with known canonical CML signaling pathways (Figure 6a).

In addition to signaling proteins, we identified proteins that regulate carbohydrate and lipid metabolism, protein synthesis, gene expression, and cellular assembly and organization.

These findings are in accord with the postulated broad roles of Hsp90 in maintaining cellular homeostasis and in being an important mediator of cell transformation (Zuehlke & Johnson, 2010; Workman et al, 2007; Dezwaan & Freeman, 2008; McClellan et al, 2007).

Following identification by MS, a number of key proteins were further validated by chemical precipitation and Western blot, in both K562 cells and in primary CML blasts (Figure 6c, left, Figures 6d, 13a, 13b). The effect of PU-H71 on the steady-state levels of these proteins was also queried to further support their Hsp90-regulated expression/stability (Figure 6c, right) (Zuehlke & Johnson, 2010). The top scoring networks enriched on the PU-beads were those used by Bcr-Abl to propagate aberrant signaling in CML: the PI3K/mTOR-, MAPK- and NFKB-mediated signaling pathways (Network 1, 22 focus molecules, score = 38 and Network 2, 22 focus molecules, score = 36, Table 5f). Connectivity maps were created for these networks to investigate the relationship between component proteins (Figures 15a, 15b). These maps were simplified for clarity, retaining only major pathway components and relationships (Figure 6b).

The PI3K/mTOR-pathwav

Activation of the PI3K/mTOR-pathway has emerged as one of the essential signaling mechanisms in Bcr-Abl leukemogenesis (Ren, 2005). Of particular interest within this pathway is the mammalian target of rapamycin (mTOR), which is constitutively activated in Bcr-Abl-transformed cells, leading to dysregulated translation and contributing to leukemogenesis. A recent study provided evidence that both the mTORCl and mTORC2 complexes are activated in Bcr-Abl cells and play key roles in mRNA translation of gene products that mediate mitogenic responses, as well as in cell growth and survival (Carayol et al, 2010). mTOR and key activators of mTOR, such as RICTOR, RAPTOR, Sinl (MAPKAP1), class 3 PI3Ks PIK3C3, also called hVps34, and PIK3R4 (VSP15) (Nobukuni et al, 2007), were identified in the PU-Hsp90 pull-downs (Tables 5a, 5d; Figures 6c, 6d, 13b). The NF-KB pathway

Activation of nuclear factor-κΒ (NF-κΒ) is required for Bcr-Abl transformation of primary bone marrow cells and for Bcr-Abl-transformed hematopoietic cells to form tumors in nude mice (McCubrey et al., 2008). PU-isolated proteins enriched on this pathway include NF-KB as well as activators of NF-kB such as IKBKAP, that binds NF-kappa-B-inducing kinase (NIK) and IKKs through separate domains and assembles them into an active kinase complex, and TBK-1 (TANK-binding kinase 1) and TAB1 (TAK1 -binding protein 1), both positive regulators of the I-kappaB kinase/NF-kappaB cascade (Hacker & Karin, 2006) (Tables 5a, 5d). Recently, Bcr-Abl-induced activation of the NF-κΒ cascade in myeloid leukemia cells was demonstrated to be largely mediated by tyrosine -phosphorylated PKD2 (or PRKD2) (Mihailovic et al, 2004) which we identify here to be a PU-H71/Hsp90 interactor (Tables 5a, 5d; Figures 6c, 6d, 13b).

The Raf/MAPK pathway

Key effectors of the MAPK pathway, another important pathway activated in CML (Ren, 2005; McCubrey et al, 2008), such as Raf-1, A-Raf, ERK, p90RSK, vav and several MAPKs were also included the PU-Hsp90-bound pool (Tables 5a, 5d; Figures 6c, 6d, 13b). In addition to the ERK signal transduction cascade, we identify components that act on activating the P38 MAPK pathway, such as MEKK4 and TAB1. IP A connects the MAPK- pathway to key elements of many different signal transduction pathways including PBK/mTOR-, STAT- and focal adhesion pathways (Figures 15a-d, 6b).

The STAT-pathwav

The STAT-pathway is also activated in CML and confers cytokine independence and protection against apoptosis (McCubrey et al., 2008) and was enriched by PU-H71 chemical precipitation (Network 8, 20 focus molecules, score = 14, Table 5f, Figure 15c). Both STAT5 and STAT3 were associated with PU-H71-Hsp90 complexes (Tables 5a, 5d; Figures 6c, 6d, 13b). In CML, STAT5 activation by phosphorylation is driven by Bcr-Abl (Ren,

2005) . Bruton agammaglobulinemia tyrosine kinase (BTK), constitutively phosphorylated and activated by Bcr-Abl in pre-B lymphoblastic leukemia cell (Hendriks & Kersseboom,

2006) , can also signal through STAT5 (Mahajan et al, 2001). BTK is another Hsp90- regulated protein that we identified in CML (Tables 5a, 5d; Figures 6c, 6d, 13b). In addition to phosphorylation, STATs can be activated in myeloid cells by calpain (CAPNl)-mediated proteolytic cleavage, leading to truncated STAT species (Oda et al, 2002). CAPN1 is also found in the PU-bound Hsp90 pulldowns, as is activated Ca(2+)/calmodulin-dependent protein kinase Ilgamma (CaMKIIgamma), which is also activated by Bcr-Abl (Si & Collins, 2008) (Tables 5a, 5d). CaMKIIgamma activity in CML is associated with the activation of multiple critical signal transduction networks involving the MAPK and STAT pathways. Specifically, in myeloid leukemia cells, CaMKIIgamma also directly phosphorylates STAT3 and enhances its transcriptional activity (Si & Collins, 2008).

The focal adhesion pathway

Retention and homing of progenitor blood cells to the marrow microenvironment are regulated by receptors and agonists of survival and proliferation. Bcr-Abl induces adhesion independence resulting in aberrant release of hematopoietic stem cells from the bone marrow, and leading to activation of adhesion receptor signaling pathways in the absence of ligand binding. The focal adhesion pathway was well represented in PU-H71 pulldowns (Network 12, 16 focus molecules, score = 13, Table 5f, Figure 15d). The focal adhesion-associated proteins paxillin, FAK, vinculin, talin, and tensin are constitutively phosphorylated in Bcr- Abl-transfected cell lines (Salgia et al, 1995), and these too were isolated in PU-Hsp90 complexes (Tables 5a, 5d and Figure 6c). In CML cells, FAK can activate STAT5 (Le et al, 2009).

Other important transforming pathways in CML, those driven by MYC (Sawyers, 1993) (Network 7, 15 focus molecules, score = 22, Figures 6a and 15e, Table 5f) and TGF-β (Naka et al, 2010) (Network 10, 13 focus molecules, score = 18, Figures 6a and 15f, Table 5f), were identified here as well. Among the identified networks were also those important for disease progression and aberrant cell cycle and proliferation of CML (Network 3, 20 focus molecules, score = 33, Network 4, 20 focus molecules, score = 33, Network 5, 20 focus molecules, score = 32, Network 6, 19 focus molecules, score = 30, Network 9, 14 focus molecules, score = 20, Network 11, 12 focus molecules, score = 17 and Network 13, 10 focus molecules, score = 12, Figure 6a and Table 5f). In summary, PU-H71 enriches a broad cross-section of proteins that participate in signaling pathways vital to the malignant phenotype in CML (Figure 6). The interaction of PU-bound Hsp90 with the aberrant CML signalosome was retained in primary CML samples (Figures 6d, 13b). PU-H71 identified proteins and networks are those important for the malignant phenotype We demomstrate that the presence of these proteins in the PU-bead pull-downs is functionally significant and suggests a role for Hsp90 in broadly supporting the malignant signalosome in CML cells. To demonstrate that the networks identified by PU-beads are important for transformation in K562, we next showed that inhibitors of key nodal proteins from individual networks (Figure 6b, yellow boxes - Bcr-Abl, NFKB, mTOR, MEK and CAMIIK) diminish the growth and proliferation potential of K562 cells (Figure 7a).

Next we demonstrated that PU-beads identified Hsp90 interactors with yet no assigned role in CML, also contribute to the transformed phenotype. The histone-arginine methyltransferase CARMl , a transcriptional co-activator of many genes (Bedford & Clarke, 2009), was validated in the PU-bead pull-downs from CML cell lines and primary CML cells (Figures 6c, 6d, 13). This is the first reported link between Hsp90 and CARMl, although other arginine methyltransferases, such as PRMT5, have been shown to be Hsp90 clients in ovarian cancer cells (Maloney et al., 2007). While elevated CARMl levels are implicated in the development of prostate and breast cancers, little is known on the importance of CARMl in CML leukomogenesis (Bedford & Clarke, 2009). We found CARMl essentially entirely captured by the Hsp90 species recognized by PU-beads (Figure 7b) and also sensitive to degradation by PU-H71 (Figure 6c, right). CARMl therefore, may be a novel Hsp90 oncoprotein in CML. Indeed, knock-down experiments with CARMl but not control shRNAs (Figure 7c), demonstrate reduced viability and induction of apoptosis in K562 cells, supporting this hypothesis.

To demonstrate that the presence of proteins in the PU-pulldowns is due to their participation in aberrantly activated signaling and not merely their abundant expression, we compared PU- bead pulldowns from K562 and Mia-PaCa-2, a pancreatic cancer cell line (Table 5a). While both cells express high levels of STAT5 protein (Figure 7d), activation of the STAT5 pathway, as demonstrated by STAT5 phosphorylation (Figure 7d) and DNA-binding (Jaganathan et al., 2010), was noted only in the K562 cells. In accordance, this protein was identified only in the K562 PU-bead pulldowns (Table 5a and Figure 7e). In contrast, activated STAT3 was identified in PU-Hsp90 complexes from both K562 (Figures 6c, 7e) and Mia-PaCa-2 cells extracts (Figures 7e, 7f).

The mTOR pathway was identified by the PU-beads in both K562 and Mia-PaCa-2 cells

(Figures 7e, 7f), and indeed, its pharmacologic inhibition by PP242, a selective inhibitor that targets the ATP domain of mTOR (Apsel et al, 2008), is toxic to both cells (Figures 7a, 7g). On the other hand, the Abl inhibitor Gleevec (Deininger & Druker, 2003) was toxic only to K562 cells (Figures 7a, 7g). Both cells express Abl but only K562 has the oncogenic Bcr- Abl (Figure 7d) and PU-beads identify Abl, as Bcr-Abl, in K562 but not in Mia-PaCa-2 cells (Figure 7e).

PU-H71 identifies a novel mechanism of oncogenic ST AT -activation

PU-bead pull-downs contain several proteins, including Bcr-Abl (Ren, 2005), CAMKIIy (Si & Collins, 2008), FAK (Salgia et al, 1995), vav-1 (Katzav, 2007) and PRKD2 (Mihailovic et al., 2004) that are constitutively activated in CML leukemogenesis. These are classical Hsp90-regulated clients that depend on Hsp90 for their stability because their steady-state levels decrease upon Hsp90 inhibition (Figure 6c) (Zuehlke & Johnson, 2010; Workman et al, 2007). Constitutive activation of STAT3 and STAT5 is also reported in CML (Ren, 2005; McCubrey et al, 2008). These proteins, however, do not fit the criteria of classical client proteins because STAT5 and STAT3 levels remain essentially unmodified upon Hsp90 inhibition (Figure 6c). The PU-pull-downs also contain proteins isolated potentially as part of an active signaling mega-complex, such as mTOR, VSP32, VSP15 and RAPTOR (Carayol et al, 2010). mTOR activity, as measured by cellular levels of p-mTOR, also appears to be more sensitive to Hsp90 inhibition than are the complex components (i.e. compare the relative decrease in p-mTOR and RAPTOR in PU-H71 treated cells, Figure 6c). Further, PU- Hsp90 complexes contain adapter proteins such as GRB2, DOCK, CRKL and EPS 15, which link Bcr-Abl to key effectors of multiple aberrantly activated signaling pathways in K562 (Brehme et al, 2009; Ren, 2005) (Figure 6b). Their expression also remains unchanged upon Hsp90 inhibition (Figure 6c). We therefore wondered whether the contribution of Hsp90 to certain oncogenic pathways extends beyond its classical folding actions. Specifically, we hypothesized that Hsp90 might also act as a scaffolding molecule that maintains signaling complexes in their active configuration, as has been previously postulated (Dezwaan & Freeman, 2008; Pratt et al, 2008).

Hsp90 binds to and influences the conformation of STAT 5

To investigate this hypothesis further we focused on STAT5, which is constitutively phosphorylated in CML (de Groot et al, 1999). The overall level of p-STAT5 is determined by the balance of phosphorylation and dephosphorylation events. Thus, the high levels of p- STAT5 in K562 cells may reflect either an increase in upstream kinase activity or a decrease in protein tyrosine phosphatase (PTPase) activity. A direct interaction between Hsp90 and p- STAT5 could also modulate the cellular levels of p-STAT5.

To dissect the relative contribution of these potential mechanisms, we first investigated the effect of PU-H71 on the main kinases and PTPases that regulate STAT5 phosphorylation in K562 cells. Bcr-Abl directly activates STAT5 without the need for JAK phosphorylation (de Groot et al, 1999). Concordantly, ST AT5 -phosphorylation rapidly decreased in the presence of the Bcr-Abl inhibitor Gleevec (Figure 8a, left, Gleevec). While Hsp90 regulates Bcr-Abl stability, the reduction in steady-state Bcr-Abl levels following Hsp90 inhibition requires more than 3 h (An et al., 2000). Indeed no change in Bcr-Abl expression (Figure 8a, left, PU- H71, Bcr-Abl) or function, as evidenced by no decrease in CRKL phosphorylation (Figure 8a, left, PU-H71, p-CRKL/CRKL), was observed with PU-H71 in the time interval it reduced p-STAT5 levels (Figure 8a, left, PU-H71, p-STAT5). Also, no change in the activity and expression of HCK, a kinase activator of STAT5 in 32Dcl3 cells transfected with Bcr-Abl Klejman et al, 2002), was noted (Figure 8a, right, HCK p-HCK).

Thus reduction of p-STAT5 phosphorylation by PU-H71 in the 0 to 90 min interval (Figure 8c, left, PU-H71) is unlikely to be explained by destabilization of Bcr-Abl or other kinases. We therefore examined whether the rapid decrease in p-STAT5 levels in the presence of PU- H71 may be accounted for by an increase in PTPase activity. The expression and activity of SHP2, the major cytosolic STAT5 phosphatase (Xu & Qu, 2008), were also not altered within this time interval (Figure 8a, right, SHP2/p-SHP2). Similarly, the levels of SOCS1 and SOCS3, which form a negative feedback loop that switches off STAT-signaling Deininger & Druker, 2003) were unaffected by PU-H71 (Figure 8a, right, SOCS 1/3).

Thus no effect on STAT5 in the interval 0-90min can likely be attributed to a change in kinase or phosphatase activity towards STAT5. As an alternative mechanism, and because the majority of p-STAT5 but not STAT5 is Hsp90 bound in CML cells (Figure 8b), we hypothesized that the cellular levels of activated STAT5 are fine-tuned by direct binding to Hsp90.

The activation/inactivation cycle of STATs entails their transition between different dimer conformations. Phosphorylation of STATs occurs in an anti-parallel dimer conformation that upon phosphorylation triggers a parallel dimer conformation. Dephosphorylation of STATs on the other hand require extensive spatial reorientation, in that the tyrosine phosphorylated STAT dimers must shift from parallel to anti-parallel configuration to expose the phospho- tyrosine as a better target for phosphatases (Lim & Cao, 2006). We find that STAT5 is more susceptible to trypsin cleavage when bound to Hsp90 (Figure 8c), indicating that binding of Hsp90 directly modulates the conformational state of STAT5, potentially to keep STAT5 in a conformation unfavorable for dephosphorylation and/or favorable for phosphorylation.

To investigate this possibility we used a pulse-chase strategy in which orthovanadate (Na ₃ V0 ₄ ), a non-specific PTPase inhibitor, was added to cells to block the dephosphorylation of STAT5. The residual level of p-STAT5 was then determined at several later time points (Figure 8d). In the absence of PU-H71, p-STAT5 accumulated rapidly, whereas in its presence, cellular p-STAT5 levels were diminished. The kinetics of this process (Figure 8d) were similar to the rate of p-STAT5 steady-state reduction (Figure 8a, left, PU-H71).

Hsp90 maintains STAT 5 in an active conformation directly within STAT 5 -containing transcriptional complexes

In addition to STAT5 phosphorylation and dimerization, the biological activity of STAT5 requires its nuclear translocation and direct binding to its various target genes (de Groot et al, 1999; Lim & Cao, 2006). We wondered therefore, whether Hsp90 might also facilitate the transcriptional activation of STAT5 genes, and thus participate in promoter-associated STAT5 transcription complexes. Using an ELISA-based assay, we found that STAT5 (Figure 8e) is constitutively active in K562 cells and binds to a STAT5 binding consensus sequence (5'-TTCCCGGAA-3'). STAT5 activation and DNA binding is partially abrogated, in a dose-dependent manner, upon Hsp90 inhibition with PU-H71 (Figure 8e). Furthermore, quantitative ChIP assays in K562 cells revealed the presence of both Hsp90 and STAT5 at the critical STAT5 targets MYC and CCND2 (Figure 8f). Neither protein was present at intergenic control regions (not shown). Accordingly, PU-H71 (1 μΜ) decreased the mRNA abundance of the STAT5 target genes CCND2, MYC, CCND1, BCL-XL and MCL1 (Katzav, 2007), but not of the control genes HPRT and GAPDH (Figure 8g and not shown).

Collectively, these data show that STAT5 activity is positively regulated by Hsp90 in CML cells (Figure 8h). Our findings are consistent with a scenario whereby Hsp90 binding to STAT5 modulates the conformation of the protein and by this mechanism it alters STAT5 phosphorylation/ dephosphorylation kinetics, shifting the balance towards increased levels of p-STAT5. In addition, Hsp90 maintains STAT5 in an active conformation directly within STAT5 -containing transcriptional complexes. Considering the complexity of the STAT- pathway, other potential mechanisms however, cannot be excluded. Therefore, in addition to its role in promoting protein stability, Hsp90 promotes oncogenesis by maintaining client proteins in an active configuration.

More broadly, the data suggest that it is the PU-H71-Hsp90 fraction of cellular Hsp90 that is most closely involved in supporting oncogenic protein functions in tumor cells, and PU-H71- Hsp90 proteomics can be used to identify a broad cross-section of the protein pathways required to maintain the malignant phenotype in specific tumor cells (Figure 9).

Discussion

It is now appreciated that many proteins that are required to maintain tumor cell survival may not present mutations in their coding sequence, and yet identifying these proteins is of extreme importance to understand how individual tumors work. Genome wide mutational studies may not identify these oncoproteins since mutations are not required for many genes to support tumor cell survival (e.g. IRF4 in multiple myeloma and BCL6 in B-cell lymphomas) (Cerchietti et al., 2009). Highly complex, expensive and large-scale methods such as RNAi screens have been the major means for identifying the complement of oncogenic proteins in various tumors (Horn et al, 2010). We present herein a rapid and simple chemical-proteomics method for surveying tumor oncoproteins regardless of whether they are mutated (Figure 9). The method takes advantage of several properties of PU-H71 which i) binds preferentially to the fraction of Hsp90 that is associated with oncogenic client proteins, and ii) locks Hsp90 in an onco-client bound configuration. Together these features greatly facilitate the chemical affinity-purification of tumor-associated protein clients by mass spectrometry (Figure 9). We propose that this approach provides a powerful tool in dissecting, tumor-by-tumor, lesions characteristic of distinct cancers. Because of the initial chemical precipitation step, which purifies and enriches the aberrant protein population as part of PU-bead bound Hsp90 complexes, the method does not require expensive SILAC labeling or 2-D gel separations of samples. Instead, protein cargo from PU-bead pull-downs is simply eluted in SDS buffer, submitted to standard SDS-PAGE, and then the separated proteins are extracted and trypsinized for LC/MS/MS analyses. While this method presents a unique approach to identify the oncoproteins that maintain the malignant phenotype of tumor cells, one needs to be aware that, similarly to other chemical or antibody-based proteomics techniques, it also has potential limitations (Rix & Superti- Furga, 2009). For example, "sticky" or abundant proteins may also bind in a nondiscriminatory fashion to proteins isolated by the PU-H71 beads. Such proteins were catalogued by several investigators (Trinkle-Mulcahy et al, 2008), and we have used these lists to eliminate them from the pull-downs with the clear understanding that some of these proteins may actually be genuine Hsp90 clients. Second, while we have presented several lines of evidence that PU-H71 is specific for Hsp90 (Figure 11; Taldone & Chiosis, 2009), one must also consider that at the high concentration of PU-H71 present on the beads, unspecific and direct binding of the drug to a small number of proteins is unavoidable.

In spite of the potential limitations described in the preceeding paragraph, we have, using this method, performed the first global evaluation of Hsp90-facilitated aberrant signaling pathways in CML. The Hsp90 interactome identified by PU-H71 affinity purification significantly overlaps with the well-characterized CML signalosome (Figure 6a), indicating that this method is able to identify a large part of the complex web of pathways and proteins that define the molecular basis of this form of leukemia. We suggest that PU-H71 chemical- proteomics assays may be extended to other forms of cancer in order to identify aberrant signaling networks that drive the malignant phenotype in individual tumors (Figure 9). For example, we show further here how the method is used to identify the aberrant protein networks in the MDA-MB-468 triple-negative breast cancer cells, the MiaPaCa2 pancreatic cancer cells and the OCI-LY1 diffuse large B-cell lymphoma cells.

Since single agent therapy is not likely to be curative in cancer, it is necessary to design rational combinatorial therapy approaches. Proteomic identification of oncogenic Hsp90- scaffolded signaling networks may identify additional oncoproteins that could be further targeted using specific small molecule inhibitors. Indeed, inhibitors of mTOR and CAMKII, which are identified by our method to contribute to the transformation of K562 CML cells and be key nodal proteins on individual networks (Figure 6b, yellow boxes), are active as single agents (Figure 7a) and synergize with Hsp90 inhibition in affecting the growth of these leukemia cells (Figure 21). When applied to less well-characterized tumor types, PU-H71 chemical proteomics might provide less obvious and more impactful candidate targets for combinatorial therapy. We exemplify this concept in the MDA-MB-468 triple-negative breast cancer cells, the MiaPaCa2 pancreatic cancer cells and the OCI-LY1 diffuse large B-cell lymphoma cells.

In the triple negative breast cancer cell line MDA-MB-468 major signaling networks identified by the method were the PI3K/AKT, IGF-IR, NRF2-mediated oxidative stress response, MYC, PKA and the IL-6 signaling pathways (Figure 22). Pathway components as identified by the method are listed in Table 3.

Table 3.

© 2000-

2012

Ingenuity

Systems,

Inc. All rights

reserved.

Entrez Gene

ID Notes Symbol Name Location Type(s) Drug(s) alpha- and

gamma-adaptin

AAGAB AAGAB binding protein Cytoplasm other

abhydrolase

domain

ABHD10 ABHD10 containing 10 Cytoplasm other

ArfGAP with

coiled-coil,

ankyrin repeat

and PH domains

ACAP2 ACAP2 2 Nucleus other

AHA1 , activator

of heat shock

90kDa protein

ATPase

homolog 1

AHSA1 AHSA1 (yeast) Cytoplasm other

A kinase

(PRKA) anchor

AKAP8 AKAP8 protein 8 Nucleus other

A kinase

(PRKA) anchor

AKAP8L AKAP8L protein 8-like Nucleus other

Aly/REF export transcription

ALYREF ALYREF factor Nucleus regulator

ankyrin repeat

ANKRD17 ANKRD17 domain 17 unknown other ankyrin repeat

ANKRD50 ANKRD50 domain 50 unknown other

acidic (leucine- rich) nuclear

phosphoprotein

32 family,

ANP32A ANP32A member A Nucleus other

ANXA1 1 ANXA1 1 annexin A1 1 Nucleus other

Plasma

ANXA2 ANXA2 annexin A2 Membrane other

Plasma

ANXA7 ANXA7 annexin A7 Membrane ion channel

ADP-ribosylation

factor GTPase

activating

ARFGAP1 ARFGAP1 protein 1 Cytoplasm transporter

ADP-ribosylation

factor guanine

nucleotide- exchange factor

2 (brefeldin A-

ARFGEF2 ARFGEF2 inhibited) Cytoplasm other

ADP-ribosylation

factor interacting

ARFIP2 ARFIP2 protein 2 Cytoplasm other

Rho GTPase

activating

ARHGAP29 ARHGAP29 protein 29 Cytoplasm other

Rho guanine

nucleotide

exchange factor

ARHGEF40 ARHGEF40 (GEF) 40 unknown other

N- acylsphingosine

amidohydrolase

(acid

ASAH1 ASAH1 ceramidase) 1 Cytoplasm enzyme atlastin GTPase

ATL3 ATL3 3 Cytoplasm other

BCL2- associated

BAG4 BAG4 athanogene 4 Cytoplasm other

BCL2- associated

BAG 6 BAG6 athanogene 6 Nucleus enzyme beclin 1 ,

autophagy

BECN1 BECN1 related Cytoplasm other

baculoviral IAP

repeat

BIRC6 BIRC6 containing 6 Cytoplasm enzyme bleomycin

BLMH BLMH hydrolase Cytoplasm peptidase

BRCA1- associated ATM

BRAT1 BRAT1 activator 1 Cytoplasm other

BRCA1/BRCA2- containing

BRCC3 BRCC3 complex, Nucleus enzyme subunit 3

bromodomain

BRD4 BRD4 containing 4 Nucleus kinase

BTAF1 RNA

polymerase II,

B-TFIID

transcription

factor- associated,

170kDa (Mot1

homolog, S. transcription

BTAF1 BTAF1 cerevisiae) Nucleus regulator budding

uninhibited by

benzimidazoles

1 homolog beta

BUB1 B BUB1 B (yeast) Nucleus kinase budding

uninhibited by

BUB3 benzimidazoles

(includes 3 homolog

BUB3 EG:12237) (yeast) Nucleus other

BYSL BYSL bystin-like Cytoplasm other

basic leucine

zipper and W2 translation

BZW1 BZW1 domains 1 Cytoplasm regulator calcyclin binding

CACYBP CACYBP protein Nucleus other

CALU CALU calumenin Cytoplasm other

calcium/calmodu

lin-dependent

protein kinase II

CAMK2G CAMK2G gamma Cytoplasm kinase cullin-associated

and neddylation- transcription

CAND1 CAND1 dissociated 1 Cytoplasm regulator

CANX CANX calnexin Cytoplasm other

CAP, adenylate

cyclase- associated Plasma

CAP1 CAP1 protein 1 (yeast) Membrane other

cell cycle

associated Plasma

CAPRI N1 CAPRIN1 protein 1 Membrane other

capping protein

(actin filament)

muscle Z-line,

CAPZA1 CAPZA1 alpha 1 Cytoplasm other

capping protein

(actin filament)

muscle Z-line,

CAPZB CAPZB beta Cytoplasm other

coactivator- associated

arginine

methyltransferas transcription

CARM1 CARM1 e 1 Nucleus regulator

CASK transcription

CASKIN1 CASKIN1 interacting Nucleus regulator protein 1

CAT CAT catalase Cytoplasm enzyme

carbonyl

CBR1 CBR1 reductase 1 Cytoplasm enzyme

coiled-coil

domain

CCDC124 CCDC124 containing 124 unknown other

coiled-coil

domain

CCDC99 CCDC99 containing 99 Nucleus other

cell division

cycle 37

homolog (S.

CDC37 CDC37 cerevisiae) Cytoplasm other

cell division

cycle 37

homolog (S.

cerevisiae)-like

CDC37L1 CDC37L1 1 Cytoplasm other

CDC42 binding

protein kinase

gamma (DMPK-

CDC42BPG CDC42BPG like) Cytoplasm kinase

cadherin 1 , type

1 , E-cadherin Plasma

CDH1 CDH 1 (epithelial) Membrane other

cyclin- dependent

CDK1 CDK1 kinase 1 Nucleus kinase flavopiridol cyclin- dependent

CDK13 CDK13 kinase 13 Nucleus kinase

cyclin- dependent PD-0332991 ,

CDK4 CDK4 kinase 4 Nucleus kinase flavopiridol cyclin- dependent BMS-387032,

CDK7 CDK7 kinase 7 Nucleus kinase flavopiridol

CTF18,

chromosome

transmission

fidelity factor 18

homolog (S.

CHTF18 CHTF18 cerevisiae) unknown other

CNDP

dipeptidase 2

(metallopeptidas

CNDP2 CNDP2 e M20 family) Cytoplasm peptidase

calponin 3,

CNN3 CNN3 acidic Cytoplasm other

CCR4-NOT

transcription

complex,

CNOT1 CNOT1 subunit 1 Cytoplasm other

CCR4-NOT

transcription

complex, transcription

CNOT2 CNOT2 subunit 2 Nucleus regulator

CNOT7 CNOT7 CCR4-NOT Nucleus transcription transcription regulator complex,

subunit 7

coproporphyrino

CPOX CPOX gen oxidase Cytoplasm enzyme cold shock

domain protein transcription

CSDA CSDA A Nucleus regulator casein kinase 1 ,

CSNK1A1 CSNK1A1 alpha 1 Cytoplasm kinase casein kinase 2,

alpha 1

CSNK2A1 CSNK2A1 polypeptide Cytoplasm kinase casein kinase 2,

alpha prime

CSNK2A2 CSNK2A2 polypeptide Cytoplasm kinase catenin

(cadherin- associated

protein), beta 1 , transcription

CTNNB1 CTNNB1 88kDa Nucleus regulator catenin

(cadherin- associated

CTNND1 CTNND1 protein), delta 1 Nucleus other

CTSB CTSB cathepsin B Cytoplasm peptidase

Plasma

CTTN CTTN cortactin Membrane other

cytosolic

thiouridylase

subunit 1

homolog (S.

CTU1 CTU1 pombe) Cytoplasm other

cytoplasmic

FMR1

interacting

CYFIP1 CYFIP1 protein 1 Cytoplasm other

DCP1

decapping

enzyme

homolog A (S.

DCP1A DCP1A cerevisiae) Nucleus other

dicer 1 ,

ribonuclease

DICER1 DICER1 type III Cytoplasm enzyme

DnaJ (Hsp40)

homolog,

subfamily A,

DNAJA1 DNAJA1 member 1 Nucleus other

DnaJ (Hsp40)

homolog,

subfamily A,

DNAJA2 DNAJA2 member 2 Nucleus enzyme

DnaJ (Hsp40)

homolog,

subfamily B,

DNAJB1 DNAJB1 member 1 Nucleus other

DnaJ (Hsp40)

DNAJB1 1 DNAJB1 1 homolog, Cytoplasm other subfamily B,

member 1 1

DnaJ (Hsp40)

homolog,

subfamily B, transcription

DNAJB6 DNAJB6 member 6 Nucleus regulator

DnaJ (Hsp40)

homolog,

subfamily C,

DNAJC7 DNAJC7 member 7 Cytoplasm other

Plasma

DSP DSP desmoplakin Membrane other

deltex 3-like

DTX3L DTX3L (Drosophila) Cytoplasm enzyme

EBNA1 binding

EBNA1 BP2 EBNA1 BP2 protein 2 Nucleus other

enhancer of

mRNA

EDC3 decapping 3

(includes homolog (S.

EDC3 EG:315708) cerevisiae) Cytoplasm other

enhancer of

mRNA

EDC4 EDC4 decapping 4 Cytoplasm other

eukaryotic

translation

elongation factor translation

EEF1 B2 EEF1 B2 1 beta 2 Cytoplasm regulator eukaryotic

translation

elongation factor translation

EEF2 EEF2 2 Cytoplasm regulator elongation factor

Tu GTP binding

domain

EFTUD2 EFTUD2 containing 2 Nucleus enzyme eukaryotic

translation

initiation factor

2B, subunit 2 translation

EIF2B2 EIF2B2 beta, 39kDa Cytoplasm regulator eukaryotic

translation

initiation factor translation

EIF3A EIF3A 3, subunit A Cytoplasm regulator eukaryotic

translation

initiation factor translation

EIF4A1 EIF4A1 4A1 Cytoplasm regulator eukaryotic

translation translation

EIF6 EIF6 initiation factor 6 Cytoplasm regulator

ELAV

(embryonic

lethal, abnormal

vision,

Drosophila)-like

ELAVL1 ELAVL1 1 (Hu antigen R) Cytoplasm other

ELP3 ELP3 elongation Nucleus enzyme protein 3

homolog (S.

cerevisiae)

EMD EMD emerin Nucleus other

tucotuzumab celmoleukin, catumaxoma epithelial cell b, adhesion Plasma adecatumum

EPCAM EPCAM molecule Membrane other ab

EPPK1 EPPK1 epiplakin 1 Cytoplasm other

epidermal

growth factor

receptor

pathway Plasma

EPS15 EPS15 substrate 15 Membrane other

epidermal

growth factor

receptor

pathway

substrate 15-like Plasma

EPS15L1 EPS15L1 1 Membrane other

epithelial

splicing

regulatory

ESRP1 ESRP1 protein 1 Nucleus other

extended

synaptotagmin-

ESYT1 ESYT1 like protein 1 unknown other

eukaryotic

translation

termination translation

ETF1 ETF1 factor 1 Cytoplasm regulator

electron- transfer- flavoprotein,

alpha

ETFA ETFA polypeptide Cytoplasm transporter

transcription

ETV3 ETV3 ets variant 3 Nucleus regulator

Fanconi anemia,

complementatio

FANCD2 FANCD2 n group D2 Nucleus other

fatty acid

FASN FASN synthase Cytoplasm enzyme

farnesyl- diphosphate TAK-475, farnesyltransfera zoledronic

FDFT1 FDFT1 se 1 Cytoplasm enzyme acid

four and a half Plasma

FHL3 FHL3 LIM domains 3 Membrane other

FK506 binding

FKBP4 FKBP4 protein 4, 59kDa Nucleus enzyme

FK506 binding

protein 9, 63

FKBP9 FKBP9 kDa Cytoplasm enzyme

FAD1 flavin

adenine

FLAD1 FLAD1 dinucleotide Cytoplasm enzyme synthetase

homolog (S.

cerevisiae)

FLNA FLNA filamin A, alpha Cytoplasm other

FLNB FLNB filamin B, beta Cytoplasm other

far upstream

element (FUSE) transcription

FUBP1 FUBP1 binding protein 1 Nucleus regulator

far upstream

element (FUSE) transcription

FUBP3 FUBP3 binding protein 3 Nucleus regulator

GAN GAN gigaxonin Cytoplasm other

glucosidase,

alpha; neutral

GANAB GANAB AB Cytoplasm enzyme

glyceraldehyde-

3-phosphate

GAPDH GAPDH dehydrogenase Cytoplasm enzyme

phosphoribosylg

lycinamide

formyltransferas

e,

phosphoribosylg

lycinamide

synthetase,

phosphoribosyla

minoimidazole

GART GART synthetase Cytoplasm enzyme LY231514 glucosidase,

GBA GBA beta, acid Cytoplasm enzyme

grancalcin, EF- hand calcium

GCA GCA binding protein Cytoplasm other

GRB10

interacting GYF

GIGYF2 GIGYF2 protein 2 unknown other

GINS complex

subunit 4 (Sld5

GINS4 GINS4 homolog) Nucleus other

galactosidase,

GLA GLA alpha Cytoplasm enzyme

galactosidase,

GLB1 GLB1 beta 1 Cytoplasm enzyme

glomulin, FKBP

associated

GLMN GLMN protein Cytoplasm other

Plasma

GPHN GPHN gephyrin Membrane enzyme

glucose-6- phosphate Extracellular

GPI GPI isomerase Space enzyme

G protein

pathway

GPS1 GPS1 suppressor 1 Nucleus other

growth factor

receptor-bound

GRB2 GRB2 protein 2 Cytoplasm other

general transcription

GTF2F1 GTF2F1 transcription Nucleus regulator factor I IF,

polypeptide 1 ,

74kDa

general

transcription

factor I IF,

polypeptide 2, transcription

GTF2F2 GTF2F2 30kDa Nucleus regulator

general

transcription transcription

GTF2I GTF2I factor Mi Nucleus regulator

H1 histone

family, member

H1 F0 H1 F0 0 Nucleus other

H1 histone

family, member

H1 FX H1 FX X Nucleus other

tributyrin, belinostat, pyroxamide, histone transcription vorinostat,

HDAC2 HDAC2 deacetylase 2 Nucleus regulator romidepsin tributyrin, belinostat, pyroxamide,

MGCD0103, histone transcription vorinostat,

HDAC3 HDAC3 deacetylase 3 Nucleus regulator romidepsin tributyrin, belinostat, pyroxamide, histone transcription vorinostat,

HDAC6 HDAC6 deacetylase 6 Nucleus regulator romidepsin hypoxia

inducible factor

1 , alpha subunit

HIF1AN HIF1AN inhibitor Nucleus enzyme

histone cluster

HIST1 H1 B HIST1 H1 B 1 , H1 b Nucleus other

histone cluster

HIST1 H1 D HIST1 H1 D 1 , H1d Nucleus other

heterogeneous

nuclear

ribonucleoprotei

HNRNPAO HNRNPAO n AO Nucleus other

17- dimethylamin oethylamino- heat shock 17- protein 90kDa demethoxyge alpha Idanamycin,

(cytosolic), class IPI-504,

HSP90AA1 HSP90AA1 A member 1 Cytoplasm enzyme cisplatin heat shock

protein 90kDa

alpha

(cytosolic), class

HSP90AA4P HSP90AA4P A member 4, unknown other pseudogene

17- dimethylamin oethylamino- heat shock 17- protein 90kDa demethoxyge alpha Idanamycin,

(cytosolic), class IPI-504,

HSP90AB1 HSP90AB1 B member 1 Cytoplasm enzyme cisplatin

17- dimethylamin oethylamino- 17- heat shock demethoxyge protein 90kDa Idanamycin, beta (Grp94), IPI-504,

HSP90B1 HSP90B1 member 1 Cytoplasm other cisplatin heat shock

HSPA4 HSPA4 70kDa protein 4 Cytoplasm other

heat shock

70kDa protein 5

(glucose- regulated

HSPA5 HSPA5 protein, 78kDa) Cytoplasm enzyme

heat shock

HSPA8 HSPA8 70kDa protein 8 Cytoplasm enzyme

heat shock

HSPB1 HSPB1 27kDa protein 1 Cytoplasm other

heat shock

60kDa protein 1

HSPD1 HSPD1 (chaperonin) Cytoplasm enzyme

heat shock

105kDa/1 10kDa

HSPH1 HSPH1 protein 1 Cytoplasm other

isocitrate

dehydrogenase

2 (NADP+),

IDH2 IDH2 mitochondrial Cytoplasm enzyme

immunoglobulin

(CD79A) binding

IGBP1 IGBP1 protein 1 Cytoplasm phosphatase

insulin-like

growth factor 2

mRNA binding translation

IGF2BP3 IGF2BP3 protein 3 Cytoplasm regulator

inhibitor of

kappa light

polypeptide

gene enhancer

in B-cells,

kinase complex- associated

IKBKAP IKBKAP protein Cytoplasm other

interleukin

enhancer

binding factor 2, transcription

ILF2 ILF2 45kDa Nucleus regulator

ILF3 ILF3 interleukin Nucleus transcription enhancer regulator

binding factor 3,

90kDa

thioguanine, VX-944,

IMP (inosine 5'- interferon monophosphate alfa- ) 2b/ribavirin, dehydrogenase mycophenolic

IMPDH1 IMPDH1 1 Cytoplasm enzyme acid, ribavirin thioguanine, VX-944,

IMP (inosine 5'- interferon monophosphate alfa- ) 2b/ribavirin, dehydrogenase mycophenolic

IMPDH2 IMPDH2 2 Cytoplasm enzyme acid, ribavirin inverted formin,

FH2 and WH2

domain

INF2 INF2 containing Cytoplasm other

integrator

complex subunit

INTS3 INTS3 3 Nucleus other

interleukin-1

receptor- associated Plasma

IRAKI IRAKI kinase 1 Membrane kinase

inositol-3- phosphate

ISYNA1 ISYNA1 synthase 1 unknown enzyme

itchy E3

ubiquitin protein

ligase homolog

ITCH ITCH (mouse) Nucleus enzyme

KH domain

containing, RNA

binding, signal

transduction transcription

KHDRBS1 KHDRBS1 associated 1 Nucleus regulator

KH-type splicing

regulatory

KHSRP KHSRP protein Nucleus enzyme

lectin,

galactoside- binding, soluble, Extracellular

LGALS3 LGALS3 3 Space other

lectin,

galactoside- binding, soluble, Plasma transmembrane

LGALS3BP LGALS3BP 3 binding protein Membrane receptor

lipase A,

lysosomal acid,

cholesterol

LIPA LIPA esterase Cytoplasm enzyme

lectin, mannose-

LMAN2 LMAN2 binding 2 Cytoplasm transporter

LMNA LMNA lamin A/C Nucleus other LPS-responsive

vesicle

trafficking,

beach and

anchor

LRBA LRBA containing Cytoplasm other

leucine-rich

PPR-motif

LRPPRC LRPPRC containing Cytoplasm other

LSM14A, SCD6

homolog A (S.

LSM14A LSM14A cerevisiae) Cytoplasm other

membrane

associated

guanylate

kinase, WW and

PDZ domain

MAG 13 MAG 13 containing 3 Cytoplasm kinase mitog en-

MAP3K7 activated protein

(includes kinase kinase

MAP3K7 EG:172842) kinase 7 Cytoplasm kinase mitog en- activated protein

MAPK1 MAPK1 kinase 1 Cytoplasm kinase mitog en- activated protein

MAPK3 MAPK3 kinase 3 Cytoplasm kinase mitog en- activated protein

MAPK9 MAPK9 kinase 9 Cytoplasm kinase minichromosom

e maintenance

complex

MCM2 MCM2 component 2 Nucleus enzyme

MEM01

(includes mediator of cell

MEM01 EG:298787) motility 1 Cytoplasm other

antigen

identified by

monoclonal

MKI67 MKI67 antibody Ki-67 Nucleus other

myeloid

leukemia factor

MLF2 MLF2 2 Nucleus other

mutS homolog 6

MSH6 MSH6 (E. coli) Nucleus enzyme

MSI1 musashi

(includes homolog 1

MSI 1 EG:17690) (Drosophila) Cytoplasm other

musashi

homolog 2

MSI2 MSI2 (Drosophila) Cytoplasm other

metastasis

associated 1

family, member transcription

MTA2 MTA2 2 Nucleus regulator deforolimus,

OSI-027, mechanistic NVP- target of BEZ235, rapamycin temsirolimus, (serine/threonin tacrolimus,

MTOR MTOR e kinase) Nucleus kinase everolimus

MTX1 MTX1 metaxin 1 Cytoplasm transporter

MYB binding

protein (P160) transcription

MYBBP1A MYBBP1A 1a Nucleus regulator

MYC binding

MYCBP2 MYCBP2 protein 2 Nucleus enzyme

nucleus

accumbens

associated 1 ,

BEN and BTB

(POZ) domain transcription

NACC1 NACC1 containing Nucleus regulator

N- acetyltransferas

e 10 (GCN5-

NAT 10 NAT 10 related) Nucleus enzyme

nuclear cap

binding protein

subunit 1 ,

NCBP1 NCBP1 80kDa Nucleus other

NCK-associated Plasma

NCKAP1 NCKAP1 protein 1 Membrane other

NCK interacting

protein with SH3

NCKIPSD NCKIPSD domain Nucleus other

NCL NCL nucleolin Nucleus other

nuclear receptor transcription

NCOR1 NCOR1 corepressor 1 Nucleus regulator

nuclear receptor transcription

NCOR2 NCOR2 corepressor 2 Nucleus regulator

nuclear factor of

kappa light

polypeptide

gene enhancer

in B-cells 2 transcription

NFKB2 NFKB2 (p49/p100) Nucleus regulator

NFKB transcription

NKRF NKRF repressing factor Nucleus regulator

non-metastatic

cells 7, protein

expressed in

(nucleoside- diphosphate

NME7 NME7 kinase) Cytoplasm kinase

nicotinamide N- methyltransferas

NNMT NNMT e Cytoplasm enzyme

nucleolar protein

family 6 (RNA-

NOL6 NOL6 associated) Nucleus other

nucleophosmin transcription

NPM1 NPM1 (nucleolar Nucleus regulator phosphoprotein

B23, numatrin)

NAD(P)H

dehydrogenase,

NQ01 NQ01 quinone 1 Cytoplasm enzyme

NAD(P)H

dehydrogenase,

NQ02 NQ02 quinone 2 Cytoplasm enzyme

NUCB1 NUCB1 nucleobindin 1 Cytoplasm other

NudC domain

NUDCD1 NUDCD1 containing 1 unknown other

NudC domain

NUDCD3 NUDCD3 containing 3 unknown other

nudix

(nucleoside

diphosphate

linked moiety X)-

NUDT5 NUDT5 type motif 5 Cytoplasm phosphatase

NUF2, NDC80

kinetochore

complex

component,

homolog (S.

