Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Malignant melanoma accounts for approximately 80 percent of deaths from skin cancer.
• melanoma is surgically curable when discovered at early stages, regional and systemic spread of the disease considerably worsens the prognosis with only 14% of metastatic melanoma patients surviving for five years (American Cancer Society. Cancer facts & figures, 2011).
• the mitogen-activated protein kinase (MAPK) pathway has recently been elucidated as a critical growth pathway in several melanoma subtypes (Lopez-Bergami P. Pigment Cell Melanoma Res. 2011, 24(5):902-921).
• BRAF v-Raf murine sarcoma viral oncogene homolog Bl
• the most frequent BRAF somatic mutation in malignant melanoma is substitution of valine at residue 600 to confer constitutive catalytic activity and signaling (Davies H, et ah, Nature. 2002; 417(6892), 949- 954.).
• the RAF kinase family is comprised of three members, ARAF, BRAF and CRAF, which play a pivotal role in transducing signals in the canonical MAPK signaling pathway from RAS to downstream kinases, MEK1/2 and ERK1/2.
• PAKs group-I p21- activated kinases
• the pathway crosstalk between PAKs and the MAPK pathway signaling in epithelial cells can be induced by a variety of conditions, including growth factor stimulation and cell adhesion to the extracellular matrix (Slack-Davis JK, et al, J Cell Biol. 2003, 162(2) :281-291; Zang M, et al, J Biol Chem. 2001, 276(27):25157- 25165; Beeser A, et al, J Biol Chem. 2005, 280(44):36609-36615).
• PAKl As a major downstream effector of the Rho family small GTPases Cdc42 and Racl, PAKl also plays a fundamental role in linking extracellular signals to changes in actin cytoskeleton organization, cell shape and adhesion dynamics (Arias-Romero LE, & Chernoff J., Biology Cell. 2008, 100(2):97-108;
• PAKl is widely expressed in a variety of normal tissues and expression is significantly increased in breast and lung cancers (Holm C, et al, J Natl Cancer Inst. 2006, 98(10):671-680; Arias-Romero LE, et al, Oncogene 2010, 29(43):5839-5849; Ong CC, et al, Proc Natl Acad Sci U.S.A. 2011, 108(17):7177-7182).
• the present invention relates to methods for treating a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is overexpressed in the tumor compared to non-cancerous skin cells.
• PAKl is amplified in the tumor.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is amplified in the melanoma.
• the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, the individual is a human. In some embodiments, the invention provides methods for treating melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods for treating a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor wherein the PAKl inhibitor is a small molecule, a nucleic acid, or a polypeptide. In some embodiments, the invention provides methods for treating a melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor wherein the PAKl inhibitor is a small molecule, a nucleic acid, or a polypeptide.
• the invention provides methods for treating a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor wherein the PAKl inhibitor is used in combination with a therapeutic agent.
• the invention provides methods for treating a melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor wherein the PAKl inhibitor is used in combination with a therapeutic agent.
• the invention provides uses of PAKl inhibitors for the treatment of melanoma in an individual.
• the invention provides uses of PAKl inhibitors in the manufacture of a medicament for the treatment of melanoma.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is overexpressed in the tumor compared to noncancerous skin cells.
• PAKl is amplified in the tumor.
• the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma.
• the melanoma is a wild-type BRAF melanoma wherein PAKl is amplified in the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is overexpressed in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, the individual is a human.
• the invention provides compositions and kits comprising a PAKl inhibitor for use in the treatment of melanoma.
• a PAKl inhibitor for use in the treatment of melanoma.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is overexpressed in the tumor compared to non-cancerous skin cells.
• PAKl is amplified in the tumor.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma.
• the melanoma is a wild-type BRAF melanoma wherein PAKl is amplified in the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is overexpressed in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, the individual is a human.
• the invention provides methods of inhibiting CRAF signaling and/or MEK signaling in a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is overexpressed in the tumor compared to non-cancerous skin cells.
• PAKl is amplified in the tumor.
• PAKl is overexpressed in the tumor compared to non-cancerous skin cells.
• PAKl is amplified in the tumor.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is amplified in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is overexpressed in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma.
• the melanoma is a mutant BRAF melanoma.
• the individual is a human.
• the invention provides methods for treating melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor.
• the invention provides, methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the BRAF genotype of the melanoma, wherein a melanoma comprising a wild type BRAF indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the expression of PAKl in the melanoma, wherein overexpression of PAKl in the melanoma compared to non-cancerous skin cells indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the copy number of PAKl in the melanoma, wherein amplification of PAKl in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the BRAF genotype of the melanoma and determining the expression of PAKl in the melanoma, wherein the presence of a wild-type BRAF and/or the overexpression of PAKl in the melanoma compared to non-cancerous skin cells indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising one or more of determining the BRAF genotype of the melanoma, determining the expression of PAKl in the melanoma, and determining the copy number of PAKl in the melanoma wherein one or more of the presence of a wild-type BRAF, the overexpression of PAKl in the melanoma compared to non-cancerous skin cells, and amplification of PAKl in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the BRAF genotype of the melanoma, wherein a melanoma comprising a wild type BRAF indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the PAKl expression level of the melanoma, wherein an overexpression of PAKl in the melanoma compared to non-cancerous cells indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the copy number of PAKl in the melanoma, wherein amplification of PAKl in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the BRAF genotype of the melanoma and PAKl expression level of the melanoma, wherein a melanoma comprising a wild type BRAF and/or overexpression of PAKl in the melanoma compared to non-cancerous cells indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on one or more of the BRAF genotype of the melanoma, PAKl expression level of the melanoma, and copy number of PAKl in the melanoma, wherein a melanoma comprising one or more of a wild type BRAF, overexpression of PAKl in the melanoma compared to non-cancerous cells, and amplification of PAKl indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods of adjusting treatment of melanoma in a patient undergoing treatment with a PAKl inhibitor, said method comprising assessing the PAKl expression in the melanoma, wherein overexpression of PAKl in the melanoma indicates that treatment of the individual is adjusted until PAKl overexpression is no longer detected.
• the melanoma is a wild- type BRAF melanoma.
• PAKl is amplified in the melanoma.
• the melanoma is a wild-type melanoma and PAKl is amplified in the melanoma.
• Figure 1 shows that PAKl is highly expressed in human melanoma.
• A Analysis of l lql3 copy number gains in human melanoma tissues. Vertical red line represents chromosome location of the PAKl gene.
• B PAKl DNA copy and mRNA expression (226507_at Affymetrix MAS 5.0 signal) correlated for melanoma tumor samples.
• C Representative images of PAKl
• Cytoplasmic expression score 0 (I), 1 (II), 2 (III) and 3 (IV). Chromogen deposition indicates immunoreactivity against a hematoxylin counterstain. PAKl expression was also seen in stromal cells (III) and cells intercalating within the epidermis that may represent Langerhan's cells (IV).
• Figure 2 demonstrates PAKl playing a critical role in proliferation of BRAF wild- type melanoma cells.
• A Proliferation of melanoma cells following transfection with siRNA oligonucleotides was measured by Cell TiterGlo ATP consumption assay. PAKl was required for cell growth and the data were normalized to control and shown as the mean + SD.
• PAK1/2 inhibition in SK-MEL23 BRAF wild-type melanoma cells decreases signaling to the cytoskeletal, MAPK, proliferation and NF- ⁇ pathways as determined via reverse phase protein array (RPPA) analysis. Normalized RPPA results are presented as mean + SD.
• siNTC non- targeting control siRNA.
• siNRAS NRAS-specific siRNA.
• siPAKl PAKl-specific siRNA.
• ⁇ 1 chromosomal deletion of PAKl gene.
• Figure 3 depicts a series of immunoblots demonstrating that PAKl is required for CRAF activation in BRAF wild- type melanoma cells.
• A PAKl- and PAK2- selective or non-targeting control (NTC) siRNA oligonucleotides were transfected into SK-MEL23 and 537MEL melanoma cells. After 48 h, endogenous MEK1 (A), MEK2 (B) or CRAF (C) proteins were immunoprecipitated and the complexes were immunoblotted to detect phosphorylation of residues critical for catalytic activation. Total protein levels in the immunocomplexes were also determined as loading controls.
• D Cells were treated with DMSO or 5 ⁇ PF-3758309 for 4 h and endogenous CRAF was immunoprecipitated and immunoblotted for Ser338
• Figure 4 contains images demonstrating PAK is required for melanoma cell migration.
