KR20140135198A - Methods of treating melanoma with pak1 inhibitors - Google Patents

Abstract

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

Description

[0001] METHODS OF TREATING MELANOMA WITH PAK INHIBITORS [0002]

The present invention relates to methods and compositions for treating melanoma using a PAK1 inhibitor.

Malignant melanoma accounts for about 80% of skin cancer deaths. Although melanoma can be surgically treated if found at an early stage, local and systemic spread of the disease significantly worsens the prognosis and only 14% of metastatic melanoma patients survive for 5 years [American Cancer Society, Cancer facts & figures (2011). The mitogen-activated protein kinase (MAPK) pathway has recently been identified as an important growth pathway in several melanoma subtypes [Lopez-Bergami P., Pigment Cell Melanoma Res. 24 (5): 902-921 (2011)). For example, from an integrated analysis of data from 4493 patients, the incidence of BRAF (v-Raf murine sarcoma virus tumor gene homolog B1) mutations is 41% in skin melanoma [Lee JH, et al., Br J Dermatol . 164 (4): 776-784 (2011)). The most common BRAF somatic mutation in malignant melanoma is the replacement of valine at residue 600, which provides constitutive catalytic activity and signaling [Davies H, et al., Nature. 417 (6892), 949-954 (2002)]. Genetic studies have confirmed that BRAF is required for initiation and maintenance of melanoma in preclinical model systems [Davies H, et al., Nature. 417 (6892), 949-954 (2002); Hoeflich KP, et al., Cancer Res. 66 (2): 999-1006 (2006); Dankort D, et al., Genes Dev. 21 (4): 379-3844-6 (2007)). The discovery facilitated a flood of new drug development activities to develop small molecule inhibitors of BRAF, including GDC-0879, PLX-4720, PLX-4032 / Bemura Fenip (Zelboraf TM) and GSK2118436 [Hoeflich KP, et al., Cancer Res. 69 (7): 3042-3051 (2009); Tsai J, et al., Proc Natl Acad Sci USA 105 (8): 3041-3046 (2008); Bollag G, et al., Nature, 467 (7315): 596-599 (2011); Ribase, & Flaherty KT., Nature Rev. 8 (7): 426-433 (2011)). The inhibitors selectively reduce the growth of BRAF oncogene tumor invading tumor cells and provide hope to patients with melanoma subset with activation mutations in BRAF oncogene [Ribas A & Flaherty KT., Nature Rev. 8 (7): 426-433 (2011)). However, much less anti-tumor efficacy has been observed using the current BRAF small molecule inhibitors for wild-type BRAF melanoma cells [Hoeflich KP, et al., Cancer Res. 69 (7): 3042-3051 (2009); Tsai J, et al., Proc Natl Acad Sci USA 105 (8): 3041-3046 (2008)], new insights into possible therapeutic targets for biology, tumor-induced signaling and disease management in all categories of melanoma patients Thereby increasing the need to identify another melanoma-related driver gene to provide. The RAF kinase family consists of three members, ARAF, BRAF and CRAF, which play a central role in signal transduction from the RAS to the downstream canine MEK1 / 2 and ERK1 / 2 canonical MAPK signaling pathways. However, another kinase has also been reported to play a role in ERK activation. In particular, the activation of the MAPK pathway by phosphorylation of both MEKl in Ser298, a region near the activation loop residue Ser217 / Ser221, which is a substrate for CRAF, and RAF kinase, in Ser338, which is a key residue for activation of the group I p21-activated kinase (PAK) Several contributing groups have been reported [King AJ, et al., Nature, 396 (6707): 180-183 (1998); Tang Y, et al., Mol Cell Biol. 19 (3): 1881-1891 (1999); Frost JA, et al., EMBO J. 16 (21): 6426-6438 (1997)]. Path crosstalk between PAK and MAPK pathway signaling in epithelial cells can be induced by a variety of conditions, including growth factor stimulation and cell attachment to extracellular matrix [Slack-Davis JK, et al ., J Cell Biol. 162 (2): 281-291 (2003); Zang M, et al., J Biol Chem. 276 (27): 25127-25165 (2001); Beesere, et al., J Biol Chem. 280 (44): 36609-36615 (2005)). As a major downstream action factor for the Rho small CTPase Cdc42 and Rac1, PAK1 also plays a key role in linking extracellular signals to alter actin cytoskeleton organization, cell morphology, and attachment dynamics [Arias-Romero LE , &Amp; Chernoff J., Biology Cell. 100 (2): 97-108 (2008); Kumar R, et al., Nat Rev Cancer, 6 (6): 459-471 (2006); Ong CC, et al., Oncotarget. 2 (6): 491-496 (2011)). PAK1 is widely expressed in various finishing tissues, and expression is significantly increased in breast and lung cancer [Holm C, et al., J Natl Cancer Inst. 98 (10): 671-680 (2006); Arias-Romero LE, et al., Oncogene, 29 (43): 5839-5849 (2010); Ong CC, et al., Proc Natl Acad Sci USA 108 (17): 7177-7182 (2011)]. Functional studies have also shown that cell transformation [Vadlamudi RK, et al., J Biol Chem. 275 (46): 36238-36244 (2000)], and tumor growth [Ong CC, et al., Oncotarget. 2 (6): 491-496 (2011); Yi C, et al., Cancer Res. 68 (19): 7932-7937 (2008); Chow HY, et al., PloS One, 5 (11): e13791 (2010)]. These results suggest that PAK1 may be responsible for tumor formation in relation to some diseases.

The invention relates to a method of treating melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In some embodiments, PAK1 is amplified in tumors. 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 PAKl 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 embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some aspects, the object is a human being. In some aspects, the invention provides a method of treating a melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor.

In some aspects, the invention provides a method of treating a melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is a small molecule, nucleic acid or polypeptide. In some aspects, the invention provides a method of treating a melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is a small molecule, nucleic acid or polypeptide. In some aspects, the invention provides a method of treating a melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is used in conjunction with the therapeutic agent. In some aspects, the invention provides a method of treating a melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is used in conjunction with the therapeutic agent.

In some embodiments, the invention provides the use of a PAKl inhibitor for the treatment of melanoma in a subject. The present invention provides the use of a PAKl inhibitor in the manufacture of a medicament for the treatment of melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In some embodiments, PAK1 is amplified in tumors. 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 PAKl 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 embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some aspects, the object is a human being.

In some embodiments, the invention provides compositions and kits comprising a PAKl inhibitor for use in the treatment of melanoma. Various aspects relating to the above methods of treatment are described herein and apply to compositions and kits. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In some embodiments, PAK1 is amplified in tumors. 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 PAKl 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 embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some aspects, the object is a human being.

In some aspects, the invention provides a method of inhibiting CRAF signaling and / or MEK signaling in melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In some embodiments, PAK1 is amplified in tumors. In some embodiments, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In some embodiments, PAK1 is amplified in tumors. 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 PAKl 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 embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some aspects, the object is a human being. In some aspects, the invention provides a method of treating a melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor.

In some embodiments, the invention includes determining a BRAF genotype of a melanoma, wherein the melanoma comprising the wild-type BRAF is a human melanoma suitable for treatment with a PAK1 inhibitor, Provide a way to identify species patients. In some embodiments, the invention includes measuring the expression of PAKl in melanoma, wherein the overexpression of PAKl in melanoma relative to non-cancerous skin cells is indicative of a PAKl inhibitor Lt; RTI ID = 0.0 > melanoma < / RTI > In some embodiments, the invention includes measuring the copy number of PAK1 in a melanoma, wherein the amplification of PAK1 in the melanoma is indicative of the patient being eligible for treatment with the PAK1 inhibitor, A method of identifying a human melanoma patient suitable for treatment. In some embodiments, the invention includes determining the BRAF genotype of the melanoma and determining the expression of PAKl in the melanoma, wherein the overexpression of PAKl in the melanoma relative to the presence of wild-type BRAF and / or non-cancerous skin cells Providing a method of identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor, wherein the patient is eligible for treatment with a PAK1 inhibitor. In some embodiments, the invention includes one or more of determining the BRAF genotype of the melanoma, measuring the expression of PAKl in the melanoma, and determining the number of copies of PAKl in the melanoma, wherein the presence of the wild-type BRAF, A human melanoma patient suitable for treatment with a PAK1 inhibitor, wherein at least one of the overexpression of PAK1 in melanoma and the amplification of PAK1 in melanoma is suitable for treatment with a PAK1 inhibitor compared to non-cancerous skin cells Provide a way to verify.

In some embodiments, the invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the BRAF genotype of the melanoma, wherein the melanoma The species indicates that the patient is suitable for treatment with a PAK1 inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

In some embodiments, the invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the level of PAK1 expression of the melanoma, wherein the non- Overexpression of PAK1 in melanoma indicates that the patient is suitable for treatment with a PAK1 inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

In some embodiments, the invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the number of copies of PAK1 in the melanoma, wherein the PAK1 in the melanoma ≪ / RTI > indicates that the patient is suitable for treatment with a PAK1 inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

In some embodiments, the invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the BRAF genotype of the melanoma and the PAK1 expression level of the melanoma, Wherein overexpression of PAK1 in melanoma relative to melanoma and / or non-cancerous cells comprising wild-type BRAF indicates that the patient is amenable to treatment with a PAK1 inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

In some embodiments, the invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) a BRAF genotype of the melanoma, a PAK1 expression level of the melanoma, and a copy number of the PAK1 in the melanoma , Wherein at least one of overexpression of PAK1 in melanoma and amplification of PAK1 relative to wild-type BRAF, non-cancerous cells indicates that the patient is suitable for treatment with a PAK1 inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

In some embodiments, the invention includes evaluating PAKl expression in a melanoma, wherein overexpression of PAKl in the melanoma is indicative of the modulation of treatment of the individual until no further PAKl overexpression is detected, Provides a method of modulating the treatment of melanoma in patients undergoing treatment. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is amplified in melanoma. In some instances, melanoma is a wild type black species and PAK1 is amplified in melanoma.

Figure 1 shows that PAK1 is highly expressed in human melanoma. (A) Analysis of 11q13 copy number increase in human melanoma tissue. The vertical red line indicates the chromosomal location of the PAK1 gene. (B) Expression of PAK1 DNA complexes and mRNAs correlated with melanoma tumor samples (226507_ in the Affymetrix MAS 5.0 signal). (C) Illustrative images of PAK1 immunohistochemistry in primary human malignant melanoma. Cellular expression scores: 0 (I), 1 (II), 2 (III) and 3 (IV). Color deposition indicates immunoreactivity to hematoxylin versus staining. PAK1 expression also appeared in cells that were inserted into the epidermis, which could also represent stromal cells (III) and Langerhan's cells (IV).
Figure 2 shows that PAK1 plays a crucial role in the proliferation of BRAF wild type melanoma cells. The proliferation of melanoma cells after transfection with (A) siRNA oligonucleotides was measured by Cell TiterGlo ATP consumption analysis. PAK1 was required for cell growth, and data were normalized to the control and expressed as mean ± standard deviation. (B) In a panel of melanoma cell lines, PAK1 inhibition was observed in cells with a BRAF (V600E) mutation (n = 9; p = 0.07; 624MEL, 888MEL, 928MEL, RPMI-7951, A375, Colo829, LOX- 3M, A375), but not BRAF (V600E) mutants (n = 5; 537MEL, Hs940T, MeWO, SK-MEL2, SK-MEL23 and SK-MEL30). (C) Inhibition of PAK1 / 2 reduced accumulation of ERK1 / 2 and MEK1 / 2 phosphorylation and cyclin D1. (D) PAK1 / 2 inhibition in SK-MEL23 BRAF wild-type melanoma cells reduces signaling to the cytoskeleton, MAPK, proliferation and NF-κB pathway as measured by reverse phase protein array (RPPA) analysis. Standardized RPPA results are expressed as mean ± standard deviation. siNTC is a non-targeted control siRNA. siRNA is an NRAS-specific siRNA. siPAK1 is a PAK1-specific siRNA. ΔPAK1 is a chromosomal deletion of the PAK1 gene.
Figure 3 shows a series of immunoblots demonstrating that PAKl is required for CRAF activation in BRAF wild type melanoma cells. (A) PAK1- and PAK2-selective or non-targeted control (NTC) siRNA oligonucleotides were transfected into SK-MEL23 and 537MEL melanoma cells. After 48 hours, endogenous MEK1 (A), MEK2 (B) or CRAF (C) proteins were immunoprecipitated and the complexes were immunoblotted to detect phosphorylation of the residues important for catalytic activation. Total protein levels in the immunocomplexes were also measured as loading controls. (D) cells were treated with DMSO or 5 [mu] M PF-3758039 for 4 hours, endogenous CRAF was immunoprecipitated and immunoblotted against Ser338 phosphorylation. Total CRAF levels in the immune complexes were also shown. (E) SK-MEL23 cells were treated with DMSO, 5 μM PF-3758309 or 20 μM IPA-3 for 4 hours. The CRAF immunoconjugate was incubated with the inactive MEK1 protein for 30 minutes in kinase buffer. Levels of phospho-MEKl (Ser217 / Ser221) were measured and CRAF catalytic activity was recorded as normalized MEKl phosphorylation level for the entire CRAF protein.
Figure 4 includes images demonstrating that PAK is required for melanoma cell migration. Confluent WM-266-4 melanoma cells were stained after transfection with non-targeted control (NTC) or PAK1 / 2 siRNA oligonucleotides for 72 hours, and when lesions occurred (dark shade) and 28 hours (Bright area) after recording. The difference in relative wound density was statistically significant (p <0.001; n = 3).
Figure 5 shows a series of immunoblots showing in vitro differential sensitivity of MAPK signaling in BRAF wild-type and BRAF (V600E) melanoma cells treated with PAK inhibitors. (A) SK-MEL23 and A375 cells were treated with DMSO, 5 [mu] M PF-3758309 or 0.2 [mu] M PLX-4720 for 4 hours and the lysates were analyzed for phosphorylation of MAPK pathway components. A brighter and darker exposure of the p-MEK1 / 2 (S217 / S221) immunoblot is shown. (B) Flag Ectopic expression of the epitope-tagged PAK1 induced MAPK pathway activation in A375 cells. Specificity was demonstrated using PF-3758309 inhibitor treatment as a control.
Figure 6 is a series of graphs showing reduced viability of BRAF wild type melanoma cells due to treatment with an in-house PAK inhibitor. The catalytic inhibition of PAK1 by treatment with I-007, I-054, I-087 and PF-3758309 was assayed using the (A) SK-MEL23 (Promega) And (B) 537 MEL cells.
Figure 7 shows in vivo differential sensitivity of MAPK signaling due to PAK inhibition in xenograft tumor mouse models of BRAF wild type and BRAF (V600E) melanoma. (A) The pharmacodynamic response of BRAF wild-type and mutant tumors measured by phosphorylation of CRAF (Ser338) after vehicle or 35 mg / kg PF-3758309 administration. (B) Anti-tumor efficacy of 10, 15 and 25 mg / kg PF-3758309 ip daily dosing in the SK-MEL23 preclinical tumor model of BRAF wild-type melanoma.
Figure 8 shows a series of graphs showing individual tumor data for the SK-MEL23 preclinical tumor model of BRAF wild-type melanoma. (A) tumor growth inhibition and (B) weight loss for individual animals treated with 10, 15, and 25 mg / kg PF-3758309. To analyze repeated measurements of tumor volume from the same animals over time, nonlinear profiles were adapted to the time course of log ₂ tumor volume at each dose level using a cubic regression spline. The non-linear profile was then associated with capacity in the mixing model. Using a cubic regression spline, nonlinear profiles were adapted to the time trend of log ₂ tumor volume at each dose level. The non-linear profile was then associated with capacity in the mixing model. Tumor growth inhibition (% TGI) as a percent of vehicle was calculated as a percentage of the area under the fit curve (AUC) for each dose group in comparison to the vehicle, using the following formula:% TGI = 100 x (1-AUC _dose / AUC _vehicle ). R version 2.8.1 and Excel version 12.0.1 (Microsoft). Data were analyzed using R version 2.8.1 (R Foundation for Statistical Computing, Vienna, Austria) and mixed models were fit in R using the nlme package, version 3.1-89 .
Figure 9 shows immunoblots showing the differential pharmacokinetic responses of BRAF wild-type tumors treated with G945 BRAF inhibitor or PF-3758309. Phosphorylation of CRAF (Ser338) was measured for SK-MEL23 xenograft tumors following administration of 35 mg / kg PF-3758309 ip or 10 mg / kg G945 (BRAF inhibitor) po compound. Tumors were collected one hour after dosing and were frozen for an instant. Each lane represents a tumor lysate from an individual xenografted mouse.
Figure 10 is a diagram showing the mechanism of actin for PAK1 in BRAF wild-type melanoma. (A) In connection with tumorigenic mutations, BRAF strongly induces activation of the MAPK signaling pathway, and these tumor cells are sensitive to inhibition of the kinase. (B) In melanomas where BRAF is not mutated, increased expression and genomic amplification of PAK1 are frequent, resulting in increased signal transduction to CRAF-MEK-ERK and potentially additional action factor pathways. The melanoma population is relatively insensitive to BRAF inhibition and the proliferative capacity is PAK1 dependent.

