Apoptin induces inhibition of Bcr-Abl kinase in CML cells PRIOR APPLICATION INFORMATION

This application claims the benefit of US Provisional Patent Application 60/983,946, filed October 31 , 2007. BACKGROUND OF THE INVENTION

Receptor Tyrosine Kinases (r-TKs) transduce the extracellular signals into the cytoplasm by autophosphorylation that drives the cells towards metabolic alteration, differentiation, proliferation and migration (Kanakura et al., 1990). Nonreceptor tyrosine kinases (nr-TK) are frequently the leading components of cellular signal transduction cascades that carry signals initiated on the cell surface and also within the cell. The nr-TK family of proteins that includes Janus kinases (Jaks), Src and AbI are also capable of phosphorylating specific target proteins. In addition to the TK domain, nr-TKs possess other domains responsible for protein- protein, protein-lipid or protein-DNA interactions (Tice et al., 1999). in nr-TKs the domains like Src homology 2 (SH2) and Src homology 3 (SH3) are most frequently associated with protein-protein interactions (Sicheri and Kuriyan, 1997).

The nr-TK AbI, which is present in both nucleus and cytoplasm, classically contains a nuclear localization signal (NLS), an actin binding domain and a DNA binding domain (McWhirter et ai., 1993). Normally AbI is involved in the regulation of the cell cycle following genotoxic stress through integrin signaling (He et al., 2002; Jacobsen et ai., 2006). The AbI protein also serves as a cellular module that integrates signals from various extracellular and intracellular sources subsequently driving the cellular decision making processes for cell cycle progression or initiation of apoptosis {Bedi et al., 1994). Normally the Abl-SH3 domain acts as an inhibitor on its own TK signaling (Cohen et al., 1995). This negative regulation on AbI-TK is lost if there is an allostoric modification of the SH3 domain following fusion with another peptide moiety. Moreover, this modified SH3 domain then functions as a positive regulator of phosphorylation for the AbI-TK (Muller et al., 1993). Following 9:22 translocation (Phildelphia chromosome, Ph+) that is observed in about 95% of chronic myeloid leukemia (CML) and about one third of adult acute lymphoblastic leukemia (ALL), the proto-oncogene AbI on chromosome 9 and the Bcr cluster gene on chromosome 22 leads to the production of fusion

protein Bcr-Abl (Deininger et a!., 2000). As mentioned, unlike normal AbI, Bcr-Abl can undergo uncontrolled autophosphoryiation activating downstream cell growth, proliferation and anti-apoptotic pathways. Among the other downstream targets, Crk and CrkL are the most prominent tyrosine-phosphoryiated proteins in Bcr-Abl transformed cells, involved in the regulation of cellular motility and integrin- mediated eel! adhesion (ten Hoeve et al., 1994). CrkL acts as an adapter molecule and also activates Ras. Ras is one of the important mediators of cell proliferation downstream of the Mitogen Activated Protein Kinase (MAPK) pathway. Similarly Bcr-Abl autophosphoryiation at tyrosine residue 177 (Tyr-177) provides a docking site for other adapter molecules like Grb-2 (and Grb-1 ) that stabilizes Ras in its active GTP-bound form (Cortez et al., 1995; Meng S, 2005; Million and Van Etten, 2000). This implies that the Ras pathway is constitutively active during the pathogenesis of CML (Kiyokawa E, 1997). Moreover, activation of the Sapk/Jnk pathway by Bcr-Abl has been demonstrated and is also required for malignant transformation (Kang et a!., 2000). Constitutive phosphorylation of STATs (Signal Transduction and Activators of Transcription, STAT1 and STAT5) has also been reported in several Bcr-Abl expressing transformed cell lines and in primary CML cells. STAT5 activation, in general, contributes to the clonal malignant transformations. However the role of STATS in Bcr-Abl transformed cells is primarily anti-apoptotic (Danial NN, 2000). Similarly, PI3-kinase (P13-K) activity is required for the proliferation of Bcr-Abi positive cells (Danial NN, 2000). Interestingly, Bcr-Abl forms complexes with PI3-K where Crk and CrkL act as adaptor molecules. PI3-K is phosphorylated in this protein complex leading to trans-activation of the serine-threonine kinase Akt that is a main down-stream substrate of PI3-K. Akt has proven role in anti-apoptotic signaling (Arslan et al., 2006; Burchert et al., 2005).

Bcr-Abl is an attractive drug target for CML. Drugs like Imatinib®, Desatinib® and Nilotinib® belong to new generation of signal transduction inhibitors (STi) that are in clinical use or in clinical trials (Inokuchi, 2006; Noble et al., 2004). However, these specific inhibitors do not work as rapid oncolytic agents during advanced disease or blast crisis, or as radiation or classical cytotoxic chemotherapy agents (Pier Paolo Piccaluga, 2007). The efficacy of ST! working on an upstream activator like Bcr-Abl may be diminished, mainly because multiple

signaling pathways are affected in advanced cancer (Jacquel et al., 2003; Krystal, 2001). Therefore, targeting multiple kinase pathways is necessary for an improved therapeutic outcome (Hu et al., 2006).

