Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF TREATING MELANOMA WITH PAK1 INHIBITORS
Malignant melanoma accounts for approximately 80 percent of deaths from skin
cancer.
Although 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). For instance, from a pooled analysis of data from 4493
patients the
occurrence of BRAF (v-Raf murine sarcoma viral oncogene homolog B1) mutation
is 41% in
cutaneous melanomas (Lee JH, et al., Br J Dennatol. 2011, 164(4):776-784). The
most frequent
BRAF somatic mutation in malignant melanoma is substitution of valine at
residue 600 to confer
constitutive catalytic activity and signaling (Davies H, et al., Nature. 2002;
417(6892), 949-
954.). Genetic studies have confirmed that BRAF is required for initiation and
maintenance of
melanoma in preclinical model systems (Davies H, et al., 2002, ibid; Hoeflich
KP, et al., Cancer
Res. 2006, 66(2):999-1006; Dankort D, et al., Genes Dev. 2007, 21(4):379-3844-
6). These
discoveries prompted a flurry of drug discovery activity to develop small
molecule inhibitors of
BRAF, including GDC-0879, PLX-4720, PLX-4032/vemurafenib (ZelborafTh4) and
GSK2118436 (Hoeflich KP, et al., Cancer Res. 2009, 69(7):3042-3051;Tsai J, et
al., Proc Natl
Acad Sci U.S.A. 2008, 105(8):3041-3046; Bollag G, et al., Nature 2011,
467(7315):596-599;
Ribas A, & Flaherty KT., Nature Rev. 2011, 8(7):426-433). These inhibitors
selectively
decrease the growth of BRAF oncogene addicted tumor cells and provide hope for
patients with
the subset of melanoma that has activating mutations in the BRAF oncogene
(Ribas A, &
Flaherty KT., 2011, ibid). However, significantly less anti-tumor efficacy
with current BRAF
small molecule inhibitors is observed for wild-type BRAF melanoma cells
(Hoeflich KP, et al.,
2009, ibid:3042-3051;Tsai J, et al., ibid), raising the need to identify
additional melanoma-
associated driver genes to provide new insights into the biology, oncogenic
signaling and
possible therapeutic targets for disease management of melanoma patients of
all classifications.
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. However, additional kinases have been
reported to
also play a role in ERK activation. In particular, several groups have
reported that group-I p21-
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activated kinases (PAKs) contribute to MAPK pathway activation via
phosphorylation of both
CRAF at Ser338, a critical residue for activation, and MEK1 at Ser298, a site
that is proximal to
the activation loop residues Ser217/Ser221 that are substrates for the RAF
kinases (King AJ, et
al., Nature. 1998; 396(6707), 180-183; Tang Y, et al., Mol Cell Biol. 1999,
19(3):1881-1891;
Frost JA, et al., EMBO J. 1997, 16(21):6426-6438). The 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). As a major
downstream
effector of the Rho family small GTPases Cdc42 and Racl, PAK1 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;
Kumar R, et al., Nat Rev Cancer 2006, 6(6):459-471; Ong CC, et al.,
Oncotarget. 2011,
2(6):491-496). PAK1 is widely expressed in a variety of normal tissues and
expression is
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). Functional studies have
also implicated
PAK1 in cell transformation (Vadlamudi RK, et al., J Biol Chem. 2000,
275(46):36238-36244)
and tumor growth (Ong CC, et al., 2011, ibid; Yi C, et al., Cancer Res. 2008,
68(19):7932-7937;
Chow HY, et al., PloS One 2010, 5(11):e13791). These findings indicate that
PAK1 may
contribute to tumorigenesis in some disease contexts.
