MX2014010953A - Methods of treating melanoma with pak1 inhibitors. - Google Patents

Abstract

The present invention provides methods and compositions for the treatment of melanoma using a PAK1 inhibitor. In some embodiments, PAK1 is overexpressed and/or amplified in the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma.

Description

METHODS FOR TREATMENT OF MELANOMA WITH PAKl INHIBITORS Malignant melanoma accounts for approximately 80 percent of deaths from skin cancer. Although melanoma is surgically curable when it is discovered in the early stages, regional and systemic dissemination of the disease considerably worsens the prognosis with only 14% of metastatic melanoma patients surviving for five years (American Cancer Society, Cancer facts & 2011). The pathway of mitogen-activated protein kinase (MAPK) has recently been elucidated as a critical growth pathway in several melanoma subtypes (López-Bergami P. Pigment Cell Melanoma Res. 2011, 24 (5): 902-921). For example, from a cumulative analysis of data from 4493 patients, the occurrence of BRAF (homologous viral oncogene of murine sarcoma v-Raf Bl) is 41% in cutaneous melanomas (Lee JH, et al., Br J Dermatol., 2011, 164 (4): 776-784). The most frequent BRAF somatic mutation in malignant melanoma is substitution of valine at residue 600 to confer constitutive catalytic activity and signaling (Davies H, et al., Nature, 2002; 417 (6892), 949-954.). Genetic studies have confirmed that BRAF is required for melanoma initiation and maintenance in preclinical model systems (Davies H, et al., 2002, ibid, Hoeflich KP, et al., Cancer Res. 2006, 66 (2): 999- 1006; Dankort D, et al., Genes Dev. 2007, 21 (4): 379-3844-6). These discoveries propelled a wave of activity from drug discovery to develop small molecule inhibitors of BRAF, including GDC-0879, PLX-4720, PLX-4032 / vemurafenib (Zelboraf ™) and GSK2118436 (Hoeflich KP, et al., Cancer Res. 2009, 69 (7): 3042-3051; Tsai J, et al., Proc Nati Acad Sci USA 2008, 105 (8): 3041-3046; Bollag G, et al., Nature 2011, 467 (7315): 596-599; Ribas A, &; Flaherty KT., Nature Rev. 2011, 8 (7): 426-433). These inhibitors selectively decrease the growth of tumor cells addicted to BRAF oncogene and provide hope for patients with the subset of melanoma that has activating mutations in the BRAF oncogene (Ribas A, &Flaherty KT., 2011, ibid). However, significantly less antitumor efficacy with current small molecule BRAF inhibitors is observed for wild type BRAF melanoma cells (Hoeflich KP, et al., 2009, ibid: 3042-3051, -Tsai J, et al., Ibid) , raising the need to identify additional driving genes associated with melanoma to promote new knowledge in biology, oncogenic signaling and possible therapeutic agents for disease management of melanoma patients of all classifications.

The RAF kinase family comprises three members, ARAF, BRAF and CRAF, which play an ivotal role to transduce signals in the canonical MAPK signaling pathway of RAS to downstream kinases, MEKl / 2 and ERKl / 2. However, additional kinases have been reported to also they play a role in ERK activation. In particular, several groups have been reported that group-I activated p21 kinases (PAKs) contribute to MAPK pathway activation by phosphorylation of both CRAF at Ser338, a critical activation residue and MEK1 at Ser298, a site that is close to the residues activation loop Ser217 / Ser221 which are substrates for RAF kinases (King AJ, et al., Nature, 1998; 396 (6707), 180-183; Tang Y, et al., Mol Cell Biol. 1999, 19 (3) : 1881-1891; Frost JA, et al., EMBO J. 1997, 16 (21): 6426-6438). The path communication between PAK signaling and MAPK in epithelial cells can be induced by a variety of conditions, including stimulation of growth factor and cell adhesion to the extracellular matrix (Slack-Davis JK, et al., J Cell Biol 2003, 162 (2): 281-291; Zang M, et al., J Biol Chem. 2001, 276 (27): 25157-25165; Beeser A, et al., J Biol Chem. 2005, 280 (44 ): 36609-36615). As a major effector downstream of the small GTPases of the Rho Cdc42 and Racl family, PAK1 also plays a pivotal role in linking extracellular signals with changes in actin cytoskeleton organization, cell shape and adhesion dynamics (Arias-Romero LE, & Chernoff J., Biology Cell, 2008, 100 (2): 97-108; Kumar R, et al., Nat Rev Cancer 2006, 6 (6): 459-471; Ong CC, et al., Oncotarget., 2011, 2 (6): 491-496). PAK1 is widely expressed in a variety of normal tissues and expression is increased significantly in breast and lung cancers (Holm C, et al., J Nati Cancer Inst. 2006, 98 (10): 671-680, Arias-Romero LE, et al., Oncogene 2010, 29 (43): 5839- 5849; Ong CC, et al., Proc Nati Acad Sci USA 2011, 108 (17): 7177-7182). Functional studies have also been implicated in transformation of PAK1 cell (Vadlamudi RK, et al., J Biol Chem. 2000, 275 (46): 36238-36244) and tumor growth (Ong CC, et al., 2011, ibid; Yi C, et al., Cancer Res. 2008, 68 (19): 7932-7937; Chow HY, et al., PloS One 2010, 5 (11): el3791). These findings indicate that PAK1 may contribute to tumorigenesis in certain disease contexts.

The present invention relates to methods for treating a melanoma in an individual, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in the tumor compared to non-cancerous skin cells. In some modalities, PAK1 is amplified in the tumor. In some embodiments, melanoma is a wild-type BRAF melanoma, where PAK1 is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma. melanoma. In some modalities, PAK1 is overexpressed in melanoma and PAK1 is amplified in melanoma. In some modalities, melanoma is a mutant BRAF melanoma. In some modalities, the individual is a human. In some embodiments, the invention provides methods for treating melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAK1 inhibitor.

In some embodiments, the invention provides methods for treating a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is a small molecule, a nucleic acid or a polypeptide. In some embodiments, the invention provides methods for treating a melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is a small molecule, a nucleic acid or a polypeptide.

In some embodiments, the invention provides methods for treating a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor wherein the PAK1 inhibitor is used in combination with a therapeutic agent < 3. In some embodiments, the invention provides methods for treating melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAKI inhibitor wherein the PAKI inhibitor is used in combination with a therapeutic agent.

In some aspects, the invention provides uses of PAKI inhibitors for the treatment of melanoma in an individual. The invention provides uses of PAKI inhibitors in the manufacture of a medicament for the treatment of melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in the tumor compared to non-cancerous skin cells. In some modalities, PAKl is amplified in the tumor. In some embodiments, melanoma is a wild type BRAF melanoma, where PAKl is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is overexpressed in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, melanoma is a mutant BRAF melanoma. In some modalities, the individual is a human.

In some aspects, the invention provides compositions and equipment comprising a PAKI inhibitor for use in the treatment of melanoma. Various modalities relating to these treatment methods are described here and apply to compositions and equipment. In some embodiments, melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in the tumor compared to non-cancerous skin cells. In some modalities, PAKl is amplified in the tumor. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAKl is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is overexpressed in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, melanoma is a mutant BRAF melanoma. In some modalities, the individual is a human.

In some embodiments, the invention provides methods for inhibiting CRAF signaling and / or MEK signaling in a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of the PAKI inhibitor. In some embodiments, melanoma is a wild-type BRAF melanoma. In some modalities, PAKl is overexpressed in the tumor and compared to non-cancerous skin cells. In some modalities, PAKl amplifies in the tumor. In some embodiments, PAK1 is overexpressed in the tumor compared to non-cancerous skin cells. In some modalities, PAK1 is amplified in the tumor. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is overexpressed in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, melanoma is a mutant BRAF melanoma. In some modalities, the individual is a human. In some embodiments, the invention provides methods for treating melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAKI inhibitor.

In some aspects, the invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor comprising determining the BRAF genotype of the melanoma, wherein the melanoma comprising a wild-type BRAF indicates that the patient is suitable for treatment with a PAKI inhibitor. In some aspects, the invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor comprising determining the expression of PAKI in the melanoma, wherein the overexpression of PAKI in the melanoma compared to non-cancerous skin cells indicates that the patient is suitable for treatment with a PAKI inhibitor. In some aspects, the invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor, which comprises determining the number of copies of PAK1 in the melanoma, wherein the amplification of PAK1 in the melanoma indicates that the patient It is suitable for treatment with a PAKI inhibitor. In some aspects, the invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor, which comprises determining the BRAF genotype of the melanoma and determining the expression of PAKI in the melanoma, wherein the presence of a BRAF of wild type and / or overexpression of PAKl in melanoma compared to non-cancerous skin cells, indicates that the patient is suitable for treatment with a PAKI inhibitor. In some aspects, the invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor, comprising one or more of determining the melanoma BRAF genotype, determining the expression of PAKI in the melanoma, and determining the number of melanoma. copy of PAKl in melanoma, where one or more of the presence of a wild-type BRAF, Overexpression of PAKI in melanoma compared to non-cancerous skin cells and amplification of PAKI in melanoma indicates that the patient is suitable for treatment with a PAKI inhibitor.

In some aspects, the invention provides methods for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient based on the BRAF genotype of melanoma, wherein a melanoma comprising a wild-type BRAF indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

In some aspects, the invention provides methods for treating a patient with human melanoma with a PAKI inhibitor comprising: (a) selecting a patient based on the level of PAK1 expression of the melanoma, wherein an over expression of PAK1 in the melanoma compared to non-cancerous cells indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

In some aspects, the invention provides methods for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient based on the number of copies of PAK1 in the melanoma, wherein the amplification of PAKl in melanoma indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

In some aspects, the invention provides methods for the treatment of a patient with human melanoma with a PAKI inhibitor, comprising: (a) selecting a patient based on the melanoma BRAF genotype and melanoma PAK1 expression level, wherein a Melanoma comprising a wild-type BRAF and / or overexpression of PAKI in melanoma compared to non-cancerous cells indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

In some aspects, the invention provides methods for treating a patient with human melanoma with a PAKI inhibitor, comprising: (a) selecting a patient based on one or more of the melanoma BRAF genotypes, melanoma PAK1 expression level and copy number of PAKl in melanoma, where a melanoma comprises one or more of wild type BRAF, overexpression of PAKl in melanoma compared to non-cancerous cells and amplification of PAKl, indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administer to the select patient an effective therapeutic amount of a PAKl inhibitor.

In some aspects, the invention provides methods for adjusting melanoma treatment in a patient undergoing treatment with a PAKI inhibitor, the method comprising estimating the expression of PAK1 in the melanoma, wherein the overexpression of PAK1 in the melanoma indicates that the treatment of the individual is adjusted until the overexpression of PAK1 is no longer detected. In some embodiments, melanoma is a wild-type BRAF melanoma. In some modalities, PAKl is amplified in melanoma. In some modalities, melanoma is a wild-type melanoma and PAK1 is amplified in melanoma.

Figure 1A-1C shows that PAK1 is highly expressed in human melanoma. (Fig.lA) Analysis of gains of number of copies llql3 in human melanoma tissues. The vertical red line represents the chromosome location of the PAKl gene. (Fig.lB) DNA copy PAKl and mRNA expression (signal 226507_at Affymetrix MAS 5.0) correlated for melanoma tumor samples. (FIG.1C) Representative images of PAKl immunohistochemistry in primary human malignant melanomas. Cytoplasmic expression score: 0 (I), 1 (II), 2 (III) and 3 (IV). Chromogen deposition indicates immunoreactivity against a contraction of hematoxylin. PAKI expression is also seen in stromal cells (III) and cells that interspersed within the epidermis that can represent Langerhan cells (IV).

Figure 2A-2D demonstrates that PAK1 plays a critical role in proliferating BRAF wild type melanoma cells. (Fig.2A) Proliferation of melanoma cells after transfection with siR A oligonucleotide was measured by the Cell TiterGlo ATP consumption assay. PAK1 is required for cell growth and the data was normalized for control and shown as the average + SD. (Fig.2B) In a panel of melanoma cell lines, PAK1 inhibition selectively impairs cell growth without BRAF mutation (V600E) (n = 5; 537 EL, HS940T, MeWO, SK-MEL2, SK-MEL23, SK -MEL30) compared to those with BRAF mutation (V600E) (n = 9, p = 0.07, 624 EL, 888MEL, 928 EL, RPMI-7951, A375, Colo829, LOX-IMVI, Malme-3M, A375). (Fig.2C) Inhibition of PAK1 / 2 decreases ERK1 / 2 and MEK1 / 2 phosphorylation and accumulation of cyclin DI. (Fig.2D) Inhibition PAKl / 2 in wild-type melanoma cells SK-MEL23 BRAF decreases signaling to the cytoskeleton, MAPK, proliferation and NF-? as determined by a reverse phase protein matrix (RPPA) analysis. The results of normalized RPPA are presented as average, + SD. siNTC = control siRNA without target, if RAS = NRAS-specific siRNA, siPAKl = specific siRNA PAK1,? 1 = chromosomal deletion of the PAK1 gene.

Figure 3A-3E illustrates a series of immuno transferences demonstrating that PAK1 is required for CRAF activation in BRAF wild type melanoma cells. (Fig.3A) siRNA control oligonucleotides -PAK1 and -PAK2selective or no target (NTC) were transfected into melanoma cells SK-MEL23 and 537MEL. After 48 h, endogenous MEKl proteins (Fig.3A), MEK2 (Fig.3B) or CRAF (Fig.3C) were immunized and the complexes were immuno-transferred to detect phosphorylation of critical residues for catalytic activation. Total protein levels in the immune complexes were also determined as loading controls. (Fig.3D) Cells were treated with DMSO or PF-3758309 5 μ? for 4 h and endogenous CRAF was immuno precipitated and immuno transferred for Ser338 phosphorylation. Total CRAF levels in immune complexes are also illustrated. (Fig.3E) SK-MEL23 cells were treated with DMSO, PF-3758309 5 μ? or IPA-3 20 μ? for 4 h. Immune CRAF complexes were incubated with inactive MEKl protein in kinase buffer for 30 minutes. Levels of phospho-MEKl (Ser217 / Ser221) were determined and catalytic activity CRAF is reported as MEKl phosphorylation levels normalized to total CRAF proteins.

Figure 4A-4B contains images demonstrating that PAK is required for migration of melanoma cells. After control without target (NTC) or transfection oligonucleotide PAKl / 2 siRNA for 72 h, confluent melanoma cells WM-266-4 were injured and images were recorded when the wounds were made (dark shading) and after incubation for 28 h (bright field). Differences in relative wound density were statistically significant (p <0.001, n = 3).

Figure 5A-5B illustrates a series of immunoblots demonstrating in vitro differential sensitivity of MAPK signaling in wild type BRAF and BRAF melanoma cells (V600E) treated with PAK inhibitors. (Fig.5A) SK-MEL23 and A375 cells were treated with DMSO, PF-3758309 5 μ? or PLX-4720 0.2 μ? for 4 h and lysates were analyzed for phosphorylation of MAPK pathway components. Lighter and darker exposures of immunoblots p-MEKl / 2 (S217 / S221) are shown. (Fig.5B) Ectopic expression PAK1 tagged with Flag epitope directed MAPK pathway activation in A375 cells. Specificity was demonstrated using treatment with an inhibitor PF-3758309 as a control.