In a search for signal transduction inhibitors that could block multiple proliferation pathways, activate apoptosis and also possibly bypass some of the above described inherent problems with STI ₁ we investigated apoptin as a candidate {Maddika et al., 2006; Poon et al., 2005b). Apoptin is a 14 kDa viral protein encoded by the VP3 gene of Chicken Anemia Virus (Adair, 2000). It is well documented that apoptin induces apoptosis in a broad range of transformed and cancer ceils but not in non-transformed or primary ceils (Heilman et al., 2006; Poon et al., 2005a). Apoptin induced apoptosis is independent of death receptor pathways (Maddika et al., 2005; Maddika et al., 2006). When applied, Apoptin remains in the cytoplasm of primary cells, but it migrates into the nucleus and induces apoptosis in transformed cells by activating the mitochondrial death pathway in a Nur77 (nuclear receptor 77) dependent manner (Maddika et al., 2005). The cellular localization of apoptin plays crucial role for its seiective toxicity (Danen-Van Oorschot et al., 2003; Maddika et al., 2007b; Poon et al., 2005a). Apoptin associates itself with the anaphase-promoting complex resulting subsequent G2/M arrest and apoptosis (Teodoro et al., 2004) and also it was reported that Sapk/Jnk signaling pathway is possibly activated during apoptin induced cell death (Ben et al., 2005).

We initially observed (protein array) that apoptin interacts with the SH3 domain of AbI. In the present study, using k562 and 32D ^p210 as cellular models of CML (human and murine) we detected by highly stringent pull-down and co- immunoprecipitation assays that apoptin interacts not only with the AbI protein isolated from a number of cell lysates, but also it has strong binding properties with the oncoprotein Bcr-Abl. From these results, we postulated, 'apoptin and Bcr-Abl interaction ¹ constitutively inhibits the phosphorylation, and thus activation of Bcr- AbI. We documented for the first time that Apoptin's interaction with Bcr-Abl kinase significantly decreases Bcr-Abl phosphorylation and activity, as a consequence CrkL, STAT5, c-Myc, SAPK/JNK, Akt and also BAD activation status is modified. This sequence of events leads to initiation of apoptotic ceil death that is more pronounced than the effects of Imatinib® on Bcr-Abl expressing cells.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method of treating ALL or CML comprising administering to an individual in need of such treatment an effective amount of apoptin or an apoptin SH3-interacting domain fragment.

According to a second aspect of the invention, there is provided a method of identifying agents capable of inhibiting Bcr-Abl kinase activity comprising: incubating Bcr-Abl, a Bcr-Abl kinase substrate and an agent of interest under conditions suitable for Bcr-Abl kinase activity; and determining if Bcr-Abl kinase activity has been inhibited, by comparing phosphorylation of the Bcr-Abl kinase substrate to a control.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Interaction of apoptin with Abi and Bcr-Abl ^pz1 °. (A)

TransSignal SH3™ Domain Arrayi was used to screen apoptin for potential interactions with SH3 domains. Positions: D3, 4 indicate the interaction of apoptin and SH3 domains of Abi. Tat-apoptin, was hybridized with TransSignal SH3™ Domain Array"! , and the image was acquired on high performance chemiiuminescence film (Hyperfilm™ECL, Amersham Biosciences). Spots with stronger intensity indicate higher binding affinity with ligand of interest to SH3 domain(s). The proteins in the array are spotted in duplicates. Histidine tagged ligands have been spotted along the bottom (row E) and in duplicate along the right side of the membrane (column 21 , 22) for alignment purpose. These results were confirmed by "pu!l-down assay", co-immunoprecipitation and imunofluorescence study as shown in B, C and D respectively. (B) Akt, Apoptin, and Bcr-Abl interaction; GST-apoptin was used in the 'pull-down assay'. The interaction was tested either on total cell Sysates from Bcr-Abl expressing 32D ^p210 cells, or on Bcr-Abl-negative 32D-DMSZ (control). Lane #1 : Molecular weight marker. #2: puil-down assay on 32D-DMSZ extract (negative control). #3: 32D ^pZ1 ° extract (positive control), #4: 32D ^p21 ° extract and pulled with glutathione sepharose bead

(beads control). #5: 32D ^p210 extract incubated with GST-apoptin and pulled with glutathione sepharose beads. The PVDF membrane was sequentially treated with three primary antibodies (mouse Bcr-Abl, mouse AbI and rabbit Akt). (C) Apoptin and Bcr-Ab! interaction as demonstrated by co-immunoprecipitation (CO-IP) assay: co-immunoprecipitatioπ was done with anti-Bcr-Ab! antibody on transiently transfected 32D ^p210 (wt) cells. The IP product was positive for apoptin (GFP- Apoptin: 40 kDa) by immunoblot with anti-apoptin antibody. Lane #1 : Molecular weight marker, #2: GST-Apoptin (positive control), #3: 32D ^p21 ° cells transfected with GFP-apoptin (CO-IP), #4: 32D ^p210 transfected with GFP-Apoptin (CO-IP supernatant), #5: 32D ^p210 transfected with GFP (CO-IP), #6: 32D ^pZ10 No transfection (CO-IP, negative control), #7: 32D transfected with GFP-Apoptin (CO- IP), #8. 32D transfected with GFP-apoptin (CO-IP). (D) immunocytochemistry staining showing nuclear iocalization of Bcr-Ab! after interacting with apoptin 32D ^P210 and 32D cells were transiently transfected for GFP-Apoptin expression. Nuclei: blue (DAPI); Bcr-Abl ^p210 with Cy3 tagged secondary antibody: Red and GFP-apoptin: Green. Merged image in lane 1 Column 4 shows nuclear co- localization of Bcr-Abl ^p210 and GFP-Apoptin in small clusters.