NUF2 NUF2 cerevisiae) Nucleus other

OTU domain,

ubiquitin

aldehyde

OTUB1 OTUB1 binding 1 unknown enzyme

OTU domain

OTUD4 OTUD4 containing 4 unknown other

proliferation- associated 2G4, transcription

PA2G4 PA2G4 38kDa Nucleus regulator proliferating cell

PCNA PCNA nuclear antigen Nucleus enzyme

PDGFA

associated

PDAP1 PDAP1 protein 1 Cytoplasm other

programmed cell

PDCD2L PDCD2L death 2-like unknown other

programmed cell

death 6

interacting

PDCD6IP PDCD6IP protein Cytoplasm other

protein disulfide

isomerase

family A,

PDIA6 PDIA6 member 6 Cytoplasm enzyme pyruvate

dehydrogenase

kinase, isozyme

PDK3 PDK3 3 Cytoplasm kinase

PDZ and LIM transcription

PDLIM1 PDLIM1 domain 1 Cytoplasm regulator

PDZ and LIM

PDLIM5 PDLIM5 domain 5 Cytoplasm other

phosphoinositid

e-3-kinase,

PIK3C2B PIK3C2B class 2, beta Cytoplasm kinase polypeptide phosphoinositid

e-3-kinase,

PIK3C3 PIK3C3 class 3 Cytoplasm kinase

phosphoinositid

e-3-kinase,

regulatory

PIK3R4 PIK3R4 subunit 4 Cytoplasm other

phospholipase

A2-activating

PLAA PLAA protein Cytoplasm other

phospholipase B

domain Extracellular

PLBD2 PLBD2 containing 2 Space other

nelarabine,

MB07133, clofarabine, polymerase cytarabine, (DNA directed), trifluridine, delta 1 , catalytic vidarabine,

POLD1 POLD1 subunit 125kDa Nucleus enzyme entecavir polymerase

(RNA) II (DNA

directed)

polypeptide A,

POLR2A POLR2A 220kDa Nucleus enzyme

peptidylprolyl

isomerase E

PPIE PPIE (cyclophilin E) Nucleus enzyme

protein

phosphatase 1 ,

catalytic subunit,

PPP1 CB PPP1 CB beta isozyme Cytoplasm phosphatase

protein

phosphatase 2,

catalytic subunit,

PPP2CA PPP2CA alpha isozyme Cytoplasm phosphatase

ISAtx-247, protein tacrolimus, phosphatase 3, pimecrolimus catalytic subunit, , cyclosporin

PPP3CA PPP3CA alpha isozyme Cytoplasm phosphatase A

protein

phosphatase 4,

PPP4C PPP4C catalytic subunit Cytoplasm phosphatase

protein

phosphatase 5,

PPP5C PPP5C catalytic subunit Nucleus phosphatase

protein

phosphatase 6,

PPP6C PPP6C catalytic subunit Nucleus phosphatase

primase, DNA,

polypeptide 2 fludarabine

PRIM2 PRIM2 (58kDa) Nucleus enzyme phosphate protein kinase,

AMP-activated,

PRKAA1 PRKAA1 alpha 1 catalytic Cytoplasm kinase subunit protein kinase,

AMP-activated,

beta 1 non-

PRKAB1 PRKAB1 catalytic subunit Nucleus kinase protein kinase,

AMP-activated,

beta 2 non-

PRKAB2 PRKAB2 catalytic subunit Cytoplasm kinase protein kinase,

AMP-activated,

gamma 1 non-

PRKAG1 PRKAG1 catalytic subunit Nucleus kinase protein kinase C

PRKCSH PRKCSH substrate 80K-H Cytoplasm enzyme protein kinase,

DNA-activated,

catalytic

PRKDC PRKDC polypeptide Nucleus kinase protein arginine

methyltransferas

PRMT1 PRMT1 e 1 Nucleus enzyme protein arginine

methyltransferas

PRMT5 PRMT5 e 5 Cytoplasm enzyme proteasome

(prosome,

macropain)

subunit, alpha

PSMA1 PSMA1 type, 1 Cytoplasm peptidase proteasome

(prosome,

macropain) 26S

subunit,

PSMC1 PSMC1 ATPase, 1 Nucleus peptidase proteasome

(prosome,

macropain) 26S

subunit, non-

PSMD1 PSMD1 ATPase, 1 Cytoplasm other

proteasome

(prosome,

macropain)

activator subunit

PSME1 PSME1 1 (PA28 alpha) Cytoplasm other

paraspeckle

PSPC1 PSPC1 component 1 Nucleus other

Pentatricopeptid

e repeat domain

PTCD3 PTCD3 3 Cytoplasm other

prostaglandin E transcription

PTGES2 PTGES2 synthase 2 Cytoplasm regulator

PTK2 PTK2 protein

(includes tyrosine kinase

PTK2 EG:14083) 2 Cytoplasm kinase pumilio homolog

PUM1 PUM1 1 (Drosophila) Cytoplasm other

RAB3D RAB3D RAB3D, Cytoplasm enzyme member RAS

oncogene family

RAB3 GTPase

activating

protein subunit 1

RAB3GAP1 RAB3GAP1 (catalytic) Cytoplasm other

RAB3 GTPase

activating

protein subunit 2

RAB3GAP2 RAB3GAP2 (non-catalytic) Cytoplasm enzyme

RAB5C,

member RAS

RAB5C RAB5C oncogene family Cytoplasm enzyme

Rab

geranylgeranyltr

ansferase, beta

RABGGTB RABGGTB subunit Cytoplasm enzyme

RAD23 homolog

RAD23B RAD23B B (S. cerevisiae) Nucleus other

RAE1 RNA

export 1

homolog (S.

RAE1 RAE1 pombe) Nucleus other

RAN binding

RANBP2 RANBP2 protein 2 Nucleus enzyme

Ran GTPase

activating

RAN GAP 1 RANGAP1 protein 1 Cytoplasm other

RanBP-type and

C3HC4-type

zinc finger transcription

RBCK1 RBCK1 containing 1 Cytoplasm regulator

RNA binding

RBM10 RBM10 motif protein 10 Nucleus other

v-rel

reticuloendotheli

osis viral

oncogene

homolog A transcription NF-kappaB

RELA RELA (avian) Nucleus regulator decoy replication factor

C (activator 1 ) 2,

RFC2 RFC2 40kDa Nucleus other

replication

protein A2,

RPA2 RPA2 32kDa Nucleus other

ribosomal

RPS6 RPS6 protein S6 Cytoplasm other

ribosomal

protein S6

kinase, 90kDa,

RPS6KA3 RPS6KA3 polypeptide 3 Cytoplasm kinase

ribosomal translation

RPSA RPSA protein SA Cytoplasm regulator

RuvB-like 1 (E. transcription

RUVBL1 RUVBL1 coli) Nucleus regulator

RuvB-like 2 (E. transcription

RUVBL2 RUVBL2 coli) Nucleus regulator

S100A8 S100A8 S100 calcium Cytoplasm other binding protein

A8

S100 calcium

binding protein

S100A9 S100A9 A9 Cytoplasm other

SAM domain

and HD domain

SAMHD1 SAMHD1 1 Nucleus enzyme

Extracellular

SELO SELO selenoprotein 0 Space enzyme

SET domain

SETD2 SETD2 containing 2 Cytoplasm enzyme transcription

SF1 SF1 splicing factor 1 Nucleus regulator

SHANK- associated RH

domain Plasma

SHARPIN SHARPIN interactor Membrane other

transcription

SIRT1 SIRT1 sirtuin 1 Nucleus regulator

SIRT3 SIRT3 sirtuin 3 Cytoplasm enzyme

SWI/SNF

related, matrix

associated, actin

dependent

regulator of

chromatin,

subfamily a, transcription

SMARCA2 SMARCA2 member 2 Nucleus regulator

SWI/SNF

related, matrix

associated, actin

dependent

regulator of

chromatin,

subfamily a, transcription

SMARCA4 SMARCA4 member 4 Nucleus regulator small nuclear

ribonucleoprotei

SNRNP200 SNRNP200 n 200kDa (U5) Nucleus enzyme

SNX9 SNX9 sorting nexin 9 Cytoplasm transporter

SON DNA

SON SON binding protein Nucleus other

SPC24, NDC80

kinetochore

complex

SPC24 component,

(includes homolog (S.

SPC24 EG:147841 ) cerevisiae) Cytoplasm other

transcription

SQSTM1 SQSTM1 sequestosome 1 Cytoplasm regulator

SRSF protein

SRPK2 SRPK2 kinase 2 Nucleus kinase suppression of

tumorigenicity

13 (colon

carcinoma)

(Hsp70

ST13 ST13 interacting Cytoplasm other protein) signal

transducing

adaptor

molecule (SH3

domain and

STAM STAM ITAM motif) 1 Cytoplasm other

signal

transducer and

activator of

transcription 3

(acute-phase transcription

STAT3 STAT3 response factor) Nucleus regulator signal

transducer and

activator of transcription

STAT5B STAT5B transcription 5B Nucleus regulator stress-induced- phosphoprotein

STIP1 STIP1 1 Cytoplasm other

serine/threonine

STK3 STK3 kinase 3 Cytoplasm kinase serine/threonine

kinase receptor

associated Plasma

STRAP STRAP protein Membrane other

STIP1 homology

and U-box

containing

protein 1 , E3

ubiquitin protein

STUB1 STUB1 ligase Cytoplasm enzyme sulfotransferase

family, cytosolic,

1A, phenol- preferring,

SULT1A1 SULT1A1 member 1 Cytoplasm enzyme sulfotransferase

family, cytosolic,

SULT2B1 SULT2B1 2B, member 1 Cytoplasm enzyme

SURF4 SURF4 surfeit 4 Cytoplasm other

TGF-beta

activated kinase

1/MAP3K7

TAB1 TAB1 binding protein 1 Cytoplasm enzyme

TBC1 domain

family, member

TBC1 D15 TBC1 D15 15 Cytoplasm other

TBC1 domain

family, member

9B (with GRAM

TBC1 D9B TBC1 D9B domain) unknown other

TANK-binding

TBK1 TBK1 kinase 1 Cytoplasm kinase transforming

growth factor

TBRG4 TBRG4 beta regulator 4 Cytoplasm other

TCEAL4 TCEAL4 transcription unknown other 19

tetratricopeptide

repeat domain

TTC35 TTC35 35 Nucleus other

tetratricopeptide

TTC5 TTC5 repeat domain 5 unknown other

flucytosine,

5-fluorouracil, plevitrexed, nolatrexed, capecitabine, trifluridine, thymidylate floxuridine,

TYMS TYMS synthetase Nucleus enzyme LY231514 ubiquitin-like

modifier

activating

UBA1 UBA1 enzyme 1 Cytoplasm enzyme

ubiquitin-like

modifier

activating

UBA7 UBA7 enzyme 7 Cytoplasm enzyme

UBA domain

UBAC1 UBAC1 containing 1 Nucleus other

ubiquitin

associated

UBAP2 UBAP2 protein 2 Cytoplasm other

ubiquitin

associated

UBAP2L UBAP2L protein 2-like unknown other

ubiquitin

associated and

SH3 domain

UBASH3B UBASH3B containing B unknown enzyme

ubiquitin protein

UBE3A UBE3A ligase E3A Nucleus enzyme

ubiquitination

UBE4B UBE4B factor E4B Cytoplasm enzyme

UBQLN1 UBQLN1 ubiquilin 1 Cytoplasm other

UBQLN2 UBQLN2 ubiquilin 2 Nucleus other

UBQLN4 UBQLN4 ubiquilin 4 Cytoplasm other

ubiquitin protein

UBR1 ligase E3

(includes component n-

UBR1 EG:197131 ) recognin 1 Cytoplasm enzyme

ubiquitin protein

ligase E3

component n-

UBR4 UBR4 recognin 4 Nucleus other

ubiquitin

carboxyl- terminal

UCHL5 UCHL5 hydrolase L5 Cytoplasm peptidase

ubiquitin fusion

degradation 1

UFD1 L UFD1 L like (yeast) Cytoplasm peptidase

unc-45 homolog Plasma

UNC45A UNC45A A (C. elegans) Membrane other ubiquitin specific

USP10 USP10 peptidase 10 Cytoplasm peptidase ubiquitin specific

USP1 1 USP1 1 peptidase 1 1 Nucleus peptidase ubiquitin specific

peptidase 13

(isopeptidase T-

USP13 USP13 3) unknown peptidase ubiquitin specific

peptidase 14

(tRNA-guanine

transglycosylase

USP14 USP14 ) Cytoplasm peptidase ubiquitin specific

USP15 USP15 peptidase 15 Cytoplasm peptidase ubiquitin specific

USP24 USP24 peptidase 24 unknown peptidase ubiquitin specific

USP28 USP28 peptidase 28 Nucleus peptidase ubiquitin specific

USP32 USP32 peptidase 32 Cytoplasm enzyme ubiquitin specific

USP34 USP34 peptidase 34 unknown peptidase ubiquitin specific

USP47 USP47 peptidase 47 Cytoplasm peptidase ubiquitin specific

peptidase 5

USP5 USP5 (isopeptidase T) Cytoplasm peptidase ubiquitin specific

peptidase 7

(herpes virus-

USP7 USP7 associated) Nucleus peptidase ubiquitin specific

peptidase 9, X- Plasma

USP9X USP9X linked Membrane peptidase vestigial like 1 transcription

VGLL1 VGLL1 (Drosophila) Nucleus regulator vacuolar protein

sorting 1 1

homolog (S.

VPS 1 1 VPS1 1 cerevisiae) Cytoplasm transporter

WW domain

WBP2 WBP2 binding protein 2 Cytoplasm other

WW domain

binding protein 4

(formin binding

WBP4 WBP4 protein 21 ) Cytoplasm other

WD repeat

WDR1 1 WDR1 1 domain 1 1 unknown other

WD repeat

WDR18 WDR18 domain 18 Nucleus other

WD repeat

WDR5 WDR5 domain 5 Nucleus other

WD repeat

WDR6 WDR6 domain 6 Cytoplasm other

WD repeat

WDR61 WDR61 domain 61 unknown other

WD repeat transcription

WDR77 WDR77 domain 77 Nucleus regulator WD repeat

WDR82 WDR82 domain 82 Nucleus other

XPA binding

XAB2 XAB2 protein 2 Nucleus other

X-linked inhibitor

XIAP XIAP of apoptosis Cytoplasm other

tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, beta transcription

YWHAB YWHAB polypeptide Cytoplasm regulator tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, epsilon

YWHAE YWHAE polypeptide Cytoplasm other

tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, gamma

YWHAG YWHAG polypeptide Cytoplasm other

tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, eta transcription

YWHAH YWHAH polypeptide Cytoplasm regulator tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, theta

YWHAQ YWHAQ polypeptide Cytoplasm other

tyrosine 3- monooxygenase

/tryptophan 5- monooxygenase

activation

protein, zeta

YWHAZ YWHAZ polypeptide Cytoplasm enzyme zinc finger,

BED-type

ZBED1 ZBED1 containing 1 Nucleus enzyme zinc finger

CCCH-type

ZC3H 13 ZC3H13 containing 13 unknown other

zinc finger

CCCH-type

ZC3H4 ZC3H4 containing 4 unknown other

zinc finger

CCCH-type, Plasma

ZC3HAV1 ZC3HAV1 antiviral 1 Membrane other zinc finger RNA

ZFR ZFR binding protein Nucleus other

zinc finger

ZNF51 1 ZNF51 1 protein 51 1 Nucleus other

ZW10,

kinetochore

associated,

homolog

ZW10 ZW10 (Drosophila) Nucleus other

Zwilch,

kinetochore

associated,

homolog

ZWILCH ZWILCH (Drosophila) Nucleus other

PI3K-AKT-mTOR pathway

Phosphatidylinositol 3 kinases (PI3K) are a family of lipid kinases whose inositol lipid products play a central role in signal transduction pathways of cytokines, growth factors and 5 other extracellular matrix proteins. PI3Ks are divided into three classes: Class I, II and III with Class I being the best studied one. It is a heterodimer consisting of a catalytic and regulatory subunit. These are most commonly found to be pi 10 and p85. Phosphorylation of phosphoinositide(4,5)bisphosphate (PIP2) by Class I PI3K generates PtdIns(3,4,5)P3. The different PI3ks are involved in a variety of signaling pathways. This is mediated through their0 interaction with molecules like the receptor tyrosine kinases (RTKs), the adapter molecules GAB1-GRB2, and the kinase JAK. These converge to activate PDK1 which then phosphorylates AKT. AKT follows two distinct paths: 1) Inhibitory role - for example, AKT inhibits apoptosis by phosphorylating the Bad component of the Bad/Bcl-XL complex, allowing for cell survival. 2) Activating role - AKT activates IKK leading to NF-KB5 activation and cell survival. By its inhibitory as well as activating role, AKT is involved in numerous cellular processes like energy storage, cell cycle progression, protein synthesis and angiogenesis.

This pathway is composed of, but not restricted to 1-phosphatidyl-D-myo-inositol 4,5- bisphosphate, 14-3-3, 14-3-3-Cdknlb, Akt, BAD, BCL2, BCL2L1, CCND1, CDC37,0 CDKN1A, CDKN1B, citrulline, CT NB1, EIF4E, EIF4EBP1, ERK1/2, FKHR, GAB1/2, GDF15, Glycogen synthase, GRB2, Gsk3, Ikb, IkB-NfkB, IKK (complex), ILK, Integrin, JAK, L-arginine, LIMS1, MAP2K1/2, MAP3K5, MAP3K8, MAPK8IP1, MCL1, MDM2, MTOR, NANOG, NFkB (complex), nitric oxide, NOS3, PI 10, p70 S6k, PDPK1, phosphatidylinositol-3,4,5-triphosphate, PI3K p85, PP2A, PTEN, PTGS2, RAF1, Ras, RHEB, SEN, SHC1 (includes EG:20416), SHIP, Sos, THEM4, TP53 (includes EG:22059), TSC1, Tscl-Tsc2, TSC2, YWHAE

IGF-IR signaling network Insulin- like growth factor- 1 (IGF-1) is a peptide hormone under control of the growth hormone. IGF-1 promotes cell proliferation, growth and survival. Six specific binding proteins, IGFBP 1-6, allow for a more nuanced control of IGF activity. The IGF-1 receptor (IGF-IR) is a transmembrane tyrosine kinase protein. IGF-1 -induced receptor activation results in autophosphorylation followed by an enhanced capability to activate downstream pathways. Activated IGF-IR phosphorylates SHC and IRS-1. SHC along with adapter molecules GRB2 and SOS forms a signaling complex that activates the Ras/Raf/MEK/ERK pathway. ERK translocation to the nucleus results in the activation of transcriptional regulators ELK-1, c-Jun and c-Fos which induce genes that promote cell growth and differentiation. IRS-1 activates pathways for cell survival via the PI3K/PDK1/AKT/BAD pathway. IRS-1 also activates pathways for cell growth via the PI3K/PDKl/p70RSK pathway. IGF-1 also signals via the JAK/STAT pathway by inducing tyrosine phosphorylation of JAK-1, JAK-2 and STAT-3. SOCS proteins are able to inhibit the JAKs thereby inhibiting this pathway. The adapter protein GRB10 interacts with IGF-IR. GRBIO also binds the E3 ubiquitin ligase NEDD4 and promotes ligand stimulated ubiquitination, internalization, and degradation of the IGF-IR as a means of long-term attenuation of signaling.

This pathway is composed of, but not restricted to 1-phosphatidyl-D-myo-inositol 4,5- bisphosphate, 14-3-3, 14-3-3-Bad, Akt, atypical protein kinase C, BAD, CASP9 (includes EG: 100140945), Ck2, ELK1, ERK1/2, FKHR, FOS, GRBIO, GRB2, IGF1, Igfl-Igf p, IGF1R, Igfbp, IRS1/2, JAK1/2, JUN, MAP2K1/2, MAPK8, NEDD4, p70 S6k, PDPK1, phosphatidylinositol-3,4,5-triphosphate, PI3K (complex), Pka, PTK2 (includes EG: 14083), PTPN11, PXN, RAF1, Ras, RASA1, SHC1 (includes EG:20416), SOCS, SOCS3, Sos, SRF, STAT3, Stat3-Stat3 NRF2-mediated Oxidative Stress Response Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids and DNA. Severe oxidative stress can trigger apoptosis and necrosis. Oxidative stress is involved in many diseases such as atherosclerosis, Parkinson's disease and Alzheimer's disease. Oxidative stress has also been linked to aging. The cellular defense response to oxidative stress includes induction of detoxifying enzymes and antioxidant enzymes. Nuclear factor- erythroid 2-related factor 2 (Nrf2) binds to the antioxidant response elements (ARE) within the promoter of these enzymes and activates their transcription. Inactive Nrf2 is retained in the cytoplasm by association with an actin-binding protein Keapl . Upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, cytochrome P450, NAD(P)H quinone oxidoreductase, heme oxygenase and superoxide dismutase.

This pathway is composed of, but not restricted to ABCC1, ABCC2, ABCC4 (includes EG: 10257), Actin, Actin-Nrf2, Afar, AKR1A1, AKT1, AOX1, ATF4, BACH1, CAT, Cbp/p300, CBR1, CCT7, CDC34, CLPP, CUL3 (includes EG:26554), Cul3-Rocl, Cypla/2a/3a/4a/2c, EIF2AK3, ENC1, EPHX1, ERK1/2, ERP29, FKBP5, FMOl (includes EG: 14261), FOS, FOSL1, FTH1 (includes EG: 14319), FTL, GCLC, GCLM, GPX2, GSK3B, GSR, GST, HERPUD1, HMOX1, Hsp22/Hsp40/Hsp90, JINK1/2, Jnkk, JUN/JUNB/JUND, KEAP1, Keapl-Nrf2, MAF, MAP2K1/2, MAP2K5, MAP3K1, MAP3K5, MAP3K7 (includes EG: 172842), MAPK14, MAPK7, MK 3/6, musculoaponeurotic fibrosarcoma oncogene, NFE2L2, NQO, PI3K (complex), Pkc(s), PMF1, PPIB, PRDX1, Psm, PTPLAD1, RAF1, Ras, RBX1, reactive oxygen species, SCARB1, SLC35A2, Sod, SQSTM1, STIP1, TXN (includes EG: 116484), TXNRD1, UBB, UBE2E3, UBE2K, USP14, VCP

Protein Kinase A signaling pathway

Protein kinase A (PKA) regulates processes as diverse as growth, development, memory, and metabolism. It exists as a tetrameric complex of two catalytic subunits (PKA-C) and a regulatory (PKA-R) subunit dimer. Type-II PKA is anchored to specific locations within the cell by AKAPs. Extracellular stimuli such as neurotransmitters, hormones, inflammatory stimuli, stress, epinephrine and norepinephrine activate G-proteins through receptors such as GPCRs and ADR-α/β. These receptors along with others such as CRHR, GcgR and DCC are responsible for cAMP accumulation which leads to activation of PKA. The conversion of ATP to cAMP is mediated by the 9 transmembrane AC enzymes and one soluble AC. The transmembrane AC are regulated by heterotrimeric G-proteins, Gas, Gaq and Gai. Gas and Gaq activate while Gai inhibits AC. ϋβ and Gy subunits act synergistically with Gas and Gaq to activate ACII, IV and VII. However the β and γ subunits along with Gai inhibit the activity of ACI, V and VI.

G-proteins indirectly influence cAMP signaling by activating PLC, which generates DAG and IP3. DAG in turn activates PKC. IP3 modulates proteins upstream to cAMP signaling with the release of Ca2+ from the ER through IP3R. Ca2+ is also released by CaCn and CNG. Ca2+ release activates Calmodulin, CamK s and CamKs, which take part in cAMP modulation by activating ACI. Gal 3 activates MEK 1 and RhoA via two independent pathways which induce phosphorylation and degradation of ΙκΒα and activation of PKA. High levels of cAMP under stress conditions like hypoxia, ischemia and heat shock also directly activate PKA. TGF-β activates PKA independent of cAMP through phosphorylation of SMAD proteins. PKA phosphorylates Phospholamban which regulates the activity of SERCA2 leading to myocardial contraction, whereas phosphorylation of Tnnl mediates relaxation. PKA also activates KDELR to promote protein retrieval thereby maintaining steady state of the cell. Increase in concentration of Ca2+ followed by PKA activation enhances eNOS activity which is essential for cardiovascular homeostasis. Activated PKA represses ER activation by inhibition of Rafl . PKA inhibits the interaction of 14-3-3 proteins with BAD and NFAT to promote cell survival. PKA phosphorylates endothelial MLCK leading to decreased basal MLC phosphorylation. It also phosphorylates filamin, adducin, paxillin and FAK and is involved in the disappearance of stress fibers and F-actin accumulation in membrane ruffles. PKA also controls phosphatase activity by phosphorylation of a specific PPtasel inhibitor, DARPP32. Other substrates of PKA include histone HI, histone H2B and CREB.

This pathway is composed of, but not restricted to 1-phosphatidyl-D-myo-inositol 4,5- bisphosphate, 14-3-3, ADCY, ADCYl/5/6, ADCY2/4/7, ADCY9, Adducin, AKAP, APC, ATF1 (includes EG: 100040260), ATP, BAD, BRAF, Ca2+, Calcineurin protein(s), Calmodulin, CaMKII, CHUK, Cng Channel, Creb, CREBBP, CREM, CT NB1, cyclic AMP, DCC, diacylglycerol, ELK1, ER l/2, Filamin, Focal adhesion kinase, G protein alphai, G protein beta gamma, G-protein beta, G-protein gamma, GLI3, glycogen, glycogen phosphorylase, Glycogen synthase, GNA13, GNAQ, GNAS, GRK1/7, Gsk3, Hedgehog, Histone HI, Histone h3, Ikb, IkB-NfkB, inositol triphosphate, ITPR, KDELR, LIPE, MAP2K1/2, MAP3K1, Mlc, myosin-light-chain kinase, Myosin2, Nfat (family), NFkB (complex), NGFR, NOS3, NTN1, Patched, Pde, Phk, Pka, Pka catalytic subunit, PKAr, Pkc(s), PLC, PLN, PP1 protein complex group, PPP1R1B, PTPase, PXN, RAFl, Rapl, RHO, RHOA, Rock, Ryr, SMAD3, Smad3-Smad4, SMAD4, SMO, TCF/LEF, Tgf beta, Tgf beta receptor, TGFBR1, TGFBR2, TH, Tni, VASP IL-6 signaling pathway

The central role of IL-6 in inflammation makes it an important target for the management of inflammation associated with cancer. IL-6 responses are transmitted through Glycoprotein 130 (GP130), which serves as the universal signal-transducing receptor subunit for all IL-6- related cytokines. IL-6-type cytokines utilize tyrosine kinases of the Janus Kinase (JAK) family and signal transducers and activators of transcription (STAT) family as major mediators of signal transduction. Upon receptor stimulation by IL-6, the JAK family of kinases associated with GP130 are activated, resulting in the phosphorylation of GP130. Several phosphotyrosine residues of GP130 serve as docking sites for STAT factors mainly STAT3 and STAT1. Subsequently, STATs are phosphorylated, form dimers and translocate to the nucleus, where they regulate transcription of target genes. In addition to the JAK STAT pathway of signal transduction, IL-6 also activates the extracellular signal-regulated kinases (ERK1/2) of the mitogen activated protein kinase (MAPK) pathway. The upstream activators of ERK1/2 include RAS and the src homology-2 containing proteins GRB2 and SHC. The SHC protein is activated by JAK2 and thus serves as a link between the IL-6 activated JAK STAT and RAS-MAPK pathways. The phosphorylation of MAPKs in response to IL-6 activated RAS results in the activation of nuclear factor IL-6 (NF-IL6), which in turn stimulates the transcription of the IL-6 gene. The transcription of the IL-6 gene is also stimulated by tumor necrosis factor (TNF) and Interleukin-1 (IL-1) via the activation of nuclear factor kappa B (NFKB).

Based on the findings by the method described here in MDA-MB-468 cells, combination of an inhibitor of components of these identified pathways, such as those targeting but not limited to AKT, mTOR, PI3K, IGFIR, IKK, Bcl2, PKA complex, phosphodiesterases are proposed to be efficacious when used in combination with an Hsp90 inhibitor.

Example of AKT inhibitors are PF-04691502, Triciribine phosphate (NSC-280594 , A- 674563. CCT128930. AT7867. PHT-427. GSK690693. MK-2206

Example of PI3K inhibitors are 2-(lH-indazol-4-yl)-6-(4-methanesulfonylpiperazin-l- ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine, BKM120, NVP-BEZ235, PX-866, SF 1126. XL147. Example of mTOR inhibitors are deforolimus, everolimus, NVP-BEZ235, OSI-027, tacrolimus, temsirolimus, Ku-0063794, WYE-354, PP242, OSI-027, GSK2126458, WAY- 600, WYE-125132

Examples of Bcl2 inhibitors are ABT-737, Obatoclax (GX15-070), ABT-263, TW-37

Examples of IGFIR inhibitors are NVP-ADW742, BMS-754807, AVE 1642, BIIB022, cixutumumab, ganitumab, IGF1, OSI-906

Examples of JAK inhibitors are Tofacitinib citrate (CP-690550), AT9283, AG-490, INCBO 18424 (Ruxolitinib), AZD1480, LY2784544, NVP-BSK805, TGI 01209, TG-101348

Examples of IkK inhibitors are SC-514, PF 184 Examples of inhibitors of phosphodiesterases are aminophylline, anagrelide, arofylline, caffeine, cilomilast, dipyridamole, dyphylline, L 869298, L-826,141, milrinone, nitroglycerin, pentoxifylline, roflumilast, rolipram, tetomilast, theophylline, tolbutamide, amrinone, anagrelide, arofylline, caffeine, cilomilast, L 869298, L-826,141, milrinone, pentoxifylline, roflumilast, rolipram, tetomilast

In the Diffuse large B-cell lymphoma (DLBCL) ceil Line OCI-LY.1 , major signaling networks identified by the method were the B cell receptor, P Cteta, PI3K/AKT, CD40, CD28 and the ERK MAPK signaling pathways (Figure 23). Pathway components as identified by the

method are listed in Table 4.

Table 4.

© 2000-

2012

Ingenuity

Systems,

Inc. All

rights

reserved.

ID Notes Symbol Entrez Gene Name Location Type(s) Drug(s)

alpha- and

gamma-adaptin

AAGAB AAGAB binding protein Cytoplasm other

ABM ABM abl-interactor 1 Cytoplasm other

active BCR-related

ABR ABR gene Cytoplasm other

AHA1 , activator of

heat shock 90kDa

protein ATPase

AHSA1 AHSA1 homolog 1 (yeast) Cytoplasm other

apoptosis-inducing

factor,

mitochondrion-

AIFM1 AIFM1 associated, 1 Cytoplasm enzyme

A kinase (PRKA)

AKAP8 AKAP8 anchor protein 8 Nucleus other

A kinase (PRKA)

anchor protein 8-

AKAP8L AKAP8L like Nucleus other

alkB, alkylation

repair homolog 8

ALKBH8 ALKBH8 (E. coli) Cytoplasm enzyme

TA 270, benoxaprofen, meclofenamic acid, zileuton, sulfasalazine, balsalazide, 5- arachidonate 5- aminosalicylic acid,

ALOX5 ALOX5 lipoxygenase Cytoplasm enzyme masoprocol

anaphase

promoting complex

ANAPC7 ANAPC7 subunit 7 Nucleus other

ankyrin repeat and

FYVE domain transcription

ANKFY1 ANKFY1 containing 1 Nucleus regulator

ankyrin repeat

ANKRD17 ANKRD17 domain 17 unknown other

acidic (leucine- rich) nuclear

phosphoprotein 32

ANP32B ANP32B family, member B Nucleus other

adaptor-related

protein complex 1 ,

AP1 B1 AP1 B1 beta 1 subunit Cytoplasm transporter

AP2A1 AP2A1 adaptor-related Cytoplasm transporter protein complex 2,

alpha 1 subunit

APAF1 interacting

APIP APIP protein Cytoplasm enzyme apolipoprotein B

imRNA editing

enzyme, catalytic

APOBEC3G APOBEC3G polypeptide-like 3G Nucleus enzyme

ADP-ribosylation

factor GTPase

ARFGAP1 ARFGAP1 activating protein 1 Cytoplasm transporter

ADP-ribosylation

factor guanine

nucleotide- exchange factor 2

(brefeldin A-

ARFGEF2 ARFGEF2 inhibited) Cytoplasm other

ADP-ribosylation

factor interacting

ARFIP2 ARFIP2 protein 2 Cytoplasm other

Rho guanine

nucleotide

exchange factor

ARHGEF1 ARHGEF1 (GEF) 1 Cytoplasm other

AT rich interactive

domain 1A (SWI- transcription

ARID1A ARID1A like) Nucleus regulator

N-acylsphingosine

amidohydrolase

(acid ceramidase)

ASAH 1 ASAH1 1 Cytoplasm enzyme acetylserotonin 0- methyltransferase-

ASMTL ASMTL like Cytoplasm enzyme arsA arsenite

transporter, ATP- binding, homolog 1

ASNA1 ASNA1 (bacterial) Nucleus transporter alveolar soft part

sarcoma

chromosome

ASPSCR1 ASPSCR1 region, candidate 1 Cytoplasm other

ataxia

telangiectasia

ATM ATM mutated Nucleus kinase ataxia

telangiectasia and

ATR ATR Rad3 related Nucleus kinase

ATXN10 ATXN10 ataxin 10 Cytoplasm other

ATXN2L ATXN2L ataxin 2-like unknown other

BRISC and

BRCA1 A complex

BABAM1 BABAM1 member 1 Nucleus other

BCL2-associated

BAG6 BAG 6 athanogene 6 Nucleus enzyme baculoviral IAP

BIRC6 BIRC6 repeat containing 6 Cytoplasm enzyme

BRCA1 -associated

BRAT1 BRAT1 ATM activator 1 Cytoplasm other BRCA1/BRCA2- containing

BRCC3 BRCC3 complex, subunit 3 Nucleus enzyme

BTAF1 RNA

polymerase II, B- TFIID transcription

factor-associated ,

170kDa (Mot1

homolog, S. transcription

BTAF1 BTAF1 cerevisiae) Nucleus regulator

Bruton

agammaglobuline

BTK BTK mia tyrosine kinase Cytoplasm kinase budding

uninhibited by

benzimidazoles 1

homolog beta

BUB1 B BUB1 B (yeast) Nucleus kinase budding

BUB3 uninhibited by

(includes benzimidazoles 3

BUB3 EG: 12237) homolog (yeast) Nucleus other

basic leucine

zipper and W2 translation

BZW1 BZW1 domains 1 Cytoplasm regulator calcyclin binding

CACYBP CACYBP protein Nucleus other

CALU CALU calumenin Cytoplasm other

calcium/calmodulin

-dependent protein

CAMK1 D CAMK1 D kinase ID Cytoplasm kinase calcium/calmodulin

-dependent protein

CAMK2D CAMK2D kinase II delta Cytoplasm kinase calcium/calmodulin

-dependent protein

CAMK2G CAMK2G kinase II gamma Cytoplasm kinase calcium/calmodulin

-dependent protein

CAMK4 CAMK4 kinase IV Nucleus kinase cullin-associated

and neddylation- transcription

CAND1 CAND1 dissociated 1 Cytoplasm regulator

CANX CANX calnexin Cytoplasm other

CAP, adenylate

cyclase-associated Plasma

CAP1 CAP1 protein 1 (yeast) Membrane other

calpain 1 , (mu/l)

CAPN1 CAPN1 large subunit Cytoplasm peptidase cell cycle

associated protein Plasma

CAPRIN1 CAPRI N1 1 Membrane other

coactivator- associated

arginine

methyltransferase transcription

CARM1 CARM1 1 Nucleus regulator

CCNY CCNY cyclin Y Nucleus other

CD38 CD38 CD38 molecule Plasma enzyme Membrane

CD74 molecule,

major

histocompatibility

complex, class II Plasma transmembrane

CD74 CD74 invariant chain Membrane receptor

cell division cycle

37 homolog (S.

CDC37 CDC37 cerevisiae) Cytoplasm other

cell division cycle

37 homolog (S.

CDC37L1 CDC37L1 cerevisiae)-like 1 Cytoplasm other

cyclin-dependent

CDK1 CDK1 kinase 1 Nucleus kinase flavopiridol cyclin-dependent PD-0332991 ,

CDK4 CDK4 kinase 4 Nucleus kinase flavopiridol cyclin-dependent BMS-387032,

CDK7 CDK7 kinase 7 Nucleus kinase flavopiridol cyclin-dependent BMS-387032,

CDK9 CDK9 kinase 9 Nucleus kinase flavopiridol chromatin

assembly factor 1 ,

CHAF1 B CHAF1 B subunit B (p60) Nucleus other

chromodomain

helicase DNA

CHD8 CHD8 binding protein 8 Nucleus enzyme

CTF18,

chromosome

transmission

fidelity factor 18

homolog (S.

CHTF18 CHTF18 cerevisiae) unknown other

CNN2 CNN2 calponin 2 Cytoplasm other

CCR4-NOT

transcription

CNOT1 CNOT1 complex, subunit 1 Cytoplasm other

2',3'-cyclic

nucleotide 3'

CNP CNP phosphodiesterase Cytoplasm enzyme

centlein,

centrosomal

CNTLN CNTLN protein unknown other

COBRA1 C0BRA1 cofactor of BRCA1 Nucleus other

COR07 C0R07 coronin 7 Cytoplasm other

v-crk sarcoma

virus CT10

oncogene homolog

CRKL CRKL (avian)-like Cytoplasm kinase

cold shock domain

containing E1 ,

CSDE1 CSDE1 RNA-binding Cytoplasm enzyme casein kinase 1 ,

CSNK1A1 CSNK1A1 alpha 1 Cytoplasm kinase casein kinase 2,

alpha 1

CSNK2A1 CSNK2A1 polypeptide Cytoplasm kinase casein kinase 2,

alpha prime

CSNK2A2 CSNK2A2 polypeptide Cytoplasm kinase

C-terminal binding transcription

CTBP2 CTBP2 protein 2 Nucleus regulator

CTSZ CTSZ cathepsin Z Cytoplasm peptidase cutC copper

transporter

CUTC CUTC homolog (E. coli) Cytoplasm other

cytochrome b5

CYB5R3 CYB5R3 reductase 3 Cytoplasm enzyme cytoplasmic FMR1

interacting protein

CYFIP1 CYFIP1 1 Cytoplasm other

cytoplasmic FMR1

interacting protein

CYFIP2 CYFIP2 2 Cytoplasm other

DBNL DBNL drebrin-like Cytoplasm other

DDB1 and CUL4

DCAF7 DCAF7 associated factor 7 Cytoplasm other

dicer 1 ,

ribonuclease type

DICER1 DICER1 III Cytoplasm enzyme

DIM1

dimethyladenosine

transferase 1

homolog (S.

DIMT1 DIMT1 cerevisiae) Cytoplasm enzyme

DIS3 mitotic

control homolog

DIS3L DIS3L (S. cerevisiae)-like Cytoplasm enzyme

DnaJ (Hsp40)

homolog,

subfamily A,

DNAJA1 DNAJA1 member 1 Nucleus other

DnaJ (Hsp40)

homolog,

subfamily A,

DNAJA2 DNAJA2 member 2 Nucleus enzyme

DnaJ (Hsp40)

homolog,

subfamily B,

DNAJB1 DNAJB1 member 1 Nucleus other

DnaJ (Hsp40)

homolog,

subfamily B,

DNAJB1 1 DNAJB1 1 member 1 1 Cytoplasm other

DnaJ (Hsp40)

homolog,

subfamily B,

DNAJB2 DNAJB2 member 2 Nucleus other

DnaJ (Hsp40)

homolog,

DNAJC10 DNAJC10 subfamily C, Cytoplasm enzyme member 10

DnaJ (Hsp40)

homolog,

subfamily C,

DNAJC21 DNAJC21 member 21 unknown other

DnaJ (Hsp40)

homolog,

subfamily C,

DNAJC7 DNAJC7 member 7 Cytoplasm other

DNA (cytosine-5-)- methyltransferase

DNMT1 DNMT1 1 Nucleus enzyme dedicator of

DOCK2 DOCK2 cytokinesis 2 Cytoplasm other

DPH5 homolog (S.

DPH5 DPH5 cerevisiae) unknown enzyme dihydropyrimidinas

DPYSL2 DPYSL2 e-like 2 Cytoplasm enzyme developmental^

regulated GTP

DRG1 DRG1 binding protein 1 Cytoplasm other deltex 3-like

DTX3L DTX3L (Drosophila) Cytoplasm enzyme

EBNA1 binding

EBNA1 BP2 EBNA1 BP2 protein 2 Nucleus other eukaryotic

translation

elongation factor 1 translation

EEF1A1 EEF1A1 alpha 1 Cytoplasm regulator

EH-domain

EHD1 EHD1 containing 1 Cytoplasm other eukaryotic

translation initiation

factor 2B, subunit translation

EIF2B2 EIF2B2 2 beta, 39kDa Cytoplasm regulator engulfment and

ELM01 ELM01 cell motility 1 Cytoplasm other ectopic P-granules

autophagy protein

5 homolog (C.