Figure 6A-6B illustrates a series of graphs demonstrating decreased viability of BRAF wild-type melanoma cells due to treatment with in-house PAK inhibitors. Catalytic inhibition of PAK1 by treatment of I-007, 1-054, 1-087 and PF-3758309 was tested in vitro using (Fig.6A) SK-MEL23 and (Fig.6B) 537MEL cells using a Cell TiterGlo viability assay (Promega) of 4-days.

Figure 7A-7B shows the differential sensitivity In vivo MAPK signaling due to PAK inhibition in mouse models of wild type BRAF melanoma and BRAF melanoma tumor xenoinj (V600E). (Fig.7A) Pharmacodynamic response of mutant and wild-type BRAF tumors measured by phosphorylation of CRAF (Ser338) following either vehicle administration or 35 mg / kg of PF-3758309. (Fig.7B) Anti-tumor efficacy of 10, 15 and 25 mg / kg of PF-3758309 i.p. Daily dosing in the SK-MEL23 pre-clinical tumor model of BRAF wild type melanoma.

Figure 8A-8B illustrates a series of graphs demonstrating individual tumor data for the SK-MEL23 pre-clinical tumor model of wild type BRAF melanoma. (Fig.8A) Inhibition of tumor growth and (Fig.8B) loss of body weight are shown for animals treated with 10, 15 and 25 mg / kg of PF-3758309. To analyze the repeated measurement of tumor volumes of the same animals over time, cubic regression splines were employed to fit a non-linear profile to the time courses of tumor volume log2 at each dose level. These non-linear profiles were then related to doses within the mixed model. Cubic regression splines were employed to fit a nonlinear profile to the tumor volume log2 time courses at each dose level. These non-linear profiles were then related to doses within the mixed model. Inhibition of tumor growth as a percentage of Vehicle (% TGI) is calculated as the percentage of the area under the adjusted curve (AUC) for the respective dose group per day in relation to the vehicle, using the formula:% TGI = 100 x (1 - AUCdose / AUCveh) · The plot was made and generated using R version 2.8.1 and Excel, version 12.0.1 (Microsoft). The data is analyzed using R version 2.8.1 (R Foundation for Statistical Computing, Vienna, Austria), and the mixed models were fitted within R using the nlme package, version 3.1-89.

Figure 9 shows immunoblots demonstrating different pharmacodynamic responses of BRAF wild type tumors treated with either G945 BRAF inhibitor or PF-3758309. Phosphorylation of CRAF (Ser338) is determined for SK-MEL23 xenograft tumors after administration of either 35 mg / kg of PF-3758309 i.p. or 10 mg / kg of G945 (BRAF inhibitor) compounds p.o. Tumors were collected 1 hour after dosing and frozen instantaneously. Each lane represents tumor lysate from an individual xenograft mouse.

Figure 10A-10B is a diagram illustrating the mechanism of action for PAK1 in the wild-type melanoma BRAF. (Fig. 10A) in the context of oncogenic mutation, BRAF strongly directs the activation of the MAPK signaling pathway and these tumor cells are sensitive to inhibition of this kinase. (Fig.lOB) In melanomas where BRAF is not mutated, elevated expression and genomic amplification of PAKI is common and results in increased signaling to CRAF-MEK-ERK and potentially in additional effector pathways. This subset of melanoma is relatively insensitive to BRAF inhibition and the proliferative capacity depends on PAK1.

The present invention provides methods and compositions for the treatment of melanoma in an individual, which comprises contacting the melanoma with a therapeutically effective amount of a PAKI inhibitor. The invention also provides these methods of treatment comprising administering to the individual, a therapeutically effective amount of a PAKI inhibitor. In some embodiments, melanoma is a wild-type BRAF melanoma. In some modalities, melanoma overexpresses PAKl compared to non-cancerous cells. In some modalities, PAKl is amplified in melanoma. In some embodiments, melanoma is a wild type BRAF melanoma and PAKl overexpressed in comparison to non-cancerous cells. In some modalities, melanoma is a wild type BRAF melanoma, melanoma overexpressed PAKl compared to non-cancerous cells, and PAKl is amplified in melanoma.

All references cited here, including patent applications, patent publications and numbers - - of Genbank Access here are incorporated by reference, as if each individual reference was indicated in a specific and individual way by reference.

Definitions The techniques and methods described or referred to herein in general are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely employed methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd . Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., Eds., (2003)); the METHOD IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CÉLULA CULTURE (R. I. Freshney, ed. (1987)); Synthesis of Oligonucleotides (M. J. Gait, ed., 1984); Method in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cells, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed. , 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Tissue and Cell Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology - - (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., Eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., Eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., Ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

Unless otherwise defined, technical and scientific terms herein employed have the same meaning as is commonly understood by a person of ordinary skill in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed. , J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed. , John Wiley & Sons (New York, N.Y. 1992), provide a person skilled in the art with a general guide to many of the terms - - employees in the present application.

"PAK," as used herein, refers to a family of non-receptor serine / threonine protein kinases (STKs). The protein kinase protein family (PAK) activated p21 serine / threonine protein kinases plays important roles in cytoskeletal organization, cell morphogenesis, cellular processes and cell survival (Daniels et al., Trends Biochem.Sci. 1999 24: 350-355; Sells et al., Trends Cell, Biol. 19977: 162-167). The PAK family consists of six members subdivided into two groups: PAK 1-3 (group I) and PAK 4-6 (group II) that are distinguished based on sequence homologies and the presence of a self-inhibitory region in group PAKs I. Activated p21 kinases (PAKs) serve as important mediators of Rae function and Cdc42 GTPase as well as routes required for Ras- directed tumorigenesis. (Manser et al., Nature 1994 367: 40-46, B Dummler et al., Cancer Metathesis Rev. 2009 28: 51-63; R. Kumar et al., Nature Rev. Cancer 2006 6: 459-473).

"PAK1" or "activated p21-activated protein kinase (Cdc42 / Rac) -1" as used herein, refers to a PAK1 native to any vertebrate source, including mammals such as primates (e.g., humans) and rodents (for example, rats and mice), unless otherwise indicated. The terms cover the location - - genomic (eg Ilql3-ql4 cytogenetic band, chromosome 11: 77033060-77185108, and / or GC11M077033), "whole length", unprocessed PAKl as well as any form of PAKl resulting in processing in the cell. The term also covers variants of natural origin of PAKl, for example splicing variant or allelic variants. The exemplary human PAKl nucleic acid sequence is NC_000011.9. An exemplary human PAKl amino acid sequence is NP_0011220921 or NP_002567.3. The exemplary mouse PAKl nucleic acid sequence is NC_000073.6 or an exemplary mouse PAKl amino acid sequence NP_035165.2. The exemplary rat PAKl nucleic acid sequence is NC_005100.2 or an exemplary rat PAKl amino acid sequence NP_058894.1. The exemplary dog PAKI nucleic acid sequence is NC_006603.3 or an exemplary dog PAKl amino acid sequence XP_849651.1. The exemplary cow PAKl nucleic acid sequence is AC_000186.1 or NC_007330.5. An exemplary cow amino acid sequence PAKl is NP_001070366.1. The exemplary rhesus monkey PAKl nucleic acid sequence is NC_007871.1. An exemplary rhesus monkey PAKl amino acid sequence is XP_001090310.1 or NP_001090423.2. The exemplary chicken PAKl nucleic acid sequence is NC_006088.3 or an exemplary chicken amino acid sequence PAKl NP_001155844.1"BRAF" or "Serine / threonine-protein kinase B-Raf", as used - - here, as used herein, refers to a BRAF native to any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., ratone and rats), unless otherwise indicated. The terms encompass genomic location [eg, cytogenetic band 7q34, chromosome 7: 140433812-140624564, and / or GC07M140424), "full length", unprocessed BRAF as well as any form of BRAF that results from processing in the cell. The term also covers variants of natural origin of BRAF, for example, splice variants or allelic variants. The exemplary human BRAF nucleic acid sequence is NC_000007.13 or an exemplary human BRAF amino acid sequence NP_004324.2. The exemplary mouse BRAF nucleic acid sequence is NC_000072.6 or an exemplary mouse BRAF amino acid sequence NP_647455.3. The sequence of an exemplary rat BRAF nucleic acid is NC_005103.2 or an exemplary rat BRAF amino acid sequence XP_231692.4. The exemplary dog BRAF nucleic acid sequence is NC_006598.3 or an exemplary dog BRAF amino acid sequence XP_532749.3. The exemplary chicken BRAF nucleic acid sequence is NC_006088.3 or an exemplary chicken BRAF amino acid sequence NP_990633.1. The sequence of an exemplary cow BRAF nucleic acid is AC_000161.1 or an exemplary cow BRAF amino acid sequence - - XP_002687048.1 The exemplary horse BRAF nucleic acid sequence is NC_009147.2 or an exemplary horse BRAF amino acid sequence XP_001496314.2.

"BRAF wild type" refers here to BRAF of natural origin (including variants of natural origin) not associated with melanoma. An example of wild type BRAF is provided by GenBank Accession Number NP_004324.2. As is known in the art, with respect to BRAF melanomas it can be categorized and classified by BRAF type: wild type BRAF and mutant BRAF.

"BRAF Mutant" as used herein refers to a BRAF protein with one or more mutations that are associated with melanoma. An example of a mutant BRAF is one in which a valine in position 600 is replaced with a glutamate (V600E). As is known in the art, melanomas can be categorized by BRAF type: wild type BRAF and mutant BRAF.

"CRAF" or "viral leukemia oncogene v-raf 1" as used herein refers to a CRAF native to any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) ), unless otherwise indicated. The terms encompass genomic location (eg, 3p25 cytogenetic band, chromosome 3: 12625100-12705700, and / or GC03M012625), "full length", unprocessed CRAF as well as - - any form of CRAF that results from processing in the cell. The term also covers variants of natural origin of CRAF, for example splicing variants or allelic variants. The exemplary human CRAF nucleic acid sequence is NC_000003.11 or an exemplary human CRAF amino acid sequence NP_002871.1.

"MEK" or "mitogen-activated protein kinase kinase", as used herein, refers to a family of kinase enzymes that phosphorylate mitogen-activated protein kinase (MAPK). There are seven genes: MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K3 (MKK3), MAP2K4 (MK4), MAP2K5 (MKK5), MAP2K6 (MKK6), and MAP2K7 (MKK7). Activators of p38 (MKK3 and MKK6), JNK (MKK4 and MKK7), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways. The exemplary human MEK1 nucleic acid sequence is NC_000015.9 or an exemplary human MEK1 amino acid sequence NP_002746.1. The exemplary human MEK2 nucleic acid sequence is NC_000019.9 or an exemplary human MEK2 amino acid sequence NP_109587.1. The nucleic acid sequence of exemplary human MEK3 is NC_000017. io An amino acid sequence of exemplary human MEK3 is NP_002747.2 or NP_659731.1. The exemplary human MEK4 nucleic acid sequence is NC_000017.10 or an exemplary human MEK4 amino acid sequence NP 003001.1. The nucleic acid sequence of human MEK5 - - exemplary is NC_000015.9. An amino acid sequence of exemplary human MEK5 is NP_001193733.1, NP_002748.1, or NP_660143.1. The nucleic acid sequence of exemplary human MEK6 is NC_000017.10 or an amino acid sequence of exemplary human MEK6 NP_002749.2. The nucleic acid sequence of exemplary human MEK7 is NC_000019.9 or an amino acid sequence of exemplary human MEK7 NP_660186.1.

"Polynucleotide" or "nucleic acid", as used herein interchangeably, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, nucleotides or modified bases and / or their analogues, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogues. If present, the modification to the nucleotide structure can be imparted before or after the assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can also be modified after synthesis, such as by conjugation with a tag. Other types of modifications include for example "caps", substitution of one or more of the nucleotides of natural origin with an analog, modifications - - internucleotide, such as for example those with uncharged bonds (eg, methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), those containing pendent portions, such as for example proteins (for example, nucleases, toxins, antibodies, signal peptides, pli-L-lysine, etc.), those with intercalators (for example, acridine, psoralen, etc.), those containing chelators (for example, example, metals, radioactive metals, boron, oxidative metals, etc.), those that contain alkylators, those with modified bonds (for example, alpha numeric nucleic acids), etc.), as well as unmodified forms of the polynucleotide (s). In addition, any of the hydroxyl groups ordinarily present in the sugars can be replaced for example by phosphonate groups, phosphonate groups, protected by standard or activated protecting groups to prepare additional bonds with additional nucleotides, or can be conjugated to solid and semi-solid supports. The terminal OH 5 'and 3' can be phosphorylated or substituted with amines or portions of the organic end termination group from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized into standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally - - they are known in the art, including for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 21-azido-ribose, carbocyclic sugar analogs,? -meric sugars, epimeric sugars such as arabinose, xyloses or lixoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abbasic nucleoside analogues such as methyl riboside. One or more phosphodiester bonds can be replaced by alternating linking groups. These alternative linking groups include but are not limited to, embodiments wherein the phosphate is replaced by P (O) S ("thioate"), P (S) S ("dithioate"), (0) NR2 ("amidate" ), P (0) R, P (0) OR ", CO or CH2 (" formacetal "), wherein each R or R 'is independently H or substituted or unsubstituted alkyl (1-20 C), which optionally contains an ether (-0-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl bond Not all bonds in a polynucleotide need to be identical The preceding description applies to all polynucleotides referred to herein, including RA and DNA. "oligonucleotide" as used herein, it generally refers to short, single-stranded polynucleotides that have but not necessarily less than about 250 nucleotides in length Oligonucleotides can be synthetic The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. previous description for polynucleotides is equal and completely applies to oligonucleotides. The term - - "primer" refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and after polymerization of a complementary nucleic acid, generally providing a free 31 -OH group.

The term "small molecule" refers to any molecule with a molecular weight of about 2000 daltons or less, preferably of about 500 daltons or less.

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures including but not limited to monoclonal antibodies, polyclonal antibodies, multispecies antibodies (eg, bispecific antibodies) and antibody fragments provided they exhibit the binding activity. of the desired antigen.

The term "detection" includes any means to detect, including direct and indirect detection.

The term "biomarker" as used herein refers to an indicator, for example predictive, diagnostic and / or prognostic, that can be detected in a sample. The biomarker can serve as an indicator of a particular subtype of a disease or disorder (eg, cancer) characterized by certain molecular, pathological, histological and / or clinical characteristics. For example, biomarkers for melanoma include but are not - - limited to the presence of wild-type BRAF, overexpression of PAK1 and amplification of PAK1.

The "quantity" or "level" of a biomarker associated with an increased clinical benefit for an individual is a detectable level in a biological sample. This can be measured by methods known to a person skilled in the art and also described herein. The level of expression or amount of biomarker that is estimated can be used to determine the response to treatment.

The expressions "level of expression" or "level expression" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" in general refers to the process by which information (encoded by genes and / or epigenetics) is converted into structures present and operating in the cell. Therefore, as used herein, "expression" can refer to transcription in a polynucleotide, translation in a polypeptide or even polynucleotide and / or polypeptide modifications (eg post-transduction modification of a polypeptide).

