CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 61/191,921, filed September 11, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] Supported by NCI grant UOl CA84244 and United States Department of Energy DE-FG02-03ER63630. The government has certain rights in this invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. [0003] Not applicable

BACKGROUND OF THE INVENTION

[0004] Cancer is a disease marked by the uncontrolled growth of abnormal cells. Cancer cells have overcome the barriers imposed on normal cells, which have a finite lifespan, to grow indefinitely. As cancerous cell growth continues, genetic alterations may mount until a cancerous cell has achieved a more aggressive growth phenotype. If left untreated, metastasis, which is the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream, may ensue. Metastasis can result in the destruction of healthy organs and tissues when cancerous cells infiltrate different areas of the body and continue growing to form new tumors.

[0005] According to a recent American Cancer Society study, approximately 1,500,000 new cancer cases are expected to be diagnosed in the United States in the year 2008 alone. Despite the availability of numerous chemotherapeutic agents and diagnostic tools, additional diagnostic methods are needed to investigate the mechanisms and pathways that lead to cancerous cell growth. Such methods may lead to early intervention that can be used effectively against various types of cancers. Specific cancers respond to certain treatments better, identification of such cancers may lead to a better cure rate. This invention caters to such needs among others.

BRIEF SUMMARY OF THE INVENTION

[0006] The present disclosure demonstrates that depletion of FBXW7 increases the levels of mTOR and p-mTOR. The enzyme mTOR (mammalian target of rapamycin) has become a major target for therapeutic intervention to treat a number of complex human diseases, including cancer and cardiovascular dysfunction. While much has been learned about the signaling mechanisms that operate upstream (Pten-PI3 K-AKT) or downstream (S6-K, 4E- BP) from mTOR, almost nothing is known about the processes that control levels of mTOR protein.

[0007] The present invention demonstrates that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7 (also known as hCDC4, FBWl and MGO). FBXW7 binds directly to mTOR, leading to increased ubiquitination and protein turnover, both in mouse and in human cells. Since deletion or mutation of human PTEN (phosphatase and tensin homologue) also leads to activation of mTOR, the genetic relationship between FBXW7 and PTEN in human cancers was also investigated.

[0008] Cell lines from human breast cancers, as well as primary breast tumors, showed a reciprocal relationship between deletion or mutation of PTEN and loss of FBXW7. Tumors that showed heterozygous loss of FBXW7 generally showed no loss or mutation of PTEN, and vice versa. Tumor cell lines harboring deletions or mutations in FBXWl are particularly sensitive to Rapamycin treatment, suggesting that loss of FBXWl may be a novel biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway.

[0009] In one aspect this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involving a step of determining if one or more copies of the Fbxw7 gene is mutated (includes deletion) in a patient sample.

[0010] In another aspect this invention provides a method of determining the risk of cancer. The method involves the step of determining the amounts of mTOR, p-mTOR and/or one or more downstream components relative to one or more upstream components, wherein the downstream and upstream components are components of the mTOR signaling pathway.

[0011] In yet another aspect this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involves a step of determining the amounts of mTOR, p-mTOR and/or one or more downstream components relative to one or more upstream components in a patient sample, wherein said downstream and upstream components are components of the mTOR signaling pathway.

[0012] In another embodiment a method of assaying for compounds that treat cancer in a subject is disclosed. The method comprising the steps of contacting a cell with one or more candidate compounds, and monitor the effect of said one or more candidate compounds on Fbxw7 gene or protein expression; thereby indentifying said one or more candidate compounds that alters mTOR expression or activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 illustrates identification of Fbxw7 novel targets by genome-wide search using CPD sequences. Comparison of minimal consensus CPD sequences found in mTOR with those of the known human target proteins cyclin E (CCNE) and C-Myc.

[0014] Figure 2 illustrates that CPD sequences of mTor are evolutionarily conserved in different species ranging from humans to zebra fish.

[0015] Figure 3 shows that depletion of FBXW7 increases mTOR and p-mTOR levels.

[0016] Figure 3 A shows that the levels of mTor and p-mTor, as well as the downstream mTor target S6-kinase (P-S6), are upregulated in Fbxw7-/- MEFs, whereas the levels of Akt and p-Akt (lower panels) were not appreciably affected. Two independent preparations of MEFs were used, with similar results in both cases.

[0017] Figure 3B illustrates elevation of mTor in tissues from Fbxw7 knockout mice. For both thymus (left panel) and spleen (right panel) Lane 1 contains extracts from wild-type mice, lane 2 from Pten+I- mice, and lane 3 from Fbxw7+/- mice. Comparing lane 1 with lane 3, mTor and p-mTor protein levels are increased in the Fbxw7+I- tissues. In contrast, levels of Akt and p-Akt are not significantly altered in the same Fbxw7+/- lysates. While loss of one copy of Pten does not significantly change mTor protein levels, both Akt and p-Akt are elevated.

[0018] Figure 3C shows that the levels of mTOR and p-mTOR, as well as the downstream mTOR target S6-kinase (P-S6), are upregulated by depletion FBXW7 in human colon cancer cell lines HCTl 16 and DLDl, whereas the levels of AKT and p-AKT (lower panels) were not appreciably affected.

[0019] Figure 3D illustrates downregulation of mTOR by expression of a dominant negative truncated FBXW7 protein. Overexpression of full length FBXW7 (HA-FBXW7) in 293T cells decreases the mTOR and p-mTOR levels (lane 2) in comparison to cells containing only the empty vector (lane 1), in contrast, overexpression of a dominant-negative form of FBXW7 (HA-FBXW7-ΔF) increases the mTOR and p-mTOR levels (Iane3).

[0020] Figure 4 shows that mTOR interacts directly with FBXW7.

[0021] Figure 4 A illustates immunoprecipitation of HA tagged FBXW7 identifies mTOR as an interacting protein. Human 293 cells were transfected with the full length or truncated (FBXW7 or FBXW7-ΔF) constructs, followed by immunoprecipitation of the HA-tagged proteins and immunoblotting with antibodies against mTOR (top panel). In the reciprocal experiment, immunoprecipitation of mTOR shows that the region of FBXW7 that interacts is the WD40 domain which is retained in the FBXW7ΔF construct (middle panel). Lower panel shows the input levels of mTOR and FBXW7 proteins in the lysates.

[0022] Figure 4B shows that the wild type fragment of human mTOR was cloned into p3XFL AG-CMV- 10 vector. The DelT631 and T631G fragments were generated by in vitro site-directed mutagenesis.

[0023] Figure 4C shows that FBXW7 binds the wild-type fragment of mTOR, but binding to the mTOR(delT631) and mTOR(T631G) mutants was dramatically reduced by about 80%.

[0024] Figure 4D shows that MCF7 breast cancer cells show increased mTOR levels upon treatment with MG-132, but this is not seen in SUM149PT cells which have no functional FBXW7 gene.

[0025] Figure 4E illustrates that the regulation of mTOR by FBXW7 is through the proteasome-dependent degradation pathway. 293 T and SUM 149PT cells were transfected with a vector containing CMV promoter-driven HA-ubiquitin. Immunoprecipitation of mTOR followed by Western blot analysis of the HA-ubiquitin showed that in 293T cells which have functional FBXW7 protein, the immunoprecipitated mTOR is ubiquitinated, whereas SUM 149PT cells, which lack FBXW7, show no mTOR ubiquitination. The two right hand lanes show overall ubiquitin levels in the same cell lysates.

[0026] Figure 4F, HCTl 16_WT and HCTl 16_FBXW7-/- cells were transfected with HA- ubiquitin. Immunoprecipitation of mTOR followed by Western blot analysis of the HA- ubiquitin showed that ubiquitination of mTOR was only seen in the HCTl 16_WT cells, and not in HCTl 16_FBXW7-/- cells. The vector lane shows HCTl 16_FBXW7-/- cells transfected with empty vector construct.

[0027] Figure 4G illustrates that ubiquitination of mTOR is restored by exogenous FBXW7 expression. HCTl 16_FBXW7-/- cells were transfected with an FBXW7-expressing construct and HA-tagged ubiquitin. Immunoprecipitation of mTOR showed increased ubiquitination compared to controls. The increased ubiquitination had only a small effect on mTOR levels in these experiments (Figure 8), probably because of the pleiotropic effects of over-expressing this potent tumor suppressor in tumor cells, which leads to growth arrest and apoptosis.

