WO2008077062A2 - Suppression of stat3 reactivation after src kinase inhibition to treat cancer - Google Patents

Abstract

Methods of treating cancer are disclosed that comprise administering an inhibitor of SFK in a therapeutically effective amount to the subject wherein STAT3 is durably inhibited. The methods include the administration of an SFK inhibitor in combination with a suitable inhibitor of STAT3 reactivation including a STAT3 inhibitor, a JAK inhibitor or any molecule that inhibits STAT3 reactivation or the compensatory pathway for cell survial after inhibition of the SFK. Also provided are therapeutic compositions comprising an SFK inhibitor in combination with at least one inhibitor of STAT3 reactivation.

Description

SUPPRESSION OF STAT3 REACTIVATION AFTER SRC KINASE INHIBITION TO TREAT CANCER

FIELD OF THE INVENTION

[0001] This invention relates to the treatment of chronic and acute cancer disorders and diseases by administering a combination of an SFK inhibitor with an anti-STAT inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Pat. App. Ser. No. 60/870,682 filed

December 19, 2006. Application is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] None.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT [0004] None.

REFERENCE TO SEQUENCE LISTING [0005] None.

BACKGROUND OF THE INVENTION

[0006] The Src family of kinases ("SFKs") are a family of non-receptor tyrosine kinases that are involved in signal transduction in cancer cells. A role for SFKs in the initiation and/or progression of cancer has been demonstrated in multiple tumor cell lines. Id. ; See also, Trevino, J. G., Summy, J. M., Lesslie, D. P., Parikh, N. U., Hong, D. S., Lee, F. Y., Donate, N. J., Abbruzzese, J. L., Baker, C. H., and Gallick, G. E., Inhibition of SRC Expression and Activity Inhibits Tumor Progression and Metastasis of Human Pancreatic Adenocarcinoma Cells in an Orthotopic Nude Mouse Model. Am J Pathol, 168: 962-972, 2006. For example, in epithelial cancers, SFKs facilitate epithelial-to-mesenchymal transition, which may be important in cancer progression. See e.g., Johnson, F. M. and Gallick, G. E., Src Family of Non-Receptor Tyrosine Kinases as Molecular Targets for Cancer Therapy. Current Medicinal Chemistry, In Press, 2006.

[0007] Inhibition of SFKs using a tyrosine kinase inhibitor has been shown to result in cytotoxicity, cell cycle arrest, and apoptosis in head and neck squamous carcinoma and non-small cell lung cancer cell lines. Johnson, F. M., Saigal, B., Talpaz, M., and Donate, N. J., Dasatinϊb (BMS-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non-small Cell Lung Cancer Cells, Clin Cancer Res, 11: 6924-6932, 2005. One such inhibitor, dasatinib (N- (2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-l-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide, BMS-354825, offered by Bristol-Myers Squibb, Wallingford, CT, is a thiazole-based dual SFK/Abl inhibitor useful in the treatment of leukemia. Talpaz, M., Shah, Ν. P., Kantarjian, H., Donato, Ν., Νicoll, J., Paquette, R., Cortes, J., O'Brien, S., Νicaise, C, Bleickardt, E., Blackwood-Chirchir, M. A., Iyer, V., Chen, T. T., Huang, F., Decillis, A. P., and Sawyers, C. L., Dasatinib in Imatinib-Resistant Philadelphia Chromosome-Positive Leukemias. Ν Engl J Med, 354: 2531-2541, 2006;

[0008] SFKs and certain growth factor receptors are overexpressed in various cancers. Halpern M. S., England J. M., Kopen G. C, Christou A. A., Taylor R. L. Jr., Endogenous c-src as a Determinant of the Tumorigenicity of src Oncogenes, Proc Natl Acad Sd U S A. 1996 93(2): 824-827. Haura, E. B., Zheng, Z., Song, L., Cantor, A., Bepler, G., Activated Epidermal Growth Factor Receptor-Stat-3 Signaling Promotes Tumor Survival In Vivo in Non-Small Cell Lung Cancer, Clin. Cancer Res. 2005, 11(23): 8288-8294. Likewise, the activation paradigm and role of STATs (signal transducers and activators of transcription proteins) in certain cancers has been reported. See Yu, H., Jove, R., The Stats of Cancer — New Molecular Targets Come of Age, Nature Rev. 2004, 4: 97-106.

[0009] At least one member of the Src family of kinases (SFKs), c-Src, reportedly induces STATs involved in the tumorigenesis process. Xi, S., Zhang, Q., Dyer, K. F., Lerner, E. C, Smithgall, T. E. Gooding, W. E., Kamens, J., Grandis, J. R., Src Kinases Mediate STAT Growth Pathways in Squamous Cell Carcinoma of the Head and Neck, J. Biol. Chem. 2003, 278(34): 31574-31583. In particular, STAT3 is a member of the signal transducer and activator of transcription protein family that regulates many aspects of cell growth, survival and differentiation. Constitutive STAT3 has been associated with various human cancers and commonly suggests poor prognosis as it has anti-apoptotic as well as proliferative effects. Yu, H. and Jove, R. The STATs of Cancer-New Molecular Targets Come of Age, Nat Rev Cancer, 4: 97-105, 2004.

[0010] Src family kinases (SFK) also mediate STAT growth pathways in various cancers. Xi, S., Zhang, Q., Dyer, K. F., Lerner, E. C, Smithgall, T. E., Gooding, W. E., Kamens, J., and Grandis, J. R., Src kinases Mediate STAT Growth Pathways in Squamous Cell Carcinoma of the Head and Neck, J Biol Chem, 278: 31574-31583, 2003. A need exists, therefore, for pharmaceutical composition and/or method of treatment for cancer that will inhibit both SFKs and STATs.

SUMMARY OF INVENTION

[0011] Methods of treating cancer in a subject in need thereof are provided, the methods comprising administering an inhibitor of SFK in a therapeutically effective amount to the subject wherein STAT3 is durably inhibited. The methods include the administration of an SFK inhibitor in combination with a suitable inhibitor of STAT3 reactivation including a STAT3 inhibitor, a JAK inhibitor or any molecule that inhibits STAT3 reactivation or the compensatory pathway for cell survial after inhibition of the SFK. Further provided, a therapeutic composition comprising an SFK inhibitor in combination with at least one inhibitor of STAT3 reactivation.

[0012] Novel pharmaceutical compositions comprising at least one compound that inhibits SFK and one or more pharmaceutical agent that inhibits the reactivation of STAT3 including one or more of inhibitors of STAT3, JAK, and/or certain growth factors are also provided.

[0013] Pharmaceutical compositions useful to treat acute and chronic cancers and/or associated disorders such as tumors are disclosed. Treatment of acute and chronic cancer in a mammal in need of such treatment is provided by combinations of at least one SFK inhibitor and one or more pharmaceutical agents to inhibit the reactivation of STAT3 in a subject by administering such combinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure IA is Western blot showing Src inhibition and STAT3 inhibition and reactivation.

[0015] Figure IB is Western blot showing Src inhibition and STAT3 inhibition and reactivation.

[0016] Figure 2A is a Western blot showing the effect of EGFR on STAT3 activation.

[0017] Figure 2B is a Western blot showing STAT3 reactivation in the presence of erlotinib, an EGFR inhibitor and no STAT3 reactivation in the presence of pyridine 6 (P6).

[0018] Figure 2C shows the inhibition of AKT, MAPK, and STAT3 in the presence of dasatinib, with and without pyridine 6 (P6).

[0019] Figure 2D is a Western blot demonstrating STATl inhibition in the presence of pyridine 6 (P6).

[0020] Figure 3A is a plot demonstrating the synergistic effect of the combination dasatinib and pyridone 6 (P6).

[0021] Figure 3B is a plot demonstrating the synergistic effect of the combination dasatinib and pyridone 6 (P6).

[0022] Figure 3 C is a plot demonstrating the synergistic effect of the combination dasatinib and pyridone 6 (P6).

[0023] Figure 3D is a plot demonstrating the synergistic effect of the combination dasatinib and pyridone 6 (P6).

[0024] Figure 3E is a Western blot showing the synergistic effect of the combination of dasatinib and pyridine 6 (P6) on HIF 1 α, cyclinDl, SOCSl and p27 concentrations.

[0025] Figure 4A demonstrates cell cycle arrest and cytotoxicity (apoptosis), respectively, for the combination dasatinib and pyridone 6 (P6).

[0026] Figure 4B demonstrates cell cycle arrest and cytotoxicity (apoptosis), respectively, for the combination dasatinib and pyridone 6 (P6). DETAILED DESCRIPTION OF THE INVENTION

[0027] Methods of treating cancer by inhibiting SFK wherein STAT3 is also durably inhibited are provided. The methods and compositions described herein provide for the inhibition SFKs and the durable inhibition of STAT3. These methods and compositions inhibit the mechanisms that underlie the reactivation of STAT3 in cancer cells treated with SFK inhibitors and the associated biological effects of inhibiting both SFKs and STAT3. Hence, this invention provides for combination therapies of SFK and inhibitors of STAT3 reactivation to prevent and treat cancer.

[0028] The methodologies and compositions provided herein can inhibit STAT3 independent of growth factor inhibition and/or simultaneously with inhibition of growth factors. The methods and compositions may also be directed to inhibit the JAK family of kinases so as to prevent or inhibit both basal STAT3 activation and reactivation and in an effort to prevent or treat various cancer indications and disorders. The methods and compositions disclosed herein further provide the combination of a JAK inhibitor and SFK inhibitor to synergistically treat cancer and tumors.