The present invention provides methods and compositions for the treatment of melanoma in a subject, wherein the melanoma is contacted with a therapeutically effective amount of a PAK1 inhibitor. The present invention also provides such a method of treatment comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, melanoma overexpresses PAKl relative to non-cancerous cells. In some embodiments, PAKl is amplified in melanoma. In some embodiments, the melanoma is a wild type BRAF melanoma and overexpresses PAKl over non-cancerous cells. In some instances, melanoma is a wild-type BRAF melanoma, melanoma over-expresses PAK1 over non-cancerous cells, and PAK1 is amplified in melanoma.

All references cited herein, including patent applications, patent publications and Genbank accession numbers, are incorporated herein by reference in their entirety, as each individual reference is specifically &lt; RTI ID = 0.0 &gt; do.

Justice

The techniques and procedures described or referred to herein are generally well understood and can be carried out by those skilled in the art using conventional methods, for example, using commonly used methods described in the following references : 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., (2003)); PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORAROTY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, 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; Cell and Tissue 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); 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 defined otherwise, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. John Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., &Lt; RTI ID = 0.0 &gt; New York, NY 1992) provides general guidance to most of the terms used in the present application to those skilled in the art.

As used herein, "PAK" refers to a class of non-receptor serine / threonine protein kinase (STK). The p21-activated protein kinase (PAK) class of serine / threonine protein kinases plays an important role in cytoskeletal organization, cell morphogenesis, cellular processes, and cell survival [Daniels et al., Trends Biochem. Sci. 24: 350-355 (1999); Sells et al., Trends Cell. Biol. 7: 162-167 (1997)). The PAK family consists of six members subdivided into two groups: PAK 1 to 3 (Group I) and PAK 4 to 6 (Group II), which are distinguished on the basis of the presence of self-suppressing regions in the sequence homology and group I PAKs. . The p21-activated kinase (PAK) functions as an important mediator of the Rac and Cdc42 GTPase functions as well as the pathway required for Ras-induced tumorigenesis [Manser et al., Nature, 367: 40-46 (1994); B Dummler et al., Cancer Metathesis Rev. 28: 51-63 (2009); R. Kumar et al., Nature Rev. Cancer, 6: 459-473 (2006)].

As used herein, "PAK1" or "p21-activated protein (Cdc42 / Rac) -activated kinase 1", unless otherwise stated, &Lt; / RTI &gt; and rats). The term refers to any untreated form of PAK1 resulting from genomic location (e.g., the 11q13-q14 cytogenetic band, chromosome 11: 77033060-77185108, and / or GC11M077033) . The term also includes naturally occurring variants of PAKl, such as splice variants or allelic variants. The sequence of an exemplary human PAK1 nucleic acid is NC_000011.9. An exemplary human PAK1 amino acid sequence is NP_0011220921 or NP_002567.3. The sequence of an exemplary mouse PAK1 nucleic acid is NC_000073.6 or an exemplary mouse PAK1 amino acid sequence NP_035165.2. The sequence of an exemplary rat PAK1 nucleic acid is NC_005100.2 or an exemplary rat PAK1 amino acid sequence NP_058894.1. The sequence of an exemplary open PAK1 nucleic acid is NC_006603.3 or an exemplary open PAK1 amino acid sequence XP_849651.1. The sequence of an exemplary small PAK1 nucleic acid is AC_000186.1 or NC_007330.5. An exemplary small PAK1 amino acid sequence is NP_001070366.1. The sequence of an exemplary rhesus monkey PAK1 nucleic acid is NC_007871.1. The exemplary rhesus monkey PAK1 amino acid sequence is XP_001090310.1 or NP_001090423.2. The sequence of an exemplary chicken PAK1 nucleic acid is NC_006088.3 or an exemplary chicken PAK1 amino acid sequence NP_001155844.1.

As used herein, "BRAF" or "serine / threonine-protein kinase B-Raf" Refers to natural BRAF from any vertebrate source, including mammals. The term includes any form of BRAF resulting from genomic location (e.g., 7q34 cytogenetics band, chromosome 7: 140433812-140624564, and / or GC07M140424), untreated BRAF of "full length" do. The term also includes natural variants of BRAF, such as splice variants or allelic variants. The sequence of an exemplary human BRAF nucleic acid is NC_000007.13 or an exemplary human BRAF amino acid sequence NP_004324.2. The sequence of an exemplary mouse BRAF nucleic acid 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. Exemplary dog BRAF nucleic acid sequences are NC_006598.3 or exemplary dog BRAF amino acid sequences XP_532749.3. The sequence of an exemplary chicken BRAF nucleic acid is NC_006088.3 or an exemplary chicken BRAF amino acid sequence NP_990633.1. Exemplary bovine BRAF nucleic acid sequences are AC_000161.1 or exemplary bovine BRAF amino acid sequence XP_002687048.1. Exemplary horses The sequence of the BRAF nucleic acid is NC_009147.2 or exemplary horse BRAF amino acid sequence XP_001496314.2.

"Wild-type BRAF" refers to natural BRAF (including native variants) not associated with melanoma herein. An example of a wild-type human BRAF is provided in Jinbank accession number NP_004324.2. As is known in the art, in connection with BRAF, melanoma can be categorized and classified by BRAF type: wild type BRAF and mutant BRAF.

As used herein, "mutant BRAF" refers to a BRAF protein having one or more mutations associated with melanoma. One example of a mutant BRAF is the mutation of valine at position 600 with glutamate (V600E). As is known in the art, melanomas can be categorized by BRAF type: wild-type BRAF and mutant BRAF.

As used herein, "CRAF" or "v-raf leukemia virus tumor gene 1" refers to a mammal such as a primate (e.g., a human) and rodents (e.g., mice and rats) Quot; natural CRAF " from any vertebrate source, including &lt; / RTI &gt; The term includes any form of CRAF resulting from genomic location (e.g., 3p25 cytogenetic band, chromosome 3: 12625100-12705700, and / or GC03M012625), untreated CRAF of "full length" do. The term also includes natural variants of CRAF, such as splice variants or allelic variants. The sequence of an exemplary human CRAF nucleic acid is NC_000003.11 or an exemplary human CRAF amino acid sequence NP_002871.1.

"MEK" or "mitogen-activated protein kinase" refers to a class of kinase enzymes that phosphorylate mitogen-activated protein kinase (MAPK), as used herein. There are seven genes: MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K3 (MKK3), MAP2K4 (MKK4), 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 signaling pathways. The sequence of an exemplary human MEK1 nucleic acid is NC_000015.9 or an exemplary human MEK1 amino acid sequence NP_002746.1. The sequence of an exemplary human MEK2 nucleic acid is NC_000019.9 or an exemplary human MEK2 amino acid sequence NP_109587.1. The sequence of an exemplary human MEK3 nucleic acid is NC_000017.10. Exemplary human MEK3 amino acid sequences are NP_002747.2 or NP_659731.1. The sequence of an exemplary human MEK4 nucleic acid is NC000017.10 or an exemplary human MEK4 amino acid sequence NP00001.1. The sequence of an exemplary human MEK5 nucleic acid is NC_000015.9. Exemplary human MEK5 amino acid sequences are NP_001193733.1, NP_002748.1, or NP_660143.1. The sequence of an exemplary human MEK6 nucleic acid is NC_000017.10 or an exemplary human MEK6 amino acid sequence NP_002749.2. The sequence of an exemplary human MEK7 nucleic acid is NC_000019.9 or an exemplary human MEK7 amino acid sequence NP_660186.1.

As used herein interchangeably, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide can be any substrate that can be incorporated into the polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or by DNA or RNA polymerases or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and analogs thereof. When present, modifications to the nucleotide structure can be made before or after the formation of the polymer. The sequence of the nucleotide can be interrupted by the non-nucleotide component. The polynucleotide can be further modified after synthesis, for example, by conjugation with a label. Other types of modifications include, for example, "caps ", substitution of one or more natural nucleotides by an analog, inter-nucleotide modifications such as uncharged linkages (e. G., Methylphosphonate, , Phosphoramidate, carbamate, and the like) and modifications by charged bonds (such as phosphorothioate, phosphorodithioate, etc.), pendant residues such as proteins (e.g., (E.g., a metal, a radioactive metal, a boron (e.g., a metal, a radioactive metal, a radioactive metal, , Oxidative metals, etc.), modifications involving alkylating agents, modifications by modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified versions of polynucleotide (s). In addition, any hydroxyl groups typically present in sugars may be substituted by, for example, a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to generate additional bonds to additional nucleotides Or may be bonded to a solid or semi-solid support. The 5 ' and 3 ' terminal OH may be phosphorylated, or substituted with an amine or organic capping moiety of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized with standard protecting groups. The polynucleotides may also be, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, analogs per carboxylic acid, Such as, for example, arabinose, xylose or ricose, pyranose sugar, furanos sugar, sedoheptuloses, acrylic analogs and non-basic abasic nucleoside analogs such as methyl riboside , And may contain analog forms of the ribose or deoxyribose sugar commonly known in the art. One or more phosphodiester linkages may be substituted by an alternative linker. P (S) S ("dithioate"), (O) NR2 ("amidate"), P (O) R, P (O) OR ', CO or CH2 ("formacetal"), wherein R or R' is independently H or optionally an ether (-O-) bond, Substituted or unsubstituted alkyl (1-20C) containing aryl, alkenyl, cycloalkyl, cycloalkenyl or aralidyl. Not all bonds in the polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

As used herein, "oligonucleotide" refers to short single-stranded polynucleotides that are generally, but not necessarily, less than about 250 nucleotides in length. The oligonucleotide may be a synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally applicable to oligonucleotides entirely.

The term "primer" generally refers to a single-stranded polynucleotide that can be hybridized to a nucleic acid after polymerization of a complementary nucleic acid by providing a free 3'-OH group.

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

The term "antibody" is used herein in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments when exhibiting the desired antigen- But are not limited to, various antibody structures.

The term "detection" includes any detection method, including direct and indirect detection.

The term "biomarker " as used herein refers to an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator that can be detected in a sample. A biomarker can serve as an indicator of a particular subtype of a disease or disease (e.g., cancer) that is defined by a particular molecular, pathological, histological, and / or clinical feature. 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 "amount" or " level "of a biomarker associated with an increased clinical benefit to an individual is detectable at a biological sample. These are well known to those skilled in the art and can be measured by the methods disclosed herein. The level or amount of expression of the evaluated biomarker can be used to measure the response to treatment.

The term " level of expression "or" expression level "is generally used interchangeably and generally refers to the amount of a biomarker in a biological sample. "Expression" refers generally to a process by which information (e.g., gene-encoding and / or epigenetic information) is converted into a structure that is present and functional in the cell. Thus, as used herein, "expression" refers to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide).

Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotides and / or polypeptide modifications (e. G., Post-translational modifications of the polypeptide) may also be used as the transcripts or transcribed products produced by alternative splicing Or from post-translational treatment of the polypeptide, for example by proteolysis, should be regarded as expressed. "Expressed gene" includes mRNAs that are transcribed into polynucleotides and then translated into polypeptides, and also those that are transcribed into RNA but not translated into a polypeptide (e.g., transcription and ribosomal RNA).

The terms "elevated expression "," elevated expression level ", "elevated levels ", and" over- expressed "refer to individuals or individuals who do not suffer from a disease, Refers to an increased expression or increased level of biomarker in an individual relative to a host marker (e.g., a housekeeping biomarker). In some instances, elevated expression or overexpression is the result of gene amplification.

"Lowered expression", "lowered expression level" or "lowered level" refers to an individual or individual that is not suffering from a control, eg, a disease or disease (eg, cancer) or an internal control (eg, Refers to a reduced expression or reduced level of a biomarker in an individual relative to a biomarker).

The term "antiferrobiomarker" refers to a group of biomarkers or biomarkers (e.g., polynucleotides and / or polypeptides) that are typically present in all cell types. In some instances, antagonistic biomarkers are "antagonistic genes". "Antagonistic gene" refers herein to a gene or a group of genes that encode a protein whose activity is essential for cell function maintenance and typically resembles all cell types.

As used herein, "amplification" refers generally to the process of generating multiple copies of a sequence of interest. "Multiplexer" refers to two or more photocopiers. A "copy" does not necessarily imply complete sequence complementarity or identity to the template sequence. For example, the radiolabel may be a nucleotide analogue such as deoxyinosine, a sequence that is intentionally altered (e. G., A sequence variation introduced through a primer that hybridizes to the template but does not complement the sequence), and / May include sequence errors that occur. Dicellular cells typically contain two copies of a given gene on each chromosome. In some embodiments of the invention, "amplification" of an intracellular chromosomal gene refers to a process in which more than two copies of a gene are present in a cell.

The term "multi-PCR" refers to a single PCR reaction performed in a nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.

The term "diagnosis" is used herein to denote identification or classification of a molecular or pathological condition, disease or disease (e.g., cancer). For example, "diagnosis" can refer to the identification of a particular type of cancer. "Diagnosis" can also refer to the classification of a particular subtype of cancer, eg, by histopathological criteria, or by molecular characteristics (eg, a combination of biomarkers or biomarkers For example, a specific gene or a protein encoded by the gene).

The term " assisted diagnosis "is used herein to describe a method that facilitates clinical measurement of the presence or characteristics of a disease or condition (e.g., cancer) of a particular type of symptom or condition. For example, a method of adjunctive diagnosis of a disease or disease (e.g., cancer) may include measuring a particular biomarker in a biological sample from an individual.

&Quot; Correlated "or" correlated "means comparing the performance and / or results of the first analysis or protocol in some way with the performance and / or outcome of the second analysis or protocol. For example, the results of the first analysis or protocol may be used to perform the second protocol, and / or the results of the first analysis or protocol may be used to determine whether a second analysis or protocol should be performed. With respect to the polynucleotide assay or protocol aspect, the results of a polynucleotide expression assay or protocol may be used to determine whether a particular therapeutic regimen should be performed.

"Individual response" or "response" includes, but is not limited to, (1) inhibition of disease progression (eg, cancer progression) to some extent, including delay and complete stopping; (2) reduction in tumor size; (3) inhibition (i. E., Decrease, delay or complete stop) of cancer cell infiltration into adjacent surrounding organs and / or tissues; (4) inhibition of metastasis (i. E., Decrease, delay or complete arrest); (5) to some extent relief of one or more symptoms associated with a disease or disease (e.g., cancer); (6) increased progression-free survival length; And / or (9) any endpoint that represents a benefit in an individual, including reduced mortality at a given point in time after treatment.