[0028] Figure 5 illustrates genetic interaction between FBXW7 and PTEN in human breast cancers. The panels show concordance of gene copy number changes in breast cancer cell lines (Figure 5A) and in 3 independent sets of primary breast tumors (Figures 5B-D). The lighter-gray bar indicates loss, the darker-gray bar indicates gain, and the black bar indicates no changes in gene copy number.

[0029] Figure 5A illustrates the data from 53 human breast cancer cell lines, ordered in the vertical axis from 1-53 (Neve R.M. et al., Cancer Cell 10:515-27 (2006)). The copy number of FBXW7 and PTEN was determined by quantitative PCR TaqMan using FBXW7 and PTEN specific probes. Lighter-gray bars indicating deletion of FBXW7 rarely occur in tumors that also show deletion of PTEN (pvalue=0.014).

[0030] Figures 5B-D illustrate three independent sets of human primary breast cancers, the DNAs of which have been analyzed by the same BAC CGH microarray platform.

(B) 185 human primary breast cancers analyzed by Climent et al. (Cancer Res. 67:818-26 (2007)).

(C) 145 human primary breast cancers analyzed by Chin et al. (Cancer Cell. 10:529-41 (2006)). (D) 67 human primary breast cancers analyzed by Fridlyand et al. (BMC Cancer. 6:96 (2006)).

The copy number of FBXWl and PTEN was determined based on the published CGH data (FBXW7 was based on BAC RPl 1-73G16, PTEN on BAC RPl 1-380G5). Loss is defined as Iog ₂ (ratio) <-0.25 and gain as Iog ₂ (ratio) >0.25.

[0031] Figure 6, RNA expression of FBXW7 and PTEN in breast cancer cell lines was analyzed by RT-PCR. The arrows indicate cell lines carrying mutations as determined by sequence analysis (see Table 1).

[0032] Figure 7 illustrates the relationship between loss of FBXW7 and sensitivity to mTOR inhibitor (rapamycin) treatment.

[0033] Figure 7A shows that the breast cancer SUM 149PT cells which have homozygous mutations in FBXW7 were killed at a rapamycin concentration of 10OnM, whereas MDA- MB453 cells with wild type FBXW7 were resistant to this treatment.

[0034] Figure 7B deals with the treatment of nude mouse xenografts with rapamycin. The SUM 149PT cells showed a relative decrease in size followed by stable tumor growth, whereas the MDAMB453 cells were unaffected by treatment.

[0035] Figure 7C shows that the deletion or mutation of FBXW7 increases sensitivity to mTOR inhibitor (rapamycin) treatment in human breast cancers. Tumor cells with deletion or mutation of FBXW7 are in Group 1, those with deletion or mutation of PTEN are Group 2, and cells with wild type copies of both genes are in Group 3.

[0036] Figures 7D-F shows that downregulation of FBXW7 using specific shRNA in rapamycin resistant MDA-MB453 (D), LY2 (E) and MCF7 (F) cells increases the sensitivity to this treatment.

[0037] Figure 8 shows that the exogenous FBXW7 expression in SUM 149PT cells reduces mTOR protein levels (lane 2), but increased mTOR levels upon treatment with MG-132 (lane 4).

[0038] Figure 9. HA-FBXW7 co-immunoprecipitates with mTOR fragment, HA-HtrA2 does not. Lane 1 : vector; Lane 2: HA-FBXW7 and Flag-mTOR fragment (wild type); and Lane 3: HA-HtrA2 and Flag-mTOR fragment (wild type).

[0039] Figure 10. HA-FBXW7 co-immunoprecipitates with endogenous mTOR, HA- HtrA2 does not. Lane 1 : vector; Lane 2: HA-FBXW7; and Lane 3: HA-HtrA2. DETAILED DESCRIPTION OF THE INVENTION INTRODUCTION

[0040] The present invention provides methods of determining whether or not a cancer patient has a mutated Fbxw7 gene (point mutations, deletions, or additions, including the absence of the gene by complete deletion and promoter silencing) and thereby determining whether or not the patient is a candidate for mTOR inhibitor therapy. Polymorphism may also be considered as a mutation for the purposes of this application. The determination involves detecting Fbxw7 DNA, RNA, or protein and determining whether or not the molecule is mutated, thereby determining whether or not the gene is mutated. Any method known to those of skill in the art to detect mutations can be used, including PCR (e.g., Taqman), sequencing techniques, Southern, western, and northern blots, microarrays, immunohistochemical techniques, ELISA, mass spectroscopy, enzymatic, binding or functional assays, and the like.

[0041] This invention also presents a method of determination of risk of cancer by comparing the relative amounts of components of the mTOR signaling pathway that are downstream to mTOR versus the amounts of components of the mTOR signaling pathway that are upstream to mTOR. Relatively higher levels of mTOR and downstream components as compared to a control are indicative of absence of the Fbxw7 protein or presence of the Fbxw7 mutated gene.

DEFINITIONS

[0042] "Mammalian target of rapamycin protein inhibitor" or "mTOR inhibitor" includes drugs such as rapamycin, deforolimus, sirolimus, temsirolimus, and everolimus that selectively inhibit the mammalian target of rapamycin (mTOR).

[0043] As used herein, the term "cancer" refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells. Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion/metastasis, limitless replicative potential, and sustained angiogenesis. The term "cancer cell" is meant to encompass both pre-malignant and malignant cancer cells. Other forms of cancer include carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, head and neck cancer, e.g. , oral cavity, pharyngeal and tongue cancer, kidney, breast, lung, kidney, bladder, colon, endometrium, T- cell acute lymphoblastic leukemia (T-ALL), ovarian, prostate, pancreas, stomach, brain, skin, melanoma, basal cell, uterine, testicular, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia, and multiple myeloma.

[0044] The term "marker" refers to a molecule (typically protein, nucleic acid, carbohydrate, or lipid) that is expressed in the cell, expressed on the surface of a cancer cell, secreted by a cancer cell or over or under expressed as compared to a non-cancer cell, and which is useful for the diagnosis of cancer, for providing a prognosis, and for preferential targeting of a pharmacological agent to the cancer cell. Oftentimes, such markers are molecules that are differentially expressed, e.g., overexpressed or underexpressed in a melanoma or other cancer cell in comparison to a normal cell, for instance, 1 -fold over/under expression, 2-fold over/under expression, 3 -fold over/under expression or more in comparison to a noncancerous cell, or a primary cancer (vs. mets). Further, a marker can be a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a non-cancerous cell.

[0045] It will be understood by the skilled artisan that markers may be used singly or in combination with other markers for any of the uses, e.g., determination of the suitability of a subject for mTOR inhibitor therapy, disclosed herein.

[0046] "Biological sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, hair, nails, skin tissue, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird; reptile; or fish.

[0047] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., skin, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy" of the tumor mass, or a "fine-needle aspiration biopsy" which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison 's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V.

Fbxw7 Mutations, Detection of Risk of Cancer and mTOR Inhibitor Therapy

[0048] In one aspect this invention provides a method of determining if a subject at risk for or diagnosed with cancer should receive mTOR inhibitor therapy, the method involving a step of determining if the Fbxw7 gene is mutated in a patient sample.

[0049] In another aspect this invention provides a method of determining if a subject is at risk for cancer is presented. The method involves determining the amounts of mTOR, p- mTOR or one or more downstream components relative to one or more upstream components in a patient sample and comparing the amounts to those obtained in a known sample. The known sample could either be a cancerous sample or a cancer-free sample. The terms upstream and downstream are used in this application are with reference to mTOR in the mTOR signaling pathway. Relatively higher levels of mTOR and /or downstream components could be interpreted as indicative of the subject being at risk for cancer (i.e. through disruption of the Fbxw7 pathway).