• NTC non-targeting control
• Figure 5 depicts a series of immunoblots demonstrating 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 ⁇ PF-3758309 or 0.2 ⁇ PLX- 4720 for 4 h and lysates were analyzed for phosphorylation of MAPK pathway components. Lighter and darker exposures of p-MEKl/2(S217/S221) immunoblots are shown.
• Figure 6 depicts a series of graphs demonstrating decreased viability of BRAF wild-type melanoma cells due to treatment with in-house PAK inhibitors.
• Catalytic inhibition of PAKl via 1-007, 1-054, 1-087 and PF-3758309 treatment was tested in vitro using (A) SK-MEL23 and (B) 537MEL cells using a 4-day Cell TiterGlo (Promega) viability assay.
• 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.
• Figure 8 depicts a series of graphs demonstrating individual tumor data for the SK-MEL23 preclinical tumor model of BRAF wild-type melanoma.
• A Tumor growth inhibition and
• B body weight loss are shown for individual animals treated with 10, 15 and 25 mg/kg PF- 3758309.
• cubic regression splines were used to fit a non linear profile to the time courses of log 2 tumor volume at each dose level. These non linear profiles were then related to dose within the mixed model.
• Cubic regression splines were used to fit a non linear profile to the time courses of log 2 tumor volume at each dose level. These non linear profiles were then related to dose within the mixed model.
• TGI Tumor growth inhibition as a percentage of Vehicle
• AUC area under the fitted curve
• Figure 9 shows immunoblots demonstrating differing pharmacodynamic responses of BRAF wild-type tumors treated with either G945 BRAF inhibitor or PF-3758309.
• Phosphorylation of CRAF(Ser338) was determined for SK-MEL23 xenograft tumors following administration of either 35 mg/kg PF-3758309 i.p. or 10 mg/kg G945 (BRAF inhibitor) p.o. compounds.
• Tumors were harvested 1 hour post dosing and flash frozen. Each lane represents tumor lysate from an individual xenograft mouse.
• FIG 10 is a diagram depicting the mechanism of action for PAK1 in BRAF wild- type melanoma.
• A In the context of oncogenic mutation, BRAF strongly drives activation of the MAPK signaling pathway and these tumor cells are sensitive to inhibition of this kinase.
• B In melanomas in which BRAF is not mutated, elevated expression and genomic amplification of PAK1 is frequent and results in increased signaling to CRAF-MEK-ERK and potentially additional effector pathways. This subset of melanoma is relatively insensitive to BRAF inhibition and proliferative capacity is dependent on PAK1.
• the present invention provides methods and compositions for the treatment of melanoma in an individual contacting the melanoma with a therapeutically effective amount of a PAK1 inhibitor.
• the invention also provides such methods of treatment comprising administering to the individual, a therapeutically effective amount of a PAK1 inhibitor.
• the melanoma is a wild-type BRAF melanoma.
• the melanoma overexpresses PAK1 compared to non-cancerous cells.
• PAK1 is amplified in the melanoma.
• the melanoma is a wild- type BRAF melanoma and overexpresses PAK1 compared to non-cancerous cells.
• the melanoma is a wild-type BRAF melanoma
• the melanoma overexpresses PAK1 compared to non-cancerous cells and PAK1 is amplified in the melanoma.
• PAK as used herein, refers to a family of non-receptor serine/threonine protein kinases
• the p21-activated protein kinase (PAK) family of serine/threonine protein kinases plays important roles in cytoskeletal organization, cellular morphogenesis, cellular processes and cell survival (Daniels et ah, Trends Biochem. Sci. 1999 24: 350-355; Sells et ah, Trends Cell. Biol. 1997 7: 162-167).
• the PAK family consists of six members subdivided into two groups: PAK 1-3 (group I) and PAK 4-6 (group II) which are distinguished based upon sequence homologies and the presence of an autoinhibitory region in group I PAKs.
• p21 -Activated kinases serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis.
• PAKl or "p21 -activated protein (Cdc42/Rac)-activated kinase 1" as used herein refers to a native PAKl from any vertebrate source, including mammals such as primates ⁇ e.g., humans) and rodents ⁇ e.g., mice and rats), unless otherwise indicated.
• the term also encompasses naturally occurring variants of PAKl, e.g., splice variants or allelic variants.
• the sequence of an exemplary human PAKl nucleic acid is NC_000011.9.
• An exemplary human PAKl amino acid sequence is NP_0011220921 or
• NP_002567.3 The sequence of an exemplary mouse PAKl nucleic acid is NC_000073.6 or an exemplary mouse PAKl amino acid sequence NP_035165.2.
• sequence of an exemplary rat PAKl nucleic acid is NC_005100.2 or an exemplary rat PAKl amino acid sequence
• NP_058894.1 The sequence of an exemplary dog PAKl nucleic acid is NC_006603.3 or an exemplary dog PAKl amino acid sequence XP_849651.1.
• the sequence of an exemplary cow PAKl nucleic acid is AC_000186.1 or NC_007330.5.
• An exemplary cow PAKl amino acid sequence is NP_001070366.1.
• the sequence of an exemplary rhesus monkey PAKl nucleic acid is NC_007871.1.
• An exemplary rhesus monkey PAKl amino acid sequence is XP_001090310.1 or NP_001090423.2.
• the sequence of an exemplary chicken PAKl nucleic acid is
• BRAF or "Serine/threonine-protein kinase B-Raf,” as used herein, refers to as used herein refers to a native BRAF from any vertebrate source, including mammals such as primates ⁇ e.g., humans) and rodents ⁇ e.g., mice and rats), unless otherwise indicated.
• GC07M 140424) "full-length,” unprocessed BRAF as well as any form of BRAF that result from processing in the cell.
• the term also encompasses naturally occurring variants of BRAF, e.g., 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.
• the sequence of an exemplary dog BRAF nucleic acid is NC_006598.3 or an exemplary dog BRAF amino acid sequence 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.
• the sequence of an exemplary cow BRAF nucleic acid is AC_000161.1 or an exemplary cow BRAF amino acid sequence XP_002687048.1.
• the sequence of an exemplary horse BRAF nucleic acid is NC_009147.2 or an exemplary horse BRAF amino acid sequence XP_001496314.2.
• Wild-type BRAF refers herein to a naturally occurring BRAF (including naturally occurring variants) not associated with melanoma.
• An example of wild-type human BRAF is provided by GenBank Accession No. NP_004324.2.
• BRAF melanomas can be categorized and classified by BRAF type: wild-type BRAF and mutant BRAF.
• Wild BRAF refers to a BRAF protein with one or more mutations which is associated with melanoma.
• An example of a mutant BRAF is one where a valine at position 600 is replaced with a glutamate (V600E).
• V600E glutamate
• melanomas can be categorized by BRAF type: wild-type BRAF and mutant BRAF.
• CRAF or "v-raf leukemia viral oncogene 1" as used herein, as used herein, as used herein refers to a native CRAF from any vertebrate source, including mammals such as primates (e.g. , humans) and rodents (e.g. , mice and rats), unless otherwise indicated.
• the terms encompass the genomic location (e.g. , 3p25 cytogenetic band, chromosome 3: 12625100-12705700, and/or
• GC03M012625 "full-length,” unprocessed CRAF as well as any form of CRAF that result from processing in the cell.
• the term also encompasses naturally occurring variants of CRAF, e.g. , 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 mitogen-activated protein kinase kinase
• MEK1 MAP2K1
• MEK2 MEK2
• MKK3 mitogen-activated protein kinase 3
• MKK4 MKK4
• MAP2K5 MKK5
• MAP2K6 MKK6
• MKK7 MKK7
• the activators of p38 MKK3 and MKK6), JNK (MKK4 and MKK7)
• ERK MEK1 and MEK2
• 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.
• An exemplary human MEK3 amino acid sequence is NP_002747.2 or NP_659731.1.
• the sequence of an exemplary human MEK4 nucleic acid is NC_000017.10 or an exemplary human MEK4 amino acid sequence NP_003001.1.
• the sequence of an exemplary human MEK5 nucleic acid is NC_000015.9.
• An exemplary human MEK5 amino acid sequence is 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.
• Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
• the nucleotides can be
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may be further modified after synthesis, such as by conjugation with a label.
• modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g. , methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g. , phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g. , nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g.
• any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
• the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.
• Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, ?-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
• One or more phosphodiester linkages may be replaced by alternative linking groups.
• alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate"), P(S)S ("dithioate”), (0)NR2 ("amidate"), P(0)R, P(0)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical.