Fragments of the transcribed polynucleotide, the translated polypeptide or polynucleotide and / or polypeptide modifications (eg, post-translational modifications of a polypeptide) will also have to be considered as expressed whether they originate from a transcript - - generated by alternating splicing or degraded transcription, or post-production processing of the polypeptide, for example by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide such as mR A and then translated into a polypeptide, and also those that are transcribed into R A but do not translate into a polypeptide (e.g., transfer and ribosomal RNAs).

"High expression", "high expression levels", "high levels" and "overexpressed" refer to an increased expression or increased levels of a biomarker in an individual relative to a control, such as an individual or individuals who do not suffer from the disease or disorder (for example, cancer) ^ or an internal control (for example, maintenance biomarker). In some examples, expression or severe elevated expression is the result of gene amplification.

"Reduced expression", "reduced expression levels" or "reduced levels" refer to diminished levels or decreased expression of a biomarker in an individual relative to a control, such as an individual or individuals who do not suffer from the disease or disorder ( for example, cancer), or an internal control (for example, maintenance biomarker).

The term "maintenance biomarker" refers to a biomarker or group of biomarkers (for - - example, polynucleotides and / or polypeptides) that are typically present in a similar manner in all cell types. In some modalities, the maintenance biomarker is a "maintenance gene". A "maintenance gene" refers herein to a gene or group of genes that encode proteins of which activities are essential for the maintenance of cellular function and which are typically present in a similar manner in all cell types.

"Amplification", as used herein, generally refers to the process of producing multiple copies of a desired sequence. "Multiple copies" means at least two copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations that are introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and / or errors of sequence that occur during amplification. Diploid cells typically contain two copies of a given gene, one on each chromosome. In some aspects of the invention "amplification" or a chromosomal gene and in a cell refers to a process wherein two or more copies of the gene are present in the cell.

- - The term "multiplex PCR" refers to a single PCR reaction that is carried out on nucleic acid that is obtained from a single source (eg, an individual) using more than one set of primers for the purpose of amplifying two or more sequences of DNA in a single reaction.

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (eg, cancer). For example, "diagnosis" can refer to the identification of a particular type of cancer. "Diagnosis" may also refer to the classification of a particular cancer subtype, for example by histopathological criteria, or by molecular characteristics (for example, a subtype characterized by expression of one or a combination of biomarkers (for example, particular genes or proteins). encoded by the genes)).

The term "diagnostic aid" is used herein to refer to methods that assist in making a clinical determination regarding the presence or nature of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method to aid diagnosis of a disease or condition (eg, cancer) may comprise measuring certain biomarkers in a biological sample of an individual.

- - By "correlates" or "correlate" is meant to compare in any way the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol can be used to carry out second protocols and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. With respect to the polynucleotide analysis modality or protocol, the results of a polynucleotide expression assay or protocol can be used to determine whether a specific therapeutic regimen should be performed. "Individual response" or "response" can be estimated using any endpoint that indicates a benefit to the individual, including without limitation, (1) inhibition, to some extent of disease progression (eg, progress of cancer), including braking and complete detention; (2) a reduction in tumor size; (3) inhibition (i.e. reduction, braking or complete stop) of infiltration of cancer cells into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, braking or complete arrest) of metastasis; (5) relief, to some extent from one or more symptoms associated with the disease or disorder (eg, cancer); (6) increase the duration - - of progress-free survival; and / or (9) decrease mortality at a certain point of time after treatment.

The term "prediction" or "forecasting" is used here to refer to the likelihood that a patient will respond either favorably or unfavorably to a particular anticancer therapy. In one modality, the prediction or forecast refers to the extent of those responses. In one embodiment, the prediction or prognosis refers to whether and / or the likelihood of a patient surviving or improving after treatment, for example, treatment with a particular therapeutic agent and for a certain period of time without recurrence of the disease. The predictive methods of the invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools for predicting whether a patient is likely to respond favorably to a treatment regimen, such as a particular therapeutic regimen, including for example administration of a particular agent or combination of therapeutics, surgical intervention, treatment with steroids, etc. or if the patient's long-term survival is likely after a therapeutic regimen.

- - The term "substantially equal" as used herein, denotes a sufficiently high degree of similarity between two numerical values, such as a person skilled in the art will consider the difference between the two values of little or no biological significance and / or statistics within of the context of the biological characteristic measured by the values (for example, values or Ka expression). The difference between the two values, for example is less than about 50%, is less than about 40%, is less than about 30%, is less than about 20%, and / or is less than about 10% as a function of the comparator / reference value.

The phrase "substantially different" as used herein, denotes a sufficiently high degree of difference between two numerical values such that a person skilled in the art considers the difference between the two values of statistical significance within the context of the biological characteristic measured by the values (for example, K¿ values). The difference between the two values for example is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value for the molecule of reference / comparator.

The word "tag" when used here, is - - refers to a detectable compound or composition. The tag is typically conjugated or fused directly or indirectly with a reagent, such as a polynucleotide probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label itself may be detectable (eg, radioisotope labels or fluorescent labels) or in the case of an enzymatic label, it may catalyze chemical alteration of a compound or substrate composition that results in a detectable product.

An "effective amount" of an agent refers to an effective amount, at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

A "therapeutically effective amount" of a substance / molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance / molecule, agonist or antagonist to produce a desired response in the individual. An effective therapeutic amount is also one in which any toxic or noxious effects of the substance / molecule, agonist or antagonist are overcome by beneficial therapeutic effects.

An "effective prophylactic amount" refers to an effective amount at a dose and for periods of time - - necessary to achieve the desired prophylactic result. Typically but not necessary, since a prophylactic dose is used in subjects before or at a previous stage of the disease, the effective prophylactic amount will be less than the effective therapeutic amount. In the case of melanoma, the effective therapeutic amount of the PAK1 inhibitor can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (eg, slow to some extent and preferably stop) tumor metastasis; inhibit or delay, to some extent, tumor growth or tumor progression; and / or alleviating to some extent one or more of the symptoms associated with the disorder. In extension the drug can prevent growth and / or exterminate existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be, for example, measured when assessing survival duration, time to disease progression (TTP), response rates (), response duration and / or quality of life.

"Reduce" or "inhibit" is to decrease or reduce an activity, function and / or quantity compared to a reference. In certain modalities, "reduce" or "inhibit" means the ability to cause a total decrease of 20% or greater. In another modality, by "reduce" or "inhibit" - - the ability to cause a total decrease of 50% or greater is understood. Still in another modality, "reduce" or "inhibit" means the ability to cause a total reduction of 75%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor or the size or number of blood vessels in angiogenic disorders.

The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to a subject to which the formulation is to be administered. These formulations can be sterile.

A "sterile" formulation is aseptic or free of all living organisms and their spores.

A "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation other than an active ingredient that is not toxic to a subject. An acceptable pharmaceutical carrier includes but is not limited to a buffer, excipient, stabilizer or preservative.

As used herein, "treatment" is an approach to obtain desired beneficial or clinical outcomes. For - - purposes of this invention, beneficial or desired clinical outcomes include but are not limited to any one or more of: alleviation of one or more symptoms, decrease in the extent of the disease, avoidance or delay of spread (e.g., metastasis, e.g. metastasis to the lung or the lymphatic node) of the disease, prevent or delay recurrence of the disease, retard or slow down the progress of the disease, improve the state of the disease and remission (either partial or total). Also encompassed by "treatment" is a reduction in the pathological consequence of a proliferative disease. The methods of the invention contemplate any of one or more of these treatment aspects.

The term "melanoma" refers to a high-malignancy tumor that starts in normal skin or moles of melanocytes and undergoes rapid and extensive metastasis. The term "melanoma" can be used intangeably with the terms "malignant melanoma", "melanocarcinoma", "melanoepithelioma" and "melanosarcoma".

"Tumor", as used herein refers to all growth and proliferation of neoplastic cells, either malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive as - - it refers here.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of these cancers include but are limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung. lung, peritoneal cancer, hepatocellular cancer, gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, carcinoma of superficial dissemination, lentigo maligna melanoma, m lentiginous acral elanomas, nodular melanomas, multiple myeloma, and B-cell lymphoma (including low-grade non-Hodgkin's lymphoma) - - grade / follicular, (NHL); Small lymphocytic NHL (SL); Follicular NHL / intermediate grade; Diffuse NHL of intermediate grade; High grade immunoblastic NHL; High grade lymphoblastic NHL; NHL of high-grade small non-dissociated cells; NHL of bulky disease; mantle cell lymphoid; lymphoma related to AIDS; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain, as well as head and neck cancer, and associated metastases. In certain embodiments, cancers that are susceptible to treatment by the antibodies of the invention include mum cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell cancer, cancer of the prostate, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma. In some embodiments, the cancer is chosen from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Without - - However, in some modalities, cancer is chosen from non-small cell lung cancer, colorectal cancer, glioblastoma, and mamma carcinoma, including metastatic forms of these cancers.

The term "anticancer therapy" refers to a therapy useful for treating cancer. Examples of anticancer therapeutics include but are not limited to chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents and other agents to treat cancer, anti-cancer antibodies. CD20, platelet-derived growth factor inhibitors (e.g., Gleevec ™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-beta, BlyS, APRIL, BCMA receptor (s), TRAlL / Apo2, and other bioactive and organic chemical agents, etc. Their combinations are also included in the invention.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The expression is intended to include radioactive isotopes (eg, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and isotopes - - radioactive), chemotherapeutic agents for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitoraicin C, chlorarabucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes , antibiotics and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or their variants and the various anti-tumor or anti-cancer agents described below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

A "toxin" is any substance capable of having a deleterious effect on the growth or proliferation of a cell.

A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXA ®); alkyl sulfonates such as busulfan, improsulphan and piposulfane; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylene-isophosphoramide and trimethylmelamine; acetogenins (especially bulatacin and bulatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachona; lapachol; Colchicines; betulinic acid; a camptothecin (including the synthetic analog topotecan (HYGAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetyl caraptothecin, scopolectin and 9-aminocamptothecin); Bryostatin; Calistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediin antibiotics (for example, calicheamicin, especially gammall calicheamicin and omegall calicheamicin (see, for example, Nicolaou et al., Angew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor, dinemycin, including dynemycin A, a esperamycin, as well as neocarzinostatin chromophore and chromophores antibiotics of chromoprotein enediin - related), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin , cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, injection of liposomes HC1 doxorubicin (DOXIL®), doxorubicin liposomal TLC D-99 (MIOCET®), doxorubicin liposoraal pegylated (CAELIX®), and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, micofebolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, chelamicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as - - aminoglutetimide, mitotane, trilostane; folic acid replenisher such as frolic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabuchil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornitin; eliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofirano; spirogermanium; tenuazonic acid; triaziquone; 2, 2 ', 2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); Dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosina; arabinoside ("Ara-C"); thiotepa; taxoid, for example, paclitaxel (TAXOL®), paclitaxel albumin engineered nanoparticle formulation (ABRAXANETM), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (for example, ELOXATIN®), and carboplatin; vincas, which prevent polymerization of tubulin that form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and - - vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; Daunomycin; aminopterin; ibandronate; Topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®) ), or risedronate (ACTONEL®); troxacitabine (a 1, 3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in proliferation of aberrant cells, such as for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; Topoisomerase 1 inhibitor (for example, LURTOTECA ®); rmRH (for example, ABARELIX®); BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SÜTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as sodium oblimers (GENASENSE®); pixantrone; EGFR inhibitors (see definition a - - continuation); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR ™); and pharmaceutically acceptable salts, acids or derivatives, of any of the foregoing; as well as combinations of two or more of the foregoing such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth. They may be the hormones themselves, including but not limited to: anti-estrogens with agonist / antagonist misto profile, including, tamoxifen (NOLVADEX®), 4-hydroxy tamoxifen, toremifen (FARESTON®), idoxifen, droloxifen, raloxifen (EVISTA®), trioxifen, keoxifen, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (these agents can block the dimerization of estrogen receptor (ER), inhibit DNA binding, - - increase turnover and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formmene and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors including vorozole (RIVISOR ®), megestrol acetate (MEGASE®), fadrozole and (5) -imidazoles; luteinizing hormone-releasing hormone agonist, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin and tripterelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all of transretionic acid and fenretinide; onapristone; anti-progesterone; regulators by decrement of estrogen receptor (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and acceptable pharmaceutical salts, acids or derivatives of any of the foregoing; as well as combinations of two or more of the above.

The term "prodrug" as used in this application refers to a precursor or derivative form of an active pharmaceutical substance that is less cytotoxic to tumor cells compared to the precursor drug and is capable of being enzymatically activated or converted to the most active precursor form. See, e.g. , Wilman, "Prodrugs in - - Cancer Chemotherapy "Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al.," Prodrugs: A Chemical Approach to Targeted Drug Delivery, "Directed Drug Delivery, Borchardt et al., (Ed. .), pp. 247-267, Humana Press (1985) Prodrugs of this invention include but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, prodrugs modified with D-amino acid, glycosylated prodrugs, prodrugs containing β-lactam, prodrugs containing optionally constituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, prodrugs of 5-fluorocytosine and others of 5-fluorouridine which can be converted into the most active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized in a prodrug form for use in this invention include but are not they are limited to those chemotherapeutic agents described above.

A "growth inhibitory agent" when used herein refers to a compound or composition that inhibits growth of a cell (e.g., a melanoma cell). Examples of growth inhibitory agents include agents that block the progress of the cell cycle (at a site other than the S phase), such as agents that - - induce Gl brake and phase M brake. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Those agents that slow Gl also spill over the S phase brake, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. The taxanes (paclitaxel and docetaxel) are anti-cancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules of tubulin dimers and stabilize microtubules by avoiding depolymerization, which results in the inhibition of mitosis in cells.

By "radiation therapy" it means the use of gamma rays or directed beta rays to induce enough damage to a cell to limit its ability to function normally or to completely destroy the cell. HE - - will appreciate that there will be many ways known in the art to determine the dose and duration of treatment. Typical treatments are given as a single administration and typical dose ranges are from 10 to 200 units (Grays) per day.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits and rodents (e.g. rats). In certain modalities, the individual or subject is a human.

Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

The term "concurrently" is used herein to refer to administration of two or more therapeutic agents, wherein at least part of the administration overlaps over time. Accordingly, concurrent administration includes a dose regimen when the administration of one or more agents continues after stopping the administration of one or more other agents.

By "reduce or inhibit" is meant the ability to cause a total decrease of 20%, 30%, 40%, 50%, - - 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

The term "package insert" is used to refer to instructions usually included in commercial packages of therapeutic products, which contain information regarding indications, use, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of these therapeutic products. In some embodiments, the package insert of the invention comprises instructions for treating melanoma with a PAK1 inhibitor.

An "article or manufacture" is any manufacture (e.g., packaging or container) or equipment that comprises at least one reagent, e.g., a medicament for the treatment of a disease or disorder (e.g., cancer), or a probe for specifically detect a biomarker described herein. In certain modalities, the manufacture or equipment is promoted, distributed or sold as a unit to perform the methods described herein.