The present invention relates to methods for treating a melanoma in an
individual comprising
contacting the melanoma with a therapeutically effective amount of a PAK1
inhibitor. In some
embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments,
PAK1 is
overexpressed in the tumor compared to non-cancerous skin cells. In some
embodiments, PAK1
is amplified in the tumor. In some embodiments, the melanoma is a wild-type
BRAF melanoma
wherein PAK1 is overexpressed in the melanoma. In some embodiments, the
melanoma is a
wild-type BRAF melanoma wherein PAK1 is amplified in the melanoma. In some
embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
overexpressed in
the melanoma and PAK1 is amplified in the melanoma. In some embodiments, PAK1
is
overexpressed in the melanoma and PAK1 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
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comprising administering to the individual a therapeutically effective amount
of a PAK1
inhibitor.
In some embodiments, the invention provides methods for treating a melanoma in
an individual
comprising contacting the melanoma with a therapeutically effective amount of
a PAK1 inhibitor
wherein the PAK1 inhibitor is a small molecule, a nucleic acid, or a
polypeptide. In some
embodiments, the invention provides methods for treating a melanoma in an
individual
comprising administering to the individual a therapeutically effective amount
of a PAK1
inhibitor wherein the PAK1 inhibitor is a small molecule, a nucleic acid, or a
polypeptide.
In some embodiments, the invention provides methods for treating a melanoma in
an individual
comprising contacting the melanoma with a therapeutically effective amount of
a PAK1 inhibitor
wherein the PAK1 inhibitor is used in combination with a therapeutic agent. 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 PAK1
inhibitor wherein the PAK1 inhibitor is used in combination with a therapeutic
agent.
In some aspects, the invention provides uses of PAK1 inhibitors for the
treatment of melanoma
in an individual. The invention provides uses of PAK1 inhibitors in the
manufacture of a
medicament for the treatment of melanoma. In some embodiments, the melanoma is
a wild-type
BRAF melanoma. In some embodiments, PAK1 is overexpressed in the tumor
compared to non-
cancerous skin cells. In some embodiments, PAK1 is amplified in the tumor. In
some
embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
overexpressed in
the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma
wherein
PAK1 is amplified in the melanoma. In some embodiments, the melanoma is a wild-
type BRAF
melanoma wherein PAK1 is overexpressed in the melanoma and PAK1 is
overexpressed in the
melanoma. In some embodiments, PAK1 is overexpressed in the melanoma and PAK1
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 aspects, the invention provides compositions and kits comprising a
PAK1 inhibitor for
use in the treatment of melanoma. Various embodiments relating to these
treatment methods are
described herein and apply to compositions and kits. In some embodiments, the
melanoma is a
wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in the
tumor
compared to non-cancerous skin cells. In some embodiments, PAK1 is amplified
in the tumor.
In some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
overexpressed in the melanoma. In some embodiments, the melanoma is a wild-
type BRAF
melanoma wherein PAK1 is amplified in the melanoma. In some embodiments, the
melanoma is
a wild-type BRAF melanoma wherein PAK1 is overexpressed in the melanoma and
PAK1 is
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overexpressed in the melanoma. In some embodiments, PAK1 is overexpressed in
the
melanoma and PAK1 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 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 PAK1 inhibitor. In some embodiments, the
melanoma is a
wild-type BRAF melanoma. In some embodiments, PAK1 is overexpressed in the
tumor
compared to non-cancerous skin cells. In some embodiments, PAK1 is amplified
in the tumor.
In some embodiments, PAK1 is overexpressed in the tumor compared to non-
cancerous skin
cells. In some embodiments, PAK1 is amplified in the tumor. In some
embodiments, the
melanoma is a wild-type BRAF melanoma wherein PAK1 is overexpressed in the
melanoma. In
some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
amplified
in the melanoma. In some embodiments, the melanoma is a wild-type BRAF
melanoma wherein
PAK1 is overexpressed in the melanoma and PAK1 is overexpressed in the
melanoma. In some
embodiments, PAK1 is overexpressed in the melanoma and PAK1 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 PAK1 inhibitor.