The term " predicting "or" predicting "is used herein to indicate the likelihood that a patient will respond favorably or adversely to a particular chemotherapeutic treatment. In one embodiment, "predicting" or "predicting" relates to the extent of the reaction. In one aspect, "predicting" or "predicting" relates to whether a patient survives or improves after treatment, e.g. The predictive methods of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment modality for any particular patient. The predictive method of the present invention can be used to determine whether a patient is likely to respond to a given therapy, including therapeutic treatment, such as administration of a given therapeutic agent or combination, surgical intervention, steroid therapy, Is a useful tool for predicting whether long-term survival is possible.

The term "substantially the same" as used herein means that a person skilled in the art will be able to determine the difference between two values in the context of the biological characteristics as measured by the values (e.g., K _d value or expression) Quot; means a sufficiently high degree of similarity between two values so as to have little or no biological and / or statistical significance. The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20% and / or less than about 10% as a function of the reference / comparison value.

As used herein, the phrase "substantially different" means that a person skilled in the art will be able to determine the difference between two values in the context of the biological characteristics measured by the values (e.g., K _d values) It means a sufficiently high degree of difference between the two values to be considered meaningful. The difference between the two values is, for example, 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 reference / comparison molecule.

The word "marker" refers to a detectable compound or composition when used herein. The label is typically conjugated or fused to a reagent, such as a polynucleotide probe or antibody, either directly or indirectly, to facilitate detection of the reagent to which it is conjugated or fused. A label may itself be detectable (e. G., A radioactive isotope label or a fluorescent label), or, if it is an enzyme label, facilitate chemical changes of the substrate compound or composition providing the detectable product.

"Effective amount" of an agent refers to an amount effective at the dosage required to achieve the desired therapeutic or prophylactic result and over the time required.

A "therapeutically effective amount" of a substance / molecule, agent, or antagonist of the present invention refers to the amount of a substance / molecule, agonist or antagonist that is capable of eliciting an objective response in a subject, And the like. A therapeutically effective amount is also an amount in which any toxic or deleterious effect of a substance / molecule, agent, or antagonist is increased by a therapeutically beneficial effect.

"Amount of prophylactic effect" refers to an amount effective at the dosage required to achieve the desired prophylactic result and for the time required. Typically, but not necessarily, the prophylactic dose will be less than the therapeutically effective amount, because the prophylactic dose is used prior to or early in the initial stage of the disease in the subject.

In the case of melanoma, a therapeutically effective amount of a PAK1 inhibitor reduces the number of cancer cells; Reduce primary tumor size; Inhibiting (i.e., delaying and preferably stopping) cancerous cell infiltration into peripheral organs; Inhibiting (i.e., delaying and preferably stopping) tumor metastasis; To some extent to inhibit or delay tumor growth or tumor progression; One or more symptoms associated with the disease may be alleviated to some extent. The drug may be cytostatic and / or cytotoxic to such an extent that the drug can inhibit growth and / or kill existing cancer cells. In the case of cancer treatment, the in vivo efficacy can be measured, for example, by assessing survival time, time to disease progression (TTP), response rate (RR), response time and / or quality of life.

"Reducing" or "inhibiting" is reducing or decreasing activity, function and / or amount relative to a reference. In certain embodiments, "reducing" or "inhibiting" means an ability to cause a total reduction of 20% or more. In yet another aspect, "reducing" or "inhibiting" means an ability to cause a total reduction of 50% or more. In another embodiment, "reducing" or "inhibiting" means an ability to cause a total reduction of 75%, 85%, 90%, 95% or more. The presence or size of the metastasis, the size of the primary tumor, or the size or number of blood vessels in an angiogenic disorder.

The term "pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient is effective and does not contain additional ingredients that are toxic enough that the formulation will not be acceptable to the recipient. The formulation can be sterilized.

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

"Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the non-toxic active ingredient to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" is an approach to obtaining beneficial or desired clinical results. In the present invention, beneficial or desired clinical results include, but are not limited to, any one or more of the following: relief of one or more symptoms, reduction in the severity of the disease, diffusion of the disease (e.g., Prevention or delay of disease recurrence, delay or delay of disease progression, improvement of disease state, and remission (whether partial or total). "Treatment" also includes a reduction in the pathological consequences of a proliferative disease. The methods of the invention contemplate any one or more of the above therapeutic embodiments.

The term "melanoma" refers to a highly malignant tumor that begins in melanocytes of normal skin or point and transitions rapidly and broadly. The term "melanoma" can be used interchangeably with the terms "malignant melanoma", "black carcinoma", "black epithelial tumor" and "black sarcoma".

As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pro-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disease", "proliferative disease" and "tumor" are not mutually exclusive when referred to herein.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal, typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. More specific examples of such cancers include, but are not limited to: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung Pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer including gastric cancer, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer and gastric cancer. , Colon cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal cancer or renal cell cancer, prostate cancer, extramural cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, superficial melanoma, malignant melanoma , Terminally black melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (low grade / follicular non-Hodgkin lymphoma (NHL); Small lymphocyte (SL) NHL; Medium grade / follicular NHL; Medium grade diffusivity NHL; High grade immunoblastic NHL; High grade lymphoid constituents NHL; High grade consumed - split cells NHL; Bulky disease NHL; Mantle cell lymphoma; AIDS-related lymphoma; And Waldenstrom ' s Macroglobulinemia); Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Shape cell leukemia; Chronic lymphocytic leukemia; And post-transplant lymphoproliferative disorders (PTLD), and abnormal vascular proliferation, edema (e.g., edema associated with brain tumors), and Meigs syndrome, brain and head and neck cancer, and associated metastasis. In certain embodiments, the cancer that is suitable for treatment with an antibody of the invention includes but is not limited to breast, colon, rectal, non-small cell lung, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell, prostate, , Soft tissue sarcoma, Kaposi sarcoma, carcinoid, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from non-small cell lung cancer, colon cancer, glioblastoma, and breast cancer, including the metastatic form of the cancer.

The term "chemotherapy" refers to therapies useful for treating cancer. Examples of chemotherapeutic agents include, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, Other agents, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g., Gleevec (TM) (Imatinib Mesylate), COX-2 inhibitors (such as celecoxib), interferon, Antagonists (e.g., neutralizing antibodies) that bind to one or more of the following target PDGFR-beta, BlyS, APRIL, BCMA receptor (s), TRAIL / Apo2, and other bioactive and organic chemical agents But are not limited to these. Combinations of these are also included in the present invention.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term refers to a radioisotope (e.g., radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu) For example, it is also possible to use other drugs such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, And fragments thereof, such as nucleic acid degrading enzymes, antibiotics and toxins, such as small molecule toxins or enzymatically active toxins (including fragments and / or variants thereof) having bacterial, fungal, plant or animal origin, And various antitumor or anti-cancer drugs described below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

A "toxin" is any substance that can have a detrimental effect on cell growth or proliferation.

"Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclosporphamides (CYTOXAN TM); Alkyl sulphonates, such as, for example, clay plates, impro sulphates and poly sulphates; Aziridines, such as benzodopa, carbobucone, metouredopa, and uredopa; Ethyleneimine and methylamelamine, including altretamine, triethylene melamine, triethylenephosphoramide, triethylene thiophosphoramide and trimethylolmelamine; Acetogenin (especially bulatacin and bulatacinone); Delta-9-tetrahydrocannabinol (doroninol, MARINOL (R)); Beta-rapacon; Rafacall; Colchicine; Betulinic acid; Camptothecin (including synthetic analogous topotecan (HYCAMTIN TM), CPT-11 (irinotecan, CAMPTOSAR TM), acetylcamptothecin, scopolylectin and 9-aminocamptothecin; Bryostatin; Calistatin; CC-1065 (including its adogelesin, chaselesin and non-gelsin analogs); Grape philatoxin; Grapefinal acid; Tenifocide; Cryptophycin (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Duocamycin (including synthetic analogs, KW-2189 and CB1-TM1); Eluterin; Pancreatistin; Sarcoctic tine; Sponge statin; Nitrogen mustards such as chlorambucil, chlorpavine, chlorophosphamide, estramustine, ifposamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novolacin, phenesterin , Fred Nimustine, Troposphamide, Uracil Mustard; Nitrosoureas such as camustine, chlorozotocin, potemustine, roomustine, nimustine and lanimustine; Antibiotics, such as, for example, endothelin antibiotics [eg, calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega I 1 (see, for example, Nicolaou et al., Angew. Chem Int. Engl., 33: 183-186 (1994)); CDP323, oral alpha-4 integrin inhibitor; Dynemycin including Dynemycin A; Esperamicin; And neocaxinostatin chromophore and related chromoprotein endian antibiotic chromophore], acclinomycin, actinomycin, otramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, caridinophilin , Doxorubicin [ADRIAMYCIN (R), morpholino-doxorubicin, cyanomolybdenum, doxorubicin, doxorubicin, (DOXIL &lt; (R) &gt;, liposomal doxorubicin TLC D-99 (MYOCET TM), PEGylated liposomal doxorubicin (E.g., CAELYX (registered trademark)) and deoxydoxorubicin], epirubicin, esorubicin, dirubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, Mechin, Pepel Antimetabolites such as methotrexate, methotrexate, methotrexate, methotrexate, methotrexate, methotrexate, methotrexate, methotrexate, methotrexate, , Gemcitabine (GEMZAR®), Tegafur (UFTORAL®), Capecitabine (XELODA®), Epothilone and 5-fluorouracil (5-FU), a folic acid analog such as denonfterin, methotrexate, proteopterin, trymetrexate, purine analogs such as fludarabine, 6-mercaptopurine, Guanine, pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, camopur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine, androgen, For example, there may be mentioned carlos teron, dromoglomerolone propionate, epithiostanol, Adrenaline, such as aminoglutethimide, mitotane, trilostane, folic acid, such as, for example, proline acid, acetic acid, aldophosphamide glycoside, amino levulin Acid, enil uracil, amsacrine, vestrabacil, bsanthrene; Edatroxate; Depopamin; Demechecine; Diazquinone; Elformin; Elifthinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Fur monotherapy; Nitraerine; Pentostatin; Phenamate; Pyra rubicin; Rosoxanthrone; 2-ethylhydrazide; Procarbazine; PSK (R) polysaccharide complex (JHS Natural Products, Eugene, OR); Lauric acid; Liqin; Xanthopyran; Spirogermanium; Tenuazonic acid; Triazinone; 2,2 ', 2'-trichlorotriethylamine; Tricothexene (particularly T-2 toxin, veraclin A, loridine A and angiidine); urethane; Vindesine (ELDISINE TM, FILDESIN TM); Takabazin; Mannostin; Mitobronitol; Mitolactol; Pipobroman; Astaxanthin; Arabinoside ("Ara-C"); Thiotepa; Taxoids such as paclitaxel (TAXOL TM), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE TM) and docetaxel (TAXOTERE TM); Clorane boiling; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN (R)) and carboplatin; (VELBAN TM), VINCYCLINE (ONCOVIN TM), VENDECIN (ELDISINE TM, FILDESIN TM) and vinorelbine (NAVELBINE (R)), which prevents tubulin polymerization from forming microtubules; Etoposide (VP-16); Iospasmide; Mitoxantrone; Leucovorin; Nobanthrone; Etrexate; Daunomaisin; Aminopterin; Ibandronate; Topoisomerase inhibitors RFS 2000; Difluoromethylorhornithine (DMFO); Retinoids, including bexarotene (TARGRETIN TM), for example retinoic acid; Bisphosphonates such as claudonate (e.g. BONEFOS TM or OSTAC TM), ethydronate (DIDROCAL TM), NE- 58095, zoledronic acid / zoledronate (ZOMETA TM, alendronate (FOSAMAX TM), pamidronate (AREDIA TM), tylurodonate (TM) Antisense oligonucleotides, particularly those involved in abnormal cell proliferation, such as SKELID (R)) or risedronate (ACTONEL TM), troxacytavin (1,3-dioxolane nucleoside cytosine analog) (EGF-R); vaccines such as the THERATOPE &lt; (R) &gt; vaccine, and the like, which inhibit gene expression in the pathogenesis pathway, such as PKC-alpha, Raf, H-Ras and epithelial growth factor receptor Gene therapy vaccines, such as ALLOVECTIN, Topoisomerase 1 inhibitors (e.g., LURTOTECAN (R)), rmRH (e.g., Avarex (R)) vaccine, ; SU-11248 (SUTENT, Pfizer); periospin, COX-2 inhibitor (&lt; RTI ID = 0.0 &gt; (Eg, celecoxib or etoricoxib), proteosome inhibitors (eg, PS341), bortezomib (VELCADE®), CCI-779, typhipanib (R11577), orapenib, ABT510; Bcl-2 inhibitors, such as, for example, sodium olebmercen (GENASENSE TM), xanthrone, EGFR inhibitors (see below), tyrosine kinase inhibitors (see below), serine-threonine kinase inhibitors Such as, for example, rapamycin (sirolimus, RAPAMUNE TM), parnesyl transferase inhibitors such as ronafarnib (SCH 6636, Sarasar (SARASAR, &lt; / RTI &gt; And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing agents; And CHOP, which is an abbreviation for the combination therapy of two or more of the above drugs, for example, combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; And FOLFOX, an abbreviation for treatment regimens using oxaliplatin (eloxatin (TM)) in combination with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include "anti-hormonal agents" or "endocrine therapies" that serve to modulate, decrease, block or suppress the effects of hormones capable of promoting cancer growth. These may be the hormone itself, including but not limited to: tamoxifen (NOLVADEX TM), 4-hydroxy tamoxifen, toremifene (FARESTON TM), doxifene Antagonist profiles with a mixed agonist / antagonist profile, including dexoxifene (EVISTA TM), trioxifen, keoxifen and selective estrogen receptor modulators (SERM) such as SERM3. (FASLODEX TM) and EM800, which block estrogen receptor (ER) dimers and are capable of blocking DNA (e. G. Inhibit binding, increase ER replacement and / or inhibit ER levels); steroidal aromatase inhibitors such as pomestan and eugemestane (AROMASIN &lt; (R) &gt;) and Non-steroidal Aromata An aromatase inhibitor including an inhibitor such as anastrazole (ARIMIDEX TM), letrozole (FEMARA TM) and aminoglutethimide, and a borozol (RIVISOR, Other aromatase inhibitors including megestrol acetate (MEGASE TM), fadoloz and 4 (5) -imidazole; leuprolide (LUPRON, Progesterone such as megestrol acetate and medroxyprogesterone acetate, estrogen, such as estrogens such as, for example, luteinizing hormone-releasing hormone agonists, including luteinizing hormone- For example, diethylstilbestrol and premarin, and androgens / retinoids, such as, for example, sex hormones including all the transretinoic acid and fenretinide; onafristone; anti-progesterone Theron; estrogen receptor down-regulators (ERD); anti-androgens, e.g., flutamide, carbonyl and imide Baikal rutile rutile imide; and any salts that are pharmaceutically acceptable, of said agent, an acid or a derivative thereof; And combinations of two or more of the foregoing agents.