A "target audience" is a group of people or an institution to whom or with whom a particular medication is promoted or intended to be promoted, such as by marketing or advertising, especially for uses, - - treatments or specific indications, such as individuals, populations, newspaper readers, medical literature, and magazines, television or internet viewers, radio or internet listeners, doctors, pharmaceutical companies, etc.

As understood by a person skilled in the art, reference to "about" a value or parameter herein includes (and describes) modalities directed to a value or parameter per se. For example, description referring to "approximately X" includes the description of "X".

It is understood that aspects and embodiments of the invention described herein include "consistent" and / or consist essentially of "aspects and modalities". As used herein, the singular form "a", "an", and "the" includes plural references unless otherwise indicated.

An "individual", "subject" or "patient" is a vertebrate. In certain modalities, the vertebrate is a mammal. Mammals include but are not limited to farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats. In certain modalities, a mammal is a human.

The term "sample" or "test sample" as used herein, refers to a composition that is obtained or - - derives from a subject of interest that contains a cellular and / or other molecular entity that will be characterized and / or identified for example based on physical, biochemical, chemical and / or physiological characteristics. For example, the phrase "sample of the disease" and its variations refers to any sample obtained from a subject of interest that will be expected or known to contain the cellular and / or molecular entity to be characterized. In one embodiment, the definition encompasses blood and other liquid samples of biological origin and tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue such as from biopsy sample or fresh, frozen and / or preserved organ or tissue aspirate; blood or blood constituents; body fluids; and cells of any time of gestation or development of the subject or plasma. Samples include but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, blood whole, cells derived from blood, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenate tissue, tumor tissue, cell extracts, Y - - your combinations The term "sample" or "test sample" includes biological samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes. For the present purposes a "section" of a tissue sample is intended as a single part or piece of a tissue sample, for example a thin slice of tissue or cells that are cut from a tissue sample. In one modality, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some modalities, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissues or fluids that are known or considered to contain the tumor cells of interest; for example skin samples.

By "tissue sample" or "cell sample" is meant a collection of similar cells that are obtained from a tissue of a subject or individual. The source of the tissue or cell sample can be such a solid tissue - - as an organ, tissue sample, biopsy and / or fresh aspirate, frozen and / or preserved; blood or any blood constituents such as plasma; body fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells of any time of gestation or development of the subject. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the cell tissue sample is obtained from a diseased tissue / organ. The tissue sample may contain compounds that do not intermix naturally with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

A "reference sample", "reference cell", "reference tissue", "control sample", "control cell", or "control tissue", as used herein, refers to a cell sample of tissue, standard or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cells, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, healthy and / or non-diseased cells or tissues adjacent to diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In - - Some modalities, the reference sample are non-cancerous skin cells. In another embodiment, a reference sample is obtained from a tissue and / or untreated cell of the body of the same subject or individual. In some embodiments, the reference sample is non-cancerous skin cells from the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissues or cells) of an individual that is not the subject or individual. In some embodiments, the reference sample is non-cancerous skin cells from an individual that is not the subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or body cell of an individual that is not the subject or individual.

In certain embodiments, a reference sample is a single sample or multiple combined samples of the same subject or patient that are obtained at one or more points at different times than when the test sample is obtained. For example, a reference sample is obtained at a previous point in time from the same subject or patient as when the test sample is obtained. This reference sample It may be useful if the reference sample is obtained during the initial diagnosis of cancer and the test sample is subsequently obtained when the cancer becomes metastatic.

In certain embodiments, a reference sample includes all types of biological samples as defined above under the term "sample" that is obtained from one or more individuals that are not the subject or patient. In certain embodiments, a reference sample is obtained from one or more individuals with an angiogenic disorder (e.g., cancer) that is not the subject or patient.

In certain embodiments, a reference sample is multiple combined samples of one or more healthy individuals that are not the subject or patient. In certain embodiments, a reference sample is multiple pooled samples of one or more individuals with a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) that are not the subject or patient. In certain embodiments, a reference sample is accumulated RNA samples from normal tissues or plasma or serum samples accumulated from one or more individuals that are not the subject or patient. In certain embodiments, a reference sample is accumulated RNA samples from tumor or plasma tissues or serum samples accumulated from one or more individuals with a disease or disorder (eg, a - - angiogenic disorder such as cancer) that are not the subject or patient.

For present purposes, a "section" of a tissue sample means a single part or piece of a tissue sample, eg, a thin slice of tissue or cells that are cut from a tissue sample. It is understood that multiple sections of tissue samples can be taken and subjected to analysis, provided that it is understood that the same section of tissue sample can be analyzed at both morphological and molecular levels, or analyzed with respect to both polypeptides and polynucleotides.

Levels / amount of expression of a gene or biomarker can be determined quantitatively and / or quantitatively based on any convenient criteria known in the art, including but not limited to mR A, cDNA, proteins, protein fragments and / or copy number of genes. In certain embodiments, expression / amount of a gene or biomarker in a first sample is increased compared to the expression / amount in a second sample. In certain embodiments, expression / amount of a gene or biomarker in a first sample is decreased compared to expression / amount in a second sample. In certain modalities, the second sample is a reference sample.

In certain modalities, the terms "increase" - - or "over-expressing" refers to a total increase of approximately either 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or greater, at the level of protein or nucleic acid, detected by methods known in the standard technique such as those described herein, compared to a reference sample. In certain modalities, the terms "increase" or "over-express" refers to the increase in the level / amount of expression of a gene or biomarker in the sample where the increase is at least approximately either 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, ???, 25x, 50x, 75x, or lOOx the level / amount of expression of the respective gene or biomarker in the reference sample.

In certain embodiments, the term "decrease" here refers to a total reduction of approximately either 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, at the level of protein or nucleic acid, detected by methods known in the standard technique such as those described herein, compared to a reference sample. In certain embodiments, the term "diminish" refers to the decrease in level / amount of expression of a gene or biomarker in the sample wherein the decrease is at least approximately either 0.9x, 0.8x, 0.7x, 0.6x, 0.5x , 0.4x, 0.3x, 0.2x, O.lx, 0.05x, or O.Olx the level / amount of expression of the gene or - - respective biomarker in the reference sample.

"Detection" includes any means to detect, including direct and indirect detection.

In certain modalities, to "correlate" or "correlate" means to compare, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol can be used to carry out a second protocol and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. With respect to the modality of the analysis or gene expression protocol, the results of the analysis or gene expression protocol can be used to determine if a specific therapeutic regimen should be performed.

The word "tag" when used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent such as a nucleic acid probe or an antibody and facilitates the detection of the reagent to which it is conjugated or fused. The label itself may be detectable (eg, radioisotope labels or fluorescent labels) or in the case of an enzymatic label, it may catalyze chemical alteration of a substrate compound or composition that is detectable.

- - The term "polypeptide" refers to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeled component. Also included within the definition for example, polypeptides containing one or more analogues of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. The term "polypeptide" as used herein specifically embraces a "protein." The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid polypeptide. An isolated nucleic acid molecule is different in shape or scope and in that it is found in nature. Isolated nucleic acid molecules therefore differ from the nucleic acid molecule as it exists in - - natural cells However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide, wherein for example the nucleic acid molecule is in a chromosomal location different from natural cells.

A "gene", "target gene", "target biomarker", "target sequence", "target nucleic acid" or "target protein", as used herein, is a polynucleotide or protein of interest, the detection of which is desired . In general, a "template", as used herein, is a polynucleotide that contains the target nucleotide sequence. In some cases, the expressions "target sequence", "template DNA", "template polynucleotide", "target nucleic acid", "target polynucleotide" and their variations are used interchangeably.

A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature. In this way, a polypeptide of native sequence can have the polypeptide amino acid sequence of natural origin of any mammal. This polypeptide of native sequence can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses truncated or - - secreted from the natural origin of the polypeptide (e.g., an extracellular domain sequence), variant forms of natural origin (e.g., alternate splice forms) and naturally occurring allelic variants of the polypeptide.

An "isolated" polypeptide or "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Pollutant components of its natural environment are materials that will interfere with diagnostic or therapeutic uses for the polypeptide and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the polypeptide will be purified (1) to more than 95% by weight of polypeptide as determined by the Lowry method, or more than 99% by weight, (2) to a sufficient degree to obtain at least 15 residues of internal or N-terminal amino acid sequence by the use of a centrifuge cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or silver staining. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the natural environment of the polypeptide will not be present. In ordinary form, however, isolated polypeptide will be prepared by at least one purification step.

A "variant" polypeptide means a polypeptide - - biologically active having at least about 80% amino acid sequence identity with the native sequence polypeptide. These variants include for example polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. Ordinarily, a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the sequence polypeptide native The term "benefit" is used in the broadest sense and refers to any desirable effect and specifically includes clinical benefit as defined herein.

Clinical benefit can be measured by estimating various extreme points, for example inhibition, to some extent, of disease progression, including braking and complete arrest; reduction in the number of episodes and / or symptoms of the disease; reduction in injury size; inhibition (i.e. reduction, braking or complete arrest) of infiltration of diseased cells into adjacent peripheral organs and / or tissues; inhibition (ie reduction, braking or complete arrest) of the spread of disease; decreased auto-immune response, which may but does not have to result in regression or ablation of the - - sick injury; relief, to some extent from one or more symptoms associated with the disorder; increase in the length of the disease-free presentation after treatment, for example, progress-free survival; increased total survival; superior response speed; and / or decreased mortality at a certain point of time after treatment.

Methods of the invention The present invention provides methods for treating melanoma in an individual, which comprises contacting the melanoma with a therapeutically effective amount of a PAKI inhibitor. In some embodiments, the method comprises administering to the individual an effective therapeutic amount of a PAKI inhibitor.

Melanoma is a malignant tumor of melanocytes, for example, cells that produce melanin, a dark pigment that is responsible for the color of the skin. Melanomas predominantly occur in the skin, but are also found in other parts of the body, including the intestines and the eye, for example, uveal melanoma). Melanoma can originate in any part of the body that contains melanocytes.

Examples of melanoma include, but are not limited to, surface dispersion melanoma, nodular melanoma, malignant melanoma, and Acral melanoma. - - lentiginous Melanomas can be classified depending on a number of criteria including size, ulceration, spread to lymph nodes, and / or spread to other tissues or organs. In some embodiments, the invention provides methods for treating a Stage I melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the invention provides methods for treating a Stage II melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the invention provides methods for treating Stage III melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the invention provides methods for treating Stage IV melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the invention provides methods for treating metastatic melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the invention provides methods for treating recurrent melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the method comprises administering to the individual an effective therapeutic amount of the PAK1 inhibitor. In some embodiments, the PAK1 inhibitor is a small molecule inhibitor of PAK1. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

In some aspects, the invention provides methods for treating melanoma in an individual, wherein the melanoma is a wild type BRAF melanoma. In some embodiments, the invention provides methods for treating wild type BRAF melanoma comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor. In some embodiments, the invention provides methods for treating wild type BRAF melanoma comprising administering to the individual an effective therapeutic amount of a PAK1 inhibitor. BRAF is a member of the Raf kinase family of protein kinases specific for serine / threonine. BRAF plays a role in regulating the MAP kinase signaling pathway / ERKs (the RAF-MEK-ERK route), which affects cell division, differentiation and secretion. Signaling RAF-MEK-ERK is frequently deregulated in cancer. More than 30 mutations of the BRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers of more than 80% in melanomas, to as little as 0-18% in other tumors, such as 1-3% in lung cancers and 5% in colorectal cancer. A common mutation found in cancers, particularly melanoma, is a substitution of valine at codon 600 with glutamate (i.e., V600E). For example, a thymine is substituted with adenine in the nucleotide 1799 that leads to the V600E mutation.

BRAF V600 mutations lead to constitutive kinase BRAF activity. Methods for determining the BRAF genotype in a melanoma are known to those skilled in the art; for example, the nucleotide sequence of the melanoma BRAF gene can be determined using standard sequencing methods or by using the KASP SNP genotyping system (KBioscience). In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the melanoma is a wild type BRAF melanoma. In some embodiments, the invention provides methods for treating melanoma in an individual wherein the melanoma is a wild type BRAF melanoma and the melanoma overexpresses PAK1 compared to non-cancerous cells. In some embodiments, the invention provides methods for treating melanoma in an individual wherein the melanoma comprises wild type BRAF and PAK1 is amplified in the melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAKl is overexpressed in melanoma compared to non-cancerous cells and PAKl is amplified in melanoma. In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the melanoma is a mutant BRAF melanoma.

In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the Melanoma is a melanoma BRAF mutant and melanoma overexpresses PAKl compared to non-cancerous cells. In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the melanoma comprises a mutant BRAF and PAK1 is amplified in the melanoma. In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the melanoma comprises a mutant BRAF wherein the mutant is not a BRAF V600E mutant. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

In some aspects, the invention provides methods of treating melanoma in an individual by contacting the melanoma with an effective therapeutic amount of PAKI inhibitor. In some aspects, the invention provides methods of treating melanoma in an individual by administering to the individual an effective therapeutic amount of the PAKI inhibitor. PAKs participate in a number of routes that are commonly deregulated in human cancer cells. PAKl is a component of mitogen-activated protein kinase (MAPK), N-terminal JUN kinase (J K), steroid hormone receptor, and nuclear factor signaling (NF) pathways, all of which have been associated with oncogenesis. PAKs activate MEK and RAF1 by phosphorylating them in serine 298 and serine 338, respectively.

- - The increase in Ras-induced transformation by PAK1 correlated with its signaling effects through the kinase MAPK pathway regulated by extracellular signal (ERK), and was dissociable from effects on the JNK or p38-MAPK pathways. (R. Kumar et al., Nature Rev. Cancer 2006 6: 459). Constitutive activation of the ERK / MEK pathway is involved in the formation, progress and survival of tumors and is also associated with an aggressive phenotype, characterized by uncontrolled proliferation, loss of control of apoptosis and poor prognosis (JA Spicer, Expert Opin. Drug Discov 2008 3: 7).

Tumor formation and progress require the inactivation of pro-apoptotic signals in cancer cells. PAK activity has been shown to downregulate several important pro-apoptotic pathways. PAK1 phosphorylation of RAF1 induces RAF1 translocation of mitochondria, where they phosphorylate the BCL2 antagonist of pro-apoptotic cell death protein (BAD). PAK1, PAK2, PAK4 and PAK5 have also been reported to directly phosphorylate and inactivate BAD in select cell types such as CV-1 (simian) of origin and transport kidney cells SV40 (COS) from Chinese hamster ovaries (CHO) ) and human embryonic kidney (HEK) 293T (R. Kumar et al., ibid). However, the relevant pathways downstream of PAK1 in human tumor cells remain only partially - - understood.

PAK1 is widely expressed in a variety of normal tissues; however, expression is significantly increased in ovarian, breast and bladder cancer. (S. Balasenthil et al., J. Biol. Chem. 2004 279: 4743; M.Ito et al., J. Urol. 2007 178: 1073; P. Schraml et al., Am. J. Pathol. : 985). In luminal breast cancer, genomic amplification of PAK1 is associated with resistance to tamoxifen therapy, possibly occurring as a result of direct phosphorylation and independent transactivation of estrogen receptor ligand by PAK1 (SK Rayala et al., Cancer Res. 2006. 66: 1694-1701).