Figure 2: Schematic depiction of apoptin mediated down -regulation of Bcr-Abl ^p210 activity and effects on other downstream pathways. Apoptin inhibits Bcr-Abl phosphorylation acting as an adaptor molecule on the AbI SH3 domain and subsequently negatively regulates the cell transformation and proliferations signals generated by Jak/Stat, MAPK pathways.

Figure 3: Apoptin inhibits Bcr-Abl phosphorylation. The experiments were performed in two cell lines (A) K562 and (B) 32D ^p210 cells. Tat-apoptin, Tat- GFP (negative control) and lmatinib (positive control) were applied (all at concentration: 1 μM) to K562 and 32D ^p210 cells 24 h after cells were seeded. Cells were harvested 16 h later, and cell lysates were checked by Western blot for phosphoryiated and total Bcr-Abl. The final quantitative data was normalized according to the loading control and expressed as a ratio of phosphorylated/total Bcr-Abl. COLUMN 1 : non-treated cells (-); COLUMN 2: Tat-GFP (TG) treated cells] COLUMN 3: lmatinib (Im) treated cells; COLUMN 4: Tat-Apoptin treated cells. Lane 1#: total Bcr-Ab!, #2: phospho-Bcr-Abl and #3: loading control elE4E. The

bands were scanned and quantified by NiH-software. Bcr-Abi phosphorylation is significantly inhibited by apoptin, and the inhibitory effect is comparable to that of Imatinib. The presented data is a summary of five independent experiments.

Figure 4: Apoptin induced inhibition of Bcr-Abl phosphorylation leads to the down-regulation of STATS phosphorylation. The experiments were performed in two cell lines (A) K562 and (B) 32D ^p210 cells. Tat-apoptin, Tat-GFP (negative control) and Imatinib (positive control) were applied (1 μM) to K562 and 32D ^p210 ceils 24 h after seeding. Cells were harvested at 16 h later, and cell lysates were checked for phosphorylated and total STAT5 by Western blot. The signals (bands) were scanned and quantified by NIH-software. The final quantitative data was normalized using the loading control and expressed as a ratio of phosphorylated and total STATS. Column (-): Untreated cells; Column (TG): Tat-GFP treated cells; Column (Im): Imatinib treated cells; Column (TA): Tat- Apoptin treated cells; Lane 1 : Total STATS, Lane 2: phospho-STAT5 and Lane 3: loading control (elF4E). STAT5 phosphorylation was significantiy lower in Tat- apoptin, and Imatinib-treated cells (see the histogram), indicating that apoptin induced inhibition of Bcr-Abl phosphorylation decreases activation of STAT5, a down-stream substrate for Bcr-Abi. Data is a summary of three independent experiments. Figure 5: Apoptin induced inhibition of Bcr-Abl phosphorylation leads to the down-regulation of CrkL phosphorylation. The experiments were performed in two eel! lines (A) K562 and (B) 32D ^p210 cells. Tat-apoptin, Tat-GFP (control) and Imatinib (positive control) were applied (1 μM) to K562 and 32D ^p210 cells 24 h after seeding. Cells were harvested 16 h later and cell lysates were checked for phosphorylated and total CrkL by Western blot. The bands were scanned and quantified by NIH-software. The quantitative data was normaiized using the loading control (elF4E) and expressed as a ratio of phosphorylated and total CrkL. COLUMN 1 : Untreated cells (-); COLUMN 2: Tat-GFP (TG) treated cells; COLUMN 3: Imatinib (Im) treated cells; COLUMN 4: Tat-apoptin (TA) treated cells. Lane #1 : Total CrkL, Lane #2: phospho- CrkL and Lane #3: loading control (elF4E). The estimated CrkL phosphorylation was significantly lower (see the

histogram), indicating that apoptin induced inhibition of Bcr-Abl phosphorylation decreases activation of CrkL, a down-stream substrate for Bcr-Abl.

Figure 6: Apoptin and Imatinib activate Akt. The experiments were performed in two cell lines (A) K562 and (B) 32D ^p21 ° cells. Tat-apoptin, Tat-GFP (control) and Imatinib (positive control) were applied (1 μM) to K562 and 32D ^p210 cells 24 h after seeding. Cells were harvested 16 h later and cell lysates were checked for phosphorylated and total Akt by Western blot. The signals (bands) were scanned and quantified by NIH-software. The quantitative data was normalized employing the loading control and expressed as a ratio of phosphoryiated/total Akt. COLUMN 1 : Untreated ceils (-); COLUMN 2: Tat-GFP (TG) treated cells; COLUMN 3: Imatinib (Im) treated cells; COLUMN 4: Jat-apoptin (TA) treated cells. Lane #1 : Total Akt, Lane #2: phospho-Akt and Lane #3: loading control (tubulin). Akt phosphorylation signals were higher (see the histogram), indicating that both Imatinib and apoptin induced Akt activation. Figure 7: Comparison of cytotoxic activity of apoptin and Imatinib on