In some aspects, the invention provides, methods of identifying a human
melanoma patient
suitable for treatment with a PAK1 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 PAK1 inhibitor. In some aspects, the invention
provides methods of
identifying a human melanoma patient suitable for treatment with a PAK1
inhibitor comprising
determining the expression of PAK1 in the melanoma, wherein overexpression of
PAK1 in the
melanoma compared to non-cancerous skin cells indicates that the patient is
suitable for
treatment with a PAK1 inhibitor. In some aspects, the invention provides
methods of identifying
a human melanoma patient suitable for treatment with a PAK1 inhibitor
comprising determining
the copy number of PAK1 in the melanoma, wherein amplification of PAK1 in the
melanoma
indicates that the patient is suitable for treatment with a PAK1 inhibitor. In
some aspects, the
invention provides methods of identifying a human melanoma patient suitable
for treatment with
a PAK1 inhibitor comprising determining the BRAF genotype of the melanoma and
determining
the expression of PAK1 in the melanoma, wherein the presence of a wild-type
BRAF and/or the
overexpression of PAK1 in the melanoma compared to non-cancerous skin cells
indicates that
the patient is suitable for treatment with a PAK1 inhibitor. In some aspects,
the invention
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provides methods of identifying a human melanoma patient suitable for
treatment with a PAK1
inhibitor comprising one or more of determining the BRAF genotype of the
melanoma,
determining the expression of PAK1 in the melanoma, and determining the copy
number of
PAK1 in the melanoma wherein one or more of the presence of a wild-type BRAF,
the
overexpression of PAK1 in the melanoma compared to non-cancerous skin cells,
and
amplification of PAK1 in the melanoma indicates that the patient is suitable
for treatment with a
PAK1 inhibitor.
In some aspects, the invention provides methods for treating a human melanoma
patient with a
PAK1 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 PAK1 inhibitor; and (b) administering to the
selected patient a
therapeutically effective amount of a PAK1 inhibitor.
In some aspects, the invention provides methods for treating a human melanoma
patient with a
PAK1 inhibitor comprising: (a) selecting a patient based on the PAK1
expression level of the
melanoma, wherein an overexpression of PAK1 in the melanoma compared to non-
cancerous
cells indicates that the patient is suitable for treatment with a PAK1
inhibitor; and (b)
administering to the selected patient a therapeutically effective amount of a
PAK1 inhibitor.
In some aspects, the invention provides methods for treating a human melanoma
patient with a
PAK1 inhibitor comprising: (a) selecting a patient based on the copy number of
PAK1 in the
melanoma, wherein amplification of PAK1 in the melanoma indicates that the
patient is suitable
for treatment with a PAK1 inhibitor; and (b) administering to the selected
patient a
therapeutically effective amount of a PAK1 inhibitor.
In some aspects, the invention provides methods for treating a human melanoma
patient with a
PAK1 inhibitor comprising: (a) selecting a patient based on the BRAF genotype
of the
melanoma and PAK1 expression level of the melanoma, wherein a melanoma
comprising a wild
type BRAF and/or overexpression of PAK1 in the melanoma compared to non-
cancerous cells
indicates that the patient is suitable for treatment with a PAK1 inhibitor;
and (b) administering to
the selected patient a therapeutically effective amount of a PAK1 inhibitor.
In some aspects, the invention provides methods for treating a human melanoma
patient with a
PAK1 inhibitor comprising: (a) selecting a patient based on one or more of the
BRAF genotype
of the melanoma, PAK1 expression level of the melanoma, and copy number of
PAK1 in the
melanoma, wherein a melanoma comprising one or more of a wild type BRAF,
overexpression
of PAK1 in the melanoma compared to non-cancerous cells, and amplification of
PAK1
indicates that the patient is suitable for treatment with a PAK1 inhibitor;
and (b) administering to
the selected patient a therapeutically effective amount of a PAK1 inhibitor.
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In some aspects, the invention provides methods of adjusting treatment of
melanoma in a patient
undergoing treatment with a PAK1 inhibitor, said method comprising assessing
the PAK1
expression in the melanoma, wherein overexpression of PAK1 in the melanoma
indicates that
treatment of the individual is adjusted until PAK1 overexpression is no longer
detected. In some
embodiments the melanoma is a wild-type BRAF melanoma. In some embodiments,
PAK1 is
amplified in the melanoma. In some embodiments, the melanoma is a wild-type
melanoma and
PAK1 is amplified in the melanoma.