EPG5 EPG5 elegans) unknown other epidermal growth

factor receptor

pathway substrate Plasma

EPS15 EPS15 15 Membrane other epidermal growth

factor receptor

pathway substrate Plasma

EPS15L1 EPS15L1 15-like 1 Membrane other eukaryotic

translation translation

ETF1 ETF1 termination factor 1 Cytoplasm regulator exosome

EXOSC2 EXOSC2 component 2 Nucleus enzyme exosome

EXOSC5 EXOSC5 component 5 Nucleus enzyme exosome

EXOSC6 EXOSC6 component 6 Nucleus other

EXOSC7 EXOSC7 exosome Nucleus enzyme component 7

Fanconi anemia,

complementation

FANCD2 FANCD2 group D2 Nucleus other

Fanconi anemia,

complementation

FAN CI FANCI group I Nucleus other

F-box and leucine- rich repeat protein

FBXL12 FBXL12 12 Cytoplasm other

FBX022 FBX022 F-box protein 22 unknown enzyme

FBX03 FBX03 F-box protein 3 unknown enzyme

FCH and double

FCHSD2 FCHSD2 SH3 domains 2 unknown other

Plasma

FCRLA FCRLA Fc receptor-like A Membrane other

farnesyl- diphosphate

farnesyltransferase TAK-475, zoledronic

FDFT1 FDFT1 1 Cytoplasm enzyme acid

FK506 binding

FKBP4 FKBP4 protein 4, 59kDa Nucleus enzyme

FK506 binding

FKBP5 FKBP5 protein 5 Nucleus enzyme

Friend leukemia transcription

FLU FLU virus integration 1 Nucleus regulator

flightless I homolog

FLU FLII (Drosophila) Nucleus other

FLNA FLNA filamin A, alpha Cytoplasm other

fructosamine 3

kinase related

FN3KRP FN3KRP protein unknown kinase

formin binding

FNBP1 FNBP1 protein 1 Nucleus enzyme

GTPase activating

protein (SH3

domain) binding

G3BP1 G3BP1 protein 1 Nucleus enzyme

GTPase activating

protein (SH3

domain) binding

G3BP2 G3BP2 protein 2 Nucleus enzyme

GTPase activating

protein and VPS9

GAPVD1 GAPVD1 domains 1 Cytoplasm other

glycyl-tRNA

GARS GARS synthetase Cytoplasm enzyme

phosphoribosylglyc

inamide

formyltransferase,

phosphoribosylglyc

inamide

synthetase,

phosphoribosylami

noimidazole

GART GART synthetase Cytoplasm enzyme LY231514

GRB10 interacting

GIGYF2 GIGYF2 GYF protein 2 unknown other glomulin, FKBP

GLMN GLMN associated protein Cytoplasm other

GLRX3 GLRX3 glutaredoxin 3 Cytoplasm enzyme

golgi

phosphoprotein 3-

GOLPH3L GOLPH3L like Cytoplasm other

G patch domain

G PATCH 8 G PATCH 8 containing 8 unknown other

general

transcription factor transcription

GTF2B GTF2B I IB Nucleus regulator

general

transcription factor

I IF, polypeptide 1 , transcription

GTF2F1 GTF2F1 74kDa Nucleus regulator

general

transcription factor

IIF, polypeptide 2, transcription

GTF2F2 GTF2F2 30kDa Nucleus regulator

general

transcription factor transcription

GTF2I GTF2I Mi Nucleus regulator

general

transcription factor

MIC, polypeptide 1 , transcription

GTF3C1 GTF3C1 alpha 220kDa Nucleus regulator

GTP binding

GTPBP4 GTPBP4 protein 4 Nucleus enzyme

histone

HAT1 HAT1 acetyltransferase 1 Nucleus enzyme

hematopoietic cell- specific Lyn transcription

HCLS1 HCLS1 substrate 1 Nucleus regulator

tributyrin, belinostat, pyroxamide, histone transcription MGCD0103, vorinostat,

HDAC1 HDAC1 deacetylase 1 Nucleus regulator romidepsin

tributyrin, belinostat, histone transcription pyroxamide, vorinostat,

HDAC2 HDAC2 deacetylase 2 Nucleus regulator romidepsin tributyrin, belinostat, pyroxamide, histone transcription MGCD0103, vorinostat,

HDAC3 HDAC3 deacetylase 3 Nucleus regulator romidepsin

tributyrin, belinostat, histone transcription pyroxamide, vorinostat,

HDAC6 HDAC6 deacetylase 6 Nucleus regulator romidepsin

high density

lipoprotein binding

HDLBP HDLBP protein Nucleus transporter

HECT domain

HECTD1 HECTD1 containing 1 unknown enzyme

hect (homologous

to the E6-AP

(UBE3A) carboxyl

terminus) domain

and RCC1

(CHCI )-like

HERC1 HERC1 domain (RLD) 1 Cytoplasm other

hypoxia inducible

factor 1 , alpha

HIF1AN HIF1AN subunit inhibitor Nucleus enzyme

HIRA interacting

HIRIP3 HIRIP3 protein 3 Nucleus other

histone cluster 1 ,

HIST1 H1 B HIST1 H1 B H1 b Nucleus other

histone cluster 1 ,

HIST1 H1 D HIST1 H1 D H1 d Nucleus other

HK2 HK2 hexokinase 2 Cytoplasm kinase

major

histocompatibility

complex, class II, Plasma

HLA-DQB1 HLA-DQB1 DQ beta 1 Membrane other

major

histocompatibility

complex, class II, Plasma transmembrane

HLA-DRA HLA-DRA DR alpha Membrane receptor

major

histocompatibility

complex, class II, Plasma transmembrane

HLA-DRB1 HLA-DRB1 DR beta 1 Membrane receptor apolizumab

heterogeneous

nuclear

HNRNPAB HNRNPAB ribonucleoprotein Nucleus enzyme A/B

heterogeneous

nuclear

ribonucleoprotein

D (AU-rich element

RNA binding transcription

HNRNPD HNRNPD protein 1 , 37kDa) Nucleus regulator

heterogeneous

nuclear

ribonucleoprotein

U (scaffold

attachment factor

HNRNPU HNRNPU A) Nucleus transporter

17- heat shock protein dimethylaminoethylami 90kDa alpha no-17- (cytosolic), class A demethoxygeldanamyc

HSP90AA1 HSP90AA1 member 1 Cytoplasm enzyme in, IPI-504, cisplatin

17- heat shock protein dimethylaminoethylami 90kDa alpha no-17- (cytosolic), class B demethoxygeldanamyc

HSP90AB1 HSP90AB1 member 1 Cytoplasm enzyme in, IPI-504, cisplatin

17- dimethylaminoethylami heat shock protein no-17- 90kDa beta demethoxygeldanamyc

HSP90B1 HSP90B1 (Grp94), member 1 Cytoplasm other in, IPI-504, cisplatin heat shock 70kDa

HSPA4 HSPA4 protein 4 Cytoplasm other

heat shock 70kDa

protein 5 (glucose- regulated protein,

HSPA5 HSPA5 78kDa) Cytoplasm enzyme

heat shock 70kDa

HSPA8 HSPA8 protein 8 Cytoplasm enzyme

heat shock 70kDa

HSPA9 HSPA9 protein 9 (mortalin) Cytoplasm other

heat shock 60kDa

protein 1

HSPD1 HSPD1 (chaperonin) Cytoplasm enzyme

heat shock

105kDa/1 10kDa

HSPH1 HSPH1 protein 1 Cytoplasm other HtrA serine

HTRA2 HTRA2 peptidase 2 Cytoplasm peptidase interferon induced

with helicase C

IFIH 1 IFIH1 domain 1 Nucleus enzyme interferon-induced

protein with

tetratricopeptide

IFIT1 IFIT1 repeats 1 Cytoplasm other

interferon-induced

protein with

tetratricopeptide

IFIT3 IFIT3 repeats 3 Cytoplasm other

immunoglobulin

(CD79A) binding

IGBP1 IGBP1 protein 1 Cytoplasm phosphatase insulin-like growth

factor 2 mRNA translation

IGF2BP3 IGF2BP3 binding protein 3 Cytoplasm regulator inhibitor of kappa

light polypeptide

gene enhancer in

B-cells, kinase

complex-

IKBKAP IKBKAP associated protein Cytoplasm other

interleukin

enhancer binding transcription

ILF2 ILF2 factor 2, 45kDa Nucleus regulator inositol

polyphosphate-5- phosphatase, Plasma

INPP5B INPP5B 75kDa Membrane phosphatase inositol

polyphosphate-5- phosphatase,

INPP5D INPP5D 145kDa Cytoplasm phosphatase

ISY1 ISY1 splicing factor

(includes homolog (S.

ISY1 EG:362394) cerevisiae) Nucleus other

itchy E3 ubiquitin

protein ligase

ITCH ITCH homolog (mouse) Nucleus enzyme integrin alpha FG- GAP repeat

ITFG2 ITFG2 containing 2 unknown other

inter-alpha-trypsin

inhibitor heavy Extracellular

ITIH3 ITIH3 chain 3 Space other

ITSN2 ITSN2 intersectin 2 Cytoplasm other

lysyl-tRNA

KARS KARS synthetase Cytoplasm enzyme potassium voltage- gated channel,

shaker-related

subfamily, beta Plasma

KCNAB2 KCNAB2 member 2 Membrane ion channel

KIAA0368 KIAA0368 KIAA0368 Cytoplasm other

KIAA0564 KIAA0564 KIAA0564 Cytoplasm other translation

KIAA0664 KIAA0664 KIAA0664 Cytoplasm regulator

KIAA1524 KIAA1524 KIAA1524 Cytoplasm other

KIAA1797 KIAA1797 KIAA1797 unknown other

KIAA1967 KIAA1967 KIAA1967 Cytoplasm peptidase

leucyl-tRNA

LARS LARS synthetase Cytoplasm enzyme

LPXN LPXN leupaxin Cytoplasm other

listerin E3 ubiquitin

LTN1 LTN1 protein ligase 1 Nucleus enzyme

Ly1 antibody

reactive homolog Plasma

LYAR LYAR (mouse) Membrane other

membrane

associated

guanylate kinase,

MAG 11 WW and PDZ

(includes domain containing Plasma

MAGI 1 EG: 14924) 1 Membrane kinase

mitogen-activated

protein kinase

MAP3K1 MAP3K1 kinase kinase 1 Cytoplasm kinase

mitogen-activated

MAPK1 MAPK1 protein kinase 1 Cytoplasm kinase mitogen-activated SCIO-469, RO-

MAPK14 MAPK14 protein kinase 14 Cytoplasm kinase 3201 195

mitogen-activated

MAPK3 MAPK3 protein kinase 3 Cytoplasm kinase

mitogen-activated

MAPK9 MAPK9 protein kinase 9 Cytoplasm kinase

minichromosome

maintenance

complex

MCM2 MCM2 component 2 Nucleus enzyme

minichromosome

maintenance

complex binding

MCMBP MCMBP protein Nucleus other

MED1

(includes mediator complex transcription

MED1 EG: 19014) subunit 1 Nucleus regulator

MEM01

(includes mediator of cell

MEM01 EG:298787) motility 1 Cytoplasm other

methylphosphate

MEPCE MEPCE capping enzyme unknown enzyme

methyltransferase

METTL15 METTL15 like 15 unknown other

mutL homolog 1 ,

colon cancer,

nonpolyposis type

MLH1 MLH1 2 (E. coli) Nucleus enzyme

MTOR associated

protein, LST8

homolog (S.

MLST8 MLST8 cerevisiae) Cytoplasm other MMS19 nucleotide

excision repair

homolog (S. transcription

MMS19 MMS19 cerevisiae) Nucleus regulator

tositumomab, membrane- rituximab, ofatumumab, spanning 4- veltuzumab, domains, subfamily Plasma afutuzumab,

MS4A1 MS4A1 A, member 1 Membrane other ibritumomab tiuxetan mutS homolog 2,

colon cancer,

nonpolyposis type

MSH2 MSH2 1 (E. coli) Nucleus enzyme

mutS homolog 6

MSH6 MSH6 (E. coli) Nucleus enzyme

musashi homolog

MSI2 MSI2 2 (Drosophila) Cytoplasm other

misato homolog 1

MST01 MST01 (Drosophila) Cytoplasm other

methylenetetrahydr

ofolate

dehydrogenase

(NADP+

dependent) 1 ,

methenyltetrahydro

folate

cyclohydrolase,

formyltetrahydrofol

MTHFD1 MTHFD1 ate synthetase Cytoplasm enzyme

mechanistic target deforolimus, OSI-027, of rapamycin NVP-BEZ235, (serine/threonine temsirolimus,

MTOR MTOR kinase) Nucleus kinase tacrolimus, everolimus myxovirus

(influenza virus)

resistance 1 ,

interferon-inducible

protein p78

MX1 MX1 (mouse) Nucleus enzyme

MYB binding transcription

MYBBP1A MYBBP1A protein (P160) 1a Nucleus regulator

MYC binding

MYCBP2 MYCBP2 protein 2 Nucleus enzyme

MYH9 MYH9 myosin, heavy Cytoplasm enzyme chain 9, non- muscle

MY09A MY09A myosin IXA Cytoplasm enzyme

NAD kinase

domain containing

NADKD1 NADKD1 1 Cytoplasm other

nuclear

autoantigenic

sperm protein

NASP NASP (histone-binding) Nucleus other

N- acetyltransferase

NAT10 NAT 10 10 (GCN5-related) Nucleus enzyme non-SMC

condensin I

complex, subunit

NCAPD2 NCAPD2 D2 Nucleus other

non-SMC

condensin II

complex, subunit

NCAPG2 NCAPG2 G2 Nucleus other

nuclear cap

binding protein

NCBP1 NCBP1 subunit 1 , 80kDa Nucleus other

NCK-associated Plasma

NCKAP1 L NCKAP1 L protein 1 -like Membrane other

NCK interacting

protein with SH3

NCKIPSD NCKIPSD domain Nucleus other

NCL NCL nucleolin Nucleus other

nuclear receptor transcription

NCOR1 NC0R1 corepressor 1 Nucleus regulator nuclear receptor transcription

NCOR2 NC0R2 corepressor 2 Nucleus regulator nudE nuclear

NDE1 distribution gene E

(includes homolog 1 (A.

NDE1 EG:54820) nidulans) Nucleus other

neural precursor

cell expressed,

developmental^

down-regulated 4-

NEDD4L NEDD4L like Cytoplasm enzyme

NIMA (never in

mitosis gene a)-

NEK9 NEK9 related kinase 9 Nucleus kinase nuclear factor of

kappa light

polypeptide gene

enhancer in B-cells transcription

NFKB1 NFKB1 1 Nucleus regulator nuclear factor of

kappa light

polypeptide gene

enhancer in B-cells transcription

NFKB2 NFKB2 2 (p49/p100) Nucleus regulator nuclear factor of

kappa light transcription

NFKBIB NFKBIB polypeptide gene Nucleus regulator enhancer in B-cells

inhibitor, beta

nuclear factor of

kappa light

polypeptide gene

enhancer in B-cells transcription

NFKBIE NFKBIE inhibitor, epsilon Nucleus regulator

Plasma transmembrane

NISCH NISCH nischarin Membrane receptor

nitric oxide

synthase

NOSIP NOSIP interacting protein Cytoplasm other

nucleophosmin

(nucleolar

phosphoprotein transcription

NPM1 NPM1 B23, numatrin) Nucleus regulator

NAD(P) dependent

steroid

dehydrogenase-

NSDHL NSDHL like Cytoplasm enzyme

NSFL1 (p97)

NSFL1 C NSFL1 C cofactor (p47) Cytoplasm other

NOP2/Sun domain

NSUN2 NSUN2 family, member 2 Nucleus enzyme

nudix (nucleoside

diphosphate linked

moiety X)-type

NUDT5 NUDT5 motif 5 Cytoplasm phosphatase

2'-5'- oligoadenylate

synthetase 2,

OAS2 OAS2 69/71 kDa Cytoplasm enzyme

oxoglutarate

(alpha- ketoglutarate)

dehydrogenase

OGDH OGDH (lipoamide) Cytoplasm enzyme

optic atrophy 1

(autosomal

OPA1 OPA1 dominant) Cytoplasm enzyme

OTU domain,

ubiquitin aldehyde

OTUB1 OTUB1 binding 1 unknown enzyme

proliferation- associated 2G4, transcription

PA2G4 PA2G4 38kDa Nucleus regulator poly(A) binding

protein, translation

PABPC1 PABPC1 cytoplasmic 1 Cytoplasm regulator poly(A)-specific

PARN PARN ribonuclease Nucleus enzyme

poly (ADP-ribose)

polymerase family,

PARP9 PARP9 member 9 Nucleus other

PARVG PARVG parvin, gamma Cytoplasm other

poly(rC) binding translation

PCBP1 PCBP1 protein 1 Nucleus regulator poly(rC) binding

PCBP2 PCBP2 protein 2 Nucleus other protocadherin

gamma subfamily

PCDHGB6 PCDHGB6 B, 6 unknown other

PCI domain transcription

PCID2 PCID2 containing 2 Nucleus regulator proliferating cell

PCNA PCNA nuclear antigen Nucleus enzyme programmed cell

PDCD2L PDCD2L death 2-like unknown other

programmed cell

death 6 interacting

PDCD6IP PDCD6IP protein Cytoplasm other

phosphodiesterase

4D interacting

PDE4DIP PDE4DIP protein Cytoplasm enzyme pyruvate

dehydrogenase

PDHB PDHB (lipoamide) beta Cytoplasm enzyme protein disulfide

isomerase family

PDIA6 PDIA6 A, member 6 Cytoplasm enzyme pyruvate

dehydrogenase

PDK1 PDK1 kinase, isozyme 1 Cytoplasm kinase pyruvate

dehyrogenase

phosphatase

PDP1 PDP1 catalytic subunit 1 Cytoplasm phosphatase pyruvate

dehydrogenase

phosphatase

PDPR PDPR regulatory subunit Cytoplasm enzyme phosphorylase

PHKB PHKB kinase, beta Cytoplasm kinase phosphatidylinosito

I 4-kinase,

PI4KA PI4KA catalytic, alpha Cytoplasm kinase phosphoinositide- 3-kinase adaptor

PIK3AP1 PIK3AP1 protein 1 Cytoplasm other

phosphoinositide- 3-kinase, class 2,

PIK3C2B PIK3C2B beta polypeptide Cytoplasm kinase phosphoinositide-

PIK3C3 PIK3C3 3-kinase, class 3 Cytoplasm kinase phosphoinositide- 3-kinase,

regulatory subunit

PIK3R4 PIK3R4 4 Cytoplasm other

phospholipase A2-

PLAA PLAA activating protein Cytoplasm other

phospholipase B

domain containing Extracellular

PLBD2 PLBD2 2 Space other

phospholipase C,

gamma 2

(phosphatidylinosit

PLCG2 PLCG2 ol-specific) Cytoplasm enzyme

PM20D2 PM20D2 peptidase M20 unknown other domain containing

2

PMS1 postmeiotic

segregation

increased 1 (S.

PMS1 PMS1 cerevisiae) Nucleus enzyme

PMS2 postmeiotic

segregation

increased 2 (S.

PMS2 PMS2 cerevisiae) Nucleus other

forodesine, 9-deaza-9- purine nucleoside (3-

PNP PNP phosphorylase Nucleus enzyme thienylmethyl)guanine

polymerase (DNA nelarabine, MB07133, directed), delta 1 , clofarabine, cytarabine, catalytic subunit trifluridine, vidarabine,

POLD1 POLD1 125kDa Nucleus enzyme entecavir

polymerase (RNA)

I polypeptide C,

POLR1 C POLR1 C 30kDa Nucleus enzyme

polymerase (RNA)

II (DNA directed)

polypeptide A,

POLR2A POLR2A 220kDa Nucleus enzyme

phosphoribosyl 6-mercaptopurine, pyrophosphate thioguanine,

PPAT PPAT amidotransferase Cytoplasm enzyme azathioprine

protein

phosphatase,

Mg2+/Mn2+

PPM1A PPM1A dependent, 1A Cytoplasm phosphatase

protein

phosphatase 1 ,

catalytic subunit,

PPP1 CC PPP1 CC gamma isozyme Cytoplasm phosphatase

protein

phosphatase 2,

regulatory subunit

PPP2R1A PPP2R1A A, alpha Cytoplasm phosphatase protein

phosphatase 3, ISAtx-247, tacrolimus, catalytic subunit, pimecrolimus,

PPP3CA PPP3CA alpha isozyme Cytoplasm phosphatase cyclosporin A

protein

phosphatase 4,

PPP4C PPP4C catalytic subunit Cytoplasm phosphatase

protein

phosphatase 5,

PPP5C PPP5C catalytic subunit Nucleus phosphatase

protein

phosphatase 6,

PPP6C PPP6C catalytic subunit Nucleus phosphatase

protein kinase,

AMP-activated,

alpha 1 catalytic

PRKAA1 PRKAA1 subunit Cytoplasm kinase

protein kinase,

AMP-activated,

beta 1 non-

PRKAB1 PRKAB1 catalytic subunit Nucleus kinase

protein kinase,

AMP-activated,

beta 2 non-

PRKAB2 PRKAB2 catalytic subunit Cytoplasm kinase

protein kinase,

AMP-activated,

gamma 1 non-

PRKAG1 PRKAG1 catalytic subunit Nucleus kinase

protein kinase C

PRKCSH PRKCSH substrate 80K-H Cytoplasm enzyme

PRKD2 PRKD2 protein kinase D2 Cytoplasm kinase

protein kinase,

DNA-activated,

catalytic

PRKDC PRKDC polypeptide Nucleus kinase

protein arginine

methyltransferase

PRMT1 PRMT1 1 Nucleus enzyme

protein arginine

methyltransferase

PRMT10 PRMT10 10 (putative) unknown other

protein arginine

methyltransferase

PRMT3 PRMT3 3 Nucleus enzyme

protein arginine

methyltransferase

PRMT5 PRMT5 5 Cytoplasm enzyme

pleckstrin and

Sec7 domain

PSD4 PSD4 containing 4 Cytoplasm other

proteasome

(prosome,

PSMA1 PSMA1 macropain) Cytoplasm peptidase subunit, alpha

type, 1

proteasome

(prosome,

macropain) 26S

PSMC1 PSMC1 subunit, ATPase, 1 Nucleus peptidase proteasome

(prosome,

macropain)

activator subunit 1

PSME1 PSME1 (PA28 alpha) Cytoplasm other

Pentatricopeptide

PTCD3 PTCD3 repeat domain 3 Cytoplasm other

prostaglandin E transcription

PTGES2 PTGES2 synthase 2 Cytoplasm regulator

PTK2

(includes PTK2 protein

PTK2 EG: 14083) tyrosine kinase 2 Cytoplasm kinase

PTK2B PTK2B protein

(includes tyrosine kinase 2

PTK2B EG: 19229) beta Cytoplasm kinase protein tyrosine

phosphatase, non¬

PTPN1 PTPN1 receptor type 1 Cytoplasm phosphatase protein tyrosine

phosphatase, non¬

PTPN6 PTPN6 receptor type 6 Cytoplasm phosphatase protein tyrosine

phosphatase, Plasma

PTPRJ PTPRJ receptor type, J Membrane phosphatase poly-U binding

splicing factor

PUF60 PUF60 60KDa Nucleus other

RAB3 GTPase

activating protein

subunit 1

RAB3GAP1 RAB3GAP1 (catalytic) Cytoplasm other

RAB3 GTPase

activating protein

subunit 2 (non-

RAB3GAP2 RAB3GAP2 catalytic) Cytoplasm enzyme

Rab

geranylgeranyltran

sferase, beta

RABGGTB RABGGTB subunit Cytoplasm enzyme

RAD23 homolog B

RAD23B RAD23B (S. cerevisiae) Nucleus other

RAD51 homolog

RAD51 RAD51 (S. cerevisiae) Nucleus enzyme

RAE1 RNA export

1 homolog (S.

RAE1 RAE1 pombe) Nucleus other

RAN binding

RANBP2 RANBP2 protein 2 Nucleus enzyme

Rap guanine

nucleotide

exchange factor Plasma

RAPGEF6 RAPGEF6 (GEF) 6 Membrane other

RARS RARS arginyl-tRNA Cytoplasm enzyme synthetase

Ras association

(RalGDS/AF-6)

domain family

RASSF2 RASSF2 member 2 Nucleus other

RanBP-type and

C3HC4-type zinc transcription

RBCK1 RBCK1 finger containing 1 Cytoplasm regulator

REST corepressor transcription

RCOR1 RCOR1 1 Nucleus regulator

v-rel

reticuloendotheliosi

s viral oncogene transcription

REL REL homolog (avian) Nucleus regulator

v-rel

reticuloendotheliosi

s viral oncogene transcription

RELA RELA homolog A (avian) Nucleus regulator NF-kappaB decoy

RAS (RAD and

GEM)-like GTP-

REM1 REM1 binding 1 unknown enzyme

RNA (guanine-9-)

methyltransferase

domain containing

RG9MTD1 RG9MTD1 1 Cytoplasm other

ring finger protein

RNF138 RNF138 138 unknown other

ring finger protein

RNF20 RNF20 20 Nucleus enzyme

ring finger protein Plasma

RNF213 RNF213 213 Membrane other

ring finger protein

RNF31 RNF31 31 Cytoplasm enzyme

RNA (guanine-7-)

RNMT RNMT methyltransferase Nucleus enzyme

replication protein

RPA1 RPA1 A1 , 70kDa Nucleus other

replication protein

RPA2 RPA2 A2, 32kDa Nucleus other

ribosomal protein

RPS6 RPS6 S6 Cytoplasm other

ribosomal protein

S6 kinase, 90kDa,

RPS6KA3 RPS6KA3 polypeptide 3 Cytoplasm kinase

reticulon 4

interacting protein

RTN4IP1 RTN4IP1 1 Cytoplasm enzyme

RuvB-like 1 (E. transcription

RUVBL1 RUVBL1 coli) Nucleus regulator

RuvB-like 2 (E. transcription

RUVBL2 RUVBL2 coli) Nucleus regulator

SAM domain and

SAMHD1 SAMHD1 HD domain 1 Nucleus enzyme

SR-related CTD-

SCAF8 SCAF8 associated factor 8 Nucleus other

sed family domain

SCFD1 SCFD1 containing 1 Cytoplasm transporter

SCPEP1 SCPEP1 serine Cytoplasm peptidase carboxypeptidase

1

SCY1-like 1 (S.

SCYL1 SCYL1 cerevisiae) Cytoplasm kinase

Sec23 homolog B

SEC23B SEC23B (S. cerevisiae) Cytoplasm transporter

SEC23 interacting

SEC23IP SEC23IP protein Cytoplasm other

selenophosphate

SEPHS1 SEPHS1 synthetase 1 unknown enzyme

Sep (0- phosphoserine)

tRNA:Sec

(selenocysteine)

SEPSECS SEPSECS tRNA synthase Cytoplasm other

SEPT2 SEPT2 septin 2 Cytoplasm enzyme

SEPT9 SEPT9 septin 9 Cytoplasm enzyme

SERPINE1 imRNA

SERBP1 SERBP1 binding protein 1 Nucleus other

serpin peptidase

inhibitor, clade B

(ovalbumin),

SERPINB9 SERPINB9 member 9 Cytoplasm other

SET nuclear

SET SET oncogene Nucleus phosphatase

SET domain

SETD2 SETD2 containing 2 Cytoplasm enzyme splicing factor 3a,

SF3A1 SF3A1 subunit 1 , 120kDa Nucleus other

splicing factor

proline/glutamine-

SFPQ SFPQ rich Nucleus other

SHANK-associated

RH domain Plasma

SHARPIN SHARPIN interactor Membrane other

SIRT3 SIRT3 sirtuin 3 Cytoplasm enzyme

SIRT5 SIRT5 sirtuin 5 Cytoplasm enzyme stem-loop binding

SLBP SLBP protein Nucleus other

solute carrier

family 1 (neutral

amino acid

transporter), Plasma

SLC1A5 SLC1A5 member 5 Membrane transporter solute carrier

family 25

(mitochondrial

carrier; phosphate

SLC25A3 SLC25A3 carrier), member 3 Cytoplasm transporter solute carrier

family 25

(mitochondrial

carrier; adenine

nucleotide

translocator),

SLC25A5 SLC25A5 member 5 Cytoplasm transporter solute carrier

family 3 (activators Plasma

SLC3A2 SLC3A2 of dibasic and Membrane transporter neutral amino acid

transport), member

2

SMAD family transcription

SMAD2 SMAD2 member 2 Nucleus regulator

SWI/SNF related,

matrix associated,

actin dependent

regulator of

chromatin,

subfamily a, transcription

SMARCA4 SMARCA4 member 4 Nucleus regulator

SWI/SNF related,

matrix associated,

actin dependent

regulator of

chromatin,

subfamily c, transcription

SMARCC2 SMARCC2 member 2 Nucleus regulator

SWI/SNF related,

matrix associated,

actin dependent

regulator of

chromatin,

subfamily d, transcription

SMARCD2 SMARCD2 member 2 Nucleus regulator structural

maintenance of

SMC1A SMC1 A chromosomes 1A Nucleus transporter structural

maintenance of

SMC2 SMC2 chromosomes 2 Nucleus transporter structural

maintenance of

SMC3 SMC3 chromosomes 3 Nucleus other

structural

maintenance of

SMC4 SMC4 chromosomes 4 Nucleus transporter smg-1 homolog,

phosphatidylinosito

I 3-kinase-related

kinase (C.

SMG1 SMG1 elegans) Cytoplasm kinase survival motor

neuron domain

SMNDC1 SMNDC1 containing 1 Nucleus other

small nuclear

ribonucleoprotein

SNRNP200 SNRNP200 200kDa (U5) Nucleus enzyme spastic paraplegia

21 (autosomal

recessive, Mast Plasma

SPG21 SPG21 syndrome) Membrane enzyme

SRSF protein

SRPK1 SRPK1 kinase 1 Nucleus kinase

SRR SRR serine racemase Cytoplasm enzyme serine/arginine-rich

SRSF7 SRSF7 splicing factor 7 Nucleus other single-stranded

DNA binding transcription

SSBP2 SSBP2 protein 2 Nucleus regulator suppression of

tumorigenicity 13

(colon carcinoma)

(Hsp70 interacting

ST13 ST13 protein) Cytoplasm other

signal transducer

and activator of

transcription 1 , transcription

STAT1 STAT1 91 kDa Nucleus regulator signal transducer

and activator of

transcription 3

(acute-phase transcription

STAT3 STAT3 response factor) Nucleus regulator signal transducer

and activator of transcription

STAT5B STAT5B transcription 5B Nucleus regulator stress-induced-

STIP1 STIP1 phosphoprotein 1 Cytoplasm other

serine/threonine

STK4 STK4 kinase 4 Cytoplasm kinase serine/threonine

kinase receptor Plasma

STRAP STRAP associated protein Membrane other

STIP1 homology

and U-box

containing protein

1 , E3 ubiquitin

STUB1 STUB1 protein ligase Cytoplasm enzyme

Plasma

STX12 STX12 syntaxin 12 Membrane other

spleen tyrosine

SYK SYK kinase Cytoplasm kinase

SYMPK SYMPK symplekin Cytoplasm other

spectrin repeat

containing, nuclear

SYNE1 SYNE1 envelope 1 Nucleus other

spectrin repeat

containing, nuclear

SYNE2 SYNE2 envelope 2 Nucleus other

TGF-beta activated

kinase 1/MAP3K7

TAB1 TAB1 binding protein 1 Cytoplasm enzyme transforming,

acidic coiled-coil

containing protein

TACC3 TACC3 3 Nucleus other

TAR (HIV-1 ) RNA transcription

TARBP1 TARBP1 binding protein 1 Nucleus regulator

TAR DNA binding transcription

TARDBP TARDBP protein Nucleus regulator tubulin folding

TBCD TBCD cofactor D Cytoplasm other

TANK-binding

TBK1 TBK1 kinase 1 Cytoplasm kinase transducin (beta)- like 1 X-linked transcription

TBL1XR1 TBL1 XR1 receptor 1 Nucleus regulator transducin (beta)-

TBL3 TBL3 Iike 3 Cytoplasm peptidase transforming

growth factor beta

TBRG4 TBRG4 regulator 4 Cytoplasm other

tuftelin interacting Extracellular

TFIP1 1 TFIP1 1 protein 1 1 Space other

TH1-like

TH1 L TH1 L (Drosophila) Nucleus other

tRNA-histidine

guanylyltransferas

e 1-like (S.

THG1 L THG1 L cerevisiae) Cytoplasm enzyme

THOC2 THOC2 THO complex 2 Nucleus other

THUMP domain

THUMPD1 THUMPD1 containing 1 unknown other

THUMP domain

THUMPD3 THUMPD3 containing 3 unknown other

translocase of

inner mitochondrial

membrane 50

homolog (S.

TIMM50 TIMM50 cerevisiae) Cytoplasm phosphatase

TIP41 , TOR

signaling pathway

regulator-like (S.

TIPRL TIPRL cerevisiae) unknown other

TKT TKT transketolase Cytoplasm enzyme transducin-like

enhancer of split 3

(E(sp1 ) homolog,

TLE3 TLE3 Drosophila) Nucleus other

Plasma

TLN1 TLN 1 talin 1 Membrane other

target of EGR1 ,

member 1

TOE1 TOE1 (nuclear) Nucleus other

translocase of

outer mitochondrial

TOMM34 TOMM34 membrane 34 Cytoplasm other

TP53 regulating

TP53RK TP53RK kinase Nucleus kinase

TPP1

(includes tripeptidyl

TPP1 EG: 1200) peptidase I Cytoplasm peptidase tripeptidyl

TPP2 TPP2 peptidase II Cytoplasm peptidase

TNF receptor- associated protein

TRAP1 TRAP1 1 Cytoplasm enzyme tripartite motif transcription

TRIM25 TRIM25 containing 25 Cytoplasm regulator tripartite motif transcription

TRIM28 TRIM28 containing 28 Nucleus regulator

TRIO TRIO triple functional Plasma kinase domain (PTPRF Membrane

interacting)

TROVE domain

TROVE2 TROVE2 family, member 2 Nucleus other tetratricopeptide

TTC1 TTC1 repeat domain 1 unknown other tetratricopeptide

TTC19 TTC19 repeat domain 19 Cytoplasm other tetratricopeptide

TTC37 TTC37 repeat domain 37 unknown other tetratricopeptide

TTC5 TTC5 repeat domain 5 unknown other

TTN

(includes

TTN EG:22138) titin Cytoplasm kinase terminal uridylyl

transferase 1 , U6

TUT1 TUT1 snRNA-specific Nucleus enzyme ubiquitin-like

modifier activating

UBA1 UBA1 enzyme 1 Cytoplasm enzyme

UBA domain

UBAC1 UBAC1 containing 1 Nucleus other ubiquitin

associated protein

UBAP2 UBAP2 2 Cytoplasm other ubiquitin

associated protein

UBAP2L UBAP2L 2-like unknown other ubiquitin- conjugating

UBE20 UBE20 enzyme E20 unknown enzyme ubiquitin protein

UBE3A UBE3A ligase E3A Nucleus enzyme

UBQLN1 UBQLN 1 ubiquilin 1 Cytoplasm other ubiquitin protein

UBR1 ligase E3

(includes component n-

UBR1 EG: 197131 ) recognin 1 Cytoplasm enzyme ubiquitin protein

ligase E3

component n-

UBR4 UBR4 recognin 4 Nucleus other ubiquitin protein

ligase E3

component n-

UBR5 UBR5 recognin 5 Nucleus enzyme

UBX domain

UBXN1 UBXN1 protein 1 Cytoplasm other ubiquitin carboxyl- terminal hydrolase

UCHL5 UCHL5 L5 Cytoplasm peptidase uridine-cytidine

UCK2 UCK2 kinase 2 Cytoplasm kinase ubiquitin fusion

degradation 1 like

UFD1 L UFD1 L (yeast) Cytoplasm peptidase

UHRF1 binding

UHRF1 BP1 UHRF1 BP1 protein 1 unknown other UPF1 regulator of

nonsense

transcripts

UPF1 UPF1 homolog (yeast) Nucleus enzyme

US01 vesicle

docking protein

US01 US01 homolog (yeast) Cytoplasm transporter ubiquitin specific

USP1 1 USP1 1 peptidase 1 1 Nucleus peptidase ubiquitin specific

peptidase 13

USP13 USP13 (isopeptidase T-3) unknown peptidase ubiquitin specific

USP15 USP15 peptidase 15 Cytoplasm peptidase ubiquitin specific

USP24 USP24 peptidase 24 unknown peptidase ubiquitin specific

USP25 USP25 peptidase 25 unknown peptidase ubiquitin specific

USP28 USP28 peptidase 28 Nucleus peptidase ubiquitin specific

USP34 USP34 peptidase 34 unknown peptidase ubiquitin specific

USP47 USP47 peptidase 47 Cytoplasm peptidase ubiquitin specific

peptidase 5

USP5 USP5 (isopeptidase T) Cytoplasm peptidase ubiquitin specific

peptidase 7

(herpes virus-

USP7 USP7 associated) Nucleus peptidase ubiquitin specific

peptidase 9, X- Plasma

USP9X USP9X linked Membrane peptidase vav 1 guanine

nucleotide transcription

VAV1 VAV1 exchange factor Nucleus regulator valosin containing

VCP VCP protein Cytoplasm enzyme voltage-dependent

VDAC1 VDAC1 anion channel 1 Cytoplasm ion channel

Vpr (HIV-1 ) binding

VPRBP VPRBP protein Nucleus other

WW domain

WBP2 WBP2 binding protein 2 Cytoplasm other

WDFY family

WDFY4 WDFY4 member 4 unknown other

WD repeat domain

WDR1 1 WDR1 1 1 1 unknown other

WD repeat domain

WDR5 WDR5 5 Nucleus other

WD repeat domain

WDR6 WDR6 6 Cytoplasm other

WD repeat domain

WDR61 WDR61 61 unknown other

WD repeat domain

WDR82 WDR82 82 Nucleus other

WD repeat domain

WDR92 WDR92 92 unknown other tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein, transcription

YWHAB YWHAB beta polypeptide Cytoplasm regulator

tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein,

YWHAE YWHAE epsilon polypeptide Cytoplasm other

tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein,

gamma

YWHAG YWHAG polypeptide Cytoplasm other

tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein, transcription

YWHAH YWHAH eta polypeptide Cytoplasm regulator

tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein,

YWHAQ YWHAQ theta polypeptide Cytoplasm other

tyrosine 3- monooxygenase/tr

yptophan 5- monooxygenase

activation protein,

YWHAZ YWHAZ zeta polypeptide Cytoplasm enzyme

zinc finger CCCH- type containing

ZC3H1 1A ZC3H 1 1A 1 1A unknown other

zinc finger CCCH-

ZC3H18 ZC3H 18 type containing 18 Nucleus other

zinc finger CCCH-

ZC3H4 ZC3H4 type containing 4 unknown other

zinc finger RNA

ZFR ZFR binding protein Nucleus other

zinc finger, FYVE

domain containing

ZFYVE26 ZFYVE26 26 Cytoplasm other

zinc finger protein

ZNF259 ZNF259 259 Nucleus other

B eel! receptor signaling

Signals propagated through the B cell antigen receptor (BCR) are crucial to the development, survival and activation of B lymphocytes. These signals also play a central role in the removal of potentially self-reactive B lymphocytes. The BCR is composed of surface-bound antigen recognizing membrane antibody and associated Ig-aand Ig-β heterodimers which are capable of signal transduction via cytosolic motifs called immunoreceptor tyrosine based activation motifs (IT AM). The recognition of polyvalent antigens by the B cell antigen receptor (BCR) initiates a series of interlinked signaling events that culminate in cellular responses. The engagement of the BCR induces the phosphorylation of tyrosine residues in the IT AM. The phosphorylation of IT AM is mediated by SYK kinase and the SRC family of kinases which include LYN, FYN and BLK. These kinases which are reciprocally activated by phosphorylated ITAMs in turn trigger a cascade of interlinked signaling pathways. Activation of the BCR leads to the stimulation of nuclear factor kappa B (NFKB). Central to BCR signaling via NF-kB is the complex formed by the Bruton's tyrosine kinase (BTK), the adaptor B-cell linker (BLNK) and phospho lipase C gamma 2 (PLCy2). Tyrosine phosphorylated adaptor proteins act as bridges between BCR associated tyrosine kinases and downstream effector molecules. BLNK is phosphorylated on BCR activation and serves to couple the tyrosine kinase SYK to the activation of PLCy2. The complete stimulation of PLCy2 is facilitated by BTK. Stimulated PLCy2 triggers the DAG and Ca2+ mediated activation of Protein kinase (PKC) which in turn activates IkB kinase (IKK) and thereafter NFKB. In addition to the activation of NFKB, BLNK also interacts with other proteins like VAV and GRB2 resulting in the activation of the mitogen activated protein kinase (MAPK) pathway. This results in the transactivation of several factors like c-JUN, activation of transcription factor (ATF) and ELK6. Another adaptor protein, B cell adaptor for phosphoinositide 3-kinase (PI3K), termed BCAP once activated by SYK, goes on to trigger a PI3K/AKT signaling pathway. This pathway inhibits Glycogen synthase kinase 3 (GSK3), resulting in the nuclear accumulation of transcription factors like nuclear factor of activated T cells (NFAT) and enhancement of protein synthesis. Activation of PI3K/AKT pathway also leads to the inhibition of apoptosis in B cells. This pathway highlights the important components of B cell receptor antigen signaling.

This pathway is composed of, but not restricted to 1-phosphatidyl-D-myo-inositol 4,5- bisphosphate, ABL1, Akt, ATF2, BAD, BCL10, BcllO-CardlO-Maltl , BCL2A1, BCL2L1,

BCL6, BLNK, BTK, Calmodulin, CaMKII, CARD 10, CD 19, CD22, CD79A, CD79B, Creb,

CSK, DAPP1, EGR1, ELK1, ERK1/2, ETS1, Fcgr2, GAB1/2, GRB2, Gsk3, Ikb, IkB-NfkB,

IKK (complex), JINK1/2, Jnkk, JUN, LYN, MALT1, MAP2K1/2, MAP3K, MKK3/4/6,

MTOR, NFAT (complex), NFkB (complex), P38 MAPK, p70 S6k, PAG1, phosphatidylinositol-3,4,5-triphosphate, PI3K (complex), PIK3AP1, ΡΚΟ(β,θ), PLCG2, POU2F2, Pp2b, PTEN, PTPN11, PTPN6, PTPRC, Rac/Cdc42, RAF1, Ras, SHC1 (includes EG:20416), SHIP, Sos, SYK, VAV PKCteia pathway

An effective immune response depends on the ability of specialized leukocytes to identify foreign molecules and respond by differentiation into mature effector cells. A cell surface antigen recognition apparatus and a complex intracellular receptor-coupled signal transducing machinery mediate this tightly regulated process which operates at high fidelity to discriminate self antigens from non-self antigens. Activation of T cells requires sustained physical interaction of the TCR with an MHC-presented peptide antigen that results in a temporal and spatial reorganization of multiple cellular elements at the T-Cell-APC contact region, a specialized region referred to as the immunological synapse or supramolecular activation cluster. Recent studies have identified PKC9, a member of the Ca-independent PKC family, as an essential component of the T-Cell supramolecular activation cluster that mediates several crucial functions in TCR signaling leading to cell activation, differentiation, and survival through IL-2 gene induction. High levels of PKC9 are expressed in skeletal muscle and lymphoid tissues, predominantly in the thymus and lymph nodes, with lower levels in spleen. T cells constitute the primary location for PKC9 expression. Among T cells, CD4+/CD8+ single positive peripheral blood T cells and CD4+/CD8+ double positive thymocytes are found to express high levels of PKC9. On the surface of T cells, TCR/CD3 engagement induces activation of Src, Syk, ZAP70 and Tec-family PTKs leading to stimulation and membrane recruitment of PLCyl, PI3K and Vav. A Vav mediated pathway, which depends on Rac and actin cytoskeleton reorganization as well as on PI3K, is responsible for the selective recruitment of PKC9 to the supramolecular activation cluster. PLCyl -generated DAG also plays a role in the initial recruitment of PKC9. The transcription factors NF-KB and AP-1 are the primary physiological targets of PKC9. Efficient activation of these transcription factors by PKC9 requires integration of TCR and CD28 co-stimulatory signals. CD28 with its CD80/CD86 (B7-1/B7-2) ligands on APCs is required for the recruitment of PKC9 specifically to the supramolecular activation cluster. The transcriptional element which serves as a target for TCR/CD28 costimulation is CD28RE in the IL-2 promoter. CD28RE is a combinatorial binding site for NF-κΒ and AP-1. Recent studies suggest that regulation of TCR coupling to NF-κΒ by PKC9 is affected through a variety of distinct mechanisms. PKC9 may directly associate with and regulate the IKK complex; PKC9 may regulate the IKK complex indirectly though CaMKII; It may act upstream of a newly described pathway involving BCL10 and MALT1, which together regulate NF-κΒ and ΙκΒ via the IKK complex. PKC9 has been found to promote Activation-induced T cell death (AICD), an important process that limits the expansion of activated antigen-specific T cells and ensures termination of an immune response once the specific pathogen has been cleared. Enzymatically active PKC9 selectively synergizes with calcineurin to activate a caspase 8- mediated Fas/FasL-dependent AICD. CD28 co-stimulation plays an essential role in TCR- mediated IL-2 production, and in its absence the T cell enters a stable state of unresponsiveness termed anergy. PKC9-mediated CREB phosphorylation and its subsequent binding to a cAMP-response element in the IL-2 promoter negatively regulates IL-2 transcription thereby driving the responding T cells into an anergic state. The selective expression of PKC9 in T-Cells and its essential role in mature T cell activation establish it as an attractive drug target for immunosuppression in transplantation and autoimmune diseases.