The term "prodrug" as used herein refers to a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to a tumor cell as compared to the parent drug and can be converted to a parent drug form that is enzymatically active or more active 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 the invention include prodrugs such as phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid- A prodrug, a glycosylation precursor drug, a beta-lactam-containing prodrug, an optionally substituted phenoxyacetamide-containing prodrug or an optionally substituted phenylacetamide-containing prodrug, 5-fluorocytosine and other 5-fluoro But are not limited to, pyrrolidine prodrugs. Examples of cytotoxic drugs that can be derivatized in the form of prodrugs for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

"Growth inhibitor" as used herein refers to a compound or composition that inhibits the growth of cells (e. G., Melanoma cells). Examples of growth inhibitory agents include agents that inhibit cell cycle progression (at a stage other than the S-group), such as agents that induce G1 inhibition and M-group inhibition. Traditional M-group blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin . Drugs that inhibit G1, such as DNA alkylating agents such as tamoxifen, prednisone, takavazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara- It goes crazy. Further information can be found in Murakami et al., Mendelsohn and Israel, eds., Chapter I, entitled "Cell cycle regulation, oncogene, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially p.13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. TAXOTERE®, Rhone-Poulenc Rorer), derived from Western blots, was obtained from paclitaxel (TAXOL®, Bristol-Myers Squibb) Lt; / RTI &gt; Paclitaxel and docetaxel promote the formation of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in suppression of mitosis in cells.

"Radiotherapy" refers to the use of directed gamma rays or beta rays that limit the ability of a normally functioning cell or induce sufficient damage to the cell to completely destroy the cell. It will be appreciated that there will be many methods known in the art for determining dosage and duration of treatment. Typical treatment is provided as a single dose, with a typical dosage being 10 to 200 units per day (gray).

"Object" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs and horses), primates (eg, non-human primates such as humans and monkeys), rabbits and rodents ), But is not limited thereto. In certain embodiments, the subject or subject is a human.

Administration "together" with one or more other therapeutic agents includes simultaneous (concurrent) and sequential or sequential administration in any order. The term "simultaneously" is used herein to denote administration of two or more therapeutic agents, at least a portion of the administration overlapping in time. Thus, concurrent administration includes dosing regimens when administration of one or more other agent (s) is discontinued after discontinuation of administration of one or more other agent (s).

"Reduce or inhibit" refers to the ability to cause a total reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% . "Reducing or inhibiting" can refer to the symptom of the disease being treated, the presence or size of the metastasis, or the size of the primary tumor.

The term "package insert" is intended to refer to instructions that are typically included in commercial packages of therapeutic products, including information on indications, usage, dosage, administration, combination therapy, contraindications, and / . In some aspects, the package insert of the present invention includes instructions for treating melanoma with a PAK1 inhibitor.

A "product" is a product for the treatment of one or more reagents, such as a disease or disease (e.g., cancer), or any product comprising a probe for specifically detecting the biomarker described herein For example, a package or a container) or a kit. In certain embodiments, the product or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

A "target audience" is a group of people or facilities, such as individuals, groups, newspapers, medical literature, or the like, for which a particular medicament is intended to be promoted or promoted specifically for a particular use, And readers of magazines, television or internet viewers, radio or internet listeners, doctors, pharmaceutical companies, and the like.

As will be understood by those skilled in the art, references herein to "approximate" values or parameters include (and describe) aspects of the values or parameters themselves. For example, the description of "about X" includes a description of "X ".

Aspects and aspects of the invention described herein are understood to include "done" or "essentially" with the aspects and aspects. As used herein, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise.

An "object", "subject" or "patient" is a vertebrate animal. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (eg, cattle), sports animals, pets (eg, cats, dogs and horses), primates, mice and rats. In certain embodiments, the mammal is a human.

As used herein, the term "sample" or "test sample" refers to a cell that is to be characterized and / or identified based on, for example, physical, biochemical, chemical and / Refers to a composition obtained or derived from a corresponding subject containing another molecular substance. For example, the phrase "disease sample" and its variants refer to any sample obtained from a subject known to contain or expect to contain a cell and / or molecular substance to be characterized. In one embodiment, the definition includes blood and other liquid samples having biological origin, and tissue samples, such as biopsy samples or tissue cultures, or cells derived therefrom. The source of tissue samples may be fresh or / and frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood components; body fluids; And from tissues or plasma at any time during pregnancy or development of the subject. The sample may be a primary or a cultured cell or a cell line, a cell supernatant, a cell lysate, a platelet, a serum, a plasma, a vitreous liquid, a lymphatic fluid, a synovial fluid, a follicular fluid, a semen, a amniotic fluid, But are not limited to, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts and mixtures thereof.

The term "sample" or "test sample" is intended to refer to a sample or a sample that has been obtained in any manner, such as by treatment with a reagent, solubilization, or in a solid or solid matrix for the purpose of concentration or fragmentation of a particular component such as a protein or polynucleotide Includes engineered biological samples. As used herein, a "section" of a tissue sample refers to a single piece or piece of tissue sample, for example, a thin slice of tissue or cell that has been cut from a tissue sample. In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used for diagnostic analysis. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain representative fragments of tumor tissue. Alternatively, the tumor cells may be obtained indirectly, for example, from a skin sample, in the form of a tissue or fluid known or thought to contain the tumor cells.

"Tissue sample" or "cell sample" refers to a collection of similar cells obtained from tissue of a subject or individual. The source of the tissue or cell sample may be fresh and / or frozen and / or preserved organs, tissue samples, biopsies and / or aspirates; Any blood component such as blood, or plasma; Body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or intercellular fluid; It may be a solid tissue, such as from a cell at any time during pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines. Alternatively, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" means a sample, cell, tissue, Level. In one embodiment, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue may comprise a healthy and / or non-diseased part of the body of the same subject or individual (e.g., a tissue or a cell) , And are obtained from healthy and / or diseased cells or tissues (e. G., Cells or tissues adjacent to the tumor) adjacent to diseased cells or tissues. In some embodiments, the reference sample is a non-cancerous skin cell. In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the same subject or individual's body. In some embodiments, the reference sample is non-cancerous skin cells of the same subject or individual's body. In another embodiment, the reference sample, the reference cell, the reference tissue, the control sample, the control cell, or the control tissue is a healthy and / or non-diseased part (e.g., tissue or cell) &Lt; / RTI &gt; In some embodiments, the reference sample is a non-cancerous skin cell of the subject or a non-individual subject. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from non-treated tissue and / or cells of the subject's non-subject individual or individual.

In certain embodiments, the reference sample is a single sample or a composite multiple sample from the same subject or patient obtained at one or more time points different from when the test sample is obtained. For example, a reference sample is obtained from the same subject or patient at a point earlier than when the test sample is obtained. The reference sample may be useful when the reference sample is obtained at the initial diagnosis of cancer and the test sample is obtained when the cancer later becomes metastatic.

In certain embodiments, the reference sample comprises all types of biological samples as defined above under the term "sample" obtained from one or more subjects that are not the subject or patient. In certain embodiments, the reference sample is obtained from one or more subjects having an angiogenic disease (e.g., cancer), but not the subject or patient.

In certain embodiments, the reference sample is a composite multiple sample obtained from one or more healthy individuals that are not the subject or the patient. In certain embodiments, the reference sample is a composite multiple sample obtained from one or more individuals having a disease or condition (e.g., an angiogenic disorder such as cancer), but not a subject or a patient. In certain embodiments, the reference sample is a collected RNA sample from normal tissue, or a collected plasma or serum sample from one or more subjects that are not the subject or the patient. In certain embodiments, the reference sample is an RNA sample collected from a tumor tissue, or a collected plasma or serum sample from one or more individuals having a disease or condition (e.g., an angiogenic disorder such as a cancer) to be.

As used herein, a "section" of a tissue sample refers to a single section or piece of tissue sample, eg, a thin slice of tissue or cell that has been cut from a tissue sample. It is understood that multiple fragments of a tissue sample can be harvested and applied to the analysis, provided that the same fragments of the tissue sample are analyzed both at the morphological and molecular levels or can be analyzed for both the polypeptide and the polynucleotide .

The level / amount of expression of the gene or biomarker may be determined qualitatively and / or quantitatively based on any suitable criteria known in the art including, but not limited to, mRNA, cDNA, protein, protein fragments and / Can be measured quantitatively. In certain embodiments, the expression / amount of gene or biomarker in the first sample is increased relative to the expression / amount in the second sample. In certain embodiments, the expression / amount of the gene or biomarker in the first sample is reduced relative to the expression / amount in the second sample. In a particular embodiment, the second sample is a reference sample.

In certain embodiments, the terms "increase" or "over-express" refer to a change in the level of protein or nucleic acid detected by standard methods known in the art, such as about 5%, 10% , Or a total increase of 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% It says. In certain embodiments, the term "increasing" or "overexpressing" refers to an increase in the level / amount of expression of a gene or biomarker in a sample wherein the increase is the level / At least about 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 25 times, 50 times, 75 times or 100 times.

In certain embodiments, the term "decreased " is used herein to refer to a decrease in protein or nucleic acid level by about 5%, 10%, 20% Refers to a total reduction of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% In certain embodiments, the term "decrease" refers to a decrease in the level / amount of expression of a gene or biomarker in a sample, wherein the decrease is at least about 0.9 times the expression level / amount of each gene or biomarker in the reference sample, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, 0.1 times, 0.05 times, or 0.01 times.

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

In certain embodiments, "correlated" or "correlated" means comparing the performance and / or results of the first analysis or protocol in some way with the performance and / or outcome of the second analysis or protocol. For example, the results of the first analysis or protocol may be used to perform the second protocol, and / or the results of the first analysis or protocol may be used to determine whether a second analysis or protocol should be performed. With regard to the aspects of gene expression analysis or protocols, the results of gene expression analysis or protocols can be used to determine whether a particular therapeutic regimen should be performed.

As 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 antibody, and which facilitates detection of a reagent to which it is conjugated or fused. A label may itself be detectable (e. G., A radioactive isotope label or fluorescent label), or, if it is an enzyme label, facilitate chemical changes in the detectable substrate compound or composition.

The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, and may include modified amino acids, and non-amino acids may be involved. The term may also be used naturally or by intervention; For example, amino acid polymers modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e. G., Conjugation with a labeling moiety. The definition is, for example, a polypeptide containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), and other variants known in the art. The term "polypeptide" as used herein specifically includes "protein ". The terms "polypeptide" and "protein ", as used herein, particularly include antibodies.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from one or more contaminant nucleic acid molecules that are typically bound in the natural source of the polypeptide nucleic acid. The isolated nucleic acid molecule differs from the form or environment found in nature. Therefore, isolated nucleic acid molecules are distinguished from nucleic acid molecules when they are present in natural cells. However, the isolated nucleic acid molecule includes, for example, a nucleic acid molecule contained in a cell that normally expresses the polypeptide, wherein the nucleic acid molecule is present at a chromosomal location different from the nucleic acid molecule of the native cell.

As used herein, a "gene", "target gene", "target biomarker", "target sequence", "target nucleic acid" or "target protein" is the polynucleotide or protein of interest . Generally, "template" as used herein is a polynucleotide containing a target nucleotide sequence. In some cases, the terms "target sequence", "template DNA", "template polynucleotide", "target nucleic acid", "target polynucleotide" and variants thereof are used interchangeably.

A "native sequence" polypeptide includes a polypeptide having the same amino acid sequence as a naturally derived polypeptide. Thus, native sequence polypeptides may have the amino acid sequence of a native polypeptide from any mammal. The native sequence polypeptides may be isolated in nature or may be produced by recombinant or synthetic means. The term "native sequence" polypeptide refers in particular to the native, truncated or secreted form of the polypeptide (e. G., Extracellular domain sequences), native variant forms (e. G., Optionally spliced forms) Allelic variants.

An "isolated" polypeptide or "isolated" antibody is one that has been identified, isolated and / or recovered from one component of its natural environment. Contaminant components of the natural environment are substances that interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a particular embodiment, the polypeptide is selected from the group consisting of: (1) at least 95%, or at least 99% by weight of the polypeptide as measured by the Lowry method; (2) N (3) homogeneously purified by SDS-PAGE using Coomassie blue or silver staining under reducing or nonreducing conditions, to a sufficient degree to obtain at least 15 residues of terminal or internal amino acid sequence will be. The isolated polypeptide comprises the polypeptide in situ within the recombinant cell, since there will be no more than one component of the native environment of the polypeptide. Typically, however, isolated polypeptides will be produced by one or more purification steps.

A polypeptide "variant" means a biologically active polypeptide having about 80% or more amino acid sequence identity with a native sequence polypeptide. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. Typically, a variant will have at least about 80% amino acid sequence identity with a native sequence polypeptide, more preferably at least about 90% amino acid sequence identity, even more preferably at least about 95% amino acid sequence identity.

The term "benefit" is used in its broadest sense and refers to any desired effect, and in particular includes clinical benefit as defined herein.

Clinical benefits include the inhibition of disease progress to some extent, including various endpoints, e.g., delay and complete inhibition; Reduction in the number of symptom manifestations and / or symptoms of the disease; Reduction of lesion size; (I. E., Decreased, delayed or complete arrest) of disease cell infiltration into adjacent organs and / or tissues; Inhibition of disease spread (i. E., Decrease, delay or complete stop); Reduction of autoimmune reactions that may cause regression or elimination of disease sites but which do not necessarily occur; Alleviating to some extent one or more symptoms associated with the disease; An increase in the length of a disease-free condition, e. G., Progression-free survival after treatment; Increased overall survival; Higher response rate; And / or by evaluating the reduced mortality at that time after treatment.

The method of the present invention

The invention provides a method of treating a melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a PAK1 inhibitor.

Melanoma is a malignant tumor of cells that produces melanin cells, for example, melanin, a dark pigment that determines skin color. Melanomas occur primarily in the skin, but are also found in other parts of the body, including intestines and eyes (eg, uveal melanoma). Melanoma can occur in any part of the body that contains melanocytes. Examples of melanoma include, but are not limited to, superficial melanoma, nodular melanoma, malignant melanoma, and terminal black malachite. Melanoma can be staged according to many criteria, including size, ulceration, spread to lymph nodes, and / or spread to other tissues or organs. In some aspects, the invention provides a method of treating a primary melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some aspects, the invention provides a method of treating a second stage melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some aspects, the invention provides a method of treating a third stage melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some aspects, the invention provides a method of treating a fourth stage melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some aspects, the invention provides a method of treating metastatic melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some aspects, the invention provides a method of treating recurrent melanoma in an individual by contacting the melanoma with a PAK1 inhibitor. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the PAK1 inhibitor is a small molecule inhibitor of PAK1. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

In some embodiments, the invention provides a method of treating a melanoma in a subject, wherein the melanoma is a wild-type BRAF melanoma. In some aspects, the invention provides a method of treating a wild-type BRAF melanoma, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some aspects, the invention provides a method of treating a wild type BRAF melanoma, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor. BRAF is a member of the Raf kinase family of serine / threonine-specific protein kinases. BRAF plays a role in regulating the MAP kinase / ERK signaling pathway (RAF-MEK-ERK pathway), which affects cell division, differentiation and secretion. RAF-MEK-ERK signaling often causes regulatory abnormalities in the cancers. More than 30 mutations in the BRAF gene associated with human cancer have been identified. The frequency of BRAF mutations varies widely from more than 80% in melanoma to 0-18% in other tumors in human cancer, for example, 1% to 3% in lung cancer and 5% in colorectal cancer. A common mutation found in cancer, particularly melanoma, is the substitution of valine by glutamate at codon 600 (i.e., V600E). For example, thymine is replaced with adenine at nucleotide 1799, leading to a V600E mutation. The V600 mutation of BRAF induces constitutive BRAF kinase activity. Methods for genotyping BRAF in melanomas are known to those of skill in the art; For example, the nucleotide sequence of the BRAF gene from a melanoma can be determined using standard sequence analysis methods or using the KASP SNP genotyping system (KBioscience). In some aspects, the invention provides a method of treating a melanoma in a subject, wherein the melanoma is a wild-type BRAF melanoma. In some aspects, the invention provides a method of treating melanoma in a subject, wherein said melanoma overexpresses PAK1 over wild-type BRAF melanoma and melanoma over non-cancerous cells. In some aspects, the invention provides a method of treating melanoma in a subject, wherein said melanoma comprises wild-type BRAF, and PAK1 is amplified in melanoma. In some embodiments, melanoma is a wild-type BRAF melanoma, wherein PAK1 is overexpressed in melanoma compared to non-cancerous cells, and PAK1 is amplified in melanoma. In some aspects, the invention provides a method of treating a melanoma in a subject, wherein the melanoma is a mutant BRAF melanoma. In some aspects, the invention provides a method of treating a melanoma in a subject, wherein the melanoma is a mutant BRAF melanoma, and the melanoma overexpresses PAKl relative to non-cancerous cells. In some aspects, the invention provides a method of treating melanoma in an individual, wherein said melanoma comprises a mutant BRAF and PAK1 is amplified in a melanoma. In some aspects, the invention provides a method of treating a melanoma in a subject, wherein the melanoma comprises a mutant BRAF, and wherein the mutant BRAF is not a V600E mutant BRAF. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