• Oligonucleotide generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
• primer refers to a single stranded polynucleotide that is capable of hybridizing to a nucleic acid and following polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.
• small molecule refers to any molecule with a molecular weight of about 2000 daltons or less, preferably of about 500 daltons or less.
• antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies,
• multispecific antibodies e.g. , bispecific antibodies
• antibody fragments so long as they exhibit the desired antigen-binding activity.
• biomarker refers to an indicator, e.g. , predictive, diagnostic, and/or prognostic, which can be detected in a sample.
• the biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g. , cancer) characterized by certain, molecular, pathological, histological, and/or clinical features.
• 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 a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to the treatment.
• level of expression or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g. , posttranslational modification of a polypeptide).
• Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g. , by proteolysis.
• "Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).
• Elevated expression refers to an increased expression or increased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g. , cancer) or an internal control (e.g., housekeeping biomarker). In some examples, elevated expression or overexpression is the result of gene amplification.
• Reduced expression refers to a decrease expression or decreased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control (e.g. , housekeeping biomarker).
• a control such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control (e.g. , housekeeping biomarker).
• housekeeping biomarker refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides) which are typically similarly present in all cell types.
• the housekeeping biomarker is a "housekeeping gene.”
• a "housekeeping gene” refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically similarly present in all cell types.
• Amplification generally refers to the process of producing multiple copies of a desired sequence.
• Multiple copies mean at least two copies.
• a “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence.
• copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and/or sequence errors that occur during amplification. Diploid cells typically contain two copies of a given gene, one on each chromosome.
• "amplification” or a chromosomal gene in a cell refers to a process where more than two copies of the gene are present in the cell.
• multiplex-PCR refers to a single PCR reaction carried out on 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.
• diagnosis is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g. , cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, e.g. , by histopathological criteria, or by molecular features (e.g. , a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).
• a method of aiding diagnosis of a disease or condition can comprise measuring certain biomarkers in a biological sample from an individual.
• correlate or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. "Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease
• progression e.g., cancer progression
• a reduction in tumor size e.g., a reduction in tumor size
• inhibition i.e. , reduction, slowing down or complete stopping
• prediction or predicting is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a particular anti-cancer therapy. In one embodiment, prediction or predicting relates to the extent of those responses. In one embodiment, the prediction or predicting relates to whether and/or the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence.
• the predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
• the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient, following a therapeutic regimen is likely.
• a treatment regimen such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient, following a therapeutic regimen is likely.
• substantially the same denotes a sufficiently high degree of similarity between two numeric values, such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values ⁇ e.g., K d values or expression).
• the difference between said 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/comparator value.
• substantially different denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values ⁇ e.g., K d values).
• the difference between said 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/comparator molecule.
• label when used herein refers to a detectable compound or composition.
• the label is typically conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused.
• the label may itself be detectable ⁇ e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which results in a detectable product.
• an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
• a “therapeutically effective amount" of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects
• prophylactic ally effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• the therapeutically effective amount of the PAK1 inhibitor may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e. , slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e. , slow to some extent and preferably stop) tumor metastasis; inhibit or delay, to some extent, tumor growth or tumor progression; and/or relieve to some extent one or more of the symptoms associated with the disorder.
• the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
• efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
• Reduce or “inhibit” is to decrease or reduce an activity, function, and/or amount as compared to a reference. In certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of the blood vessels in angiogenic disorders.
• pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.
• a "sterile" formulation is aseptic or free from all living microorganisms and their spores.
• a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
• a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
• treatment is an approach for obtaining beneficial or desired clinical results.
• beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).
• treatment is a reduction of pathological consequence of a proliferative disease.
• the methods of the invention contemplate any one or more of these aspects of treatment.
• melanoma refers to a tumor of high malignancy that starts in melanocytes of normal skin or moles and metastasizes rapidly and widely.
• melanoma can be used interchangeably with the terms “malignant melanoma”, “melanocarcinoma”,
• Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but not limited to, squamous cell cancer (e.g.
• lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade/folli
• lung cancer including small-cell lung cancer, non-
• CLL chronic lymphocytic leukemia
• ALL acute lymphoblastic leukemia
• PTLD post-transplant lymphoproliferative disorder
• phakomatoses abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases.
• cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.
• the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.
• the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers.
• anti-cancer therapy refers to a therapy useful in treating cancer.
• anticancer therapeutic agents include, but are limited to, e.g. , chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer , anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g. , GleevecTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g. , celecoxib), interferons, cytokines, antagonists (e.g.
• neutralizing antibodies that bind to one or more of the following targets PDGFR-beta, BlyS, APRIL, BCMA receptor(s), TRAIL/ Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also included in the invention.
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes (e.g. , At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g.
• methotrexate adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
• a tumoricidal agent causes destruction of tumor cells.
• a "toxin” is any substance capable of having a detrimental effect on the growth or proliferation of a cell.
• chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer.
• examples of chemotherapeutic agents include alkylating agents such as thiotepa and
• cyclosphosphamide CYTOXAN®
• alkyl sulfonates such as busulfan, improsulfan and piposulfan
• aziridines such as benzodopa, carboquone, meturedopa, and uredopa
• ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine
• acetogenins especially bullatacin and bullatacinone
• delta-9-tetrahydrocannabinol (dronabinol, MARINOL®)
• beta-lapachone CYTOXAN®
• alkyl sulfonates such as busulfan, improsulfan and piposulfan
• aziridines such as benzodopa, carboquone, meturedopa, and uredopa
• lapachol lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN® ) , CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chloropho
• calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (
• ADRIAMYCIN® morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,
• mycophenolic acid nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine
• androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
• elfomithine elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (
• paclitaxel TAXOL®
• albumin-engineered nanoparticle formulation of paclitaxel ABRAXANETM
• docetaxel TXOTERE®
• chloranbucil 6-thioguanine
• mercaptopurine methotrexate
• platinum agents such as cisplatin, oxaliplatin (e.g.
• vincas which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine
• NAVELBINE® etoposide
• VP-16 etoposide
• ifosfamide mitoxantrone; leucovorin; novantrone
• edatrexate edatrexate
• daunomycin edatrexate
• aminopterin ibandronate
• topoisomerase inhibitor RFS 2000 edatrexate
• DMFO difluoromethylornithine
• retinoids such as retinoic acid, including bexarotene
• Target® bisphosphonates such as clodronate (for example, BONEFOS® or
• OSTAC® etidronate
• DIDROCAL® etidronate
• ZOMETA® alendronate
• AREDIA® pamidronate
• SKELID® tiludronate
• ACTONEL® risedronate
• troxacitabine a 1,3-dioxolane nucleoside cytosine analog
• oligonucleotides particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and
• VAXID® vaccine topoisomerase 1 inhibitor (e.g. , LURTOTECAN®); rmRH (e.g. ,
• ABARELIX® BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. , celecoxib or etoricoxib), proteosome inhibitor (e.g.
• Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4- hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as
• prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al, "Prodrugs: A Chemical
• the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
• cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
• a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell ⁇ e.g., a melanoma call).
• growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
• Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
• DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:
• the taxanes are anticancer drugs both derived from the yew tree.
• Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
• radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.
• mammals include, but are not limited to,
• the individual or subject is a human.
• Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.
• concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
• Reduce or inhibit is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
• package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
• the invention package insert comprises instructions to treat melanoma with a PAK1 inhibitor.
• An "article of manufacture” is any manufacture (e.g.
• a package or container or kit comprising at least one reagent, e.g. , a medicament for treatment of a disease or disorder (e.g. , cancer), or a probe for specifically detecting a biomarker described herein.
• the manufacture 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 an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individuals, populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.
• an “individual,” “subject,” or “patient” is a vertebrate.
• the vertebrate is a mammal.
• Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats.
• a mammal is a human.
• sample refers to a composition that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
• disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
• the definition encompasses blood and other liquid samples of biological origin and tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom.
• the source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids; and cells from any time in gestation or development of the subject or plasma.
• Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.
• sample includes biological samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semisolid or solid matrix for sectioning purposes.
• a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample.
• the sample is a clinical sample.
• the sample is used in a diagnostic assay.
• the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a
• tumor cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumor cells of interest; for instance, skin samples.
• tissue sample or “cell sample” is meant a collection of similar cells obtained from a tissue of a subject or individual.
• the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
• the tissue sample may also be primary or cultured cells or cell lines.