[0050] In yet another aspect of the invention a method of determining if a subject is at risk for cancer should receive mTOR inhibitor therapy is presented. The method involves determining the amounts of mTOR, p-mTOR or one or more downstream components relative to one or more upstream components in a patient sample and comparing the amounts to those obtained in a known sample. The known sample could either be a cancerous sample or a cancer-free sample. Relatively higher levels of mTOR and/or downstream components may be used as diagnostic tool for presenting mTOR inhibitors. [0051] If a ratio (quantity of the protein mTOR or its downstream components to amount of upstream components) in a patient sample appears to be greater that that found in a known non-cancerous sample, then the subject may be at a risk for cancer. Preferably the ratio in a positive patient sample are at least 1.5 times those found in a known non-cancerous sample. More preferably they are at least 2.0 times as large and most preferably they are 5.0 times as large.

[0052] In one embodiment the patient sample includes any biological sample as described above in the definitions section. Preferably the sample is a biopsy or blood, or saliva. The biological sample may be collected by any of the multiple means known to one of skilled in the art, few of such collection methods are discussed above.

Upstream and Downstream Components of mTOR in the mTOR Signaling Pathway

[0053] In one embodiment of the invention the upstream component(s) is/are preferably selected from p-Akt, Akt, and PDK and the downstream component(s) is/are preferably selected from p-S6K, 4E-BP and downstream markers of protein translation activity, such as Eif4 family members, Mnkl or Mnk2 .

Screening for Active Molecules

[0054] In another embodiment a method of assaying for compounds that treat cancer in a subject is disclosed. The method comprising the steps of contacting a cell with one or more candidate compounds, and monitor the effect of said one or more candidate compounds on Fbxw7 gene or protein expression; thereby indentifying said one or more candidate compounds that alters mTOR expression or activity.

[0055] The test molecules may be chosen from a compound libraries or from known anticancer agents. Combinations may also be used in the above disclosed screening method.

mTOR Inhibitors and Other Anti-cancer Treatments

[0056] In one embodiment of the invention typical mTOR inhibitors can be selected preferably from rapamycin, deforolimus, sirolimus, temsirolimus, everolimus, polymorphs, prodrugs and derivatives thereof. mTOR inhibitors could be as discussed in US Patent Publication no. 2007/0167478. Suitable derivatives of rapamycin could also be as discussed in WO 94/09010, WO 95/16691, WO 96/41807, U.S. Pat. No. 5,362,718 or WO 99/15530. All of the documents listed above are incorporated herein by reference. In addition to the mTOR inhibitors the subject may receive additional treatments if such a treatments are deemed necessary. Additional treatments may involve the use of surgery, anti-cancer agents, radiation, hormonal therapy or immunotherapy.

[0057] Examples of anti -cancer agents acceptable for use in the present invention include, without limitation, alkylating agents, anti-metabolites, plant alkaloids and terpenoids, topoisomerase inhibitors, antineoplastics, hormone therapeutics, photosensitizers, kinase inhibitors, etc.

[0058] Examples of alkylating agents include, without limitation, cisplatin, caroplatin, oxaliplatin, mechlorethamine, cyclophophamide, chlorambucil, busulfan, hexamethylmelamine, thiotepa, cyclophohphamine, uramustine, melphalan, ifosfamide, carmustine, streptozocin, dacarbazine, temozolomide, etc.

[0059] Examples of anti-metabolite agents include, without limitation, Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine, Cytarabine, Decitabine, Fluorouracil, Floxuridine, Gemcitabine, etc.

[0060] Examples of plant alkaloids and terpenoids include, without limitation, Docetaxel, Larotaxel, Paclitaxel, Vinblastine, Vincristine, Vindesine, Vinorelbine, etc.

[0061] Examples of topoisomerase inhibitors include, without limitation, Camptothecin, Topotecan, Irinotecan, Rubitecan, Etoposide, Teniposide, etc.

[0062] Examples of antineoplastics include, without limitation, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Pixantrone, Valrubicin, Actinomycin, Bleomycin, Mitomycin, Plicamycin, etc.

[0063] Examples of photosensitizer agents include, without limitation, Aminolevulinic acid, Methyl aminolevulinate, Porfimer sodium, Verteporfin, etc. [0064] Examples of kinase inhibitors include, without limitation, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, Vandetanib, Seliciclib, etc.

[0065] Examples of other anti-cancer agents include, without limitation, Alitretinoin, Tretinoin, Aflibercept, Altretamine, Amsacrine, Anagrelide, Arsenic trioxide, Pegaspargase, Bexarotene, Bortezomib, Celecoxib, Denileukin diftitox, Elesclomol, Estramustine, Irofulven, Ixabepilone, Masoprocol, Mitotane, Oblimersen, Testolactone, Tipifarnib, Trabectedin.

Detection of Fbxw7 Gene Mutation

[0066] In another embodiment determining Fbxw7 gene mutation can be performed in multiple methods, preferred methods could involve methods such as analysis of the patient DNA, RNA and protein.

[0067] In yet another embodiment analysis of the DNA, RNA and protein can be carried out by various means. Preferred means include PCR (e.g., Taqman), sequencing techniques, Southern, western, and northern blots, microarrays, immunohistochemical techniques, ELISA, mass spectroscopy, enzymatic, binding or functional assays.

[0068] Preferred methods for the detection and quantification of mTOR, upstream and downstream proteins include western blot, immunohistochemical techniques, ELISA and mass spectroscopy.

RNA and DNA Detection

® [0069] PCR assays such as Taqman allelic discrimination assay, available from Applied

Biosystems, can be used to identify RNA. In another embodiment, mass spectroscopy can be used to detect either nucleic acid or protein. Any antibody-based technique for determining a level of expression of a protein of interest can be used. For example, immunoassays such as ELISA, Western blotting, flow cytometry, immunofluorescence, and immunohistochemistry can be used to detect protein in patient samples. Combinations of the above methods, such as

® those employed in the Luminex xMAP technology can also be used in the present invention.

Analysis of a protein or nucleic acid can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.).

[0070] Applicable PCR amplification techniques are described in, e.g., Ausubel et al and Innis et al , supra. General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences {e.g., genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, "Microarrays Methods and Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al, "DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge University Press, 2002.

[0071] Analysis of nucleic acid markers can be performed using techniques known in the art including, without limitation, sequence analysis, and electrophoretic analysis. Non- limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods MoI Cell Biol, 3:39-42 (1992)), single base extension sequencing (SBE), pyrosequencing (Ronaghi et al, Science, 281(5375):363-365 (1998)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al, Science, 274:610-614 (1996); Drmanac et al, Science, 260:1649- 1652 (1993); Drmanac et al, Nat. Biotechnol, 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.

Analysis of Proteins

[0072] In another embodiment, antibody reagents can be used in assays to detect expression levels of protein biomarkers of the invention in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al., Curr. Opin. BiotechnoL, 7:60-65 (1996)). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. ScL, 699:463-80 (1997)). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997)). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).

[0073] Specific immunological binding of the antibody to a protein can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine- 125

12 ₅

( I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), y- galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea- bromocresol purple (Sigma Immunochemicals; St. Louis, MO).

[0074] A signal from a direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

[0075] The antibodies can be immobilized onto a variety of solid supports, such as polystyrene beads, magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0076] Immunohistochemical (IHC) methods, which are well known by those skilled in the art, may be used . See, for example, U.S. Pat. No. 6,441,143 to Koski et al., U.S. Pat. No. 6,376,201 to Miron et al., U.S. Pat. No. 5,876,712 to Cheever et al., U.S. Pat. No. 5,854,009 to Klug, and U.S. Pat. No. 5,843,684 to Levine et al., U.S. Pat. No. 4,968,603 to Slamon et al. and "DAKO anti-Her2 IHC System for Immunoenzymatic Staining" (Package Insert) DAKO Corporation. As described in U.S. Pat. No. 6,573,043 to Cohen et al, two general methods of IHC are available: direct and indirect assays. According to the first assay, binding of an antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. The fluorescent tag or label can be fluorescein. The enzymatic label can be horseradish peroxidase or alkaline phosphatase.

[0077] In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate can be added to provide visualization of the antigen. Such are described above. Signal amplification may occur because several secondary antibodies may react with different epitopes on the primary antibody. The primary and/or secondary antibody used for immunohistochemistry typically can be labeled with a detectable moiety. IHC techniques are further described in Immunohistochemical Staining Methods. Thomas Boenisch, ed. (3rd ed. 2001).