This pathway is composed of, but not restricted to Apl, BCL10, Bel 10-Card 11 -Malt 1, Calcineurin protein(s), CaMKII, CARD11, CD28, CD3, CD3-TCR, CD4, CD80 (includes EG: 12519), CD86, diacylglycerol, ERKl/2, FOS, FYN, GRAP2, GRB2, Ikb, IkB-NfkB, Ikk (family), IL2, inositol triphosphate, JUN, LAT, LCK, LCP2, MALT1, MAP2K4, MAP3K, MAPK8, MHC Class II (complex), Nfat (family), NFkB (complex), phorbol myristate acetate, PI3K (complex), PLC gamma, POU2F1, PRKCQ, Rac, Ras, Sos, TCR, VAV, voltage-gated calcium channel, ZAP70

CD40 signaling

CD40 is a member of the tumor necrosis factor superfami!y of cell surface receptors that transmits survival signals to B cells. Upon li garni binding, canonical signaling evoked by cel l-surface CD40 follows a muitistep cascade requiring cytoplasmic adaptors (called TNF- receptor-associated factors [TRAFs], which are recruited by CD 0 in the lipid rafts) and the IKK complex. Through NF-κΒ activation, the CD40 signalosome activates transcription of mutiple genes involved in B~cell growth and survival. Because the CD40 signalosome is active in aggressive lymphoma and contributes to tumor growth, irnnrunotherapeutie strategies directed against CD40 are being designed and currently tested in clinical trials [Bayes 2007 and Fanale 2007). CD40-mediated signal transduction induces the transcription of a large number of genes implicated in host defense against pathogens. This is accomplished by the activation of multiple pathways including NF-κΒ, MAPK and STAT3 which regulate gene expression through activation of c-Jun, ATF2 and Rel transcription factors. Receptor clustering of CD40L is mediated by an association of the ligand with p53, a translocation of ASM to the plasma membrane, activation of ASM, and formation of ceramide. Ceramide serves to cluster CD40L and several TRAF proteins (including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6) with CD40. TRAF2, TRAF3 and TRAF6 bind to CD40 directly. TRAF1 does not directly bind CD40 but is recruited to membrane micro domains through heterodimerization with TRAF2. Analogous to the recruitment of TRAF 1, TRAF 5 is also indirectly recruited to CD40 in a TRAF3 -dependent manner. Actl links TRAF proteins to TAK1/IK to activate NF-KB/I-KB, and MK complex to activate JNK, p38 MAPK and ERK1/2. NIK also plays a leading role in activating IKK. Actl -dependent CD40-mediated NF-κΒ activation protects cells from CD40L-induced apoptosis. On stimulation with CD40L or other inflammatory mediators, Ι-κΒ proteins are phosphorylated by IKK and NF-κΒ is activated through the Actl-TAKl pathway. Phosphorylated Ι-κΒ is then rapidly ubiquitinated and degraded. The liberated NF-κΒ translocates to the nucleus and activates transcription. A20, which is induced by TNF inhibits NF-κΒ activation as well as TNF-mediated apoptosis. TRAF3 initiates signaling pathways that lead to the activation of p38 and JNK but inhibits Actl -dependent CD40-mediated NF-κΒ activation and initiates CD40L-induced apoptosis. TRAF2 is required for activation of SAPK pathways and also plays a role in CD40-mediated surface upregulation, IgM secretion in B-Cells and up-regulation of ICAMl . CD40 ligation by CD40L stimulates MCP1 and IL-8 production in primary cultures of human proximal tubule cells, and this occurs primarily via recruitment of TRAF6 and activation of the ERKl/2, SAPK/ JNK and p38 MAPK pathways. Activation of SAPK JNK and p38 MAPK pathways is mediated via TRAF6 whereas ERKl/2 activity is potentially mediated via other TRAF members. However, stimulation of all three MAPK pathways is required for MCP1 and IL-8 production. Other pathways activated by CD40 stimulation include the JAK3-STAT3 and PI3K-Akt pathways, which contribute to the anti-apoptotic properties conferred by CD40L to B-Cells. CD40 directly binds to JAK3 and mediates STAT3 activation followed by upregulation of ICAMl, CD23, and LT-a.

This pathway is composed of, but not restricted to Actl, Apl, ATF1 (includes EG: 100040260), CD40, CD40LG, ERKl/2, FCER2, I kappa b kinase, ICAMl, Ikb, IkB- NfkB, JAK3, Jnk, LTA, MAP3K14, MAP3K7 (includes EG: 172842), MAPKAPK2, Mek, NFkB (complex), P38 MAPK, PI3K (complex), STAT3, Stat3-Stat3, TANK, TNFAIP3, TRAF1, TRAF2, TRAF3, TRAF5, TRAF6

CD28 signaling pathway CD28 is a co-receptor for the TCR/CD3 and is is a major positive co-stimulatory molecule. Upon ligation with CD80 and CD86, CTLA4 provides a negative co-stimulatory signal for the termination of activation. Further binding of CD28 to Class-I regulatory PI3K recruits PI3K to the membrane, resulting in generation of PIP3 and recruitment of proteins that contain a pleckstrin-homology domain to the plasma membrane, such as PIK3C3. PI3K is required for activation of Akt, which in turn regulates many downstream targets that to promote cell survival. In addition to NFAT, NF-κΒ has a crucial role in the regulation of transcription of the IL-2 promoter and anti-apoptotic factors. For this, PLC-γ utilizes PIP2 as a substrate to generate IP3 and DAG. IP3 elicits release of Ca2+ via IP3R, and DAG activates PKC-Θ. Under the influence of RLK, PLC-γ, and Ca2+; PKC-Θ regulates the phosphorylation state of IKK complex through direct as well as indirect interactions. Moreover, activation of CARMA1 phosphorylates BCL10 and dimerizes MALT1, an event that is sufficient for the activation of IKKs. The two CD28 -responsive elements in the IL-2 promoter have NF-KB binding sites. NF-κΒ dimers are normally retained in cytoplasm by binding to inhibitory I- KBS. Phosphorylation of I-KBS initiates its ubiquitination and degradation, thereby freeing NF-KB to translocate to the nucleus. Likewise, translocation of NFAT to the nucleus as a result of calmodulin-calcineurin interaction effectively promotes IL-2 expression. Activation of Vavl by TCR-CD28-PI3K signaling connects CD28 with the activation of Rac and CDC42, and this enhances TCR-CD3-CD28 mediated cytoskeletal re -organization. Rac regulates actin polymerization to drive lamellipodial protrusion and membrane ruffling, whereas CDC42 generates polarity and induces formation of filopodia and microspikes. CDC42 and Rac GTPases function sequentially to activate downstream effectors like WASP and PAK1 to induce activation of ARPs resulting in cytoskeletal rearrangements. CD28 impinges on the Rac/PAKl -mediated IL-2 transcription through subsequent activation of MEKKl, MKKs and JNKs. JNKs phosphorylate and activate c-Jun and c-Fos, which is essential for transcription of IL-2. Signaling through CD28 promotes cytokine IL-2 mRNA production and entry into the cell cycle, T-cell survival, T-Helper cell differentiation and Immunoglobulin isotype switching. This pathway is composed of, but not restricted to 1,4,5-IP3, 1-phosphatidyl-D-myo-inositol 4,5-bisphosphate, Akt, Apl, Arp2/3, BCL10, Ca2+, Calcineurin protein(s), Calmodulin, CARD11, CD28, CD3, CD3-TCR, CD4, CD80 (includes EG: 12519), CD86, CDC42, CSK, CTLA4, diacylglycerol, FOS, FYN, GRAP2, GRB2, Ikb, IkB-NfkB, IKK (complex), IL2, ITK, ITPR, Jnk, JUN, LAT, LCK, LCP2, MALT1, MAP2K1/2, MAP3K1, MHC Class II (complex), Nfat (family), NFkB (complex), PAK1, PDPK1, phosphatidylinositol-3,4,5- triphosphate, PI3K (complex), PLCG1, PRKCQ, PTPRC, RAC1, SHP, SYK, TCR, VAV1, WAS, ZAP70

ERK-MAPK pathway

The ERK (extracellular-regulated kinase)/MAPK (mitogen activated protein kinase) pathway is a key pathway that transduces cellular information on meiosis/mitosis, growth, differentiation and carcinogenesis within a cell. Membrane bound receptor tyrosine kinases (RTK), which are often growth factor receptors, are the starting point for this pathway. Binding of ligand to RTK activates the intrinsic tyrosine kinase activity of RTK. Adaptor molecules like growth factor receptor bound protein 2 (GRB2), son of sevenless (SOS) and She form a signaling complex on tyrosine phosphorylated RTK and activate Ras. Activated Ras initiates a kinase cascade, beginning with Raf (a MAPK kinase kinase) which activates and phosphorylates MEK (a MAPK kinase); MEK activates and phosphorylates ERK (a MAPK). ERK in the cytoplasm can phosphorylate a variety of targets which include cytoskeleton proteins, ion channels/receptors and translation regulators. ERK is also translocated across into the nucleus where it induces gene transcription by interacting with transcriptional regulators like ELK-1, STAT-1 and -3, ETS and MYC. ERK activation of p90RSK in the cytoplasm leads to its nuclear translocation where it indirectly induces gene transcription through interaction with transcriptional regulators, CREB, c-Fos and SRF. RTK activation of Ras and Raf sometimes takes alternate pathways. For example, integrins activate ERK via a FAK mediated pathway. ERK can also be activated by a CAS-CRK-Rapl mediated activation of B-Raf and a PLCy-PKC-Ras-Raf activation of ERK. This pathway is be composed of, but not restricted to 1,4,5-IP3, 1-phosphatidyl-D-myo- inositol 4,5-bisphosphate, 14-3-3(β,γ,θ,η,ζ), 14-3-3(η,θ,ζ), ARAF, ATF1 (includes EG: 100040260), BAD, BCAR1, BRAF, c-Myc/N-Myc, cAMP-Gef, CAS-Crk-DOCK 180, Cpla2, Creb, CRK/CRKL, cyclic AMP, diacylglycerol, DOCK1, DUSP2, EIF4E, EIF4EBP1, ELK1, ER 1/2, Erkl/2 dimer, ESR1, ETS, FOS, FYN, GRB2, Histone h3, Hsp27, Integrin, KSRl, LAMTOR3, MAP2K1/2, MAPKAPK5, MKPl/2/3/4, MNKl/2, MOS, MSKl/2, NFATC1, Pak, PI3K (complex), Pka, PKC (α,β,γ,δ,ε,ι), PLC gamma, PP1/PP2A, PPARG, PTK2 (includes EG: 14083), PTK2B (includes EG: 19229), PXN, Rac, RAF1, Rapl, RAPGEFl, Ras, RPS6KA1 (includes EG:20111), SHCl (includes EG:20416), Sos, SRC, SRF, Statl/3, Talin, VRK2

Based on the findings by the method described here in the DLBCL OCI-LY1, combination of an inhibitor of components of these pathways, such as those targeting but not limited to SYK, BTK, mTOR, PI3K, Ikk, CD40, MEK, Raf, JAK, the MHC complex components, CD80, CD3 are proposed to be efficacious when used in combination with an Hsp90 inhibitor.

Examples of BTK inhibitors are PCI-32765

Examples of SYK inhibitors are R-406, R406, R935788 (Fostamatinib disodium) Examples of CD40 inhibitors are SGN-40 (anti-huCD40 mAb)

Examples of inhibitors of the CD28 pathway are abatacept, belatacept, blinatumomab, muromonab-CD3, visilizumab.

Example of inhibitors of major histocompatibility complex, class II are apolizumab

Example of PI3K inhibitors are 2-(lH-indazol-4-yl)-6-(4-methanesulfonylpiperazin-l- ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine, BKM120, NVP-BEZ235, PX-866, SF 1126, XL147.

Example of mTOR inhibitors are deforolimus, everolimus, NVP-BEZ235, OSI-027, tacrolimus, temsirolimus, Ku-0063794, WYE-354, PP242, OSI-027, GSK2126458, WAY- 600, WYE-125132 Examples of JAK inhibitors are Tofacitinib citrate (CP-690550), AT9283, AG-490,

INCBO 18424 (Ruxolitinib), AZD1480, LY2784544, NVP-BSK805, TGI 01209, TG-101348

Examples of IkK inhibitors are SC-514, PF 184 Example of inhibitors of Raf are sorafenib, vemurafenib, GDC-0879, PLX-4720, PLX4032 (Vemura/enib), NVP-BHG712, SB590885, AZ628, ZM 336372

Example of inhibitors of SRC are AZM-475271, dasatinib, saracatinib

In the MiaPaCa2 pancreatic cancer cell line major signaling networks identified by the method were the PBK/AKT, IGFl, cell cycie-G2/M DNA damage checkpoint regulation, ER ^' MAPK and the PKA signaling pathways (Figure 24).

Interactions between the several network component proteins are exemplified in Figure 16.

Pancreatic adenocarcinoma continues to be one of the most lethal cancers, representing the fourth leading cause of cancer deaths in the United States. More than 80% of patients present with advanced disease at diagnosis and therefore, are not candidates for potentially curative surgical resection. Gemcitabine-based chemotherapy remains the main treatment of locally advanced or metastatic pancreatic adenocarcinoma since a pivotal Phase III trial in 1997. Although treatment with gemcitabine does achieve significant symptom control in patients with advanced pancreatic cancer, its response rates still remain low and is associated with a median survival of approximately 6 months. These results reflect the inadequacy of existing treatment strategies for this tumor type, and a concerted effort is required to develop new and more effective therapies for patients with a pancreatic cancer.

A current review of Pub Med. literature, clinical trial database (clinicaltrials.gov), American Society of Clinical Oncology (ASCO) and American Association of Cancer Research (AACR) websites, concluded that the molecular pathogenesis of a pancreatic cancer involves multiple pathways and defined mutations, suggesting this complexity as a major reason for failure of targeted therapy in this disease. Faced with a complex mechanism of activating oncogenic pathways that regulate cellular proliferation, survival and metastasis, therapies that target a single activating molecule cannot thus, overpower the multitude of aberrant cellular processes, and may be of limited therapeutic benefit in advanced disease. Based on the findings by the method described here in MiaPaCa2 cells, combination of an inhibitor of components of these identified pathways, such as those targeting but not limited to AKT, mTOR, PI3K, JAK, STAT3, IKK, Bcl2, PKA complex, phosphodiesterases, ERK, Raf, JNK are proposed to be efficacious when used in combination with an Hsp90 inhibitor.

Example of AKT inhibitors are PF-04691502, Triciribine phosphate (NSC-280594), A- 674563, CCT128930, AT7867, PHT-427, GSK690693, MK-2206 dihydrochloride

Example of PI3K inhibitors are 2-(lH-indazol-4-yl)-6-(4-methanesulfonylpiperazin-l- ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine, BKM120, NVP-BEZ235, PX-866, SF 1126. XL147.

Example of mTOR inhibitors are deforolimus, everolimus, NVP-BEZ235, OSI-027, tacrolimus, temsirolimus, Ku-0063794, WYE-354, PP242, OSI-027, GSK2126458, WAY- 600, WYE-125132

Examples of Bcl2 inhibitors are ABT-737, Obatoclax (GX15-070), ABT-263, TW-37

Examples of JAK inhibitors are Tofacitinib citrate (CP-690550), AT9283, AG-490, INCBO 18424 (Ruxolitinib), AZD1480, LY2784544, NVP-BSK805, TGI 01209, TG-101348

Examples of IkK inhibitors are SC-514, PF 184

Examples of inhibitors of phosphodiesterases are aminophylline, anagrelide, arofylline, caffeine, cilomilast, dipyridamole, dyphylline, L 869298, L-826,141, milrinone, nitroglycerin, pentoxifylline, roflumilast, rolipram, tetomilast, theophylline, tolbutamide, amrinone, anagrelide, arofylline, caffeine, cilomilast, L 869298, L-826,141, milrinone, pentoxifylline, roflumilast, rolipram, tetomilast

Indeed, inhibitors of mTOR, which is identified by our method to potentially contribute to the transformation of MiaPaCa2 cells (Figure 7e), are active as single agents (Figure 7f) and synergize with Hsp90 inhibition in affecting the growth of these pancreatic cancer cells (Figure 17). Quantitative analysis of synergy between mTOR and Hsp90 inhibitors: To determine the drug interaction between pp242 (mTOR inhibitor) and PU-H71 (Hsp90 inhibitor), the combination index (CI) isobologram method of Chou-Talalay was used as previously described. This method, based on the median-effect principle of the law of mass action, quantifies synergism or antagonism for two or more drug combinations, regardless of the mechanisms of each drug, by computerized simulation. Based on algorithms, the computer software displays median-effect plots, combination index plots and normalized isobolograms (where non constant ratio combinations of 2 drugs are used). PU-H71 (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0125 μΜ) and pp242 (0.5, 0.125, 0.03125, 0.0008, 0.002, 0.001 μΜ) were used as single agents in the concentrations mentioned or combined in a non constant ratio (PU-H71 : pp242; 1 : 1, 1 :2, 1 :4, 1 :7.8, 1 : 15.6, 1 : 12.5). The Fa (fraction killed cells) was calculated using the formulae Fa=l-Fu; Fu is the fraction of unaffected cells and was used for a dose effect analysis using the computer software (CompuSyn, Paramus,New Jersey, USA).

In a similar fashion, inhibitors of the PI3K-AKT-mTOR pathway which is identified by our method to contribute to the transformation of MDA-MB-468 cells, are more efficacious in the MDA-MB-468 breast cancer cells when combined with the Hsp90 inhibitor.

Cell cycle: G2/M DNA Damage checkpoint regulation

G2/M checkpoint is the second checkpoint within the cell cycle. This checkpoint prevents cells with damaged DNA from entering the M phase, while also pausing so that DNA repair can occur. This regulation is important to maintain genomic stability and prevent cells from undergoing malignant transformation. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and rad3 related (ATR) are key kinases that respond to DNA damage. ATR responds to UV damage, while ATM responds to DNA double-strand breaks (DSB). ATM and ATR activate kinases Chkl and Chk2 which in turn inhibit Cdc25, the phosphatase that normally activates Cdc2. Cdc2, a cyclin-dependent kinase, is a key molecule that is required for entry into M phase. It requires binding to cyclin Bl for its activity. The tumor suppressor gene p53 is an important molecule in G2/M checkpoint regulation. ATM, ATR and Chk2 contribute to the activation of p53. Further, pl9Arf functions mechanistically to prevent MDM2's neutralization of p53. Mdm4 is a transcriptional inhibitor of p53. DNA damage-induced phosphorylation of Mdm4 activates p53 by targeting Mdm4 for degradation. Well known p53 target genes like Gadd45 and p21 are involved in inhibiting Cdc2. Another p53 target gene, 14-3-3σ, binds to the Cdc2-cyclin B complex rendering it inactive. Repression of the cyclin Bl gene by p53 also contributes to blocking entry into mitosis. In this way, numerous checks are enforced before a cell is allowed to enter the M phase.

This pathway is composed of, but not limited to 14-3-3, 14-3-3 (β,ε,ζ), 14-3-3-Cdc25, ATM, ATM/ATR, BRCA1, Cdc2-CyclinB, Cdc2-CyclinB-Sfn, Cdc25B/C, CDK1, CDK7, CDK 1A, CDK 2A, Cdkn2a-Mdm2, CHEK1, CHEK2, CKS1B, CKS2, Cyclin B, EP300, Ep300/Pcaf, GADD45A, KAT2B, MDM2, Mdm2-Tp53-Mdm4, MDM4, PKMYT1, PLK1, PRKDC, RPRM, RPS6KA1 (includes EG:20111), Scf, SFN, Top2, TP53 (includes EG:22059), WEE1

Based on the findings by the method described here, combination of an inhibitor of components of this pathway, such as those targeting CDK1, CDK7, CHEK1, PLK1 and TOP2A(B) are proposed to be efficacious when used in combination with an Hsp90 inhibitor.

Examples of inhibitors are AQ4N, becatecarin, BN 80927, CPI-0004Na, daunorubicin, dexrazoxane, doxorubicin, elsamitrucin, epirubicin, etoposide, gatifloxacin, gemifloxacin, mitoxantrone, nalidixic acid, nemorubicin, norfloxacin, novobiocin, pixantrone, tafluposide, TAS-103, tirapazamine, valrubicin, XK469, BI2536

PU-beads also identify proteins of the DNA damage, replication and repair, homologous recombination and cellular response to ionizing radiation as Hsp90-regulated pathways in select CML, pancreatic cancer and breast cancer cells. PU-H71 synergized with agents that act on these pathways.

Specifically, among the Hsp90-regulated pathways identified in the K562 CML cells, MDA-

MB-468 breast cancer cells and the Mia-PaCa-2 pancreatic cancer cells are several involved in DNA damage, replication and repair response and/or homologous recombination (Tables

3, 5a-5f). Hsp90 inhibition may synergize or be additive with agents that act on DNA damage and/or homologous recombination (i.e. potentiate DNA damage sustained post treatment with

IR/chemotherapy or other agents, such as PARP inhibitors that act on the proteins that are important for the repair of double-strand DNA breaks by the error-free homologous recombinational repair pathway). Indeed, we found that PU-H71 radiosensitized the Mia- PaCa-2 human pancreatic cancer cells. We also found PU-H71 to synergize with the PARP inhibitor olaparib in the MDA-MB-468 and HCC1937 breast cancer cells (Figure 25).

Identification of Hsp90 clients required for tumor cell survival may also serve as tumor- specific biomarkers for selection of patients likely to benefit from Hsp90 therapy and for pharmacodynamic monitoring of Hsp90 inhibitor efficacy during clinical trials (i.e. clients in Figure 6, 20 whose expression or phosphorylation changes upon Hsp90 inhibition). Tumor specific Hsp90 client profiling could ultimately yield an approach for personalized therapeutic targeting of tumors (Figure 9). This work substantiates and significantly extends the work of Kamal et al, providing a more sophisticated understanding of the original model in which Hsp90 in tumors is described as present entirely in multi-chaperone complexes, whereas Hsp90 from normal tissues exists in a latent, uncomplexed state (Kamal et al., 2003). We propose that Hsp90 forms biochemically distinct complexes in cancer cells (Figure 11a). In this view, a major fraction of cancer cell Hsp90 retains "house keeping" chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl) (Zuehlke & Johnson, 2010; Workman et al, 2007). We now present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes. Herein we describe such a role for Hsp90 in maintaining constitutive STAT5 signaling in CML (Figure 8h). These data are consistent with previous work in which we showed that Hsp90 was required to maintain functional transcriptional repression complexes by the BCL6 oncogenic transcriptional repressor in B cell lymphoma cells (Cerchietti et al, 2009). In sum, our work uses chemical tools to provide new insights into the heterogeneity of tumor associated Hsp90 and harnesses the biochemical features of a particular Hsp90 inhibitor to identify tumor- specific biological pathways and proteins (Figure 9). We believe the functional proteomics method described here will allow identification of the critical proteome subset that becomes dysregulated in distinct tumors. This will allow for the identification of new cancer mechanisms, as exemplified by the STAT mechanism described herein, the identification of new onco-proteins, as exemplified by CARM1 described herein, and the identification of therapeutic targets for the development of rationally combined targeted therapies complementary to Hsp90.

Materials and Methods

Cell Lines and Primary Cells

The CML cell lines K562, Kasumi-4, MEG-01 and KU182, triple -negative breast cancer cell line MDA-MB-468, HER2+ breast cancer cell line SKBr3, melanoma cell line SK-Mel-28, prostate cancer cell lines LNCaP and DU145, pancreatic cancer cell line Mia-PaCa-2, colon fibroblast, CCCDI8C0 cell lines were obtained from the American Type Culture Collection. The CML cell line KCL-22 was obtained from the Japanese Collection of Research Bioresources. The NIH-3T3 fibroblast cells were transfected as previously described (An et al, 2000). Cells were cultured in DMEM/F12 (MDA-MB-468, SKBr3 and Mia-PaCa-2), RPMI (K562, SK-Mel-28, LNCaP, DU145 and NIH-3T3) or MEM (CCDI8C0) supplemented with 10% FBS, 1% L-glutamine, 1% penicillin and streptomycin. Kasumi-4 cells were maintained in IMDM supplemented with 20% FBS, 10 ng/ml Granulocyte macrophage colony-stimulating factor (GM-CSF) and l xPen/Strep. PBL (human peripheral blood leukocytes) and cord blood were obtained from patient blood purchased from the New York Blood Center. Thirty five ml of the cell suspension was layered over 15 ml of Ficoll- Paque plus (GE Healthcare). Samples were centrifuged at 2,000 rpm for 40 min at 4 °C, and the leukocyte interface was collected. Cells were plated in RPMI medium with 10% FBS and used as indicated. Primary human blast crisis CML and AML cells were obtained with informed consent. The manipulation and analysis of specimens was approved by the University of Rochester, Weill Cornell Medical College and University of Pennsylvania Institutional Review Boards. Mononuclear cells were isolated using Ficoll-Plaque (Pharmacia Biotech, Piscataway, NY) density gradient separation. Cells were cryopreserved in freezing medium consisting of Iscove's modified Dulbecco medium (IMDM), 40%> fetal bovine serum (FBS), and 10% dimethylsulfoxide (DMSO) or in CryoStor™ CS-10 (Biolife). When cultured, cells were kept in a humidified atmosphere of 5% C0 ₂ at 37°C.

Cell lysis for chemical and immuno-precipitation

121 Cells were lysed by collecting them in Felts Buffer (HEPES 20mM, KC1 50mM, MgCl ₂ 5mM, NP40 0.01%, freshly prepared Na ₂ Mo0 ₄ 20mM, pH 7.2-7.3) with added l^g/pL of protease inhibitors (leupeptin and aprotinin), followed by three successive freeze (in dry ice) and thaw steps. Total protein concentration was determined using the BCA kit (Pierce) according to the manufacturer's instructions.

Immunoprecipitation

The Hsp90 antibody (H9010) or normal IgG (Santa Cruz Biotechnology) was added at a volume of 10 to the indicated amount of cell lysate together with 40 of protein G agarose beads (Upstate), and the mixture incubated at 4°C overnight. The beads were washed five times with Felts lysis buffer and separated by SDS-PAGE, followed by a standard western blotting procedure.

Chemical precipitation

Hsp90 inhibitors beads or Control beads, containing an Hsp90 inactive chemical (ethanolamine) conjugated to agarose beads, were washed three times in lysis buffer. Unless otherwise indicated, the bead conjugates (80μί) were then incubated at 4°C with the indicated amounts of cell lysates (120-500 μg), and the volume was adjusted to 200 with lysis buffer. Following incubation, bead conjugates were washed 5 times with the lysis buffer and proteins in the pull-down analyzed by Western blot. For depletion studies, 2-4 successive chemical precipitations were performed, followed by immunoprecipitation steps, where indicated.

Additional methods are also described herein at pages 173-183.

Supplementary Materials

Table 5 Legend

Table 5. (a-d) List of proteins isolated in the PU-beads pull-downs and identified as indicated in Supplementary Materials and Methods, (e) Dataset of mapped proteins used for analysis in the Ingenuity Pathway, (f) Protein regulatory networks generated by bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IPA) software. Proteins listed in Table 5e were analyzed by IPA. Table 5a. Putative Hsp90 interacting proteins identified using the QSTAR-Elite hybrid quadrupole time-of-flight mass spectrometer (QTof MS) (AB/MDS Sciex)

#GChiosis_K562andMiPaca2_AII, Samples Report created on 08/05/2010

GChiosis_K562andMiPaca2_AII

Displaying:Number of Assigned Spectra

Isoform 1 of RAF proto- oncogene serine/threonine-

RAF1 P04049 protein kinase IPI00021786 73 kDa 5 1 1

A-Raf proto-oncogene

serine/threonine-protein IPI00020578

ARAF P10398 kinase (+1 ) 68 kDa 2 0 1

VAV1 P15498 Proto-oncogene vav IPI0001 1696 98 kDa 3 1 0

Tyrosine-protein kinase

BTK Q06187 BTK IPI00029132 76 kDa 1 1 8 0

PTK2; Isoform 1 of Focal adhesion IPI00012885 1 19

FAK1 Q05397 kinase 1 (+1 ) kDa 4 5 4

Tyrosine-protein

phosphatase non-receptor 179

PTPN23 Q9H3S7 type 23 IPI00034006 kDa 8 8 2

Isoform Del-701 of Signal

transducer and activator of IPI00306436

STAT3 P40763 transcription 3 (+2) 88 kDa 15 4 6 interleukin-1 receptor- associated kinase 1 isoform IPI00060149

IRAKI P51617 3 (+3) 68 kDa 7 2 1

MAPK1 ; Mitogen-activated protein

ERK2 P28482 kinase 1 , ERK2 IPI00003479 41 kDa 23 5 14

Isoform A of Mitogen-

MAP3K4; activated protein kinase IPI00186536 182

MEKK4 Q9Y6R4 kinase kinase 4 (+2) kDa 3 7 0

Mitogen-activated protein

kinase kinase kinase 7- IPI00019459

TAB1 Q 15750 interacting protein 1 (+1 ) 55 kDa 1 3 2

MAPK14; Isoform CSBP2 of Mitogen- IPI00002857

p38 Q 16539 activated protein kinase 14 (+1 ) 41 kDa 1 0 0

Isoform 3 of Dual specificity

MAP2K3; mitogen-activated protein

MEK3 P46734 kinase kinase 3 IPI00220438 39 kDa 0 0 2

CAPN1 P07384 Calpain-1 catalytic subunit IPI0001 1285 82 kDa 10 1 1 0

Isoform 1 of Insulin-like

growth factor 2 mRNA-

IGF2BP2 000425 binding protein 3 IPI00658000 64 kDa 18 14 20

Insulin-like growth factor 2

IGF2BP1 088477 mRNA-binding protein 1 IPI00008557 63 kDa 1 1 19 0

CAPNS1 P04632 Calpain small subunit 1 IPI00025084 28 kDa 0 0 3

RUVBL1 Q9Y265 Isoform 1 of RuvB-like 1 IPI00021 187 50 kDa 10 17 30

RUVBL2 Q9Y230 RuvB-like 2 IPI00009104 51 kDa 20 30 26

MYCBP Q99417 MYCBP protein IPI00871 174 14 kDa 2 0 3

AKAP8 043823 A-kinase anchor protein 8 IPI00014474 76 kDa 4 0 0

A-kinase anchor protein 8-

AKAP8L Q9ULX6 like IPI00297455 72 kDa 3 3 2

Isoform 2 of IPI00220740

NPM1 PQ8748 Nucleophosmin (+1 ) 29 kDa 8 4 49

Isoform 1 of Histone- arginine methyltransferase IPI00412880

CARM1 Q86X55 CARM1 (+1 ) 63 kDa 12 16 9

CALM P62158 Calmodulin IPI00075248 17 kDa 0 0 34

Calcium/calmodulin- dependent protein kinase

CAMK1 Q14012 type 1 IPI00028296 41 kDa 0 0 3

Isoform 4 of

Calcium/calmodulin- IPI00172450

CAMK2G Q 13555 dependent protein kinase (+1 1 ) 60 kDa 2 3 0 type II gamma chain

Non-receptor tyrosine- 134

TYK2 P29597 protein kinase TYK2 IPI00022353 kDa 2 0 0

Serine/threonine-protein

TBK1 Q9UHD2 kinase TBK1 IPI00293613 84 kDa 10 0 0

Isoform 1 of

Phosphatidylinositol 4- 231

PI4KA P42356 kinase alpha IPI00070943 kDa 15 4 0

Isoform 3 of

Serine/threonine-protein IPI00183368 341

SMG1 Q96Q15 kinase SMG1 (+5) kDa 1 9 0

Isoform 4 of Phosphorylase

b kinase regulatory subunit IPI00181893 124

PHKB Q93100 beta (+1 ) kDa 10 3 9 cDNA FLJ56439, highly

similar to Pantothenate

PANK4 Q9NVE7 kinase 4 IPI00018946 87 kDa 7 7 0

Isoform 2 of cAMP- dependent protein kinase IPI00217960

PRKACA P17612 catalytic subunit alpha, PKA (+1 ) 40 kDa 0 0 4 protein kinase, AMP- activated, alpha 1 catalytic IPI00410287

PRKAA1 Q13131 subunit isoform 2 (+3) 66 kDa 1 1 6 1 cDNA FLJ40287 fis, clone

TESTI2027909, highly

similar to 5 -AMP- ACTIVATED PROTEIN KINASE, GAM MA- 1 IPI00473047

PRKAG1 Q8N7V9 SUBUNIT (+1 ) 39 kDa 10 0 1

Isoform 4 of N-terminal IPI00062264

SCYL1 Q96KG9 kinase-like protein (+5) 86 kDa 8 2 0

351

ATM Q13315 Serine-protein kinase ATM IPI00298306 kDa 2 4 1

Isoform 1 of

Serine/threonine-protein IPI00412298 301

ATR Q13535 kinase ATR (+1 ) kDa 5 0 3 cDNA FLJ51909, highly

similar to Serine-threonine

kinase receptor-associated

STRAP Q9Y3F4 protein IPI00294536 40 kDa 13 0 4

Serine/threonine-protein

RIOK2 Q9BVS4 kinase RI02 IPI00306406 63 kDa 7 6 1 cDNA FLJ60070, highly

similar to Serine/threonine- IPI00009334

PRKD2 Q9BZL6 protein kinase D2 (+1 ) 98 kDa 4 0 0

Isoform 2 of Casein kinase I

CSNK1A1 P48729 isoform alpha IPI00448798 42 kDa 5 0 1

Casein kinase II subunit IPI00010865

CSNK2B P67870 beta (+1 ) 25 kDa 1 0 1

Isoform 2 of Kinase IPI00013384

KSR1 Q8IVT5 suppressor of Ras 1 (+1 ) 97 kDa 3 0 0

Isoform 1 of BMP-2- 129

BMP2K Q9NSY1 inducible protein kinase IPI00337426 kDa 4 3 0

Isoform 2 of

Serine/threonine-protein IPI00290439

SRPK1 Q96SB4 kinase SRPK1 (+1 ) 74 kDa 1 1 2 7

Serine/threonine-protein IPI00333420

SRPK2 P78362 kinase SRPK2 (+3) 78 kDa 1 1 0 Serine/threonine-protein IPI00021248

PLK1 P53350 kinase PLK1 (+1 ) 68 kDa 3 0 0

Cell division protein kinase

CDK7 P50613 7 IPI00000685 39 kDa 2 0 1

Isoform 1 of Cell division

cycle 2-related protein IPI00021 175 164

CDK12 Q9NYV4 kinase 7 (+1 ) kDa 0 0 3

Cell division cycle and

apoptosis regulator protein 133

CCAR1 Q8IX12 1 IPI00217357 kDa 3 0 0

Cell division cycle protein IPI00294575

CDC27 P30260 27 homolog (+1 ) 92 kDa 7 2 1

CDC23 Q9UJX2 cell division cycle protein 23 IPI00005822 69 kDa 1 4 4

Isoform 1 of Cell division IPI00301923

CDK9 P5G750 protein kinase 9 (+1 ) 43 kDa 3 0 1

Isoform 1 of Mitotic

checkpoint

serine/threonine-protein 120

BUB1 B 060566 kinase BUB1 beta IPI00141933 kDa 3 1 0

Mitotic checkpoint

serine/threonine-protein 122

BUB1 043683 kinase BUB1 IPI00783305 kDa 1 0 0

Anaphase-promoting 217

ANAPC1 Q9H1A4 complex subunit 1 IPI00033907 kDa 12 6 7 anaphase-promoting

complex subunit 7 isoform IPI00008248

ANAPC7 Q9UJX3 a (+1 ) 67 kDa 3 8 0

Isoform 1 of Anaphase- promoting complex subunit

ANAPC5 Q9UJX4 5 IPI00008247 85 kDa 9 3 0

Isoform 1 of Anaphase- promoting complex subunit

ANAPC4 Q9UJX5 4 IPI00002551 92 kDa 3 0 0

Serine/threonine-protein 107

NEK9 Q8TD19 kinase Nek9 IPI00301609 kDa 3 3 5

IPI00025695

CDC45 075419 CDC45-related protein (+2) 66 kDa 7 7 0

CRKL P46109 Crk-like protein IPI00004839 34 kDa 5 0 0

Isoform 1 of Dedicator of 212

DOCK2 Q92608 cytokinesis protein 2 IPI00022449 kDa 2 3 1

Isoform 2 of Dedicator of IPI00183572 241

DOCK7 Q96N67 cytokinesis protein 7 (+5) kDa 2 0 0

Putative uncharacterized IPI0041 1452 238

DOCK1 1 Q5JSL3 protein DOCK1 1 (+1 ) kDa 0 0 1

Isoform 1 of Epidermal

growth factor receptor

EPS15 P42566 substrate 15 IPI00292134 99 kDa 23 26 3

Isoform 1 of Growth factor IPI00021327

GRB2 P62993 receptor-bound protein 2 (+1 ) 25 kDa 5 1 2

Isoform 1 of Transcription IPI00221035

BTF3 P20290 factor BTF3 (+1 ) 22 kDa 0 0 3

LGALS3 P17931 Galectin-3 IPI00465431 26 kDa 0 0 9

Non-POU domain- containing octamer-binding

NONO Q 15233 protein IPI00304596 54 kDa 0 0 4

Inosine triphosphate

ITPA Q9BY32 pyrophosphatase IPI00018783 21 kDa 0 0 5

RBX1 P62877 RING-box protein 1 IPI00003386 12 kDa 0 0 5 Receptor-interacting

serine/threonine-protein

RIPK1 Q 13546 kinase 1 IPI00013773 76 kDa 2 0 0

Histidine triad nucleotide-

HINT1 P49773 binding protein 1 IPI00239077 14 kDa 0 0 9

GSE1 Isoform 1 of Genetic IPI00215963 136

KIAA0182 Q 14687 suppressor element 1 (+1 ) kDa 1 1 2 0

28 kDa heat- and acid-

PDAP1 Q 13442 stable phosphoprotein IPI00013297 21 kDa 0 0 5

Isoform 1 of IPI00179473

SQSTM1 Q13501 Sequestosome-1 (+1 ) 48 kDa 3 5 1

F-box-like/WD repeat- containing protein

TBL1XR1 Q9BZK7 TBL1XR1 IPI00002922 56 kDa 3 12 3

Protein arginine N-

PRMT5 014744 methyltransferase 5 IPI00441473 73 kDa 12 1 1 3

Protein arginine N- IPI00102128

PRMT6 Q96LA8 methyltransferase 6 (+1 ) 42 kDa 2 0 0

IPI00103026

PRMT3 Q8WUV3 PRMT3 protein (Fragment) (+2) 62 kDa 6 1 1

Isoform 1 of Autophagy- IPI00304926 213

ATG2A Q2TAZ0 related protein 2 homolog A (+1 ) kDa 2 3 0

Isoform 2 of Activating

molecule in BECN1- regulated autophagy IPI00106552 136

AMBRA1 Q9C0C7 protein 1 (+3) kDa 2 2 1

Isoform Long of Autophagy

ATG5 Q9H1Y0 protein 5 IPI00006800 32 kDa 2 1 0

YWHAE P62258 14-3-3 protein epsilon IPI00000816 29 kDa 13 1 13

Isoform 1 of Myb-binding IPI00005024 149

MYBBP1A Q9BQG0 protein 1A (+1 ) kDa 4 4 29

Cell differentiation protein

RQCD1 Q92600 RCD1 homolog IPI00023101 34 kDa 5 1 8

YWHAQ P27348 14-3-3 protein theta IPI00018146 28 kDa 0 0 4

DNA damage-binding 127

DDB1 Q16531 protein 1 IPI00293464 kDa 25 15 2

Nuclease-sensitive

YBX1 P67809 element-binding protein 1 IPI00031812 36 kDa 6 13 40

RCOR1 Q9UKL0 REST corepressor 1 IPI00008531 53 kDa 9 5 0

HDAC1 Q 13547 Histone deacetylase 1 IPI00013774 55 kDa 10 1 1 1

Isoform 2 of Lysine-specific IPI00217540

KDM1A 060341 histone demethylase 1 (+1 ) 95 kDa 13 4 0 cDNA FLJ56474, highly

similar to Histone 133

HDAC6 Q9UBN7 deacetylase 6 IPI0000571 1 kDa 4 6 2

Histone-binding protein IPI00395865

RBBP7 Q 16576 RBBP7 (+2) 48 kDa 5 4 3

HIST1 H1 C P16403 Histone H1.2 IPI00217465 21 kDa 1 0 7

HDAC2 Q92769 histone deacetylase 2 IPI00289601 66 kDa 2 3 1

HIST1 H1 B P16401 Histone H1.5 IPI00217468 23 kDa 0 0 5

H1 FX Q92522 Histone H1x IPI00021924 22 kDa 0 0 3

SWI/SNF complex subunit 123

SMARCC1 Q92922 SMARCC1 IPI00234252 kDa 15 17 0

Isoform 2 of SWI/SNF IPI00150057 125

SMARCC2 Q8TAQ2 complex subunit SMARCC2 (+1 ) kDa 6 7 0

Tumor necrosis factor,

TNFAIP2 Q03169 alpha-induced protein 2 IPI00304866 73 kDa 2 1 0 Isoform 2 of

Phosphatidylinositol-binding IPI00216184

PICALM Q 13492 clathrin assembly protein (+5) 69 kDa 1 7 0

Isoform 1 of Protein 103

KIAA1967 Q8IM163 KIAA1967 IPI00182757 kDa 17 23 3

DNA replication licensing IPI00018350

MCM5 P33992 factor MCM5 (+2) 82 kDa 24 18 2

Transferrin receptor protein

TFRC P02786 1 IPI00022462 85 kDa 25 7 0

Isoform 1 of Transcription

TRIM28 Q13263 intermediary factor 1-beta IPI00438229 89 kDa 16 14 4

270

TLN 1 Q9Y490 Talin-1 IPI00298994 kDa 12 12 0

Kinetochore protein NDC80

NDC80 014777 homolog IPI00005791 74 kDa 13 4 0

Isoform 1 of Ras GTPase- activating-like protein 181

IQGAP2 Q13576 IQGAP2 IPI00299048 kDa 18 21 1

Macrophage migration

MIF P14174 inhibitory factor IPI00293276 12 kDa 3 0 25

Proliferation-associated

PA2G4 Q9UQ80 protein 2G4 IPI00299000 44 kDa 3 8 14

Isoform 1 of Cytoplasmic IPI00644231 145

CYFIP1 Q7L576 FMR1 -interacting protein 1 (+1 ) kDa 8 4 4

Proliferating cell nuclear

PCNA P12004 antigen IPI00021700 29 kDa 9 3 10 tRNA (cytosine-5-)-

NSUN2 Q08J23 methyltransferase NSUN2 IPI00306369 86 kDa 1 1 8 5

Isoform 1 of Nuclear IPI00289344 270

NCOR1 075376 receptor corepressor 1 (+1 ) kDa 1 1 13 1

Isoform 1 of Nuclear 275

NCOR2 Q9Y618 receptor corepressor 2 IPI00001735 kDa 8 5 2

Isoform 1 of Interleukin

ILF3 Q 12906 enhancer-binding factor 3 IPI00298788 95 kDa 25 16 20

Interleukin enhancer-

ILF2 Q12905 binding factor 2 IPI00005198 43 kDa 8 1 1 18

Isoform 1 of KH domain- containing, RNA-binding,

signal transduction-

KHDRBS1 Q07666 associated protein 1 IPI00008575 48 kDa 8 15 2

576

RNF213 Q9HCF4 Isoform 1 of Protein AL017 IPI00642126 kDa 12 49 16

Metastasis-associated

MTA2 094776 protein MTA2 IPI00171798 75 kDa 14 12 3

TRMT1 12 Q9UI30 TRM1 12-like protein IPI00009010 14 kDa 0 0 3

Enhancer of rudimentary

ERH P84090 homolog IPI00029631 12 kDa 0 0 3

Isoform 1 of F-box only

FBX022 G8NEZ5 protein 22 IPI00183208 45 kDa 0 0 3

Isoform 1 of Tumor protein IPI00301360

TP63 Q9H3D4 63 (+5) 77 kDa 0 0 3

Serine/threonine-protein

PPP5C P53041 phosphatase 5 IPI00019812 57 kDa 3 1 0

Isoform 1 of Protein IPI00852685 141

DIAPH1 060610 diaphanous homolog 1 (+1 ) kDa 6 7 0

Replication protein A 70

RPA1 P27694 kDa DNA-binding subunit IPI00020127 68 kDa 22 8 0 Isoform 3 of Plasminogen

activator inhibitor 1 RNA-

SERBP1 Q8NC51 binding protein IPI00470498 43 kDa 0 6 16

Serine/threonine-protein

phosphatase 2A 56 kDa

regulatory subunit epsilon IPI00002853

PPP2R5E Q16537 isoform (+1 ) 55 kDa 0 0 2

Isoform 1 of

Serine/threonine-protein

phosphatase 2A 65 kDa

regulatory subunit A beta IPI00294178

PPP2R1 B P30154 isoform (+3) 66 kDa 3 2 0

Serine/threonine-protein

phosphatase 2A 55 kDa

regulatory subunit B alpha

PPP2R2A P63151 isoform IPI0033251 1 52 kDa 9 1 5

Isoform 1 of

Serine/threonine-protein

phosphatase 6 regulatory IPI00402008 103

PPP6R1 Q9UPN7 subunit 1 (+1 ) kDa 5 2 5

Transforming growth factor- beta receptor-associated

TGFBRAP1 Q8WUH2 protein 1 IPI00550891 97 kDa 1 0 0

Isoform 1 of Obg-like

OLA1 Q9NTK5 ATPase 1 IPI00290416 45 kDa 8 4 3

IPI00295741

CTSB P07858 Cathepsin B (+2) 38 kDa 0 0 2

IPI00002745

CTSZ Q9UBR2 Cathepsin Z (+1 ) 34 kDa 1 0 0

ARFGAP with coiled-coil,

ANK repeat and PH

ACAP2 Q 15057 domain-containing protein 2 IPI00014264 88 kDa 3 2 1

Isoform 1 of ARF GTPase- IPI00384861

GIT1 Q9Y2X7 activating protein GIT1 (+2) 84 kDa 2 0 0

Isoform 2 of Rho guanine

nucleotide exchange factor IPI00339379

ARHGEF1 Q92888 1 (+2) 99 kDa 4 3 0

Isoform 1 of Rho guanine

nucleotide exchange factor 1 12

ARHGEF2 Q92974 2 IPI00291316 kDa 14 7 2

Ran GTPase-activating

RAN GAP 1 P46060 protein 1 IPI00294879 64 kDa 13 4 1

Isoform 6 of GTPase- activating protein and VPS9 IPI00292753 166

GAPVD1 Q14C86 domain-containing protein 1 (+4) kDa 4 6 6

Isoform 1 of Rab3 GTPase- activating protein catalytic 1 1 1

RAB3GAP1 Q 15042 subunit IPI00014235 kDa 9 6 3

GTP-binding nuclear IPI00643041

RAN P62826 protein Ran (+1 ) 24 kDa 7 2 6

SAR1A Q9NR31 GTP-binding protein SAR1a IPI00015954 22 kDa 3 1 1

Ras-related protein Rab- IPI00020436

RAB1 1 B Q15907 1 1 B (+1 ) 24 kDa 6 1 0

TBC1 domain family,

TBC1 D15 Q8TC07 member 15 isoform 3 IPI00794613 80 kDa 6 4 4

Telomere length regulation

TEL02 Q9Y4R8 protein TEL2 homolog IPI00016868 92 kDa 1 1 1 1

Isoform 1 of Telomere- IPI00293845 274

RIF1 Q5UIP0 associated protein RIF1 (+1 ) kDa 2 0 2 Telomerase Cajal body

WRAP53 Q9BUR4 protein 1 IPI00306087 59 kDa 3 0 0

Isoform 1 of 182 kDa IPI00304589 182

TNKS1 BP1 Q9C0C2 tankyrase-1 -binding protein (+1 ) kDa 23 79 12 programmed cell death 4 IPI00240675