Bcr-Abl positive and negative cells. (A) 32D ^p210 cells were grown in a 96-well plate (10,000 cells/well). Tat-apoptin, Imatinib (positive control) and Tat-GFP (negative controi) were applied (in triplicates, 1 μM) at 0, 4, 8, 12, 18 and 24 h. The % of living cells was assessed MTT assay. (B) 32D-DSMZ cells and 32Dp210 cells were treated with apoptin, Imatinib or both. Each of the treatment groups were analyzed for apoptosis after 24 h by Flow cytometry (Nicoletti method). 10,000 cells where analyzed per sample, M2 indicates dead cells and M1 indicates living cells. The data from 3 independent experiments was compiled and shown in the attached diagram. Cell growth without any treatment was considered as 100% proliferation (control). Normalized values, expressed as percent of cell survival, indicates Imatinib has minimum effect on the survival of Bcr-Abl -negative 32D- DSMZ cells that is comparable to the untreated control group whereas when applied to 32D ^p210 cells, imatinib alone killed about 50% of the cells. Apoptin alone or in combination with Imatinib was strongly toxic to both 32D-DSMZ and 32D ^pz10 cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

The non-receptor tyrosine kinase activity of fusion gene Bcr-Abl derived oncoproteins is the key factor responsible for development and progress of Philadelphia-chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) and Ph+ Acute Lymphoblastic Leukemia (ALL). In the search for a peptide-based inhibitor of Bcr-Abl tyrosine kinase, we investigated a naturally occurring molecule caiied Apoptin. 'Apoptin' is a 14 kDa viral protein (chicken anemia virus protein-3, CAV-VP3) and known to induce apoptosis in a wide range of transformed but not in primary celts. During the initial phase of our study, an array-based analysis demonstrated that Apoptin interacts with the SH3 domain of AbI. We further investigated the role of Apoptin on Bcr-Abl. High stringent pull-down and co- immunoprecipitation assays revealed that Apoptin strongly interacts with the fusion protein Bcr-Abl (p210). We also identified Apoptin's ability to significantly inhibit Bcr-Abl kinase and presumably indirectly a series of downstream targets (e.g. CrkL, STAT5, c-Myc, etc.). In comparison studies, using lmatinib ^® we discovered that apoptin has a significant killing efficacy on human and mouse CML cell lines expressing Bcr-Abl. We postulated that the interacting segment of the Apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase. The obtained data provides foundation for the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

Targeting protein kinase pathways activated in malignant cells is one of the most active research areas focused on anti-cancer drug development, and Bcr-Abl being uniquely expressed in CML is an attractive therapeutic target. As an anti- apoptotic protein, Bcr-Abl significantly contributes to the development and proliferative potential of CML. Apoptin is a potent anti-cancer agent that selectively induces apoptosis in transformed cells but not in primary cells. This selective toxic

nature is believed to be due to apoptin's ability to target multiple signal transduction proteins and relocate them into specific cellular compartments.

In the current study, we have documented for the first time that Tat-Apoptin, a celi-penetrating conjugate of Apoptin, strongly binds to the SH3 domain of Bcr- Abi and modifies the phosphorylation of Bcr-Abl and several of its downstream targets that lead to an anti-proliferative effect and induction of intrinsic apoptotic pathways in the rapidly growing CML cells. The analysis of human CML cells K562 and Bcr-Abl (p210) expressing murine cell line 32D ^p210 , we observed that these cells are significantly responsive to apoptin, and that it favorably compares to Imatinib. As these human and murine cell lines are rapidly growing with a high cytoplasmic Bcr-Abl (p210) pool, the cell culture conditions mimic blast crisis stage of CML, since, as it is in CML ₁ the centra! mitogenic Ras-MAPK cascade is also activated in these cell lines. Furthermore, previous studies with smaller but high affinity peptides blocking the N-terminal SH3 domain of Grb2 peptide indicated that peptide based inhibitor of Bcr-Abl kinase or its down-stream targets could be a valuable anti-CML tool if combined with conventional cytotoxic therapy (Kardinal et al., 2001 ).

STATs act as regulators of cell proliferation (Meyer et al., 1998). The N- terminal regulatory region of AbI protein contains SH2 and SH3 domains which are important for the regulation of its activity in vivo (Nam et al., 1996). We have previously reported that apoptin productively interacts with SH3 domain of p85 subunit of PI3-K (Maddika et al., 2008). In the current work, we reported for the first time that apoptin inhibits STATS activation in Bcr-Abl expressing cell lines. This observation strongly supports apoptin's role as a proliferation inhibitor of CML cells. STATS also regulates the BfM family gene A1 that reportedly collaborates with c-Myc and is required for Bcr-Abl transformation (Sawyers et al., 1992; van Lohuizen et al., 1989). In the current study, we also demonstrated for the first time that Bcr-Abl induced activation of c-Myc is downregulated in the presence of apotin. The gene encoding CrkL is located near the bcr locus of chromosome 22

(band q1 1) and forms strong complexes with Bcr-Abl (ten Hoeve et al., 1993). These interactions and trans-activation of CrkL and c-Crk Il by activated Bcr-Abl

kinase and their functional consequences are already well documented (Feller, 2001 ; Posern et al., 2000; Voss et al., 2000). The resulting abnormally high TK activity of CrkL is maintained by activated Bcr-Abl causing reduced cell adhesion, increased proliferation and resistance against apoptotic cell death. In the current study, we demonstrated for the first time that Apoptin inhibits phosphorylation of CrkL in Bcr-Abl expressing cells. This observation indicates that Apoptin can indirectly affect growth-supportive role of phosphoryiated CrkL by inhibiting Bcr-Abl kinase. Overall, these observations signify apoptin as a down-regulator of Bcr-Abl co-activated multiple cell proliferation and anti-apoptotic pathways. We also repeatedly observed apoptin-induced activation of Akt in Bcr-Abl expressing cells. Akt is a downstream target for Bcr-Abl kinase and known to interact with Apoptin. However it also functions independently of Bcr-Abl, for example via activation over the PI3-K/PDK1 -2 pathway. Direct apoptin-Akt interaction initiates nuclear trafficking of Akt that instead of activating an anti-apoptotic response initiates apoptosis by a process that is only partially understood (Maddika et al., 2007b). We have observed the nuclear transport of apoptin-iπteracting proteins including Bcr-Abl. We hypothesize that nuclear re-location of Bcr-Abl may markedly affects its biologic properties.