• the tissue or cell sample is obtained from a disease tissue/organ.
• the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
• a “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g. , tissue or cells) of the same subject or individual.
• healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue e.g., cells or tissue adjacent to a tumor.
• the reference sample is non-cancerous skin cells.
• a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual.
• the reference sample is non-cancerous skin cells of the body of the same subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual.
• the reference sample is non-cancerous skin cells of an individual who is not the subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.
• a reference sample is a single sample or combined multiple samples from the same subject or patient that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample is obtained at an earlier time point from the same subject or patient than when the test sample is obtained. Such reference sample may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
• a reference sample includes all types of biological samples as defined above under the term "sample” that is obtained from one or more individuals who is not the subject or patient. In certain embodiments, a reference sample is obtained from one or more individuals with an angiogenic disorder (e.g. , cancer) who is not the subject or patient.
• an angiogenic disorder e.g. , cancer
• a reference sample is a combined multiple samples from one or more healthy individuals who are not the subject or patient. In certain embodiments, a reference sample is a combined multiple samples from one or more individuals with a disease or disorder (e.g. , an angiogenic disorder such as, for example, cancer) who are not the subject or patient. In certain embodiments, a reference sample is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or patient. In certain embodiments, a reference sample is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) who are not the subject or patient.
• a disease or disorder e.g., an angiogenic disorder such as, for example, cancer
• a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis, provided that it is understood that the same section of tissue sample may be analyzed at both morphological and molecular levels, or analyzed with respect to both polypeptides and polynucleotides.
• Expression levels/amount of a gene or biomarker can be determined qualitatively and/or quantitatively based on any suitable criterion known in the art, including but not limited to mRNA, cDNA, proteins, protein fragments and/or gene copy number.
• expression/amount of a gene or biomarker in a first sample is increased as compared to expression/amount in a second sample.
• expression/amount of a gene or biomarker in a first sample is decreased as compared to expression/amount in a second sample.
• the second sample is reference sample.
• the terms “increase” or “overexpress” refer to an overall increase of about any of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of protein or nucleic acid, detected by standard art known methods such as those described herein, as compared to a reference sample.
• the terms “increase” or “overexpress” refer to the increase in expression level/amount of a gene or biomarker in the sample wherein the increase is at least about any of 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 25x, 50x, 75x, or lOOx the expression level/amount of the respective gene or biomarker in the reference sample.
• the term "decrease” herein refers to an overall reduction of about any of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of protein or nucleic acid, detected by standard art known methods such as those described herein, as compared to a reference sample.
• the term decrease refers to the decrease in expression level/amount of a gene or biomarker in the sample wherein the decrease is at least about any of 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, O. lx, 0.05x, or O.Olx the expression level/amount of the respective gene or biomarker in the reference sample.
• Detection includes any means of detecting, including direct and indirect detection.
• correlate or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of gene expression analysis or protocol, one may use the results of the gene expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.
• label when used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused.
• the label may itself be detectable (e.g. , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
• polypeptide refers to polymers of amino acids of any length.
• the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
• polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
• the term “polypeptide” as used herein specifically encompasses a "protein”.
• polypeptide and “protein” as used herein specifically encompass antibodies.
• an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide nucleic acid.
• An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
• an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
• a “gene,” “target gene,” “target biomarker,” “target sequence,” “target nucleic acid” or “target protein,” as used herein, is a polynucleotide or protein of interest, the detection of which is desired.
• a “template,” as used herein, is a polynucleotide that contains the target nucleotide sequence.
• target sequence is a polynucleotide that contains the target nucleotide sequence.
• the terms “target sequence,” “template DNA,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” and variations thereof, are used interchangeably.
• a “native sequence” polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature.
• a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any mammal.
• Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means.
• the term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g. , an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.
• an “isolated” polypeptide or “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• the polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, or more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or silver stain.
• Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
• a polypeptide "variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide.
• variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide.
• a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the native sequence polypeptide.
• Clinical benefit can be measured by assessing various endpoints, e.g. , inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and/or symptoms; reduction in lesion size; inhibition (i.e. , reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread;
• endpoints e.g. , inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and/or symptoms; reduction in lesion size; inhibition (i.e. , reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread;
• the present invention provides methods for treating melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the method comprises administering to the individual a therapeutically effective about of a PAKl inhibitor.
• Melanoma is a malignant tumor of melanocytes, e.g., cells that produce melanin, a dark pigment which is responsible for the color of skin. Melanomas predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye e.g. uveal melanoma).
• Melanoma can originate in any part of the body that contains melanocytes. Examples of melanoma includes, but are not limited to superficial spreading melanoma, nodular melanoma, Lentigo maligna melanoma, and Acral lentiginous melanoma. Melanoma may be staged depending on a number of criteria including size, ulceration, spread to lymph nodes, and/or spread to other tissues or organs. In some embodiments, the invention provides methods of treating a Stage I melanoma in an individual by contacting the melanoma with an inhibitor of PAKl.
• the invention provides methods of treating a Stage II melanoma in an individual by contacting the melanoma with an inhibitor of PAKl. In some embodiments, the invention provides methods of treating Stage III melanoma in an individual by contacting the melanoma with an inhibitor of PAKl. In some embodiments, the invention provides methods of treating Stage IV melanoma in an individual by contacting the melanoma with an inhibitor of PAKl. In some embodiments, the invention provides methods of treating metastatic melanoma in an individual by contacting the melanoma with an inhibitor of PAKl. In some embodiments, the invention provides methods of treating recurrent melanoma in an individual by contacting the melanoma with an inhibitor of PAKl. In some embodiments, the method comprises
• the PAKl inhibitor is a small molecule inhibitor of PAKl.
• the individual is a mammal. In some embodiments the individual is a human.
• the invention provides methods of treating melanoma in an individual wherein the melanoma is a wild-type BRAF melanoma. In some embodiments, the invention provides methods of treating wild-type BRAF melanoma comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor. In some embodiments, the invention provides methods of treating wild-type BRAF melanoma comprising administering to the individual a therapeutically effective amount of a PAKl 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/ERKs signaling pathway (the RAF-MEK-ERK pathway), which affects cell division, differentiation, and secretion.
• RAF-MEK-ERK signaling is frequently dysregulated in cancer. More than 30 mutations of the BRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers from more than 80% in melanomas, to as little as 0-18% in other tumors, such as 1-3% in lung cancers and 5% in colorectal cancer.
• a common mutation found in cancers, particularly melanoma is a substitution of valine at codon 600 with glutamate (i.e., V600E).
• a thymine is substituted with adenine at nucleotide 1799 which leads to the V600E mutation.
• V600 mutations of BRAF lead to constitutive BRAF kinase activity.
• Methods to determine the genotype of BRAF in a melanoma are known to those in the art; for example, the nucleotide sequence of the BRAF gene from the melanoma may be determined using standard sequencing methods or by using the KASP SNP genotyping system (KBioscience).
• the invention provides methods of treating melanoma in an individual wherein the melanoma is a wild-type BRAF melanoma.
• the invention provides methods of treating melanoma in an individual wherein the melanoma is a wild-type BRAF melanoma and the melanoma overexpresses PAKl compared to non-cancerous cells.
• the invention provides methods of treating melanoma in an individual wherein the melanoma comprises a wild- type BRAF and PAKl is amplified in the melanoma.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma compared to non-cancerous cells and PAKl is amplified in the melanoma.
• the invention provides methods of treating melanoma in an individual wherein the melanoma is a mutant BRAF melanoma. In some embodiments the invention provides methods of treating melanoma in an individual wherein the melanoma is a mutant BRAF melanoma and the melanoma overexpresses PAKl compared to non-cancerous cells. In some embodiments the invention provides methods of treating melanoma in an individual wherein the melanoma comprises a mutant BRAF and PAKl is amplified in the melanoma.
• the invention provides methods of treating melanoma in an individual wherein the melanoma comprises a mutant BRAF wherein the mutant BRAF is not a V600E mutant BRAF.
• the individual in a mammal.
• the individual is a human.
• the invention provides methods of treatment of melanoma in an individual by contacting the melanoma with a therapeutically effective amount of PAKl inhibitor.
• the invention provides methods of treatment of melanoma in an individual by administering to the individual a therapeutically effective amount of PAKl inhibitor.
• PAKs participate in a number of pathways that are commonly deregulated in human cancer cells.