PDCD4 Q53EL6 isoform 2 (+1 ) 51 kDa 2 5 3

Isoform 2 of Fermitin family IPI00216699

FERMT3 Q86UX7 homolog 3 (+1 ) 75 kDa 8 0 0

Isoform 1 of Protein

tyrosine kinase 2 beta; IPI00029702 1 16

PTK2B Q 14289 PYK2; FAK2 (+1 ) kDa 2 0 0

IPI00023461 207

MLLT4 P55196 Isoform 4 of Afadin (+1 ) kDa 1 2 0

Isoform 1 of Tripartite motif- IPI00514832

TRIM56 Q9BRZ2 containing protein 56 (+1 ) 81 kDa 0 0 3

Hypoxia up-regulated IPI00000877 1 1 1

HYOU1 Q9Y4L1 protein 1 (+1 ) kDa 0 3 0

Zymogen granule protein

ZG16B Q96DA0 16 homolog B IPI00060800 23 kDa 0 3 0

Isoform 3 of Type I inositol- 3,4-bisphosphate 4- IPI00044388 109

INPP4A Q96PE3 phosphatase (+3) kDa 3 0 0

Putative uncharacterized IPI00872508

INF2 Q27J81 protein INF2 (+3) 55 kDa 0 0 3

IPI00384745

GNL1 P36915 HSR1 protein (+1 ) 62 kDa 2 1 0

SAM domain and HD

SAMHD1 Q9Y3Z3 domain-containing protein 1 IPI00294739 72 kDa 1 1 2 6

Isoform Long of Tight IPI00216219 195

TJP1 Q07157 junction protein ZO-1 (+2) kDa 6 3 0

Isoform 1 of Large proline- IPI00465128 1 19

BAT3 P46379 rich protein BAT3 (+4) kDa 4 5 3 spectrin, alpha, erythrocytic 280

SPTA1 D3DVD8 1 IPI00220741 kDa 43 62 0

IPI00302592 280

FLNA P21333 Isoform 2 of Filamin-A (+2) kDa 26 91 0

IPI00178352 291

FLNC Q14315 Isoform 1 of Filamin-C (+1 ) kDa 55 183 0

Isoform 2 of LisH domain

and HEAT repeat- containing protein 139

KIAA1468 Q9P260 KIAA1468 IPI00023330 kDa 0 0 3

Isoform 1 of HEAT repeat-

HEATR2 Q86Y56 containing protein 2 IPI00242630 94 kDa 5 2 1 1

HEAT repeat-containing 129

HEATR6 Q6A108 protein 6 IPI00464999 kDa 2 1 0

Basement membrane- specific heparan sulfate 469

HSPG2 P98160 proteoglycan core protein IPI00024284 kDa 4 9 0

IPI00029601

CTTN Q14247 Src substrate cortactin (+1 ) 62 kDa 6 6 2

AH receptor-interacting

AIP 000170 protein IPI00010460 38 kDa 10 0 0

1 16

NAT 10 Q9H0A0 N-acetyltransferase 10 IPI00300127 kDa 8 3 1

219

DICER1 Q9UPY3 dicerl IPI00219036 kDa 8 3 1

Isoform A of Constitutive IPI00472054 122

FAM120A Q9NZB2 coactivator of PPAR- (+1 ) kDa 1 1 12 gamma-like protein 1

Isoform 2 of Nuclear mitotic IPI00006196 237

NUMA1 Q 14980 apparatus protein 1 (+2) kDa 4 4 4

Isoform 1 of Thyroid

receptor-interacting protein

TRIP13 Q 15645 13 IPI00003505 49 kDa 3 3 8

Isoform 1 of Protein IPI00006050 102

FAM1 15A Q9Y4C2 FAM1 15A (+3) kDa 9 1 0

ATP-dependent RNA

helicase SUPV3L1 ,

SUPV3L1 Q81YB8 mitochondrial IPI00412404 88 kDa 8 3 0

LTV1 Q96GA3 Protein LTV1 homolog IPI00153032 55 kDa 5 6 0

Cell growth-regulating

LYAR Q9NX58 nucleolar protein IPI00015838 44 kDa 1 2 6

ASAH 1 Q13510 Acid ceramidase IPI00013698 45 kDa 8 1 0

Isoform 3 of Pre-mRNA 3'- IPI00008449

FIP1 L1 Q6UN15 end-processing factor FIP1 (+3) 58 kDa 6 3 0

Isoform 1 of Tumor

suppressor p53-binding IPI00029778 214

TP53BP1 Q 12888 protein 1 (+3) kDa 0 6 3

Isoform Epsilon of IPI00071059

BAX Q07812 Apoptosis regulator BAX (+3) 18 kDa 3 0 6

Adenine

APRT P07741 phosphoribosyltransferase IPI00218693 20 kDa 0 0 6

FH1/FH2 domain- 127

FHOD1 Q9Y613 containing protein 1 IPI00001730 kDa 5 2 0

CPNE3 075131 Copine-3 IPI00024403 60 kDa 4 5 0

Isoform 2 of Transducin-like IPI00177938

TLE1 Q04724 enhancer protein 3 (+4) 82 kDa 5 2 1

Putative uncharacterized IPI00554538

TPP1 014773 protein TPP1 (+2) 60 kDa 4 1 1

Isoform 1 of Serologically

defined colon cancer 123

SDCCAG1 060524 antigen 1 IPI00301618 kDa 2 2 3

Isoform 1 of Nck-associated IPI00031982 129

NCKAP1 Q9Y2A7 protein 1 (+1 ) kDa 5 1 2

Nucleoporin 54kDa variant

NUP54 Q7Z3B4 (Fragment) IPI00172580 56 kDa 1 7 0

NUP85 Q9BW27 Nucleoporin NUP85 IPI00790530 75 kDa 14 2 0

162

NUP160 Q12769 nucleoporin 160kDa IPI00221235 kDa 13 1 0

Isoform 1 of Nucleolar

NOP14 P78316 protein 14 IPI00022613 98 kDa 9 2 0

Isoform 1 of U4/U6 small

Q8WWY nuclear ribonucleoprotein IPI00292000

PRPF31 3 Prp31 (+1 ) 55 kDa 3 2 0

Isoform 1 of U4/U6 small

nuclear ribonucleoprotein IPI00005861

PRPF3 043395 Prp3 (+1 ) 78 kDa 3 0 0

Isoform 1 of CCR4-NOT

transcription complex 267

CNOT1 A5YKK6 subunit 1 IPI00166010 kDa 53 73 23

Leucine-rich repeat-

LRRC40 Q9H9A6 containing protein 40 IPI00152998 68 kDa 4 3 0

PHB2 Q99623 Prohibitin-2 IPI00027252 33 kDa 8 0 0

VAC 14 Q08AM6 Protein VAC 14 homolog IPI00025160 88 kDa 5 2 0 Putative uncharacterized IPI00294891

NOP2 P46087 protein N0P2 (+2) 94 kDa 0 0 7

NOB1 Q9ULX3 RNA-binding protein NOB1 IPI00022373 48 kDa 5 0 0

Isoform 1 of Sterile alpha

and TIR motif-containing

SARM1 Q6SZW1 protein 1 IPI00448630 79 kDa 0 0 5

FtsJ methyltransferase

FTSJD2 Q8N1 G2 domain-containing protein 2 IPI00166153 95 kDa 3 1 0

Isoform 2 of Nuclear factor IPI00292537 105

NFKB1 P19838 NF-kappa-B p105 subunit (+1 ) kDa 1 0 2

4F2 cell-surface antigen IPI00027493

SLC3A2 PQ8195 heavy chain (+5) 58 kDa 3 0 0

Putative uncharacterized IPI00914992

WIGB Q9BRP8 protein WIBG (Fragment) (+2) 23 kDa 0 0 4

Diablo homolog, IPI00008418

DIABLO Q9NR28 mitochondrial precursor (+4) 36 kDa 1 0 2

Isoform 1 of Apoptosis- inducing factor 1 , IPI00000690

AIFM1 095831 mitochondrial (+1 ) 67 kDa 2 0 0

Isoform 1 of Zinc finger

CCCH-type antiviral protein 101

ZC3HAV1 Q7Z2W4 1 IPI00410067 kDa 7 0 0

Isoform 1 of Paraspeckle IPI00103525

PSPC1 Q8WXF1 component 1 (+1 ) 59 kDa 5 2 0

STRN 043815 Isoform 1 of Striatin IPI00014456 86 kDa 5 1 0

IPI00017334

PHB P35232 Prohibitin (+1 ) 30 kDa 5 0 0

Serum deprivation-

SDPR 095810 response protein IPI00005809 47 kDa 0 0 4

G protein pathway IPI00012301

GPS2 Q13227 suppressor 2 (+1 ) 37 kDa 5 0 0

Isoform Long of Cold shock

domain-containing protein IPI00470891

CSDE1 075534 E1 (+2) 89 kDa 4 0 0

Isoform 1 of

Chromodomain-helicase- IPI00000846 218

CHD4 Q14839 DNA-binding protein 4 (+1 ) kDa 12 45 2

Isoform 1 of AT-rich

interactive domain- 242

RID1A 014497 containing protein 1A IPI00643722 kDa 20 37 0

Protein tyrosine

phosphatase-like protein IPI00008998

PTPLAD1 Q9PQ35 PTPLAD1 (+1 ) 43 kDa 2 0 0 hypothetical protein

PLBD1 Q6P4A8 LOC79887 IPI00016255 63 kDa 0 0 2

Isoform 1 of Mucosa- associated lymphoid tissue

lymphoma translocation IPI00009540

MALT1 Q9UDY8 protein 1 (+2) 92 kDa 0 0 2

Isoform 1 of B-cell

CLL/lymphoma 7 protein IPI00006266

BCL7C Q8WUZ0 family member C (+2) 23 kDa 2 0 0

IPI00294618

PRCC Q92733 Proline-rich protein PRCC (+2) 52 kDa 2 0 0

Wiskott-Aldrich syndrome

WASF2 Q9Y6W5 protein family member 2 IPI00472164 54 kDa 2 0 0

Isoform 1 of PH and SEC7 IPI00304670 1 16

PSD4 Q8NDX1 domain-containing protein 4 (+2) kDa 2 0 0 Zinc finger BED domain-

ZBED1 096006 containing protein 1 IPI00006203 78 kDa 2 0 0

IPI00021983

NCSTN Q92542 Isoform 1 of Nicastrin (+3) 78 kDa 2 0 0

IPI00431697

CT45A5 Q6NSH3 Cancer/testis antigen 45-5 (+4) 21 kDa 2 0 0

Isoform 1 of Mps one

binder kinase activator-like IPI00386122

MOBKL3 Q9Y3A3 3 (+2) 26 kDa 0 0 1

Isoform 2 of S-phase IPI00172421

SKP1 P63208 kinase-associated protein 1 (+1 ) 18 kDa 0 0 4

186

KIF14 Q 15058 Kinesin-like protein KIF14 IPI00299554 kDa 1 1 0

Isoform 1 of Activating

signal cointegrator 1

ASCC2 Q9H1 I8 complex subunit 2 IPI00549736 86 kDa 0 0 1

Isoform 1 of Zinc finger ZZ- type and EF-hand domain- IPI00385631 331

ZZEF1 043149 containing protein 1 (+1 ) kDa 0 0 1

MLF2 Q 15773 Myeloid leukemia factor 2 IPI00023095 28 kDa 2 0 1 preferentially expressed IPI00893980

PRAME P78395 antigen in melanoma (+3) 21 kDa 4 0 0

15 kDa selenoprotein

060613 isoform 1 precursor IPI00030877 18 kDa 0 0 2

Table 5b. Putative Hsp90 interacting co-chaperones identified using the QSTAR-Elite hybrid quadrupole time-of-flight mass spectrometer (QTof MS) (AB/MDS Sciex)

1 1

DnaJ homolog

subfamily B member

DNAJB1 P25685 1 IPI00015947 38 kDa 3 0 1

DnaJ homolog

subfamily C member

DNAJC13 075165 13 IPI00307259 254 kDa 0 0 3

DnaJ homolog

subfamily C member

DNAJC8 075937 8 IPI00003438 30 kDa 1 0 0

DnaJ homolog

subfamily C member

DNAJC9 Q8WXX5 9 IPI00154975 30 kDa 3 0 1

IPI00784002

SACS Q9NZJ4 Isoform 2 of Sacsin (+1 ) 505 kDa 2 1 0

Peptidyl-prolyl cis-

PPIB P23284 trans isomerase B IPI00646304 24 kDa 4 0 0 PPIase

Isoform 1 of (peptid

Peptidyl-prolyl cis- yiproly trans isomerase-like lisome

PPIL1 Q9Y3C6 2 IPI00003824 59 kDa 13 1 0 rase)

Peptidyl-prolyl cis-

PPIA P62937 trans isomerase A IPI00419585 18 kDa 0 0 6

40 kDa peptidyl- prolyl cis-trans

PPID Q08752 isomerase IPI00003927 41 kDa 3 1 0

Isoform A of

Peptidyl-prolyl cis- IPI00009316

PPIE Q9UNP9 trans isomerase E (+2) 33 kDa 0 0 3

Protein disulfide-

P4HB P07237 isomerase IPI00010796 57 kDa 11 36 1

FK506-binding

FKBP4 Q02790 protein 4 IPI00219005 52 kDa 21 12 8

FK506-binding

FKBP10 Q96AY3 protein 10 IPI00303300 64 kDa 0 0 7

FK506-binding IPI00182126

FKBP9 095302 protein 9 (+1 ) 63 kDa 1 0 0

BAG family

molecular

chaperone regulator IPI00030695

BAG4 095429 4 (+1 ) 50 kDa 4 0 0 BAG

BAG family

molecular

chaperone regulator

BAG2 095816 2 IPI00000643 24 kDa 1 1 3

Tetratricopeptide

TTC27 Q6P3X3 repeat protein 27 IPI00183938 97 kDa 13 3 2

Tetratricopeptide IPI00000606

TTC4 095801 repeat protein 4 (+1 ) 45 kDa 1 0 0

Tetratricopeptide IPI00170855

TTC19 Q6DKK2 repeat protein 19 (+1 ) 56 kDa 2 0 0

Pentatricopeptide

repeat-containing

PTCD1 075 27 protein 1 IPI00171925 79 kDa 2 0 0

Isoform 1 of TPR

repeat-containing

B3KU92 protein LOC90826 IPI00395476 95 kDa 3 0 0

Isoform 1 of

Mitochondrial import

receptor subunit

TOMM40 O96Q08 TOM40 homolog IPI00014053 38 kDa 3 0 0 TOM40 Isoform 2 of Protein

UNC45B Q8IWX7 unc-45 homolog A IPI00735181 102 kDa 33 6 2 UNC45

Stress-70 protein,

mitochondrial;

HSPA9 P38646 GRP75 IPI00007765 74 kDa 19 25 4 GRP75

60 kDa heat shock

protein,

mitochondrial;

HSPD1 P10809 HSP60 I PI 00784154 61 kDa 19 29 1 HSP60

*Grp94 and Trap-1 are Hsp90 iso forms to which PU-H71 binds directly

Table 5c. Putative Hsp90 interacting proteins acting in the proteasome pathway identified using the QSTAR-Elite hybrid quadrupole time-of-flight mass spectrometer (GT of MS) (AB/MDS Sciex)

Probable E3 ubiquitin-

HERC1 Q15751 protein ligase HERC1 IPI00022479 532 kDa 1 2 0

E3 ubiquitin-protein ligase

KCMF1 Q9P0J7 KCMF1 IPI00306661 42 kDa 1 0 0

TRIP12 protein; Probable

E3 ubiquitin-protein ligase IPI00032342

TRIP12 Q14669 TRIP12 (+1 ) 226 kDa 0 0 6

Isoform 1 of Ubiquitin

carboxyl-terminal

USP47 Q96K76 hydrolase 47 IPI00607554 157 kDa 1 1 8 2

Isoform 1 of Ubiquitin

carboxyl-terminal IPI00297593

USP34 Q70CQ2 hydrolase 34 (+2) 404 kDa 15 6 3

Isoform 1 of Ubiquitin

carboxyl-terminal

USP15 Q9Y4E8 hydrolase 15 IPI00000728 1 12 kDa 12 10 2 ubiquitin specific protease IPI00003964

USP9X Q93008 9, X-linked isoform 4 (+1 ) 290 kDa 24 52 9

Isoform 1 of Ubiquitin-

UBAP2L Q14157 associated protein 2-like IPI00514856 1 15 kDa 9 12 17

Ubiquitin-like modifier-

UBA1 P22314 activating enzyme 1 IPI00645078 1 18 kDa 6 6 26

Isoform 2 of Ubiquitin

carboxyl-terminal IPI00219512

UCHL5 Q9Y5K5 hydrolase isozyme L5 (+6) 36 kDa 12 0 5

Ubiquitin carboxyl-terminal IPI00003965

USP7 Q93009 hydrolase 7 (+1 ) 128 kDa 8 3 0

Ubiquitin carboxyl-terminal

USP10 Q14694 hydrolase 10 IPI00291946 87 kDa 5 2 2

Ubiquitin carboxyl-terminal IPI00185661

USP32 Q8NFA0 hydrolase 32 (+1 ) 182 kDa 5 1 2

Isoform 1 of Ubiquitin

carboxyl-terminal IPI00045496

USP28 Q96RU2 hydrolase 28 (+1 ) 122 kDa 1 1 2

Ubiquitin carboxyl-terminal IPI00219913

USP14 P54578 hydrolase 14 (+2) 56 kDa 2 2 0

Isoform 1 of Cell division IPI00022091

CDC16 Q 13042 cycle protein 16 homolog (+3) 72 kDa 1 3 0 Table 5d. Putative Hsp90 interacting proteins identified using the Waters Xevo QTof MS

^N in gray are proteins for which the excized gel size fails to mach the reported MW

Table 5e. Function, pathway and network analysis eligible proteins selected for processing by Ingenuity Pathway from the input list

© 2000-2010 Ingenuity Systems, Inc. All rights reserved.

signal transducer and transcription

P51692 STAT5B activator of transcription 5B Nucleus regulator

v-raf-1 murine leukemia

P04049 RAF1 viral oncogene homolog 1 Cytoplasm kinase sorafenib

v-raf murine sarcoma 361 1

P10398 ARAF viral oncogene homolog Cytoplasm kinase

vav 1 guanine nucleotide transcription

P 15498 VAV1 exchange factor Nucleus regulator

Bruton

agammaglobulinemia

Q06187 BTK tyrosine kinase Cytoplasm kinase

PTK2 protein tyrosine

Q05397 PTK2 kinase 2 Cytoplasm kinase

protein tyrosine

phosphatase, non-receptor

Q9H3S7 PTPN23 type 23 Cytoplasm phosphatase

signal transducer and

activator of transcription 3

(acute-phase response transcription

P40763 STAT3 factor) Nucleus regulator

interleukin-1 receptor- Plasma

P51617 IRAKI associated kinase 1 Membrane kinase

mitogen-activated protein

P28482 MAPK1 kinase 1 Cytoplasm kinase

mitogen-activated protein

Q9Y6R4 MAP3K4 kinase kinase kinase 4 Cytoplasm kinase

TGF-beta activated kinase

1/MAP3K7 binding protein

Q15750 TAB1 1 Cytoplasm enzyme

mitogen-activated protein

Q16539 MAPK14 kinase 14 Cytoplasm kinase SCIO-469, RO-3201 195 calpain 1 , (mu/l) large

P07384 CAPN1 subunit Cytoplasm peptidase

insulin-like growth factor 2 translation

000425 IGF2BP3 mRNA binding protein 3 Cytoplasm regulator

insulin-like growth factor 2 translation

088477 IGF2BP1 mRNA binding protein 1 Cytoplasm regulator

Q9Y6M1 IGF2BP2 Cytoplasm

insulin-like growth factor 2 translation imRNA binding protein 2 regulator transcription

Q9Y265 RUVBL1 RuvB-like 1 (E. coli) Nucleus regulator transcription

Q9Y230 RUVBL2 RuvB-like 2 (E. coli) Nucleus regulator transcription

Q99417 MYCBP c-myc binding protein Nucleus regulator

A kinase (PRKA) anchor

043823 AKAP8 protein 8 Nucleus other

A kinase (PRKA) anchor

Q9ULX6 AKAP8L protein 8-like Nucleus other

NPM1 nucleophosmin (nucleolar

(includes phosphoprotein B23, transcription

P06748 EG:4869) numatrin) Nucleus regulator coactivator-associated

arginine methyltransferase transcription

Q86X55 CARM1 1 Nucleus regulator calcium/calmodulin- dependent protein kinase II

Q13555 CAMK2G gamma Cytoplasm kinase

Plasma

P29597 TYK2 tyrosine kinase 2 Membrane kinase

Q9UHD2 TBK1 TANK-binding kinase 1 Cytoplasm kinase phosphatidylinositol 4-

P42356 PI4KA kinase, catalytic, alpha Cytoplasm kinase

SMG1 homolog,

phosphatidylinositol 3- kinase-related kinase (C.

Q96Q15 SMG1 elegans) Cytoplasm kinase

Q93100 PHKB phosphorylase kinase, beta Cytoplasm kinase

Q9NVE7 PANK4 pantothenate kinase 4 Cytoplasm kinase protein kinase, AMP- activated, alpha 1 catalytic

Q13131 PRKAA1 subunit Cytoplasm kinase protein kinase, AMP- activated, gamma 1 non-

Q8N7V9 PRKAG1 catalytic subunit Nucleus kinase

060341 KDM1A demethylase 1A Nucleus enzyme tributyrin, belinostat, transcription pyroxamide, vorinostat,

Q9UBN7 HDAC6 histone deacetylase 6 Nucleus regulator romidepsin

retinoblastoma binding transcription

Q16576 RBBP7 protein 7 Nucleus regulator tributyrin, belinostat, transcription pyroxamide, vorinostat,

Q92769 HDAC2 histone deacetylase 2 Nucleus regulator romidepsin

SWI/SNF related, matrix

associated, actin

dependent regulator of

chromatin, subfamily c, transcription

Q92922 SMARCC1 member 1 Nucleus regulator

SWI/SNF related, matrix

associated, actin

SMARCC2 dependent regulator of

(includes chromatin, subfamily c, transcription

Q8TAQ2 EG:6601 ) member 2 Nucleus regulator

tumor necrosis factor, Extracellular

Q03169 TNFAIP2 alpha-induced protein 2 Space other

phosphatidylinositol binding

Q13492 PI CALM clathrin assembly protein Cytoplasm other

Q8N163 KIAA1967 KIAA1967 Cytoplasm peptidase

minichromosome

maintenance complex

P33992 MCM5 component 5 Nucleus enzyme

transferrin receptor (p90, Plasma

P02786 TFRC CD71 ) Membrane transporter

transcription

Q13263 TRIM28 tripartite motif-containing 28 Nucleus regulator

Plasma

Q9Y490 TLN1 talin 1 Membrane other

NDC80 homolog,

kinetochore complex

014777 NDC80 component (S. cerevisiae) Nucleus other

IQ motif containing GTPase

Q13576 IQGAP2 activating protein 2 Cytoplasm other

Q8TC07 TBC1D15 member 15 Cytoplasm other TEL2, telomere

maintenance 2, homolog

Q9Y4R8 TEL02 (S. cerevisiae) unknown other

RAP1 interacting factor

Q5UIP0 RIF1 homolog (yeast) Nucleus other

WD repeat containing,

Q9BUR4 WRAP53 antisense to TP53 unknown other

tankyrase 1 binding protein

Q9C0C2 TNKS1 BP1 1 , 182kDa Nucleus other

programmed cell death 4

(neoplastic transformation

Q53EL6 PDCD4 inhibitor) Nucleus other

fermitin family homolog 3

Q86UX7 FERMT3 (Drosophila) Cytoplasm enzyme

PTK2B protein tyrosine

Q14289 PTK2B kinase 2 beta Cytoplasm kinase myeloid/lymphoid or mixed- lineage leukemia (trithorax

homolog, Drosophila);

P55196 MLLT4 translocated to, 4 Nucleus other

Q9Y4L1 HYOU1 hypoxia up-regulated 1 Cytoplasm other

zymogen granule protein

Q96DA0 ZG16B 16 homolog B (rat) unknown other

inositol polyphosphates- phosphatase, type I,

Q96PE3 INPP4A 107kDa Cytoplasm phosphatase guanine nucleotide binding

P36915 GNL1 protein-like 1 unknown other

SAM domain and HD

Q9Y3Z3 SAMHD1 domain 1 Nucleus enzyme tight junction protein 1 Plasma

Q07157 TJP1 (zona occludens 1 ) Membrane other

HLA-B associated

P46379 BAT3 transcript 3 Nucleus enzyme

P21333 FLNA filamin A, alpha Cytoplasm other

Q14315 FLNC filamin C, gamma Cytoplasm other

Q86Y56 HEATR2 HEAT repeat containing 2 unknown other containing 1

075131 CPNE3 copine III Cytoplasm kinase transducin-like enhancer of

split 1 (E(sp1 ) homolog, transcription

Q04724 TLE1 Drosophila) Nucleus regulator

014773 TPP1 tripeptidyl peptidase I Cytoplasm peptidase serologically defined colon

060524 SDCCAG1 cancer antigen 1 Nucleus other

Plasma

Q9Y2A7 NCKAP1 NCK-associated protein 1 Membrane other

Q7Z3B4 NUP54 nucleoporin 54kDa Nucleus transporter

Q9BW27 NUP85 nucleoporin 85kDa Cytoplasm other

Q12769 NUP160 nucleoporin 160kDa Nucleus transporter

CCR4-NOT transcription

A5YKK6 CNOT1 complex, subunit 1 unknown other

leucine rich repeat

Q9H9A6 LRRC40 containing 40 Nucleus other

transcription

Q99623 PHB2 prohibitin 2 Cytoplasm regulator

Vac14 homolog (S.

Q08AM6 VAC 14 cerevisiae) unknown other

NIN1/RPN12 binding

protein 1 homolog (S.

Q9ULX3 N0B1 cerevisiae) Nucleus other

PRAME

(includes preferentially expressed

P78395 EG:23532) antigen in melanoma Nucleus other

FtsJ methyltransferase

Q8N1 G2 FTSJD2 domain containing 2 unknown other

nuclear factor of kappa light

polypeptide gene enhancer transcription

P19838 NFKB1 in B-cells 1 Nucleus regulator solute carrier family 3

(activators of dibasic and

neutral amino acid Plasma

P08195 SLC3A2 transport), member 2 Membrane transporter

096006 ZBED1 containing 1 Nucleus enzyme

Table 5f. Significant networks and associated biofunctions assigned by Ingenuity Pathways Core Analysis to proteins isolated by PU-H71 in the K562 cell line

© 2000-2010 Ingenuity Systems, Inc. All rights reserved.

ARID1A, atypical protein kinase C, CARM1, Cbp/p300,

CHD4, ERK1/2, Esr1-Esr1 -estrogen-estrogen, GIT1, GPS2, Hdac1/2, HISTONE, Histone h3, Histone h4,

Gene Expression, KDM1A, Mi2, MTA2, MYBBP1A, N-cor, NCOR1, NCOR2, Cellular Assembly NCoR/SMRT corepressor, NuRD, PHB2, PHB (includes and Organization, EG:5245), Rar, RBBP7, RCOR1 , Rxr, SLC3A2,

Cellular SMARCC1, SMARCC2 (includes EG:6601), Sos,

6 30 19 Compromise TBL1XR1, TIP60, TRIM28

AKAP8, AKAP14, ALDH1B1, CDCA7, CEPT1, CIT, CNBP, CPNE3, DISC1, DOCK11, FTSJD2, HTT, IFNA2, IGF2BP3, IQGAP3, KIF14, LGMN, MIR124, MIR129-2 (includes EG:406918), MIRN339, MYC, MYCBP, NEK9 (includes EG:91754), NFkB (complex), NUP160, PANK4,

Cell Cycle, PEA15, PRPF40B, RNF213, SAMHD1, SCAMP5, TPP1,

7 22 15 Development TRIM56, WRAP53, YME1L1

Cellular BCR, BTK, Calpain, CAPN1, CAPNS1, Collagen type I, Compromise, CRKL, DOCK2, Fcerl, GNRH, Ige, JAK, KSR1, MAPK1, Hypersensitivity NCK, NFAT (complex), Pdgf, PHKB, Pkg, PLC gamma, Response, Ptk, PTK2B, STAT, STAT1/3/5, STAT1/3/5/6, STAT3/5, Inflammatory STAT5A, STAT5a/b, STAT5B, SYK/ZAP, Talin, TLN1,

8 20 14 Response TYK2, VAV, VAV1

ABLIM, ACAP2, AKR1C14, ARF6, ARPC1A, ATP9A, BUB1, CREBL2, DHRS3, DYRK3, FHOD1, FLNC, FSH,

Cell Morphology, GK7P, GNL1, GRB2, HEATR2, Lh, LOC81691, NCSTN, Cellular NDC80, PDGF BB, PI4K2A, PRMT6, PTP4A1, QRFP,

Development and RAB11B, RQCD1, SCARB2, SLC2A4, THBS1, TP53I11,

9 20 14 Function TRIP13, Vegf, ZBED1

AGT, AGTRAP, ATG5 (includes EG:9474), Cathepsin, COL4A6, CORIN, ENPP1, FAM120A, GATM, H1FX, HSPG2 (includes EG:3339), IGF2BP2, ITPA, KIAA0182, LPCAT3, MCPT1, MIR17 (includes EG:406952), MYL3, NOS1 , NSUN2, PFK, PLA1A, RPS6, SCYL1, SDPR, SERBP1, SMOC2, SRF, SRFBP1, STOML2, TGFB1,

10 18 13 Cell Morphology TGFBRAP1, TMOD3, VAC 14, WIBG

AMBRA1, AR, CDC45L, CDCA7L, CLDND1, CTDSP2, FAM115A, HEATR6, HNF4A, HYAL3, KIAA1468, LRRC40, MIR124-1 (includes EG:406907), NUP54, PECI, PERP, POLR3G, PRCC, PTPN4, PTPN11, RIOK2, RNF6,

Gene Expression, RNPEPL1, SF3B4, SLC17A5, SLC25A20, SLC30A7,

Developmental SLC39A7, SSFA2, STK19, SUPV3L1, TBC1D15, TCF19,

11 17 12 Disorder ZBED3, ZZEF1

Actin, AIFM1, Arp2/3, CD3, CTTN, CYFIP1, DIAPH1,

Cell Morphology, Dynamin, ERK, F Actin, FERMT3, Focal adhesion kinase, Cellular Assembly Gpcr, Growth hormone, Integrin, IQGAP2, Jnk, Lfa-1, and Organization, MLF2, MLLT4, NCKAP1, Nfat (family), Pak, PI3K, PI3K Cellular p85, Pkc(s), PPP5C, PTK2, Rac, Rap1, Ras homolog,

12 16 13 Development Rsk, TCR, TJP1, WASF2 ANKRD2, APRT, ARL6IP1 , BANP, C1 10RF82, CAMK1 ,

CKMT1 B, CNOT1 , CTSZ (includes EG: 1522), DOCK7

(includes EG:85440), FIP1 L1 , GART, GH1 , GIP2, GSK3B,

HDAC5, Hla-abc, IFNG, MAN2B1 , NAPSA, NTHL1 ,

NUP85, ORM2, PTPN23, SLC5A8, SLC6A6, TBX3,

Cancer, Cell Cycle, TNKS1 BP1 , TOB1 , TP53, TRIM22, UNC5B, VPS33A,

13 12 10 Gene Expression YBX1 , YWHAZ

*IPA computes a score for each possible network according to the fit of that network to the inputted proteins. The score is calculated as the negative base- 10 logarithm of the p-value that indicates the likelihood of the inputted proteins in a given network being found together due to random chance. Therefore, scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone.

Supplementary Materials and Methods

Reagents

The Hsp90 inhibitors, the solid-support immobilized and the fluorescein-labeled derivatives were synthesized as previously reported (Taldone et al, 2011, Synthesis and Evaluation of Small...; Taldone et al., 2011, Synthesis and Evaluation of Fluorescent...; He et al., 2006). We purchased Gleevec from LC Laboratories, AS703026 from Selleck, K -93 from Tocris, and PP242, BMS-345541 and sodium vanadate from Sigma. All compounds were used as DMSO stocks. Western Blotting

Cells were either treated with PU-H71 or DMSO (vehicle) for 24 h and lysed in 50 mM Tris, pH 7.4, 150 mM NaCl and 1% NP40 lysis buffer supplemented with leupeptin (Sigma Aldrich) and aprotinin (Sigma Aldrich). Protein concentrations were determined using BCA kit (Pierce) according to the manufacturer's instructions. Protein lysates (15-200 μg) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane and probed with the following primary antibodies against: Hsp90 (1 :2000, SMC-107A/B; StressMarq), Bcr-Abl (1 :75, 554148; BD Pharmingen), PI3K (1 : 1000, 06-195; Upstate), mTOPv (1 :200, Sc-1549; Santa Cruz), p-mTOR (1 : 1000, 2971; Cell Signaling), STAT3 (1 : 1000, 9132; Cell Signaling), p-STAT3 (1 :2000, 9145; Cell Signaling), STAT5 (1 :500, Sc- 835; Santa Cruz), p-STAT5 (1 : 1000, 9351; Cell Signaling), RICTOR (1 :2000, NB100-611; Novus Biologicals), RAPTOR (1 : 1000, 2280; Cell Signaling), P90RSK (1 : 1000, 9347; Cell Signaling), Raf-1 ( 1 :300, Sc-133; Santa Cruz), CARM1 (1 : 1000, 09-818; Millipore), CRKL (1 :200, Sc-319; Santa Cruz), GRB2 (1 : 1000, 3972; Cell Signaling), FAK (1 : 1000, Sc-1688; Santa Cruz), BTK (1 : 1000, 3533; Cell Signaling), A-Raf (1 : 1000, 4432; Cell Signaling), PRKD2 (1 :200, sc-100415, Santa Cruz), HCK (1 :500, 06-833; Milipore), p-HCK (1 :500, ab52203; Abeam) and β-actin (1 :2000, A1978; Sigma). The membranes were then incubated with a 1 :3000 dilution of a corresponding horseradish peroxidase conjugated secondary antibody. Detection was performed using the ECL-Enhanced Chemiluminescence Detection System (Amersham Biosciences) according to manufacturer's instructions.

Densitometry

Gels were scanned in Adobe Photoshop 7.0.1 and quantitative densitometric analysis was performed using Un-Scan-It 5.1 software (Silk Scientific). Nano-LC-MS/MS

Lysates prepared as mentioned above were first pre-cleaned by incubation with control beads overnight at 4°C. Pre-cleaned K562 cell extract (1,000 μg) in 200 μΐ Felts lysis buffer was incubated with PU-H71 or control-beads (80 μΐ) for 24 h at 4°C. Beads were washed with lysis buffer, proteins eluted by boiling in 2% SDS, separated on a denaturing gel and Coomassie stained according to manufacturer's procedure (Biorad). Gel-resolved proteins from pull-downs were digested with trypsin, as described (Winkler et al, 2002). In-gel tryptic digests were subjected to a micro-clean-up procedure (Erdjument-Bromage et al, 1998) on 2 μΐ, bed-volume of Poros 50 R2 (Applied Biosystems - ΆΒ') reversed-phase beads, packed in an Eppendorf gel-loading tip, and the eluant diluted with 0.1% formic acid (FA). Analyses of the batch purified pools were done using a QSTAR-Elite hybrid quadrupole time-of-flight mass spectrometer (QTof MS) (AB/MDS Sciex), equipped with a nano spray ion source. Peptide mixtures (in 20 are loaded onto a trapping guard column (0.3x5-mm PepMap CI 8 100 cartridge from LC Packings) using an Eksigent nano MDLC system (Eksigent Technologies, Inc) at a flow rate of 20 μΕ/ηιίη. After washing, the flow was reversed through the guard column and the peptides eluted with a 5-45% MeCN gradient (in 0.1 % FA) over 85 min at a flow rate of 200 nL/min, onto and over a 75-micron x 15 -cm fused silica capillary PepMap C18 column (LC Packings); the eluant is directed to a 75-micron (with 10-micron orifice) fused silica nano-electrospray needle (New Objective). Electrospray ionization (ESI) needle voltage was set at about 1800 V. The mass analyzer is operated in automatic, data- dependent MS/MS acquisition mode, with the threshold set to 10 counts per second of doubly or triply charged precursor ions selected for fragmentation scans. Survey scans of 0.25 sec are recorded from 400 to 1800 amu; up to 3 MS/MS scans are then collected sequentially for the selected precursor ions, recording from 100 to 1800 amu. The collision energy is automatically adjusted in accordance with the m/z value of the precursor ions selected for MS/MS. Selected precursor ions are excluded from repeated selection for 60 sec after the end of the corresponding fragmentation duty cycle. Initial protein identifications from LC- MS/MS data was done using the Mascot search engine (Matrix Science, version 2.2.04; www.matrixscience.com) and the NCBI (National Library of Medicine, NIH - human taxonomy containing, 223,695 protein sequences) and IPI (International Protein Index, EBI,

Hinxton, UK - human taxonomy, containing 83,947 protein sequences) databases. One missed tryptic cleavage site was allowed, precursor ion mass tolerance = 0.4Da fragment ion mass tolerance = 0.4 Da, protein modifications were allowed for Met-oxide, Cys-acrylamide and N-terminal acetylation. MudPit scoring was typically applied with 'require bold red' activated, and using significance threshold score p<0.05. Unique peptide counts (or 'spectral counts') and percent sequence coverages for all identified proteins were exported to Scaffold Proteome Software (version 2 06 01, www.proteomesoftware.com) for further bioinformatic analysis (Table 5a). Using output from Mascot, Scaffold validates, organizes, and interprets mass spectrometry data, allowing more easily to manage large amounts of data, to compare samples, and to search for protein modifications. Findings were validated in a second MS system, the Waters Xevo QTof MS instrument (Table 5d). Potential unspecific interactors were identified and removed from further analyses as indicated (Trinkle-Mulcahy et al., 2008).

Bioinformatic pathways analysis

Proteins were analyzed further by bioinformatic pathways analysis (Ingenuity Pathway Analysis 8.7 [IP A]; Ingenuity Systems, Mountain View, CA, www. ingenuity . com) (Munday et al., 2010; Andersen et al., 2010). IPA constructs hypothetical protein interaction clusters based on a regularly updated "Ingenuity Pathways Knowledge Base". The Ingenuity Pathways Knowledge Base is a very large curated database consisting of millions of individual relationships between proteins, culled from the biological literature. These relationships involve direct protein interactions including physical binding interactions, enzyme substrate relationships, and cis-trans relationships in translational control. The networks are displayed graphically as nodes (individual proteins) and edges (the biological relationships between the nodes). Lines that connect two molecules represent relationships. Thus any two molecules that bind, act upon one another, or that are involved with each other in any other manner would be considered to possess a relationship between them. Each relationship between molecules is created using scientific information contained in the Ingenuity Knowledge Base. Relationships are shown as lines or arrows between molecules. Arrows indicate the directionality of the relationship, such that an arrow from molecule A to B would indicate that molecule A acts upon B. Direct interactions appear in the network diagram as a solid line, whereas indirect interactions as a dashed line. In some cases a relationship may exist as a circular arrow or line originating from one molecule and pointing back at that same molecule. Such relationships are termed "self-referential" and arise from the ability of a molecule to act upon itself. In practice, the dataset containing the

UniProtKB identifiers of differentially expressed proteins is uploaded into IPA. IPA then builds hypothetical networks from these proteins and other proteins from the database that are needed fill out a protein cluster. Network generation is optimized for inclusion of as many proteins from the inputted expression profile as possible, and aims for highly connected networks. Proteins are depicted in networks as two circles when the entity is part of a complex; as a single circle when only one unit is present; a triangle pointing up or down to describe a phosphatase or a kinase, respectively; by a horizontal oval to describe a transcription factor; and by circle to depict "other" functions. IPA computes a score for each possible network according to the fit of that network to the inputted proteins. The score is calculated as the negative base- 10 logarithm of the p-value that indicates the likelihood of the inputted proteins in a given network being found together due to random chance. Therefore, scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone. All the networks presented here were assigned a score of 10 or higher (Table 5f). Radioisotope binding studies and Hsp90 quantification studies

Saturation studies were performed with ¹³¹ I-PU-H71 and cells (K562, MDA-MB-468, SKBr3, LNCaP, DU-145, MRC-5 and PBL). Briefly, triplicate samples of cells were mixed with increasing amount of ¹³¹ I-PU-H71 either with or without 1 μΜ unlabeled PU-H71. The solutions were shaken in an orbital shaker and after 1 hr the cells were isolated and washed with ice cold Tris-buffered saline using a Brandel cell harvester. All the isolated cell samples were counted and the specific uptake of ¹³¹ I-PU-H71 determined. These data were plotted against the concentration of ¹³¹ I-PU-H71 to give a saturation binding curve. For the quantification of PU-bound Hsp90, 9.2xl0 ⁷ K562 cells, 6.55xl0 ⁷ KCL-22 cells, 2.55xl0 ⁷ KU182 cells and 7.8xl0 ⁷ MEG-01 cells were lysed to result in 6382, 3225, 1349 and 3414 μg of total protein, respectively. To calculate the percentage of Hsp90, cellular Hsp90 expression was quantified by using standard curves created of recombinant Hsp90 purified from HeLa cells (Stressgen#ADI-SPP-770).

Pulse-Chase

K562 cells were treated with Na ₃ V0 ₄ (1 mM) with or without PU-H71 (5 μΜ), as indicated. Cells were collected at indicated times and lysed in 50 mM Tris pH 7.4, 150 mM NaCl and 1% NP-40 lysis buffer, and were then subjected to western blotting procedure.

Tryptic digestion K562 cells were treated for 30 min with vehicle or PU-H71 (50 μΜ). Cells were collected and lysed in 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40 lysis buffer. STAT5 protein was immunoprecipitated from 500 μg of total protein lysate with an anti-STAT5 antibody (Santa Cruz, sc-835). Protein precipitates bound to protein G agarose beads were washed with trypsin buffer (50 mM Tris pH 8.0, 20 mM CaCl ₂ ) and 33 ng of trypsin has been added to each sample. The samples were incubated at 37°C and aliquots were collected at the indicated time points. Protein aliquots were subjected to SDS-PAGE and blotted for STAT5.