In some embodiments, the invention provides a method of treating melanoma in a subject by contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the invention provides a method of treating a melanoma in a subject by administering to the subject a therapeutically effective amount of a PAKl inhibitor. PAK is involved in many pathways that are not normally regulated in human cancer cells. PAK1 is a component of both the mitogen-activated protein kinase (MAPK), JUN N-terminal kinase (JNK), steroid hormone receptor and nuclear factor (NF) signaling pathways associated with tumorigenesis. PAK activates MEK and RAF1 by phosphorylating them on serine 298 and serine 338, respectively. Increased Ras-induced transduction by PAK1 was correlated with its effect on signaling through the extracellular signal-regulated kinase (ERK) -MAPK pathway, and is independent of its effect on the JNK or p38-MAPK pathway [R. Kumar et al., Nature Rev. Cancer, 6: 459 (2006)]. Constitutive activation of the ERK / MEK pathway is involved in tumor formation, progression, and survival, and is also associated with aggressive phenotypes characterized by uncontrolled proliferation, loss of control of apoptosis and poor prognosis [J. Spicer, Expert Opin. Drug Discov. 3: 7 (2008). Tumorigenesis and progression requires the inactivation of pro-apoptotic signals in cancer cells. PAK activity has been shown to down-regulate several important pro-apoptotic pathways. PAK1 phosphorylation of RAF1 induces RAF1 translocation to the mitochondria, where the mitochondria phosphorylate apo-apoptotic protein BCL2-antagonist (BAD). PAK1, PAK2, PAK4 and PAK5 may also be selected, for example, in selected strains, including CV-1 (monkey) in origin and SV40 (COS) kidney, Chinese hamster ovary (CHO) and human fetal kidney Direct phosphorylation and inactivation of BAD in cell types [R. Kumar et al., Nature Rev. Cancer, 6: 459 (2006)]. However, the appropriate pathway downstream of PAK1 in human tumor cells is only partially understood.

PAK1 is widely expressed in a variety of normal tissues; Expression is significantly increased in ovarian, breast, and bladder cancer [S. Balasenthil et al., J. Biol. Chme. 279: 4743 (2004); M. Ito et al., J. Urol. 178: 1073 (2007); P. Schraml et al., Am. J. Pathol. 163: 985 (2003)). In luminal breast cancer, genomic amplification of PAK1 is associated with resistance to tamoxifen therapy, possibly as a result of direct phosphorylation of the estrogen receptor by PAK1 and ligand-independent transfer activity [S. Rayala et al., Cancer Res. 66: 1694-1701 (2006)).

In some embodiments, the invention provides a method of treating melanoma in a subject by contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the invention provides a method of treating melanoma in a subject by administering a therapeutically effective amount of a PAKl inhibitor in the subject. In some embodiments, the PAK1 gene is amplified in melanoma. In some embodiments, the number of copies of PAK1 in melanoma is greater than about 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or 5.0. Methods for measuring the number of copies of the PAK1 gene in melanomas are known in the art. For example, the number of copies of the PAK1 gene can be measured using an SNP array such as the Affymetrix 500K SNP array assay. In some aspects, the invention provides a method of treating melanoma in an individual, wherein the number of copies of PAK1 in the melanoma is greater than about 2.5. In some aspects, the invention provides a method of measuring the number of copies of PAKl in a melanoma following treatment with a PAKl inhibitor. In some embodiments, the number of copies of PAKl in melanoma is non-cancerous; For example, it is compared to the number of copies of PAK1 in non-cancerous skin cells. In some embodiments, PAK1 is amplified in melanoma and melanoma overexpresses PAK1. In some embodiments, PAK1 is amplified in melanoma and melanoma is wild-type BRAF melanoma. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

In some embodiments, the invention provides a method of treating a melanoma in a subject by contacting the melanoma with a therapeutically effective amount of the PAK1 inhibitor, wherein the PAKl is overexpressed in the melanoma. In some embodiments, the invention provides a method of treating a melanoma in a subject by administering to the subject a therapeutically effective amount of a PAKl inhibitor, wherein the PAKl is overexpressed in the melanoma. Methods for measuring the expression of PAKl are known in the art. Examples of methods for determining the level of expression of PAK1 in melanoma include, but are not limited to, immunohistochemistry, reverse phase protein arrays (RPPA), quantitative PCR, immunoassays, and the like. PAK1 expression levels can be compared to other tumors and cells using the Gene Expression Omnibus (GEO) database.

In some aspects, the invention provides a method of treating a melanoma in a subject by contacting the melanoma with a PAK1 inhibitor, wherein the PAK1 is overexpressed in the melanoma relative to the non-cancerous cell. In some embodiments, the invention provides a method of treating a melanoma in a subject by administering to the subject a therapeutically effective amount of a PAKl inhibitor, wherein the PAKl is overexpressed in the melanoma. In some embodiments, the expression of PAKl in melanoma is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 % Or more. In some embodiments, the expression of PAK1 in melanoma is about 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0 times, 8.0 times, 9.0 times, 10 times, or 10 times. In some embodiments, melanoma overexpresses PAKl over non-cancerous cells, while melanoma is a wild-type BRAF melanoma. In some instances, melanoma overexpresses PAK1 over non-cancerous cells, and PAK1 is amplified in melanoma. In some embodiments, melanoma overexpresses PAK1 over non-cancerous cells, melanoma is a wild-type BRAF black species, and PAK1 is amplified in melanoma. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

In some embodiments, the invention provides a method of inhibiting CRAF signaling in a melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the invention provides a method of inhibiting CRAF signaling in a melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor. Methods for measuring CRAF signaling are known in the art. For example, CRAF activation can be measured by immunoblotting of CRAF isolated from melanoma from a subject before and / or after treatment with a PAKl inhibitor. Activation of CRAF can be measured using a phospho-CRAF (Ser338) antibody. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in melanoma. In some embodiments, the melanoma is a wild type BRAF melanoma, wherein PAK1 is overexpressed in melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma, wherein PAK1 is amplified in melanoma. In some embodiments, the melanoma is a wild type BRAF black paper, wherein PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

In some embodiments, the invention provides a method of inhibiting MEK signaling in melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the invention provides a method of inhibiting MEK signaling in melanoma in a subject, comprising administering to the subject a therapeutically effective amount of a PAK1 inhibitor. Methods for measuring MEK signaling are known in the art. For example, MEK activation can be measured by immunoblotting of MEK isolated from melanoma from a subject before and / or after treatment with a PAK1 inhibitor. Activation of MEK can be measured using a phospho-MEK1 / 1 (Ser217 / Ser221) antibody. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in melanoma. In some embodiments, the melanoma is a wild type BRAF melanoma, wherein PAK1 is overexpressed in melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma, wherein PAK1 is amplified in melanoma. In some embodiments, the melanoma is a wild type BRAF black paper, wherein PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the subject is a mammal. In some aspects, the object is a human being.

PAK1 Inhibitor of

Provided herein are inhibitors of PAKl useful in the methods described herein (e. G., PAK1 antagonists). In some embodiments, the PAK1 inhibitor is a small molecule, nucleic acid, polypeptide, or antibody. Examples of PAK inhibitors are provided in WO 2007/072153 and WO 2010/07184, which are incorporated herein by reference.

Small molecule

There is provided herein a small molecule for use as a PAK1 inhibitor for the treatment of melanoma. The small molecule is preferably a binding polypeptide or an organic molecule other than an antibody, as defined herein, which binds to PAKl or interferes with PAKl signaling as described herein. Bound organic small molecules can be identified and chemically synthesized using known methods (see, for example, PCT Publication Nos. WO 00/00823 and WO 00/39585). The binding organic small molecule is typically less than about 2000 daltons in size or less than about 1500, 750, 500, 250 or 200 daltons in size, wherein the polypeptide is preferably conjugated to a polypeptide as described herein The organic small molecule can be identified without undue experimentation using known techniques. In this regard, it is noted that techniques for searching organic small molecule libraries for molecules capable of binding to a polypeptide target are known in the art (see, for example, PCT Publication Nos. WO 00/00823 and WO 00/39585 Reference). The binding organic small molecule may be, for example, an aldehyde, ketone, oxime, hydrazone, semicarbazone, carbazide, primary amine, secondary amine, tertiary amine, N- substituted hydrazine, The present invention relates to a process for the production of a compound of the general formula I wherein R 1 is selected from the group consisting of ether, thiol, thioether, disulfide, carboxylic acid, ester, amide, urea, carbamate, carbonate, ketal, thioketal, acetal, thioacetal, allyl halide, arylsulfonate, Compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, aminoalcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, Diazo compounds, acid chlorides, and the like. Small molecule inhibitors of PAK kinase have been described (see WO 2006072831, WO 2007023382, WO 2007072153, WO 2010/071846, US 20090275570).

(I)

&Lt; RTI ID = 0.0 &

A series of PAK1 selective inhibitors prepared on 2-aminopyrido [2,3-d] pyrimidin-7 (8H) -one (I) scaffold were purchased from Afraxis, Inc. (WO 2009086204, WO 2010071846, WO 2011044535, WO 2011156646, WO 2011156786, WO 2011156640, WO 2011156780, WO 2011156775, WO 2011044264). AstraZeneca discloses bicyclic heterocyclic PAK1 inhibitors of formula II (see WO 2006106326).

(III)

(IV)

(V)

Pfizer may be prepared by reacting 1 H-thieno [3,2-c] pyrazole (III), 3-amino-tetrahydropyrrolo [3,4- c] pyrazole (IV) -3-yl) pyrimidine-2,4-diamine (V) (see WO 2004007504, WO 2007023382, WO 2007072153 and WO 2006072831).

(VI)

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

(VII)

A series of N2-bicyclic indolyl, indazolyl and benzimidazolyl derivatives of N4- (1H-pyrazol-3-yl) pyrimidine-2,4-diamine (VII) (U.S. Serial No. 61 / 527,453, filed December 22, 2011) and its azela-indolyl, indazolyl and benzimidazolyl derivatives (U.S. Serial No. 61 / 579,227, filed December 22, 2011) All are incorporated by reference in their entirety (A is indolyl, indazolyl, and benzimidazolyl or an aza derivative thereof).

Nucleic acid

The present invention provides herein polynucleotide agonists of PAK1 for the treatment of melanoma in a subject. The polynucleotide may be an RNAi such as siRNA or miRNA, an antisense oligonucleotide, an RNAzyme, a DNAzyme, an oligonucleotide, a nucleotide, or a single-stranded or double-stranded nucleic acid, , Or genomic or synthetic origin DNA, which may represent a sense or antisense strand for any DNA-like or RNA-like substance of natural or synthetic origin, including iRNA, ribonucleoproteins (e.g., iRNP) And may be any fragment thereof, including RNA (e.g., mRNA, rRNA, tRNA). In some embodiments, the polynucleotide targets PAKl expression (e. G., Targets PAK1 mRNA). The polynucleotide may be an antisense nucleic acid and / or a ribozyme. The antisense nucleic acid comprises a sequence complementary to at least a portion of the RNA transcript of PAK1. Absolute complementarity, though desirable, is not required.

As used herein, the term "complementary to at least a portion of the RNA" means a sequence that is sufficiently complementary to form a stable duplex by hybridization with RNA; In the case of double-stranded PAK1 antisense nucleic acid, therefore, a single strand of duplex DNA can be examined, or triploid formation can be analyzed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridized nucleic acid, the more base mismatches with the PAK1 RNA, so that the nucleic acid contains and can still form a stable duplex (or in some cases triplicate). Those skilled in the art will be able to ascertain an acceptable degree of mismatch using standard procedures for measuring the melting point of a hybridized complex.

Polynucleotides complementary to the 5 'tail of the delivery cipher, such as the 5' untranslated sequence to the codon, including the AUG start codon, should work most effectively in suppressing translation. However, sequences complementary to the 3 'untranslated sequence of mRNA have also been found to be effective in inhibiting translation of mRNA (see generally Wagner, R., Nature 372: 333-335 (1994)). Thus, oligonucleotides complementary to the 5'- or 3'-non-translated, non-coding region of the PAK1 gene can be used in an antisense approach to inhibit translation of endogenous PAK1 mRNA. A polynucleotide complementary to the 5 'untranslated region of the mRNA must contain a complement of the AUG start codon. Antisense polynucleotides complementary to the mRNA encoding region are less effective translational inhibitors, but may be used in accordance with the present invention. Whether designed to hybridize to either the 5'-, 3'-, or the coding region of PAK1 mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, preferably an oligonucleotide at a length in the range of 6 to about 50 nucleotides . In certain embodiments, the oligonucleotide is 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 present invention is produced intracellularly by transcription from an endogenous sequence. For example, a vector or part thereof is transcribed to generate an antisense nucleic acid (RNA) of the PAK1 gene. The vector contains a sequence encoding a PAK1 antisense nucleic acid. The vector may be maintained as an episome or integrated into a chromosome as long as it can be transcribed to produce the desired antisense RNA. The vector may be constructed by recombinant DNA technology, which is standard in the art. The vector may be a plasmid, a virus, or others known in the art, used for replication and expression in vertebrate cells. Expression of a sequence encoding PAK1, or a fragment thereof, can be by any promoter known to act in vertebrate, preferably human, cells. The promoter may be derived or a component. The promoter is a promoter contained in the SV40 early expression promoter region [Bernoist and Chambon, Nature 29: 304-310 (1981)], the 3 'long terminal repeat sequence of Rous sarcoma virus [Yamamoto et al., Cell 22: 787-797 (1980)], the herpes thymidine promoter [Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445 (1981)], the regulatory sequence of the metallothionein gene [Brinster, et al., Nature 296: 39-42 (1982)], and the like.

Small inhibitory RNA (siRNA) can also serve as a PAK1 inhibitor for use in the treatment of melanoma. PAKl expression can be achieved by contacting a melanoma with a vector or construct that results in the production of small double-stranded RNA (dsRNA), or small double-stranded RNA, so that expression of PAKl is specifically inhibited. Methods for selecting suitable dsRNA or dsRNA-encoding vectors are known in the art (Tuschi, T et al., Genes Dev. 13 (24): 3191-3197 (1999); Elbashir, SM et al. McManus MT and Sharp PA, Nature Reviews Genetics 3: 737-747 (2002); Bremmelkamp, TR et al., Science (2002); Nature 411: 494-498 (2001); Hannon, GF, Nature 418: 244-251 296: 550-553 (2002); U.S. Patent Nos. 6,573,099 and 6,506,559 and International Patent Publications WO 01/36646, 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, and 4) GGTTTATGATTAAGGGTTT, all obtained from Dharmacon, Inc.