In some aspects, the invention provides methods for treating melanoma in an individual by contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor. In some aspects, the invention provides methods for treating melanoma in an individual by administering to the individual an effective therapeutic amount of a PAK1 inhibitor. In some embodiments, the PAK1 gene is amplified in melanoma. In some embodiments, the copy number of PAK1 in the melanoma is approximately either 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or greater than 5.0. Methods for determining the copy number of the PAK1 gene in a melanoma are known in the art. For example, the copy number of the PAK1 gene can be determined by using matrices - - SNP such as Affymetrix 500K SNP array analysis. In some embodiments, the invention provides methods for treating melanoma in an individual, wherein the copy number of PAK1 in the melanoma is greater than approximately 2.5. In some embodiments, the invention provides methods for determining the copy number of PAKI in a melanoma subsequent to treatment with a PAKI inhibitor. In some embodiments, the copy number of PAK1 in a melanoma is compared to the copy number of PAK1 in non-cancerous cells; for example, non-cancerous skin cells. In some modalities, PAKl is amplified in melanoma and melanoma overexpressed PAKl. In some modalities, PAKl is amplified in melanoma and melanoma is a wild type BRAF melanoma. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

In some aspects, the invention provides methods for treating melanoma in an individual by contacting the melanoma with an effective therapeutic amount of a PAKI inhibitor wherein PAK1 is overexpressed in the melanoma. In some aspects, the invention provides methods for treating melanoma in an individual by administering to the individual an effective therapeutic amount of a PAKI inhibitor wherein PAK1 is overexpressed in the melanoma. Methods for determining expression of PAKl are known in the art. Examples of methods to determine levels of - - PAKI expression in a melanoma include but are not limited to immunohistochemistry, reverse phase protein matrix (RPPA), quantitative PCR, immunoassays and the like. Levels of PAKl expression can be compared with other tumors and cells by using the Gene Expression Omnibus (GEO) database.

In some embodiments, the invention provides methods for treating melanoma in an individual by contacting melanoma with a PAKI inhibitor wherein PAK1 is overexpressed in the melanoma compared to non-cancerous cells. In some aspects, the invention provides methods for treating melanoma in an individual, by administering to the individual an effective therapeutic amount of a PAKI inhibitor wherein PAK1 is overexpressed in the melanoma. In some embodiments, expression of PAK1 in melanoma is approximately either 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% expression in non-cancerous cells. In some embodiments, the expression of PAKl in melanoma is approximately either 1.5-fold, 2.0-fold, 2.5-fold, 3, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0 -Times, 8.0-times, 9.0-times, 10-times or greater than 10-times compared to PAKI expression in non-cancerous cells. In some modalities, melanoma overexpresses PAKl compared to non-cancerous cells and melanoma is a BRAF melanoma of type - - wild. In some modalities, melanoma overexpresses PAK1 compared to non-cancerous cells and PAK1 is amplified in melanoma. In some embodiments, melanoma overexpresses PAK1 compared to non-cancerous cells and melanoma is a wild type BRAF melanoma and PAK1 is amplified in melanoma. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

In some aspects, the invention provides methods for inhibiting CRAF signaling in a melanoma in an individual comprising contacting the melanoma with an effective therapeutic amount of a PAKI inhibitor. In some aspects, the invention provides methods for inhibiting CRAF signaling in a melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAKI inhibitor. Methods for measuring CRAF signaling are known in the art. For example, CRAF activation can be determined by immunoblotting CRAF isolated from a melanoma of an individual before and / or after treatment with a PAKI inhibitor. Activation of CRAF can be measured using phospho-CRAF antibodies (Ser338). In some embodiments, melanoma is a wild-type BRAF melanoma. In some modalities, PAKl is overexpressed in melanoma. In some modalities, melanoma is a wild type BRAF melanoma where PAKl - - it is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is overexpressed in melanoma and PAK1 is amplified in melanoma. In some modalities, PAK1 is overexpressed in melanoma and PAK1 is amplified in melanoma. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

In some aspects, the invention provides methods for inhibiting MEK signaling in a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor. In some aspects, the invention provides methods for inhibiting MEK signaling in a melanoma in an individual, comprising administering to the individual an effective therapeutic amount of a PAK1 inhibitor. Methods for measuring MEK signaling are known in the art. For example, MEK activation can be determined by immunoblotting of MEK isolated from a melanoma of an individual before and / or after treatment with a PAK1 inhibitor. The activation of MEK can be measured using phospho-MEK1 / 1 antibodies (Ser217 / Ser221). In some embodiments, melanoma is a wild-type BRAF melanoma. In some modalities, PAK1 is overexpressed in melanoma. In some modalities, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, the individual is a mammal. In some modalities, the individual is a human.

PAKI inhibitors Here PAKI inhibitors (e.g., PAKl antagonists) useful in the methods described herein are provided. In some embodiments, the PAKI inhibitor is a small molecule, a nucleic acid, a polypeptide or an antibody. Examples of PAK inhibitors are provided in WO 2007/072153, and WO 2010/07184 both of which are incorporated herein by reference.

Small molecules Here small molecules are provided for use as PAKI inhibitors for the treatment of melanoma.

Small molecules preferably are organic molecules other than polypeptides or binding antibodies as defined herein that bind to PAKI or interfere with - - PAK1 signage as described here. Small organic linker molecules can be identified and synthesized chemically using known methodology (see, for example, PCT Publication Numbers WO 00/00823 and WO 00/39585). Small organic linker molecules are usually less than about 2000 daltons in size, alternately less than about 1500, 750, 500, 250 or 200 daltons in size, wherein these small organic molecules are capable of binding specifically to a specific polypeptide as described herein, can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening libraries of small organic molecules for molecules that are capable of binding to a polypeptide target are well known in the art (see, for example, PCT Publication Numbers WO 00/00823 and WO 00 / 39585). Small organic linking molecules can be for example aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatics, heterocyclic compounds, anilines, - - alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides or the like.

Inhibitors of small molecules of PAK have been described (see O2006072831, WO2007023382, WO2007072153, WO2010 / 071846, US20090275570). (i) (II) A series of selective PAK1 inhibitors made on scaffold 2-aminopyrido [2,3-d] pyrimidin-7 (8H) -one (I) have been described by Afraxis, Inc., in a series of patent applications (WO2009086204, WO2010071846, WO2011044535, WO2011156646, WO2011156786, WO2011156640, WO2011156780, WO2011156775, WO2011044264).

AstraZeneca has described bicyclic heterocyclic PAK1 inhibitors of the formula II (see WO2006106326).

Pfizer has described PAK inhibitors made in 1H-thieno [3, 2-c] pyrazole (III), 3-amino-tetrahydropyrrolo [3, -c] pyrazole (IV) and N4- (lH-pyrazol-3-yl) pyrimidine -2,4-diamine (V) (see WO 2004007504, WO 2007023382, WO2007072153, and WO2006072831).

PF-3758309 (VI) is a potent ATP-competitive inhibitor of PAK1, 4, 5 and 6 that has been used in clinical trials. (BW Murray et al., Proc. Nati. Acad. Sci USA 2010 107 (20): 9446; Rosen L et al., Phase 1, dose escalation, safety, pharmacokinetic and pharmacodynamic study of single agent PF-03758309, oral PAK inhibitor, in patients with advanced solid tumors [abstract] .In: Proceedings of te AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2011 Nov 12-16; San Francisco, CA Philadelphia (PA): AACR; Cancer Ter 2011; 10 (11 - - Suppl): Abstract nr A177.

A series of indolyl, indazolyl and benzimidazolyl N2-bicyclic derivatives of N4- (lH-pyrazol-3-yl) pyrimidine-2,4-diamines (VII) (U.S. Patent Application Serial Number: 61 / 527,453 filed in 08/25/2011) and its derivatives aza-indolyl, indazolyl and benzimidazolyl (US Patent Application Serial Number 61 / 579,227, filed on 12/22/2011) have been described and this reference is incorporated by reference in its entirety (A = indolyl, indazolyl and benzimidazolyl or aza derivatives thereof).

Nucleic acids The invention provides here PAKI polynucleotide antagonists for the treatment of melanoma in an individual. The polynucleotide can be RNAi such as siRNA or miRNA, an antisense oligonucleotide, RNAzymes, DNAzymes, oligonucleotides, nucleotides or any fragments thereof, including DNA or RNA (eg, mRNA, rRNA, tRNA) of genomic or synthetic origin, which can be of one - - strand or double strand and can represent a sense strand or antisense nucleic acid peptide (PNA), or to any DNA or RNA type material, of natural or synthetic origin, including, for example, iRNA, ribonucleoproteins (e.g., iRNPs). In some embodiments, the polynucleotide targets PAK1 expression (e.g., targets PAK1 mRNA).

The polynucleotide can be an antisense and / or ribosomal nucleic acid. The antisense nucleic acids comprise a sequence complementary to at least a portion of an RNA transcript of PAK1. However, absolute complementarity is not required, although it is preferred.

A sequence "complementary to at least a portion of an RNA" referred to herein means a sequence that has sufficient complementarity to be able to hybridize with RNA, forming a stable duplex; in the case of double-stranded PAK1 antisense nucleic acids, a single strand of the duplex DNA can thus be tested or assayed by triplex formation. The ability to hybridize will depend both on the degree of complementarity and the length of the antisense nucleic acid.

In general, the higher the hybridizing nucleic acid, the more mismatched base with PAK1 RNA may contain and still form a stable duplex (or triplex as the case may be). A person with skill in the art - - can assess a tolerable degree of mismatching by the use of standard procedures to determine the melting point of the hybridized complex.

Polynucleotides that are complementary to the 5 'end of the message, for example the 5' untranslated sequence up to and including the AUG start codon, should work more efficiently to inhibit translation. However, sequences complementary to the 3 'sequences without translation of mRNAs have been shown to be effective in inhibiting translation of mRNAs equally. See generally, Wagner, R., 1994, Wature 372: 333-335. In this way, oligonucleotides complementary to any of the untranslated 5 'or 3' untranslated regions of the PA 1 gene can be employed in an antisense approach to inhibit translation of endogenous PAK1 mRNA. Polynucleotides complementary to the untranslated region of 51 mRNA should include the complement of the AUG start codon. Antisense polynucleotides complementary to mRNA coding regions are less efficient translation inhibitors but may be employed according to the invention. Whether designed to hybridize the 5'-, 3'- or coding region of PAKl mRNA, antisense nucleic acids should be at least six nucleotides in length and preferably are oligonucleotides in the range of 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide has at least 10 - - nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

In one embodiment, the PAK1 antisense nucleic acid of the invention is produced intracellularly by transcription of an exogenous sequence. For example, a vector or its portion is transcribed, producing an antisense nucleic acid (RNA) of the PAK1 gene. This vector will contain a sequence encoding the PAK1 antisense nucleic acid. This vector can remain episomal or be chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. These vectors can be constructed by standard recombinant DNA technology methods in the art. Vectors can be plasmid, viral or others known in the art, used for replication and expression in vertebrate cells. The sequence expression encoding PAK1, or its fragments can be by any promoter known in the art to act on vertebrate cells preferably of humans. These promoters can be inducible or constitutive. These promoters include but are not limited to the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310 (1981), the promoter contained in the long 3 'terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22 : 787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Nati. Acad. Sci. USA - - 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296: 39-42 (1982)), etc.

Small inhibitory RNAs (siR As) may also function as PAK1 inhibitors for use in the treatment of melanoma. The expression PAK1 can be by contacting the melanoma with a small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, such that the expression of PAK1 is specifically inhibited. Methods for selecting a dsRNA or vector encoding appropriate dsRNA are well known in the art (Tuschi, T et al (1999) Genes Dev. 13 (24) .3191-3197; Elbashir, SM et al., (2001) Nature 411 : 494-498; Hannon, GF (2002) Nature 418: 244-251; McManus MT and Sharp, PA (2002) Nature Reviews Genetics 3: 737-747; Bremmelkamp, TR et al. (2002) Science 296: 550- 553, U.S. Patent Nos. 6,573,099 and 6,506,559 and International Patent Publications WOOl / 36646, WO 99/32619 and WO 01/68836.

Examples of PAK1 siRNA oligonucleotide sequences include but are not limited to 1) GAAGAGAGGTTCAGCTAAA, 2) GGAGAAATTACGAAGCATA, 3) ACCCAAACATTGTGAATTA, 4) GGTTTATGATTAAGGGTTT, all obtained from Dharmacon, Inc.

Polypeptides The invention provides polypeptide inhibitors of - - PAKl activity for the treatment of melanoma in an individual. For example, binding polypeptides are polypeptides that bind preferentially and specifically to PAK1 as described herein. In some embodiments, the linker polypeptides are PAKl antagonists. Linker polypeptides can be chemically synthesized using known polypeptide synthesis methodology or can be prepared and purified using recombinant technology. Linker polypeptides are usually at least about 5 amino acids in length, in alternating form at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, or 100 amino acids in length or more, wherein these binding polypeptides are capable of specifically binding to a PAKI, as described herein. Linker polypeptides can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening polypeptide libraries to bind polypeptides that are capable of specifically binding to a target polypeptide are well known in the art.

- - (See, for example, U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143, PCT Publication Numbers WO 84/03506 and W 084/03564, Geysen et al., Proc. Nati. Acad Sci. USA, 81: 3998-4002 (1984), Geysen et al., Proc. Nati, Acad. Sci. USA, 82: 178-182 (1985), Geysen et al., In Synthetic Peptides as Antigens, 130-149 (1986), Geysen et al., J. Immunol., Meth., 102: 259-274 (1987), Schoofs et al., J. Immunol., 140: 611-616 (1988), Cwirla, SE. et al. (1990) Proc. Nati, Acad. Sci. USA, 87: 6378; Lowman, HB et al. (1991) Biochemistry, 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Nati. Acad. Sci. USA, 88: 8363, and Smith, GP ( 1991) Current Opin, Biotechnol., 2: 668).

In this regard, expression of bacteriophage (phage) is a well-known technique that allows to screen large polypeptide libraries to identify the member or members of these libraries that are capable of binding specifically to a target PAK1. Phage expression is a technique by which variant polypeptides are displayed as fusion proteins to the coating protein on the surface of bacteriophage particles (Scott, J.K. and Smith, G.P. (1990) Science, 249: 386). The utility of phage display lies in the fact that large libraries of protein variants - - Selectively randomized (or randomly cloned cDNAs) can be classified quickly and efficiently by those sequences that bind to a target molecule with high affinity. Expression of peptide libraries (Cwirla, SE et al (1990) Proc. Nati, Acad. Sci. USA, 87: 6378) or protein (Lowman, HB et al. (1991) Biochemistry, 30: 10832; Clackson, T et al. (1991) Nature, 352: 624; Marks, J. D et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Nati. Acad. Sci. USA, 88: 8363) in phage have been used to screen millions of polypeptides or oligopeptides by those with specific binding properties (Smith, GP (1991) Current Opin, Biotechnol., 2: 668). Classifying phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a method for affinity purification using the target receptor, and means for evaluating the results of link enrichments. US Patents Numbers 5,223,409, 5,403,484, 5,571,689, and 5,663,143.