Different components of the Bcr-Abl downstream pathways are involved in the pathogenesis of CM L and are highly active compared to normal cells. Hyper- activation of STATs, Ras-MAPK or CrkL-lntegrin pathways lead to the development of characteristics of CML morphology. Apoptin targets many of these signaling pathways, thus it is well suited for targeting the cellular signaling environment of Bcr-Abl positive cancer cells. We consider here apoptin as a model/lead molecule for the development of smaller peptides or peptidomimetics that would target multiple cell proliferation and anti-apoptotic pathways. This may be of advantage also for CML-treatment, as advanced, highly mutated CML-ceils may no longer solely rely on Bcr-Abl as the driver of cell proliferation. Since apoptin attacks multiple targets related to ceil proliferation, either alone, or in combination with Imatinib/Gleevec it offers much higher curative potential than Gleevec alone.

As discussed in co-pending PCT application CA2007/000681 , it has been shown that amino acids 81-86 of apoptin form a near perfect SH3-interacting

domain. Accordingly, in some embodiments of the invention, an 'apoptin SH3- interacting domain fragment ¹ comprising amino acids 81-86 of apoptin is fused to a transmembrane transport domain. As will be appreciated by one of skill in the art, the apoptin SH3-interacting domain fragment comprising amino acids 81-86 may comprise additional native apoptin residues adjacent thereto or may comprise non- native residues. In other embodiments, the apoptin SH3-interacting domain fragment may comprise amino acids 74-89 of apoptin. In other embodiments, the apoptin SH3-interacting domain fragment may consist of or may consist essentially of amino acids 74-89 of apoptin or amino acids 81-86 of apoptin. It is of note that such fragments may be easily determined by one of skill in the art through routine experimentation by testing apoptin fragments of interest for loss of SH3-interacting activity. in other embodiments, a fusion protein comprising apoptin or an apoptin SH3-interacting domain fragment and a transmembrane transport domain. The transmembrane transport domain may be for example TAT, TAT and Antennapedia, Penetratin, VP22 and poly-arginine as well as other suitable cell- penetrating peptides known in the art.

Accordingly, in one embodiment of the invention, there is provided a method of treating ALL or CML comprising administering to an individual in need of such treatment an effective amount of apoptin or an apoptin SH3-interacting domain fragment, in other embodiments, an effective amount of the fusion peptide as described above is administered.

In yet other embodiments, there is provided the use of a fusion peptide as discussed above for the treatment of ALL or CML. In other embodiments, there is provided a method of preparing a pharmaceutical composition or medicament for the treatment of CML or ALL comprising admixing a fusion peptide as described above with a suitable excipient.

In another embodiment of the invention, there is provided a method of inhibiting Bcr-Abl kinase activity in a Bcr-Abi positive cancer cell comprising administering to said ceil an effective amount of apoptin, an apoptin SH3- interacting domain fragment or a fusion peptide as described above.

As will be appreciated by one of skill in the art, an effective amount of apoptin or an apoptin SH3-interacting domain fragment or a fusion protein thereof

as described herein is the amount required to inhibit Bcr-Abl kinase activity as discussed above.

In another aspect of the invention, there is provided a method of identifying agents capable of inhibiting Bcr-Abl kinase activity. As will be appreciated by one of skill in the art, such agents preferably have three-dimensional structures similar to apoptin and/or an apoptin SH3-interacting domain fragment. It is of note that such agents may be prepared by careful substitution of the native amino acid sequence with various naturally occurring and synthetic amino acid analogues and/or chemically modifying the agent and then determining what effect the modification has on Bcr-Abi inhibition, for example, by comparing inhibition by the agent with inhibition by a known Bcr-Abl inhibitor, for example, the Tat-apoptin fusion described herein although other suitable apoptin- based agents described herein may be used as well. It is of note that Bcr-Abl substrates are well-known in the art and accordingly determining the kinase activity of Bcr-Abl on such a substrate, for example but by no means limited to, CrkL, STATS, c-Myc, PI3K/Akt and the like, in the presence of the agent of interest and comparing that activity to the kinase activity of Bcr-Abi in the presence of a known inhibitor such as Tat-apoptin, as discussed above.

In other embodiments of the invention, there is provided a method of identifying agents capable of inhibiting Bcr-Abl kinase activity comprising: incubating Bcr-Abl, a Bcr-Abl kinase substrate and an agent of interest under conditions suitable for Bcr-Abl kinase activity; and determining if Bcr-Abl kinase activity has been inhibited by comparing phosphorylation of the Bcr-Abl kinase substrate to a control. As will be appreciated by one of skill in the art, the agent of interest may be based on apoptin or a fragment thereof as discussed above or may be taken from a library or panel of compounds. Furthermore, the control may be a negative control or a mock-treated control that comprises Bcr-Abl and the Bcr-Abi kinase substrate incubated under the same or similar suitable conditions for Bcr-Abl kinase activity. Alternatively, the control may comprise the apoptin-TAT fusion, Bcr-Abl and the Bcr-Abl kinase substrate incubated under the same or simiSar suitable conditions for Bcr-Abl kinase activity. In these embodiments, agents having greater inhibition than apotpin-TAT may be isolated.