Polypeptide

The present invention provides a polypeptide inhibitor of PAK1 activity for the treatment of melanoma in a subject. For example, binding polypeptides are polypeptides that bind, preferably specifically bind, PAKl as described herein. In some embodiments, the binding polypeptide is a PAK1 antagonist. Binding polypeptides can be chemically synthesized using known polypeptide synthesis methods or can be prepared and purified using recombinant techniques. Binding polypeptides are typically at least about five amino acids in length or 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, , 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, , 97, 98, 99, or 100 amino acids, wherein the binding polypeptide can bind, preferably specifically bind, PAK1 as described herein. Binding polypeptides can be identified without undue experimentation using known techniques. In this regard, techniques for retrieving polypeptide libraries for binding polypeptides that are capable of specifically binding to a polypeptide target are known to those skilled 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 and 5,663,143, PCT publications WO 84/03506 and WO 84/03564, Geysen et al., Proc. Natl. Acad. Sci. USA, 81: 3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985), Geysen et al., 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., Proc. Natl Acad Sci USA, (1991); Marks, JD et al., J. MoI. Biol., &Lt; RTI ID = 0.0 &gt; Kang, AS et al., Proc. Natl. Acad. Sci. USA, 88: 8363 (1991); a nd Smith, G. P., Current Opin. Biotechnol., 2: 668 (1991)).

In this regard, bacteriophage (phage) display is one known technique for enabling the detection of a large-scale polypeptide library to identify the member (s) of the library capable of specifically binding to the target PAK1. Phage Disinte is a technique for expressing a mutant polypeptide as a fusion protein to a coat protein on the surface of bacteriophage particles [Scott, J.K. and Smith, G. P., Science, 249: 386 (1990)]. The usefulness of phage display lies in the fact that a large library of selective random protein variants (or randomly cloned cDNAs) can be rapidly and effectively categorized against sequences that bind to the target molecule under high affinity. The display of peptides [Cwirla, S.E. et al., Proc. Natl. Acad. Sci. USA, 87: 6378 (1990)] or proteins on phage [Lowman, HB et al. et al., Biochemistry, 30: 10832 (1991); Clackson, T. et al., Nature, 352: 624 (1991); Marks, J.D. et al., J. Mol. Biol., 222: 581 (1991); Kang, A.S. et al., Proc. Natl. Acad. Sci. USA, 88: 8363 (1991)] libraries have been used to search for millions of polypeptides or oligopeptides for having specific binding properties (Smith, GP, Current Opin. Biotechnol., 2: 668 (1991)]. Classifying the phage libraries of random mutants requires a method for making and propagating a large number of variants, an affinity purification procedure using a target receptor, and means for evaluating the results of binding enhancement (U.S. Patent No. 5,223,409, 5,403,484, 5,571,689 and 5,663,143).

Although most of the phage display methods have used the phage phage, the phage phage display system has been widely used in the fields of lambdoid phage display systems (WO 95/34683, US 5,627,024), T4 phage display systems (Ren et al., Gene, 215: ); 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 (1996); Ewimov et al., Virus Genes, 10: 173 (1995)] and T7 phage display systems (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); US 5,766,905) are also known.

A further enhancement enhances the ability of the display system to display a functional library that has the potential to search the peptide library for binding to a selected target molecule and to search for those proteins with the desired properties. Combination reaction devices for phage display reactions have been developed (WO 98/14277), biomolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036 ) Was used to compare and contrast the phage display library. WO 97/35196 discloses a phage display library in which one ligand binds to a target molecule and an affinity ligand which is contacted with a second solution in which the affinity ligand will not bind to the target molecule, Describes a method for selectively separating a ligand. WO 97/46251 discloses a method of biopanning a random phage display library using an affinity purified antibody and then separating the binding phage and then performing a micropanning process using a microplate well, Describes a method for separating phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported [Li et al., Mol Biotech., 9: 187 (1998)]. WO 97/47314 describes the use of a substrate exclusion library to distinguish enzyme specificities using a combinatorial library, which may be a phage display library. A method for screening enzymes suitable for use in detergents using phage display is described in WO 97/09446. Other methods of screening for specific binding proteins are described in U.S. Patent Nos. 5,498,538, 5,432,018 and WO 98/15833. Methods for constructing peptide libraries and searching for these libraries are also disclosed in U.S. Patent 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.

Antibody

In some aspects of the invention, the PAK1 inhibitor for the treatment of melanoma in a subject is a separate antibody that binds to PAK1. In some embodiments, the antibody is humanized. In another embodiment of the invention, the anti-PAK1 antibody is an antibody that inhibits PAK1 function. In some embodiments, the antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some embodiments, the antibody is an antibody fragment, such as an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is a full-length antibody, such as 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 binding polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody and / or binding polypeptide. Amino acid sequence variants of antibodies and / or binding polypeptides may be produced by introducing appropriate modifications into the nucleotide sequence encoding the antibody and / or binding polypeptide, or by peptide synthesis. Such modifications include, for example, deletion from the amino acid sequence of the antibody and / or binding polypeptide, and / or insertion into the sequence and / or substitution of the residue in the sequence. Any combination of deletion, insertion and substitution can be performed to achieve the final structure if the final structure has the desired properties, e.g., target-binding.

In certain embodiments, antibody variants and / or binding polypeptide variants having one or more amino acid substitutions are provided. The corresponding sites for substitutional mutagenesis include HVR and FR. An amino acid substitution is introduced into the antibody and / or binding polypeptide and the product can be retrieved for the desired activity, e. G., Maintenance / improved antigen binding, reduced immunogenicity or improved ADCC or CDC. One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the generated variant (s) selected for further study will have a change (eg, improvement) in specific biological properties (eg, increased affinity, reduced immunogenicity) relative to the parent antibody and / Will have a substantially retained specific biological property of the antibody. Exemplary substitution variants are affinity enhancing antibodies, which can be conveniently made using, for example, phage display-based affinity enhancing techniques as described herein. Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on the digestion and retrieved for specific biological properties (e.g., binding affinity).

Mutations (e. G., Substitutions) can be performed, for example, in HVR to improve antibody affinity. The mutation may be a HVR "hotspot ", i.e., a residue encoded by a codon that mutates at a high frequency during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 107: 179-196 )), And SDR (a-CDR), and the generated mutation VH or VL is assayed for binding affinity. Affinity enhancement by constructing and re-selecting the second library is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 178: 1-37 (O'Brien et al., Ed., Human In some aspects of affinity enhancement, any of a variety of methods (e. G., Error-prone PCR, shuffling or oligonucleotides &lt; RTI ID = 0.0 &gt; -Induced mutagenesis) can be introduced into the variable gene selected for maturation. Next, a second library is constructed. The library is then searched to identify any antibody variants with the desired affinity Another method of introducing a change involves an HVR-directed approach, where multiple HVR residues (e. G., 4 to 6 residues at the same time) are randomly selected. For example, Turning to mutagenesis or modeling can be identified specifically by using this CDR-H3 and CDR-L3 is often particularly targeted.

In certain embodiments, substitutions, insertions or deletions can occur within one or more HVRs, so long as the mutations do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative variations (e. G., Conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVR. The variation may be outside the HVR "hotspot" or the SDR. In certain embodiments of the provided variant VH and VL sequences, each HVR is unmodified or contains no more than 1, 2, or 3 amino acid substitutions.

Combination therapy

The PAK1 inhibitor of the methods described herein can be used alone or in combination with other medicaments for treatment of melanoma. For example, the PAK1 inhibitor described herein may be co-administered with one or more other therapeutic agents comprising another PAK1 inhibitor. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of Aldesleukin, Dacarbazine, DTIC-Dome (Dacarbazine), Ipilimumab, Proleukin (Aldelucin) Vemurafenib, Yervoy (eicilimumab) and / or Zelboraf (bemurafenip). An example of the use of PAK1 inhibitors in combination therapy is provided in PCT / EP2011 / 070008, filed November 14,

The above-mentioned combination therapy includes multiple administrations (when two or more therapeutic agents are included in the same or separate formulations), and separate administration, and in the case of separate administration, administration of the PAK1 inhibitor results in administration of another therapeutic agent and / Prior to, concurrent with, and / or after administration of the compound of the invention. In some embodiments, the PAK1 inhibitor is used for treatment of melanoma in a subject in conjunction with radiation therapy. In some embodiments, the PAK1 inhibitor is used for the treatment of melanoma in a subject, together with the surgical removal of all or part of the melanoma from the subject.

In some aspects of the invention, the subject has previously been treated for melanoma, for example, using chemotherapy. In one example, chemotherapy is surgery. In another embodiment, the subject can be further treated by additional chemotherapy prior to administration of the PAKl inhibitor, at the time of administration (e.g., concurrently) or after administration. Examples of chemotherapy include, but are not limited to, surgery, radiation therapy (radiation therapy), biotherapy, immunotherapy, chemotherapy or a combination of the above treatments.

Route of administration

The route of administration may be by injection, either single or multiple bolus or infusion, for example, subcutaneously, intravenously, intraperitoneally, intramuscularly, intraarterially, intralesional or intraarticularly, for a prolonged period of time, Such as by injection, topical administration, inhalation, or sustained or sustained release means. In some aspects, the invention provides a method of treating a melanoma in a subject using a PAKl inhibitor, wherein the PAKl inhibitor is administered intravenously to the subject. In another aspect, the invention provides a method of treating a melanoma in a subject using a PAKl inhibitor, wherein the PAKl inhibitor is administered topically to the subject.

Pharmaceutical composition

In the methods of the present invention, the therapeutic formulations of the present invention comprise a PAK1 inhibitor having the desired degree of purity as a selective physiologically acceptable carrier, excipient or stabilizer [Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, including: buffering agents such as phosphates, citrates and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, Cyclohexanol, 3-pentanol, and m-cresol); Low-Jarang (less than 10 residues) polypeptides; Proteins, such as cerium albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Other carbohydrates including monosaccharides, daisaccharides, and glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter-ions such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as TWEEN TM, PLURONICS TM or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound, as required for the particular indication being treated, preferably those having complementary activity that do not adversely affect each other. For example, it may be desirable to provide an additional immunosuppressive agent. The molecules are suitably present together in an effective amount for the intended purpose.

The active ingredients may also be incorporated into microcapsules made, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) Each of the microcapsules may be encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. This technique is described in Remington ' s Pharmaceutical Science 16th edition, Osol, A. Ed. (1980).

Sustained-release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol), polylactides (U.S. Patent No. 3,773,919), L - copolymers of glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (lactic acid-glycolic acid copolymer And poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid have been shown to release the molecules for 100 days As long as the encapsulated antibody is retained in the body for an extended period of time, they are denatured or aggregated as a result of exposure to moisture at &lt; RTI ID = 0.0 &gt; 37 C, &lt; A loss of water activity and possibly a change in immunogenicity can be induced. [0060] A reasonable method for stabilization can be devised depending on the mechanism involved. For example, when the flocculation mechanism is intermolecularly via the thio-disulfide exchange, S bond formation, stabilization can be achieved by modifying the sulfhydryl moiety, lyophilizing from an acidic solution, adjusting the moisture content, using suitable additives, and developing specific polymer matrix compositions.

In some embodiments, the invention provides a composition comprising a PAKl inhibitor for use in melanoma treatment. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma that is overexpressed in melanoma compared to non-cancerous cells, for example, non-cancerous skin cells. In some embodiments, the melanoma is a wild-type BRAF melanoma, wherein PAK1 is amplified in melanoma. In some embodiments, the melanoma is a wild type BRAF black paper, wherein PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, melanoma is a mutant BRAF melanoma, melanoma overexpresses PAK1 over non-cancerous cells and / or PAK1 is amplified in melanoma. In some aspects, the invention provides a composition comprising a PAKl inhibitor for use in the treatment of melanoma in a mammal. In some aspects, the invention provides a composition comprising a PAKl inhibitor for use in the treatment of melanoma in a human.

In some embodiments, the invention provides the use of a PAKl inhibitor in the manufacture of a medicament for the treatment of melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is overexpressed in melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma that is overexpressed in melanoma compared to non-cancerous cells, for example, non-cancerous skin cells. In some embodiments, the melanoma is a wild-type BRAF melanoma, wherein PAK1 is amplified in melanoma. In some embodiments, the melanoma is a wild type BRAF black paper, wherein PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, PAKl is overexpressed in melanoma and PAKl is amplified in melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, melanoma is a mutant BRAF melanoma, melanoma overexpresses PAK1 over non-cancerous cells and / or PAK1 is amplified in melanoma. In some aspects, the invention provides the use of a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma in a mammal. In some aspects, the invention provides the use of a PAKl inhibitor in the manufacture of a medicament for the treatment of melanoma in humans.

Kit

The present invention also provides kits, agents, compositions and unit dosage forms for use in any of the methods described herein.

The kits of the present invention comprise one or more containers (or unit dosage forms and / or products) comprising a PAKl inhibitor and, in some aspects, additional instructions for use in the treatment of melanoma according to any of the methods described herein . The kit may also include instructions for selecting individuals suitable for treatment (e.g., screening based on the BRAF genotype). The instructions supplemented with the kits of the present invention are typically written instructions on a label or package insert (e.g., a print sheet included in the kit), but may be stored in a machine-readable manual (e.g., ) Is also allowed. In some embodiments, the kit also comprises another therapeutic agent.

The kit of the present invention is in a suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kit may optionally provide additional components such as buffering and analysis information. Accordingly, the present application also provides a product comprising a vial (e.g., a sealed vial), a bottle, a jar, a flexible packaging, and the like.

Melanoma Biomarker  And treatment

The present invention provides a method for identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor by measuring the presence of one or more melanoma biomarkers. In some embodiments, melanoma biomarkers are overexpression of PAKl in melanoma, amplification of PAKl in melanoma, and / or the presence of wild-type BRAF in melanoma. In some embodiments, overexpression of PAKl is measured by comparison with non-cancerous tissue, for example, non-cancerous skin tissue. In some embodiments, the biomarker is detected in a test sample obtained from an individual. In some embodiments, the presence of the biomarker is measured by comparing the test sample with a reference sample.

In one aspect, the invention provides a method of identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor by measuring the expression of PAK1 in a melanoma, wherein overexpression of PAK1 in the melanoma relative to non- Lt; RTI ID = 0.0 &gt; inhibitor. &Lt; / RTI &gt; In some embodiments, more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 100% of the PAK1 Overexpression indicates that the patient is amenable to treatment with a PAK1 inhibitor. 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, and 8.0 times the PAK1 expression in non-cancerous cells, 9.0- or 10-fold over-expression of PAK1 indicates that the patient is amenable to treatment with a PAK1 inhibitor. Methods for measuring the expression of PAKl are known in the art. Examples of methods for determining the level of expression of PAK1 in melanoma include, but are not limited to, immunohistochemistry, reverse phase protein arrays (RPPA), quantitative PCR, immunoassays, and the like. PAK1 expression levels can be compared to other tumors and cells using a gene expression omnibus (GEO) database.

In yet another aspect, the present invention provides a method of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor by measuring the amplification of PAKl in a melanoma, wherein the amplification of the PAKl gene in the melanoma results in the patient being treated with a PAKl inhibitor &Lt; / RTI &gt; In some embodiments, the number of copies of PAK1 greater than about 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 10 in melanoma Indicating that it is suitable for treatment with a PAK1 inhibitor. Methods for measuring amplification of a gene are known in the art. For example, the number of copies of the PAK1 gene can be determined by using an SNP array such as the Apifetrix 500K SNP array assay.

In another aspect, the invention provides a method of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor by detecting the genotype of BRAF in the melanoma, wherein the wild-type BRAF in the melanoma is suitable for treatment with a PAKl inhibitor And the like. Methods for genotyping BRAF genes in melanomas are known in the art; For example, the nucleotide sequence of the BRAF gene from melanoma can be determined using standard sequence analysis methods or using the KASP SNP genotyping system (KBioscience).