Although most phage display methods have employed filamentous phage, lambdoid phage display systems (O 95/34683; US 5,627,024), T4 phage expression systems (Ren et al., Gene, 215: 439 (1998)).; Zhu et al., Cancer Research, 58 (15): 3209-3214 (1998); Jiang et al., Infection &Immunity, 65 (11): 4770-4777 (1997); Ren et al., Gene, 195 (2): 303-311 (1997); Ren, Protein Sci., 5: 1833 - - (nineteen ninety six); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage expression systems (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. Patent Number 5,766,905), are also known. .

Further enhancements increase the ability of expression systems to screen peptide libraries to bind select target molecules and display functional proteins with the screening potential of those proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control biomolecular interactions (WO 98/20169; WO 98/20159) and properties of helical peptides. restricted (WO 98/20036). WO 97/35196 describes a method for isolating an affinity ligand wherein a phage display library is contacted with a solution wherein the ligand will bind to a target molecule and a second solution wherein the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 discloses a method for biocribing a random phage display library with an affinity purified antibody and then isolating the binding phage, followed by a micro-labeling process using microplate wells to isolate high affinity binding phage. The use of protein A from Staphlylococcus aureus as a label - - affinity has also been reported (Li et al. (1998) Mol Biotech., 9: 187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library that can be a phage display library. A method for selecting enzymes suitable for use in detergents employing phage display is described in WO 97/09446. Additional methods for selecting specific binding proteins are described in U.S. Patents. Numbers 5,498,538, 5,432,018, and WO 98/15833.

Methods for generating peptide libraries and screening these libraries are also described in U.S. Patents. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.

Antibodies In some embodiments of the invention, the PAK1 inhibitor for the treatment of melanoma in an individual are isolated antibodies that bind to PAK1. In some embodiments, the antibody is humanized. In a further aspect of the invention, an anti-PAKI antibody or an antibody that inhibits PAK1 function. In some embodiments, the antibody is a monoclonal antibody, including a humanized or humanized chimeric antibody. In some embodiments, the antibody is a fragment of antibody, for example, an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is an integral length antibody, for example, an "intact" IgGl antibody or another antibody class or isotype as defined herein.

In certain embodiments, amino acid sequence variants of the antibodies and / or the binding polypeptides provided herein are contemplated. For example, it may be convenient to improve the binding affinity and / or other biological properties of the antibody and / or binding polypeptide. Amino acid sequence variants of an antibody and / or binding polypeptide can be prepared by introducing appropriate modifications in the nucleotide sequences encoding the antibody and / or binding polypeptide or by peptide synthesis. These modifications include, for example, deletions of and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody and / or binding polypeptide. Any combination of deletion, insertion and substitution can be made to arrive at the final construction, provided that the final construction possesses the desired characteristics, for example, target link.

In certain embodiments, antibody variants and / or linker polypeptide variants having one or more amino acid substitutions are provided. Sites of Interest for substitutional mutagenesis include HVRs and FRs. Amino acid substitutions can be introduced into an antibody and / or binding polypeptide of interest and the products screened for a desired activity, for example retained / improved antigen binding, decreased immunogenicity or improved ADCC or CDC.

One type of substitution variant involves replacing one or more hypervariable region residues of a precursor antibody (eg, a humanized or human antibody). In general, the resultant variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the precursor antibody and / or will have certain substantially retained biological properties of the precursor antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies display in phage and screen for a particular biological activity (e.g., binding affinity).

Alterations (for example, substitutions) can be made in HVRs, for example, to improve affinity of - - antibody. These alterations can be made in HVR "hot spots", ie, coding residues codon that undergo high frequency mutation during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196 ( 2008)), and / or SDRs (a-CDRs), and the resulting variant VH or VL is tested by binding affinity. Maturation of affinity for construction and reselection of secondary libraries have been described for example in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some affinity maturation modalities, diversity is introduced into the select variable genes for maturation by any of a variety of methods (e.g., error-prone PCR, chain intermixing or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then sieved to identify any antibody variants with the desired affinity. Another method to introduce diversity involves approaches directed to HVR, where several HVR residues (for example, 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example using modeling or alanine scanning mutagenesis. CDR-H3 and CDR-L3 in particular are often targets.

In certain modalities, substitutions, insertions or deletions may occur within one or more HVRs as long as said alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce the binding affinity can be made in HVRs. These alterations may be outside the "hot spots" HVR or SDRs. In certain embodiments of the VH and VL variant sequences that are provided above, each HVR either is unaltered or contains no more than one, two or three amino acid substitutions.

Combination therapy The PAKI inhibitors of the methods described herein can be used either alone or in combination with other agents in a therapy for the treatment of melanoma. For example, a PAKI inhibitor described herein can be co-administered with at least one additional therapeutic agent including another PAKI inhibitor. In certain embodiments, an additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent may be Aldesleukin, Dacarbazine, DTIC-Dome (Dacarbazine), Ipilimumab, Proleukin (Aldesleukin), Vemurafenib, Yervoy (Ipilimumab), and / or Zelboraf (Vemurafenib). For example the use of PAKI inhibitors in combination therapies is provided by PCT / EP2011 / 070008 filed on November 14, 2011.

- - These combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case administration of the PAKI inhibitor may occur prior to, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvant. In some embodiments, PAKI inhibitors are used for the treatment of melanoma in an individual, in combination with radiation therapy. In some embodiments, PAKI inhibitors are employed for the treatment of melanoma in an individual, in combination with surgical separation of all or a portion of the individual's melanoma.

In some embodiments of the invention, the individual has been previously treated for melanoma, for example using anticancer therapy. In one example, anticancer therapy is surgery. In another embodiment, the subject can be further treated with an additional anti-cancer therapy before, during (eg, simultaneously) or after administration of the PAKI inhibitor. Examples of anticancer therapies include without limitation, surgery, radiation therapy (radiotherapy), biotherapy, immunotherapy, chemotherapy or a combination of these therapies.

Administration route The administration route is according to methods - - known and accepted, such as by single or multiple bolus or infusion over a prolonged period of time in a convenient manner, for example by injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by means of sustained release or extended release. In some embodiments, the invention provides methods for the treatment of melanoma in an individual with a PAK1 inhibitor wherein the PAK1 inhibitor is administered intravenously to the individual. In other embodiments, the invention provides methods for the treatment of melanoma in an individual with a PAK1 inhibitor wherein the PAK1 inhibitor is administered topically to the individual.

Pharmaceutical compositions For the methods of the invention, therapeutic formulations of the invention are prepared for storage by mixing the PAK1 inhibitor having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are not toxic to containers at the doses and concentrations employed and include buffers such - - as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. ); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysis; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; against salt-forming ions such as sodium; metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

The present formulation may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be convenient to also provide an immunosuppressive agent. These molecules are conveniently present in combination in amounts that - - they are effective for the intended purpose.

The active ingredients may also be entrapped in microcapsule prepared, for example by coacervation or interfacial polymerization techniques, for example, hydroxymethyl cellulose or gelatin microcapsule and poly- (methyl methacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. These techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations can be made. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides (U.S. Patent Number 3,773,919), L-glutamic acid copolymers, and? ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres composed of copolymers of lactic acid-glycolic acid and acetate) - - leuprolide), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a long time, they can become denatured or aggregated as a result of exposure to humidity at 37 ° C, resulting in loss of biological activity and possible changes in immunogenicity.Risk strategies can be designed for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilized from acidic solutions, controlling moisture content, using additives. and developing specific polymer matrix compositions.

In some aspects, the invention provides a composition comprising a PAK1 inhibitor for use in the treatment of melanoma. In some modalities, melanoma is a wild type BRAF melanoma. In some modalities, PAK1 is over expressed in melanoma. In some modalities, melanoma is a wild type BRAF melanoma in which PAK1 is overexpressed in melanoma in - - comparison with non-cancerous cells; for example, non-cancerous skin cells. In some modalities, melanoma is a wild type BRAF melanoma in which PAKl is amplified in melanoma. In some modalities, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, melanoma is a BRAF mutant melanoma. In some modalities, melanoma is a BRAF mutant melanoma and melanoma overexpressed PAKl compared with non-cancerous cells and / or PAKl is amplified in melanoma. In some embodiments, the invention provides a composition comprising PAKI inhibitor for use in the treatment of melanoma in a mammal. In some embodiments, the invention provides composition comprising PAKI inhibitor for use in the treatment of melanoma in a human.

In some aspects, the invention provides a use for a PAKI inhibitor in the manufacture of a medicament for the treatment of melanoma. In some modalities, melanoma is a wild type BRAF melanoma. In some modalities, PAKl is overexpressed in melanoma. In some embodiments, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma compared to non-cancerous cells; for example cells of - - non-cancerous skin. In some modalities, melanoma is a wild type BRAF melanoma in which PAKl is amplified in melanoma. In some modalities, melanoma is a wild type BRAF melanoma in which PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some modalities, melanoma is a BRAF mutant melanoma. In some modalities, melanoma is a BRAF mutant melanoma and melanoma overexpressed PAKl compared with non-cancerous cells and / or PAKl is amplified in melanoma. In some embodiments, the invention provides a use for a PAKI inhibitor in the manufacture of a medicament for the treatment of melanoma in a mammal. In some embodiments, the invention provides a use for a PAKI inhibitor in the manufacture of a medicament for the treatment of melanoma in a human.

Equipment The invention also provides equipment, medicines, compositions, and unit dosage forms for use in any of the methods described herein.

The kits of the invention include one or more containers comprising a PAKI inhibitor (or unit dosage forms and / or articles of manufacture) and in some embodiments, further comprising instructions for use in - - Melanoma treatment according to any of the methods described herein. The equipment may also comprise a description of the selection of an individual suitable for treatment (for example selection based on the BRAF genotype). Instructions provided on the equipment of the invention are typically instructions written on a label or packaging insert (eg, a sheet of paper that is included in the equipment), but machine-readable instructions (eg, instructions that transport a disc optical or magnetic storage) are also acceptable. In some modalities, the team also understands another therapeutic agent.

The equipment of the invention is in convenient packaging. Convenient packaging includes, but is not limited to, ampoules, bottles, jars, flexible packaging (e.g., sealed plastic or sealing bags) and the like. Teams can optionally provide additional components such as dampers and interpretive information. The present application in this manner also provides articles of manufacture, which include ampoules (such as sealed ampoules), bottles, jars, flexible packaging and the like.

Melanoma biomarkers and treatment The invention provides methods for identifying human melanoma patients suitable for treatment with - - a PAKI inhibitor when determining the presence of one or more melanoma biomarkers. In some modalities, the melanoma biomarker is overexpression of PAKl in melanoma, amplification of PAKl in melanoma, and / or the presence of wild type BRAF in melanoma. In some embodiments, expression of PAKl is determined by comparison with non-cancerous tissue; for example non-cancerous skin tissue. In some modalities, biomarkers are detected in a test sample that is obtained from the individual. In some embodiments, the presence of the biomarker is determined in comparison to a test sample with a reference sample.

In one embodiment, the invention provides methods for identifying human melanoma patients suitable for treatment with a PAKI inhibitor by determining expression of PAKI in melanoma wherein overexpression of PAKI in melanoma compared to non-cancerous cells indicates that the patient is adequate for treatment with a PAKI inhibitor. In some embodiments, overexpression of PAKl in approximately any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% in melanoma in Comparison with non-cancerous cells indicates that the patient is suitable for treatment with a PAKI inhibitor. In some modalities, overexpression of PAKl in about any of 1.5-fold, 2.0-fold, 2.5- - - times, 3, 3.5-times, 4.0-times, 4.5-times, 5.0-times, 6.0-times, 7.0-times, 8.0-times, 9.0-times, or 10-times compared to PAKI expression in non-cancerous cells indicates that the patient is suitable for treatment with a PAKI inhibitor. Methods for determining expression of PAKl are known in the art. Examples of methods for determining expression levels of PAKI in a melanoma include, but are not limited to, immunohistochemistry, reverse phase protein matrix (RPPA), quantitative PCR, immunoassays and the like. Levels of PAKl expression can be compared with other tumors and cells by using the Gene Expression Omnibus (GEO) database.

In another embodiment, the invention provides methods for identifying human melanoma patients suitable for treatment with a PAKI inhibitor by detecting the amplification of PAKI in melanoma wherein amplification of the PAKI gene in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor. In some embodiments, a PAKl copy number of approximately either 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 or greater than 10 in the Melanoma indicates that the patient is suitable for treatment with a PAKI inhibitor. Methods for determining amplification of a gene are known in the art. For example, the copy number of the PAKl gene may be - - determined using SNP arrays such as Affymetrix 500K SNP array analysis.

In another embodiment, the invention provides methods for identifying human melanoma patients suitable for treatment with a PAK1 inhibitor by detecting the BRAF genotype in melanoma wherein wild-type BRAF in melanoma indicates that the patient is suitable for treatment with an inhibitor. PAK1. Methods for determining the genotype of the BRAF gene in melanoma are known in the art; for example, the nucleotide sequence of the BRAF gene of melanoma can be determined using standard sequencing methods or by using the KASP SNP genotyping system (KBioscience).

The invention provides methods for treating melanoma in a patient provided that the patient has been found to have a melanoma biomarker selected for overexpression of PAK1 in the melanoma, amplification of PAK1 in the melanoma and / or the presence of wild type BRAF in the melanoma.; the method comprising administering to the patient an effective therapeutic amount of a PAK1 inhibitor. In some modalities, the patient is a human patient. In some embodiments of the above embodiment, at least one of the biomarkers is overexpression of PAK1 wherein PAK1 is overexpressed in approximately any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 100% or greater in melanoma in - - comparison with non-cancerous cells. In some embodiments, the expression of PAKl in melanoma is greater than approximately either 1.5-fold, 2.0-fold, 2.5-fold, 3, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold , 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold compared to the expression of PAKl in non-cancerous cells. Methods for determining PAK1 expression are known in the art. Examples of methods for determining expression levels of PAKI in a melanoma include, but are not limited to immunohistochemistry, reverse phase protein matrix (RPPA), quantitative PCR, immunoassays and the like. Levels of PAKl expression can be compared with other tumors and cells by using the Gene Expression Omnibus (GEO) database.

In some embodiments of the above embodiment, at least one of the biomarkers is amplification of PAKI in melanoma wherein a copy number of PAK1 is approximately any of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, or greater than 10 in melanoma. Methods for determining amplification of a gene are known in the art. For example, the number of copies of the PAKl gene can be determined by using SNP arrays such as Affymetrix 500K SNP array analysis.

In some modalities of the previous modality, - - minus one of the biomarkers is the BRAF genotype in melanoma where the patient has a melanoma that contains a wild-type melanoma.

In some embodiments of the above modality, the presence of melanoma biomarkers in the patient has been previously determined before treatment with the PAKI inhibitor.