Activated STAT5 DNA binding assay

The DNA-binding capacity of STAT5a and STAT5b was assayed by an ELISA-based assay (TransAM, Active Motif, Carlsbad, CA) following the manufacturer instructions. Briefly, 5xl0 ⁶ K562 cells were treated with PU-H71 1 and 10 μΜ or control for 24 h. Ten micrograms of cell lysates were added to wells containing pre-adsorbed STAT consensus oligonucleotides (5'-TTCCCGGAA-3'). For control treated cells the assay was performed in the absence or presence of 20 pmol of competitor oligonucleotides that contains either a wild- type or mutated STAT consensus binding site. Interferon-treated HeLa cells (5 μg per well) were used as positive controls for the assay. After incubation and washing, rabbit polyclonal anti-STAT5a or anti-STAT5b antibodies (1 : 1000, Active Motif) was added to each well, followed by HPR-anti-rabbit secondary antibody (1 : 1000, Active Motif). After HRP substrate addition, absorbance was read at 450 nm with a reference wavelength of 655 nm (Synergy4, Biotek, Winooski, VT). In this assay the absorbance is directly proportional to the quantity of DNA-bound transcription factor present in the sample. Experiments were carried out in four replicates. Results were expressed as arbitrary units (AU) from the mean absorbance values with SEM.

Quantitative Chromatin Immunoprecipitation (Q-ChIP)

Q-ChIP was made as previously described with modifications (Cerchietti et al, 2009). Briefly, 10 ⁸ K562 cells were fixed with 1% formaldehyde, lysed and sonicated (Branson sonicator, Branson). STAT5 N20 (Santa Cruz) and Hsp90 (Zymed) antibodies were added to the pre-cleared sample and incubated overnight at 4 °C. Then, protein-A or G beads were added, and the sample was eluted from the beads followed by de-crosslinking. The DNA was purified using PCR purification columns (Qiagen). Quantification of the ChIP products was performed by quantitative PCR (Applied Biosystems 7900HT) using Fast SYBR Green (Applied Biosystems). Target genes containing STAT binding site were detected with the following primers: CCND2 (5-GTTGTTCTGGTCCCTTTAATCG and 5- ACCTCGCATACCCAGAGA), MYC (5 -ATGCGTTGCTGGGTT ATTTT and 5- CAGAGCGTGGGATGTTAGTG) and for the intergenic control region (5- CCACCTGAGTCTGCAATGAG and 5 -C AGTCTCC AGCCTTTGTTCC) .

Real time QPCR

R A was extracted from PU-H71 -treated and control K562 cells using R easy Plus kit (Qiagen) following the manufacturer instructions. cDNA was synthesized using High Capacity RNA-to-cDNA kit (Applied Biosystems). We amplified specific genes with the following primers: MYC (5-AGAAGAGCATCTTCCGCATC and 5- CCTTTAAACAGTGCCCAAGC), CCND2 (5 -TGAGCTGCTGGCT AAGATC A and 5- ACGGTACTGCTGCAGGCTAT), BCL-XL (5- CTTTTGTGGAACTCTATGGGAACA and 5-CAGCGGTTGAAGCGTTCCT), MCL1 (5-AGACCTTACGACGGGTTGG and 5- ACATTCCTGATGCCACCTTC), CCND 1 (5-CCTGTCCTACTACCGCCTCA and 5- GGCTTCGATCTGCTCCTG), HPRT (5- CGTCTTGCTCGAGATGTGATG and 5- GCACACAGAGGGCTACAATGTG), GAPDH (5-CGACCACTTTGTCAAGCTCA and 5- CCCTGTTGCTGTAGCCAAAT), RPL13A (5- TGAGTGAAAGGGAGCCAGAAG and 5- CAGATGCCCCACTCACAAGA). Transcript abundance was detected using the Fast SYBR Green conditions (initial step of 20 sec at 95 °C followed by 40 cycles of 1 sec at 95 °C and 20 sec at 60 °C). The CT value of the housekeeping gene (RPL13A) was subtracted from the correspondent genes of interest (ACT). The standard deviation of the difference was calculated from the standard deviation of the CT values (replicates). Then, the ACT values of the PU-H71 -treated cells were expressed relative to their respective control-treated cells using the AACT method. The fold expression for each gene in cells treated with the drug relative to control treated cells is determined by the expression: 2 ^"AACT . Results were represented as fold expression with the standard error of the mean for replicates.

Hsp70 knock-down

Transfections were carried out by electroporation (Amaxa) and the Nucleofector Solution V (Amaxa), according to manufacturer's instructions. Hsp70 knockdown studies were performed using siRNAs designed as previously reported (Powers et al, 2008) against the open reading frame of Hsp70 (HSPA1 A; accession number NM 005345). Negative control cells were trans fected with inverted control siR A sequence (Hsp70C; Dharmacon RNA technologies). The active sequences against Hsp70 used for the study are Hsp70A (5'- GGACGAGUUUGAGCACAAG-3 ') and Hsp70B (5'- CCAAGCAGACGCAGAUCUU-3'). Sequence for the control is Hsp70C (5 '-GGACGAGUUGUAGCACAAG-3 '). Three million cells in 2 mL media (RPMI supplemented with 1% L-glutamine, 1% penicillin and streptomycin) were transfected with 0.5 μΜ siRNA according to the manufacturer's instructions. Transfected cells were maintained in 6-well plates and at 84h, lysed followed by standard Western blot procedures. Kinase screen (Fabian et al., 2005)

For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E.coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32°C until lysis (90-150 min). The lysates were centrifuged (6,000 x g) and filtered (0.2μιη) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in lx binding buffer (20 % SeaBlock, 0.17x PBS, 0.05 % Tween 20, 6 mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05 % Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05 % Tween 20, 0.5 μιη non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. KINOME^can's selectivity score (S) is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that bind to the compound by the total number of distinct kinases tested, excluding mutant variants. TKEEspot™ is a proprietary data visualization software tool developed by KJNOMEscan (Fabian et al., 2005). Kinases found to bind are marked with red circles, where larger circles indicate higher-affinity binding. The kinase dendrogram was adapted and is reproduced with permission from Science and Cell Signaling Technology, Inc.

Lentiviral vectors, lentiviral production and K562 cells transduction

Lentiviral constructs of shRNA knock-down of CARMl were purchased from the TRC lentiviral shRNA libraries of Openbiosystem: pLKO. l-shCARMl-KDl (catalog No: RHS3979-9576107) and pLK0.1-shCARMl-KD2 (catalog No: RHS3979-9576108). The control shRNA (shRNA scramble) was Addgene plasmid 1864. GFP was cloned in to replace puromycin as the selection marker. Lentiviruses were produced by transient transfection of 293T as in the previously described protocol (Moffat et al., 2006). Viral supernatant was collected, filtered through a 0.45-μιη filter and concentrated. K562 cells were infected with high-titer lentiviral concentrated suspensions, in the presence of 8 μg/ml polybrene (Aldrich). Transduced K562 cells were sorted for green fluorescence (GFP) after 72 hours transfection. RNA extraction and quantitative Real-Time PCR (qRT-PCR)

For qRT-PCR, total RNA was isolated from 10 ⁶ cells using the RNeasy mini kit (QIAGEN, Germany), and then subjected to reverse-transcription with random hexamers (Superscript III kit, Invitrogen). Real-time PCR reactions were performed using an ABI 7500 sequence detection system. The PCR products were detected using either Sybr green I chemistry or TaqMan methodology (PE Applied Biosystems, Norwalk, CT). Details for real-time PCR assays were described elsewhere (Zhao et al, 2009). The primer sequences for CARMl qPCR are TGATGGCCAAGTCTGTCAAG(forward) and

TG AAAGC AAC GTC AAAC C AG(reverse) . Cell viability, Apoptosis, and Proliferation assay

Viability assessment in K562 cells untransfected or transfected with CARMl shRNA or scramble was performed using Trypan Blue. This chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells that exclude the dye are viable. Apoptosis analysis was assessed using fluorescence microscopy by mixing 2 of acridine orange (100 μg/mL), 2 μΕ of ethidium bromide (100 μg/mL), and

20 of the cell suspension. A minimum of 200 cells was counted in at least five random fields. Live apoptotic cells were differentiated from dead apoptotic, necrotic, and normal cells by examining the changes in cellular morphology on the basis of distinctive nuclear and cytoplasmic fluorescence. Viable cells display intact plasma membrane (green color), whereas dead cells display damaged plasma membrane (orange color). An appearance of ultrastructural changes, including shrinkage, heterochromatin condensation, and nuclear degranulation, are more consistent with apoptosis and disrupted cytoplasmic membrane with necrosis. The percentage of apoptotic cells (apoptotic index) was calculated as: % Apoptotic cells = (total number of cells with apoptotic nuclei/total number of cells counted) x 100. For the proliferation assay, 5 x 10 ³ K562 cells were plated on a 96-well solid black plate (Corning). The assay was performed according to the manufacturer's indications (CellTiter- Glo Luminescent Cell Viability Assay, Promega). All experiments were repeated three times. Where indicated, growth inhibition studies were performed using the Alamar blue assay. This reagent offers a rapid objective measure of cell viability in cell culture, and it uses the indicator dye resazurin to measure the metabolic capacity of cells, an indicator of cell viability. Briefly, exponentially growing cells were plated in microtiter plates (Corning # 3603) and incubated for the indicated times at 37 °C. Drugs were added in triplicates at the indicated concentrations, and the plate was incubated for 72 h. Resazurin (55 μΜ) was added, and the plate read 6 h later using the Analyst GT (Fluorescence intensity mode, excitation 530nm, emission 580nm, with 560nm dichroic mirror). Results were analyzed using the Softmax Pro and the GraphPad Prism softwares. The percentage cell growth inhibition was calculated by comparing fluorescence readings obtained from treated versus control cells. The IC ₅₀ was calculated as the drug concentration that inhibits cell growth by 50%.

Quantitative analysis of synergy between mTOR and Hsp90 inhibitors

To determine the drug interaction between pp242 (mTOR inhibitor) and PU-H71 (Hsp90 inhibitor), the combination index (CI) isobologram method of Chou-Talalay was used as previously described (Chou, 2006; Chou & Talalay, 1984). This method, based on the median-effect principle of the law of mass action, quantifies synergism or antagonism for two or more drug combinations, regardless of the mechanisms of each drug, by computerized simulation. Based on algorithms, the computer software displays median-effect plots, combination index plots and normalized isobolograms (where non constant ratio combinations of 2 drugs are used). PU-H71 (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0125 μΜ) and pp242 (0.5, 0.125, 0.03125, 0.0008, 0.002, 0.001 μΜ) were used as single agents in the concentrations mentioned or combined in a non constant ratio (PU-H71 : pp242; 1 : 1, 1 :2, 1 :4,

1 :7.8, 1 : 15.6, 1 : 12.5). The Fa (fraction killed cells) was calculated using the formulae Fa=l-

Fu; Fu is the fraction of unaffected cells and was used for a dose effect analysis using the computer software (CompuSyn, Paramus, New Jersey, USA). Flow cytometry

CD34 isolation - CD34+ cell isolation was performed using CD34 MicroBead Kit and the automated magnetic cell sorter autoMACS according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA). Viability assay - CML cells lines were plated in 48-well plates at the density of 5x 10 ⁵ cells/ml, and treated with indicated doses of PU-H71. Cells were collected every 24 h, stained with Annexin V-V450 (BD Biosciences) and 7-AAD (Invitrogen) in Annexin V buffer (10 mM HEPES/NaOH, 0.14 M NaCl, 2.5 mM CaCl ₂ ). Cell viability was analyzed by flow cytometry (BD Biosciences). For patient samples, primary CML cells were plated in 48-well plates at 2x 10 ⁶ cells/ml, and treated with indicated doses of PU-H71 for up to 96 h. Cells were stained with CD34-APC, CD38-PE-CY7 and CD45-APC- H7 antibodies (BD Biosciences) in FACS buffer (PBS, 0.05% FBS) at 4 °C for 30 min prior to Annexin V/7-AAD staining. PU-H71 binding assay - CML cells lines were plated in 48- well plates at the density of 5x l0 ⁵ cells/ml, and treated with 1 μΜ PU-H71-FITC. At 4 h post treatment, cells were washed twice with FACS buffer. To measure PU-H71-FITC binding in live cells, cells were stained with 7-AAD in FACS buffer at room temperature for 10 min, and analyzed by flow cytometry (BD Biosciences). Alternatively, cells were fixed with fixation buffer (BD Biosciences) at 4°C for 30 min, permeabilized in Perm Buffer III (BD Biosciences) on ice for 30 min, and then analyzed by flow cytometry. At 96 h post PU-H71- FITC treatment, cells were stained with Annexin V-V450 (BD Biosciences) and 7-AAD in Annexin V buffer, and subjected to flow cytometry to measure viability. To evaluate the binding of PU-H71-FITC to leukemia patient samples, primary CML cells were plated in 48- well plates at 2x 10 ⁶ cells/ml, and treated with 1 μΜ PU-H71-FITC. At 24 h post treatment, cells were washed twice, and stained with CD34-APC, CD38-PE-CY7 and CD45-APC-H7 antibodies in FACS buffer at 4°C for 30 min prior to 7-AAD staining. At 96 h post treatment, cells were stained with CD34-APC, CD38-PE-CY7 and CD45-APC-H7 antibodies followed by Annexin V-V450 and 7-AAD staining to measure cell viability. For competition test, CML cell lines at the density of 5 ^χ 10 ⁵ cells/ml or primary CML samples at the density of 2x l0 ⁶ cells/ml were treated with 1 μΜ unconjugated PU-H71 for 4 h followed by treatment of 1 μΜ PU-H71-FITC for 1 h. Cells were collected, washed twice, stained for 7-AAD in

FACS buffer, and analyzed by flow cytometry. Hsp90 staining - Cells were fixed with fixation buffer (BD Biosciences) at 4°C for 30 min, and permeabilized in Perm Buffer III

(BD Biosciences) on ice for 30 min. Cells were stained with anti-Hsp90 phycoerythrin conjugate (PE) (F-8 clone, Santa Cruz Biotechnologies; CA) for 60 minutes. Cells were washed and then analyzed by flow cytometry. Normal mouse IgG2a-PE was used as isotype control.

Statistical Analysis

Unless otherwise indicated, data were analyzed by unpaired 2-tailed t tests as implemented in GraphPad Prism (version 4; GraphPad Software). A P value of less than 0.05 was considered significant. Unless otherwise noted, data are presented as the mean±SD or mean±SEM of duplicate or triplicate replicates. Error bars represent the SD or SEM of the mean. If a single panel is presented, data are representative of 2 or 3 individual experiments.

Maintenance of the B Cell Receptor Pathway and COP9 Signalosome by Hsp90 Reveals

Novel Therapeutic Targets in Diffuse Large B Cell Lymphoma

Experimental Outline

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, the substrate proteins of which are involved in cell survival, proliferation and angiogenesis. Hsp90 is expressed constitutively and can also be induced by cellular stress, such as heat shock. Because it can chaperone substrate proteins necessary to maintain a malignant phenotype, Hsp90 is an attractive therapeutic target in cancer. In fact, inhibition of Hsp90 results in degradation of many of its substrate proteins. PUH71, an inhibitor of Hsp90, selectively inhibits the oncogenic Hsp90 complex involved in chaperoning onco-proteins and has potent anti-tumor activity diffuse large B cell lymphomas (DLBCLs). By immobilizing PUH71 on a solid support, Hsp90 complexes can be precipitated and analyzed to identify substrate oncoproteins of Hsp90, revealing known and novel therapeutic targets. Preliminary data using this method identified many components of the B cell receptor (BCR) pathway as substrate proteins of Hsp90 in DLBCL. BCR pathway activation has been implicated in lymphomagenesis and survival of DLBCLs. In addition to this, many components of the COP9 signalosome (CSN) were identified as substrates of Hsp90 in DLBCL. The CSN has been implicated in oncogenesis and activation of NF-κΒ, a survival mechanism of DLBCL. Based on these findings, we hypothesize that combined inhibition of Hsp90 and BCR pathway components and/or the CSN will synergize in killing DLBCL. Therefore, our specific aims are:

Specific Aim 1: To determine whether concomitant modulation of Hsp90 and BCR pathways cooperate in killing DLBCL cells in vitro and in vivo

Immobilized PU-H71 will be used to pull down Hsp90 complexes in DLBCL cell lines to detect interactions between Hsp90 and BCR pathway components. DLBCL cell lines treated with increasing doses of PU-H71 will be analyzed for degradation of BCR pathway components DLBCL cell lines will be treated with inhibitors of BCR pathway components alone and in combination with PU-H71 and assessed for viability. Effective combination treatments will be investigated in DLBCL xenograft mouse models. Specific Aim 2: To evaluate the role of the CSN in DLBCL

Subaim 1: To determine whether the CSN can be a therapeutic target in DLBCL

CPs and treatment with PU-H71 will validate the CSN as a substrate of Hsp90 in DLBCL cell lines. The CSN will be genetically ablated alone and in combination with PU-H71 in DLBCL cell lines to demonstrate DLBCL dependence on the CSN for survival. Mouse xenograft models will be treated with CSN inhibition, alone and in combination with PU-H71, to show effect on tumor growth and animal survival.

Subaim 2: To determine the mechanism of CSN in the survival of DLBCL

Immunoprecipitations (IPs) of the CSN will be used to demonstrate CSN-CBM interaction. Genetic ablation of the CSN will be used to demonstrate degradation of BcllO and ablation of NF-κΒ activity in DLBCL cell lines.

Background and Significance

1. DLBCL Classification

DLBCL is the most common form of non-Hodgkin's lymphoma. In order to improve diagnosis and treatment of DLBCL, many studies have attempted to classify this molecularly heterogeneous disease. One gene expression profiling study divided DLBCL into two major subtypes (Alizadeh et al, 2000). Germinal center (GC) B cell like (GCB) DLBCL can be characterized by the expression of genes important for germinal center differentiation including BCL6 and CD 10, whereas activated B cell like (ABC) DLBCL can be distinguished by a gene expression profile resembling that of activated peripheral blood B cells. The NF-κΒ pathway is more active and often mutated in ABC DLBCL. Another classification effort using gene expression profiling identified three major classes of DLBCL. OxPhos DLBCL shows significant enrichment of genes involved in oxidative phosphorylation, mitochondrial function, and the electron transport chain. BCR/proliferation DLBCL can be characterized by an increased expression of genes involved in cell-cycle regulation. Host response (HR) DLBCL is identified based on increased expression of multiple components of the T-cell receptor (TCR) and other genes involved in T cell activation (Monti et al, 2005).

These prospective classifications were made using patient samples and have not been the final answer for diagnosis or treatment of patients. Because patient samples are comprised of heterogeneous populations of cells and tumor microenvironment plays a role in the disease, (de Jong and Enblad, 2008), DLBCL cell lines do not classify as well as patient samples. However, well-characterized cell lines can be used as models of the different subtypes of DLBCL in which to investigate the molecular mechanisms behind the disease. 2. DLBCL: Need for novel therapies

Standard chemotherapy regimens such as the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) cure about 40% of DLBCL patients, with 5- year overall survival rates for GCB and ABC patients of 60%> and 30%>, respectively (Wright et al., 2003). The addition of rituximab immunotherapy to this treatment schedule (R-CHOP) increases survival of DLBCL patients by 10 to 15% (Coiffier et al, 2002). However, 40% of DLBCL patients do not respond to R-CHOP, and the side effects of this combination chemoimmunotherapy are not well tolerated, emphasizing the need for identifying novel targets and treatments for this disease. Classification of patient tumors has advanced the understanding of the molecular mechanisms underlying DLBCL to a degree. Until these details are better understood, treatments cannot be individually tailored. Preclinical studies of treatments with new drugs alone and in combination treatments and the investigation of new targets in DLBCL will provide new insight on the molecular mechanisms behind the disease.

3. Hsp90: A promising target

Hsp90 is an emerging therapeutic target for cancer. The chaperone protein is expressed constitutively, but can also be induced upon cellular stress, such as heat shock. Hsp90 maintains the stability of a wide variety of substrate proteins involved in cellular processes such as survival, proliferation and angiogenesis (Neckers, 2007). Substrate proteins of Hsp90 include oncoproteins such as NPM-ALK in anaplastic large cell lymphoma, and BCR-ACL in chronic myelogenous leukemia (Bonvini et al, 2002; Gorre et al, 2002). Because Hsp90 maintains the stability of oncogenic substrate proteins necessary for disease maintenance, it is an attractive therapeutic target. In fact, inhibition of Hsp90 results in degradation of many of its substrate proteins (Bonvini et al, 2002; Caldas-Lopes et al, 2009; Chiosis et al, 2001; Neckers, 2007; Nimmanapalli et al., 2001). As a result, many inhibitors of Hsp90 have been developed for the clinic (Taldone et al, 2008). 4. PU-H71: A novel Hsp90 inhibitor

A novel purine scaffold Hsp90 inhibitor, PU-H71, has been shown to have potent anti-tumor effects with an improved pharmacodynamic profile and less toxicity than other Hsp90 inhibitors (Caldas-Lopes et al, 2009; Cerchietti et al, 2010a; Chiosis and Neckers, 2006). Studies from our laboratory have shown that PU-H71 potently kills DLBCL cell lines, xenografts and ex vivo patient samples, in part, through degradation of BCL-6, a transcriptional repressor involved in DLCBL proliferation and survival (Cerchietti et al., 2010a) . A unique property of PU-H71 is its high affinity for tumor related-Hsp90, which explains why the drug been shown to accumulate preferentially in tumors (Caldas-Lopes et al, 2009; Cerchietti et al., 2010a). This property of PU-H71 makes it a useful tool in identifying novel targets for cancer therapy. By immobilizing PU-H71 on a solid support, a chemical precipitation (CP) of tumor-specific Hsp90 complexes can be obtained, and the substrate proteins of Hsp90 can be identified using a proteomics approach. Preliminary experiments using this method in DLBCL cell lines have revealed at least two potential targets that are stabilized by Hsp90 in DLBCL cells: the BCR pathway and the COP9 signalosome (CSN).

5. Combination Therapies in Cancer

Identifying rational combination treatments for cancer is essential because single agent therapy is not curative (Table 6). Monotherapy is not effective in cancer because of tumor cell heterogeneity. Although tumors grow from a single cell, their genetic instability produces a heterogeneous population of daughter cells that are often selected for enhanced survival capacity in the form of resistance to apoptosis, reduced dependence on normal growth factors, and higher proliferative capacity (Hanahan and Weinberg, 2000). Because tumors are comprised of heterogeneous populations of cells, a single drug will kill not all cells in a given tumor, and surviving cells cause tumor relapse. Tumor heterogeneity provides an increased number of potential drug targets and therefore, the need for combining treatments. Table 6. Multiple therapeutic agents are required for tumor cure. (Kufe DW, 2003)

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Exposure to chemotherapeutics can give rise to resistant populations of tumor cells that can survive in the presence of drug. Avoiding this therapeutic resistance is another important rationale for combination treatments.

Combinations of drugs with non-overlapping side effects can result in additive or synergistic anti-tumor effect at lower doses of each drug, thus lowering side effects. Therefore, the possible favorable outcomes for synergism or potentiation include i) increasing the efficacy of the therapeutic effect, ii) decreasing the dosage but increasing or maintaining the same efficacy to avoid toxicity, iii) minimizing the development of drug resistance, iv) providing selective synergism against a target (or efficacy synergism) versus host. Drug combinations have been widely used to treat complex diseases such as cancer and infectious diseases for these therapeutic benefits.

Because inhibition of Hsp90 kills malignant cells and results in degradation of many of its substrate proteins, identification of tumor-Hsp90 substrate proteins may reveal additional therapeutic targets. In this study, we aim to investigate the BCR pathway and the CSN, substrates of Hsp90, in DLBCL survival as potential targets for combination therapy with Hsp90 inhibition. We predict that combined inhibition of Hsp90 and its substrate proteins will synergize in killing DLBCL, providing increased patient response with decreased toxicity. 6. Synergy between inhibition of Hsp90 and its substrate BCL6: Proof of principle

The transcriptional repressor BCL6, a signature of GCB DLBCL gene expression, is the most commonly involved oncogene in DLBCL. BCL6 forms a transcriptional repressive complex to negatively regulate expression of genes involved in DNA damage response and plasma cell differentiation of GC B cells. BCL6 is required for B cells to undergo immunoglobulin affinity maturation (Ye et al., 1997), and somatic hypermutation in germinal centers. Aberrant constitutive expression of BCL6 (Ye et al., 1993), may lead to DLBCL as shown in animal models. A peptidomimetic inhibitor of BCL6, RIBPI, selectively kills BCL-6- dependent DLBCL cells (Cerchietti et al., 2010a; Cerchietti et al., 2009b) and is under development for the clinic.

CPs using PU-H71 beads reveal that BCL6 is a substrate protein of Hsp90 in DLBCL cell lines, and treatment with PU-H71 induces degradation of BCL6 (Cerchietti et al, 2009a) (Figure 18). RI-BPI synergizes with PU-H71 treatment to kill DLBCL cell lines and xenografts (Cerchietti et al., 2010b) (Figure 18). This finding acts as proof of principal that targets in DLBCL can be identified through CPs of tumor-Hsp90 and that combined inhibition of Hsp90 and its substrate proteins synergize in killing DLBCL.

7. BCR Signaling

The BCR is a large transmembrane receptor whose ligand-mediated activation leads to an extensive downstream signaling cascade in B cells (outlined in Figure 19). The extracellular ligand-binding domain of the BCR is a membrane immunoglobulin (mlg), most often mlgM or mlgD, which, like all antibodies, contains two heavy Ig (IgH) chains and two light Ig (IgL) chains. The Iga/IgP (CD79a/CD79b) heterodimer is associated with the mlg and acts as the signal transduction moiety of the receptor. Ligand binding of the BCR causes aggregation of receptors, inducing phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) found on the cytoplasmic tails of CD79a/CD79b by src family kinases (Lyn, Blk, Fyn). Syk, a cytoplasmic tyrosine kinase is recruited to doubly phosphorylated ITAMs on CD79a/CD79b, where it is activated, resulting in a signaling cascade involving Bruton's tyrosine kinase (BTK), phospholipase Cy (PLCy), and protein kinase CP (PKC-β). BLNK is an important adaptor molecule that can recruit PLCy, phosphatidylinositol-3 -kinase (PI3-K) and Vav. Activation of these kinases by BCR aggregation results in formation of the BCR signalosome at the membrane, comprised of the BCR, CD79a/CD79b heterodimer, src family kinases, Syk, BTK, BLNK and its associated signaling enzymes. The BCR signalosome mediates signal transduction from the receptor at the membrane to downstream signaling effectors.

Signals from the BCR signalosome are transduced to extracellular signal-related kinase (ERK) family proteins through Ras and to the mitogen activated protein kinase (MAPK) family through Rac/cdc43. Activation of PLCy causes increases in cellular calcium (Ca ²⁺ ), resulting in activation of Ca ²⁺ -calmodulin kinase (CamK) and NFAT. Significantly, increased cellular Ca ²⁺ activates PKC-β, which phosphorylates Carmal (CARD11), an adaptor protein that forms a complex with BCL10 and MALT1. This CBM complex activates ΙκΒ kinase (IKK), resulting in phosphorylation of ΙκΒ, which sequesters NF-κΒ subunits in the cytosol. Phosphorylated ΙκΒ is ubiquitinylated, causing its degradation and localization of NF-KB subunits to the nucleus. Many other downstream effectors in this complex pathway (p38 MAPK, ERK1/2, CaMK) translocate to the nucleus to affect changes in transcription of genes involved in cell survival, proliferation, growth, and differentiation (NF-κΒ, NFAT). Syk also activates phosphatidylinositol 3-kinase (PI3K), resulting in increased cellular PIP ₃ . This second messenger activates the acutely transforming retrovirus (Akt)/mammalian target of rapamycin (mTOR) pathway which promotes cell growth and survival (Dal Porto et al., 2004).

8. Aberrant BCR signaling in DLBCL

BCR signaling is necessary for survival and maturation of B cells (Lam et al, 1997), particularly survival signaling through NF-κΒ. In fact, constitutive NF-κΒ signaling is a hallmark of ABC DLBCL (Davis et al, 2001). Moreover, mutations in the BCR and its effectors contribute to the enhanced activity of NF-κΒ in DLBCL, specifically ABC DLBCL.

It has been shown that mutations in the ITAMs of the CD79a/CD79b heterodimer associated with hyperresponsive BCR activation and decreased receptor internalization in DLBCL (Davis et al., 2010). CD79 ITAM mutations also block negative regulation by Lyn kinase. Lyn phosphorylates immunoreceptor tyrosine-based inactivation motifs (ITIMs) on CD22 and the Fc γ-receptor, membrane receptors that communicate with the BCR. After docking on these phosphorylated ITIMs, SHPl dephosphorylates CD79 ITAMs causing downmodulation of BCR signaling. Lyn also phosphorylates Syk at a negative regulatory site, decreasing its activity (Chan et al., 1997). Taken together, mutations in CD79 ITAMs, found in both ABC and GCB DLBCL, result in decreased Lyn kinase activity and increased signaling through the BCR. Certain mutations in the BCR pathway components directly enhance NF-κΒ activity. Somatic mutations in the CARD 11 adaptor protein result in constitutive activation of IKK causing enhanced NF-κΒ activity even in the absence of BCR engagement (Lenz et al., 2008). A20, a ubiquitin-editing enzyme, terminates NF-κΒ signaling by removing ubiquitin chains from IKK. Inactivating mutations in A20 remove this brake from NF-κΒ signaling in ABC DLBCL (Compagno et al, 2009).

This constitutive BCR activity in ABC DLBCL has been referred to as "chronic active BCR signaling" to distinguish it from "tonic BCR signaling." Tonic BCR signaling maintains mature B cells and does not require CARD 11 because mice deficient in CBM components have normal numbers of B cells (Thome, 2004). Chronic active BCR signaling, however, requires the CBM complex and is distinguished by prominent BCR clustering, a characteristic of antigen- stimulated B cells and not resting B cells. In fact, knockdown of CARD11, MALT1, and BCL10 is preferentially toxic for ABC as compared to GCB DLBCL cell lines (Ngo et al, 2006). Chronic active BCR signaling is associated mostly with ABC DLBCL, however CARD 11 and CD79 IT AM mutations do occur in GCB DLBCL (Davis et al, 2010; Lenz et al, 2008), suggesting that BCR signaling is a potential target across subtypes of DLBCL.

9. Targeting the BCR pathway in DLBCL

Because it promotes cell growth, proliferation and survival, BCR signaling is an obvious target in cancer. Mutations in the BCR pathway in DLBCL (described above) highlight its relevance as a target in the disease. In fact, many components of the BCR have been targeted in DLBCL, and some of these treatments have already translated to patients. Overexpression of protein tyrosine phosphatase (PTP) receptor-type O truncated (PTPROt), a negative regulator of Syk, inhibits proliferation and induces apoptosis in DLBCL, identifying Syk as a target in DLBCL (Chen et al, 2006). Inhibition of Syk by small molecule fostamatinib disodium (R406) blocks proliferation and induces apoptosis in DLBCL cell lines (Chen et al, 2008). This orally available compound has also shown significant clinical activity with good tolerance in DLBCL patients (Friedberg et al., 2010).

An RNA interference screen revealed Btk as a potential target in DLBCL. Short hairpin RNAs (shRNAs) targeting Btk are highly toxic for DLBCL cell lines, specifically ABC DLBCL. A small molecule irreversible inhibitor of Btk, PCI-32765 (Honigberg et al., 2010), potently kills DLBCL cell lines, specifically ABC DLBCL (Davis et al, 2010). The compound is in clinical trials and has shown efficacy in B cell malignancies with good tolerability (Fowler et al., 2010).

Constitutive activity of NF-κΒ makes it a rational target in DLBCL. NF-κΒ can be targeted through different approaches. Inhibition of IKK blocks phosphorylation of ΙκΒ, preventing release and nuclear translocation of NF-κΒ subunits. MLX105, a selective IKK inhibitor, potently kills ABC DLBCL cell lines (Lam et al, 2005). NEDD8-activating enzyme (NAE) regulates the CRL1 ^|3TRCP ubiquitination of phosphorylated ΙκΒ, resulting in its degradation and the release of NF-κΒ subunits. Inhibition of NAE by small molecules such as MLN4924 induces apoptosis in ABC DLBCL and shows strong tumor burden regression in DLBCL and patient xenograft models. MLN4924 shows more potency in ABC DLBCL, which is expected because of the higher dependence on constitutive NF-κΒ activity for survival in this subtype (Milhollen et al., 2010). Because it activates IKK, inhibiting PKC-β is another approach to block NF-κΒ activity. Specific PKC-β inhibitors, such as Ly379196, kill both ABC and GCB DLBCL cell lines, albeit at high doses (Su et al, 2002).

These approaches to targeting NF-κΒ activity are promising therapies for DLBCL. Inhibition of IKK and NAE is most potent in ABC DLBCL, but less potent effect was also seen in GCB DLBCL. These studies suggest that combining NF-κΒ activity with other targeted therapies may produce a more robust effect across DLBCL subtypes.

The PI3K/Akt/mTOR pathway is deregulated in many human diseases and is constitutively activated in DLBCL (Gupta et al., 2009). Because malignant cells exploit this pathway to promote cell growth and survival, small molecule inhibitors of the pathway have been heavily researched. Rapamycin (sirolimus), a macrolide antibiotic that targets mTOR, is an FDA approved oral immunosuppressant (Yap et al., 2008). Everolimus, an orally available rapamycin analog, has also been approved as a transplant immunosuppressant (Hudes et al, 2007). These compounds have antitumor activity in DLBCL cell lines and patient samples (Gupta 2009), but their effect is mostly antiproliferative and only narrowly cytotoxic. To achieve cytotoxicity, rapamycin and everolimus have been evaluated in combination with many other therapeutic agents (Ackler et al., 2008; Yap et al., 2008). Phase II clinical studies of everolimus in DLBCL have been moderately successful with an ORR of 35% (Reeder C, 2007). Everolimus has also been shown to sensitize DLBCL cell lines to other cytotoxic agents (Wanner et al., 2006). These findings clearly demonstrate the therapeutic potential of mTOR inhibition in DLBCL, especially in combination therapies. Inhibition of Akt is also a promising cancer therapy and can be targeted in many ways. Lipid based inhibitors block the PIP3 -binding PH domain of Akt to prevent its translocation to the membrane. One such drug, perifosine, has shown antitumor activity both in vitro and in vivo.

Overall, the compound has shown only partial responses, prompting combination with other targeted therapies (Yap et al, 2008). Small molecule inhibitors of Akt, such as GSK690693, cause growth inhibition and apoptosis in lymphomas and leukemias, specifically ALL (Levy et al., 2009), and may be effective in killing DLBCL as a monotherapy or in combination with other targeted therapies. The MAPK pathway is another interesting target in cancer therapeutics. The oncogene MCT- 1 is highly expressed in DLBCL patient samples and is regulated by ERK. Inhibition of ERK causes apoptosis in DLBCL xenograft models (Dai et al., 2009). Small molecule inhibitors of ERK and MEK have been developed and demonstrate excellent safety profiles and tumor suppressive activity in the clinic. The response to these drugs, however, has not been robust with four partial patient responses observed and stable disease reported in 22% of patients (Friday and Adjei, 2008). Inhibition of MEK alone may be insufficient to cause cytotoxicity because the upstream regulators of the MAPK pathway, namely Ras and Raf, are most frequently mutated in cancer and may regulate other kinases that maintain cell survival despite MEK inhibition. In the face of these pitfalls, MEK inhibitors such as AZD6244 have entered the clinic. The partial response to MEK inhibition suggests that combinations of these inhibitors with other targeted therapies may reveal a more robust patient response (Friday and Adjei, 2008).

10. The CSN: Structure and Function The CSN was first discovered in Aradopsis in 1996 as a negative regulator of photomorphogenesis (Chamovitz et al., 1996). The complex is highly conserved from yeast to human and is comprised of eight subunits, CSN1-CSN8, numbered in size from largest to smallest (Deng et al., 2000). Most of the CSN subunits are more stable as part of the eight subunit holocomplex, but some smaller complexes, such as the mini-CSN, containing CSN4- 7, have been reported (Oron et al, 2002; Tomoda et al, 2002). CSN5, first identified as Junactivation-domain-binding protein (Jabl), functions independently of the holo-CSN, and has been shown to interact with many cellular signaling mediators (Kato and Yoneda-Kato, 2009). The molecular constitution and functionality of these complexes are not yet clearly understood.

CSN5 and CSN6 each contain an MPRl-PADl-N-terminal (MPN) domain, but only CSN5 contains a JAB1 MPN domain metalloenzyme motif (JAMM/MPN+ motif). The other six subunits contain a proteasome-COP9 signalosome-initiation factor 3 domain (PCI (or PINT)) (Hofmann and Bucher, 1998). Though the exact function of these domains is not yet fully understood, they bear an extremely similar homology to the lid complex of the proteasome and the eIF3 complex (Hofmann and Bucher, 1998), suggesting that the function of the CSN relates to protein synthesis and degradation. The best characterized function of the CSN is the regulation of protein stability. The CSN regulates protein degradation by deneddylation of cullins. Cullins are protein scaffolds at the center of the ubiquitin E3 ligase. They also serve as docking sites for ubiquitin E2 conjugating enzymes and protein substrates targeted for degradation. The cullin-RING-E3 ligases (CRLs) are the largest family of ubiquitin ligases. Post-translational modification of the cullin subunit of a CRL by conjugation of Nedd8 is required for CRL activity (Chiba and Tanaka, 2004; Ohh et al, 2002). The CSN 5 JAMM motif catalyzes removal of Nedd8 from CRLs; this deneddylation reaction requires an intact CSN holocomplex (Cope et al, 2002; Sharon et al, 2009). Although cullin deneddylation inactivates CRLs, the CSN is required for CRL activation (Schwechheimer and Deng, 2001), and may prevent CRL components from self-destruction by autoubiquitinylation (Peth et al., 2007).

The CSN has many other biological functions, including apoptosis and cell proliferation.

Knockout of CSN components 2, 3, 5, and 8 in mice causes early embryonic death due to massive apoptosis with CSN5 knockout exhibiting the most severe phenotype (Lykke- Andersen et al., 2003; Menon et al., 2007; Tomoda et al., 2004; Yan et al., 2003). These functions may be related to the complex's role in protein stability and degradation because the phenotypes in these knockout animals parallel the phenotype of NAE knockout mice (Tateishi et al., 2001) and knockout mice of various cullins (Dealy et al., 1999; Li et al., 2002; Wang et al, 1999).

Ablation of CSN5 in thymocytes results in apoptosis as a result of increased expression of proapoptotic BCL2-associated X protein (Bax) and decreased expression of anti-apoptotic Bcl-xL protein (Panattoni et al., 2008). The interaction of CSN5 with the cyclin-dependent kinase (CDK) inhibitor p27 suggests its role in cell proliferation (Tomoda et al, 1999). CSN5 knockout thymocytes display G2 arrest (Panattoni et al., 2008), while CSN8 plays a role in T cell entry to the cell cycle from quiescence (Menon et al, 2007).

11. The CSN and cancer

The involvement of the CSN in such cellular functions as apoptosis, proliferation and cell cycle regulation suggest that it may play a role in cancer. In fact, overexpression of CSN5 is observed in a variety of tumors (Table 7), and knockdown of CSN5 inhibits the proliferation of tumor cells (Fukumoto et al, 2006). CSN5 is also involved in myc-mediated transcriptional activation of genes involved in cell proliferation, invasion and angiogenesis (Adler et al, 2006). CSN2 and CSN3 are identified as putative tumor suppressors due to their ability to overcome senescence (Leal et al, 2008), and inhibit the proliferation of mouse fibroblasts (Yoneda-Kato et al, 2005), respectively.

Table 7. CSN5 Overexpression Correlating Tumor Progression or Clinical Outcome (Richardson and Zundel, 2005)

Knockdown of CSN5 in xenograft models significantly decreases tumor growth (Supriatno et al., 2005). Derivatives of the natural product curcumin inhibit the growth of pancreatic cancer cells by inhibition of CSN5 (Li et al, 2009). Taken together, these findings indicate that the CSN is a good therapeutic target in cancer.

12. The CSN and NF-KB activation: A role in DLBCL?

The CSN regulates NF-κΒ activity differently in different cellular contexts. In TNFa- stimulated synviocytes of rheumatoid arthritis patients, knockdown of CSN5 abrogates TNFRl-ligationdependent ΙκΒα degradation and NF-κΒ activation (Wang et al., 2006). Ablation of CSN subunits in TNFa-stimulated endothelial cells, however, results in stabilization of ΙκΒα and sustained nuclear translocation of NF-κΒ (Schweitzer and Naumann, 2010).

Studies of the CSN in T cells demonstrate its critical role in T cell development and survival. Thymocytes from CSN5 null mice display cell cycle arrest and increased apoptosis. Importantly, these cells show accumulation of ΙκΒα, reduced nuclear NF-κΒ accumulation, and decreased expression of anti-apoptotic NF-κΒ target genes (Panattoni et al., 2008), suggesting that CSN5 regulates T-cell activation. In fact, the CSN interacts with the CBM complex in activated T cells. T-cell activation stimulates interaction of the CSN with MALT1 and CARD11 and with BCL10 through MALT1. CSN2 and CSN 5 stabilize the CBM by deubiquitinylating BCL10. Knockdown of either subunit causes rapid degradation of BcllO and also blocks IKK activation in TCR-stimulated T cells, suggesting that CSN may regulate NF-KB activity through this mechanism (Welteke et al., 2009). The exact function of the CSN in NF-κΒ regulation is not well defined, and may differ depending on cell type. The involvement of the CSN in NF-κΒ regulation, particularly in T cells and through the stabilization of the CBM, suggests that it may play a role in DLBCL pathology. Preliminary Results

CPs were performed in OCI-Lyl and OCI-Ly7 DLBCL cell lines. Cells were lysed, and cytosolic and nuclear lysates were extracted. Lysates were incubated with either control or agarose beads coated with PUH71 overnight, then washed to remove non-specifically bound proteins. Tightly binding proteins were eluted by boiling in SDS/PAGE loading buffer, separated by SDS/PAGE and visualized by colloidal blue staining. Gel lanes were cut into segments and analyzed by mass spectroscopy by our collaborators. Proteins that were highly represented (determined by number of peptides) in PUH71 pulldowns but not control pulldowns are candidate DLBCL-related Hsp90 substrate proteins. After excluding common protein contaminants and the agarose proteome, we obtained 80% overlapping putative client proteins (N=~200) in both cell lines represented by multiple peptides. One of the pathways highly represented among PU-H71 Hsp90 clients in these experiments is the BCR pathway (23 proteins out of 200, shown in grey in Figure 19 and Figure 23). We have begun validating this finding. Preliminary data shows that Syk and Btk are both degraded with increasing PU-H71 and are both pulled down with PU-H71 in CPs of DLBCLs. PU-H71 synergizes with R406, a Syk inhibitor, to kill DLBCL cell lines (Figure 20).

Experimental Approach

AIMl: To determine whether concomitant modulation of Hsp90 and BCR pathways cooperate in killing DLBCL cells in vitro and in vivo

Our preliminary data identified many components of the BCR pathway as substrate proteins of Hsp90 in DLBCL. The BCR pathway has been implicated in oncogenesis and DLBCL survival. We hypothesize that combined inhibition of Hsp90 and components of the BCR pathway will synergize in killing DLBCL.

Experimental Design and Expected Outcomes

DLBCL cell lines will be maintained in culture. GCB DLBCL cell lines will include OCI- Lyl, OCI-Ly7, and Toledo. ABC DLBCL cell lines will include OCI-Ly3, OCI-LylO, HBL- 1, TMD8. Cell lines OCI-Lyl, OCI-Ly7, and OCI-LylO will be maintained in 90% Iscove's modified medium containing 10% FBS and supplemented with penicillin and streptomycin. Cell lines Toledo, OCI-Ly3, and HBL-1 will be grown in 90% RPMI and 10% FBS supplemented with penicillin and streptomycin, L-glutamine, and HEPES. The TMD8 cell line will be grown in medium containing 90% mem-alpha and 10% FBS supplemented with penicillin and streptomycin.

Components of the BCR pathway were identified as subtrate proteins of Hsp90 in a preliminary experiment of a proteomics analysis of PU-H71 CPs in two DLBCL cell lines.

To verify that the components of the BCR pathway are stabilized by Hsp90, CPs will be performed using DLBCL cell lines and analyzed by western blot using commercially available antibodies to BCR pathway components, including CD79a, CD79b, Syk, Btk, PLCy2, AKT, mTOR, CAMKII, p38 MAPK, p40 ERK1/2, p65, Bcl-XL, Bcl6. CPs will be performed with increasing salt concentrations to show the affinity of Hsp90 for these substrate proteins. Because some proteins are expressed at low levels, nuclear/cytosolic separation of cell lysates will be performed to enrich for Hsp90 substrate proteins that are not readily detected using whole cell lysate.