The present invention provides a method of treating a melanoma in a patient comprising administering to the mammal a therapeutically effective amount of a biologically active agent selected from the group consisting of overexpression of PAK1 in melanoma, amplification of PAK1 in melanoma, and / It must have been identified as having a marker; The method comprises administering to the patient a therapeutically effective amount of a PAK1 inhibitor. In some embodiments, the patient is a human patient. In some aspects of the foregoing aspects, at least one of the biomarkers is overexpressed of PAKl, wherein PAKl is present in the melanoma at about 10%, 20%, 30%, 40%, 50%, 60%, 70% %, 80%, 90%, 100% or more. In some embodiments, the expression of PAK1 in melanoma is approximately 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5.0, 6.0, or 1.5 times higher than that of PAK1 in non-cancerous cells Fold, 7.0 fold, 8.0 fold, 9.0 fold, or 10 fold. Methods for measuring the expression of PAKl are known in the art. Examples of methods for determining the level of expression of PAK1 in melanoma include, but are not limited to, immunohistochemistry, reverse phase protein arrays (RPPA), quantitative PCR, immunoassays, and the like. PAK1 expression levels can be compared to other tumors and cells using a gene expression omnibus (GEO) database.

In some aspects of the foregoing aspects, at least one of the biomarkers is an amplification of PAK1 in melanoma, wherein the number of copies of PAK1 is from about 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 10. Methods for measuring amplification of a gene are known in the art. For example, the number of copies of the PAK1 gene can be determined by using an SNP array such as the Apifetrix 500K SNP array assay.

In some aspects of the above aspects, at least one of the biomarkers has a genotype of BRAF in melanoma, wherein the patient has melanoma containing wild type melanoma. In some aspects of the foregoing aspects, the presence of melanoma biomarkers in a patient has already been measured prior to treatment with a PAK1 inhibitor.

The present invention provides a method of modulating the treatment of melanoma in a patient undergoing treatment with a PAK1 inhibitor, which method comprises measuring the expression of PAK1 in a melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments, overexpression of PAKl in melanoma indicates that treatment with a PAKl inhibitor can continue. In some instances, the expression of PAKl in melanoma is monitored over time in patients undergoing treatment with PAKl. In some embodiments, the expression of PAKl in melanoma is monitored at least daily, at least weekly, at least monthly. In some instances, the expression of PAKl in melanoma is monitored over time in patients undergoing treatment with PAKl. When PAKl expression is increased during the course of treatment with a PAKl inhibitor, the amount of PAKl inhibitor administered to the patient is increased or remained the same. In some embodiments, the amount of PAKl inhibitor administered to the patient is increased until the level of PAKl expression is reduced or is no longer detected. When PAKl expression is reduced during the course of treatment with a PAKl inhibitor, the amount of PAKl inhibitor administered to the patient is reduced or remained the same. In some embodiments, PAKl expression is monitored over time in melanomas of patients undergoing treatment with a PAKl inhibitor, wherein treatment with the PAKl inhibitor continues until the expression of PAKl in the melanoma is no longer detectable.

An exemplary sun

In some aspects, the invention provides a method of treating melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. In another embodiment, the melanoma is a wild-type BRAF melanoma. In another embodiment, PAK1 is overexpressed in tumors relative to non-cancerous skin cells. In another embodiment of any of the above, PAK1 is amplified in a tumor. In another embodiment, the number of copies of PAK1 in the tumor is greater than about 2.5.

In another embodiment of any of the above aspects, the inhibitor is a small molecule, nucleic acid or polypeptide. In some embodiments, the small molecule is PF-3758309. In some embodiments, the small molecule is a compound of formula VII:

(VII)

In another embodiment, the small molecule is a compound of formula VII, wherein A is 4-indolyl, 5-indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazolyl or 5-benzimidazolyl; R ^a, R ^1a and R ^1b are independently hydrogen or C _{1 -3} alkyl; R ⁵ is hydrogen or C _{1 -6} alkyl; R ⁶ is hydrogen, halogen or C _{1 -6} alkyl; R &lt; ⁷ &gt; is cycloalkyl optionally substituted by fluorine.

In another embodiment of any of the above aspects, the subject is a human.

In another embodiment of any of the above aspects, the PAK1 inhibitor is used in combination with a therapeutic agent.

The present invention provides the use of a PAK1 inhibitor for the treatment of melanoma in a subject. In some embodiments of the foregoing uses, the melanoma is a wild-type BRAF melanoma.

The present invention provides a composition comprising a PAK1 inhibitor for use in the treatment of melanoma. In some aspects of the composition, the melanoma is a wild-type BRAF melanoma. In some embodiments, the compositions also include pharmaceutically acceptable excipients.

The present invention provides the use of a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma. In some embodiments of the foregoing uses, the melanoma is a wild-type BRAF melanoma.

The present invention provides a kit comprising a PAK1 inhibitor and a PAK1 inhibitor for use in treating melanoma, comprising instructions for use in the treatment of melanoma. In some aspects of the kit, the melanoma is a wild-type BRAF melanoma.

The present invention provides a method of inhibiting CRAF signaling in melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor.

The present invention provides a method of inhibiting MEK signaling in melanoma tumors, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor.

The present invention provides a method of identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor, comprising measuring the BRAF genotype of the melanoma, wherein the black paper patient comprising the wild type BRAF is suitable for treatment with a PAK1 inhibitor .

The invention provides a method of identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor, comprising measuring the expression of PAK1 in a melanoma, wherein the overexpression of PAK1 in the melanoma relative to the non- Lt; RTI ID = 0.0 &gt; inhibitor. &Lt; / RTI &gt; In some aspects of the method, overexpression of PAKl in melanoma is 2.5 times greater than expression of PAKl in non-cancerous skin cells. The present invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the BRAF genotype of the melanoma, wherein the melanoma comprising the wild-type BRAF is administered to a patient Lt; RTI ID = 0.0 &gt; PAK1 &lt; / RTI &gt;inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient.

The present invention provides a method of treating a human melanoma patient with a PAK1 inhibitor, comprising: (a) selecting a patient based on the PAK1 expression level of the melanoma, wherein the overexpression of PAK1 in the melanoma Lt; RTI ID = 0.0 &gt; PAK1 &lt; / RTI &gt;inhibitor; (b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient. In some aspects of the method, overexpression of PAKl in melanoma is 2.5 times greater than expression of PAKl in non-cancerous skin cells.

The present invention provides a method of treating a human melanoma patient comprising administering a therapeutically effective amount of a PAK1 inhibitor to a selected individual whose melanoma genotype is determined to be wild-type for BRAF.

The present invention provides a method of treating a human melanoma patient comprising administering a therapeutically effective amount of a PAK1 inhibitor to a patient determined to be overexpressing PAKl relative to a black paper non-cancerous skin cell. In some aspects of the method, overexpression of PAKl in melanoma is 2.5 times greater than expression of PAKl in non-cancerous skin cells.

The present invention includes evaluating PAKl expression in a melanoma wherein the overexpression of PAKl in the melanoma is being treated with a PAKl inhibitor indicating that treatment of the individual is modulated until PAKl overexpression is no longer detected A method of modulating the treatment of melanoma in a patient is provided.

All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with alternative features providing the same, equal, or similar purpose. Accordingly, unless expressly stated otherwise, each feature disclosed is but one example of a comprehensive set of equivalent or similar features.

Other details of the invention are illustrated by the following non-limiting examples. The disclosures of all references in the specification are specifically incorporated herein by reference.

Example

The following examples are for the purpose of illustrating the present invention only and should not be construed as limiting the present invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation.

Example  1: in melanoma Elevated PAK1  Protein expression and Genome  Amplification

To determine the possible degree of PAK1 dysregulation in human melanoma, primary tumor tissues from 87 melanoma patients were evaluated for DNA replication number changes using a high resolution single nucleotide polymorphism (SNP) array. The Apimetrix 500K SNP array assay, genomic DNA preparation, chip processing and data analysis were performed as previously described to measure the increase in 11q13 of the region of chromosome 11 with the PAK1 gene in sampled melanoma tumors [Harvey PM, et al., Genes Chromosomes Cancer, 47 (6): 530-542 (2008)). To collect expression array data for the matched tumor samples, RNA was extracted from the frozen tumor tissue and applied to the apimetrix (Santa Clara, CA) HGU133 gene expression microarray. The frequency of PAK1 amplification was 9% (8 out of 87 samples with a replica of 2.5) on the tumor panel (FIG. 1A). RNA was purified from 42 melanoma tumors and cell line samples, and the increased number of PAK1 copies correlated with mRNA expression (Pearson correlation coefficient = 0.75; FIG. 1B). Unregulated PAK1 expression is more frequent than anticipated by genomic amplification alone, suggesting that additional transcriptional or regulatory mechanisms in this citation increase PAK1 expression [Reddy SD, et al., Cancer Res, 68 20): 8195-8200 (2008); and de la Torre-Ubieta L, et al., Genes Dev, 24 (8): 799-813 (2010)]. Increased expression of PAK1 in melanoma compared to normal skin tissue has also been demonstrated using gene expression data deposited in the gene expression omnibus database (GSE4587). Interestingly, PAK1 gene amplification was preferentially observed in tumors lacking an activating mutation in the BRAF oncogene at 22% versus 0% for each BRAF wild type or mutant (p = 0.005, bilateral t-test; Fig. 1B) . PAK1 mRNA expression levels were different between wild type and BRAF (V600E) or BRAF (V600M) genotypes (p = 0.006 and p = 0.125, respectively, Fig. Collectively, this suggests that PAK1 may be a tumor-promoting "driver" gene among a subpopulation of BRAF wild-type melanomas.

To further assess the extent of PAK1 modulation in human melanoma, PAK1 protein expression levels and subcellular localization were confirmed by immunohistochemistry (IHC) staining of separate sets 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 included 23 nodules, 3 malignant surplus, 45 sclerotic scalpels, 3 connective tissue formation, 5 terminal surplus and 13 inextensible melanoma specimens. Four cancers were pT1, 17 were pT2, 28 were pT3, and 35 were pT4. Eight cases were not able to determine the exact stage. Tissue microarrays (TMA) were assembled as previously described (Bubendorf L., et al., J Pathol, 195 (1): 72-79 (2001)). Approved for use of all human tissues from the Regional Research Ethics Committee (C02.216). Immunohistochemistry (IHC) was performed as previously described [Ong CC et al., PNAS, 108 (17): 7177-7182 (2011)]. The intensity of PAK1 expression scored separately in the cytoplasm and nucleus of neoplastic cells to a grade of 0-3. The highest intensity score among the replica cores was used as a score for each patient. The same pathologist who did not know about clinical data scored in all cases. The association between categorical variables was assessed using a chi-squared test. The strong and selective IHC reactivity of PAK1 antibodies has already been demonstrated [Ong CC, et al., PNAS, 108 (17): 7177-7182 (2011)]. In malignant melanoma, 46 (52%) of 92 primary tumor samples were positive for PAK1 expression, and 26% of all cases showed intermediate (2+) or strong (3+) intensity staining in malignant cells (Fig. 1C, panels III and IV; table 1). The nucleus position of PAK1 was confirmed only in a very small proportion of melanomas. The same results were obtained using an alkaline phosphatase label and a rapid red color instead of a wasp peroxidase label and brown diaminobenzidine. PAK1 was weakly expressed in basal keratinocytes in normal skin, and lymphocytes and putative Langerhans cells were positive for PAK1 expression (Fig. 1C, panel IV). Together, the data show that PAK1 DNA replication number, mRNA and protein expression are approximately upregulated in human melanoma.

Example  2: In primary melanoma PAK1  Overexpression BRAF  Negative association between mutations

(JA, et al., Br J Dermatol, 164 (4): 776-784 (2011)), melanoma tissues were identified as BRAF (codon 600) and melanoma The known hot spot mutations in NRAS (codon 12, 13, 61 and 146) genes were genotyped. The mutation status was measured for BRAF codon 600 and NRAS codons 12, 13, 61 and 146 by KASPar (KBioscience, Hertz, UK) and the conventional Sanger DNA sequencing method. BRAF (39 Val600Glu, 1 Val600Lys and 46 wild type) and NRAS (1 Gln61His, 7 Gln61Lys, 1 Gln61Lys + Gln61Arg + Leu59Ala, 1 Gln61Leu, 19 Gln61Arg, 2 Gln61Arg + Gln61Lys and 53 wild- ) Were available for 86 and 84 tumors, respectively, consistent with the previously reported mutation frequency range for subcutaneous melanoma [Lee JH, et al., Br J Dermatol, 164 (4): 776-784 (2011)]. PAK1 IHC staining was scored unknowingly for clinicopathological details and mutation status, and the results are summarized in Table 1. Significantly, PAK1 protein expression was selectively regulated in BRAF wild-type tumors compared to melanoma expressing tumor-induced V600E or V600K mutants (4 out of 40 tumors under high IHC staining) (19 of 46 were resistant to PAK1 Positive for IHC staining). The negative association between PAK1 expression 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 either mutation was also negatively associated with PAK1 protein expression (p = 0.004, Chi square 8.128). A similar trend was observed when the samples were divided only into NRAS mutants and non-mutant status (p = 0.45, chi-square 0.569), although not statistically significant. (P = 0.61 Student's T-test), pathologic tumor (pT) staging (p = 0.14 chi square), Breslow thickness (p = 0.85 Student T- Or ulceration (p = 0.91 chi-square). Collectively, these results provide evidence that PAK1 modulatory disorders are strongly associated with subcutaneous melanoma deficient in tumor-induced mutations of BRAF, and define subgroups of human melanoma without effective targeting therapies.

Overexpression of PAK1 protein in BRAF wild-type melanoma MAPK
Activator genotype PAK1 IHC P-value Chi-square 0,1 2,3 BRAF WT 27 19 0.001 10.702 Mutant 36 4 NRAS WT 37 16 0.45 0.569 Mutant 24 7

Example  3: PAK1 silver BRAF  Required for the proliferation of wild-type melanoma cells

Taking into account genomic and histological data for increased PAKl expression in subpopulation of wild-type human melanoma for BRAF, PAK1 expression and RNAi of PAK1 in a panel of melanoma cell lines to clarify the contribution of PAKl to tumor cell proliferation The effect of mediated knockdown was investigated. Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va.) And incubated at 37 ° C and 5% CO ₂ with Dulbecco's Modification containing 10% fetal bovine serum and 4 mM L-glutamine Dulbecco &apos; s Modified Eagle Medium (DMEM) or the Roswell Park Memorial Institute 1640 (RPMI 1640) medium. (Dharmacon RNAi Technologies, Chicago, Illinois, USA), already characterized for efficacy and selectivity of PAK1 and PAK2 knockdown [Ong CC, et al., PNAS, 108 (17): 7177-7182 (SiRNA) < / RTI &gt; oligonucleotide duplexes. Cell viability was assessed by ATP content using a Cytoter-Glycolysis assay (Promega, Madison, Wis.) And the results represent the mean ± SD from three experiments. Increased PAK1 protein expression in melanoma expressing wild-type BRAF as compared to mutant BRAF was also observed for immortalized cell lines in cultures. Cell viability analysis showed that the 537 MEL, MeWo, SK-MEL23 and SK-MEL30 melanoma cells expressed high levels of the PAK1 protein and that the transient knockdown of PAK1 through multiple PAK1-selective siRNA oligonucleotides was negative for the non-targeted negative control siRNA oligonucleotides Showed a 1.8- to 4.3-fold reduction in cell viability when compared to transfected cells (p &lt;0.0001; Fig. 2A). In addition, inhibition of PAK1 generally reduced the proliferation of BRAF wild-type melanoma cells (p &lt;0.07; n = 14) compared to BRAF ^V600E cells, further supporting the role for PAK1 as a driver of proliferation in these melanoma subtypes (Fig. 2B). To better assess the mechanism by which PAK1 contributes to proliferation, PAK-dependent cell signaling was assessed in 537MEL and SK-MEL23 cells. 1 mM phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitor cocktail 1/2 (Sigma Aldrich, St. Louis, Mo., USA), Cell Extraction Buffer (Invitrogen, Carlsbad, CA) Protein extracts from cell lysates at 4 ° C using one complete EDTA-free Mini® Protease Inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) tablet, . For Western blot analysis, the proteins were separated by 4-12% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore Corp., Villerica, Mass., USA). Immunoblotting was performed using the indicated primary antibodies and analyzed using secondary antibodies against enhanced chemiluminescence (ECL). MAPK pathway activation was markedly inhibited by PAK knockdown when measured by phosphorylation of ERK and MEK (Figure 2C). Consistent with the above results, the level of cyclin D1 (essential for regulating cyclin-dependent kinase and G1 / S progression) was also reduced as a result of PAKl clearance. PAK1 signaling was further investigated in BRAF wild-type melanoma cells using a reverse phase protein array (RPPA) phosphoproteomics platform. All samples were first diluted to a final concentration of 0.5 mg / mL and protein lysates were analyzed by RPPA (Theranostics Health, LLC). The sample dilution was double-pressed onto the slide, Was first applied to immunostaining using panels of antibodies that were predominantly directed against specific phosphorylated or cleaved proteins. The antibodies were tested for both phosphorylation and protein specificity using single band detection at appropriate molecular weights by immunoblotting The intensity values for each end point were calculated for each of the samples present in the linear dynamic range of staining after background removal at each point (for slides stained only in the slide topical background and also only the secondary antibody) Dilution &lt; / RTI &gt; curve for each dilution curve. Sypro Ruby) (Invitrogen) RPPA data were processed by log ₂ conversion and linear scaling (z-score conversion) to ensure regularity and linearity. The RPPA assay showed reduced signal transduction from the BRAF wild-type (SK-MEL23) to MAPK, nuclear factor-kappaB (NF-kB) and cytoskeletal pathway after PAK1 inhibition, but BRAF mutation (A375) (Fig. 2D).