The invention provides methods for using melanoma treatment in a patient undergoing treatment with a PAKI inhibitor wherein the expression of PAK1 in the melanoma is determined. In some modalities, melanoma is a wild type BRAF melanoma. In some embodiments, overexpression of PAKl in melanoma indicates that treatment with the PAKI inhibitor may continue. In some embodiments, the expression of PAK1 in a melanoma in a patient undergoing treatment with PAK1 is monitored over time. In some modalities, the expression of PAKl in melanoma is monitored at least daily, at least weekly, at least monthly. In some embodiments, the expression of PAK1 in a melanoma in a patient undergoing treatment with a PAK1 inhibitor is monitored over time. If the expression of PAKI increases with the course of treatment with the PAKI inhibitor, the amount of PAKI inhibitor administered to the patient increases or remains the same. In some modalities, the amount of PAKI inhibitor - - administered to the patient is increased until the level of PAK1 expression decreases or is no longer detectable. If the expression of PAK1 decreases in the course of treatment with the PAKI inhibitor, the amount of PAKI inhibitor administered to the patient is decreased or remains the same. In some embodiments, the expression of PAKl in a melanoma of a patient undergoing treatment with a PAKI inhibitor is monitored over time where treatment with the PAKI inhibitor is continued until PAK1 expression in the melanoma is no longer detected plus.

Exemplary Modalities In some embodiments, the invention provides methods for treating a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAKI inhibitor. In additional embodiments, the melanoma is a wild-type BRAF melanoma. In yet further modalities, PAKl is overexpressed in the tumor compared to non-cancerous skin cells. In additional modalities of any of the above modalities, PAK1 is amplified in the tumor. In additional embodiments, the copy number of PAK1 in the tumor is greater than about 2.5.

In additional embodiments of any of the above embodiments, the inhibitor is a small molecule, a nucleic acid or a polypeptide. In some modalities, - - the small molecule is PF-3758309. In some embodiments, the small molecule is a compound of formula I.

In further embodiments, the small molecule is a compound of formula I and A is 4-indolyl, 5-indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazolyl or 5-benzimidazolyl; Ra, Rla and Rlb are independently hydrogen or Ci-3 alkyl; R5 is hydrogen or Ci-6 alkyl; R6 is hydrogen, halogen or Ci-e alkyl; and, R7 is cycloalkyl optionally substituted by fluorine.

In additional modalities of any of the foregoing modalities, the individual is a human.

In additional embodiments of any of the above embodiments, the PAK1 inhibitor is used in combination with a therapeutic agent.

The invention provides the use of a PAK1 inhibitor for the treatment of melanoma in an individual. In some modes of use, melanoma is a wild type BRAF melanoma.

The invention provides compositions that - - comprise a PAK1 inhibitor for use in the treatment of melanoma. In some embodiments of the composition, the melanoma is a wild type BRAF melanoma. In some embodiments, the composition further comprises an acceptable pharmaceutical excipient.

The invention provides the use of a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma. In some modes of use, melanoma is a wild type BRAF melanoma.

The invention provides kits comprising a PAK1 inhibitor for use in the treatment of melanoma comprising PAK1 inhibitor and instructions for use in the treatment of melanoma. In some equipment modalities, melanoma is a wild type BRAF melanoma.

The invention provides methods for inhibiting CRAF signaling in a melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor.

The invention provides methods for inhibiting MEK signaling in a melanoma tumor comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor.

The invention provides methods for identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor, which comprises determining the BRAF genotype - - of melanoma, wherein a melanoma comprising a wild-type BRAF indicates that the patient is suitable for treatment with a PAKI inhibitor.

The invention provides methods for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor comprising determining the expression of PAKI in melanoma, wherein the overexpression of PAKI in the melanoma compared with non-cancerous skin cells indicates that the Patient is suitable for treatment with a PAKI inhibitor. In some modalities of the method, overexpression of PAK1 in melanoma is 2.5 times greater than expression of PAK1 in non-cancerous skin cells. The invention provides methods for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient b on the BRAF genotype of the melanoma, wherein a melanoma comprising a wild-type BRAF indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

The invention provides methods for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient b on the level of PAKl expression of the melanoma, wherein the overexpression of PAK1 in the melanoma indicates that the patient is suitable for - - treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor. In some embodiments of the methods, overexpression of PAKI in melanoma is 2.5 times greater than PAKI expression in non-cancerous skin cells.

The invention provides methods for treating a patient with human melanoma comprising administering to the select individual an effective therapeutic amount of a PAKI inhibitor, wherein the melanoma genotype has been determined to be a wild type of BRAF.

The invention provides methods for treating a patient with human melanoma comprising administering to the patient an effective therapeutic amount of a PAKI inhibitor, wherein the melanoma has been determined to overexpress PAK1 compared to non-cancerous skin cells. In some embodiments of the methods, overexpression of PAK1 in melanoma is 2.5 times greater than expression of PAK1 in non-cancerous skin cells.

The invention provides methods for adjusting melanoma treatment in a patient undergoing treatment with a PAKI inhibitor, the method comprising estimating the expression of PAK1 in the melanoma, wherein the overexpression of PAK1 in the melanoma indicates that the treatment of the individual is adjusts until the overexpression of PAKl is no longer detected.

- - All the features described in this specification can be combined in any combination. Each characteristic described in this specification can be replaced by an alternating characteristic that serves the same, equivalent or similar purpose. In this way, unless otherwise expressly stated, each characteristic described is only an example of a generic series of equivalents or similar characteristics.

Additional details of the invention are illustrated by the following non-limiting Examples. The descriptions of all references in the specification are expressly incorporated herein by reference.

EXAMPLES The examples below are intended as purely exemplary of the invention and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

Example 1: Expression of elevated PAK1 protein and genomic amplification in melanoma.

To determine the possible extent of PAK1 deregulation in human melanoma, primary tumor tissue from 87 melanoma patients was assayed for changes in DNA copy number using polymorphism matrices - - of simple high resolution nucleotides (SNP). SNP Affimetrix 500K matrix analysis, genomic DNA preparation, chip processing and data analysis were performed as previously published (Harvey PM, et al., (2008) Genes Chromosomes Cancer, 47 (6): 530-542) for measure gains of copies of llql3, the region of chromosome 11 that hosts the PAKI gene, in the melanoma tissue sampled. To collect expression matrix data by paired tumor samples, RNA was extracted from frozen tumor tissue and applied to HGU133 Affimetrix gene expression microarrays (Santa Clara, CA). The PAKI amplification frequency was 9% (8 of 87 specimens with copy number = 2.5) in this tumor panel (Figure 1A). RNA was purified from 48 specimens of cell lines and melanoma tumor and the increased PAKI copy number was correlated with mRNA expression (Pearson correlation = 0.75, Figure IB). Expression of deregulated PAK1 was more frequent than would be predicted by genomic amplification alone, thus suggesting that additional regulatory or transcriptional mechanisms increase PAK1 expression in this indication (Reddy SD, et al, (2008) Cancer Res, 68 (20): 8195-8200 and de la Torre-Ubieta L, et al., (2010) Genes Dev, 24 (8): 799-813). Elevated expression of PAKl in melanoma compared to normal skin tissues was also demonstrated using gene expression data deposited in the Gene database - - Expression Omnibus (GSE4587). Interestingly, PAKI gene amplification is preferably observed in tumors lacking activating mutations in the BRAF oncogene at 22% against 0% for wild-type or mutant BRAF, respectively (p = 0.005, two-sided t-test; IB). The expression levels of PAKl mRNA differ between wild type and BRAF genotypes (V600E) or BRAF (V600M) (p = 0.006 and p = 0.125, respectively, Figure IB). Taken together, this suggests that PAKl may be a "driving" gene that promotes tumor in a subset of wild-type BRAF melanoma.

To further evaluate the extent of PAK1 deregulation in human melanomas, level of PAKI protein expression and subcellular localization were evaluated by immunohistochemical staining (IHC) of a different set of tissue microarrays. Briefly, formalin-fixed paraffin-embedded tissue blocks and corresponding pathology reports were obtained for 92 primary melanomas resected between 1993 and 2009 (Oxford Radcliffe Hospitals, Oxford, UK). The melanoma series includes 23 nodular melanoma specimens, 3 malignant melanoma specimens, 45 superficial dissemination specimens, 3 desmoplastic specimens, 5 lentiginous acral specimens, and 13 non-classifiable specimens. Four cancers were stage pTl, 17 were stage pT2, 28 were stage pT3, 35 were stage pT4 and 8 cases could not be classified in precise form. Tissue microarrays (TMAs) were assembled as previously described (Bubendorf L, et al., (2001) J Pathol, 195 (1): 72-79). Approval was obtained for the use of all human tissue from the Local Research Ethics Committee (C02.216).

Immunohistochemistry (IHC) was performed as previously described (Ong CC, et al., (2011) PNAS, 108 (17): 7177-7182). The PAK1 expression intensity was scored separately in the cytoplasm and nuclei of neoplastic cells on a scale of 0 to 3. The highest intensity score between replicated nuclei was used as the rating for each patient. The same pathologist qualified all cases blind to clinical data. The chi square test was used to evaluate associations between categorical variables. Robust and selective IHC reactivity of PAKI antibody was previously demonstrated (Ong CC, et al., (2011) PNAS, 108 (17) .7177-7182). In malignant melanoma, 46 of 92 (50%) primary tumor samples were positive for PAKI expression and 26% of all cases showed moderate (2+) or strong (3+) intensity staining in malignant cells (Figure 1C) , panels III and IV, Table 1). Nuclear localization of PAKl was only evident in a very small proportion of melanomas. Identical results are seen with an alkaline phosphatase label and fast red chromogen instead of horseradish peroxidase label and - - diaminobenzidine coffee. PAK1 was weakly expressed in basal keratinocytes in normal skin, and putative Langerhans cells and cells were positive for PAK1 expression (Figure 1C, panel IV). Taken together, these data show that the DNA copy number PAK1, mR A and protein expression are up-regulated largely in human melanoma.

Example 2: Negative association between overexpression of PAK1 and BRAF mutation in primary melanomas.

Given the prevalence of oncogenic mutation of BRAF and NRAS in melanoma (Lee JH, et al., (2011) Br J Dermatol, 164 (4): 776-784), melanoma tissues were genotyped for known hot spot mutations in genes BRAF (codon 600) and NRAS (codons 12, 13, 61 and 146). Mutation status was determined for BRAF codon 600 and NRAS codons 12, 13 61 and 146 by KASPar (KBioscience, Herts, England) and conventional Sanger DNA sequencing methods. Genotype data for BRAF (39 Val600Glu, 1 Val600Lys and 46 wild type) and NRAS (1 Gln61His, 7 Gln61Lis, 1 Gln61Lis + Gln61Arg + Leu59Ala, 1 Gln61Leu, 19 Gln61Arg, 2 Gln61Arg + Gln61Lis and 53 wild type) were available for 86 and 84 tumors respectively and were consistent with the mutation frequency ranges that have been previously published for cutaneous melanoma (Lee JH, et al., (2011) Br J Dermatol, 164 (4): 776-784). Staining PAK1 IHC was classified blind to clinicopathological details and mutation status and the - - The result is summarized in Table 1. Notably, PAKI protein expression was selectively deregulated in wild-type BRAF tumors (19 of 46 were positive for strong IHC staining of PAKl) compared to melanomas expressing V600E or V600K oncogenic mutants ( 4 of 40 tumors with high IHC staining). This negative correlation between expression of PAK1 and BRAF mutation was statistically significant (p <; 0.001, Chi-square 10.702). BRAF and NRAS mutations were not mutually exclusive, and the presence of tumors with any mutation was also negatively associated with PAKl protein expression (p = 0.004, Chi-square 8.128). A similar trend, although not statistically significant, was observed when samples dichotomized in only mutant or non-mutant NRAS state (p = 0.45, Chi-square 0.569). There was no significant association between protein PAK1 expression and mitotic count (p = 0.61 Student's T test), pathological tumor stage (pT) (p = 0.14 Chi-square), Breslow thickness (p = 0.85 Student's T test) or ulceration (p = 0.91 Chi-square). Taken together, these results provide evidence that PAK1 deregulation is strongly associated with cutaneous melanomas that lack oncogenic BRAF mutation and define a subset of human melanoma for which there is no effective targeted therapy.

Table 1. Overexpression of PAKl protein in melanoma of type - - wild BRAF Example 3: PAK1 is required for proliferation of BRAF wild type melanoma cells Given the genomic and histological data for elevated PAK1 expression in the subset of human melanoma that is wild-type for BRAF, PAK1 expression and the RNAi-mediated gene interference effect of PAK1 was examined in a panel of melanoma cell lines in order to clarify the contribution of PAK1 towards proliferation of tumor cells. American Type Culture Collection cell lines (ATCC, Manassas, VA) were purchased and maintained at 37 degrees C and 5% C02 in Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute 1640 medium (RPMI 1640) with bovine serum. fetal at 10% and L-glutamine at 4 mM.

- - Cell lines were transfected with oligonucleotide duplex of short interfering RNA (siRNA) commercially available from Dharmacon RNAi Technologies (Chicago, IL) that were previously characterized by efficiency and selectivity of PAK1 and PAK2 gene interference (Ong CC, et al., (2011 ) PNAS, 108 (17): 7177-7182). Cell viability was estimated by ATP content using the CellTiter-Glo Luminescent Assay (Promega, Madison, I) and the results represent average + standard deviation of three experiments. Increased expression of PAKI protein in melanomas expressing wild type against mutant BRAF is also observed for cell lines immortalized in culture. Cell viability analysis demonstrates that melanoma cells 537MEL, eWo, SK-MEL23 and SK-MEL30 express high levels of PAKl protein and transient PAKI gene interference by accumulating multiple siRNA selective oligonucleotides of PAKl resulting in a reduction of 1.8- a 4.3-fold in cell viability when compared to cells transfected with non-target negative control siRNA oligonucleotide (p <0.0001, Figure 2A). In addition, inhibition of PAKI generally reduces proliferation of BRAF wild-type melanoma cells relative to BRAFV600E cells (p <0.07; n = 14), further supporting a role for PAKI as a driver for proliferation in this melanoma subtype (FIG. 2B). To better estimate the - - mechanism by which PAK1 contributes to proliferation, cellular signaling dependent on PAK is estimated in 537MEL and SK-MEL23 cells. Protein extracts from cell lysates are prepared at 4 degrees C with Cell Extraction Buffer (Invitrogen, Carlsbad, CA), 1 mM phenylmethylsulfonyl fluoride (PMSF), Phosphatase Inhibitor Cocktail 1/2 (Sigma Aldrich, St. Louis, MO), and a complete EDTA free protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN). For Western Blot analysis, proteins were resolved by SDS-PAGE 4-12% and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, MA). Intra-transfer was performed using the indicated primary antibodies and analyzed using secondary antibodies for enhanced chemiluminescence (ECL). Activation of the MAPK pathway, as determined by phosphorylation of ERK and MEK, was dramatically inhibited by PAK gene interference (Figure 2C). According to this result, cyclin DI levels (which are essential to regulate cyclin dependent kinases and Gl / S progress) were also decreased as a consequence of PAK1 ablation. PAK1 signaling in BRAF wild type melanoma cells was further investigated using a reverse phase protein matrix phosphoprotein platform (RPPA). Protein lysates are analyzed by RPPA (Theranostics Health, LLC) upon first diluting all - - samples at a final concentration of 0.5 mg / mL. The sample dilutions were printed in duplicate on slides that were then subjected to immunostaining with a panel of antibodies primarily directed against specific phosphorylation or dissociated proteins. Each of these antibodies has previously been subjected to extensive validation for both phosphorylation and protein specificity using single band detection at the appropriate molecular weight by immunoblotting. The intensity value for each endpoint is determined by identifying points for each duplicate dilution curve for each sample that were within the linear dynamic range of the staining after background subtraction with each point (within the local background of the object holder). and also against an object holder stained with only secondary antibody). Each value was normalized to the total protein intensity value for that sample derived from an object holder stained with Sypro Ruby (Invitrogen). RPPA data were processed by log2 transformation and linear scale (z score conversion) to ensure normality and linearity. RPPA analysis showed decreased signaling to MAPK, nuclear factor-? (NF- ??) and cytoskeletal pathways following PAK1 inhibition in wild type BRAF (SK-MEL23), but not BRAF mutant (A375), melanoma cells (Figure 2D).