[0029] The Src family of kinases ("SFKs") have multiple substrates that lead to diverse biologic effects including changes in proliferation, motility, invasion, survival and angiogenesis. The role of SFKs in the initiation and/or progression of cancer has been demonstrated in colon cancer, pancretic cancer, breast cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), prostate cancer, other solid tumors, several hematologic malignancies, hepatic cancer, certain B-cell leukemias and lymphomas. Talamonti et al., J. Clin. Invest., 91, 53 (1993); Lutz et al., Biochem. Biophys. Res. 243, 503 (1998); Rosen et al., J. Biol. Chem., 261, 13754 (1986); Bolen et al., Proc. Natl. Acad. Sci. USA, 84, 2251 (1987); Masaki et al., Hepatology, 27, 1257 (1998); Biscardi et al., Adv. Cancer Res., 76, 61 (1999); and Lynch et al., Leukemia, 7, 1416 (1993). The methods and compositions described herein may be used in any one or more cancers or carcinoma disorders.

[0030] A tyrosine kinase is an enzyme that transfers a phosphate group from ATP to a tyrosine residue in a protein. Tyrosine kinases are a subgroup of the larger class of protein kinases. Fundamentally, a protein kinase is an enzyme that modifies a protein by chemically adding phosphate groups to a hydroxyl or phenolic functional group. Such modification often results in a functional change to the target protein or substrate by altering the enzyme structure, activity, cellular location or association with other proteins. Chemically, the kinase removes a phosphate group from ATP and covalently attaches it to one of three amino acids (serine, threonine or tyrosine) that have a free hydroxyl group. Many kinases act on both serine and threonine, and certain others, tyrosine. There are also a number of kinases that act on all three of these amino acids.

[0031] Tyrosine kinases are divided into two groups: cytoplasmic proteins and transmembrane receptor kinases. In humans, there are 32 cytoplasmic protein tyrosine kinases and 48 receptor-linked protein-tyrosine kinases.

[0032] Generally, tyrosine kinases play critical roles in signaling between cells.

Basically, the activation of cell surface receptors (e.g., the epidermal growth factor (EGF) receptor) by extracellular ligands results in the activation of tyrosine kinases. Then, the tyrosine kinase generates phosphotyrosine residues in the cell. The phosphotyrosine residue acts as a "beacon" and attracts signaling proteins to the receptor via SH2 domains. Hence, one important aspect of the signaling mechanism of a tyrosine kinase is the recognition of the phosphotyrosine by SH2 domains (also referred to herein as Src homology domain 2 or Src homology-2).

[0033] Generally, kinases are enzymes known to regulate the majority of cellular pathways, especially pathways involved in signal transduction or the transmission of signals within a cell. Because protein kinases have profound effect on a cell, kinase activity is highly regulated. Kinases can be turned on or off by phosphorylation (sometimes by the kinase itself -cis- phosphorylation/autophosphorylation) and by binding to activator proteins, inhibitor proteins or small molecules.

[0034] Deregulated kinase activity is a frequent cause of disease, particularly cancer where kinases regulate many aspect that control cell growth, movement and death. For example, neoplastic transformation in which multiple genetic defects such as translocation, mutations within oncogenes and the like, have been implicated in the development of leukemia. Many of these genetic defects have been identified as key components of signaling pathways responsible for proliferation and differentiation. [0035] The Src family of kinases, "SFKs," are also referred to as the transforming

(sarcoma inducing) gene of Rous sarcoma virus. SFKs are cytoplasmic proteins with tyrosine-specific protein kinase activity that associates with the cytoplasmic face of the plasma membrane. Silverman L., Sigal C. T., Resh M. D., Binding of ppδOv-src to Membranes: Evidence for Multiple Membrane Interactions, Biochem Cell Biol 1992 70(10- 11):1187-92. There are 9 Src kinases in the human genome: v-Src, c-Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and BIk. These proteins are all closely related to each other and share the same regulatory mechanism. Brickell, P. M, The p60c-src Family of Protein-Tyrosine Kinases: Structure, Regulation, and Function, Crit Rev Oncog. 1992;3(4):401-46. More specifically, Src kinases are 52-62 kD proteins having six distinct functional domains: SH4 (src homology 4), a unique domain, SH3, SH2, SHl and a C-terminal regulatory region. Brown, M. T., Cooper, J. A., Regulations, Substrates, and Functions of Src, Biochim. Biophys. Acta. 1996, 1287(2-3): 121-49.

[0036] SH4 domain contains the myristylation signals that guide the Src molecule to the cell membrane. The N-terminal half of Src kinase contains the site(s) for its tyrosine phosphorylation, and phosphorylation of tyrosine (Y) 416 regulates the catalytic activity of Src. Thomas, S. M., Brugge, J. S., Cellular Functions Regulated By Src Family Kinases, Ann. Rev. Cell Dev. Biol., 1997, 13: 513-609. Because the N-terminal region of the Src kinase is myristylated, Src can be associated with the cell membrane. This domain is responsible for the specific interaction of Src with particular receptors and protein targets. Id. The C-terminal has a phosphotyrosine residue (Tyr 527).

[0037] The modulating regions, SH3 and SH2, control intra- as well as intermolecular interactions with protein substrates which affect Src catalytic activity, localization and association with protein targets. Pawson, T., Grish, G. D., SH2 and SH3 Domains: From Structure to Function, Cell, 1992, 71: 359-362. The SH3 domain recognizes polyproline helices. The kinase domain, SHl, also known as the tyrosine kinase domain and/or catalytic binding domain, is found in all proteins of the Src family and is responsible for the tryosine kinase activity. The SHl domain has a central role in binding of substrates.

[0038] The Src kinases (herein also referred to as: "Src family of kinases," "Src proteins," and "SFKs") are normally kept off by an autoinhibitory interaction between the phosphotyrosine-binding module (SH2) that is located within the protein before the catalytic kinase domain, and its C-terminal phosphotyrosine (Tyr 527). One form of Src kinase, v-Src, encoded by Rous Sarcoma virus is, however, constitutively active. The v-src gene encodes the protein (v-Src) that on its own can induce the morphological and tumor causing potential of the virus in culture cells, and is indeed, the first of many tumor-causing genes (oncogenes) to be isolated from viruses that have normal counterparts in animal genomes. Takeya, T., Hanafusa, H. Structure and Sequence of the Cellular Gene Homologous to the RSV src Gene and the Mechanism for Generating the Transforming Virus Cell, 1983, 32: 881-890. The oncogenic properties of the v-Src protein arise from disruptions in an internal control mechanism that normally prevents the activation of the protein in the absence of external signals.

[0039] The protein encoded by the cellular counterpart of v-Src is the protein, c-Src.

By contrast, the normal cellular Src, c-Src, is usually inactive until appropriately activated. Fukami, Y., Sato, K., Ikeda, K., Kamisango, K., Koizumi, K., Matsuno, T., Evidence for Autoinhϊbitory Regulation of the c-src Gene Product. A Possible Interaction Between the src Homology 2 Domain and Autophosphorylation Site, J. Biol. Chem., 1993 268(2), 1132-1140. c-Src participates in the signal transduction pathways of receptors that regulate cell growth in animal cell. v-Src differs from cellular Src (c-Src) on the basis of the structural differences in C-terminal region responsible for regulation of kinase activity. V-Src always exists in opened, active conformation, whereas c-Src is flexible and normally inactive. Thomas et al., Ann. Rev. Cell Dev. Biol., at 513-609. Activation of c-Src is reportedly involved in carcinoma cell migration and metastasis. Sakamoto, M., Takamura, M., Ino, Y., Miura, A., Genda, T. Hirohashi, S., Involvement of c-Src in Carcinoma Cell Motility and Metastasis, Cancer Science, 2001 92(9): 941-946.

[0040] Recently, small-molecule tyrosine kinase inhibitors have been identified as a potent inhibitor of Src kinases. In head and neck squamous carcinoma and non-small cell lung cancer cell lines, dastinib results in cytotoxicity, cell cycle arrest and apoptosis. However, despite the durable inhibition of SFKs and initial inhibition of STAT3, STAT3 is not durably inhibited. [0041] Of the various STAT pathways, STAT3 has been identified as a mediator cell proliferation. Inhibition of SFKs does not durably inhibit STAT3. While the SFK inhibitor may initially inhibit STAT3, within a short period of time, STAT3 subsequently re-activiates and is expressed. Johnson, F.M., Saigal, B, Talpaz, M. and Donate, N.J., Dasatinϊb (BMS- 354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non-Small Cell Lung Cancer Cells, Clin. Cancer Res. 11:6924-6932,2005; Nam, S., Kim, D., Cheng, J. Q., Zhang, S., Lee, J. H., Buettner, R., Mirosevich, J., Lee, F. Y., and Jove, R., Action of the Src Family Kinase Inhibitor, Dasatinib (BMS-354825), on Human Prostate Cancer Cells, Cancer Res, 65: 9185- 9189, 2005; Donate, N. J., Wu, J. Y., Stapley, J., Lin, H., Arlinghaus, R., Aggarwal, B. B., Shishodia, S., Albitar, M., Hayes, K., Kantarjian, H., and Talpaz, M., Imatinib Mesylate Resistance Through BCR-ABL Independence in Chronic Myelogenous Leukemia, Cancer Res, 64: 672-677, 2004; and Hambek, M., Baghi, M., Strebhardt, K., May, A., Adunka, O., Gstottner, W., and Knecht, R., STAT 3 Activation in Head and Neck Squamous Cell Carcinomas is Controlled by the EGFR, Anticancer Res, 24: 3881-3886, 2004.

[0042] The STAT (Signal Transducers and Activators of Transcription) proteins are transcription factors specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. STAT proteins act as signal transducers in the cytoplasm and transcription activators in the nucleus. Kisseleva T., Bhattacharya S., Braunstein J., Schindler C. W., Signaling Through the JAKJSTAT Pathway, Recent Advances and Future Challenges, Gene 285: 1-24 (2002).