• PAKl is a component of the mitogen-activated protein kinase (MAPK), JUN N-terminal kinase (JNK), steroid hormone receptor, and nuclear factor (NF) signaling pathways, which all have been associated with oncogenesis.
• PAKs activate MEK and RAFl by phosphorylating them on serine 298 and serine 338, respectively.
• the increase of Ras-induced transformation by PAKl correlated with its effects on signaling through the extracellular signal-regulated kinase (ERK)- MAPK pathway, and was dissociable from effects on the JNK or p38-MAPK pathways. (R. Kumar et al. Nature Rev. Cancer 2006 6:459).
• ERK/MEK pathway Constitutive activation of the ERK/MEK pathway is implicated in the formation, progression and survival of tumors and furthermore is associated with an aggressive phenotype, characterized by uncontrolled proliferation, loss of control of apoptosis and poor prognosis (J. A. Spicer, Expert Opin. Drug Discov. 2008 3:7). Tumor formation and progression require the inactivation of pro-apoptotic signals in cancer cells. PAK activity has been shown to downregulate several important pro-apoptotic pathways. PAKl phosphorylation of RAFl induces RAFl translocation to mitochondria, where it phosphorylates the pro-apoptotic protein BCL2-antagonist of cell death (BAD).
• BAD pro-apoptotic protein BCL2-antagonist of cell death
• PAKl, PAK2, PAK4 and PAK5 have also been reported to directly phosphorylate and inactivate BAD in selected cell types, such as CV-1 (simian) in origin and carrying the SV40 (COS) kidney, Chinese hamster ovarian (CHO) and human embryonic kidney (HEK) 293T cells (R. Kumar et ah, ibid).
• CV-1 simian
• COS Chinese hamster ovarian
• HEK human embryonic kidney
• PAKl is widely expressed in a variety of normal tissues; however, expression is significantly increased in ovarian, breast and bladder cancer.
• genomic amplification of PAKl is associated with resistance to tamoxifen therapy, possibly occurring as a result of direct phosphorylation and ligand- independent transactivation of estrogen receptor by PAKl (S. K. Rayala et ah, Cancer Res. 2006. 66: 1694-1701).
• the invention provides methods of treating melanoma in an individual by contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor. In some aspects, the invention provides methods of treating melanoma in an individual by administering to the individual a therapeutically effective amount of a PAKl inhibitor.
• the PAKl gene is amplified in the melanoma. In some embodiments, the copy number of the PAKl in the melanoma is about any of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or greater than 5.0. Methods of determining the copy number of the PAKl gene in a melanoma are known in the art.
• the copy number of the PAKl gene may be determined by using SNP arrays such as the Affymetrix 500K SNP array analysis.
• the invention provides methods of treating melanoma in an individual wherein the copy number of PAKl in the melanoma is greater than about 2.5.
• the invention provides methods of determining the copy number of PAKl in a melanoma subsequent to treatment with a PAKl inhibitor.
• the copy number of PAKl in a melanoma is compared to the copy number of PAKl in non-cancerous cells; for example, non-cancerous skin cells.
• PAKl is amplified in the melanoma and the melanoma overexpresses PAKl. In some embodiments, PAKl is amplified in the melanoma and the melanoma is a wild- type BRAF melanoma. In some embodiments the individual is a mammal. In some embodiments, the individual is a human.
• the invention provides methods of treating melanoma in an individual by contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor wherein PAKl is overexpressed in the melanoma. In some aspects, the invention provides methods of treating melanoma in an individual by administering to the individual a therapeutically effective amount of a PAKl inhibitor wherein PAKl is overexpressed in the melanoma.
• Methods to determine expression of PAKl are known in the art. Examples of methods to determine expression levels of PAKl in a melanoma include, but are not limited to immunohistochemistry, reverse-phase protein array (RPPA), quantitative PCR, immunoassays, and the like. Levels of PAKl expression can be compared to other tumors and cells by using the Gene Expression Omnibus (GEO) database.
• GEO Gene Expression Omnibus
• the invention provides methods for treating melanoma in an individual by contacting the melanoma with a PAKl inhibitor wherein PAKl is overexpressed in the melanoma compared to non-cancerous cells. In some aspects, the invention provides methods of treating melanoma in an individual by administering to the individual a therapeutically effective amount of a PAKl inhibitor wherein PAKl is overexpressed in the melanoma. In some embodiments, expression of PAKl in the melanoma is about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% expression in non-cancerous cells.
• expression of PAKl in the melanoma is about any of 1.5-fold, 2.0-fold, 2.5-fold, 3, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10-fold or greater than 10-fold compared to expression of PAKl in non-cancerous cells.
• the melanoma overexpresses PAKl compared to non-cancerous cells and the melanoma is a wild- type BRAF melanoma.
• the melanoma overexpresses PAKl compared to non-cancerous cells and PAKl is amplified in the melanoma.
• the melanoma overexpresses PAKl compared to non-cancerous cells and the melanoma is a wild- type BRAF melanoma amd PAKl is amplified in the melanoma.
• the individual is a mammal. In some embodiments, the individual is a human.
• the invention provides methods of inhibiting CRAF signaling in a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor. In some aspects, the invention provides methods of inhibiting CRAF signaling in a melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor.
• Methods of measuring CRAF signaling are known in the art. For example, CRAF activation can be determined by immunoblot of CRAF isolated from a melanoma from an individual before and/or after treatment with a PAKl inhibitor. Activation of CRAF may be measured using phospho-CRAF(Ser338) antibodies.
• the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is over expressed in the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is amplified in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, the individual
• the invention provides methods of inhibiting MEK signaling in a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor. In some aspects, the invention provides methods of inhibiting MEK signaling in a melanoma in an individual comprising administering to the individual a therapeutically effective amount of a PAKl inhibitor.
• Methods of measuring MEK signaling are known in the art. For example, MEK activation can be determined by immunoblot of MEK isolated from a melanoma from an individual before and/or after treatment with a PAKl inhibitor. Activation of MEK may be measured using phospho-MEKl/l(Ser217/Ser221) antibodies.
• the melanoma is a wild-type BRAF melanoma. In some embodiments, PAKl is over expressed in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is amplified in the melanoma. In some embodiments, the melanoma is a wild- type BRAF melanoma wherein PAKl is
• PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma.
• PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma.
• the individual is a mammal. In some embodiments, the individual is a human. Inhibitors of PAKl
• PAKl inhibitors useful in the methods described herein.
• the PAKl inhibitor is a small molecule, a nucleic acid, a polypeptide or an antibody.
• PAK inhibitors are provided in WO 2007/072153, and WO 2010/07184 both of which are incorporated herein by reference.
• small molecules for use as PAKl inhibitors for the treatment of melanoma are preferably organic molecules other than binding polypeptides or antibodies as defined herein that bind to PAKl or interfere with PAKl signaling as described herein.
• Binding organic small molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). Binding organic small molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules that are capable of binding, preferably specifically, to a polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic small molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g. , PCT Publication Nos.
• Binding organic small molecules may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines
• AstraZeneca has disclosed bicyclic heterocyclic PAKl inhibitors of formula II (see
• Pfizer has disclosed PAK inhibitors elaborated on lH-thieno[3,2-c]pyrazole (III), 3-amino- tetrahydropyrrolo[3,4-c]pyrazole (IV) and N4-(lH-pyrazol-3-yl)pyrimidine-2,4-diamine (V) (see WO 2004007504, WO 2007023382, WO2007072153, and WO2006072831).
• PF-3758309 (VI) is a potent ATP-competitive inhibitor of PAK1, 4, 5 and 6 that has been in clinical testing.
• PA 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.
• the invention provides herein polynucleotide antagonists of PAK1 for the treatment of melanoma in an individual.
• the polynucleotide can be an RNAi such as siRNA or miRNA, an antisense oligonucleotides, an RNAzymes, a DNAzymes, an oligonucleotides, a nucleotides, or any fragments of these, including DNA or RNA ⁇ e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins ⁇ e.g., iRNPs).
• RNAi such as siRNA or miRNA
• an antisense oligonucleotides such as
• the polynucleotide targets PAK1 expression ⁇ e.g. targets PAK1 mRNA).
• the polynucleotide may be an antisense nucleic acid and/or a ribozyme.
• the antisense nucleic acids comprise a sequence complementary to at least a portion of an RNA transcript of PAKl. However, absolute complementarity, although preferred, is not required.
• a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded PAKl antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
• the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with an PAKl RNA it may contain and still form a stable duplex (or triplex as the case may be).