- - PAK1 has been shown to phosphorylate both CRAF (Ser338) and MEKl (Ser298) (17, 29-31). Therefore, the molecular mechanism by which PAK1 triggers the activation of the MAPK pathway in wild type melanoma BRAF cells was investigated. Since phospho-specific antibodies that develop at Ser217 / Ser221 activation loop sites in MEK proteins do not distinguish between MEK1 and MEK2, the MEK isoforms were immunoprecipitated from transfected cells with either control or oligonucleotide if NA selective PAK as previously described (Hatzivassiliou G, et al., (2010) Nature, 464 (7287): 431-435) and MEK activation was detected by immunoblotting with phospho-MEK1 / 2 antibodies (Ser217 / Ser221). The PAK gene interference decreased both phosphorylation of MEK1 (Figure 3A) and MEK2 (Figure 3B) in 537MEL and SK-MEL23 cells. Since the phosphorylation site Ser298 in MEK1 is not conserved in MEK2, the PAK-dependent activation of both MEK isoforms will suggest that upstream signaling to CRAF may be a driver of MAPK pathway regulation in BRAF wild type melanoma cells. CRAF was immunoprecipitated from either transfected control cells or selective siRNA oligonucleotides PAK as previously described (Hatzivassiliou G, et al., (2010) Nature, 464 (7287): 431-435) and CRAF activation was detected by immunoblotting with phospho-antibodies -CRAF (Ser338). Western analysis showed that the - - PAK ablation reduces phosphorylation of CRAF in Ser338, a critical residue for complete activation of this kinase (Figure 3C). The dependence on CRAF phosphorylation (Ser338) (Figure 3D) and CRAF effector signaling (Figure 3E) on PAK catalytic activity was also confirmed using PF-3758309, an inhibitor of PAKs currently in clinical development (Murray BW, et al, (2010) PNAS, 107 (20): 9446-9451), and IPA-3, an allosteric inhibitor that binds to PAK1-3 and prevents activation by Rho family GTPases (Deacon SW, et al., (2008) Chemistry & Biology, 15 (4): 322-331).

Additional loss-of-function studies to analyze the role of PAKl in silvetre BRAF melanoma cells are performed by investigating the contribution of PAK1 to melanoma cell migration. Briefly, WM-266-4 melanoma cells are transfected with non-target control (NTC) or oligonucleotide PAKl / 2 siRNA for 72 h and confluent melanoma cells WM-266-4 subsequently injured. Images were recorded when the wounds were made (dark shadow) and 28 h after injury (bright field). Differences in relative wound density were statistically significant (p <0.001; n = 3) revealing a requirement for PAK1 in migration of melanoma cells (Figure 4A-B). Taken together, the functional sequelae of PAK1 blockade in BRAF wild type melanoma cells encompass pronounced cytostatic effects - - by means of reduced CRAF activation and subsequent MAPK route signaling.

Example 4: Differential sensitivity of BRAF wild type melanoma cells to PAK and BRAF inhibition To more closely investigate the activity and cellular mechanism of action of PAK signaling within sensitive and insensitive tumor types, small molecule measurement of PAK and BRAF is compared using wild type SK-MEL23 BRAF and A375 BRAF (V600E) cells. For route modulation analysis, cells were treated with PF-3758309 5 μ? or with PLX-4720 0.2 μ ?, a BRAF inhibitor, for 4 hours before cell lysates were analyzed by phosphorylation of MAPK pathway components. Administration of PF-3758309 resulted in deep MAPK path modulation in SK-MEL23 cells (lane 2), but not A375 cells (lane 5), as determined by measuring ERKl / 2 and MEK1 / 2 phosphorylation in loop residues. kinases that are critical for catalytic activity (Figure 5A). In comparison, analysis of signaling changes mediated by PLX-4720 revealed only modest inhibition of ERK1 / 2 and MEK1 / 2 phosphorylation in SK-MEL23 cells (lane 3), where the same treatment conditions potently inhibit MAPK activation in BRAF cells (V600E) (lane 6). As a control, no differences were noted for total protein levels ERK1 / 2 or MEK1 / 2 in this experiment. Consistent with reports - - Previously, MEK1-Ser298 was confirmed as a phosphorylation site specific for PAK but Ser298 phosphorylation was not linked to the looping phosphorylation of MEK in BRAF melanoma cells (V600E) (lane 5). The biological consequence of PAK1 phosphorylation of MEK1-Ser298 is not currently well understood, however it has been shown that PAK1-EK1 signaling can be mediated by cell-cell contacts and adhesion (Slack-Davis JK, et al., (2003) J Cell Biol, 162 (2): 281-291). PAK signaling was also induced by ectopic expression of Flag-PAK1 in BRAF cells (V600E) with only moderate endogenous expression of PAKI. PAKl signaling elevated in A375 cells resulted in a significant increase in CRAF and MEK phosphorylation that was reversible by addition of PF-3758309 (Figure 5B), suggesting that the acquisition of PAKl over-expression may be another mechanism to overcome dependence on oncogenic BRAF in melanoma (Johannessen CM, et al., (2011) Nature, 468 (7326): 968-972).

To determine if PAK inhibitors decrease cell viability of wild type BRAF melanoma cells, SK-MEL23 and 537MEL cells were tested with the CellTiter-Glo Luminescent Assay (Promega, Madison, WI) after treatment with PF-3758309 or with (S) ) -N2- (1- (1H-indol-5-yl) ethyl) -N4- (5-cyclopropyl-lH-pyrazol-3-yl) -6-methyl-pyrimidine-2,4-diamine (1-007) ), N2- ((lH-indol-4-yl) methyl) -N4- - - (5-cyclopropyl-lH-pyrazol-3-yl) -6-methyl-pyrimidine-2,4-diamine (1-054) and N2- ((4-chloro-?? -benzo [d] imidazole-5-) il) methyl) -N 4 - (5-cyclopropyl-1 H -pyrazol-3-yl) -N 2 -methyl-pyrimidine-2,4-diamine (1-087). Inhibition of PAK1 by treatment with all tested PAK inhibitors, significantly reduces cell proliferation indicating that the inhibition of PAK signaling is a target for treatment of wild type melanoma BRAF (Figures 6A and B).

To extend in vitro observations, pharmacodynamic modulation by PAK small molecule inhibitors is evaluated using tumor xenograft models. Cultured cells SK-MEL-23, A2058.X1 and A375.X1 are removed from culture, suspended in Hank's buffered saline solution (HBSS), 1: 1 mixture with Matrigel (BD Biosciences, USA), and implanted subcutaneously on the flank. right of nude female NCR mice without pretreatment (Taconic Farms, Hudson, NY) or XID Nude Beige (Harían Laboratories, CA). Animals with tumors with an average volume of approximately 250 mm3 were grouped into treatment cohorts. Tumor volumes were calculated by following the formula: Tumor Volume = 0.5 x (a x b2), where ¾a 'is the largest tumor diameter and * b' is the perpendicular tumor diameter. Tumor volume results are presented as average tumor volumes ± the standard error of the mean (SEM). Percent growth inhibition - - (% INH) at the end of the study (EOS) is calculated as% INH = 100 [(EOS Vehicle EOS Treatment) / (EOS Vehicle)]. Data analysis and p-value generation using the Dunnett t test was performed using the JMP program (SAS Institute, Cary, NC). All experimental procedures conformed to the guiding principles of the American Physiology Society and were approved by the Institutional Genentech Animal Care and Use Committee. After establishment of the tumor, the animals were already administered with saline or PF-3758309 (25 mg / kg, i.p.) and the tumors were harvested 1 h after dose. Tumors were frozen and sprayed on dry ice using a small Bessman tissue sprayer (Spectrum Laboratories, Rancho Dominguez, CA) and protein extracts were prepared at 4 degrees C with Cell Extraction Buffer (Invitrogen, Carlsbad, CA), phenylmethylsulfonyl fluoride 1 mM (PMSF), Phosphatase Inhibitor 1/2 cocktail (Sigma Aldrich, St. Louis, MO), and a complete EDTA-free Mini Protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN). Proteins were subsequently resolved by SDS-PAGE 4-12% and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, MA) for immunoblotting with the indicated antibodies.

Treatment with PF-3758309 resulted in a substantial decrease in CRAF phosphorylation, MEK1 / 2 and ERK1 / 2 in SK-MEL23 tumors (Figure 7A). In tumors A2058.X1 BRAF (V600E), decreased phosphorylation of CRAF (Ser338) was observed after dose PF-3758309. The effect of PF-3758309 on growth and maintenance of BRAF wild type tumors was also evaluated in an efficacy experiment for 21 days (Figure 7B and Figure 8A-B). Treatment with 10, 15 and 25 mg / kg PF-3758309 significantly impairs tumor growth (74%, 76% and 91% inhibition with respect to the control cohort, respectively) relative to the vehicle cohort as measured by the final day of the dose (t Dunnett test, p <0.0001). In comparison, minimal anti-tumor efficacy and inhibition of CRAF phosphorylation are observed for SK-EL23 tumors treated with a potent RAF inhibitor in vivo (Figure 9B) (Hoeflich KP, et al., (2009) Cancer Res, 69 (7) : 3042-3051). In addition, phosphoprotein analysis of mutant BRAF (A375) and wild-type cells (SK-MEL23) with inhibitor PLX-4720 BRAF demonstrated that these subtypes of melanoma cells exhibit different signaling responses due to inhibition of BRAF (Figure 9A).

Overall, the magnitude of route inactivation MAPK by PF-3758309 correlates with anti-tumor efficacy in the xenograft model in BRAF wild type melanoma and these data support the conclusion that interfering with PAK signaling may have therapeutic efficacy in this subset of melanoma (model illustrated in Figure 10).

Claims (33)

1. A method for treating melanoma in an individual, comprising contacting the melanoma with an effective therapeutic amount of a PAK1 inhibitor.

2. The method in accordance with the claim 1, wherein the melanoma is a wild type BRAF melanoma.

3. The method according to claim 1 or 2, wherein the PAK1 is overexpressed in the tumor compared to non-cancerous skin cells.

4. The method according to any of claims 1 to 3, wherein PAK1 is amplified in the tumor.

5. The method according to claim 4, wherein the copy number of PAK1 in the tumor is greater than about 2.5.

6. The method according to any of claims 1-5, wherein the inhibitor is a small molecule, a nucleic acid or a polypeptide.

7. The method according to claim 6, wherein the small molecule is PF-3758309.

8. The method according to claim 6, wherein the small molecule is a compound of the formula VII.

9. The method according to claim 8, wherein the small molecule is a compound of the formula VII and A is 4-indolyl, 5-indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazolyl or 5-benzimidiazolyl; Ra, Rla and Rlb are independently hydrogen or Ci-3 alkyl; R5 is hydrogen or Ci-6 alkyl; R6 is hydrogen, halogen or Ci-6 alkyl; and R7 is cycloalkyl optionally substituted by fluorine.

10. The method according to any of claims 1-9, wherein the individual is a human.

11. The method according to any of claims 1-10, wherein the PAK1 inhibitor is used in combination with a therapeutic agent.

12. Use of a PAK1 inhibitor for the treatment of melanoma in an individual.

13. The use of claim 12, wherein the melanoma is a wild-type BRAF melanoma.

1 . A composition comprising a PAK1 inhibitor for use in the treatment of melanoma.

15. The composition according to claim 14, wherein the melanoma is a wild type BRAF melanoma.

16. The composition according to claim 14 or 15, wherein it further comprises a pharmaceutically acceptable excipient.

17. Use of a PAKI inhibitor in the manufacture of a medicament for the treatment of melanoma.

18. The use according to claim 17, wherein the melanoma is a wild-type BRAF melanoma.

19. A kit comprising a PAKI inhibitor for use in the treatment of melanoma comprising PAKI inhibitor and instructions for use in the treatment of melanoma.

20. The kit according to claim 19, wherein the melanoma is a wild-type BRAF melanoma.

21. Method for inhibiting CRAF signaling in a melanoma in an individual comprising contacting the melanoma with an effective therapeutic amount of a PAKI inhibitor.

22. A method for inhibiting MEK signaling in a melanoma tumor comprising contacting the melanoma with a therapeutically effective amount of an inhibitor PAK1.

23. Method for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor comprising determining the BRAF genotype of melanoma, wherein a melanoma comprising a wild-type BRAF indicates that the patient is suitable for treatment with a PAKI inhibitor.

24. Method for identifying a human melanoma patient suitable for treatment with a PAKI inhibitor comprising determining the expression of PAKI in melanoma, wherein the overexpression of PAKI in the melanoma compared to non-cancerous skin cells, indicates that the patient is suitable for treatment with a PAKI inhibitor.

25. The method in accordance with the claim 24, where the overexpression of PAKI in melanoma is X% greater than the expression of PAKI in non-cancerous skin cells.

26. Method for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient based on the BRAF genotype of melanoma, wherein a melanoma comprising a wild-type BRAF indicates that the patient is suitable for the treatment with a PAKI inhibitor; and (b) administering to the select patient an effective therapeutic amount of a PAKI inhibitor.

27. Method for treating a human melanoma patient with a PAKI inhibitor comprising: (a) selecting a patient based on the level of PAKl expression of the melanoma, wherein an overexpression of PAK1 in the melanoma indicates that the patient is suitable for treatment with a PAKI inhibitor; and (b) administering to the select patient a therapeutically effective amount of a PAKI inhibitor.

28. The method according to claim 27, wherein the overexpression of PAKI in melanoma is 2.5-fold higher than the expression of PAKI in non-cancerous skin cells.

29. Method for treating a patient of human melanoma, comprising administering to the selected individual, a therapeutically effective amount of a PAKI inhibitor, wherein the melanoma genotype has been determined to be wild type for BRAF.

30. Method for treating a human melanoma patient comprising administering to the patient a therapeutically effective amount of a PAKI inhibitor, wherein the melanoma has been determined to overexpress PAKI as compared to non-cancerous skin cells.

31. The method according to claim 30, wherein the overexpression of PAKI in melanoma is 2.5-fold greater than the expression of PAKI in non-cancerous skin cells.

32. Method for adjusting the treatment of melanoma in a patient undergoing treatment with a PAKI inhibitor, the method includes estimating PAK1 expression in melanoma, where the overexpression of PAK1 in melanoma indicates that the treatment of the individual is adjusted until Overexpression of PAKl is no longer detected.

33. The invention as previously described.