Figure 1 shows that PAK1 is highly expressed in human melanoma. (A) Analysis
of 11q13 copy
number gains in human melanoma tissues. Vertical red line represents
chromosome location of
the PAK1 gene. (B) PAK1 DNA copy and mRNA expression (226507_at Affymetrix MAS
5.0
signal) correlated for melanoma tumor samples. (C) Representative images of
PAK1
immunohistochemistry in primary human malignant melanomas. Cytoplasmic
expression score:
0 (I), 1 (II), 2 (III) and 3 (IV). Chromogen deposition indicates
immunoreactivity against a
hematoxylin counterstain. PAK1 expression was also seen in stromal cells (III)
and cells
intercalating within the epidermis that may represent Langerhan's cells (IV).
Figure 2 demonstrates PAK1 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. PAK1 was
required
for cell growth and the data were normalized to control and shown as the mean
SD. (B) In a
panel of melanoma cell lines, PAK1 inhibition selectively impaired growth of
cells without
BRAF(V600E) mutation (n = 5; 537MEL, Hs940T, MeWO, SK-MEL2, SK-MEL23, SK-
MEL30) compared to those with BRAF(V600E) mutation (n = 9; p = 0.07; 624MEL,
888MEL,
928MEL, RPMI-7951, A375, Co1o829, LOX-TM VI, Malme-3M, A375). (C) Inhibition
of
PAK1/2 decreased ERK1/2 and MEK1/2 phosphorylation and accumulation of cyclin
Dl. (D)
PAK1/2 inhibition in SK-MEL23 BRAF wild-type melanoma cells decreases
signaling to the
cytoskeletal, MAPK, proliferation and NF-KB 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. siPAK1 = PAK1-specific
siRNA.
APAK1 = chromosomal deletion of PAK1 gene.
Figure 3 depicts a series of immunoblots demonstrating that PAK1 is required
for CRAF
activation in BRAF wild-type melanoma cells. (A) PAK1- 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
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determined as loading controls. (D) Cells were treated with DMSO or 51AM PF-
3758309 for 4 h
and endogenous CRAF was immunoprecipitated and immunoblotted for Ser338
phosphorylation. Total CRAF levels in the immunocomplexes are also shown. (E)
SK-MEL23
cells were treated with DMSO, 51AM PF-3758309 or 201AM IPA-3 for 4 h. CRAF
immunocomplexes were incubated with inactive MEK1 protein in kinase buffer for
30 minutes.
Levels of phospho-MEK1 (Ser217/Ser221) were determined and CRAF catalytic
activity is
reported as the levels of MEK1 phosphorylation normalized to total CRAF
protein.
Figure 4 contains images demonstrating PAK is required for melanoma cell
migration.
Following non-targeting control (NTC) or PAK1/2 siRNA oligonucleotide
transfection for 72 h,
confluent WM-266-4 melanoma cell were wounded and images were recorded when
wounds
were made (dark shading) and after incubation for 28 h (bright field).
Differences in relative
wound density were statistically significant (p < 0.001; n=3).
Figure 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 [1M PF-3758309 or 0.2
[1M PLX-
4720 for 4 h and lysates were analyzed for phosphorylation of MAPK pathway
components.
Lighter and darker exposures of p-MEK1/2(S217/S221) immunoblots are shown. (B)
Ectopic
expression of Flag epitope-tagged PAK1 drove MAPK pathway activation in A375
cells.
Specificity was demonstrated using PF-3758309 inhibitor treatment as a
control.