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• Polynucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
• sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
• oligonucleotides complementary to either the 5'- or 3'-non-translated, non-coding regions of the PAKl gene could be used in an antisense approach to inhibit translation of endogenous PAKl mRNA.
• Polynucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
• Antisense polynucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of PAKl mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• the PAKl antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
• a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the PAKl gene.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the PAKl antisense nucleic acid.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells.
• Expression of the sequence encoding PAKl, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells.
• Such promoters can be inducible or constitutive.
• Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat 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 sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
• Small inhibitory RNAs can also function as PAKl inhibitors for use in the treatment of melanoma.
• PAKl expression can be by contacting the melanoma with a small double stranded RNA (dsRNA) or a vector or construct that causes the production of small double- stranded RNA, such that expression of PAKl is specifically inhibited.
• dsRNA small double stranded RNA
• Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art (Tuschi, T et al (1999) Genes Dev.
• PAKl siRNA oligonucleotide sequences include, but are not limited to 1)
• GAAGAGAGGTTCAGCTAAA 2) GGAGAAATTACGAAGCATA, 3)
• binding polypeptides are polypeptides that bind, preferably and specifically to PAKl as described herein.
• the binding polypeptides are PAKl antagonists. Binding polypeptides may be chemically synthesized using known polypeptide synthesis methodology or may be prepared and purified using recombinant technology.
• Binding polypeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such binding polypeptides that are capable of binding, preferably specifically, to a PAKl, as described here
• Binding polypeptides may be identified without undue experimentation using well known techniques.
• techniques for screening polypeptide libraries for binding polypeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and W 084/03564; Geysen et al, Proc. Natl. Acad. Sci.
• bacteriophage (phage) display is one well known technique which allows one to screen large polypeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a target PAK1.
• Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J.K. and Smith, G. P. (1990) Science, 249: 386).
• the utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al.
• Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments.
• WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands.
• WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage.
• Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9: 187).
• WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library.
• a method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Patent Nos. 5,498,538, 5,432,018, and WO 98/15833.
• the PAK1 inhibitor for the treatment of melanoma in an individual is an isolated antibodies that bind to PAK1.
• the antibody is humanized.
• an anti-PAKl antibody or an antibody that inhibits PAK1 function is a monoclonal antibody, including a chimeric, humanized or human antibody.
• the antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
• the antibody is a full length antibody, e.g., an intact IgGl" antibody or other antibody class or isotype as defined herein.
• amino acid sequence variants of the antibodies and/or the binding polypeptides provided herein are contemplated.
• Amino acid sequence variants of an antibody and/or binding polypeptides may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody and/or binding polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody and/or binding polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g. , target-binding.
• antibody variants and/or binding polypeptide variants having one or more amino acid substitutions are provided.
• Sites of interest for substitutional mutagenesis include the HVRs and FRs.
• Amino acid substitutions may be introduced into an antibody and/or binding polypeptide of interest and the products screened for a desired activity, e.g. ,
• substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. , a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g. , improvements) in certain biological properties (e.g. , increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
• An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g. , using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).
• Alterations may be made in HVRs, e.g. , to improve antibody affinity.
• Such alterations may be made in HVR "hotspots," i.e. , residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g. , Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
• HVR "hotspots” i.e. , residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g. , Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
• Affinity maturation by constructing and reselecting from secondary libraries has been described, e.
• affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g. , error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
• a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
• Another method to introduce diversity involves HVR- directed approaches, in which several HVR residues (e.g. , 4-6 residues at a time) are
• HVR residues involved in antigen binding may be specifically identified, e.g. , using alanine scanning mutagenesis or modeling.
• CDR-H3 and CDR-L3 in particular are often targeted.
• substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
• conservative alterations e.g. , conservative substitutions as provided herein
• Such alterations may be outside of HVR "hotspots" or SDRs.
• each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
• PAKl inhibitors of the methods described herein can be used either alone or in combination with other agents in a therapy for the treatment of melanoma.
• a PAKl inhibitor described herein may be co-administered with at least one additional therapeutic agent including another PAKl inhibitor.
• an additional therapeutic agent is a
• the additional therapeutic agent may be Aldesleukin, dacarbazine, DTIC-Dome (Dacarbazine), Ipilimumab, Proleukin (Aldesleukin), Vemurafenib, Yervoy (Ipilimumab), and/or Zelboraf (Vemurafenib).
• Aldesleukin dacarbazine, DTIC-Dome (Dacarbazine), Ipilimumab, Proleukin (Aldesleukin), Vemurafenib, Yervoy (Ipilimumab), and/or Zelboraf (Vemurafenib).
• combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the PAKl inhibitor can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
• PAKl inhibitors are used for the treatment of melanoma in an individual in combination with radiation therapy.
• PAKl inhibitors are used for the treatment of melanoma in an individual in combination with surgical removal of all or a portion of the melanoma from the individual.
• the individual has been previously treated for melanoma, for example, using an anti-cancer therapy.
• the anti-cancer therapy is surgery.
• the subject can be further treated with an additional anti-cancer therapy before, during (e.g. , simultaneously), or after administration of the PAKl inhibitor.
• anti-cancer therapies include, without limitation, surgery, radiation therapy (radiotherapy), biotherapy, immunotherapy, chemotherapy, or a combination of these therapies.
• the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g. , injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.
• the invention provides for methods for the treatment of melanoma in an individual with a PAK1 inhibitor wherein the PAK1 inhibitor is administered intravenously to the individual.
• the invention provides for methods for the treatment of melanoma in an individual with a PAK1 inhibitor wherein the PAKlinhibitor is administered topically to the individual.
• therapeutic formulations of the invention are prepared for storage by mixing the PAK1 inhibitor having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as
• octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorb
• the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an
• immunosuppressive agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
• the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules
• Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3-hydroxybutyric acid.
• polyesters for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)
• polylactides U.S. Pat. No. 3,773,919
• encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity.
• Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
• the invention provides a composition comprising a PAKl inhibitor for use in the treatment of melanoma.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is over expressed in the melanoma.
• the melanoma is a wild-type BRAF melanoma wherein PAKl is overexpressed in the melanoma compared to non-cancerous cells; for example, non-cancerous skin cells.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is amplified in the melanoma.
• the melanoma is a wild- type BRAF melanoma wherein PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, PAKl is overexpressed in the melanoma and PAKl is amplified in the melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma. In some embodiments, the melanoma is a mutant BRAF melanoma and the melanoma overexpresses PAKl compared to non-cancerous cells and/or PAKl is amplified in the melanoma.
• the invention provides a composition comprising PAKl inhibitor for use in the treatment of melanoma in a mammal. In some embodiments, the invention provides a composition comprising PAKl inhibitor for use in the treatment of melanoma in a human. In some aspects, the invention provides a use for a PAK1 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 over expressed in the melanoma.
• the melanoma is a wild-type BRAF melanoma wherein PAK1 is overexpressed in the melanoma compared to non-cancerous cells; for example, non-cancerous skin cells.
• the melanoma is a wild- type BRAF melanoma wherein PAK1 is amplified in the melanoma.
• the melanoma is a wild- type BRAF melanoma wherein PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma.
• PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma.
• the melanoma is a mutant BRAF melanoma.
• the melanoma is a mutant BRAF melanoma and the melanoma overexpresses PAK1 compared to non-cancerous cells and/or PAK1 is amplified in the melanoma.
• the invention provides a use for a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma in a mammal.
• the invention provides a use for a PAK1 inhibitor in the manufacture of a medicament for the treatment of melanoma in a human.
• the invention also provides kits, medicines, compositions, and unit dosage forms for use in any of the methods described herein.
• Kits of the invention include one or more containers comprising a PAK1 inhibitor (or unit dosage forms and/or articles of manufacture) and in some embodiments, further comprise instructions for use in the treatment of melanoma in accordance with any of the methods described herein.
• the kit may further comprise a description of selection an individual suitable or treatment (e.g. selection based on BRAF genotype).
• Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
• the kit further comprises another therapeutic agent.
• kits of the invention are in suitable packaging.
• suitable packaging include, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
• the present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
• Melanoma biomarkers and treatment The invention provides methods to identify human melanoma patients suitable for treatment with a PAKl inhibitor by determining the presence of one or more melanoma biomarkers.
• the melanoma biomarker is overexpression of PAKl in the melanoma, amplification of PAKl in the melanoma, and/or the presence of wild- type BRAF in the melanoma.
• the overexpression of PAKl is determined by comparison to non-cancerous tissue; for example non-cancerous skin tissue.