Physical Formats

[0078] Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers. Such formats include protein microarrays, or "protein chips" (see, e.g., Ng et al., J. Cell MoI. Med., 6:329- 340 (2002)) and certain capillary devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more protein markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more protein markers for detection.

[0079] Analysis of the level of a biomarker can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion.

[0080] Alternatively, the antibodies or nucleic acid probes of the invention can be applied to sections of patient biopsies immobilized on microscope slides. The resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.

EXAMPLES

Example 1

[0081] Genome-wide search: The Fbxw7 gene has been identified as a p53-deρendent tumor suppressor gene that undergoes deletion and/or mutation in tumors from a wide range of human tissues, including breast, colon, endometrium, stomach, lung, ovary, pancreas, and prostate (Welcker M. et al., Nat Rev Cancer. 8:83-93 (2008); and Akhoondi S. et al, Cancer Res. 67:9006-12 (2007)). Point mutations in FBXW7 were reported to be the most common genetic lesions in human T-cell acute lymphoblastic leukemia (T-ALL), indicating a wide role for this protein in suppression of most forms of human malignancies. The overall point mutation frequency of FBXW7 is about 6% (Akhoondi S. et al., Cancer Res. 67:9006-12 (2007)), but since Fbxw7 is a haplo-insufficient tumor suppressor (Mao J. H. et al., Nature 432:775-9 (2004)) and deletions at 4q are relatively frequent in human cancers (Mitelman F. et al., Mitelman Database of Chromosome Aberrations in Cancer (2007)), the impact of loss of FBXW7 in human cancers may have been underestimated.

[0082] The mechanism that is primarily responsible for suppression of tumor development by FBXW7 remains largely unclear. FBXW7 encodes an F-box protein essential for ubiquitination of several well-defined oncoproteins including Myc, Cyclin E, c-Jun, Notch and Aurora-A (Welcker M et al., Nat Rev Cancer. 8:83-93 (2008)). While loss 0ΪFBXW7 has been associated with increased genetic instability through deregulation of Cyclin E (Rajagopalan H. et al., Nature 428: 77-81 (2004)), c-Myc (Yada M. et al., EMBO J. 23: 2116- 25 (2004); and Welcker M. et al., Proc Natl Acad Sci U S A. 101 :9085-90 (2004)) or Aurora- A (Mao J. H. et al., Nature 432:775-9 (2004)), some studies failed to find this correlation, suggesting that additional mechanisms may involve other pathways (Kemp Z. et al., Cancer Res. 65:11361-6 (2005)). Therefore a genome-wide search was carried out for potentially novel Fbxw7 ubiquitination targets using the consensus CDC phosphodegron (CPD) sequence I/L-I/L/P-T-P-XXXX (where lysine and arginine are unfavorable in the X locations) (Welcker M. et al., Nat Rev Cancer. 8:83-93 (2008)) in a mouse protein database (http://www.ensembl.org/Mus_musculus). This search revealed a strong match to the consensus sequence within the HEAT domain of mTor (Figure 1). The mTor CPD region is evolutionarily conserved in different species ranging from humans to zebra fish (Figure 2), suggesting its potential functional importance.

Example 2

[0083] Depletion of Fbxw7 leads to increase of mTOR: mTOR is a central component of several complex signaling networks that regulate cell growth, metabolism and proliferation. Deregulation of mTOR is emerging as common theme in diverse human conditions including cancer, cardiovascular disease and ageing, and several drugs targeting this pathway are presently in clinical trials. The possibility that Fbxw7 may directly regulate levels of mTor was tested by analyzing two independent preparations of mouse embryonic fibroblasts (MEFs) from Fbxw7+/- mice (Figure 3A). Depletion of Fbxw7 increased the levels of both total mTor and phosphorylated mTor (p-mTor), as well as the downstream mTor target S6- kinase (P-S6). In contrast, there was no appreciable effect on upstream components of the mTor signaling pathway such as Akt and phosphorated Akt (p-Akt) (Figure 3 A, lower panels).

[0084] Depletion of Fbxw7 in vivo also leads to an increase in both mTor and p-mTor protein levels. Thymus and spleen from FbXwT ^+1' mice (lanes 3, Figure 3B) showed increased mTor and p-mTor in comparison with control wild type tissue (lanes 1, Figure 3B). In contrast, levels of total Akt and p-Akt (at Ser437) do not change appreciably (Figure 3B). However in Pten ^+/~ mice (lanes 2, Figure 3B), both thymus and spleen showed an increase in p-mTor without any obvious change in total mTor protein level. In agreement with these observations, p-Akt levels were elevated in thymus and spleen from Pten ^+I~ mice and presumably were responsible for activation of mTor signaling (Figure 3B).

[0085] A similar trend was seen in human cells from which the FBXW7 gene has been deleted by homologous recombination (Rajagopalan H. et al., Nature 428: 77-81 (2004)). Both HCTl 16 and DLDl cells that lack FBXW7 have slightly higher levels of mTOR and p- mTOR, as well as increased levels of p-S6K (Figure 3C). Again, no effect was seen on the upstream targets AKT and p-AKT (Figure 3C, lower panels). The effect seen in these human cells is weaker than in the mouse, possibly because these are fully transformed human tumor cell lines that can upregulate mTOR levels by additional mechanisms.

[0086] The possibility that downregulation of FBXW7 using a dominant negative approach would lead to increased levels of mTOR was also tested. Human 293 T cells were transfected with a dominant-negative form of FBXW7 comprising a truncated FBXW7 protein that contains only the WD40 domain (HA-FBXW7-ΔF) (Wu G. et al., J. MoI Cell Biol. 21:7403- 15 (2001)). Cells transfected with HA-FBXW7-ΔF showed an increase in the level of both total mTOR and p-mTOR (Figure 3D). Cells expressing the transfected normal HA-FBXW7 protein reproducibly had lower levels of both total mTOR and p-mTOR in comparison to cells containing only the empty vector (Figure 3D). In contrast, levels of AKT and p-AKT were not affected by overexpression of FBXW7 (Figure 3D).

Example 3

[0087] Interactions between mTOR and FBXW7 proteins: Possible direct interactions between mTOR and FBXW7 proteins were ivestigated. A vector encoding HA-tagged FBXW7 was transfected into 293T cells, followed by immunoprecipitation using antibodies directed against the HA tag, and Western blot analysis using anti-mTOR antibodies. The reciprocal experiment was also carried out in which mTOR was immunoprecipitated, followed by blotting with the anti-HA antibody. Both approaches demonstrated direct interaction between the mTOR and FBXW7 proteins (Figure 4A). To further confirm that the interaction site of FBXW7 is within the WD40 domain, 293T cells were transfected with the truncated construct HA-FBXW7-ΔF. Immunoprecipitation of one protein followed by Western blot analysis of the second revealed a strong interaction between mTOR and FBXW7-ΔF (Figure 4A), suggesting that the WD40 domain of FBXW7 is the interaction site. [0088] The consensus CPD site in human mTOR is located around T631 (Figure 1). A fragment of mTOR (from 1 to 898 AAs) that contains the putative CPD was cloned, two mutants were generated, one carrying a deletion of T631 [mTOR(delT631)], and the other a point mutation converting T631 to G [mTOR(T63 IG)] (all with Flag tags) (Figure 4B). The wild type and mutant versions of this mTOR fragment were co-expressed with HA-FBXW7 in 293T cells, followed by immunoprecipitation with antibodies against one protein and Western blot analysis of the second. Although the wild type fragment of mTOR immunoprecipitated efficiently with FBXW7, binding to the mTOR (delT631) and mTOR(T63 IG) mutants was dramatically reduced (Figure 4C). In repeat experiments by two different investigators, both mutants retained some residual binding to FBXW7 in these co- transfection assays, suggesting it is possible to present an additional, but weaker, consensus binding site in mTOR.. These data demonstrate that FBXW7 binds mTOR predominantly through the major conserved CPD site. Example 4