[0043] STAT proteins regulate many aspects of cell growth, survival and differentiation. Quadros, M. R., Peruzzi, F., Kari, C, and Rodeck, U., Complex Regulation of Signal Transducers and Activators of Transcription 3 Activation in Normal and Malignant Keratinocytes, Cancer Res, 64: 3934-3939, 2004. The seven mammalian STAT family members identified are: STATl, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.

[0044] STAT proteins play a critical role in regulating innate and acquired host immune responses. Dysregulation of at least two STAT signaling cascades (i.e. Stat3 and Stat5) is associated with cellular transformation. Bromberg, J., Darnell, J. E. Jr., The Role of STATs in Transcriptional Control and Their Impact on Cellular Function, Oncogene, 2000, 19(21): 2468-2473. The seven STAT proteins identified in mammals range in size from 750 and 850 amino acids. The chromosomal distribution of these STATs, as well as the identification of STATs in more primitive eukaryotes, suggest that this family arose from a single primordial gene.

[0045] STAT3 can be activated by growth factor receptors, cytokine receptors and non-receptor tyrosine kinases (Src or JAK family kinases). As reported, STAT3 activation mediated by EGFR, EPO-R, and IL-6 R via c-Src or JAK2. See e.g., Lai, S. Y., Childs, E. E., Xi, S., Coppelli, F. M., Gooding, W. E., Wells, A., Ferris, R. L., and Grandis, J. R., Erythropoietin-Mediated Activation of JAK-STAT Signaling Contributes to Cellular Invasion in Head and Neck Squamous Cell Carcinoma, Oncogene, 24: AAA2-ΛAA9, 2005; Siavash, H., Nikitakis, N. G., and Sauk, J. J., Abrogation of IL-6-Mediated JAK Signalling by the Cyclopentenone Prostaglandin 15d-PGJ(2) in Oral Squamous Carcinoma Cells, Br J Cancer, 91: 1074-1080, 2004; & Quadros, M. R., Peruzzi, F., Kari, C, and Rodeck, U., Complex Regulation of Signal Transducers and Activators of Transcription 3 Activation in Normal and Malignant Keratinocytes, Cancer Res, 64: 3934-3939, 2004. MAPK activation can lead to decreased STAT3 phosphorylation. In solid tumors, PDGFR and c-Met can also activate STAT3 via c-Src. IGFRl and EGFR can active STAT3 in a JAK-independent manner. STAT3 activation can lead to activation of several downstream target genes including Bel- XL, cyclin Dl and VEGF.

[0046] STATs share structurally and functionally conserved domains including: an N- terminal domain that strengthens interactions between STAT dimers on adjacent DNA- binding sites; a coiled-coil STAT domain that is implicated in protein-protein interactions; a DNA-binding domain with an immunoglobulin-like fold similar to p53 tumor suppressor protein; an EF-hand-like linker domain connecting the DNA-binding and SH2 domains; an SH2 domain that acts as a phosphorylation-dependent switch to control receptor recognition and DNA-binding; and a C-terminal transactivation domain. Chen X., Vinkemeier U., Zhao Y., Jeruzalmi D., Darnell J.E., Kuriyan J., Crystal Structure of a Tyrosine Phosphorylated STAT-I Dimer Bound to DNA, Cell 93: 827-839 (1998).

[0047] STAT signaling has been implicated in various cancers. Song, J. I. and

Grandis, J. R., STAT Signaling in Head and Neck Cancer, Oncogene, 19: 2489-2495, 2000. In particular, STAT3 is tyrosine-phosphorylated and activated by a number of kinases. STAT3 activation is known to abrogate growth factor dependence which contributes to certain carcinoma tumor growth. Kijima, T., Niwa, H., Steinman, R. A., Drenning, S. D., Gooding, W. E., Wentzel, A. L., Xi, S., and Grandis, J. R., STAT3 Activation Abrogates Growth Factor Dependence And Contributes To Head And Neck Squamous Cell Carcinoma Tumor Growth In Vivo, Cell Growth Differ, 13: 355-362, 2002. Activation of STAT3 is also reported to regulate survival in human non-small cell carcinoma cells. Song, L., Turkson, J., Karras, J. G., Jove, R., and Haura, E. B., Activation OfStat3 By Receptor Tyrosine Kinases And Cytokines Regulates Survival In Human Non-Small Cell Carcinoma Cells, Oncogene, 22: 4150-4165, 2003.

[0048] Binding of cytokines or growth factors to cell-surface receptors leads to activation of cytoplasmic tyrosine kinases, such as the JAK family, which subsequently leads to phosphorylation of STAT monomers. Gadina, M., Hilton, D., Johnston, J. A., Morinobu, A., Lighvani, A., Zhou, Y. J., Visconti, R., O'Shea, J. J. Signaling by Type I and Type II Cytokine Receptors: Ten Years After, Curr. Opin. Immunol. 2001, 13: 363. The STAT proteins are activated by this phosphorylation causing them to dimerize and translocate to the nucleus, where they bind to specific promoter sequences in target genes. Horvath, C. M., The Jak-STAT Pathway Stimulated by Interferon Gamma, Science, STKE, 2004, 260: tr8.

[0049] In particular, the binding of IL-6 family cytokines (including IL-6, oncostatin

M and leukemia inhibitory factor) to the gp 130 receptor triggers STAT3 phosphorylation by JAK2. Boulton, TG, Zhong, Z, Wen, Z, Darnell, Jr, JE, Stahl, N, and Yancopoulos, GD, STAT3 Activation by Cytokines Utilizing gpl30 and Related Transducers Involves a Secondary Modification Requiring an H7-Sensitive Kinase Proc Natl Acad Sci U S A. 92(15): 6915-6919. EGF-R and certain other receptor tyrosine kinases, such as c-MET phosphorylate STAT3 in response to their ligands. Yuan ZL et al., (2004) MoI. Cell Biol. Vol. 24 (21), pages 9390-9400. STAT3 is also a target of the c-src non-receptor tyrosine kinase. Silva CM. (2004) Oncogene Vol. 23 (48), pages 8017-8023.

[0050] Janus kinases (Jak) play an important role in the initial steps of cytokine receptor signaling. While the specificity of the four members of the Jak family (Jakl, Jak2, Jak3, and Tyk2) for different cytokine receptors is not fully understood, studies report that certain specific cytokine receptors can activate one or more Jak. O'shea, J. J., Pesu, M., Borie, D. C, Changelian, P. S., A New Modality for Immunosuppression: Targeting the JAK/STAT Pathway, Nature Rev. Drug Disc. 2004 (3): 555-564. For example, the growth hormone receptor (GHR) is believed to interact predominantly with Jak2, but studies on cell lines have shown that it may also induce phosphorylation of Jakl and Jak3. Hellgren, G., Jansson, J. O., Carlsson, L. M., Carlsson, B., The Growth Hormone Receptor Associates with Jakl, Jak2 and Tyk2 in Human Liver, Growth Horm. IGF Res. 1999 9(3):212-8.

[0051] The binding of a ligand to the cell surface cytokine receptor triggers activation of JAKs. With increased kinase activity, JAK phosphorylates tyrosine residues on the receptor and creates sites for interaction with proteins that contain phosphotyrosine-binding SH2 domain. STATs possessing SH2 domains capable of binding these phosphotyrosine residues are recruited to the receptors and are tyrosine-phosphorylated by JAKs. These phosphotyrosines then act as docking sites for SH2 domains of other STATs, mediating their dimerization. Different STATs form hetero- as well as homodimers. Activated STAT dimers accumulate in the cell nucleus and activate transcription of their target genes. Hebenstreit D. et al. (2005) Drug News Perspect. Vol. 18 (4), pages 243-249. STATs may also be tyrosine-phosphorylated by other non-receptor tyrosine kinases, such as c-src, as well as receptor tyrosine kinases, such as the epidermal growth factor receptor.

[0052] The JAK-STAT pathway is negatively regulated on multiple levels. Protein tyrosine phosphatases remove phosphates from cytokine receptors as well as activated STATs Hebenstreit D. et al. (2005) Drug News Perspect. Vol. 18 (4), pages 243-249. More recently, identified Suppressors of Cytokine Signaling (SOCS) inhibit STAT phosphorylation by binding and inhibiting JAKs or competing with STATs for phosphotyrosine binding sites on cytokine receptors. Krebs, L. et al. (2001) Stem Cells Vol. 19, pages 378-387. STATs are also negatively regulated by Protein Inhibitors of Activated STATs (PIAS), which act in the nucleus through several mechanisms. Shuai, K. (2006) Vol. 16 (2), pages 196-202. For example, PIASl and PIAS3 inhibit transcriptional activation by STATl and STAT3 respectively by binding and blocking access to the DNA sequences they recognize.

[0053] The JAK-STAT signaling pathway takes part in the regulation of cellular responses to cytokines and growth factors. Employing Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs), the pathway transduces the signal carried by these extracellular polypeptides to the cell nucleus, where activated STAT proteins modify gene expression. Although STATs were originally discovered as targets of Janus kinases, it is now reported that certain stimuli can activate them independent of JAKs. D W Leaman, S Pisharody, T W Flickinger, M A Commane, J Schlessinger, I M Kerr, D E Levy, and G R Stark Roles of JAKs in Activation of STATs and Stimulation of c-fos Gene Expression by Epidermal Growth Factor, MoI Cell Biol. 1996 16(1): 369-375. This pathway plays a central role in principal cell fate decisions, regulating the processes of cell proliferation, differentiation and apoptosis.