• the biomarkers are detected in a test sample obtained from the individual.
• the presence of the biomarker is determines by comparison of a test sample with a reference sample.
• the invention provides methods to identify human melanoma patients suitable for treatment with a PAKl inhibitor by determining the expression of PAKl in the melanoma wherein overexpression of PAKl in the melanoma compared to non-cancerous cells indicated that the patient is suitable for treatment with a PAKl inhibitor.
• overexpression of PAKl by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% in the melanoma compared to non-cancerous cells indicates that the patient is suitable for treatment with a PAKl inhibitor.
• overexpression of PAKl by about any of 1.5-fold, 2.0-fold, 2.5-fold, 3, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0- fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold compared to expression of PAKl in non-cancerous cells indicates that the patient is suitable for treatment with a PAKl inhibitor.
• Methods to determine expression of PAKl are known in the art. Examples of methods to determine expression levels of PAKl in a melanoma include, but are not limited to immunohistochemistry, reverse-phase protein array (RPPA), quantitative PCR, immunoassays, and the like. Levels of PAKl expression can be compared to other tumors and cells by using the Gene Expression Omnibus (GEO) database.
• GEO Gene Expression Omnibus
• the invention provides methods to identify human melanoma patients suitable for treatment with a PAKl inhibitor by detecting the amplification of PAKl in the melanoma wherein amplification of the PAKl gene in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor.
• a copy number of PAKl of about any of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, or greater than 10 in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor.
• Methods to determine amplification of a gene are known in the art.
• the copy number of the PAKl gene may be determined by using SNP arrays such as the Affymetrix 500K SNP array analysis.
• the invention provides methods to identify human melanoma patients suitable for treatment with a PAKl inhibitor by detecting the genotype of BRAF in the melanoma wherein wild-type BRAF in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor.
• Methods to determine the genotype of the BRAF gene in the melanoma are known in the art; for example, the nucleotide sequence of the BRAF gene from the melanoma may be determined using standard sequencing methods or by using the KASP SNP genotyping system (KBioscience).
• the invention provides methods of treating melanoma in a patient provided that the patient has been found to have a biomarker for melanoma selected from overexpression of PAKl in the melanoma, amplification of PAKl in the melanoma and/or the presence of wild- type BRAF in the melanoma; the method comprising administering to the patient a therapeutically effective amount of a PAKl inhibitor.
• the patient is a human patient.
• at least one of the biomarkers is overexpression of
• PAKl wherein PAKl is overexpressed by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater in the melanoma compared to non-cancerous cells.
• expression of PAKl in the melanoma is greater than about any of 1.5-fold, 2.0- fold, 2.5-fold, 3, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10- fold compared to expression of PAKl in non-cancerous cells.
• Methods to determine expression of PAKl are known in the art.
• Examples of methods to determine expression levels of PAKl in a melanoma include, but are not limited to immunohistochemistry, reverse-phase protein array (RPPA), quantitative PCR, immunoassays, and the like. Levels of PAKl expression can be compared to other tumors and cells by using the Gene Expression Omnibus (GEO) database.
• GEO Gene Expression Omnibus
• at least one of the biomarkers is amplification of PAKl in the melanoma wherein a copy number of PAKl of about any of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, or greater than 10 in the melanoma.
• the copy number of the PAKl gene may be determined by using SNP arrays such as the Affymetrix 500K SNP array analysis.
• At least one of the biomarkers is the genotype of BRAF in the melanoma wherein the patient has a melanoma containing a wild-type melanoma. In some embodiments of the above embodiment, the presence of melanoma biomarkers in the patient had been previously determined prior to treatment with the PAKl inhibitor.
• the invention provides methods of adjusting treatment of melanoma in a patient undergoing treatment with a PAKl inhibitor wherein the expression of PAKl in the melanoma is
• the melanoma is a wild-type BRAF melanoma.
• the overexpression of PAKl in the melanoma indicates that treatment with the PAKl inhibitor may continue.
• the expression of PAKl in a melanoma in a patient undergoing treatment with PAKl is monitored over time.
• the expression of PAKl in the melanoma is monitored at least daily, at least weekly, at least monthly.
• the expression of PAKl in a melanoma in a patient undergoing treatment with a PAKl inhibitor is monitored over time.
• PAKl expression increases over the course of treatment with the PAKl inhibitor, the amount of PAKl inhibitor administered to the patient is increased or remains the same. In some embodiments, the amount of PAKl inhibitor administered to the patient is increased until the level of PAKl expression decreases or is no longer detected. If PAKl expression decreases over the course of treatment with the PAKl inhibitor, the amount of PAKl inhibitor administered to the patient is decreased or remains the same. In some embodiments, the expression of PAKl expression in a melanoma of a patient undergoing treatment with a PAKl inhibitor is monitored over time where treatment with the PAKl inhibitor is continued until PAKl expression in the melanoma is no longer detected.
• the invention provides methods for treating a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the melanoma is a wild-type BRAF melanoma.
• PAKl is overexpressed in the tumor compared to non-cancerous skin cells.
• PAKl is amplified in the tumor.
• the copy number of the PAKl in the tumor is greater than about 2.5.
• the inhibitor is a small molecule, a nucleic acid, or a polypeptide.
• the small molecule is PF-3758309.
• the small molecule is a com ound of formula I.
• the small molecule is a compound of formula I and A is 4-indolyl, 5- indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazolyl or 5-benzimidiazolyl; R a , R la and R lb are independently hydrogen or C 1-3 alkyl; R 5 is hydrogen or Ci_6 alkyl; R 6 is hydrogen, halogen or Ci-6 alkyl; and, R is cycloalkyl optionally substituted by fluorine.
• the individual is a human.
• the PAKl inhibitor is used in combination with a therapeutic agent.
• the invention provides the use of a PAKl inhibitor for the treatment of melanoma in an individual.
• the melanoma is a wild-type BRAF melanoma.
• the invention provides compositions comprising a PAKl inhibitor for use in the treatment of melanoma.
• the melanoma is a wild-type BRAF melanoma.
• the composition further comprises a pharmaceutically acceptable excipient.
• the invention provides the use of a PAKl inhibitor in the manufacture of a medicament for the treatment of melanoma.
• the melanoma is a wild- type BRAF melanoma.
• kits comprising a PAKl inhibitor for use in treating melanoma comprising PAKl inhibitor and directions for use in the treatment of melanoma.
• the melanoma is a wild-type BRAF melanoma.
• the invention provides methods of inhibiting CRAF signaling in a melanoma in an individual comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods of inhibiting MEK signaling in a melanoma tumor comprising contacting the melanoma with a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the BRAF genotype of the melanoma, wherein a melanoma comprising a wild type BRAF indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the invention provides methods of identifying a human melanoma patient suitable for treatment with a PAKl inhibitor comprising determining the expression of PAKl in the melanoma, wherein overexpression of PAKl in the melanoma compared to non-cancerous skin cells indicates that the patient is suitable for treatment with a PAKl inhibitor.
• the overexpression of PAKl in the melanoma is 2.5-fold greater than the expression of PAKl in the non-cancerous skin cells
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the BRAF genotype of the melanoma, wherein a melanoma comprising a wild type BRAF indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the invention provides methods for treating a human melanoma patient with a PAKl inhibitor comprising: (a) selecting a patient based on the PAKl expression level of the melanoma, wherein a overexpression of PAKl in the melanoma indicates that the patient is suitable for treatment with a PAKl inhibitor; and (b) administering to the selected patient a therapeutically effective amount of a PAKl inhibitor.
• the overexpression of PAKl in the melanoma is 2.5-fold greater than the expression of PAKl in the non-cancerous skin cells.
• the invention provides methods for treating a human melanoma patient comprising
• a therapeutically effective amount of a PAKl inhibitor wherein the genotype of the melanoma had been determined to be wild type for BRAF.
• the invention provides methods for treating a human melanoma patient comprising
• the overexpression of PAKl in the melanoma is 2.5-fold greater than the expression of PAKl in the non-cancerous skin cells.
• the invention provides methods of adjusting treatment of melanoma in a patient undergoing treatment with a PAKl inhibitor, said method comprising assessing the PAKl expression in the melanoma, wherein overexpression of PAKl in the melanoma indicates that treatment of the individual is adjusted until PAKl overexpression is no longer detected.
• Example 1 Elevated PAKl protein expression and genomic amplification in melanoma.