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 PAK1 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. (A)
Pharmacodynamic response of BRAF wild-type and mutant tumors measured by
phosphorylation of CRAF(5er338) following either vehicle or 35 mg/kg PF-
3758309
administration. (B) Anti-tumor efficacy of 10, 15 and 25 mg/kg PF-3758309 i.p.
daily dosing in
the SK-MEL23 preclinical tumor model of BRAF wild-type 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. To analyze the repeated measurement of tumor volumes from the same
animals over
time, cubic regression splines were used to fit a non linear profile to the
time courses of log2
tumor volume at each dose level. These non linear profiles were then related
to dose within the
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mixed model. Cubic regression splines were used to fit a non linear profile to
the time courses of
log2 tumor volume at each dose level. These non linear profiles were then
related to dose within
the mixed model. Tumor growth inhibition as a percentage of Vehicle (%TGI) was
calculated as
the percentage of the area under the fitted curve (AUC) for the respective
dose group per day in
relation to the vehicle, using the formula: %TGI = 100 x (1 - AUCdose/AUCveh).
Plotting was
performed and generated using R version 2.8.1 and Excel, version 12Ø1
(Microsoft). Data were
analyzed using R version 2.8.1 (R Foundation for Statistical Computing;
Vienna, Austria), and
the mixed models were fit within R using the nlme package, version 3.1-89.
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(5er338) 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.
Figure 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. In some
embodiments, the
melanoma is a wild-type BRAF melanoma. In some embodiments, the melanoma
overexpresses
PAK1 compared to non-cancerous cells. In some embodiments, PAK1 is amplified
in the
melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma and
overexpresses PAK1 compared to non-cancerous cells. In some embodiments, the
melanoma is
a wild-type BRAF melanoma, the melanoma overexpresses PAK1 compared to non-
cancerous
cells and PAK1 is amplified in the melanoma.
All references cited herein, including patent applications, patent
publications, and Genbank
Accession numbers are herein incorporated by reference, as if each individual
reference were
specifically and individually indicated to be incorporated by reference.
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Definitions
The techniques and procedures described or referenced herein are generally
well understood and
commonly employed using conventional methodology by those skilled in the art,
such as, for
example, the widely utilized methodologies described in Sambrook et al.,
Molecular Cloning: A
Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press,
Cold Spring
Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al.
eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2:
A
PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)),
Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL
CELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J.
Gait, ed.,
1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J.
E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney),
ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998)
Plenum Press;
Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and
D. G. Newell, eds.,
1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and
C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and
M. P. Cabs,
eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);
Current Protocols
in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular
Biology (Wiley
and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997);
Antibodies (P. Finch,
1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-
1989); Monoclonal
Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford
University Press,
2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold
Spring Harbor
Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds.,
Harwood Academic
Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T.
DeVita et al., eds.,
J.B. Lippincott Company, 1993).
Unless defined otherwise, technical and scientific terms used herein have the
same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.
Wiley & Sons
(New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions,
Mechanisms and
Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one
skilled in the art with
a general guide to many of the terms used in the present application.
"PAK," as used herein, refers to a family of non-receptor serine/threonine
protein kinases
(STKs). The p21-activated protein kinase (PAK) family of serine/threonine
protein kinases
plays important roles in cytoskeletal organization, cellular morphogenesis,
cellular processes and
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cell survival (Daniels et al., Trends Biochem. Sci. 1999 24: 350-355; Sells et
al., 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 (PAKs) serve as important mediators of Rac and Cdc42 GTPase function
as well as
pathways required for Ras-driven tumorigenesis. (Manser et al., Nature 1994
367:40-46; B
Dummler et al., Cancer Metathesis Rev. 2009 28:51-63; R. Kumar et al., Nature
Rev. Cancer
2006 6:459-473).