The Src homology domain 3 of Bcr-Abl interacts with the proline rich domain of Apoptin: To investigate possible interactions of Apoptiπ with the SH3 domains of a series of proteins we first performed a protein array-based study. Several well-characterized SH3 domains were previously identified as the potential sites critical to ligand binding on the basis of alignment with their structures (Lim and Richards, 1994). We performed a high stringency SH3 domain interaction array screening indicated that apoptin strongly interacts with the SH3 domain of some proteins including AbI (Fig. 1A, panel: D3-4). The above observation was further confirmed by 'pull-down assay' and co-immunoprecipitation studies (CO-iP) using both Bcr-Abl-positive (32Dp210), and -negative (32D-DSMZ) cell lines (Fig. 1 BC). Furthermore, our imunofluorescence studies, where GFP tagged apoptin was expressed in 32D ^p210 cells, not only confirmed this fact further, but interestingly it shows, that Bcr-Abl when expressed together with GFP-apoptin, is found predominantly in the nucleus (Fig. 1A). This observation corroborates well with an earlier discovery in our lab, that apoptin interacts and ferry activated Akt to the nucleus of cancer cells (Maddika et a!., 2007b).

Apoptin down-regulates Bcr-Abl kinase and alters the activation of downstream kinases: To study the down-stream effects of apoptin and Bcr-Abl interaction we checked the expression and phosphorylation status of Bcr-Abl (p210) and other major down-stream Bcr-Abi target molecules like STAT5, CrkL, c- Myc and Akt in both murine and human cell lines. Experiments were performed in triplicates to measure phosphorylated and total proteins. The overall results indicate, that an apoptin induced inhibition of Bcr-Abl phosphorylation downregulates STAT5, CrkL but it activates Akt. The observed here activation of Akt may be either dependent or independent of Bcr-Abl, since Akt activation by apoptin, lmatinib and some other cytotoxic agents was previously reported (Maddika et al., 2007b; Tang et a!., 2001). Modifications within these signal transduction cascades counteract CML proliferatory capacity and induce apoptosis (Fig. 2). To study the inhibition of activated Bcr-Abl by apoptin, we quantified the relative phophoryiaiton level of Bcr-Abl (p210) by immunoblotting with Bcr-Ab! specific phospho-antibodies. Experiments were performed on using K562 and 32QP ²¹⁰ _{gs moc} j _e | CML cell lines. Average values expressed as ratio of

phosphorylated and total Bcr-Abl from three independent experiments are presented in figure 3A (K562) and 3B (32D ^p210 ). The inhibition of Bcr-Abl activity by apoptin is highly significant (p < 0.01 - 0.04) in both of the cells lines.

STAT kinases serve the dual role of signal transducers and activators of transcription. Among the large family of over 30 STAT proteins, STATS has been identified as a key factor involved in anti-apoptotic signaling and malignant transformation in CIVIL Here we show that STAT5 phosphorylation was markedly reduced in K562 cells, Tat-apoptin treatment, and the effect was favorably comparable to that of lmatinib (Fig. 3A). Similar results, with statistical significance (p < 0.03) were also observed when experiments were performed in Bcr-Abi (p210) expressing mouse cell line 32D ^p210 (Fig. 3B).

To study other downstream consequences of apoptin's inhibition of Bcr-Abi phosphorylation we checked the phosphorylation status of CrkL (39 kDa) that is involved in β-integrin signaling and is a prominent substrate for activated Bcr-Abl kinase (Feller, 2001). We observed that CrkL phosphorylation is significantly inhibited (p < 0.04) in K562 cells, 14-16 h after addition of 1 μM apoptin in the culture system and this result is comparable to that of lmatinib treated cells (p < 0.02) (Fig. 4A). Similar marked inhibition of CrkL phosphorylation was observed when experiments were repeated on Bcr-Abl ^p21 ° expressing 32D ^p210 (Fig. 4B). To further characterize the pro-apoptotic effect of apoptin on Bcr-Abl expressing cells we analyzed its effect on the signaling protein Akt and its downstream target BAD. As a positive control, we used Imatinib, a known inhibitor of Bcr-Abl kinase, interestingly, although Akt is know as a mediator of cell survival, we repeatedly observed marked augmentation of Akt phosphorylation 14-16 h after either apoptin or Imatinib treatment of K562 and 32D ^p210 cells (Fig. 6AB). However, the activated Akt may still act anti-apoptotic in Bcr-Abl expressing cells if re-located to the nucleus, as previously proposed (Maddika et al., 2007a; Maddika et al., 2007b; Maddika et al., 2008; Trotman et al., 2006). We also observed, the stress-activated Jun-family kinase SAPK/JNK is activated by similar treatment reflecting the ongoing cellular stress (see supplementary data). When active, SAPK/JNK is translocated to the nucleus where it regulates transcription (Leppa and Bohmann, 1999).

Apoptin induces apoptosis in both, Bcr-Abl positive and negative celis, and small proline rich synthetic peptide resembling the interacting region of apoptin exerts a similar effect: To study the biological activity of the cell-penetrating Tat-apoptin on 32 ^p210 cells expressing Bcr-Abl (p210), we treated with Tat-apoptin (1 μM), and cell survival was assessed by MTT assay at different time points. In some other experiments cells were stained according to the Nicoietti method and 24 h later analyzed by flow-cytometry. The results are summarized in figure 7AB.