[0089] Regulation of mTOR by FBXW7: In order to determine whether the regulation of mTOR by FBXW7 is through the proteasome-dependent degradation pathway, MCF7 and SUM149PT cells were treated with the proteasome inhibitor MG-132. MCF7 cells are widely used breast epithelial cells that express a wild type FBXWl gene, while SUM 149PT cells were derived from a breast carcinoma and have undergone homozygous mutation of FBXWl (Strohmaier H. et al., Nature. 413, 316-22 (2001) and Table 1). Proteasome inhibition caused a dramatic increase in the mTOR levels in MCF7 cells, which retain FBXWl, but not in SUM147PT cells (Figure 4D), suggesting that degradation of mTOR is FBXW7 -dependent. Whether the ubiquitination status of mTOR was FBXW7-dependent was also examined. In these experiments, 293T and SUM 149PT were transfected with a vector containing CMV promoter-driven HA-ubiquitin. Immunoprecipitation of mTOR followed by Western blot analysis of the HA-ubiquitin showed that mTOR ubiquitination was found only in cells retaining a functional FBXWl gene (Figure 4E). To exclude the possibility that other differences between 293T and SUM 149PT cells may account for this effect, the experiments using HCTl 16_WT and HCTl 16_FBXW7-/- cells (Figure 4F) were repeated. The results showed again that efficient ubiquitination of mTOR was only seen in the HCTl 16_WT cells, and not in HCTl 16_FBXW7-/- cells. Finally, SUM149PT cells were co-transfected with constructs encoding both FBXW7 and HA-ubiquitin, and it was found that ubiquitination of mTOR was restored by exogenous FBXW7 expression (Figure 4G). It is concluded that ubiquitination of mTOR is largely, if not exclusively, mediated by binding to FBXW7.

Table 1

Note: NC = no change *position start from ATG

[0090] The above results have revealed a possible genetic and functional relationship between Fbxw7 and the tumor suppressor Pten. Both genes can be deleted in human or mouse tumors, and deletion of either gene leads to activation and/or over-expression of mTOR. To explore the genetic relationship between FBXWl and PTEN in human cancers, the genetic status of both genes in a panel of 53 breast cancer cell lines that have been extensively characterized by Comparative Genomic Hybridization (CGH) and gene expression microarray analyses (Neve R. M., Cancer Cell 10:515-27 (2006)) were examined. Quantitative TaqMan assays for both FBXWl and PTEN genes were developed to quantify the number of copies in each of the cell lines. The TaqMan results were in good concordance with data found by BAC CGH Microarray using the BAC clones that lie close to the locations OΪFBXW7 and PTEN in the human genome (see Table 1). Surprisingly, it was found that tumors with deletions of one copy of PTEN frequently had no deletion at the FBXW7 locus, and vice versa. The data in Figure 3 A show that most of the breast cancer cell lines that exhibited loss of a single copy of FBXW7 (23 out of 53, shown in lighter-gray in the FBXW7 column, Figure 5A) did not show corresponding loss of PTEN. In contrast, of the 14 out of 55 lines that showed loss of a single copy of PTEN (lighter-gray bars in the PTEN column, Figure 5A), only one had also lost a copy of FBXW7, suggesting FBXW7 and PTEN show some functional redundancy in tumor development. Some of the cell lines showed copy number gains (in darker-gray) of either FBXW7 or PTEN.

[0091] To confirm these observations in human primary breast cancers, the gene copy number status of FBXW7 and PTEN in three independent human primary breast cancer sets were analyzed; which have been analyzed using the same BAC CGH microarray platform (Climent J. etal., Cancer Res. 67:818-26 (2007); Chin K., Cancer Cell. 10:529-41 (2006); and Fridlyand J., BMC Cancer. 6:96 (2006)). As shown in Figures 5B to 5D, essentially the same patterns were found in all of these tumor panels. In the breast cancers (Figures 5B, C, and D) a total of only 4 tumors had lost a copy of both genes, as shown by the juxtaposition of the lighter-gray bars in both FBXW7 and PTEN columns. The chance of this occurring as a consequence of random genetic alterations in the genomes of a total of 450 tumor and cell line DNA samples is extremely small (p= 4.9 x 10 ^"7 ). A similar trend was seen in a panel of 113 primary colon tumors, although the significance of these correlations was not as strong (p=0.025; Kim, I-J. et al, data not shown).

[0092] The possibility that other somatic changes such as point mutations or gene silencing events could impact the results was also considered. RNA expression levels for FBXW7 and PTEN in 25 breast cancer cell lines were analyzed and sequence analysis of the complete coding regions of both genes in these cell lines was also carried out. In all cases, the FBXW7 gene continued to be expressed (Figure 6), indicating that no gene silencing had occurred, although very low levels were found in 5 cells lines (lanes 10, 13, 14, 16, 20; Figure 6). All of these lines had lost one copy of the FBXW7 gene except one (SUM 149PT, lane 16), in which a point mutation was detected (table 1). The PTEN gene was found to be silent in two cell lines (Figure 6, lane 11 and 12), and both had lost one copy of the PTEN gene. Three mutations in PTEN were found (in cell lines MDA-MB-415, BT549 and MDA-MB-468, lanes 15, 17 and 24, Figure 6, and table 1). All of these data, taken together, suggest that gene silencing (for example by promoter methylation) or point mutations in FBXW7 and PTEN axe relatively rare mechanisms of inactivation of these genes, in comparison to single copy deletions. These data are further compatible with the identification of both genes as haplo-insufficient tumor suppressors (Mao J. H., Nature 432:775-9 (2004); and Di Cristofano A. et al., Nat Genet. 19:348-55 (1998)) that generally do not require complete inactivation of both gene copies.

Example 5

[0093] Rapamycin treatment: On the basis of the observation that deletion or mutation of FBXW7 in human breast cancer cells leads to increased levels of mTOR, the possibility that cells harboring these deletions may show increased sensitivity to the mTOR inhibitor rapamycin was tested. Two breast cancer cell lines, SUM 149PT cells (homozygous FBXWl mutations) and MDA-MB453 cells (wild type FBXW7) were treated with rapamycin and counted numbers of viable cells using the CellTiter-Glo Luminescent assay. SUM 149PT cells proved to be very sensitive to this treatment (IC ₅₀ < 200 nM), whereas MDA-MB453 cells were relatively resistant (IC ₅₀ > 2 μM). This relative difference in sensitivity in vitro was also apparent in nude mouse xenografts of both cell lines (Figure 7B). Groups of 5 mice were injected with both cell lines, one on each flank, and were treated by intraperitoneal injection with Rapamycin over an 11-day time period, starting 8 days after tumor inoculation. The SUM 149PT cells showed a relative decrease in size followed by stable tumor growth, whereas the MDAMB453 cells were relatively unaffected by treatment.

[0094] An additional set of 10 breast cancer cell lines were treated with rapamycin at concentrations of 200 nM and 400 nM. Interestingly, cells with deletion or mutation of FBXW7 (HBLlOO, 600MPE, SUM149PT, HCC3153 and HCCl 143) showed significant sensitivity to killing by rapamycin, although the magnitude of the effect varied. Some cells with loss or mutation of PTEN (HCC1937 and HCC3153) were sensitive to rapmycin, consistent with what has been reported in literature (Figure 7C). To establish a direct link between loss of FBXW7 and rapamycin sensitivity, shRNA specific for FBXWl were used (Welcker M. et al., Curr Biol. 14:1852-7 (2004)) to downregulate expression levels of the target protein in the rapamycin-resistant MDA-MB453 cells. As shown in Figure 4D, MDA- MB453 cells expressing FBXW7-specific shRNA are markedly more sensitive to rapamycin treatment (IC ₅₀ < 0.8μM) than the parental cells. Similar results were obtained with two additional cell lines expressing the FBXW7 shRNA (Figures 7E to F). [0095] These findings implicate FBXW7 in an evolutionarily conserved pathway that controls regulation of mTOR protein levels. Since Fbxw7 is itself a transcriptional target of p53, these data establish another link between the p53 and Pten signaling networks. Since FBXW7 is a haploinsufficient tumor suppressor that undergoes heterozygous loss in a substantial proportion of human tumours, the data suggest new approaches to reduce mTOR levels in cancers by the use of drugs that may re-activate the remaining copy of Fbxw7 in a similar way that nutlins have been shown to activate wild type copies of p53 in human tumors (Buolamwini J. K. et al., Curr Cancer Drug Targets. 5:57-68 (2005)). Loss of FBXW7 may also be a useful biomarker for sensitivity of human tumors to inhibitors of the mTOR pathway.