"PAK1" or "p21-activated protein (Cdc42/Rac)-activated kinase 1" as used
herein refers to a
native PAK1 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. 1 1q13-q14 cytogenetic band, chromosome 11:77033060-77185108,
and/or
GC11M077033), "full-length," unprocessed PAK1 as well as any form of PAK1 that
result from
processing in the cell. The term also encompasses naturally occurring variants
of PAK1, e.g.,
splice variants or allelic variants. The sequence of an exemplary human PAK1
nucleic acid is
NC_000011.9. An exemplary human PAK1 amino acid sequence is NP_0011220921 or
NP_002567.3. The sequence of an exemplary mouse PAK1 nucleic acid is
NC_000073.6 or an
exemplary mouse PAK1 amino acid sequence NP_035165.2. The sequence of an
exemplary rat
PAK1 nucleic acid is NC_005100.2 or an exemplary rat PAK1 amino acid sequence
NP_058894.1. The sequence of an exemplary dog PAK1 nucleic acid is NC_006603.3
or an
exemplary dog PAK1 amino acid sequence XP_849651.1. The sequence of an
exemplary cow
PAK1 nucleic acid is AC_000186.1 or NC_007330.5. An exemplary cow PAK1 amino
acid
sequence is NP_001070366.1. The sequence of an exemplary rhesus monkey PAK1
nucleic acid
is NC_007871.1. An exemplary rhesus monkey PAK1 amino acid sequence is
XP_001090310.1
or NP_001090423.2. The sequence of an exemplary chicken PAK1 nucleic acid is
NC_006088.3 or an exemplary chicken PAK1 amino acid sequence NP_001155844.1
"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. The
terms encompass the
genomic location (e.g., 7q34 cytogenetic band, chromosome 7:140433812-
140624564, and/or
GC07M140424), "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
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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. As is known in the art, with respect to
BRAF
melanomas can be categorized and classified by BRAF type: wild-type BRAF and
mutant
BRAF.
"Mutant BRAF" as used herein 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). As is known in the art, 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
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" or "mitogen-activated protein kinase kinase," as used herein, refers to
a family of kinase
enzymes which phosphorylate mitogen-activated protein kinase (MAPK). There are
seven
genes: MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K3 (MKK3), MAP2K4 (MKK4),
MAP2K5 (MKK5), MAP2K6 (MKK6), and MAP2K7 (MKK7). The activators of p38 (MKK3
and MKK6), JNK (MKK4 and MKK7), and ERK (MEK1 and MEK2) define independent MAP
kinase signal transduction pathways. The sequence of an exemplary human MEK1
nucleic acid
is NC_000015.9 or an exemplary human MEK1 amino acid sequence NP_002746.1. The
sequence of an exemplary human MEK2 nucleic acid is NC_000019.9 or an
exemplary human
MEK2 amino acid sequence NP_109587.1. The sequence of an exemplary human MEK3
nucleic acid is NC_000017.10. An exemplary human MEK3 amino acid sequence is
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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
deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or
their analogs, or
any substrate that can be incorporated into a polymer by DNA or RNA
polymerase, or by a
synthetic reaction. 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. Other types of 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., acridine, psoralen, etc.), those containing
chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those containing
alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as
unmodified forms of the
polynucleotide(s). Further, 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
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groups. These 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. The
preceding description applies to all polynucleotides referred to herein,
including RNA and DNA.
"Oligonucleotide," as used herein, 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.
The term "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.
The term "small molecule" refers to any molecule with a molecular weight of
about 2000 daltons
or less, preferably of about 500 daltons or less.
The term "antibody" 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), and antibody fragments
so long as they
exhibit the desired antigen-binding activity.
The term "detection" includes any means of detecting, including direct and
indirect detection.
The term "biomarker" as used herein 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. For example, biomarkers
for melanoma
include, but are not limited to, the presence of wild-type BRAF,
overexpression of PAK1 and
amplification of PAK1.
The "amount" or "level" of a biomarker associated with an increased clinical
benefit to an
individual is 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.
The terms "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
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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 (e.g., posttranslational modification of a
polypeptide) 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," "elevated expression levels," "elevated levels" and
"overexpressed"
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," "reduced expression levels," or "reduced levels" 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).
The term "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. In
some embodiments, 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," as used herein 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.
For example,
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. In some aspects of the invention, "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.
The term "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.
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The term "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)).