As shown in figure 7A, treatment of 32 ^pZ1 ° cell lines with either Tat-Apoptin or the positive control imatinib caused significant cell death (p < 0.03) as compared to the negative control group receiving Tat-GFP treatment (Fig. 7B.). Notably, Imatinib was only effective against the 32D ^p210 cells whereas it did not show any effect on 32D-DSMZ cells that do not express Bcr-Abl ¹³²¹⁰ , whereas when both cell lines were exposed to apoptin, they both were killed to similar degree. This result further confirms the very nature of anti-proliferative effect of apoptin that does not rely on a single target, but it instead affects multiple cell growth pathways and thus the development of apoptin resistance is less likely.

Antibodies and Reagents: Unless otherwise indicated all chemicals were purchased from Sigma-Aidrich® Inc. (St. Louis, MO. USA). All antibodies were purchased from Sigma-Aldrich. lnc (St. Louis, MO. USA), Abeam® Inc. (Cambridge, MA. USA) or Cell Signaling Technology®, Inc. (Danvers, MA. USA). The following antibodies were used: murine/rabbit anti-Bcr-Abl/anti-Bcr (monoclonal: Abeam® Inc.), murine anti-Abl (monoclonal), murine Anti-Akt (monoclonal), rabbit anti-c-Myc-phospho and murine anti-c-Myc (monoclonal: Abeam® Inc.), Rabbit anti-BAD, anti-SAPK/JNK and anti-cleaved PARP-1 (polyclonal: Cell Signaling Technology®, Inc.), Rabbit anti-CrkL (polyclonal: Sigma-Aldrich. Inc), (Rabbit-anti-phospho-Bcr-Abl, anti-phospho-STAT5 and anti- phospho-CrkL were purchased as a polyclonal antibody cocktail; Multiplex Western detection kit from Cell Signaling Technology®, Inc), anti-rabbit Cy3, anti- mouse Cy3 (all from Sigma, Oakville, ON) and murine monoclonal anti-Apoptin antibody (kind gift from Dr. D. Jans, Australia). Bcr-Abl kinase Inhibitor Imatinib® (Novartis, Dorval QC. Canada) tablet 400 mg was dissolved into sterile phosphate

buffer solution (PBS) in a concentration of 1 mg/ml and filtered by 0.2 μm syringe filter (Fisherbrand®). The final concentration in cell culture applications was brought down to 1 μM.

Plasmids: The following plasmids were used: GFP, GFP-Apoptin (apoptin cloned into pEGFP-C1 vector, Clontech), GST, GST-apoptin {apoptin cloned into PGEX-2T vector, Amersham Biosciences). pTat-GFP, pTat-Apoptin (kindiy provided by Dr. M. Tavassoli, London, UK). All the plasmids were propagated in BL21(DE3)pLysS E. CoIi bacterial strains, after transforming the respective competent bacterial cells using standard CaCI ₂ mediated chemical transformation. The plasmid isolations from the positive clones were done using Qiagen Mini-prep and Maxi-prep kits using their standard protocols.

Cells and cell culture: Unless indicated otherwise atl cell culture media and supplements were from Gibco BRL. 32D-DSMZ and 32D ^p210 wt:b3:a2/e13:a (will be referred as 32D ^p210 in this paper) (Hallek et al., 1996) Bcr-Abl variant were grown in RPMI-1640 cell culture medium 500 ml supplemented with 20% FBS (Hyclone), 0.05g penicillin/streptomycin, 1OmM HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid), 2 mM L-Glutamine, 0.13mM L-Asparagin, 0.05 nM β-Mercaptoethanol, 1 mM Na-Pyruvate, and 3 ml 100x non-essentia! amino acids. 32D-DSMZ cells are strictly dependent on murine interleukin 3 (mlL3) so the media was supplemented with 10 % supernatant from WEHI-3B cells as described elsewhere (McCubrey et al., 1991). The model human CML cell line K562 (ATCC® # CCL-243™) was cultured in ATCC recommended Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10% FCS (v/v) and antibiotics (Klein et al., 1976; Lozzio and Lozzio, 1975). All cells were grown at 37°C with 5% CO ₂ in a humidified incubator and maintained in a logarithmic growth.

Tat-mediated protein transduction: The recombinant Tat-GFP and Tat- Apoptin proteins were expressed in BL21 bacterial cells and purified as described before (Guelen et al., 2004). Cells were seeded in either 6 well or 12 well culture plates (Corning Incorporated COSTER©). Tat-fusion proteins were added directly to the cells in culture medium reaching to the desired final concentration, 1 μM. Cells were incubated with Tat-fusion proteins at 37 ⁰ C in a 5% CO ₂ humidified

incubator for 16 h.

TransSignal SH3™ Domain Array: The SH3 domain array was performed according to the manufacturer's protocol (Panomics, inc. Redwood City, CA, USA). In brief, purified Tat-Apoptin was hybridized with TransSignal SH3™ Domain Arrayi membrane, and after necessary washing steps the image was acquired on high performance chemiluminescence film (Hyperfilm™ECL, Amersham Biosciences). The proteins in the array are spotted in duplicates. Histidine tagged ligands has been spotted along the bottom and in duplicate along the right side of the membrane for alignment purpose. GST-pull down assay and protein identification: The GST and the recombinant GST-apoptin proteins were purified according to the manufacturer's protocol using glutathione sepharose beads (Amersham Biosciences®). See the supplementary data for details. The GST-pull down assay was performed to detect Apoptin's interacting partners. Briefly, either purified GST or GST-apoptin along with total 32Dp210 or K562 cell lysate (by sonification) were immobilized on glutathione sepharose beads overnight at 4°C in IP-Buffer containing protease and phosphatase inhibitors (5OmM Tris-Hcl pH 8.0, NaCI 15OmM, ND-40 0.5%, EDTA 1mM, PMSF 1mM, NaF 1OmM, Na ₃ VO ₄ 1mM, β-glycerophosphate 25mM). The beads were washed six times with ice-cold lyses buffer and the bound proteins were separated on SDS-PAGE to be identified by immunobiotting.