Hsp90 stabilization of BCR pathway components will also be demonstrated by treatment of DLBCL cell lines with increasing doses of PU-H71. Levels of the substrate proteins listed above will be determined by western blot. Substrate proteins are expected to be degraded by exposure to PU-H71 in a dose-dependent and time-dependent manner.

Viability of DLBCL cell lines will be assessed following treatment with PU-H71 or inhibitors of BCR pathway components (Syk, Btk, PLCy2, AKT, mTOR, p38 MAPK, p40 ERK1/2, NF-KB). Inhibitors of BCR pathway components will be selected and prioritized based on reported data in DLBCL and use in clinical trials. For example, the Melnick lab has MTAs in place to use PCI-32765 and MLN4924 (described above). Cells will be plated in 96-well plates at concentrations sufficient to keep untreated cells in exponential growth for the duration of drug treatment. Drugs will be administered in 6 different concentrations in triplicate wells for 48 hours. Cell viability will be measured with a fluorometric resazurin reduction method (CellTiter-Blue, Promega).

Fluorescence (560 _e xcitation/590 _e mission) will be measured using the Synergy4 microplate reader in the Melnick lab (BioTek). Viability of treated cells will be normalized to appropriate vehicle controls, for example, water, in the case of PU-H71. Dose-effect curves and calculation of the drug concentration that inhibits the growth of the cell line by 50% compared to control (GI50) will be performed using CompuSyn software (Biosoft). Although many of these findings may be confirmatory of published data, instituting effective methods with these inhibitors and determining their dose-responses in our cell lines will be necessary for later combination treatment experiments demonstrating the effect of combined inhibition of Hsp90 and the BCR pathway. Once individual dose-response curves and GI50s for BCR pathway inhibitors have been established, DLBCL cells will be treated with both PU-H71 and single inhibitors of the BCR pathway to demonstrate the effect of the combination on cell killing. Experiments will be performed in 96-well plates using the conditions described above. Cells will be treated with 6 different concentrations of combination of drugs in constant ratio in triplicate with the highest dose being twice the GI50 of each drug as measured in individual dose-response experiments. Drugs will be administered in different sequences in order to determine the most effective treatment schedule: PU-H71 followed by drug X after 24 hours, drug X followed by PU-H71 after 24 hours, and PU-H71 with drug X. Viability will be determined after 48 hours using the assays mentioned above. Isobologram analysis of cell viability will be performed using Compusyn software.

Combination treatments in DLBCL cell lines proposed above will guide experiments in xenograft models in terms of dose and schedule. The drug schedules that exhibit the best cell killing effect will be translated to xenograft models. DLBCL cell lines will be injected subcutaneously into SCID mice, using two cell lines expected to respond to drug and one cell line expected not to respond as a negative control. Tumor growth will be monitored every other day until palpable (about 75-100 mm ³ ). Animals (n=20) will be randomly divided into the following groups: control, PU-H71, BCR pathway inhibitor (drug X), and PU-H71 + drug X with five animals per group. To measure drug effect on tumor growth, tumor volume will be measured with Xenogen IVIS system every other day after drug administration. After ten days, all animals will be sacrificed, and tumors will be assayed for apoptosis by TUNEL. To assess drug effect on survival, a second cohort of animals as specified above will be treated and sacrificed when tumors reach 1000mm ³ in size. Tumors will be analyzed biochemically to demonstrate that the drugs hit their targets, by ELISA for NF-κΒ activity or phosphorlyation of downstream targets, for example. We will perform toxicity studies established in the Melnick lab (Cerchietti et al., 2009a) in treated mice including physical examination, macro and microscopic tissue examination, serum chemistries and CBCs. Alternatives and Pitfalls

If the fluorescence assay used to detect cell viability is incompatible with some cell lines (due to acidity of media, for example,) an ATP -based luminescent method (CellTiter-Glo,

Promega) will be used. Also, because some drugs may not kill cells in 48 hours, higher drug doses and longer drug incubations will be performed if necessary to determine optimal drug treatments. It is possible inhibition of some BCR pathway components will not demonstrate an improved effect in killing DLBCL when combined with inhibition of Hsp90, but based on preliminary data shown above, we believe that some combinations will be more effective than either drug alone.

AIM 2: To evaluate the role of the CSN in DLBCL

Subaim 1: To determine whether the CSN can be a therapeutic target in DLBCL

Our preliminary data has identified subunits of the CSN as substrate proteins of Hsp90 in DLBCL. The CSN has been implicated in cancer and may play a role in DLBCL survival. We hypothesize that DLBCL requires the CSN for survival and that combined inhibition of Hsp90 and the CSN will synergize in killing DLBCL.

Experimental Design and Expected Outcomes

Expression of CSN subunits in DLBCL cell lines (described above) will be verified. DLBCL cell lines will be lysed for protein harvest and analyzed by SDS-PAGE and western blotting using commercially available antibodies to the CSN subunits and actin as a loading control.

The CSN was identified as a substrate protein of Hsp90 in a preliminary proteomics analysis of PU-H71 CPs in two DLBCL cell lines. To verify that Hsp90 stabilizes the CSN, CPs will be performed as described above using DLBCL cell lines and analyzed by western blot. Hsp90 stabilization of the CSN will also be demonstrated by treatment of DLBCL cell lines with increasing PU-H71 concentration. Protein levels of CSN subunits will be determined by western blot. CSN subunits are expected to be degraded upon exposure to PU-H71 in a dose- dependent and time-dependent manner.

DLBCL cells lines will be infected with lentiviral pLKO. l vectors containing short hairpin

(sh)RNAs targeting CSN2 or CSN5 and selected by puromycin resistance. These vectors are commercially available through the RNAi Consortium. These subunits will be used because knockdown of one CSN subunit can affect expression of other CSN subunits (Menon et al, 2007; Schweitzer et al, 2007; Schweitzer and Naumann, 2010), and knockdown of CSN2 ablates formation of the CSN holocomplex. CSN5 knockdown will be used because this subunit contains the enzymatic domain of the CSN. A pool of 3 to 5 shRNAs will be tested against each target to obtain the sequence with optimal knockdown of the target protein.

Empty vector and scrambled shRNAs will be used as controls. Because we predict that knockdown of CSN subunits will kill DLBCL cells, and we aim to measure cell viability, tetracycline (tet) inducible constructs will be used. This method may also allow us to establish conditions for dose-dependent knockdown of CSN subunits using a titration of shRNA induction. Knockdown efficiency will be assessed by western blot following infection and tet induction. Cells will be assessed for viability using the methods described in Aim 1 following infection. We predict that knockdown the CSN will kill DLBCLs, and ABC DLBCLs are expected to depend on the CSN for survival more than GCB DLBCLs because of the CSN's role in stabilizing the CBM complex. Following CSN monotherapy experiments in DLBCL, induction of CSN knockdown will be combined with PU-H71 treatment in DLBCL cell lines. shRNA constructs that demonstrate effective dose dependent CSN knock down in 48 hours (as evaluated in earlier experiments) will be used in order to perform 48 hour cell viability experiments. Control shRNAs as described above will be used. Control cells and cells infected with tet-inducible shRNA constructs targeting CSN subunits will be treated with different doses of tet and PU-H71 in constant ratio in triplicate. Drugs will be administered in different sequences in order to determine the most effective treatment schedule: PU-H71 followed by tet, tet followed by PU-H71, and PU-H71 with tet. Cell viability will be measured as described in Aim 1. Combined inhibition of the CSN and Hsp90 is expected to synergize in killing DLBCL, specifically ABC DLBCL.

Combined inhibition of the CSN and Hsp90 in DLBCL cell lines proposed above will guide experiments in xenograft models. The most effective combination of PU-H71 and CSN knockdown from in vitro experiments will be used in xenograft experiments. Control and inducible-knockout-CSN DLBCL cells will be used for xenograft, using two cell lines expected to respond to treatment and one cell line expected not to respond to treatment as a negative control. Animals will be treated with vehicle, PU-H71, or tet, using the dose and schedule of the most effective combination of PU-H71 and tet as determined by in vitro experiments. Tumor growth, animal survival and toxicity will be assayed as described in Aim 1.

Alternatives and Pitfalls

Accomplishing dose-dependent knockdown of the CSN by titration of tetracycline induction may prove difficult. If this occurs, in order to demonstrate proof of principle, shRNAs with different knockdown efficiencies will be used to simulate increasing inhibition of the CSN as a monotherapy and in combination with different doses of PU-H71.

Subaim 2: To determine the mechanism of DLBCL dependence on the CSN

Since the CSN has been shown to interact with the CBM complex and activate IKK in stimulated T-cells, we hypothesize that the CSN interacts with the CBM, stabilizes Bel 10, and activates NF-κΒ in DLBCL.

Experimental Design and Expected Outcomes

DLBCL cell lysates will be incubated with an antibody to CSN1 that effectively precipitates the whole CSN complex (da Silva Correia et al, 2007; Wei and Deng, 1998). Precipitated CSN1 complexes will be separated by SDS-PAGE and analyzed for interaction with CBM components by western blot using commercially available antibodies to the different components of the CBM: CARD11, BCL10, and MALT1. Based on reported experiments in T cells, we expect the CSN to interact preferentially with CARD 11 and MALT1 in ABC DLBCL cell lines as opposed to GCB DLBCL cell lines because of the chronic active BCR signaling in ABC DLBCL.

Because the CSN, specifically CSN5, has been shown to regulate BcllO stability and degradation in activated T-cells, we hypothesize that the CSN stabilizes BcllO in DLBCL. DLBCL cells lines will be infected with short hairpin (sh)RNAs targeting CSN subunits as described above. Cells will be treated with tet to induce CSN subunit knockdown and BcllO protein levels in infectedand induced cells will be quantified by western blot. We expect BcllO levels to be degraded with CSN subunit knockdown in a dose-dependent and time- dependent manner. To demonstrate that reduction in BcllO protein is not a result of cell death, cell viability will be measured by counting viable cells with Trypan blue before cell lysis. CSN subunit knockdown will be combined with proteasome inhibition to demonstrate that BcllO degradation is a specific effect of CSN ablation. Knockdown of CSN2 or CSN5 is expected to abrogate NF-κΒ activity in DLBCL cell lines.

Using DLBCL cell lines infected with control shRNAs or shRNAs to CSN2 or CSN5, control and infected cells will be assayed for NF-κΒ activity in several ways. First, lysates will be analyzed by western blot to determine levels of ΙκΒα protein. Second, nuclear translocation of the NF-KB subunits p65 and c-Rel will be measured by western blot of nuclear and cytosolic fractions of lysed cells or by plate-based EMSA of nuclei from control and infected cells. Finally, NF-κΒ target gene expression of these cells will be evaluated at the transcript and protein level by quantitative PCR of cDNA prepared by reverse transcriptase PCR (RT- PCR) and western blot, respectively.

Alternatives and Pitfalls

Because the CSN was shown to interact with the CBM in TCR-stimulated T cells, we predict that the CSN interacts with the CBM in DLBCL, especially in ABC DLBCL because this subtype exhibits chronic active BCR signaling. If CSN-CBM interaction is not apparent in DLBCL, then cells will be stimulated with IgM in order to activate the BCR pathway and stimulate formation of the CBM. To determine the kinetics of the CSN interaction with the CBM, cellular IPs as described above will be performed over a time course from the point of IgM stimulation. To correlate CSN-CBM interaction with the kinetics of CBM formation, BCL10 IP will be performed to demonstrate BCLIO-CARDI 1 interaction over the same time course.

Conclusions and Future Directions

The development of PU-H71 as a new therapy for DLBCL is promising, but combination treatments are likely to be more potent and less toxic. PU-H71 can also be used as a tool to identify substrate proteins of Hsp90. In experiments using this method, the BCR pathway and the CSN were identified as substrates of Hsp90 in DLBCL. The BCR plays a role in DLBCL oncogenesis and survival, and efforts to target components of this pathway have been successful. We predict that combining PU-H71 and inhibition of BCR pathway components will be a more potent and less toxic treatment approach. Identified synergistic combinations in cells and xenograft models will be evaluated for translation to clinical trials, and ultimately advance patient treatment toward rationally designed targeted therapy and away from chemotherapy.

The CSN has been implicated in cancer and NF-κΒ activation, indicating that it may be a good target in DLBCL. We hypothesize that the CSN stabilizes the CBM complex, promoting NF-κΒ activation and DLBCL survival. Therefore, we predict that combined inhibition of Hsp90 and the CSN will synergize in killing DLBCL. These studies will act as proof of principle that new therapeutic targets can be identified using the proteomics approach described in this proposal. Future studies will identify compounds that target the CSN, and ultimately bring CSN inhibitors to the clinic as an innovative therapy for DLBCL. Determining downstream effects of CSN inhibition, such as CBM stabilization and NF-κΒ activation may reveal new opportunities for additional combinatorial drug regimens of three drugs. Future studies will evaluate combinatorial regimens of three drugs inhibiting Hsp90, the CSN and its downstream targets together.

The most effective drug combinations with PU-H71 found in this study will be performed using other Hsp90 inhibitors in clinical development such as 17-DMAG to demonstrate the broad clinical applicability of identified effective drug combinations.

DLBCL, the most common form of non-Hodgkins lymphoma, is an aggressive disease that remains without cure. The studies proposed herein will advance the understanding of the molecular mechanisms behind DLBCL and improve patient therapy.

Here, we report on the design and synthesis of molecules based on purine, purine-like isoxazole and indazol-4-one chemical classes attached to Affi-Gel ^® 10 beads (Figures 30, 32, 33, 35, 38) and on the synthesis of a biotinylated purine, purine-like, indazol-4-one and isoxazole compounds (Figures 31, 36, 37, 39, 40). These are chemical tools to investigate and understand the molecular basis for the distinct behavior of Hsp90 inhibitors. They can be also used to better understand Hsp90 tumor biology by examining bound client proteins and co-chaperones. Understanding the tumor specific clients of Hsp90 most likely to be modulated by each Hsp90 inhibitor could lead to a better choice of pharmacodynamic markers, and thus a better clinical design. Not lastly, understanding the molecular differences among these Hsp90 inhibitors could result in identifying characteristics that could lead to the design of an Hsp90 inhibitor with most favorable clinical profile.

Methods of Synthesizing of Hsp90 Probes

6.1. General 1H and ¹³ C NMR spectra were recorded on a Bruker 500 MHz instrument. Chemical shifts were reported in δ values in ppm downfield from TMS as the internal standard. 1H data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constant (Hz), integration. ¹³ C chemical shifts were reported in δ values in ppm downfield from TMS as the internal standard. Low resolution mass spectra were obtained on a Waters Acquity Ultra Performance LC with electrospray ionization and SQ detector. High-performance liquid chromatography analyses were performed on a Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge CI 8, 4.6 x 150 mm, 5 μιη) using a gradient of (a) H ₂ 0 + 0.1% TFA and (b) CH ₃ CN + 0.1% TFA, 5 to 95% b over 10 minutes at 1.2 mL/min. Column chromatography was performed using 230-400 mesh silica gel (EMD). All reactions were performed under argon protection. Affi-Gel ^® 10 beads were purchased from Bio-Rad (Hercules, CA). EZ-Link ^® Amine-PE0 ₃ -Biotin was purchased from Pierce (Rockford, II). PU-H71 (He et al, 2006) and NVP-AUY922 (Brough et al, 2008) were synthesized according to previously published methods. GM was purchased from Aldrich.

6.2. Synthesis

6.2.1. 9-(3-Bromopropyl)-8-(6-iodobenzo[d] [l,3]dioxol-5-ylthio)-9H-purin-6-amine (2) 1 (He et al, 2006) (0.500 g, 1.21 mmol) was dissolved in DMF (15 mL). Cs ₂ C0 ₃

(0.434 g, 1.33 mmol) and 1,3-dibromopropane (1.22 g, 0.617 mL, 6.05 mmol) were added and the mixture was stirred at rt for 45 minutes. Then additional Cs ₂ C0 ₃ (0.079 g, 0.242 mmol) was added and the mixture was stirred for 45 minutes. Solvent was removed under reduced pressure and the resulting residue was chromatographed (CH ₂ Cl ₂ :MeOH:AcOH, 120: 1 :0.5 to 80: 1 :0.5) to give 0.226 g (35%) of 2 as a white solid. 1H NMR (CDCl ₃ /MeOH- d ₄ ) δ 8.24 (s, 1H), 7.38 (s, 1H), 7.03 (s, 1H), 6.05 (s, 2H), 4.37 (t, J= 7.1 Hz, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.41 (m, 2H); MS (ESI): m/z 534.0/536.0 [M+H] ⁺ .

6.2.2. terf-Butyl 6-aminohexylcarbamate (3) (Hansen et al, 1982)

1,6-diaminohexane (10 g, 0.086 mol) and Et ₃ N (13.05 g, 18.13 mL, 0.129 mol) were suspended in CH ₂ C1 ₂ (300 mL). A solution of di-tert-butyl dicarbonate (9.39 g, 0.043 mol) in

CH ₂ C1 ₂ (100 mL) was added dropwise over 90 minutes at rt and stirring continued for 18 h.

The reaction mixture was added to a seperatory funnel and washed with water (100 mL), brine (100 mL), dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The resulting residue was chromatographed [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 70: 1 to 20: 1] to give 7.1 g (76%) of 3. 1H NMR (CDC1 ₃ ) δ 4.50 (br s, 1H), 3.1 1 (br s, 2H), 2.68 (t, J = 6.6 Hz, 2H), 1.44 (s, 13H), 1.33 (s, 4H); MS (ESI): m/z 217.2 [M+H] ⁺ . 6.2.3. terf-Butyl 6-(3-(6-amino-8-(6-iodobenzo[d] [l,3]dioxol-5-ylthio)-9H-purin-9- yl)propylamino)hexylcarbamate (4)

2 (0.226 g, 0.423 mmol) and 3 (0.915 g, 4.23 mmol) in DMF (7 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH:MeOH-NH ₃ (7N), 100:7:3] to give 0.255 g (90%) of 4. 1H NMR (CDC1 ₃ ) δ 8.32 (s, 1H), 7.31 (s, 1H), 6.89 (s, 1H), 5.99 (s, 2H), 5.55 (br s, 2H), 4.57 (br s, 1H), 4.30 (t, J = 7.0 Hz, 2H), 3.10 (m, 2H), 2.58 (t, J = 6.7 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 1.99 (m, 2H), 1.44 (s, 13H), 1.30 (s, 4H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 156.0, 154.7, 153.0, 151.6, 149.2, 149.0, 146.3, 127.9, 120.1 , 1 19.2, 1 12.4, 102.3, 91.3, 79.0, 49.8, 46.5, 41.8, 40.5, 31.4, 29.98, 29.95, 28.4, 27.0, 26.7; HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₂₆ H ₃₇ IN ₇ 0 ₄ S, 670.1673; found 670.1670; HPLC: t _R = 7.02 min.

6.2.4. N ¹ -(3-(6-Amino-8-(6-iodobenzo[d] [l,3]dioxol-5-ylthio)-9H-purin-9- yl)propyl)hexane-l,6-diamine (5)

4 (0.310 g, 0.463 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue chromatographed [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 20: 1 to 10: 1] to give 0.37 g of a white solid. This was dissolved in water (45 mL) and solid Na ₂ C0 ₃ added until pH~12. This was extracted with CH ₂ C1 ₂ (4 x 50 mL) and the combined organic layers were washed with water (50 mL), dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to give 0.200 g (76%) of 5. 1H NMR (CDC1 ₃ ) δ 8.33 (s, 1H), 7.31 (s, 1H), 6.89 (s, 1H), 5.99 (s, 2H), 5.52 (br s, 2H), 4.30 (t, J = 6.3 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.59 (t, J = 6.3 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 1.99 (m, 2H), 1.44 (s, 4H), 1.28 (s, 4H); ¹³ C NMR (125 MHz, CDCl ₃ /MeOH-<¾) δ 154.5, 152.6, 151.5, 150.0, 149.6, 147.7, 125.9, 1 19.7, 1 19.6, 1 13.9, 102.8, 94.2, 49.7, 46.2, 41.61 , 41.59, 32.9, 29.7, 29.5, 27.3, 26.9; HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₂ iH ₂₉ IN ₇ 0 ₂ S, 570.1 148; found 570.1 124; HPLC: t _R = 5.68 min.

6.2.5. PU-H71-Affi-Gel 10 beads (6)

4 (0.301 g, 0.45 mmol) was dissolved in 15 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue dried under high vacuum overnight. This was dissolved in DMF (12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed, 3 x 50 mL DMF) in a solid phase peptide synthesis vessel. 225 of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (0.085 g, 97 μΐ, 1.13 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ Cl ₂ :Et ₃ N (9: 1, 4 x 50 mL), DMF (3 x 50 mL), Felts buffer (3 x 50 mL) and z ^' -PrOH (3 x 50 mL). The beads 6 were stored in z ^' -PrOH (beads: z-PrOH (l :2), v/v) at -80°C. 6.2.6. PU-H71-biotin (7)

2 (4.2 mg, 0.0086 mmol) and EZ-Link ^® Amine-PE0 ₃ -Biotin (5.4 mg, 0.0129 mmol) in DMF (0.2 mL) was stirred at rt for 24 h. The reaction mixture was concentrated and the residue chromatographed [CHCl ₃ :MeOH-NH ₃ (7N), 5: 1] to give 1.1 mg (16%) of 7. 1H NMR (CDC1 ₃ ) δ 8.30 (s, 1H), 8.10 (s, 1H), 7.31 (s, 1H), 6.87 (s, 1H), 6.73 (br s, 1H), 6.36 (br s, 1H), 6.16 (br s, 2H), 6.00 (s, 2H), 4.52 (m, 1H), 4.28-4.37 (m, 3H), 3.58-3.77 (m, 10H), 3.55 (m, 2H), 3.43 (m, 2H), 3.16 (m, 1H), 2.92 (m, 1H), 2.80 (m, 2H), 2.72 (m, 1H), 2.66 (m, 2H), 2.17 (t, J = 7.0 Hz, 2H), 2.04 (m, 2H), 1.35-1.80 (m, 6H); MS (ESI): m/z 872.2 [M+H] ⁺ .

6.2.7. terf-Butyl 6-(4-(5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-3- (ethylcarbamoyl)isoxazol-4-yl)benzylamino)hexylcarbamate (9)

AcOH (0.26 g, 0.25 mL, 4.35 mmol) was added to a mixture of 8 (Brough et al, 2008) (0.5 g, 0.87 mmol), 3 (0.56 g, 2.61 mmol), NaCNBH ₃ (0.11 g, 1.74 mmol), CH ₂ C1 ₂ (21 mL) and 3 A molecular sieves (3 g). The reaction mixture was stirred for 1 h at rt. It was then concentrated under reduced pressure and chromatographed [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 100: 1 to 60: 1] to give 0.50 g (75%) of 9. 1H NMR (CDC1 ₃ ) δ 7.19-7.40 (m, 12H), 7.12-7.15 (m, 2H), 7.08 (s, 1H), 6.45 (s, 1H), 4.97 (s, 2H), 4.81 (s, 2H), 3.75 (s, 2H), 3.22 (m, 2H), 3.10 (m, 3H), 2.60 (t, J= 7.1 Hz, 2H), 1.41-1.52 (m, 13H), 1.28-1.35 (m, 4H), 1.21 (t, J= 7.2 Hz, 3H), 1.04 (d, J= 6.9 Hz, 6H); MS (ESI): m/z 775.3 [M+H] ⁺ . 6.2.8. 4-(4-((6-Aminohexylamino)methyl)phenyl)-5-(2,4-dihydroxy-5-i sopropylphenyl)- N-ethylisoxazole-3-carboxamide (10)

To a solution of 9 (0.5 g, 0.646 mmol) in CH ₂ C1 ₂ (20 mL) was added a solution of BC1 ₃ (1.8 mL, 1.87 mmol, 1.0 M in CH ₂ C1 ₂ ) and this was stirred at rt for 10 h. Saturated NaHC0 ₃ was added and CH ₂ C1 ₂ was evaporated under reduced pressure. The water was carefully decanted and the remaining yellow precipitate was washed a few times with EtOAc and CH ₂ C1 ₂ to give 0.248 g (78%) of 10. 1H NMR (CDCl ₃ /MeOH-<¾) δ 7.32 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 6.94 (s, 1H), 6.25 (s, 1H), 3.74, (s, 2H), 3.41 (q, J = 7.3 Hz, 2H), 3.08 (m, 1H), 2.65 (t, J = 7.1 Hz, 2H), 2.60 (t, J= 7.1 Hz, 2H), 1.40-1.56 (m, 4H), 1.28- 1.35 (m, 4H), 1.21 (t, J = 7.3 Hz, 3H), 1.01 (d, J = 6.9 Hz, 6H); ¹³ C NMR (125 MHz, CDCl ₃ /MeOH-<¾) δ 168.4, 161.6, 158.4, 157.6, 155.2, 139.0, 130.5, 129.5, 128.71, 128.69, 127.6, 116.0, 105.9, 103.6, 53.7, 49.2, 41.8, 35.0, 32.7, 29.8, 27.6, 27.2, 26.4, 22.8, 14.5; HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₂₈ H ₃₉ N ₄ 0 ₄ , 495.2971; found 495.2986; HPLC: t _R = 6.57 min.

6.2.9. NVP-AUY922-Affi-Gel 10 beads (11)

10 (46.4 mg, 0.094 mmol) was dissolved in DMF (2 mL) and added to 5 mL of Affi- Gel 10 beads (prewashed, 3 x 8 mL DMF) in a solid phase peptide synthesis vessel. 45 μΐ of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (17.7 mg, 21 μΐ, 0.235 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ C1 ₂ (3 x 8 mL), DMF (3 x 8 mL), Felts buffer (3 x 8 mL) and i- PrOH (3 x 8 mL). The beads 11 were stored in z-PrOH (beads: z-PrOH, (1 :2), v/v) at -80°C. 6.2.10. N'-(3,3-Dimethyl-5-oxocyclohexylidene)-4-methylbenzenesulfon ohydrazide (14)

(Hiegel & Burk, 1973)

10.00 g (71.4 mmol) of dimedone (13), 13.8 g (74.2 mmol) of tosyl hydrazide (12) and /^-toluene sulfonic acid (0.140 g, 0.736 mmol) were suspended in toluene (600 mL) and this was refluxed with stirring for 1.5 h. While still hot, the reaction mixture was filtered and the solid was washed with toluene (4 x 100 mL), ice-cold ethyl acetate (2 x 200 mL) and hexane (2 x 200 mL) and dried to give 19.58 g (89%) of 14 as a solid. TLC (100% EtOAc) R _f = 0.23; 1H NMR (DMSO-<¾) δ 9.76 (s, 1H), 8.65 (br s, 1H), 7.69 (d, J= 8.2 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 5.05 (s, 1H), 2.39 (s, 3H), 2.07 (s, 2H), 1.92 (s, 2H), 0.90 (s, 6H); MS (ESI): m/z 309.0 [M+H] ⁺ .

6.2.11. 6,6-Dimethyl-3-(trifluoromethyl)-6,7-dihydro-lH-indazol-4(5H )-one (15)

To 5.0 g (16.2 mmol) of 14 in THF (90 mL) and Et ₃ N (30 mL) was added trifluoroacetic anhydride (3.4 g, 2.25 mL, 16.2 mmol) in one portion. The resulting red solution was heated at 55°C for 3 h. After cooling to rt, methanol (8 mL) and 1M NaOH (8 mL) were added and the solution was stirred for 3 h at rt. The reaction mixture was diluted with 25 mL of saturated NH ₄ C1, poured into a seperatory funnel and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (3 x 50 mL), dried over Na ₂ S0 ₄ and concentrated under reduced pressure to give a red oily residue which was chromatographed (hexane:EtOAc, 80:20 to 60:40) to give 2.08 g (55%) of 15 as an orange solid. TLC (hexane:EtOAc, 6:4) R _f = 0.37; 1H NMR (CDC1 ₃ ) δ 2.80 (s, 2H), 2.46 (s, 2H), 1.16 (s, 6H); MS (ESI): m/z 231.0 [M-H] ^" .

6.2.12. 2-Bromo-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-te trahydro-lH- indazol-l-yl)benzonitrile (16)

To a mixture of 15 (0.100 g, 0.43 mmol) and NaH (15.5 mg, 0.65 mmol) in DMF (8 mL) was added 2-bromo-4-fluorobenzonitrile (86 mg, 0.43 mmol) and heated at 90°C for 5 h. The reaction mixture was concentrated under reduced pressure and the residue chromatographed (hexane:EtOAc, 10: 1 to 10:2) to give 0.162 g (91%) of 16 as a white solid. 1H NMR (CDCI3) δ 7.97 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 8.4, 2.1 Hz, 1H), 2.89 (s, 2H), 2.51 (s, 2H), 1.16 (s, 6H); MS (ESI): m/z 410.0/412.0 [M-H] ^" .

6.2.13. 2-(irans-4-Aminocyclohexylamino)-4-(6,6-dimethyl-4-oxo-3-(tr ifluoromethyl)- 4,5,6,7-tetrahydro-lH-indazol-l-yl)benzonitrile (17)

A mixture of 16 (0.200 g, 0.485 mmol), NaOtBu (93.3 mg, 0.9704 mmol), Pd ₂ (dba) ₃

(88.8 mg, 0.097 mmol) and DavePhos (38 mg, 0.097 mmol) in 1 ,2-dimethoxy ethane (15 mL) was degassed and flushed with argon several times, trans- 1 ,4-Diaminocyclohexane (0.166 g, 1.456 mmol) was added and the flask was again degassed and flushed with argon before heating the reaction mixture at 50°C overnight. The reaction mixture was concentrated under reduced pressure and the residue purified by preparatory TLC (CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1) to give 52.5 mg (24%) of 17. Additionally, 38.5 mg (17%) of amide 18 was isolated for a total yield of 41%. 1H NMR (CDC1 ₃ ) δ 7.51 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 6.70 (dd, J = 8.3, 1.8 Hz, 1H), 4.64 (d, J = 7.6 Hz, 1H), 3.38 (m, 1H), 2.84 (s, 2H), 2.81 (m, 1H), 2.49 (s, 2H), 2.15 (d, J = 11.2 Hz, 2H), 1.99 (d, J = 11.0 Hz, 2H), 1.25-1.37 (m, 4H), 1.14 (s, 6H); MS (ESI): m/z 446.3 [M+H] ⁺ .

6.2.14. 2-(irans-4-Aminocyclohexylamino)-4-(6,6-dimethyl-4-oxo-3-(tr ifluoromethyl)- 4,5,6,7-tetrahydro-lH-indazol-l-yl)benzamide (18) A solution of 17 (80 mg, 0.18 mmol) in DMSO (147 μΐ), EtOH (590 μΐ), 5N NaOH (75 μΐ) and H ₂ 0 ₂ (88 μΐ) was stirred at rt for 3 h. The reaction mixture was concentrated under reduced pressure and the residue purified by preparatory TLC [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 10: 1] to give 64.3 mg (78%) of 18. 1H NMR (CDC1 ₃ ) δ 8.06 (d, J = 7.5 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.62 (dd, J = 8.4, 2.0 Hz, 1H), 5.60 (br s, 2H), 3.29 (m, 1H), 2.85 (s, 2H), 2.77 (m, 1H), 2.49 (s, 2H), 2.13 (d, J = 11.9 Hz, 2H), 1.95 (d, J = 11.8 Hz, 2H), 1.20-1.42 (m, 4H), 1.14 (s, 6H); MS (ESI): m/z 464.4 [M+H] ⁺ ; HPLC: t _R = 7.05 min. 6.2.15. tert- utyl 6-(irans-4-(2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-(trifluorome thyl)- 4,5,6,7-tetrahydro-lH-indazol-l-yl)phenylamino)cyclohexylami no)-6- oxohexylcarbamate (19)

To a mixture of 18 (30 mg, 0.0647 mmol) in CH ₂ C1 ₂ (1 ml) was added 6-(Boc- amino)caproic acid (29.9 mg, 0.1294 mmol), EDCI (24.8 mg, 0.1294 mmol) and DMAP (0.8 mg, 0.00647 mmol). The reaction mixture was stirred at rt for 2 h then concentrated under reduced pressure and the residue purified by preparatory TLC [hexane:CH ₂ Cl ₂ :EtOAc:MeOH-NH ₃ (7N), 2:2: 1 :0.5] to give 40 mg (91%) of 19. 1H NMR (CDCl ₃ /MeOH-<¾) δ 7.63 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 1.7 Hz, 1H), 6.61 (dd, J = 8.4, 2.0 Hz, 1H), 3.75 (m, 1H), 3.31 (m, 1H), 3.06 (t, J = 7.0 Hz, 2H), 2.88 (s, 2H), 2.50 (s, 2H), 2.15 (m, 4H), 2.03 (d, J = 11.5 Hz, 2H), 1.62 (m, 2H), 1.25-1.50 (m, 17H), 1.14 (s, 6H); ¹³ C NMR (125 MHz, CDCl ₃ /MeOH-^) δ 191.5, 174.1, 172.3, 157.2, 151.5, 150.3, 141.5, 140.6 (q, J = 39.4 Hz), 130.8, 120.7 (q, J = 268.0 Hz), 116.2, 114.2, 109.5, 107.3, 79.5, 52.5, 50.7, 48.0, 40.4, 37.3, 36.4, 36.0, 31.6, 31.3, 29.6, 28.5, 28.3, 25.7, 25.4; HRMS (ESI) m/z [M+Na] ⁺ calcd. for C ₃₄ H ₄ 7F ₃ N ₆ 0 ₅ Na, 699.3458; found 699.3472; HPLC: t _R = 9.10 min.

6.2.16. 2-(trans-4-(6- Aminohexanamido)cyclohexylamino)-4-(6,6-dimethyl-4-oxo-3- (trifluoromethyl)-4,5,6,7-tetrahydro-lH-indazol-l-yl)benzami de (20)

19 (33 mg, 0.049 mmol) was dissolved in 1 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 45 min. Solvent was removed under reduced pressure and the residue purified by preparatory TLC [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 6: 1] to give 24 mg (86%) of 20. 1H

NMR (CDCl ₃ /MeOH-<¾) δ 7.69 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 1.9 Hz, 1H), 6.64 (dd, J =

8.4, 1.9 Hz, 1H), 3.74 (m, 1H), 3.36 (m, 1H), 2.92 (t, J = 7.5 Hz, 2H), 2.91 (s, 2H), 2.51 (s,

2H), 2.23 (t, J = 7.3 Hz, 2H), 2.18 (d, J = 10.2 Hz, 2H), 2.00 (d, J = 9.1 Hz, 2H), 1.61-1.75

(m, 4H), 1.34-1.50 (m, 6H), 1.15 (s, 6H); ¹³ C NMR (125 MHz, MeOH-^) δ 191.2, 173.6, 172.2, 151.8, 149.7, 141.2, 139.6 (q, J = 39.5 Hz), 130.3, 120.5 (q, J = 267.5 Hz), 115.5, 114.1, 109.0, 106.8, 51.6, 50.0, 47.8, 39.0, 36.3, 35.2, 35.1, 31.0, 30.5, 26.8, 26.7, 25.4, 24.8; HRMS (ESI) m/z [M+H] ⁺ calcd. for C ₂ 9H ₄ 0F3N6O3, 577.3114; found 577.3126; HPLC: t _R = 7.23 min.

6.2.17. SNX-2112-Affi-Gel 10 beads (21)

19 (67 mg, 0.0992 mmol) was dissolved in 3.5 mL of CH ₂ C1 ₂ :TFA (4: 1) and the solution was stirred at rt for 20 min. Solvent was removed under reduced pressure and the residue dried under high vacuum for two hours. This was dissolved in DMF (2 mL) and added to 5 mL of Affi-Gel 10 beads (prewashed, 3 x 8 mL DMF) in a solid phase peptide synthesis vessel. 45 μΐ of Ν,Ν-diisopropylethylamine and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then 2-methoxyethylamine (18.6 mg, 22 μΐ, 0.248 mmol) was added and shaking was continued for 30 minutes. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ C1 ₂ (3 x 8 mL), DMF (3 x 8 mL) and z ^' -PrOH (3 x 8 mL). The beads 21 were stored in z ^' -PrOH (beads: z ^' -PrOH, (1 :2), v/v) at - 80°C.

6.2.18. N-Fmoc-frans-4-aminocyclohexanol (22) (Crestey et al, 2008)

To a solution of trans -4-aminocyclohexanol hydrochloride (2.0 g, 13.2 mmol) in dioxane:water (26:6.5 mL) was added Et ₃ N (1.08 g, 1.49 mL, 10.7 mmol) and this was stirred for 10 min. Then Fmoc-OSu (3.00 g, 8.91 mmol) was added over five minutes and the resulting suspension was stirred at rt for 2 h. The reaction mixture was concentrated to ~5 mL, then some CH ₂ C1 ₂ was added. This was filtered and the solid was washed with H ₂ 0 (4 x 40 mL) then dried to give 2.85 g (95%) of 22 as a white solid. Additional 0.100 g (3%) of 22 was obtained by extracting the filtrate with CH ₂ C1 ₂ (2 x 100 mL), drying over Na ₂ S0 ₄ , filtering and removing solvent for a combined yield of 98%. TLC (hexane:EtOAc, 20:80) R _f = 0.42; 1H NMR (CDC1 ₃ ) δ 7.77 (d, J= 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J= 7.4 Hz, 2H), 4.54 (br s, 1H), 4.40 (d, J= 5.6 Hz, 2H), 4.21 (t, J= 5.6 Hz, 1H), 3.61 (m, 1H), 3.48 (m, 1H), 1.9-2.1 (m, 4H), 1.32-1.48 (m, 2H), 1.15-1.29 (m, 2H); MS (ESI): m/z 338.0 [M+H] ⁺ .

6.2.19. N-Fmoc-frans-4-aminocyclohexanol tetrahydropyranyl ether (23)

1.03 g (3.05 mmol) of 22 and 0.998 g (1.08 mL, 11.86 mmol) of 3,4-dihydro-2H- pyran (DHP) was suspended in dioxane (10 mL). Pyridinium /?-toluenesulfonate (0.153 g, 0.61 mmol) was added and the suspension stirred at rt. After 1 hr additional DHP (1.08 mL, 11.86 mmol) and dioxane (10 mL) were added and stirring continued. After 9 h additional DHP (1.08 mL, 11.86 mmol) was added and stirring continued overnight. The resulting solution was concentrated and the residue chromatographed (hexane:EtOAc, 75:25 to 65:35) to give 1.28 g (100%) of 23 as a white solid. TLC (hexane:EtOAc, 70:30) R _f = 0.26; 1H NMR (CDCls) δ 7.77 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (dt, J= 7.5, 1.1 Hz, 2H), 4.70 (m, 1H), 4.56 (m, 1H), 4.40 (d, J= 6.0 Hz, 2H), 4.21 (t, J= 6.1 Hz, 1H), 3.90 (m, 1H), 3.58 (m, 1H), 3.45-3.53 (m, 2H), 1.10-2.09 (m, 14H); MS (ESI): m/z 422.3 [M+H] ⁺ .

6.2.20. frans-4-Aminocylohexanol tetrahydropyranyl ether (24)

1.28 g (3.0 mmol) of 23 was dissolved in CH ₂ C1 ₂ (20 mL) and piperidine (2 mL) was added and the solution stirred at rt for 5 h. Solvent was removed and the residue was purified by chromatography [CH ₂ Cl ₂ :MeOH-NH ₃ (7N), 80: 1 to 30: 1] to give 0.574 g (96%) of 24 as an oily residue which slowly crystallized. 1H NMR (CDC1 ₃ ) δ 4.70 (m, 1H), 3.91 (m, 1H), 3.58 (m, 1H), 3.49 (m, 1H), 2.69 (m, 1H), 1.07-2.05 (m, 14H); MS (ESI): m/z 200.2 [M+H] ⁺ .

6.2.21. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro -lH-indazol-l-yl)-2- (irans-4-(tetrahydro-2H-pyran-2-yloxy)cyclohexylamino)benzon itrile (25)

A mixture of 16 (0.270 g, 0.655 mmol), NaOtBu (0.126 g, 1.31 mmol), Pd ₂ (dba) ₃

(0.120 g, 0.131 mmol) and DavePhos (0.051 g, 0.131 mmol) in 1 ,2-dimethoxy ethane (20 mL) was degassed and flushed with argon several times. 24 (0.390 g, 1.97 mmol) was added and the flask was again degassed and flushed with argon before heating the reaction mixture at 60°C for 3.5 h. The reaction mixture was concentrated under reduced pressure and the residue purified by preparatory TLC [hexane:CH ₂ Cl ₂ :EtOAc:MeOH-NH ₃ (7N), 7:6:3: 1.5] to give 97.9 mg (28%>) of 25. Additionally, 60.5 mg (17%>) of amide 26 was isolated for a total yield of 45%. 1H NMR (CDC1 ₃ ) δ 7.52 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 1.7 Hz, 1H), 6.72 (dd, J = 8.3, 1.8 Hz, 1H), 4.72 (m, 1H), 4.67 (d, J= 7.6 Hz, 1H), 3.91 (m, 1H), 3.68 (m, 1H), 3.50 (m, 1H), 3.40 (m, 1H), 2.84 (s, 2H), 2.49 (s, 2H), 2.06-2.21 (m, 4H), 1.30-1.90 (m, 10H), 1.14 (s, 6H); MS (ESI): m/z 529.4 [M-H] ^~ .

6.2.22. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro -lH-indazol-l-yl)-2- (irans-4-(tetrahydro-2H-pyran-2-yloxy)cyclohexylamino)benzam ide (26) A solution of 25 (120 mg, 0.2264 mmol) in DMSO (220 μΐ), EtOH (885 μΐ), 5N NaOH (112 μΐ) and H ₂ 0 ₂ (132 μΐ) was stirred at rt for 4 h. Then 30 mL of brine was added and this was extracted with EtOAc (5 x 15 mL), dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by preparatory TLC [hexane:CH ₂ Cl ₂ :EtOAc:MeOH-NH ₃ (7N), 7:6:3: 1.5] to give 102 mg (82%) of 26. 1H NMR (CDC1 ₃ ) δ 8.13 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.63 (dd, J = 8.4, 2.0 Hz, 1H), 5.68 (br s, 2H), 4.72 (m, 1H), 3.91 (m, 1H), 3.70 (m, 1H), 3.50 (m, 1H), 3.34 (m, 1H), 2.85 (s, 2H), 2.49 (s, 2H), 2.05-2.19 (m, 4H), 1.33-1.88 (m, 10H), 1.14 (s, 6H); MS (ESI): m/z 547.4 [M-H] ^~ .

6.2.23. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro -lH-indazol-l-yl)-2- (frans-4-hydroxycyclohexylamino)benzamide (SNX-2112)

26 (140 mg, 0.255 mmol) and pyridinium /?-toluenesulfonate (6.4 mg, 0.0255 mmol) in EtOH (4.5 mL) was heated at 65°C for 17 h. The reaction mixture was concentrated under reduced pressure and the residue purified by preparatory TLC [hexane:CH ₂ Cl ₂ :EtOAc:MeOH-NH ₃ (7N), 2:2:1 :0.5] to give 101 mg (85%) of SNX-2112. 1H NMR (CDC1 ₃ ) δ 8.10 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 1.3 Hz, 1H), 6.60 (dd, J = 8.4, 1.6 Hz, 1H), 5.97 (br s, 2H), 3.73 (m, 1H), 3.35 (m, 1H), 2.85 (s, 2H), 2.48 (s, 2H), 2.14 (d, J= 11.8 Hz, 2H), 2.04 (d, J= 11.1 Hz, 2H), 1.33-1.52 (m, 4H), 1.13 (s, 6H); ¹³ C NMR (125 MHz, CDCl ₃ /MeOH-<¾) δ 191.0, 171.9, 151.0, 150.0, 141.3, 140.3 (q, J = 39.6 Hz), 130.4, 120.3 (q, J = 270.2 Hz), 115.9, 113.7, 109.2, 107.1, 69.1, 52.1, 50.2, 40.1, 37.0, 35.6, 33.1, 30.2, 28.0; MS (ESI): m/z 463.3 [M-H] ^" , 465.3 [M+H] ⁺ ; HPLC: t _R = Ί .91 min. 6.2.24. Preparation of control beads

DMF (8.5 mL) was added to 20 mL of Affi-Gel 10 beads (prewashed, 3 x 40 mL DMF) in a solid phase peptide synthesis vessel. 2-Methoxyethylamine (113 mg, 129 μΐ _^ , 1.5 mmol) and several crystals of DMAP were added and this was shaken at rt for 2.5 h. Then the solvent was removed and the beads washed for 10 minutes each time with CH ₂ C1 ₂ (4 x 35 mL), DMF (3 x 35 mL), Felts buffer (2 x 35 mL) and z ^' -PrOH (4 x 35 mL). The beads were stored in z-PrOH (beads: z-PrOH (1 :2), v/v) at -80°C.