[0054] Hence, methods of treating cancer in a subject in need thereof comprise administering an inhibitor of SFK to the subject in a therapeutically effective amount wherein STAT3 is durably inhibited. The methods of the present include the administration of an SFK inhibitor in combination with a suitable inhibitor of STAT3 reactivation including a STAT3 inhibitor, a JAK inhibitor or any molecule that inhibits STAT3 reactivation or the compensatory pathway for cell survial after inhibition of the SFK.

[0055] A therapeutic composition comprising an anti-SFK inhibitor in combination with at least one inhibitor of STAT3 reactivation is also provided. A pharmaceutical formulation comprising the therapeutic composition, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients is further provided herein. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.

[0056] The therapeutic formulations include those suitable for oral, parenteral

(including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. [0057] Once formulated, the therapeutic composition can be administered directly to the subject in need thereof. The subjects to be treated can be animals. However, it is preferred that the compositions are adapted for administration to humans. As noted above, direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. Dosage treatment may be a single dose schedule or a multiple dose schedule. The therapeutic compositions and methods are administered in therapeutically effective amounts.

[0058] The term "therapeutically effective amount" refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. The therapeutic effective amount for a human will depend upon the severity of the disease state, the general health, age, weight and gender of the human, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgment of the clinician.

[0059] The multiple therapeutic inhibitors may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic inhibitors may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic inhibitors may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may be any duration of time ranging from a few minutes to four weeks.

[0060] Disorders or conditions advantageously treated by the methods and compositions include the prevention or treatment of cancer, such as colorectal cancer, and cancer of the breast, lung, prostate, bladder, cervix and skin. The methods and compositions may be used in the treatment and prevention of neoplasias including but not limited to brain cancer, bone cancer, a leukemia, a lymphoma, epithelial cell-derived neoplasia (epithelial carcinoma) such as basal cell carcinoma, adenocarcinoma, gastrointestinal cancer such as lip cancer, mouth cancer, esophogeal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that effect epithelial cells throughout the body. The neoplasia can be selected from gastrointestinal cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, prostate cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers. The present compositions and methods can also be used to treat the fibrosis that occurs with radiation therapy.

[0061] The present compositions and methods can be used to treat subjects having adenomatous polyps, including those with familial adenomatous polyposis (FAP). Additionally, these compositions and methods can be used to prevent polyps from forming in subjects at risk of FAP. Specific treatable neoplasms include systemic mast cell disorders, seminoma, acute myelogenous leukemia (AML), gastrointestinal stromal tumors (GISTs) or hypopigmentary disorders.

[0062] SFK inhibitors have been developed that exhibit favorable pharmacokinetics when administered orally to humans and appear tolerated in humans without severe hemtologic or bone toxicity. As mentioned above, one such inhibitor is dastinib, a thiazole- based dual SFK/Abl inhibitor. A wide variety of SFK inhibitors may be useful in the practice of the methodologies and compositions disclosed herein. The following examples are not intended to be exhaustive. Dasatinib, available from Bristol-Myers Squibb Company, Wallingford, CT, is a small molecule inhibitor. US Patent No. 6,723,694 incorporated herein by reference discloses other SFKs modulators. US Patent No. 6,610,688, incorporated herein by reference, teaches 4-substituted 7-aza-indolin-2-ones that are inhibitors of c-src. Similarly, U.S. Patent No. 6,455,270 incorporated herein by reference teaches lichen-derived organic acids such as vulpinic acid and usnic acid which have been found to be effective inhibitors of eukaryotic protein kinase activity, including c-src. U.S. Patent No. 6,150,359 incorporated herein by reference teaches naphthyridinones that inhibit protein tyrosine kinase and cell cycle kinase mediated cellular proliferation that may be useful in the application of the disclosed methodologies and compositions. More recently, U.S Published Patent Application, US20060258642, incorporated herein by reference, teaches quinazoline derivatives have been used for the treatment of tumors. The target kinase disclosed in this recent published application is the Src family kinases, especially c-Src. PP2 (4-amino-5-(4- chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) is a potent, Src family-selective tyrosine kinase inhibitor. Hanke, J. H. et al. 1996: J Biol Chem 271, 695-701. Other Src inhibitors that may be used are taught in US Patent Applications including US20060074094, US20060058341, US20060035897, US20060004043, US20050153955, US20040186157, and US20040072836, each of which is incorporated by reference.

[0063] Since the elucidation of the details of the JAK/STAT pathways, many JAK family inihibtors have been disclosed. Jak inhibitors include, but are not limited to the JAK2 specific inhibitor AG490 and 2-fe^butyl-9-fluoro-3,6-dmydro-7H-benz[h]-imidaz[4,5- /Jisoquinoline-7-one (pyridone 6 or P6). See Burdelya, L., Catlett-Falcone, R., Levitzki, A., Cheng, F., Mora, L. B., Sotomayor, E., Coppola, D., Sun, J-Z., Sebti, S., Dalton, W. S., Jove, R., and Yu, Η., Combination Therapy with AG-490 and Interleukin 12 Achieves Greater Antitumor Effects than Either Agent Alone, 2002 (1): 893-899 and Pedranzini, L., Dechow, T., Berishaj, M., Comenzo, R., Zhou, P., Azare, J., Bornmann, W., Bromberg, Pyridone 6, A Pan-Janus-Activated Kinase Inhibitor, Induces Growth Inhibition of Multiple Myeloma Cells, Cancer Res. 2006, 66(19): 9714-9721. A large array of pyrazole-containing compounds have been used as JAK inhibitors such as those taught in U.S. Patent. Nos. 6,699,865, 6,593,357, 6,528,509, incorporated herein by reference. For examples of useful peptide-based inhibitor see U.S. Pat. No. 6,723,830 incorporated herein by reference. Other small molecule inhibitors of the JAK family have been taught in U.S. Patent Nos. 6,861,418, 6,689,772, 6,683,082, 6,677,368, 6,610,688, 6,608,048, 6,521,618, 6,506,763, 6,486,185, 6,316,635, and 6, 133,305, each of which are incorporated herein by reference.

[0064] Useful STAT3 inhibitors include the STAT3 inhibitor described in PNAS vol.

102 I no. 17 I 5998-6003 "Indirubin derivatives inhibit STAT3 signaling and induce apoptosis in human cancer cells" Sangkil Nam, RaIf Buettner, James Turkson, Donghwa Kim, Jin Q. Cheng, Stephan Muehlbeyer, Frankie Ηippe, Sandra Vatter, Karl-Ηeinz Merz, Gerhard Eisenbrand, and Richard Jove.

[0065] Other STAT3 inhibitors that may be used in connection with the methods and compositions disclosed herein may vary widely in structure and include the use of anti-sense oligonucleotides. These STAT3 inhibitors are disclosed U.S. patent 6,159,694 and U.S. Patent Application US20060217339, each of which is incorporated by reference. Useful peptide and peptide mimetic inhibitors may also be used. See, Coleman et al., Investigation of the Binding Determinants of Phosphopeptides Targeted to the Src Homology 2 Domain of the Signal Transducer and Activator of Transcription 3. Development of a High Affinity Peptide Inhibitor, J. Med Chem. 2005, 48, 6661-6670 and Turkson et al. Novel Peptidomimetic Inhibitors of Signal Transducer and Activator of Transcription 3 Dimerization and Biological Activity, MoI. Cancer. Ther. 2004, 261-269. Furthermore, useful small molecule STAT3 inhibitors of varied structure include those with platinum complexes. See for example, Turkson, et al., Inhibition of Constitutive Signal Transducer and Activator of Transcription 3 Activation by Novel Platinum Complexes with Potent Antitumor Activity, MoI. Cancer. Ther. 2004, 1533-1542. Other STAT3 small molecule inhibitors are taught in U.S. Patent No. 5,731,155 and U.S. Patent Application Nos. US20060247318, US20060210536, US20060030536, and US20050049299, each of which are incorporated herein by reference. Finally, assays have been developed for the screening of modulators of STATs. Assays that may be useful in connection with the methods and compositions disclosed herein are taught in U.S. Pat. Nos. 6,391,572 and 6,821,737, each of which is incorporated by reference.

[0066] Various inhibitors of growth factors may beneficially be used in combination with the methods and compositions disclosed. See Exhibit A, Johnson, F. et al., Abrogation ofSTAT3 Reactivation After Src Kinase Inhibition Results in Synergistic Anti-Tumor Effects, (unpublished), for more discussion on growth factor combination therapies.

[0067] Many commercially available assays for kinase activity can be used to construct screens for small molecule inhibitors; such assay techniques are well known to those skilled in the art. The hits from such screens can then be profiled against arrays of other kinases to identify selective inhibitors.

Example 1 Identification of the mechanism of STAT3 reactivation

The reactivation of STAT3 after durable inhibition of SFKs is shown as a compensatory mechanism for cell survival. Experimental Design: The effect of inhibition of molecules known to be upstream of STAT3 on its reactivation was assessed with Western blotting and a quantitative bioplex phosphoprotein assay. The biological effects of SFK and JAK inhibition were assayed with an MTT assay to assess cytotoxicity and propidium iodine/annexin V staining with FACS analysis to evaluate cell cycle and apoptosis. Cytokines were quantitated using a multiplexed, particle-based FACS analysis with monoclonal antibodies to 25 known cytokines. The combination index (CI) was calculated by the Chou-Talalay equation. Results: In all cell lines, c-Src and several downstream signaling molecules (e.g. AKT, STAT5, FAK) were rapidly and durably inhibited by dasatinib. However, STAT3 was initially inhibited but reactivated by 24 h in 14 solid tumor cell lines. This reactivation was observed with 3 different SFK inhibitors. We investigated several growth factor pathways known to affect STAT3 and found that its reactivation was not mediated by EGFR, IGFR, MAPK, COX2, or cytokine/growth factor release. The addition of JAK inhibitors (AG490 or pyridone 6) to dasatinib resulted in sustained inhibition of STAT3. The combination of pyridone 6 and dasatinib was synergistic in all four cell lines tested with CI that ranged from 0.09 to 0.66. The combination led to increased apoptosis. Conclusions: The reactivation of STAT3 after SFK inhibition is a compensatory pathway that allows cancer cell survival. Abrogation of this pathway using JAK inhibitors results in synergistic cytotoxicity. Given that STAT3 was reactivated in 14 of 15 solid tumor cell lines, this combination may have widespread applicability for cancer treatment.