Materials and Methods Cell culture

[0096] Breast cancer cell lines were obtained from Dr. Joe Gray (Neve R.M. et al., Cancer Cell 10:515-27 (2006)). Stocks of the earliest-passage cells have been stored. HCTl 16, HCTl 16 FBXW7-/-, DLDl, and DLDl FBXW7-/- cell lines were a gift from Dr. Bert Vogelstein.

Western blotting

[0097] Total protein extracts were prepared from MEFs, cancer cells, and tissues with RIPA lysis buffer. For western blots, 50μg of protein extracts per lane were electrophoresed, transferred to PVDF membranes (Millipore), and immunoblotted with AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), mTOR (Cell Signaling Technology), p- mTOR (Cell Signaling Technology), p-S6 (Cell Signaling Technology), and HA (Covance) antibodies; as control, the same membranes were stripped and immunoblotted again with anti-β-actin antibody (AC-15, Sigma). The membranes were washed and treated with rat anti- species IgG ^κ -chain secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia). The antigen -antibody reactions were visualized by using an enhanced chemiluminesence assay ECL (Amersham Pharmacia) and exposed to enhanced chemiluminesence film.

Immunoprecipitation

[0098] 293T cells were transiently transfected with pcDNA3.1 expressing HA tagged FBXW7 and FBXW7-ΔF respectively using Fugene (Roche). The cells were harvested 24 hours after the transfection and lysed in lysis buffer (50 mM Hepes pH 7.5, 250 mM NaCl, 0.5% NP40 or 0.25% dodecyl beta D maltoside in Ix PBS with protease inhibitors) incubated on ice for 60 min and centrifuged at 13,000g for 15 min. The supernatant was collected and protein concentration was estimated with the Pierce BCA assay reagent. 500μg of lysates were precleared with protein A-sepharose beads (Invitrogen) and incubated with 2 μg of antibody overnight. Addition of protein A sepharose beads with incubation for 3-4 hour followed. After the incubation, the beads were washed three times with the lysis buffer described above and the precipitates were subjected to Western blotting as below. For the immunoprecipitation with anti HA antibody, Profound HA Tag IP/Co-IP kit (Pierce) was used according to the instruction manual. TaqMan assay

[00991 We designed TaqMan primers and probes using the Primer Express Oligo Design Software vl.O (Applied Biosystems). Probes are FAM probes designed specifically for TaqMan (Applied Biosystems). All primer sets were used to perform amplifications in triplicate on the ABI 7700 instrument (Applied Biosystems, Foster City, CA, USA). Reactions (50 μl) were performed in 1 xTaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1.6 nM primer, 0.4 nM probe, 12.5 ng DNA. Cycling parameters were as follows: 95°C for 12 min χl cycle, (95°C for 20 s, 60°C for 20 s, 72°C for 60 s) χ40 cycles. Copy number was determined from the PCR cycle number (CT) at which DNAs reach a threshold amount of fluorescence above background. To normalize for differences in the amount of total input DNA, amplification at a reference locus is performed once per plate in triplicate for each individual DNA. The CT values for each set of triplicates are averaged. The Ct of the pooled reference is subtracted from the CT for each locus to obtain the ΔCT (ΔCT=CT (locus)-CT (reference)). ΔCt values are determined for locus in tumor samples and a set of six normal genomic DNAs. The average of the six ΔCT values (ΔCT(normal)) measured from the normal DNAs was calculated once for each locus in the study and used in the subsequent calculations for all experiments performed on a single ABI 7700. ΔΔCT-ΔCt (tumor)-Average ΔCT (normal). If ΔΔCT>0.5, it means gain; If ΔΔCT<- 0.5, it means loss.

Viable cell count analysis

[0100] Cell number was measured using the CellTiter-Glo Luminescent assay (Promega) according to the manufacturer's instructions, and luminescence was recorded with a luminometer (BioTek FLx800, BioTek Instruments, Inc.). The cells are seeded in 96 well plates and exposed to mTOR inhibitor rapamycin (100 nM to 2 μM) for 72 hours. The number of cells used per experiment is determined empirically. Each cell line and drug concentration will be set up in 6 replicate wells and repeated at least 3 times. shRNA constructs and retrovirus production

[0101] A shRNA with high FBXW7 knockdown efficiency was cloned into pSuper retrovirus cassettes (OligoEngine). The shRNA sequences for FBXW7 are

GTGTGGAATGCAGAGACTGGAGA (17). The breast cancer cell lines (MDA-MB453, LY2 and MCF7) were infected with high-titre retroviral stocks produced by the transient transfection of 293T ecotropic Phonix cells. After infection with the pSuper retrovirus allowing the expression of shRNA molecules, cells were selected with l-2μg /ml of puromycin in the culture medium. The control of the experiment was pSuper vector without RNAi.

Plasmids and mutagenesis

[0102] The fragment of human mTOR was amplified from human cDNA by PCR using the primers (CCCAAGCTTGAACCTCAGGGCAAGATGCT) and

(GCTCTAGATTAGATCCAAAGCCCCTAAAAGC). The fragment was digested with HindIII and Xbal, and cloned into the Hindlll-Xbal site of the p3XFLAG-CMV-10 expression vector. The two mutants (delT631 and T631G) were generated by in vitro site- directed mutagenesis using QuickChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA). The primers for delT631 mutagenesis are

TCCCGCCTGCTCCCCTCCATCCAC and GTGGATGGAGGGGAGCAGGCGGGA, for T631G, CTCCCGCCTGCTCGGACCCTCCATCCAC and GTGGATGGAGGGTCCGAGCAGGCGGGAG. All constructs were verified by sequencing.

Nude mice injection

[0103] 5xlO ⁶ SUM149PT cells and 5xlO ⁶ MDA-Mb453 cells was injected subcutaneously in 10 nude mice in opposing flanks. 8 days after inoculation, 10 nude mice was divided into two groups, one was treated with rapamycin daily (4mg per kg body weight) for 11 days, the other with placebo. Tumor sizes were measured with a caliper every two days. The tumor volume will be calculated using the formula: Volume= S ^χ S*L/2, where s is the short length of the tumor in mm and L is the long length of the tumor in mm.

[0104] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

SEQUENCES

LOCUS NM_033632 3896 bp mRNA linear PRI 24 -AUG-

2008

DEFINITION Homo sapiens F-box and WD repeat domain containing 7 (FBXW7) , transcript variant 1, mRNA. ACCESSION NM_033632

VERSION NM_033632.2 GI: 61743923 KEYWORDS

SOURCE Homo sapiens (human) ORGANISM Homo sapiens

Eukaryota,- Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;

Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;

Catarrhini ; Hominidae; Homo.

REFERENCE 1 (bases 1 to 3896) AUTHORS Finkin,S., Aylon,Y., Anzi,S., Oren,M. and Shaulian,E. TITLE Fbw7 regulates the activity of endoreduplication mediators and the p53 pathway to prevent drug-induced polyploidy

JOURNAL Oncogene 27 (32), 4411-4421 (2008) PUBMED 18391985 REMARK GeneRIF: Fbw7 regulates the activity of endoreduplication mediators and the p53 pathway to prevent drug-induced polyploidy.

REFERENCE 2 (bases 1 to 3896) AUTHORS Grim, J. E., Gustafson, M. P. , Hirata,R.K., Hagar,A.C, Swanger,J., Welcker,M., Hwang, H. C, Ericsson, J., Russell, D. W. and

Clurman,B. E.

TITLE Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiguitin ligase

JOURNAL J. Cell Biol. 181 (6), 913-920 (2008) PUBMED 18559665 REMARK GeneRIF: These data suggest that oscillations in cyclin

E-CDK2 -specific activity during the cell cycle regulate the timing of cyclin E degradation.