The term "aiding diagnosis" is used herein to refer to methods that assist in
making a clinical
determination regarding the presence, or nature, of a particular type of
symptom or condition of a
disease or disorder (e.g., cancer). For example, a method of aiding diagnosis
of a disease or
condition (e.g., cancer) can comprise measuring certain biomarkers in a
biological sample from
an individual.
By "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), including slowing down and complete
arrest; (2) a
reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or
complete stopping) of
cancer cell infiltration into adjacent peripheral organs and/or tissues; (4)
inhibition (i.e.
reduction, slowing down or complete stopping) of metastasis; (5) relief, to
some extent, of one or
more symptoms associated with the disease or disorder (e.g., cancer); (6)
increase in the length
of progression free survival; and/or (9) decreased mortality at a given point
of time following
treatment.
The term "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
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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.
The term "substantially the same," as used herein, 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., Kd
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.
The phrase "substantially different," as used herein, 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., Kd 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.
The word "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
A "prophylactically 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
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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.
In the case of melanoma, 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. To the extent the drug may prevent growth and/or kill existing
cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, 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.
To "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.
The term "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.
As used herein, "treatment" is an approach for obtaining beneficial or desired
clinical results.
For purposes of this invention, 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). Also
encompassed by "treatment" is a reduction of pathological consequence of a
proliferative
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disease. The methods of the invention contemplate any one or more of these
aspects of
treatment.
The term "melanoma" refers to a tumor of high malignancy that starts in
melanocytes of normal
skin or moles and metastasizes rapidly and widely. The term "melanoma" can be
used
interchangeably with the terms "malignant melanoma", "melanocarcinoma",
"melanoepithelioma", and "melanosarcoma".
"Tumor," as used herein, refers to all neoplastic cell growth and
proliferation, whether malignant
or benign, and all pre-cancerous and cancerous cells and tissues. The terms
"cancer",
"cancerous", "cell proliferative disorder", "proliferative disorder" and
"tumor" are not mutually
exclusive as referred to herein.
The terms "cancer" and "cancerous" refer to or describe the physiological
condition in mammals
that is typically characterized by unregulated cell growth. Examples of cancer
include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid
malignancies.
More particular examples of such cancers include, but not limited to, squamous
cell cancer (e.g.,
epithelial squamous cell cancer), 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/follicular non-Hodgkin's lymphoma
(NHL); small
lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small
non-cleaved
cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic
leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-
transplant
lymphoproliferative disorder (PTLD), as well as 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. In certain
embodiments, 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
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sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer,
mesothelioma, and
multiple myeloma. In some embodiments, the cancer is selected from: small cell
lung cancer,
glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer,
colorectal cancer
(CRC), and hepatocellular carcinoma. Yet, in some embodiments, the cancer is
selected from:
non-small cell lung cancer, colorectal cancer, glioblastoma and breast
carcinoma, including
metastatic forms of those cancers.
The term "anti-cancer therapy" refers to a therapy useful in treating cancer.
Examples of anti-
cancer 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.
The term "cytotoxic agent" as used herein 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., At211 , 1131, 1125, y90, Re186, Re188, sm153, Bi212, 1,32
r 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.