6.3. Competition assay For the competition studies, fluorescence polarization (FP) assays were performed as previously reported (Du et al, 2007). Briefly, FP measurements were performed on an Analyst GT instrument (Molecular Devices, Sunnyvale, CA). Measurements were taken in black 96-well microtiter plates (Corning # 3650) where both the excitation and the emission occurred from the top of the wells. A stock of 10 μΜ GM-cy3B was prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl ₂ , 20 mM Na ₂ Mo0 ₄ , and 0.01% NP40 with 0.1 mg/mL BGG). To each 96-well were added 6 nM fluorescent GM (GM-cy3B), 3 μg SKBr3 lysate (total protein), and tested inhibitor (initial stock in DMSO) in a final volume of 100 HFB buffer. Drugs were added in triplicate wells. For each assay, background wells (buffer only), tracer controls (free, fluorescent GM only) and bound GM controls (fluorescent GM in the presence of SKBr3 lysate) were included on each assay plate. GM was used as positive control. The assay plate was incubated on a shaker at 4°C for 24 h and the FP values in mP were measured. The fraction of tracer bound to Hsp90 was correlated to the mP value and plotted against values of competitor concentrations. The inhibitor concentration at which 50% of bound GM was displaced was obtained by fitting the data. All experimental data were analyzed using SOFTmax Pro 4.3.1 and plotted using Prism 4.0 (Graphpad Software Inc., San Diego, CA).

6.4. Chemical Precipitation, Western blotting and Flow Cytometry

The leukemia cell lines K562 and MV4-11 and the breast cancer cell line MDA-MB- 468 were obtained from the American Type Culture Collection. Cells were cultured in RPMI (K562), in Iscove's modified Dulbecco's media (MV4-11) or in DME/F12 (MDA-MB-468) supplemented with 10%> FBS, 1%> L-glutamine, 1%> penicillin and streptomycin, and maintained in a humidified atmosphere of 5% C0 ₂ at 37°C. Cells were lysed by collecting them in Felts buffer (HEPES 20 mM, KCl 50 mM, MgCl ₂ 5 mM, NP40 0.01%, freshly prepared Na ₂ Mo0 ₄ 20 mM, pH 7.2-7.3) with added 10 μg/μL of protease inhibitors (leupeptin and aprotinin), followed by three successive freeze (in dry ice) and thaw steps. Total protein concentration was determined using the BCA kit (Pierce) according to the manufacturer's instructions.

Hsp90 inhibitor beads or control beads containing an Hsp90 inactive chemical (2- methoxyethylamine) conjugated to agarose beads were washed three times in lysis buffer.

The bead conjugates (80 μΐ, or as indicated) were then incubated overnight at 4°C with cell lysates (250 μg), and the volume was adjusted to 200-300 μΐ, with lysis buffer. Following incubation, bead conjugates were washed 5 times with the lysis buffer and analyzed by Western blot, as indicated below.

For treatment with PU-H71, cells were grown to 60-70% confluence and treated with inhibitor (5 μΜ) for 24h. Protein lysates were prepared in 50 mM Tris pH 7.4, 150 mM NaCl and 1 % NP-40 lysis buffer.

For Western blotting, protein lysates (10-50 μg) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane and probed with a primary antibody against Hsp90 (1 :2000, SMC-107A/B, StressMarq), anti-IGF-IR (1 : 1000, 3027, Cell Signaling) and anti-c-Kit (1 :200, 612318, BD Transduction Laboratories). The membranes were then incubated with a 1 :3000 dilution of a corresponding horseradish peroxidase conjugated secondary antibody. Detection was performed using the ECL-Enhanced Chemiluminescence Detection System (Amersham Biosciences) according to manufacturer's instructions.

To detect the binding of PU-H71 to cell surface Hsp90, MV4-11 cells at 500,000 cells/ml were incubated with the indicated concentrations of PU-H71-biotin or D-biotin as control for 2 h at 37°C followed by staining of phycoerythrin (PE) conjugated streptavidin (SA) (BD Biosciences) in FACS buffer (PBS + 0.5% FBS) at 4°C for 30 min. Cells were then analyzed using the BD-LSRII flow cytometer. Mean fluorescence intensity (MFI) was used to calculate the binding of PU-H71-biotin to cells and values were normalized to the MFI of untreated cells stained with SA-PE.

6.5. Docking

Molecular docking computations were carried out on a HP workstation xw8200 with the Ubuntu 8.10 operating system using Glide 5.0 (Schrodinger). The coordinates for the

Hsp90a complexes with bound inhibitor PU-H71 (PDB ID: 2FWZ), NVP-AUY922 (PDB

ID: 2VCI) and 27 (PDB ID: 3D0B) were downloaded from the RCSB Protein Data Bank. For docking experiments, compounds PU-H71, NVP-AUY922, 5, 10, 20 and 27 were constructed using the fragment dictionary of Maestro 8.5 and geometry-optimized using the Optimized Potentials for Liquid Simulations- All Atom (OPLS-AA) force field (Jorgensen et al., 1996) with the steepest descent followed by truncated Newton conjugate gradient protocol as implemented in Macromodel 9.6, and were further subjected to ligand preparation using default parameters of LigPrep 2.2 utility provided by Schrodinger LLC. Each protein was optimized for subsequent grid generation and docking using the Protein Preparation Wizard provided by Schrodinger LLC. Using this tool, hydrogen atoms were added to the proteins, bond orders were assigned, water molecules of crystallization not deemed to be important for ligand binding were removed, and the entire protein was minimized. Partial atomic charges for the protein were assigned according to the OPLS-2005 force field. Next, grids were prepared using the Receptor Grid Generation tool in Glide. With the respective bound inhibitor in place, the centroid of the workspace ligand was chosen to define the grid box. The option to dock ligands similar in size to the workspace ligand was selected for determining grid sizing.

Next, the extra precision (XP) Glide docking method was used to flexibly dock compounds PU-H71 and 5 (to 2FWZ), NVP-AUY922 and 10 (to 2VCI), and 20 and 27 (to 3D0B) into their respective binding site. Although details on the methodology used by Glide are described elsewhere (Patel et al, 2008; Friesner et al, 2004; Halgren et al, 2004), a short description about parameters used is provided below. The default setting of scale factor for van der Waals radii was applied to those atoms with absolute partial charges less than or equal to 0.15 (scale factor of 0.8) and 0.25 (scale factor of 1.0) electrons for ligand and protein, respectively. No constraints were defined for the docking runs. Upon completion of each docking calculation, at most 100 poses per ligand were allowed to generate. The top- scored docking pose based on the Glide scoring function (Eldridge et al, 1997) was used for our analysis. In order to validate the XP Glide docking procedure the crystallographic bound inhibitor (PU-H71 or NVP-AUY922 or 27) was extracted from the binding site and re-docked into its respective binding site. There was excellent agreement between the localization of the inhibitor upon docking and the crystal structure as evident from the 0.098 A (2FWZ), 0.313 A (2VCI) and 0.149 A (3D0B) root mean square deviations. Thus, the present study suggests the high docking reliability of Glide in reproducing the experimentally observed binding mode for Hsp90 inhibitors and the parameter set for the Glide docking reasonably reproduces the X-ray structure.

Compound IC50 (nM)

GM 15.4

PU-H71 22.4

5 19.8

7 67.1

NVP-AUY922 4.1

10 7.0

SNX-2112 15.1

18 210.1 20 24.7

Table 8. Binding affinity for Hsp90 from SKBr3 cellular extracts.

References

1. Ackler, S., Xiao, Y., Mitten, M.J., Foster, K., Oleksijew, A., Refici, M., Schlessinger, S., Wang, B., Chemburkar, S.R., Bauch, J., et al. (2008). ABT-263 and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo. Mol Cancer Ther 7, 3265- 3274.

2. Adler, A.S., Lin, M., Horlings, H., Nuyten, D.S., van de Vijver, M.J., and Chang, H.Y.

(2006). Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet

38, 421-430.

3. Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C, Lossos, I.S., Rosenwald, A., Boldrick, J.C., Sabet, H., Tran, T., Yu, X., et al. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503-511.

4. An, W.G., Schulte, T.W. & Neckers, L.M. (2000). The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src proteins before their degradation by the proteasome. Cell Growth Differ. 11, 355-360.

5. Andersen, J.N. et al. (2010). Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors. Sci. Transl. Med. 2, 43ra55.

6. Apsel, B., et al. (2008). Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nature Chem. Biol. 4, 691-699.

7. Ashman, K. & Villar, E.L. (2009). Phosphoproteomics and cancer research. Clin.

Transl. Oncol. 11, 356-362.

8. Barril, X.; Beswick, M. C; Collier, A.; Drysdale, M. J.; Dymock, B. W.; Fink, A.;

Grant, K.; Howes, R.; Jordan, A. M.; Massey, A.; Surgenor, A.; Wayne, J.; Workman, P.; Wright, L., Bioorg. Med. Chem. Lett. 2006, 16, 2543-2548.

9. Barta, T. E.; Veal, J. M.; Rice, J. W.; Partridge, J. M.; Fadden, R. P.; Ma, W.; Jenks, M.; Geng, L.; Hanson, G. J.; Huang, K. H.; Barabasz, A. F.; Foley, B. E.; Otto, J.; Hall, S. E., Bioorg. Med. Chem. Lett. 2008, 18, 3517-3521.

10. Bedford, M.T. & Clarke, S.G. (2009). Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1-13. Bonvini, P., Gastaldi, T., Falini, B., and Rosolen, A. (2002). Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPMALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin. Cancer Res 62, 1559-1566.

Brehme, M. et al. (2009). Charting the molecular network of the drug target Bcr-Abl. Proc. Natl. Acad. Sci. USA 106, 7414-7419.

Brough, P. A., Aherne, W., Barril, X., Borgognoni, J., Boxall, K., Cansfield, J. E., Cheung, K.-M. J., Collins, I., Davies, N. G. M., Drysdale, M. J., Dymock, B., Eccles, S. A., Finch, H., Fink, A., Hayes, A., Howes, R., Hubbard, R. E., James, K., Jordan, A. M., Lockie, A., Martins, V., Massey, A., Matthews, T. P., McDonald, E., Northfield, C. J., Pearl, L. H., Prodromou, C, Ray, S., Raynaud, F. I., Roughley, S. D., Sharp, S. Y., Surgenor, A., Walmsley, D. L., Webb, P., Wood, M., Workman, P., and Wright, L. (2008). J. Med. Chem. 51, 196-218.

Burke, B,A. & Carroll, M. (2010). BCR-ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia. Leukemia 24, 1105-1112.

Caldas-Lopes, E., Cerchietti, L., Ahn, J.H., Clement, C.C., Robles, A.I., Rodina, A., Moulick, K., Taldone, T., Gozman, A., Guo, Y., et al. (2009). Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models. Proc Natl Acad Sci U S A 106, 8368-8373.

Carayol, N. et al. (2010). Critical roles for mTORC2- and rapamycin-insensitive mTORCl -complexes in growth and survival of BCR-ABL-expressing leukemic cells. Proc. Natl. Acad. Sci. USA. 107, 12469-12474.

Carden, C. P., Sarker, D., Postel-Vinay, S., Yap, T. A., Attard, G., Banerji, U., Garrett, M. D., Thomas, G. V., Workman, P., Kaye, S. B., and de Bono, J. S. (2010). Drug Discov. Today 15, 88-97.

Cerchietti, L.C., Ghetu, A.F., Zhu, X., Da Silva, G.F., Zhong, S., Matthews, M., Bunting, K.L., Polo, J.M., Fares, C, Arrowsmith, C.H., et al. (2010a). A small- molecule inhibitor of BCL6 kills DLBCL cells in vitro and in vivo. Cancer Cell 17, 400-411.

Cerchietti, L.C., Hatzi, K., Caldas-Lopes, E., Yang, S.N., Figueroa, M.E., Morin, R.D., Hirst, M., Mendez, L., Shaknovich, R., Cole, P.A., et al. (2010b). BCL6 repression of EP300 in human diffuse large B cell lymphoma cells provides a basis for rational combinatorial therapy. J Clin Invest. 20. Cerchietti, L.C., Lopes, E.C., Yang, S.N., Hatzi, K., Bunting, K.L., Tsikitas, L.A., Mallik, A., Robles, A.I., Walling, J., Varticovski, L., et al. (2009a). A purine scaffold Hsp90 inhibitor destabilizes BCL-6 and has specific antitumor activity in BCL-6- dependent B cell lymphomas. Nat Med 15, 1369-1376.

21. Cerchietti, L.C., Yang, S.N., Shaknovich, R., Hatzi, K., Polo, J.M., Chadburn, A., Dowdy, S.F., and Melnick, A. (2009b). A peptomimetic inhibitor of BCL6 with potent antilymphoma effects in vitro and in vivo. Blood 113, 3397-3405.

22. Chamovitz, D.A., Wei, N., Osterlund, M.T., von Arnim, A.G., Staub, J.M., Matsui, M., and Deng, X.W. (1996). The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch. Cell 86, 115-121.

23. Chan, V.W., Meng, F., Soriano, P., DeFranco, A.L., and Lowell, C.A. (1997).

Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity 7, 69-81.

24. Chandarlapaty, S., Sawai, A., Ye, Q., Scott, A., Silinski, M., Huang, K., Fadden, P., Partdrige, J., Hall, S., Steed, P., Norton, L., Rosen, N., and Solit, D. B. (2008). Clin.

Cancer Res. 14, 240-248.

25. Chen, L., Juszczynski, P., Takeyama, K., Aguiar, R.C., and Shipp, M.A. (2006). Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates SYK phosphorylation, proximal Bcell-receptor signaling, and cellular proliferation. Blood 108, 3428-3433.

26. Chen, L., Monti, S., Juszczynski, P., Daley, J., Chen, W., Witzig, T.E., Habermann, T.M., Kutok, J.L., and Shipp, M.A. (2008). SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma. Blood 111, 2230-2237.

27. Cheung, K. M.; Matthews, T. P.; James, K.; Rowlands, M. G.; Boxall, K. J.; Sharp, S.

Y.; Maloney, A.; Roe, S. M.; Prodromou, C; Pearl, L. H.; Aherne, G. W.; McDonald, E.; Workman, P., Bioorg. Med. Chem. Lett. 2005, 15, 3338-3343.

28. Chiba, T., and Tanaka, K. (2004). Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system. Curr Protein Pept Sci 5, 177-184.

29. Chiosis, G., and Neckers, L. (2006). Tumor selectivity of Hsp90 inhibitors: the explanation remains elusive. ACS Chem Biol 1, 279-284.

30. Chiosis, G., Timaul, M.N., Lucas, B., Munster, P.N., Zheng, F.F., Sepp-Lorenzino, L., and Rosen, N. (2001). A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells. Chem Biol 8, 289-299.

Chou, T.C. (2006). Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621— 681.

Chou, T.C. & Talalay, P. (1984). Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27- 55.

Claramunt, R. M.; Lopez, C; Perez-Medina, C; Pinilla, E.; Torres, M. R.; Elguero, J., Tetrahedron 2006, 62, 11704-11713.

Clevenger, R. C; Raibel, J. M.; Peck, A. M.; Blagg, B. S. J., J. Org. Chem. 2004, 69, 4375-4380.

Coiffier, B., Lepage, E., Briere, J., Herbrecht, R., Tilly, H., Bouabdallah, R., Morel, P., Van Den Neste, E., Salles, G., Gaulard, P., et al. (2002). CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 346, 235-242.

Compagno, M., Lim, W.K., Grunn, A., Nandula, S.V., Brahmachary, M., Shen, Q., Bertoni, F., Ponzoni, M., Scandurra, M., Califano, A., et al. (2009). Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma. Nature 459, 717-721.

Cope, G.A., Suh, G.S., Aravind, L., Schwarz, S.E., Zipursky, S.L., Koonin, E.V., and Deshaies, R.J. (2002). Role of predicted metalloprotease motif of Jabl/Csn5 in cleavage ofNedd8 from Cull . Science 298, 608-611.

Crestey, F.; Ottesen, L. K.; Jaroszewski, J. W.; Franzyk, H., Tetrahedron Lett. 2008, 49, 5890-5893.

da Silva Correia, J., Miranda, Y., Leonard, N., and Ulevitch, R.J. (2007). The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis. J Biol Chem 282, 12557- 12565.

Dai, B., Zhao, X.F., Hagner, P., Shapiro, P., Mazan-Mamczarz, K., Zhao, S., Natkunam, Y., and Gartenhaus, R.B. (2009). Extracellular signal-regulated kinase positively regulates the oncogenic activity of MCT-1 in diffuse large B-cell lymphoma. Cancer Res 69, 7835-7843.

Dal Porto, J.M., Gauld, S.B., Merrell, K.T., Mills, D., Pugh-Bernard, A.E., and

Cambier, J. (2004). B cell antigen receptor signaling 101. Mol Immunol 41, 599-613. 42. Davis, R.E., Brown, K.D., Siebenlist, U., and Staudt, L.M. (2001). Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med 194, 1861-1874.

43. Davis, R.E., Ngo, V.N., Lenz, G., Tolar, P., Young, R.M., Romesser, P.B., Kohlhammer, H., Lamy, L., Zhao, H., Yang, Y., et al. (2010). Chronic active B-cell- receptor signalling in diffuse large B-cell lymphoma. Nature 463, 88-92.

44. de Groot, R.P., Raaijmakers, J.A., Lammers, J.W., Jove, R. & Koenderman, L. (1999).

STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells. Blood 94, 1108-1112.

45. de Jong, D., and Enblad, G. (2008). Inflammatory cells and immune microenvironment in malignant lymphoma. J Intern Med 264, 528-536.

46. da Rocha Dias, S. et al. (2005). Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 65, 10686-10691.

47. Dealy, M.J., Nguyen, K.V., Lo, J., Gstaiger, M., Krek, W., Elson, D., Arbeit, J.,

Kipreos, E.T., and Johnson, R.S. (1999). Loss of Cull results in early embryonic lethality and dysregulation of cyclin E. Nat Genet 23, 245-248.

48. Deininger, M.W. & Druker, B.J. (2003). Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol. Rev. 55, 401-423.

49. Deng, X.W., Dubiel, W., Wei, N., Hofmann, K., and Mundt, K. (2000). Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet 16, 289.

50. Dezwaan, D.C. & Freeman, B.C. (2008). HSP90: the Rosetta stone for cellular protein dynamics? Cell Cycle 7, 1006-1012.

51. Dierov, J., Dierova. R. & Carroll, M. (2004). BCR/ABL translocates to the nucleus and disrupts an ATR-dependent intra-S phase checkpoint. Cancer Cell 5, 275-85.

52. Du, Y.; Moulick, K.; Rodina, A.; Aguirre, J.; Felts, S.; Dingledine, R.; Fu, FL; Chiosis, G., J. Biomol. Screen. 2007, 12, 915-924.

53. Dymock, B. W.; Barril, X.; Brough, P. A.; Cansfield, J. E.; Massey, A.; McDonald, E.;

Hubbard, R. E.; Surgenor, A.; Roughley, S. D.; Webb, P.; Workman, P.; Wright, L.;

Drysdale, M. J., J. Med. Chem. 2005, 48, 4212-4215.

54. Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P., J. Comput.- AidedMol. Des. 1997, 11, 425-445. Erdjument-Bromage, H. et al. (1998). Micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J. Chromatogr. A 826, 167- 181.

Fabian, M.A. et al. (2005). A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329-336.

Fowler, N., Sharman, J., Smith, S., Boyd, T., Grant, B., Kolibaba, K., Furman, R., Buggy, J., Loury, D., Hamdy, A., et al. (2010). The Btk Inhibitor, PCI-32765, Induces Durable Responses with Minimal Toxicity In Patients with Relapsed/Refractory B-Cell Malignancies: Results From a Phase I Study 52nd ASH Annual Meeting and Exposition.

Friday, B.B., and Adjei, A.A. (2008). Advances in targeting the Ras/Raf/MEK/Erk mitogenactivated protein kinase cascade with MEK inhibitors for cancer therapy. Clin Cancer Res 14, 342-346.

Friedberg, J.W., Sharman, J., Sweetenham, J., Johnston, P.B., Vose, J.M., Lacasce, A., SchaeferCutillo, J., De Vos, S., Sinha, R., Leonard, J.P., et al. (2010). Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood 115, 2578-2585.

Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S., J. Med. Chem. 2004, 47, 1739-1749.

Fukumoto, A., Tomoda, K., Yoneda-Kato, N., Nakajima, Y., and Kato, J.Y. (2006). Depletion of Jabl inhibits proliferation of pancreatic cancer cell lines. FEBS Lett 580, 5836-5844.

Gorre, M.E., Ellwood-Yen, K., Chiosis, G., Rosen, N., and Sawyers, C.L. (2002). BCR- ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90. Blood 100, 3041-3044.

Grbovic, O.M. et al. (2006). V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc. Natl. Acad. Sci. USA 103, 57-62. Gupta, M., Ansell, S.M., Novak, A.J., Kumar, S., Kaufmann, S.H., and Witzig, T.E. (2009). Inhibition of histone deacetylase overcomes rapamycin-mediated resistance in diffuse large Bcell lymphoma by inhibiting Akt signaling through mTORC2. Blood 114, 2926-2935. 65. Hacker, H. & Karin, M. (2006). Regulation and function of IKK and IKK-related kinases. Sci. STKE. (357):rel3.

66. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.;

Banks, J. L., J. Med. Chem. 2004, 47, 1750-1759.

67. Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57-70.

68. Hanash, S. & Taguchi, A. (2010). The grand challenge to decipher the cancer proteome.

Nat. Rev. Cancer 10, 652-660.

69. Hansen, J. B.; Nielsen, M. C; Ehrbar, U.; Buchardt, O., Synthesis 1982, 404-405.

70. He, H. et al. (2006). Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90. J. Med. Chem. 49, 381-390.

71. Hendriks, R.W. & Kersseboom, R. (2006). Involvement of SLP-65 and Btk in tumor suppression and malignant transformation of pre-B cells. Semin. Immunol. 18, 67-76.

72. Hiegel, G. A.; Burk, P., J. Org. Chem. 1973, 38, 3637-3639.

73. Hofmann, K., and Bucher, P. (1998). The PCI domain: a common theme in three multiprotein complexes. Trends Biochem Sci 23, 204-205.

74. Honigberg, L.A., Smith, A.M., Sirisawad, M., Verner, E., Loury, D., Chang, B., Li, S., Pan, Z., Thamm, D.H., Miller, R.A., et al. (2010). The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A 107, 13075-13080.

75. Horn, T., Sandmann, T. & Boutros, M. (2010). Design and evaluation of genome -wide libraries for RNA interference screens. Genome Biol. 11(6):R61.

76. Huang, K. H., Veal, J. M., Fadden, R. P., Rice, J. W., Eaves, J., Strachan, J. -P., Barabasz, A. F., Foley, B. E., Barta, T. E., Ma, W., Silinski, M. A., Hu, M., Partridge, J. M., Scott, A., DuBois, L. G., Freed, T., Steed, P. M., Ommen, A. J., Smith, E. D., Hughes, P. F., Woodward, A. R., Hanson, G. J., McCall, W. S., Markworth, C. J.,

Hinkley, L., Jenks, M., Geng, L., Lewis, M., Otto, J., Pronk, B., Verleysen, K., and Hall, S. E. (2009) J. Med. Chem. 52, 4288-4305.

77. Hudes, G., Carducci, M., Tomczak, P., Dutcher, J., Figlin, R., Kapoor, A., Staroslawska, E., Sosman, J., McDermott, D., Bodrogi, I., et al. (2007). Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 356, 2271-

2281.

78. Hurvitz, S. A.; Finn, R. S., Future Oncol. 2009, 5, 1015-1025.

79. Immormino, R. M.; Kang, Y.; Chiosis, G.; Gewirth, D. T., J. Med. Chem. 2006, 49, 4953-4960. 80. Jaganathan, S., Yue, P. & Turkson, J. (2010). Enhanced sensitivity of pancreatic cancer cells to concurrent inhibition of aberrant signal transducer and activator of transcription 3 and epidermal growth factor receptor or Src. J. Pharmacol. Exp. Ther. 333, 373-381.

81. Janin, Y.L. (2010). ATPase inhibitors of heat-shock protein 90, second season. Drug Discov. Today 15, 342-353.

82. Jorgensen, W. L.; Maxwell, D.; Tirado-Rives, J., J. Am. Chem. Soc. 1996, 118, 11225- 11236.

83. Kamal, A. et al. (2003). A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors, Nature 425, 407-410.

84. Kato, J.Y., and Yoneda-Kato, N. (2009). Mammalian COP9 signalosome. Genes Cells 14, 1209-1225.

85. Katzav, S. (2007). Flesh and blood: the story of Vavl, a gene that signals in hematopoietic cells but can be transforming in human malignancies. Cancer Lett. 255, 241-254.

86. Klejman, A. et al. (2002). The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells. EMBO J. 21, 5766-5774.

87. Kolch, W. & Pitt, A. (2010). Functional proteomics to dissect tyrosine kinase signalling pathways in cancer. Nat. Rev. Cancer 10, 618-629.

88. Kufe DW, P.R., Weichselbaum PR, Bast RC, Gansler TS, Holland JF, Frei E (2003).

Cancer Medicine 6, Vol Two (Hamilton, BC Decker).

89. Lam, K.P., Kuhn, R., and Rajewsky, K. (1997). In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90, 1073-1083.

90. Lam, L.T., Davis, R.E., Pierce, J., Hepperle, M., Xu, Y., Hottelet, M., Nong, Y., Wen, D., Adams, J., Dang, L., et al. (2005). Small molecule inhibitors of IkappaB kinase are selectively toxic for subgroups of diffuse large B-cell lymphoma defined by gene expression profiling. Clin Cancer Res 11, 28-40.

91. Law, J. H.; Habibi, G.; Hu, K.; Masoudi, H.; Wang, M. Y.; Stratford, A. L.; Park, E.;

Gee, J. M.; Finlay, P.; Jones, H. E.; Nicholson, R. I.; Carboni, J.; Gottardis, M.; Pollak, M.; Dunn, S. E., Cancer Res. 2008, 68, 10238-10246.

92. Le, Y. et al. (2009). FAK silencing inhibits leukemogenesis in BCR/ABL-transformed hematopoietic cells. Am. J. Hematol. 84, 273-278.

93. Leal, J.F., Fominaya, J., Cascon, A., Guijarro, M.V., Blanco-Aparicio, C, Lleonart, M.,

Castro, M.E., Ramon, Y.C.S., Robledo, M., Beach, D.H., et al. (2008). Cellular senescence bypass screen identifies new putative tumor suppressor genes. Oncogene 27, 1961-1970.

94. Lenz, G., Davis, R.E., Ngo, V.N., Lam, L., George, T.C., Wright, G.W., Dave, S.S., Zhao, H., Xu, W., Rosenwald, A., et al. (2008). Oncogenic CARD11 mutations in human diffuse large B cell lymphoma. Science 319, 1676-1679.

95. Levy, D.S., Kahana, J.A., and Kumar, R. (2009). AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines. Blood 113, 1723-1729.

96. Ley, T.J. et al. (2008). DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66-72.

97. Li, B., Ruiz, J.C., and Chun, K.T. (2002). CUL-4A is critical for early embryonic development. Mol Cell Biol 22, 4997-5005.

98. Li, J., Wang, Y., Yang, C, Wang, P., Oelschlager, D.K., Zheng, Y., Tian, D.A., Grizzle, W.E., Buchsbaum, D.J., and Wan, M. (2009). Polyethylene glycosylated curcumin conjugate inhibits pancreatic cancer cell growth through inactivation of Jabl . Mol Pharmacol 76, 81-90.

99. Lim, CP. & Cao, X. (2006). Structure, function and regulation of STAT proteins. Mol.

BioSyst, 2, 536-550.

100. Luo, W.; Dou, F.; Rodina, A.; Chip, S.; Kim, J.; Zhao, Q.; Moulick, K.; Aguirre, J.;

Wu, N.; Greengard, P.; Chiosis, G., Proc. Natl. Acad. Sci. USA 2007, 104, 9511-9518.

101. Luo, W.; Rodina, A.; Chiosis, G., BMC Neurosci. 2008, 9(Suppl 2):S7.

102. Luo, W.; Sun, W.; Taldone, T.; Rodina, A.; Chiosis, G., Mol Neurodegener. 2010, 5:

24.

103. Lykke-Andersen, K., Schaefer, L., Menon, S., Deng, X.W., Miller, J.B., and Wei, N.

(2003). Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. Mol Cell Biol 23, 6790-6797.

104. Mahajan, S. et al. (2001). Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells. J. Biol. Chem. 276, 31216-31228.

105. Maloney, A. et al. (2007). Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17- demethoxygeldanamycin. Cancer Res. 67, 3239-3253.

106. Marubayashi, S.; Koppikar, P.; Taldone, T.; Abdel-Wahab, O.; West, N.; Bhagwat, N.;

Lopes-Vazquez, E. C; Ross, K. N.; Gonen, M.; Gozman, A.; Ahn, J.; Rodina, A.;

zzo Ouerfelli, O.; Yang, G.; Hedvat, C; Bradner, J. E.; Chiosis, G.; Levine, R. L., J. Clin. Invest. 2010, 120, 3578-3593.

107. McClellan, A.J. et al. (2007). Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell 131, 121-135.

108. McCubrey, J. A. et al. (2008). Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy. Leukemia 22, 708-722.

109. Menon, S., Chi, H., Zhang, H., Deng, X.W., Flavell, R.A., and Wei, N. (2007). COP9 signalosome subunit 8 is essential for peripheral T cell homeostasis and antigen receptor-induced entry into the cell cycle from quiescence. Nat Immunol 8, 1236-1245.

110. Mihailovic, T. et al. (2004). Protein kinase D2 mediates activation of nuclear factor kappaB by Bcr-Abl in Bcr-Abl+ human myeloid leukemia cells. Cancer Res. 64, 8939- 8944.

111. Milhollen, M.A., Traore, T., Adams-Duffy, J., Thomas, M.P., Berger, A. J., Dang, L., Dick, L.R., Garnsey, J.J., Koenig, E., Langston, S.P., et al. (2010). MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF- {kappa} B-dependent lymphoma. Blood 116, 1515-1523.

112. Moffat, J. et al. (2006). A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell 124, 1283-1298.

113. Monti, S., Savage, K.J., Kutok, J.L., Feuerhake, F., Kurtin, P., Mihm, M., Wu, B., Pasqualucci, L., Neuberg, D., Aguiar, R.C., et al. (2005). Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood 105, 1851-1861.

114. Munday, D. et al. (2010). Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus. Mol. Cell Proteomics 9, 2438-2459.

115. Naka, K. et al. (2010). TGF-beta-FOXO signaling maintains leukemia-initiating cells in chronic myeloid leukemia. Nature 463, 676-678.

116. Neckers, L. (2007). Heat shock protein 90: the cancer chaperone. J Biosci 32, 517-530.

117. Ngo, V.N., Davis, R.E., Lamy, L., Yu, X., Zhao, H., Lenz, G., Lam, L.T., Dave, S., Yang, L., Powell, J., et al. (2006). A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106-110.

118. Nimmanapalli, R., O'Bryan, E., and Bhalla, K. (2001). Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr-Abl levels and induces apoptosis and differentiation of Bcr-Abl-positive human leukemic blasts. Cancer Res 61, 1799-1804.

119. Nishiya, Y.; Shibata, K.; Saito, S.; Yano, K.; Oneyama, C; Nakano, H.; Sharma, S. V., Anal. Biochem. 2009, 385, 314-320.

120. Nobukuni, T., Kozma, S.C. & Thomas, G. (2007). hvps34, an ancient player, enters a growing game: mTOR Complexl/S6Kl signaling. Curr. Opin. Cell Biol. 19, 135-141.

121. Nomura, D.K., Dix, M.M. & Cravatt, B.F. (2010). Activity-based protein profiling for biochemical pathway discovery in cancer. Nat. Rev. Cancer 10, 630-638.

122. Obermann, W. M. J., Sondermann, H., Russo, A. A., Pavletich, N. P., and Hartl, F. U.

(1998). J. Cell Biol. 143, 901-910.

123. Oda, A., Wakao, H. & Fujita, H. (2002). Calpain is a signal transducer and activator of transcription (STAT) 3 and STAT5 protease. Blood 99, 1850-1852.

124. Ohh, M., Kim, W.Y., Moslehi, J.J., Chen, Y., Chau, V., Read, M.A., and Kaelin, W.G., Jr. (2002). An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells. EMBO Rep 3, 177-182.

125. Old, D. W.; Wolfe, J. P.; Buchwald, S. L., J. Am. Chem. Soc. 1998, 120, 9722-9723.

126. Oron, E., Mannervik, M., Rencus, S., Harari-Steinberg, O., Neuman-Silberberg, S., Segal, D., and Chamovitz, D.A. (2002). COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster. Development 129, 4399- 4409.

127. Panaretou, B., Prodromou, C, Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1998). EMBO 17, 4829-4836.

128. Panattoni, M., Sanvito, F., Basso, V., Doglioni, C, Casorati, G., Montini, E., Bender, J.R., Mondino, A., and Pardi, R. (2008). Targeted inactivation of the COP9 signalosome impairs multiple stages of T cell development. J Exp Med 205, 465-477.

129. Parsons, D.W. et al. (2008). An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807-1812.

130. Patel, P. D.; Patel, M. R.; Kaushik-Basu, N.; Talele, T. T., J. Chem. Inf. Model. 2008,

48, 42-55.

131. Paukku, K. & Silvennoinen, O. (2004). STATs as critical mediators of signal transduction and transcription: lessons learned from STAT5. Cytokine Growth Factor Rev. 15, 435-455. 132. Peth, A., Berndt, C, Henke, W., and Dubiel, W. (2007). Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability. BMC Biochem 8, 27.

133. Powers, M.V., Clarke, P.A., Workman, P. (2008). Dual targeting of Hsc70 and Hsp72 inhibits Hsp90 function and induces tumor-specific apoptosis. Cancer Cell 14, 250-262.

134. Pratt, W.B., Morishima, Y. & Osawa, Y. (2008). The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts. J. Biol. Chem. 283, 22885- 22889.

135. Reeder C, G.M., Habermann T, Ansell S, Micallef I, Porrata L, Johnston P, Maurer M, LaPlant B, Kabat B, Inwards D, Colgan J, Call T, Markovic S, Zent C, Zeldenrust S, Tun H, and Witzig T (2007). A Phase II Trial of the Oral mTOR Inhibitor Everolimus (RAD001) in Relapsed Aggressive Non-Hodgkin Lymphoma (NHL). Blood (ASH Annual Meeting Abstracts) 110, 121.

136. Ren, R. (2005). Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat. Rev. Cancer 5, 172-183.

137. Richardson, K.S., and Zundel, W. (2005). The emerging role of the COP9 signalosome in cancer. Mol Cancer Res 3, 645-653.

138. Rix, U. & Superti-Furga, G. (2009). Target profiling of small molecules by chemical proteomics. Nat. Chem. Biol. 5, 616-624.

139. Roe, S. M.; Prodromou, C; O'Brien, R.; Ladbury, J. E.; Piper, P. W.; Pearl, L. H., J.

Med. Chem. 1999, 42, 260-266.

140. Salgia, R. et al. (1995). Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL. Oncogene 11, 1149-1155.

141. Sawyers, C.L. (1993). The role of myc in transformation by Bcr-Abl. Leuk. Lymphoma.

11, 45-46.

142. Schrodinger, L. L. C, New York.

143. Schwechheimer, C, and Deng, X.W. (2001). COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol 11, 420-426.

144. Schweitzer, K., Bozko, P.M., Dubiel, W., and Naumann, M. (2007). CSN controls NF- kappaB by deubiquitinylation of IkappaBalpha. EMBO J 26, 1532-1541.

145. Schweitzer, K., and Naumann, M. (2010). Control of NF-kappaB activation by the COP9 signalosome. Biochem Soc Trans 38, 156-161.

146. Serenex; Huang, K.; Ommen, A. J.; Barta, T. E.; Hughes, P. F.; Veal, J. M.; Ma, W.;

Smith, E. D.; Woodward, A. R.; McCall, W. S., WO-2008130879A2 2008. 147. Serenex; Huang, K.; Ommen, A. J.; Barta, T. E.; Hughes, P. F.; Veal, J. M.; Ma, W.; Smith, E. D.; Woodward, A. R.; McCall, W. S., US-20080269193A1 2008.

148. Sharon, M., Mao, H., Boeri Erba, E., Stephens, E., Zheng, N., and Robinson, C.V.

(2009). Symmetrical modularity of the COP9 signalosome complex suggests its multifunctionality. Structure 17, 31-40.

149. Si, J. & Collins, S.J. (2008). Activated Ca2+/calmodulin-dependent protein kinase Ilgamma is a critical regulator of myeloid leukemia cell proliferation. Cancer Res. 68, 3733-3742.

150. Sidera, K.; Patsavoudi, E., Cell Cycle 2008, 7, 1564-1568.

151. Solit, D. B., Zheng, F. F., Drobnjak, M., Munster, P. N., Higgins, B., Verbel, D., Heller, G., Tong, W., Cardon-Cardo, C, Agus, D. B., Scher, H. I., and Rosen, N. (2002). Clin. Cancer Res. 8, 986-993.

152. Stebbins, C. E.; Russo, A. A.; Schneider, C; Rosen, N.; Hartl, F. U.; Pavletich, N. P., Cell 1997, 89, 239-250.

153. Su, T.T., Guo, B., Kawakami, Y., Sommer, K., Chae, K., Humphries, L.A., Kato, R.M., Kang, S., Patrone, L., Wall, R., et al. (2002). PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling. Nat Immunol 3, 780-786.

154. Supriatno, Harada, K., Yoshida, H., and Sato, M. (2005). Basic investigation on the development of molecular targeting therapy against cyclin-dependent kinase inhibitor p27Kipl in head and neck cancer cells. Int J Oncol 27, 627-635.

155. Taldone, T. & Chiosis, G. (2009). Purine-scaffold hsp90 inhibitors. Curr. Top. Med.

Chem. 9, 1436-1446.

156. Taldone, T., Gozman, A., Maharaj, R., and Chiosis, G. (2008). Targeting Hsp90: small- molecule inhibitors and their clinical development. Curr Opin Pharmacol 8, 370-374. 157. Taldone, T., Sun, W., Chiosis, G. (2009). Bioorg. Med. Chem. 17, 2225-2235.

158. Taldone T, Zatorska D, Patel PD, Zong H, Rodina A, Ahn JH, Moulick K, Guzman ML, Chiosis G. Design, synthesis, and evaluation of small molecule Hsp90 probes. Bioorg Med Chem. 2011 Apr 15; 19(8):2603-14. Epub 2011 Mar 12. PMID: 21459002

159. Taldone T, Gomes-DaGama EM, Zong H, Sen S, Alpaugh ML, Zatorska D, Alonso- Sabadell R, Guzman ML, Chiosis G. Synthesis of purine-scaffold fluorescent probes for heat shock protein 90 with use in flow cytometry and fluorescence microscopy. Bioorg Med Chem Lett. 2011 Sep 15; 21(18):5347-52. Epub 2011 Jul 14. PMID: 21802945 160. Tateishi, K., Omata, M., Tanaka, K., and Chiba, T. (2001). The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice. J Cell Biol 155, 571-579.

161. Thome, M. (2004). CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat Rev Immunol 4, 348-359.

162. Tomoda, K., Kubota, Y., Arata, Y., Mori, S., Maeda, M., Tanaka, T., Yoshida, M., Yoneda-Kato, N., and Kato, J.Y. (2002). The cytoplasmic shuttling and subsequent degradation of p27Kipl mediated by Jabl/CSN5 and the COP9 signalosome complex. J Biol Chem 277, 2302-2310.

163. Tomoda, K., Kubota, Y., and Kato, J. (1999). Degradation of the cyclin-dependent- kinase inhibitor p27Kipl is instigated by Jabl . Nature 398, 160-165.

164. Tomoda, K., Yoneda-Kato, N., Fukumoto, A., Yamanaka, S., and Kato, J.Y. (2004).

Multiple functions of Jabl are required for early embryonic development and growth potential in mice. J Biol Chem 279, 43013-43018.

165. Trinkle-Mulcahy, L., et al. (2008). Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell. Biol. 183, 223-239.

166. Tsaytler, P.A., Krijgsveld, J., Goerdayal, S.S., Rudiger, S. & Egmond, M.R. (2009).

Novel Hsp90 partners discovered using complementary proteomic approaches. Cell Stress Chaperones 14, 629-638.

167. Wang, J., Li, C, Liu, Y., Mei, W., Yu, S., Liu, C, Zhang, L., Cao, X., Kimberly, R.P., Grizzle, W., et al. (2006). JAB1 determines the response of rheumatoid arthritis synovial fibroblasts to tumor necrosis factor-alpha. Am J Pathol 169, 889-902.

168. Wang, Y., Penfold, S., Tang, X., Hattori, N., Riley, P., Harper, J.W., Cross, J.C., and Tyers, M. (1999). Deletion of the Cull gene in mice causes arrest in early embryogenesis and accumulation of cyclin E. Curr Biol 9, 1191-1194.

169. Wanner, K., Hipp, S., Oelsner, M., Ringshausen, I., Bogner, C, Peschel, C, and Decker, T. (2006). Mammalian target of rapamycin inhibition induces cell cycle arrest in diffuse large B cell lymphoma (DLBCL) cells and sensitises DLBCL cells to rituximab. Br J Haematol 134, 475-484.

170. Wei, N., and Deng, X.W. (1998). Characterization and purification of the mammalian COP9 complex, a conserved nuclear regulator initially identified as a repressor of photomorphogenesis in higher plants. Photochem Photobiol 68, 237-241. 171. Welteke, V., Eitelhuber, A., Duwel, M., Schweitzer, K., Naumann, M., and Krappmann, D. (2009). COP9 signalosome controls the Carmal-BcllO-Maltl complex upon T-cell stimulation. EMBO Rep 10, 642-648.

172. Whitesell, L. & Lindquist, S. L. (2005). Nat. Rev. Cancer 5, 761-772.

173. Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and Neckers, L. M. (1994).

Proc. Natl. Acad. Sci. USA 91, 8324-8328.

174. Winkler, G S. et al. (2002). Isolation and mass spectrometry of transcription factor complexes. Methods 26, 260-269.

175. Workman, P., Burrows, F., Neckers, L. & Rosen, N. (2007). Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann. N. Y. Acad. Sci. 1113, 202-216.

176. Wright, G., Tan, B., Rosenwald, A., Hurt, E.H., Wiestner, A., and Staudt, L.M. (2003).

A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 100, 9991-9996.

177. Xu, D. & Qu, C.K. (2008). Protein tyrosine phosphatases in the JAK/STAT pathway.

Front. Biosci. 13, 4925-4932.

178. Yan, J., Walz, K., Nakamura, H., Carattini-Rivera, S., Zhao, Q., Vogel, H., Wei, N., Justice, M.J., Bradley, A., and Lupski, J.R. (2003). COP9 signalosome subunit 3 is essential for maintenance of cell proliferation in the mouse embryonic epiblast. Mol Cell Biol 23, 6798-6808.

179. Yap, T.A., Garrett, M.D., Walton, M.I., Raynaud, F., de Bono, J.S., and Workman, P.

(2008). Targeting the PI3K-AKT-mTOR pathway: progress, pitfalls, and promises. Curr Opin Pharmacol 8, 393-412.

180. Ye, B.H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard, R., Leung, C, Nouri- Shirazi, M., Orazi, A., Chaganti, R.S., et al. (1997). The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat Genet 16, 161-170.

181. Ye, B.H., Lista, F., Lo Coco, F., Knowles, D.M., Offit, K., Chaganti, R.S., and Dalla- Favera, R. (1993). Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large- cell lymphoma. Science 262, 747-750.

182. Yoneda-Kato, N., Tomoda, K., Umehara, M., Arata, Y., and Kato, J.Y. (2005). Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3.

EMBO J 24, 1739-1749.

183. Zhao, X. et al. (2009). Methylation of RUNXl by PRMTl abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev. 22, 640-653. Zuehlke, A. & Johnson, J.L. (2010). Hsp90 and co-chaperones twist the functions of diverse client proteins. Biopolymers 93, 211-217.