REFERENCE 3 (bases 1 to 3896) AUTHORS Lee , J . W . , Jeong , E . G . , Lee , S . H . , Yoo , N . J . and Lee , S . H . TITLE hCDC4 gene mutation is rare in colorectal carcinomas in Korean patients

JOURNAL Pathology 40 (3), 305 (2008) PUBMED 18428053 REMARK GeneRIF: could not confirm that hCDC4 is frequently mutated in colon cancers .

REFERENCE 4 (bases 1 to 3896) AUTHORS Welcker,M. and Clurman,B.E. TITLE FBW7 ubiguitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation

JOURNAL Nat. Rev. Cancer 8 (2), 83-93 (2008) PUBMED 18094723 REMARK GeneRIF: FBW7 has a role in the development of cancer

Review article

REFERENCE 5 (bases 1 to 3896) AUTHORS Jeong, E. G., Kim, M. S., Kim, S. H., Yoo, N. J. and Lee, S. H. TITLE FBXW7 gene mutation is rare in acute leukemia samples of Korean patients JOURNAL Acta Haematol. 118 (4), 200-202 (2007)

PUBMED 17992009 REMARK GeneRIF: There were no mutations in exons 8 or 9 in 160 acute leukemia samples from Korea, in contrast to earlier reports suggesting a role in T-ALL.

REFERENCE 6 (bases 1 to 3896) AUTHORS Koepp,D.M., Schaefer,L.K. , Ye, X., Keyomarsi,K. , Chu,C, Harper, J. W. and Elledge,S.J.

TITLE Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase

JOURNAL Science 294 (5540) , 173-177 (2001) PUBMED 11533444 REMARK GeneRIF: associates specifically with phosphorylated cyclin E, and

SCFFbw7 catalyzes cyclin E ubiquitination in vitro

REFERENCE 7 (bases 1 to 3896) AUTHORS Oberg,C, Li, J., Pauley,A. , Wolf, E., Gurney,M. and Lendahl,U. TITLE The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian SeI -10 homolog

JOURNAL J. Biol. Chem. 276 (38), 35847-35853 (2001)

PUBMED 11461910

REFERENCE 8 (bases 1 to 3896)

AUTHORS Gupta-Rossi, N. , Le Bail,θ., Gonen,H., Brou,C, Logeat,F., Six, E. ,

Ciechanover, A. and Israel, A.

TITLE Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notchl receptor

JOURNAL J. Biol. Chem. 276 (37), 34371-34378 (2001)

PUBMED 11425854

REFERENCE 9 (bases 1 to 3896)

AUTHORS Winston, J. T. , Koepp,D.M., Zhu,C, Elledge,S.J. and Harper , J . W .

TITLE A family of mammalian F-box proteins

JOURNAL Curr. Biol. 9 (20), 1180-1182 (1999)

PUBMED 10531037

REFERENCE 10 (bases 1 to 3896)

AUTHORS Robertson, N. G. , Khetarpal,U. , Gutierrez-Espeleta,G.A. , Bieber,F.R. and Morton,CC.

TITLE Isolation of novel and known genes from a human fetal cochlear CDNA library using subtractive hybridization and differential screening JOURNAL Genomics 23 (1) , 42-50 (1994) PUBMED 7829101 COMMENT REVIEWED REFSEQ: This record has been curated by NCBI staff. The reference sequence was derived from BF514288.1, W39194.1,

AY049984.1 and AC080078.7.

On Mar 24, 2005 this sequence version replaced gi: 16117780.

Summary: This gene encodes a member of the F-box protein family which is characterized by an approximately 40 amino acid motif, the

F-box. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKPl-cullin-F- box) , which function in phosphorylation-dependent ubiquitination. The F-box proteins are divided into 3 classes: Fbws containing WD-

40 domains, FbIs containing leucine-rich repeats, and Fbxs containing either different protein-protein interaction modules or no recognizable motifs. The protein encoded by this gene was previously referred to as FBX30, and belongs to the Fbws class; in addition to an F-box, this protein contains 7 tandem WD40 repeats. This protein binds directly to cyclin E and probably targets cyclin E for ubiguitin-mediated degradation. Mutations in this gene are detected in ovarian and breast cancer cell lines, implicating the gene ' s potential role in the pathogenesis of human cancers .

Three transcript variants encoding three different isoforms have been found for this gene, [provided by RefSeq] .

Transcript Variant: This variant (1) represents the longest transcript and encodes the longest isoform (1) .

Publication Note: This RefSeq record includes a subset of the publications that are available for this gene. Please see the

Entrez Gene record to access additional publications.

COMPLETENESS: complete on the 3' end.

PRIMARY REFSEQ_SPAN PRIMARY_IDENTIFIER PRIMARY_SPAN COMP

1-70 BF514288.1 308-377 C

71-272 W39194.1 138-339 c

273-2335 AY049984.1 193-2255

2336-3896 AC080078.7 47601-49161

FEATURES Location/Qualifiers source 1..3896

/organism="Homo sapiens"

/mol_type= "mRNA"

/db_xref= " taxon : 9606 "

/chromosome="4 "

/map="4g31.3" gene 1..3896

/gene="FBXW7"

/synonym="AGO, CDC4, FBW6, FBW7, FBX30, FBXW6, SELlO,

FBXO30, SEL-10, DKFZp686F23254 "

/note= "F-box and WD repeat domain containing 7"

/db_xref="GeneID:55294"

/db_xref= "HGNC : 16712 "

/db_xref="HPRD: 05888"

/db_xref="MIM: 606278" exon 1..80

/gene="FBXW7"

/inference= "alignment: SpIign"

/number=l exon 81..650

/gene="FBXW7 ^»

/inference="alignment : Splign"

/number=2

CDS 150..2273 /gene= " FBXW7 "

/note="isoform 1 is encoded by transcript variant 1; F-box protein SEL-10; homolog of C elegans sel-10; archipelago,

Drosophila, homolog of; F-box protein FBW7; F-box and

WD-40 domain protein 7 (archipelago homolog, Drosophila) "

/codon_start=l

/product=" F-box and WD repeat domain containing 7 isoform

1"

/protein_id="NP_361014.1"

/db_xref="GI: 16117781"

/db_xref="CCDS:CCDS3777.1"

/db_xref="GeneID: 55294"

/db_xref="HGNC: 16712"

/db_xref= "HPRD: 05888"

/db_xref="MIM: 606278"

/translation "MNQELLSVGSKRRRTGGSLRGNPSSSQVDEEQMNRWEEEQQQQ

LRQQEEEHTARNGEWGVEPRPGGQNDSQQGQLEENNNRFISVDEDSSGNQEEQEEDE EHAGEQDEEDEEEEEMDQESDDFDQSDDSSREDEHTHTNSVTNSSSIVDLPVHQLSSP FYTKTTKMKRKLDHGSEVRSFSLGKKPCKVSEYTSTTGLVPCSATPTTFGDLRAANGQ GQQRRRITSVQPPTGLQEWLKMFQSWSGPEKLLALDELIDSCEPTQVKHMMQVIEPQF QRDFISLLPKELALYVLSFLEPKDLLQAAQTCRYWRILAEDNLLWREKCKEEGIDEPL HIKRRKVIKPGFIHSPWKSAYIRQHRIDTNWRRGELKSPKVLKGHDDHVITCLQFCGN RIVSGSDDNTLKVWSAVTGKCLRTLVGHTGGVWSSQMRDNIIISGSTDRTLKVWNAET GECIHTLYGHTSTVRCMHLHEKRWSGSRDATLRVWDIETGQCLHVLMGHVAAVRCVQ YDGRRWSGAYDFMVKVWDPETETCLHTLQGHTNRVYSLQFDGIHWSGSLDTSIRVW DVΈTGNCIHTLTGHQSLTSGMELKDNILVSGNADSTVKIWDIKTGQCLQTLQGPNKHQ

SAVTCLQFNKNFVITSSDDGTVKLWDLKTGEFIRNL VTLESGGSGGWWRIRASNTKL

VCAVGSRNGTEETKLLVLDFDVDMK " exon 651 . . 733

/gene= " FBXW7 "

/inference="alignment .-Splign"

/number=5 exon 734..875

/gene="FBXW7"

/inference=" alignment : Splign"

/number=6 exon 876..1010

/gene="FBXW7"