Example 2 Effect of SKF Inhibition on Downstream Pathways

Figure 2 shows the effect of SFK inhibition on downstream pathways. (A) TuI 67 cells were treated with 100 nM dasatinib for the indicated times, lysed, and analyzed by Western blotting with the indicated antibodies. Dasatinib led to durable inhibition of c-Src, FAK, AKT, and STAT5, but STAT3 was not durably inhibited. (B) Tul67 cells were treated with one of three different SFK inhibitors (dasatinib, PPl, or SKI606) for 24 hours then lysed, and analyzed by Western blotting with the indicated antibodies. All three SFK inhibitors led to durable c-Src inhibition but STAT3 was not inhibited at 24 hours.

Example 3 The Effect of the combination of SFK, JAK and EGFR Inhibition

Figure 2 depicts the effect of the combination of SFK, JAK, and EGFR inhibition on downstream pathways. (A) TuI 67 cells were treated with 100 nM dasatinib or vehicle for 30 minutes then 2 nM EGF was added for 5 minutes, cell were lysed, and assayed with Western blotting to determine the level of EGFR activation. Dasatinib did not affect EGFR activation. (B) Tu 167 cells were treated with dasatinib, erlotinib, pyridone 6 (P6) or a combination of these agents for 24 hours. In two samples EGF was added for 5 minutes prior to cell lysis. The cells were then lysed, and analyzed by Western blotting with the indicated antibodies. Erlotinib and EGF did not significantly affect STAT3 activation, but P6 completely inhibited STAT3 activation. (C) TuI 67 cells were treated with dasatinib, pyridone 6 (P6) or both for 24 hours. Phosphoproteins were assayed and quantitated using the bioplex phosphoprotein assay. Bars represent standard deviation. This confirms and quantitates the results of the Western blot demonstrating inhibition of AKT and MAPK with dasatinib, but reactivation of STAT3 at 24 h. There is sustained STAT3 inhibition by P6. (D) TuI 67 cells were treated with dasatinib, pyridone 6 (P6) or both for 24 hours. STATl activation was assayed with Western blotting with a phosphospecific antibody. SFK inhibition did not significantly affect STATl activation, but P6 completely inhibited STATl activation similar to the findings with STAT3.

Example 4 Combination of SFK and JAK Inhibition

Figure 3 shows the combination of SFK and JAK inhibition results in synergistic antitumor effects in vitro. Tul67 (A), Tu686 (B), A549 (C), and H226 (D) cells were treated with pyridone 6 alone, dasatinib alone, or the two agents combined in a fixed ratio at the indicated doses. The number of viable cells was determined by MTT assay and is expressed a fold control (vehicle alone). In all cell lines, the combination results in significantly more cytotoxicity than single agents. TuI 67 cells were treated with dasatinib, pyridone 6 or both for 24 hours and downstream mediators of SFK and STAT3 were assayed by Western blotting with the indicated antibodies. The combination resulted in more inhibition of HIF-I- alpha, cyclin Dl, and SOCSl and an upregulation on p27.

Example 5 The Effect of SFK and JAK Inhibition on Cell Cycle Apoptosis

Figure 4 shows the effect of SFK and JAK inhibition on cell cycle and apoptosis. (A) TuI 67 cells were treated with dasatinib, pyridone 6, or both for 24 h and 48 h, stained with PI and analyzed with FACS to determine the proportion of cells in each phase on the cell cycle. Nocadazole treatment was added as a positive control that is known to cause G2/M arrest. (B) TuI 67 cells were treated with dasatinib, pyridone 6, or both for 6 h and stained with PI and annexin V to estimate the number of necrotic cells (PI positive) and those undergoing early apoptosis (annexin V positive). The combination resulted in more apoptosis than either drug alone.

Example 6 Biological Effects of Inhibiting SFKs and STAT3

The reactivation of STAT3 diminishes the pro-apoptotic and anti-proliferative effects of SFK inhibition. We sought to determine the mechanism that underlies the reactivation of STAT3 in cancer cells treated with SFK inhibitors and assess the biological effects of inhibiting both SFKs and STAT3. Since EGFR is a major growth factor pathway in epithelial cancers, particularly HNSCC and NSCLC, we initially investigated this pathway and found that STAT3 reactivation was not mediated by EGFR or MAPK. We also found no evidence that SFK inhibition led to the release of a soluble factor or cytokine. Inhibition of the JAK family of kinases, however, did inhibit both basal STAT3 activation and reactivation. The combination of a JAK inhibitor and SFK inhibitor was synergistic in all cell lines tested. Given the consistent finding of STAT3 reactivation in the cancer cell lines that we tested, the combination of STAT and SFK inhibition may have wide therapeutic applicability.

MATERIALS AND METHODS

[0068] Materials. Dasatinib was provided by Bristol-Myers Squibb (New York, NY) and was prepared as a 10 mM stock solution in DMSO. Antibodies used in Western blotting included phosphorylated MAPK (Promega, Madison, WI); AKT and phosphorylated AKT (New England Biolabs, Beverly, MA); Src (Santa Cruz Biotechnology, Santa Cruz, CA); pY419-c-Src, pY705-STAT3, pY694-STAT5, total EGFR, pEGFR (845, 992, 1148), pSTATl, HIF-1-alpha, cyclin Dl (Cell Signaling Technology, Beverly, MA); pY861-FAK (Biosource, Camarillo, CA); pTyrosine (Upstate Biotechnology, Lake Placid, NY); and actin (Sigma Chemical, St. Louis, MO). Pyridone 6, AG490, and PPl were purchased from EMD Bioscience (La Jolla, CA). SKI-606 was a gift from Wyeth pharmaceuticals.

[0069] Cell Culture. Fifteen human cancer cell lines were used in this study: six

HNSCC cell lines (obtained from Dr. J. Myers and Dr. G. dayman of The University of Texas M. D. Anderson Cancer Center), four NSCLC cell lines (obtained from American Type Culture Collection, Manassas, VA), three mesothelioma cell lines (obtained from American Type Culture Collection), and three squamous skin cancer cell lines (obtained from Dr. J. Myers). Cells were grown in monolayer cultures in Dulbecco's modified Eagle's medium (HNSCC and skin cancer cell lines) or RPMI 1640 medium (NSCLC and mesothelioma cell lines) containing 10% fetal bovine serum and 2 mM glutamine at 37⁰C in a humidified atmosphere of 95% air and 5% CO₂.

[0070] Western Blot. Detached cells from each cell culture plate were collected by centrifugation, washed in PBS, and added to the cell lysate from their corresponding plates. Adherent cells were rinsed with ice-cold PBS and lysed in the cell culture plate for 20 min on ice in lysis buffer consisting of 50 mM Trizma base (ph 8; Sigma Chemical Company), 1% Triton X-100, 150 mM NaCl, 20 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride, and 1 mM sodium vanadate. Lysates were spun in a centrifuge at 14,000 rpm for 5 min, and the supernatant was collected. Equal protein aliquots were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, immunoblotted with primary antibody, and detected with horseradish peroxidase-conjugated secondary antibody (BioRad Laboratories, Hercules, CA) and ECL reagent (Amersham Biosciences, Piscataway, NJ).

[0071] Quantitative Bioplex Phosphoprotein Assay. Cells at 5 x 10⁵ per milliliter were treated with p6, dasatinib or both for 24 hours. Protein lysates were prepared by using cell lysis buffer with PMSF (Bio-Rad laboratories, Life Science Research Group, Hercules, CA, USA) on samples collected. Phosphorylated proteins were detected by Bio-Rad phosphoprotein immunoassay kit using Bio-Plex 100 system with workstation (Bio-Rad) according to the manufacturer's protocol. The targeted phophorylated proteins included the following: Akt (Ser473), ERK- 1/2 (Thr202/Tyr204) and STAT3 (Tyr705). Briefly, 50 μl of cell lysate (adjusted to a concentration of 200-900 μg/ml of protein) was plated in the 96 well filter plate coated with anti-phospho-protein antibodies coupled beads and allowed to incubate overnight (16 hours) on a platform shaker at 300 rpm at room temperature. After vacuum-filter and washing the wells; 1 microliter of detection antibodies (25x) were added, vortexed and then incubated for 30 minutes. After additional vacuum-filter and washing of the wells, 0.5 microliter streptavidin-PE (10Ox) was added to each well and allowed to incubate for 10 minutes. After vacuum-filter and washing the wells, 125 microliter of resuspension buffer was added to each well and allowed to incubate for 30 seconds. Data acquisition and analysis was completed by using Bio-Plex manager (V4.1.1. software). [0072] MTT assay. The MTT assay was used to assess cytotoxicity of drugs and drug combinations. Cells were plated into 96-well plates and incubated for 24 h using the conditions described above for standard cell culture maintenance. The cells were subsequently exposed to dasatinib, pyridone 6, or both at various concentrations for 72 h. Eight wells were treated at each concentration. After treatment, 25 μl of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was added to each well and incubated for 3 h. The medium was then removed and 100 μL Of Me₂SO was added. The absorbance of individual wells was read at 570 nM.