Co-immunoprecipitation (CO-IP): Depending on the protein, 100-500 μg of cell lysate (by sonication) was added with 2-5 μg of appropriate antibody in IP buffer and incubated for 4 h at 4°C agitating on a rotary shaker. Subsequently, 100 μl of 50% protein-G Sepharose beads (Amersham Pharmacia Biotech®) washed three times with PBS was added to the protein-antibody immune complexes and further incubated for 1 h at 4 ⁰ C. The beads were then washed six times with the lyses buffer, each time centrifuging at 4°C and removing the supernatant. After final wash, the beads were suspended in 50 μl of 5x SDS loading dye, denatured for 5 mtn at 95 ⁰ C and the released proteins were collected by brief centrifugation at 1300Og. The proteins were resolved on SDS-PAGE (8%) and detected by immunobiotting with appropriate antibodies.

Immunobiotting: The protein concentration in the cell lysate was estimated

by Bradford assay. Then, 30 μg of the protein lysate was mixed with 5x SDS loading dye and heated to denature for 5 min at 99°C. The protein samples were resolved by SDS PAGE and transferred to PVDF-membrane (Amersham Biosciences®) using a Wet transfer apparatus (BioRad®) for 1 h at constant current of 85 mA. Membranes were blocked for 1 h with 5% non-fat dry milk powder in Tris-buffered saline with 0.25% v/v Tween-20 (TBS-T) and then incubated overnight with the appropriate primary antibody diluted in TBS-T containing 1 % non-fat dry milk powder or 5% bovine serum albumin (BSA). Membranes were washed three times for 5 min in TBS-T and later incubated with an appropriate secondary antibody conjugated with horseradish peroxidase (HRP) for 45 min at room temperature. Following the repeated washing steps (15mm, x3) the specific proteins on the membrane were detected by using enhanced chemiluminescent (ECL) staining (Amersham Biosciences®). lmmunocytochemistry and fluorescent imaging: Cells were grown overnight in 10 ml Petri dish and then transfected with appropriate plasmids, following iipofectamine protocol (Invirogen®). After 16-18 h of incubation the cells were collected and washed with PBS and then fixed in 4% w/v paraformaldehyde in PBS. Thin smear of cells were made on pre-washed standard microscope glass slides and then air-dried. The cells were permeabilized with permeablization buffer (0.1 % triton X-100 in PBS) and blocked with 5% BSA/PBS for 1 h and subsequently incubated overnight at 4°C with an appropriate primary antibody diluted in the blocking buffer. An appropriate secondary antibody conjugated with Cy3 or FITC depending on the experiment were used following adequate washing steps. Slides were mounted with Vectashϋd® containing DAPI. The slides were then imaged for fluorescent signals and analyzed using Zeiss fluorescent microscope and Zeiss Axiovision 3.1 software.

Bcr-Abl multiplex kinase assays: The in vitro kinase assays were performed using a non-radioactive method. Briefly, the phosphorylation status of Bcr-Abl, STAT5, CrkL and Akt were measured by scanning the signal strength of phosphorylated proteins on Western blot membranes. The kinase reaction was performed in K562 and 32D ^p210 cells by overnight (16 h) incubation with Tat- Apoptin (test), Imatinib® (positive control) Tat-GFP, or no treatment (negative

controls). The cell extracts were resolved by SDS-PAGE (10%) and detected by immunobiotting using their respective phospho-specific antibodies or antibodies detecting both phosphorylated and noπ-phosphorylated kinase. A high-resolution scanner (STORM 860: Molecular Dynamics®: Amersham Pharmacia Biotech) was used to scan the immunoblot membranes after ECL treatment and individual band strengths were measured by Image Quant 5.2 software (Molecular Dynamics®). The final results were normalized according to respective loading controls and expressed as a ratio of measured values for phosphorylated proteins vs total protein bands. Statistical Analysis: Unless stated otherwise, all normalized band intensity data were statistically analyzed by students t test assuming equal variance using Microsoft® Excel software. The variance patterns in each set of data were previously checked by ANOVA from Excel data analysis tool package.

Cell death and cell proliferation assays: Cell proliferation was assessed by MTT assay, whereas cell death was measured by propidium iodide uptake. Cells subjected to appropriate experimental conditions of apoptosis were washed once with PBS and collected after centrifugation at 80Og for 10 min at room temperature. The cells were suspended in PBS and propidium iodide was added <1 μg/mi) and the propidium iodide (Pl) fluorescence was measured directly by flow cytometry using FL-2 channel. Living cells with intact cell membrane were Pl- negative, whereas dead cells with a permeable membrane were Pl positive with a stronger fluorescence signal. The Pl-positive cells were gated using 'BD CellquestPro' software and represented in terms of percentage of cell death. The MTT assays were performed as previously described (Hashemi et al., 2007; Mosmann, 1983).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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