• RNA was extracted from frozen tumor tissue and applied to Affymetrix (Santa Clara, CA) HGU133 gene expression microarrays. The frequency of PAK1 amplification was 9% (8 of 87 specimens with copy number > 2.5) in this tumor panel ( Figure 1A). RNA was purified from 42 melanoma tumor and cell lines specimens and increased PAK1 copy number was correlated with mRNA expression (Pearson correlation 0.75; Figure IB).
• PAK1 protein expression level and subcellular localization were ascertained via immunohischemistry (IHC) staining of a distinct set of tissue microarrays. Briefly, formalin-fixed paraffin-embedded tissue blocks and corresponding pathology reports were obtained for 92 primary melanomas resected between 1993 and 2009 (Oxford Radcliffe Hospitals, Oxford, UK). The melanoma series comprised 23 nodular, 3 lentigo maligna, 45 superficial spreading, 3 desmoplastic, 5 acral lentiginous and 13 unclassifiable melanoma specimens.
• Tissue microarrays were assembled as described previously (Bubendorf L, et al., (2001) J Pathol, 195(l):72-79). Approval was obtained for the use of all human tissue from the local research ethics committee (C02.216). Immunohistochemistry (IHC) was performed as described previously (Ong CC, et al., (2011) PNAS, 108(17):7177-7182). Intensity of PAK1 expression was scored separately in the cytoplasm and nuclei of neoplastic cells on a scale of 0 to 3.
• PAKl was weakly expressed in basal keratinocytes in normal skin, and lymphocytes and presumed Langerhans cells were positive for PAKl expression (Figure 1C, panel IV). Together, these data show that PAKl DNA copy number, mRNA and protein expression are broadly upregulated in human melanoma.
• melanoma tissues were genotyped for known hotspot mutations in BRAF (codon 600) and NRAS (codons 12, 13, 61 and 146) genes. Mutation status was determined for BRAF codon 600 and NRAS codons 12, 13 61 and 146 via KASPar (KBioscience, Herts, England) and conventional Sanger DNA sequencing methods.
• BRAF wild-type tumors (19 of 46 were positive for strong IHC staining of PAKl) compared to melanomas expressing oncogenic V600E or V600K mutants (4 of 40 tumors with high IHC staining).
• This negative correlation between PAKl expression and BRAF mutation was statistically significant (p ⁇ 0.001, Chi-squared 10.702).
• a similar trend, albeit not statistically significant, was observed when dichotomizing samples into only NRAS mutant and non-mutant status (p 0.45, Chi-squared 0.569).
• Example 3 PAKl is required for proliferation of BRAF wild- type melanoma cells
• RNAi-mediated knockdown of PAKl was examined in a panel of melanoma cell lines in order to clarify the contribution of PAKl towards tumor cell proliferation.
• Cell lines were acquired from the American Type Culture Collection (ATCC; Manassas, VA) and maintained at 37°C and 5% C02 in Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI 1640) media with 10% fetal bovine serum and 4 mM L-glutamine.
• ATCC American Type Culture Collection
• DMEM Dulbecco's Modified Eagle Medium
• RPMI 1640 Roswell Park Memorial Institute 1640
• RNAi Technologies Dharmacon RNAi Technologies (Chicago, IL) that were previously characterized for efficiency and selectivity of PAKl and PAK2 knockdown (Ong CC, et al, (2011) PNAS, 108(17):7177-7182).
• Cellular viability was assessed via ATP content using the CellTiter-Glo Luminescent Assay (Promega, Madison, WI) and results represent mean + standard deviation from three experiments.
• Increased PAKl protein expression in melanomas expressing wild-type versus mutant BRAF was also observed for immortalized cell lines in culture.
• Protein extracts from cell lysates were prepared at 4°C with Cell Extraction Buffer (Invitrogen, Carlsbad, CA), 1 mM phenylmethylsulphonyl fluoride (PMSF), Phosphatase Inhibitor Cocktail 1/2 (Sigma Aldrich, St. Louis, MO), and one tablet of Complete EDTA-free MiniTM protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN).
• Cell Extraction Buffer Invitrogen, Carlsbad, CA
• PMSF phenylmethylsulphonyl fluoride
• Phosphatase Inhibitor Cocktail 1/2 Sigma Aldrich, St. Louis, MO
• Roche Diagnostics Indianapolis, IN
• Western blot analysis proteins were resolved by 4-12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, MA). Immunoblotting was performed using the indicated primary antibodies and analyzed using secondary antibodies for enhanced chemiluminescence (ECL).
• PAK1 signaling in BRAF wild-type melanoma cells was further investigated using a reverse-phase protein array (RPPA) phosphoproteomics platform. Protein lysates were analyzed by RPPA (Theranostics Health, LLC) by first diluting all samples to a final concentration of 0.5 mg/mL.
• RPPA reverse-phase protein array
• the sample dilutions were printed in duplicate on slides that were then subjected to immuno staining with a panel of antibodies primarily directed against specific phosphorylated or cleaved proteins. Each of these antibodies had previously undergone extensive validation for both phosphorylation and protein specificity using single band detection at the appropriate molecular weight by immunoblotting.
• the intensity value for each end point was determined by identifying spots for each duplicate dilution curve for each sample that were within the linear dynamic range of the staining after background subtraction with each spot (within slide local background and also against a slide stained with secondary antibody only). Each value was normalized relative to the total protein intensity value for that sample derived from a slide stained with Sypro Ruby (Invitrogen).
• RPPA data were processed by log 2 transformation and linear scaling (z-score conversion) to ensure normality and linearity.
• RPPA analysis showed a decreased signaling to MAPK, nuclear factor- ⁇ (NF- ⁇ ) and cytoskeletal pathways following PAK1 inhibition in BRAF wild-type (SK-MEL23), but not BRAF mutant (A375), melanoma cells (Figure 2D).
• PAK1 has been shown to phosphorylate both CRAF (Ser338) and MEKl(Ser298) (17, 29-31). Hence, the molecular mechanism by which PAK1 triggers activation of the MAPK pathway in BRAF wild-type melanoma cells was investigated.
• MEK isoforms were immunoprecipitated from cells transfected with either control or PAK-selective siRNA oligonucleotides as previously described (Hatzivassiliou G, et al, (2010) Nature, 464(7287):431-435) and MEK activation was detected via immunoblotting with phospho-MEKl/2(Ser217/Ser221) antibodies. PAK knockdown diminished both MEK1 ( Figure 3A) and MEK2 ( Figure 3B) phosphorylation in 537MEL and SK-MEL23 cells.
• CRAF was immunoprecipitated from cells transfected with either control or PAK- selective siRNA oligonucleotides as previously described (Hatzivassiliou G, et al., (2010) Nature, 464(7287):431-435) and CRAF activation was detected via immunoblotting with phospho- CRAF(Ser338) antibodies.
• Example 4 Differential sensitivity of BRAF wild-type melanoma cells to PAK and BRAF inhibition
• PAK1 phosphorylation of MEK1-Ser298 is presently not well understood, however it has been shown that PAK1-MEK1 signaling can be mediated by cell-cell contact and adhesion (Slack- Davis JK, et al, (2003) J Cell Biol, 162(2):281-291). PAK signaling was also induced via ectopic expression of Flag-PAKl in BRAF(V600E) cells with only moderate endogenous expression of PAK1.
• SK- MEL23 and 537MEL cells were assayed with the CellTiter-Glo Luminescent Assay (Promega, Madison, WI) after treatment with PF-3758309 or with (S)-N 2 -(l-(lH-indol-5-yl)ethyl)-N 4 -(5- cyclopropyl-lH-pyrazol-3-yl)-6-methylpyrimidine-2,4-diamine (1-007), N -((lH-indol-4- yl)methyl)-N 4 -(5-cyclopropyl-lH-pyrazol-3-yl)-6-methylpyrimidine-2,4-diamine (1-054) and N 2 -
• HBSS HBSS
• Matrigel BD Biosciences, USA
• Data analysis and generation of p values using the Dunnett t test was done using JMP software (SAS Institute, Cary, NC). All experimental procedures conformed to the guiding principles of the American Physiology Society and were approved by Genentech's Institutional Animal Care and Use Committee. Following tumor establishment, animals were either administered saline or PF- 3758309 (25 mg/kg, i.p.) and tumors were harvested 1 h after dosing. Tumors were frozen and pulverized on dry ice using a small Bessman tissue pulverizer (Spectrum Laboratories, Collinso Dominguez, CA) and protein extracts were prepared at 4°C with Cell Extraction Buffer

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