[0073] Determination of Synergism and Antagonism. The combination index (CI) was calculated by the Chou-Talalay equation, which takes into account both potency (Dm or IC₅o) and the shape of the dose-effect curve. See, Chou, T. C. and Talalay, P., Quantitative Analysis Of Dose-Effect Relationships: The Combined Effects Of Multiple Drugs Or Enzyme Inhibitors, Adv Enzyme Regul, 22: 27-55, 1984; Chou, T. C, Riedeout, D., Chou, J., Bertino, J. R., and Dulbecco, R., Chemotherapeutic Synergism, Potential And Antagonism, Vol. 2, p. 371-379. San Diego, CA: Academic Press, 1991; Chou, T. C. and Rideout, D. C, The Median-Effect Principle And The Combination Index For Quantitation Of Synergism And Antagonism, p. 61-102. San Diego, CA: Academic Press, 1991. The general equation for the classic isobologram (CI = 1) is given by: CI = (D)_\l(Dx)_\ + (D)₂/(Dx)₂ ; where (Dx)i and (Dx)₂ in the denominators are the doses (or concentrations) for Di (dasatinib) and D₂ (another drug) alone that gives x % inhibition, whereas (D) ₁ and (ZJ)₂ in the numerators are the doses of dasatinib and another drug in combination that also inhibited x % (i.e., isoeffective). CI < 1, CI = 1, CI > 1 indicate synergism, additive effect, and antagonism, respectively http://cancerres.aacηournals.org/cgi/content/full/62/23/ - Bl 3. Nonexclusive competitors are defined as inhibitors binding to different targets or different sites of the same target. The inputs are the concentrations of single inhibitors, the combination doses at different ratios or at fixed ratios, and the fractional inhibition; ie, fraction affected (Fa) of single drugs and combinations. Fa - (Drug A control - Drug A treated) / Drug A control). Fraction of unaffected cells (Fu) = 1 - Fa. The (Dx)i or (Dx)₂ can be readily calculated from the median- effect equation of Chou et al. (3 - 4). Dx = Dm[FaI (l-Fa)]^lm; where Dm is the median-effect dose that is obtained from the antilogof the X-intercept of the median-effect plot, X = log (D) versus Y = log [IaI(I - ia)] or Dm = io-(^r-"^!te'^"c^^/n!, and m is the slope of median-effect plot. Calcusyn software (Biosoft, Ferguson, MO) allows automated calculation of m, Dm, Dx, and CI values. From (Dm)ι, (Dx)₂, and Dl + Dl, isobolograms can be constructed based on the first equation.

[0074] Cytokine Profiling. Cell media were collected after treatment with 100 nM dasatinb or vehicle control and frozen at - 80° C until analysis. 100 μL of cell media was used in each well plate. A validated panel of 25 human cytokines/chemokines (Cytokine 25- plex antibody bead kit) was measured in duplicate using the Bioplex Protein Array Luminex 100 system (Biosource, Invitrogen Corp, Carlsbad, California), according to manufacture's instructions. These included interleukin-1 beta (IL-I β), IL-lra, IL-2, IL-2R, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-10, IL-12p40, IL- 13, IL- 15, IL- 17, tumor necrosis factor-alpha (TNF-α), interferon-alpha (IFN- α), (IFN-γ), granulocyte-monocyte colony stimulating factor (GM- CSF), macrophage chemoattractant protein- 1 (MCP-I), macrophage inflammatory protein lα (MIP-I α), MIP-I β, inducible protein-10 (IP-10), MIG, Eotaxin, and RANTES. This is a multiplexed, particle-based, flow cytometric assay that utilizes anti-cytokine monoclonal antibodies linked to microspheres incorporating distinct proportions of two fluorescent dyes. For each cytokine calibration curves, eight standards ranged from 2.0 to 32,000 pg/mL.

[0075] Cell Cycle and Apoptosis Analysis. Subconfluent cells were treated with 100 nM dasatinib, 2.5 μ M pyridine 6, or both for 6 h (apoptosis) or 24 and 48 h (cell cycle). Cells were also treated with nocadazole as a positive control for G2/M arrest. For cell cycle, cells were harvested, washed in phosphate-buffered saline (PBS), fixed in 1% paraformaldehyde, rewashed in PBS, and resuspended in 70% ethanol at -20⁰C overnight. Cells were washed twice with PBS and stained with 20 μ g/ml propidium iodide (PI). DNA content was analyzed on a cytofluorimeter by fluorescence-activated cell sorting analysis (FACScan; Becton Dickinson and Company, San Jose, CA) using ModFit software (Verity Software House, Turramurra, NSW, Australia). For apoptosis, treated cells were then harvested and stained with annexin V and PI and analyzed on a cytofluorimeter by FACScan using ModFit software.

RESULTS

[0076] Src inhibition leads to initial STAT3 inhibition and later reactivation in multiple cancer cell types in culture. Fifteen human cancer cell lines were treated with 100 nM dasatinib for 0, 2 h, 6 h, and 24 h. Protein expression was measured by Western blot. In all cell lines c-Src was rapidly and durably inhibited. Additionally, several molecules downstream of Src (AKT, STAT5, and FAK) were also durably inhibited. In 14 of 15 cell lines tested [HNSCC (6/6), NSCLC (3/4), mesothelioma (3/3), and squamous skin carcinoma (3/3)] STAT3 activation was intitally inhibited but levels of pSTAT3 (Yl 05) returned to or above baseline by 24 h (Fig. IA, Johnson, F.M. et al., Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non-Small Cell Lung Cancer Cells, Clin Cancer Res, 11:6924-2932,2005, and data not shown). One representative cell line was chosen for further investigation. In TuI 67 cells (HNSCC cell line), treatment with 3 distinct SFK inhibitors all resulted in rapid (within 15 min, data not shown) and durable c-Src inhibition, but a re-activation of STAT3 (Y105) by 24 h. See Figure IB.

[0077] Reactivation of STAT3 is not mediated by activation of the EGFR pathway.

STAT3 can be activated by growth factor or cytokine receptors coupled to the Src or JAK families of kinases. Yu, H. and Jove, R., Nat Rev Cancer, 4: 97-105, 2004. Dasatinib does not have any known direct stimulatory effect on growth factor or cytokine receptors Lombardo, L. J., Lee, F. Y., Chen, P., Norris, D., Barrish, J. C, Behnia, K., Castaneda, S., Cornelius, L. A., Das, J., Doweyko, A. M., Fairchild, C, Hunt, J. T., Inigo, L₅ Johnston, K., Kamath, A., Kan, D., Klei, H., Marathe, P., Pang, S., Peterson, R., Pitt, S., Schieven, G. L., Schmidt, R. J., Tokarski, J., Wen, M. L., Wityak, J., and Borzilleri, R. M., Discovery ofN-(2- chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-l-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a Dual Src/Abl Kinase Inhibitor With Potent Antitumor Activity In Preclinical Assays, J Med Chem, 47: 6658-6661, 2004. We examined the effects of dasatinib on EGFR because this is a key growth factor pathway in several epithelial rumors and because of the extensive research that demonstrates that EGFR activation leads to STAT3 activation in HNSCC. Song, J. I. and Grandis, J. R., STAT Signaling in Head and Neck Cancer, Oncogene, 19: 2489-2495, 2000. Dasatinib treatment for 15 min with or without EGF did not affect EGFR activation in intact cells (Fig. 2A) which demonstrates that dasatinib does not directly affect EGFR in intact cells and confirms the in vitro kinase assay data. We hypothesized that SFK inhibition might lead to the indirect stimulation of EGFR and subsequent STAT3 reactivation based on the observation that in HNSCC cells treated with dasatinib, MAPK was transiently activated. Johnson, F. M., Saigal, B., Talpaz, M., and Donato, N. J., Clin Cancer Res, 11: 6924-6932, 2005. Tul67 cells were treated with an inhibitor of EGFR (erlotinib) which did not affect the STAT3 reactivation by dasatinib (Fig. 2B). Additionally, treatment of TuI 67 cells with EGF only led to a slight increase in STAT3 activation. In contrast, MAPK was markedly activated by EGF. This suggests that STAT3 is not significantly affected by EGFR in these cells. In order to determine if MAPK activation lead to STAT3 activation in cells treated with dasatinib, cells were treated with an inhibitor of MAPK (PD98059) with no effect on STAT3 reactivation (data not shown). We also examined the effect of COX-2 inhibition because COX-2 can activate STAT3, but found no effect of COX-2 inhibitors on STAT3 baseline activation or re-activation at 24 h in these cells (data not shown). Dalwadi, H., Krysan, K., Heuze-Vourc'h, N., Dohadwala, M., Elashoff, D., Sharma, S., Cacalano, N., Lichtenstein, A., and Dubinett, S, Cyclooxygenase-2-Dependent Activation Of Signal Transducer And Activator Of Transcription 3 By Interleukin-6 In Non-Small Cell Lung Cancer, Clin Cancer Res, 11: 7674-7682, 2005. Stimulation of cells with insulin-like growth factor did not lead to significant activation of STAT3 nor did dasatinib affect IGFlR activation.