PAK1 was found to phosphorylate both CRAF (Ser338) and MEK1 (Ser298) (17, 29-31). Thus, we have investigated the molecular mechanism by which PAK1 induces activation of the MAPK pathway in BRAF wild type melanoma cells. Since the phospho-specific antibody raised to the Ser217 / Ser221 activation loop site on the MEK protein is not distinguishable between MEK1 and MEK2, it can be used as a control or as previously described [Hatzivassiliou G, et al., Nature, 464 (7287): MEK isoforms were immunoprecipitated from cells transfected with PAK-selective siRNA oligonucleotides and MEK activation was detected by immunoblotting with a phospho-MEK1 / 2 (Ser217 / Ser221) antibody Respectively. PAK knockdown reduced both MEKl (Fig. 3A) and MEK2 (Fig. 3B) phosphorylation in 537MEL and SK-MEL23 cells. Since the Ser298 phosphorylation site on MEK1 is not translated in MEK2, PAK-dependent activation of both MEK isoforms suggests that upstream signaling to CRAF may be a driver of MAPK pathway regulation in BRAF wild-type melanoma cells. CRAF was immunoprecipitated from control or cells transfected with the PAK-selective siRNA oligonucleotides as previously described [Hatzivassiliou G, et al., Nature, 464 (7287): 431-435 (2010)] and phospho-CRAF And activation of CRAF was detected by immunoblotting using antibody (Ser338). Western analysis demonstrated that PAK elimination reduced the phosphorylation of CRAF on Ser338, an important moiety for the complete activation of the kinase (Fig. 3C). The dependence of CRAF (Ser338) phosphorylation (Fig. 3D) and CRAF action factor signaling (Fig. 3E) on PAK catalytic activity was also measured using PF-3758309 [Murray BW, et al., PNAS, IPA-3, which is an allosteric inhibitor that binds to PAK1-3 and prevents activation by the Rho class GPTase [Deacon SW, et al., Chemistry & Biology, 15 (4): 322-331 (2008)].

Another dysfunctional study was performed to analyze the role of PAK1 in BRAF wild-type melanoma cells by examining the contribution of PAK1 to melanoma cell migration. Briefly, WM-266-4 melanoma cells were transfected with non-targeted control (NTC) or PAK1 / 2 siRNA oligonucleotides for 72 hours and confluent WM-266-4 melanoma cells were subsequently challenged. Images were recorded when a wound occurred (dark shade) and 28 hours after injury (bright area). The difference in relative wound density was statistically significant (p <0.001; n = 3), indicating the need for PAK1 in melanoma cell migration (Fig. 4). Collectively, the functional consequences of PAKl blockade in BRAF wild-type melanoma cells include reduced CRAF activation and prominent cell proliferation inhibitory effects by subsequent MAPK pathway signaling.

Example  4: PAK  And BRAF  For inhibition BRAF  Differential sensitivity of wild-type melanoma cells

Small molecule inhibition of PAK and BRAF was compared using SK-MEL23 BRAF wild-type and A375 BRAF (V600E) cells to further investigate the activity and cellular mechanism of PAK signaling activity within sensitive and non-sensitive tumor types. For analysis of pathway regulation, cells were treated with 5 μM PF-3758309 or 0.2 μM PLX-4720, BRAF inhibitor for 4 h before analyzing cell lysates for phosphorylation of MAPK pathway components. Administration of PF-3758309 caused sufficient MAPK pathway regulation in SK-MEL23 cells (lane 2) as measured by ERK1 / 2 and MEKl / 2 phosphorylation on kinase loop residues important for catalytic activity, but A375 cells Lane 5) (Fig. 5A). In comparison, the analysis of PLX-4720-mediated signal transduction changes showed only modest inhibition of ERK1 / 2 and MEK1 / 2 phosphorylation in SK-MEL23 cells (lane 3), while the same treatment conditions resulted in BRAF (V600E) MAPK activation was strongly inhibited in the cells (lane 6). As a control, no difference was noted for the total ERK1 / 2 or MEK1 / 2 protein levels in the experiments. Consistent with previous reports, MEK1-Ser298 was identified as a PAK-specific phosphorylation site, but Ser298 phosphorylation was not associated with MEK activation loop phosphorylation in BRAF (V600E) melanoma cells (lane 5). Although the biological consequences of PAKl phosphorylation of MEKl-Ser298 are currently poorly understood, it has been found that PAKl-MEKl signaling can be mediated by cell-cell contact and attachment (Slack-Davis JK, et al., J Cell Biol , 162 (2): 281-291 (2003)). PAK signaling was also induced by ectopic expression of the flag-PAK1 in BRAF (V600E) under only moderate endogenous expression of PAK1. Increased PAK1 signaling in A375 cells resulted in a significant increase in CRAF and MEK phosphorylation that could be reversed by the addition of PF-3758309 (Fig. 5B), suggesting that acquisition of PAK1 overexpression overcomes the dependence on tumor- induced BRAF in melanoma (Johannessen CM, et al., Nature, 468 (7326): 968-972 (2011)).

PAK inhibitor is to determine whether to reduce the cell viability of the wild type BRAF melanoma cells, the SK-MEL23 and 537MEL cells, as PF-3758309 or ^{(S) -N 2 - (1-} (1H- indol-5-yl ) ethyl) -N ⁴ - (5- cyclopropyl -1H- pyrazol-3-yl) -6-methyl-pyrimidine-2,4-diamine ^{(I-007), N 2} - ((1H- indol- 4- yl) methyl) -N ⁴ - (5- cyclopropyl -1H- pyrazol-3-yl) -6-methyl-pyrimidine-2,4-diamine (I-054) and N ² - ((4 -chloro -1H- benzo [d] imidazol-5-yl) methyl) -N ⁴ - (5- cyclopropyl -1H- pyrazol-3-yl) -N ^{2-methyl-pyrimidine-2,4-} Diamine (I-087) and analyzed by a celite-glow emission assay (Promega, Madison, WI). Inhibition of PAK1 by treatment with all tested PAK inhibitors significantly reduced cell proliferation, suggesting that inhibition of PAK signaling is a target for the treatment of BRAF wild-type melanoma (FIGS. 6A and B).

In order to expand the in vitro observations, pharmacodynamic modulation by PAK small molecule inhibitors was evaluated using a tumor xenograft model. The cultured SK-MEL-23, A2058.X1 and A375.X1 cells were removed from the culture, suspended in Hank's buffered saline solution (HBSS), and cultured in Matrigel (BD Biosciences) (Taconic Farms, Hudson, United States of America) or Beige Nude XID (Harlan Laboratories, Inc., USA) in a 1: ), California, USA) mice were subcutaneously implanted in the right flank. Animals with an average volume of tumors of about 250 mm &lt; ³ &gt; were classified as treatment cohorts. Tumor volume was calculated by the following equation: Tumor volume = 0.5 x (axb ² ) where 'a' is the maximum tumor diameter and 'b' is the vertical tumor diameter. The percent inhibition of percent growth (% INH) at the end of the study (SEM) was calculated as% INH = 100 [(EOS Vehicle EOS Treatment) / (EOS Vehicle)] using the Dunnett t test Data analysis and p-value calculations were performed using JMP software (SAS Institute, Carrie, NC). All experimental procedures were followed by the American Physiology Society's guidance, After tumor formation, the animals were either given saline or PF-3758309 (25 mg / kg, ip), and the tumor was administered 1 It was collected after hours. The sheep were frozen and ground on dry ice using a small Bessman tissue grinder (Spectrum Laboratories, Rancho Domingue, CA) and incubated in cell extraction buffer (Invitrogen, USA ), 1 mM phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitor cocktail 1/2 (Sigma Aldrich, St. Louis, Missouri, USA) and a complete EDTA-free Mini (TM) protease inhibitor Protein extracts were prepared at 4 DEG C using one cocktail (Roche Diagnostics, Indianapolis, IN) tablet. Proteins were separated by 4-12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corporation, Villerica, MA) for immunoblotting with the indicated antibodies.

Treatment with PF-3758309 resulted in a substantial reduction in CRAF, MEK1 / 2 and ERK1 / 2 phosphorylation in SK-MEL23 tumors (Fig. 7A). In A2058.X1 BRAF (V600E) tumors, reduced phosphorylation of CRAF (Ser338) was not observed after administration of PF-3758309. The effect of PF-3758309 on growth and maintenance of BRAF wild-type tumors was also evaluated in efficacy experiments for 21 days (Figure 7B and Figures 8A-B). Treatment with 10, 15 and 25 mg / kg PF-3758309 impaired tumor growth (74%, 76% and 91% inhibition, respectively, compared to the control cohort) compared to the vehicle cohort as measured on the last day of administration - black, p < 0.0001). In comparison, minimal anti-tumor efficacy and inhibition of CRAF phosphorylation was observed for SK-MEL23 tumors treated with strong RAF inhibitors in vivo (Hoeflich KP, et al., Cancer Res, 69 (7): 3042-3051 (2009)]. In addition, phosphoproteomic analysis of BRAF mutant (A375) and wild type (SK-MEL23) cells treated with PLX-4720 BRAF inhibitor showed that these cell subtypes of melanoma showed different signaling responses due to BRAF inhibition (Fig. 9A). Overall, the degree of MAPK pathway inactivation by PF-3758309 correlated with anti-tumor efficacy in the BRAF wild-type melanoma xenograft model, and the data suggest that interfering with PAK signaling may have therapeutic efficacy in the melanoma subpopulation (The model shown in Fig. 10).

Claims (33)

A method of treating melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. The method according to claim 1,
Wherein the black paper is a wild-type BRAF melanoma. 3. The method according to claim 1 or 2,
PAK1 is overexpressed in tumors relative to non-cancerous skin cells. 4. The method according to any one of claims 1 to 3,
How PAK1 is amplified in tumors. 5. The method of claim 4,
Wherein the number of copies of PAK1 in the tumor is greater than about 2.5. 6. The method according to any one of claims 1 to 5,
Wherein the inhibitor is a small molecule, nucleic acid or polypeptide. The method according to claim 6,
Wherein the small molecule is PF-3758309. The method according to claim 6,
Wherein the small molecule is a compound of formula VII:
(VII)

9. The method of claim 8,
Wherein the small molecule is a compound of formula VII and A is 4-indolyl, 5-indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazolyl or 5-benzimidazolyl; R ^a, R ^1a and R ^1b is independently hydrogen or C _{1 -3} alkyl; R ⁵ is hydrogen or C _{1 -6} alkyl; R ⁶ is hydrogen, halogen or C _{1 -6} alkyl; Wherein R &lt; ⁷ &gt; is cycloalkyl optionally substituted by fluorine. 10. The method according to any one of claims 1 to 9,
Wherein the object is a human. 11. The method according to any one of claims 1 to 10,
Wherein the PAK1 inhibitor is used in combination with the therapeutic agent. The use of a PAK1 inhibitor for the treatment of melanoma in an individual. 13. The method of claim 12,
Use for black paper is a wild type BRAF melanoma. A composition comprising a PAK1 inhibitor, for use in the treatment of melanoma. 15. The method of claim 14,
Wherein the black paper is a wild type BRAF melanoma. 16. The method according to claim 14 or 15,
A composition comprising a pharmaceutically acceptable excipient. Use of a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma. 18. The method of claim 17,
Use for black paper is a wild type BRAF melanoma. A kit comprising a PAK1 inhibitor for use in treating melanoma, comprising instructions for use in the treatment of PAK1 inhibitors and melanoma. 20. The method of claim 19,
Kit is a black paper wild type BRAF melanoma. A method of inhibiting CRAF signaling in a melanoma in a subject, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. A method of inhibiting MEK signaling in melanoma tumors, comprising contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor. A method of identifying a human melanoma patient suitable for treatment with a PAK1 inhibitor, comprising determining the BRAF genotype of the melanoma, wherein the melanoma comprising the wild type BRAF indicates that the patient is suitable for treatment with a PAK1 inhibitor. Wherein the overexpression of PAK1 in the melanoma relative to the non-cancerous skin cells is indicative of the patient being suitable for treatment with a PAK1 inhibitor, wherein the PAK1 inhibitor is selected from the group consisting of human melanoma How to identify a sick patient. 25. The method of claim 24,
Overexpression of PAK1 in melanoma is X% greater than expression of PAK1 in non-cancerous skin cells. A method of treating a human melanoma patient with a PAK1 inhibitor, comprising:
(a) selecting the patient based on the BRAF genotype of the melanoma, wherein the melanoma comprising the wild-type BRAF indicates that the patient is suitable for treatment with a PAK1 inhibitor;
(b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient. A method of treating a human melanoma patient with a PAK1 inhibitor, comprising:
(a) selecting patients based on PAK1 expression levels of melanoma, wherein overexpression of PAK1 in melanoma indicates that the patient is amenable to treatment with a PAK1 inhibitor;
(b) administering a therapeutically effective amount of a PAK1 inhibitor to the selected patient. 28. The method of claim 27,
Overexpression of PAK1 in melanoma is 2.5 times greater than expression of PAK1 in non-cancerous skin cells. A method of treating a human melanoma patient comprising administering to a selected individual a therapeutically effective amount of a PAK1 inhibitor, wherein the genotype of the melanoma is determined to be wild type for BRAF. A method of treating a human melanoma patient, comprising administering to the patient a therapeutically effective amount of a PAK1 inhibitor, wherein the PAK1 inhibitor is measured to be overexpressing PAKl compared to a black paper non-cancerous skin cell. 31. The method of claim 30,
Overexpression of PAK1 in melanoma is 2.5 times greater than expression of PAK1 in non-cancerous skin cells. Wherein the overexpression of PAK1 in the melanoma is indicative of treatment of the individual until the PAK1 overexpression is no longer detected, in a patient undergoing treatment with a PAK1 inhibitor, How to adjust the treatment of a species. The present invention as described above.

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