/inference="alignment : Splign"

/number=7 exon 1011..1134

/gene= " FBXW7 "

/inference="alignment : Splign"

/number=8 exon 1135..1271 /gene="FBXW7"

/inference="alignment : SpIign"

/number=9 exon 1272..1385

/gene="FBXW7"

/inference= "alignment : Splign"

/number=10 exon 1386..1567

/gene="FBXW7"

/inference= "alignment : Splign"

/number=ll exon 1568..1793

/gene= " FBXW7 "

/inference="alignment: Splign"

/number=12 exon 1794..2004

/gene="FBXW7"

/inference="alignment : Splign"

/number=13 exon 2005..3896

/gene="FBXW7"

/inference="alignment : Splign"

/number=14

STS 3629..3842

/gene= " FBXW7 "

/standard_name=" SHGC- 67303"

/db_xref="UniSTS: 84198" polyA_signal 3873..3878

/gene="FBXW7" polyA_site 3896

/gene="FBXW7"

ORIGIN

1 ccttccgcag ctgccgcttc agtccgaagg aggaagggaa ccaacccact ttctcggcgc

61 cgcggctctt ttctaaaagt aatgtgaaaa cctttgcatc ttctgatagt ctagccaagg

121 tccaagaagt agcaagctgg cttttggaaa tgaatcagga actgctctct gtgggcagca

181 aaagacgacg aactggaggc tctctgagag gtaacccttc ctcaagccag gtagatgaag

241 aacagatgaa tcgtgtggta gaggaggaac agcaacagca actcagacaa caagaggagg

301 agcacactgc aaggaatggt gaagttgttg gagtagaacc tagacctgga ggccaaaatg

361 attcccagca aggacagttg gaagaaaaca ataatagatt tatttcggta gatgaggact

421 cctcaggaaa ccaagaagaa caagaggaag atgaagaaca tgctggtgaa caagatgagg

481 aggatgagga ggaggaggag atggaccagg agagtgacga ttttgatcag tctgatgata

541 gtagcagaga agatgaacat acacatacta acagtgtcac gaactccagt agtattgtgg

601 acctgcccgt tcaccaactc tcctccccat tctatacaaa aacaacaaaa atgaaaagaa

661 agttggacca tggttctgag gtccgctctt tttctttggg aaagaaacca tgcaaagtct

721 cagaatatac aagtaccact gggcttgtac catgttcagc aacaccaaca acttttgggg

781 acctcagagc agccaatggc caagggcaac aacgacgccg aattacatct gtccagccac

841 ctacaggcct ccaggaatgg ctaaaaatgt ttcagagctg gagtggacca gagaaattgc

901 ttgctttaga tgaactcatt gatagttgtg aaccaacaca agtaaaacat atgatgcaag

961 tgatagaacc ccagtttcaa cgagacttca tttcattgct ccctaaagag ttggcactct

1021 atgtgctttc attcctggaa cccaaagacc tgctacaagc agctcagaca tgtcgctact

1081 ggagaatttt ggctgaagac aaccttctct ggagagagaa atgcaaagaa gaggggattg

1141 atgaaccatt gcacatcaag agaagaaaag taataaaacc aggtttcata cacagtccat

1201 ggaaaagtgc atacatcaga cagcacagaa ttgatactaa ctggaggcga ggagaactca

1261 aatctcctaa ggtgctgaaa ggacatgatg atcatgtgat cacatgctta cagttttgtg

1321 gtaaccgaat agttagtggt tctgatgaca acactttaaa agtttggtca gcagtcacag

1381 gcaaatgtct gagaacatta gtgggacata caggtggagt atggtcatca caaatgagag

1441 acaacatcat cattagtgga tctacagatc ggacactcaa agtgtggaat gcagagactg

1501 gagaatgtat acacacctta tatgggcata cttccactgt gcgttgtatg catcttcatg

1561 aaaaaagagt tgttagcggt tctcgagatg ccactcttag ggtttgggat attgagacag

1621 gccagtgttt acatgttttg atgggtcatg ttgcagcagt ccgctgtgtt caatatgatg

1681 gcaggagggt tgttagtgga gcatatgatt ttatggtaaa ggtgtgggat ccagagactg 1741 aaacctgtct acacacgttg caggggcata ctaatagagt ctattcatta cagtttgatg 1801 gtatccatgt ggtgagtgga tctcttgata catcaatccg tgtttgggat gtggagacag 1861 ggaattgcat tcacacgtta acagggcacc agtcgttaac aagtggaatg gaactcaaag 1921 acaatattct tgtctctggg aatgcagatt ctacagttaa aatctgggat atcaaaacag 1981 gacagtgttt acaaacattg caaggtccca acaagcatca gagtgctgtg acctgtttac 2041 agttcaacaa gaactttgta attaccagct cagatgatgg aactgtaaaa ctatgggact 2101 tgaaaacggg tgaatttatt cgaaacctag tcacattgga gagtgggggg agtgggggag 2161 ttgtgtggcg gatcagagcc tcaaacacaa agctggtgtg tgcagttggg agtcggaatg 2221 ggactgaaga aaccaagctg ctggtgctgg actttgatgt ggacatgaag tgaagagcag 2281 aaaagatgaa tttgtccaat tgtgtagacg atatactccc tgcccttccc cctgcaaaaa 2341 gaaaaaaaga aaagaaaaag aaaaaaatcc cttgttctca gtggtgcagg atgttggctt 2401 ggggcaacag attgaaaaga cctacagact aagaaggaaa agaagaagag atgacaaacc 2461 ataactgaca agagaggcgt ctgctgtctc atcacataaa aggcttcact tttgactgag 2521 ggcagctttg caaaatgaga ctttctaaat caaaccaggt gcaattattt ctttattttc 2581 ttctccagtg gtcattgggc agtgttaatg ctgaaacatc attacagatt ctgctagcct 2641 gttcttttac cactgacagc tagacaccta gaaaggaact gcaataatat caaaacaagt 2701 actggttgac tttctaatta gagagcatct gcaacaaaaa gtcatttttc tggagtggaa 2761 aagcttaaaa aaattactgt gaattgtttt tgtacagtta tcatgaaaag cttttttttt 2821 tttttttttg ccaaccattg ccaatgtcaa tcaatcacag tattagcctc tgttaatcta 2881 tttactgttg cttccatata cattcttcaa tgcatatgtt gctcaaaggt ggcaagttgt 2941 cctgggttct gtgagtcctg agatggattt aattcttgat gctggtgcta gaagtaggtc 3001 ttcaaatatg ggattgttgt cccaaccctg tactgtactc ccagtggcca aacttattta 3061 tgctgctaaa tgaaagaaag aaaaaagcaa attatttttt tttatttttt ttctgctgtg 3121 acgttttagt cccagactga attccaaatt tgctctagtt tggttatgga aaaaagactt 3181 tttgccactg aaacttgagc catctgtgcc tctaagaggc tgagaatgga agagtttcag 3241 ataataaaga gtgaagtttg cctgcaagta aagaattgag agtgtgtgca aagcttattt 3301 tcttttatct gggcaaaaat taaaacacat tccttggaac agagctatta cttgcctgtt 3361 ctgtggagaa acttttcttt ttgagggctg tggtgaatgg atgaacgtac atcgtaaaac 3421 tgacaaaata ttttaaaaat atataaaaca caaaattaaa ataaagttgc tggtcagtct 3481 tagtgtttta cagtatttgg gaaaacaact gttacagttt tattgctctg agtaactgac 3541 aaagcagaaa ctattcagtt tttgtagtaa aggcgtcaca tgcaaacaaa caaaatgaat 3601 gaaacagtca aatggtttgc ctcattctcc aagagccaca actcaagctg aactgtgaaa 3661 gtggtttaac actgtatcct aggcgatctt ttttcctcct tctgtttatt tttttgtttg 3721 ttttatttat agtctgattt aaaacaatca gattcaagtt ggttaatttt agttatgtaa 3781 caacctgaca tgatggagga aaacaacctt taaagggatt gtgtctatgg tttgattcac 3841 ttagaaattt tattttctta taacttaagt gcaataaaat gtgttttttc atgtta