[0078] Reactivation of STAT3 is not mediated by cytokine release. In order to examine the effect of SFK inhibition on cytokine production, we examined the effect of 100 nM dasatinib on the production of 25 different cytokines in both serum-free and complete medium after 6 and 24 h of treatment. The results were similar in all treatment groups. The majority of cytokines and growth factors were undetectable [interleukin (IL)-2, IL-4, IL-5, IL-7, IL-13, IL- 17, interferon-gamma, granulocyte-monocyte colony stimulating factor, macrophage inflammatory protein 1 alpha, macrophage inflammatory protein 1 beta, eotaxin, macrophage chemoattractant protein-1] or unaffected [IL-lbeta, IL-12p40, IL-15, tumor necrosis factor (TNF)-alpha, interferon-alpha, inducible protein- 10, MIG, RANTES, IL-IO]. Two cytokines (IL-6, IL-8) were decreased by treatment with dasatinib (Table 1). No cytokine or growth factor assayed was significantly increased by dasatinib treatment. In addition, we transferred conditioned medium from TuI 67 cells treated with dasatinib for 24 h to fresh cells and did not observe any STAT3 activation (data not shown). In toto, these data suggest that the reactivation of STAT3 is not mediated by a soluble factor, although an autocrine effect mediated by a secreted but unstable or cell-bound factor cannot be excluded. [0079] JAK family kinase inhibitors block the reactivation of STAT3. One major pathway for STAT activation is through the JAK family of kinases that includes JAKl, JAK2, JAK3, and TYK2. By Western blot, we detected expression of JAK2 and TYK2 but not JAKl or JAK3 in TuI 67 cells (data not shown), which is an expected expression pattern in an epithelial solid tumor. Next we sought to determine if JAK inhibition would abrogate the STAT3 re-activation by dasatinib. Cells were treated with dasatinib, pyridone 6 (P6), or both and protein expression was assayed by Western blotting and bioplex phosphoprotein assay (Figures 2B and 2C). Thompson et al., Photochemical Preparation of a Pyridone Containing Tetracycle: a Jak Protein Kinase Inhibitor, Bioorg Med Chem Lett, 12:1219- 1223, 2002; Pedranzini et al., Pyridone 6, a Pan-Janus-Activataed Kinase Inhibitor, Induces Growth Inhibition of Multiple Myeloma Cells, Cancer Res, 66:9714-9721, 2006. In contrast to the EGFR, MAPK, COX2, and SFK inhibitors, pyridone 6 completely and durably blocked the basal activation of STAT3 and the reactivation of STAT3 in cells treated with dasatinib. Pyridone 6 did not inhibit the activity of c-Src, AKT, or MAPK consistent with the published in vitro kinase assay data. Thompson, J. E., Cubbon, R. M., Cummings, R. T., Wicker, L. S., Frankshun, R., Cunningham, B. R., Cameron, P. M., Meinke, P. T., Liverton, N., Weng, Y., and DeMartino, J. A., Photochemical Preparation of a Pyridone Containing Tetracycle: a Jak Protein Kinase Inhibitor, Bioorg Med Chem Lett, 12: 1219-1223, 2002. Identical effects on pSTAT3 were observed with another JAK inhibitor, AG490 (data not shown). Pyridone 6 also inhibited STATl activation that was only minimally affected by dasatinib (Figure 2D). Dasatinib does not have any direct inhibitory or stimulatory effects on any JAK family member at this concentration (100 nM) as determined by in vitro kinase assays on isolated proteins (Francis Lee, personal communication). In contrast, pyridone 6 has been shown to inhibit all JAK family members in vitro with IC50's of 1 to 15 nM.

[0080] Blocking the reactivation of STAT3 leads to enhanced cytotoxicity and effects on downstream mediators of proliferation and angiogenesis. The reactivation of STAT3 is likely to oppose the anti-tumor effects of SFK inhibitors. We hypothesized that blocking the reactivation of STAT3 would enhance the anti-tumor effects of SFK inhibitors. To test this hypothesis, we combined dasatinib with pyridone 6 in HNSCC and NSCLC cell lines at various concentrations and tested resulting cell viability using the MTT assay (Figures 3A- D). The combination of pyridone 6 and dasatinib were synergistic in all cell lines tested regardless of their basal sensitivity to either drug. In many cases, the combination indicies were less significantly less than 1 for the combination (Table 2). Consistent with our previous data, cell cycle analysis revealed that treatment with dasatinib alone caused an increase of cells G0/G1 fraction with a corresponding decrease of cells in the S and G2/M phases. Treatment with pyridone 6 led to an increase of cells in G2/M and a corresponding decrease of cells in the G0/G1 fraction. The combination of the two drugs led to a significant increase in cells in the sub-GO fraction and a decrease in the G0/G1 (Fig. 4A). Consistent with this, the combination caused a higher proportion of apoptotic cells (Fig. 4B). Downstream targets of SFKs and STAT3 (cyclinDl, HIF-I alpha, SOCSl) were also significantly inhibited by the combination and p27 upregulated, consistent with the enhanced biological effects seen in the biological assays (Figure 3E). Xu, Q., Briggs, J., Park, S., Niu, G., Kortylewski, M., Zhang, S., Gritsko, T., Turkson, J., Kay, H., Semenza, G. L., Cheng, J. Q., Jove, R., and Yu, H., Targeting Stat3 Blocks Both HIF-I And VEGF Expression Induced By Multiple Oncogenic Growth Signaling Pathways, Oncogene, 24: 5552-5560, 2005; Gray, M. J., Zhang, J., Ellis, L. M., Semenza, G. L., Evans, D. B., Watowich, S. S., and Gallick, G. E. HIF-lalpha, STAT3, CBP/P300 And Ref-1/APE Are Components OfA Transcriptional Complex That Regulates Src-Dependent Hypoxia-Induced Expression Of VEGF In Pancreatic And Prostate Carcinomas, Oncogene, 24: 3110-3120, 2005.

[0081] In this study, we demonstrated that SFK inhibition leads to initial STAT3 inhibition but a reactivation of STAT3 at later time points in 14 of 15 cell lines tested including HNSCC, NSCLC, mesothelioma, and squamous carcinoma of the skin. STAT3 is reported to be activated by growth factor and cytokine receptors via SFKs or JAK kinases. We initially focused on the EGFR pathway because it is known to activate STAT3 in HNSCC and NSCLC and because of the transient activation of MAPK previously demonstrated in these cell lines.

[0082] However, the mechanism of STAT3 reactivation does not involve the activation of EGFR or MAPK. Treatment of cells with JAK inhibitors did lead to the durable inhibition of STAT3. The combination of pyridone 6 with dasatinib was synergistic in vitro and led to significantly more apoptosis. Consistent with the increased biological effects, we also observed enhanced effects on downstream signaling molecules including p27, cyclin Dl, SOCSl, and HIF-I alpha consistent with the combined effects on both SFKs and STAT3. [0083] Given the intimate relationship between SFKs and STAT3 in HNSCC, the lack of sustained STAT3 inhibition with dasatinib treatment was surprising. The mechanism for STAT3 reactivation has not been fully elucidated. This may be a compensatory pathway activated by the cells to promote survival in the face of sustained SFK inhibition. The effect of both AG490 and pyridine 6 on STAT3 activity suggests that STAT3 is reactivated through a JAK-dependent mechanism. However, we did not identify a growth factor, cytokine or receptor that led to JAK activation after dasatinib treatment. It is also possible that some of the activation of STAT3 by c-Src may be at least partially kinase-independent and that by inhibiting only its kinase activity, we may have not completely inhibited c-Src's effects on STAT3. Schlaepfer, D. D., Broome, M. A., and Hunter, T., Fibronectin-Stimulated Signaling From A Focal Adhesion Kinase-C-Src Complex: Involvement Of The Grb2, P130cas, And Nek Adaptor Proteins, MoI Cell Biol, 17: 1702-1713, 1997. Alternatively, the reactivation of STAT3 may be due to the effects of dasatinib on other targets. Although this would not be predicted by dasatinib 's known targets, unpredicted molecular and biological effects do occur with other selective kinase inhibitors. For example, imatinib treatment can lead to MAPK activation in chronic myelogenous leukemia (CML) cells and to the release of HB-EGF and the subsequent activation of EGFR and MAPK in HNSCC cells. Yu, C, Krystal, G., Varticovksi, L., McKinstry, R., Rahmani, M., Dent, P., and Grant, S., Pharmacologic Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Mitogen-Activated Protein Kinase Inhibitors Interact Synergistically With STI571 To Induce Apoptosis In Bcr/Abl-Expressing Human Leukemia Cells, Cancer Res, 62: 188-199, 2002; Johnson, F. M., Saigal, B., and Donato, N. J., Induction Of Heparin-Binding EGF-Like Growth Factor And Activation Of EGF Receptor In Imatinib Mesylate-Treated Squamous Carcinoma Cells, J Cell Physiol, 205: 218-227, 2005. Imatinib also reverses multi-drug resistance of CML cells by an unknown mechanism that requires prolonged exposure. Yeheskely-Hayon, D., Regev, R., Eytan, G. D., and Dann, E. J., The Tyrosine Kinase Inhibitors Imatinib And AG957 Reverse Multidrug Resistance In A Chronic Myelogenous Leukemia Cell Line, Leuk Res, 29: 793-802, 2005. The re-activation of STAT3 after treatment with distinct SFK inhibitor suggests that this is a target-specific effect; however none of these inhibitors is completely specific for SFKs. [0084] Another surprising finding in this study was that EGFR activation or inhibition did not significantly affect STAT3 or c-Src in HNSCC cells. EGFR stimulation and inhibition did lead to expected MAPK (ERKl /2) activation and inhibition respectively. EGFR activation is linked to c-Src and STAT3 activation in other HNSCC cell lines and in patient tissues. STAT3 activation, demonstrated by increased dimer formation (STAT3:STAT3 and STAT3:STAT1) and increased phosphorylation, is common in HNSCC tissue specimens. Abrogation of either EGFR or TGF-alpha led to decreased STAT3 activation in HNSCC cell lines in vitro and in vivo. Hambek, M., Baghi, M., Strebhardt, K., May, A., Adunka, O., Gstottner, W., and Knecht, R., STAT 3 Activation In Head And Neck Squamous Cell Carcinomas Is Controlled By The EGFR, Anticancer Res, 24: 3881-3886, 2004; Song, J. I. and Grandis, J. R., Oncogene, 19: 2489-2495, 2000. However, c-Src and STAT3 activation are not always dependent on EGFR. In a panel of NSCLC cell lines that included those with both mutant or with EGFR, the effect of SFK inhibition (dasatinib) on STAT3 activation was modest to absent. Song, L., Morris, M., Bagui, T., Lee, F. Y., Jove, R., and Haura, E. B., Dasatinib (BMS-354825) Selectively Induces Apoptosis In Lung Cancer Cells Dependent On Epidermal Growth Factor Receptor Signaling For Survival, Cancer Res, 66: 5542-5548, 2006; Alvarez, J. V., Greulich, H., Sellers, W. R., Meyerson, M., and Frank, D. A., Signal Transducer And Activator Of Transcription 3 Is Required For The Oncogenic Effects Of Non-Small-Cell Lung Cancer-Associated Mutations Of The Epidermal Growth Factor Receptor, Cancer Res, 66: 3162-3168, 2006. The inhibition of EGFR did not significantly affect c-Src or STAT3 activation in the EGFR wt cell lines, but did significantly inhibit c-Src activation in the mutant EGFR cell lines. Id.; Zhang, J., Kalyankrishna, S., Wislez, M., Thilagananthan, N., Saigal, B., Wei, W., Long, M., Wistuba, L, Johnson, F. M., and Kurie, J. M., Src-Family Kinases are Activated in Non-Small Cell Lung Cancer and Promote the Survival of EGFR-dependent Cell Lines, American Journal of Pathology, In Press, 2006; Haura, E. B., Zheng, Z., Song, L., Cantor, A., and Bepler, G., Activated Epidermal Growth Factor Receptor-Stat-3 Signaling Promotes Tumor Survival In Vivo In Non-Small Cell Lung Cancer, Clin Cancer Res, 11: 8288-8294, 2005. NSCLC cells that are dependent on EGFR pathways (mutant EGFR) for survival and proliferation are more sensitive to the proapoptotic effects of SFK inhibition in vitro. Synergy between Src and EGFR inhibitors has been observed in these cell lines. This suggests that if STAT3 activation is driven by EGFR in patients with NSCLC or HNSCC, then the addition of EGFR inhibitors to SFK inhibitors should result in enhanced cytotoxic effects. This is clearly the case in NSCLC cell lines with EGFR mutations. In our cell lines, the addition of an EGFR inhibitor had additive to modest synergistic effects in two HNSCC cell lines tested (data not shown). None of the cell lines used in the current study has a known EGFR mutation.

[0085] Although the mechanism of STAT3 reactivation has not been fully elucidated, it is clear that there are synergistic anti-tumor effects with SFK and JAK inhibitors. With the combination treatment, we observe durable inhibition of several pathways known to be important for cancer cell survival and proliferation: MAPK, AKT, c-Src, and STAT3. There are also enhanced effects on cyclin Dl, HIF-I -alpha, and p27. Most likely, the enhanced biological and signaling effects are mediated by the durable inhibition of both SFKs and STAT3. The use of rationally designed combinations of targeted agents based on pathway elucidation is appealing in tumors that are not driven by a single genetic mutation. There has been a renewed interest in SFK inhibitors recently because of the development of specific, potent agents such as dasatinib, the drug used in the current study. Dasatinib is orally bioavailable, well tolerated in patients, and has recently been approved for the treatment of CML. Specific STAT3 inhibitors are being actively developed, but none are yet in clinical development. Turkson, J., Kim, J. S., Zhang, S., Yuan, J., Huang, M., Glenn, M., Haura, E., Sebti, S., Hamilton, A. D., and Jove, R., Novel Peptidomimetic Inhibitors Of Signal Transducer And Activator Of Transcription 3 Dimerization And Biological Activity, MoI Cancer Ther, 3: 261-269, 2004; Turkson, J., Zhang, S., Palmer, J., Kay, H., Stanko, J., Mora, L. B., Sebti, S., Yu, H., and Jove, R., Inhibition Of Constitutive Signal Transducer And Activator Of Transcription 3 Activation By Novel Platinum Complexes With Potent Antitumor Activity, MoI Cancer Ther, 3: 1533-1542, 2004; Coleman, D. R. t, Ren, Z., Mandal, P. K., Cameron, A. G., Dyer, G. A., Muranjan, S., Campbell, M., Chen, X., and McMurray, J. S., Investigation Of The Binding Determinants Of Phosphopeptides Targeted To The SRC Homology 2 Domain Of The Signal Transducer And Activator Of Transcription 3, Development OfA High-Affinity Peptide Inhibitor, J Med Chem, 48: 6661-6670, 2005.

[0086] JAK inhibitors, such as the one used in these studies, are being developed for clinical use, but none are in clinical trials for oncology as of yet. JAK3 inhibitors, most of which also inhibit JAK2, have been designed to prevent organ allograft rejection and at least one is in early clinical development in this setting. Changelian, P. S., Flanagan, M. E., Ball, D. J., Kent, C. R., Magnuson, K. S., Martin, W. H., Rizzuti, B. J., Sawyer, P. S., Perry, B. D., Brissette, W. H., McCurdy, S. P., Kudlacz, E. M., Conklyn, M. J., Elliott, E. A., Koslov, E. R., Fisher, M. B., Strelevitz, T. J., Yoon, K., Whipple, D. A., Sun, J., Munchhof, M. J., Doty, J. L., Casavant, J. M., Blumenkopf, T. A., Hines, M., Brown, M. F., Lillie, B. M., Subramanyam, C, Shang-Poa, C, Milici, A. J., Beckius, G. E., Moyer, J. D., Su, C, Woodworth, T. G., Gaweco, A. S., Beals, C. R., Littman, B. H., Fisher, D. A., Smith, J. F., Zagouras, P., Magna, H. A., Saltarelli, M. J., Johnson, K. S., Nelms, L. F., Des Etages, S. G., Hayes, L. S., Kawabata, T. T., Finco-Kent, D., Baker, D. L., Larson, M., Si, M. S., Paniagua, R., Higgins, J., Holm, B., Reitz, B., Zhou, Y. J., Morris, R. E., O'Shea, J. J., and Borie, D. C, Prevention Of Organ Allograft Rejection By A Specific Janus Kinase 3 Inhibitor, Science, 302: 875-878, 2003; O'Shea, J. J., Pesu, M., Borie, D. C, and Changelian, P. S, A New Modality For Immunosuppression: Targeting The JAKISTAT Pathway, Nat Rev Drug Disco v, 3: 555-564, 2004.

[0087] The data as set out in the Figures and Tables 1 and 2 clearly demonstrates the potential of combined JAK/STAT and SFK inhibition in HNSCC, NSCLC, and possibly multiple other cancer types.

Table 1: Effect of Dasatinib on Cytokine Production

Table 2: Median effects of Dasatinib and Pyridone 6 as single agents and in combination.

* Extrapolated

Claims

CLAIMSWe claim:

1. A method of treating cancer or an associated disorder in a subject in need thereof comprising administering a SFK inhibitor and a pharmaceutical agent that inhibits the reactivation of STAT3 to the subject in a therapeutically effective amount, wherein STAT3 is durably inhibited.

2. A pharmaceutical composition for treating cancer or an associated disorder in a subject in need thereof comprising a SFK inhibitor and a pharmaceutical agent that inhibits the reactivation of STAT3 effective amount, wherein STAT3 is durably inhibited.

3. A kit that is suitable for use in the treatment, prevention of inhibition of cancer or cancer associated disorder comprising a first dosage form of a SFK inhibitor and a second dosage form of a pharmaceutical agent that inhibits the reactivation of STAT3, said first dosage and second dosage provided in a quantity which comprise a therapeutically effective amount for the treatment, prevention or inhabitation of oncological disease or disorder.

4. A pharmaceutical composition comprising SFK inhibitor and a pharmaceutical agent selected from the group consisting of STAT3 inhibitor, JAK-STAT3 signaling pathway inhibitor, JAK inhibitor or an inhibitor of the STAT3 compensatory pathway for cell survival wherein said composition inhibits the reactivation of STAT3.

5. The methods of claims 1, 2, 3 and 4 wherein the inhibition of the reactivation of STAT3 is independent of a growth factor modulation or inhibition.

6. A method of treating cancer or associated disorder to a subject in need thereof comprising administering a SFK inhibitor and a JAK inhibitor wherein STAT3 reactivation inhibited.

7. A method of treating cancer or associated disorder to a subject in need thereof comprising administering a SFK inhibitor and a inhibitor of the JAK STAT signaling pathway wherein STAT3 reactivation inhibited.

8. A method of treating cancer or associated disorder to a subject in need thereof comprising administering a SFK inhibitor and a inhibitor of the STAT3 compensatory pathway wherein STAT3 is durably inhibited.

9. A method of treating cancer or associated disorder to a subject in need thereof comprising administering a SFK inhibitor and a inhibitor of the STAT3 wherein STAT3 is durably inhibited upon SFK inhibition.

10. A method of inhibiting reactivation of STAT3 after SFK inhibition comprising administering to a subject in need thereof a therapeutic amount of a SFK inhibitor and a pharmaceutical agent selected from the group consisting of STAT3 inhibitor, JAK-STAT3 signaling pathway inhibitor, JAK inhibitor or an inhibitor of the STAT3 compensatory pathway for cell survival

11. A method of treating an oncological disease or disorder in a subject in need thereof comprising:

(a) preparing a therapeutic composition comprising a therapeutic effective amount of an SFK inhibitor and at least one inhibitor of STAT3 reactivation; and

(b) administering in a repetitive dosing regimen the therapeutic composition to the subject.