A "chemotherapeutic agent" refers to a chemical compound useful in the
treatment of cancer.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa
and
cyclosphosphamide (CYTOXAMD); 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, MARINOUD); beta-
lapachone;
lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic
analogue topotecan
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(HYCAMTINIO), CPT-11 (irinotecan, CAMPTOSAWD), 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,
chlorophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as
carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and
ranimnustine; antibiotics such
as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin
gammal I and
calicheamicin omegaIl (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 (including
ADRIAMYCIN , morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-
doxorubicin, doxorubicin HClliposome injection (DOXIUD), liposomal doxorubicin
TLC D-99
(MYOCETIO), peglylated liposomal doxorubicin (CAELYVD), 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 (GEMZAR0), tegafur
(UFTORAUD),
capecitabine (XELODA10), 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,
floxuridine;
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;
elfornithine; 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;
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rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-
trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine
(ELDISINE , FILDESINI0); dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g.,
paclitaxel (TAXOUD),
albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and
docetaxel
(TAXOTERED); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate;
platinum agents
such as cisplatin, oxaliplatin (e.g., ELOXATINIO), and carboplatin; vincas,
which prevent
tubulin polymerization from forming microtubules, including vinblastine
(VELBANIO),
vincristine (ONCOVINIO), vindesine (ELDISINE , FILDESINIO), and vinorelbine
(NAVELBINECI); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin;
novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS
2000;
difluoromethylornithine (DMF0); retinoids such as retinoic acid, including
bexarotene
(TARGRETINC)); bisphosphonates such as clodronate (for example, BONEFOS or
OSTA00), etidronate (DIDROCAUD), NE-58095, zoledronic acid/zoledronate
(ZOMETA10),
alendronate (FOSAMAX0), pamidronate (AREDIA10), tiludronate (SKELID10), or
risedronate
(ACTONEUD); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense
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., LURTOTECANC)); 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., PS341);
bortezomib (VELCADRO); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bc1-2
inhibitor
such as oblimersen sodium (GENASENSED); pixantrone; EGFR inhibitors (see
definition
below); tyrosine kinase inhibitors (see definition below); serine-threonine
kinase inhibitors such
as rapamycin (sirolimus, RAPAMUNECI); farnesyltransferase inhibitors such as
lonafarnib
(SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or
derivatives of any
of the above; as well as combinations of two or more of the above such as
CHOP, an
abbreviation for a combined therapy of cyclophosphamide, doxorubicin,
vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen with
oxaliplatin
(ELOXATINTM) combined with 5-FU and leucovorin.
Chemotherapeutic agents as defined herein include "anti-hormonal agents" or
"endocrine
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:
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anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen
(NOLVADEVD), 4-
hydroxytamoxifen, toremifene (FARESTON10), idoxifene, droloxifene, raloxifene
(EVISTA10),
trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such
as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant (FASLODEVD),
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 formestane and exemestane (AROMASINIO), and nonsteroidal
aromatase
inhibitors such as anastrazole (ARIIVIIDEVD), letrozole (FEMARAIO) and
aminoglutethimide,
and other aromatase inhibitors include vorozole (RIVISOR0), megestrol acetate
(MEGASE10),
fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone
agonists, including
leuprolide (LUPRON and ELIGARD10), goserelin, buserelin, and tripterelin; sex
steroids,
including progestines such as megestrol acetate and medroxyprogesterone
acetate, estrogens
such as diethylstilbestrol and premarin, and androgens/retinoids such as
fluoxymesterone, all
transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen
receptor down-
regulators (ERDs); anti-androgens such as flutamide, nilutamide and
bicalutamide; and
pharmaceutically acceptable salts, acids or derivatives of any of the above;
as well as
combinations of two or more of the above.
The term "prodrug" as used in this application refers to a precursor or
derivative form of 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 Belfast (1986) and Stella et al., "Prodrugs: A
Chemical
Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al.,
(ed.), pp. 247-
267, Humana Press (1985). 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, 13-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.
Examples of 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). Examples of growth
inhibitory agents include
agents that block cell cycle progression (at a place other than S phase), such
as agents that induce
G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and
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vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin,
epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase
arrest, for example, 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:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel)
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 (TAXOUD, 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.
By "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.
An "individual" or "subject" is a mammal. Mammals include, but are not limited
to,
domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates
(e.g., humans and non-
human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
In certain
embodiments, 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.
The term "concurrently" is used herein to refer to administration of two or
more therapeutic
agents, where at least part of the administration overlaps in time.
Accordingly, 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).
By "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.
The term "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. In some embodiments, the invention package insert
comprises
instructions to treat melanoma with a PAK1 inhibitor.
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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. In certain
embodiments, 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 intern&
viewers, radio or intern&
listeners, physicians, drug companies, etc.
As is understood by one skilled in the art, reference to "about" a value or
parameter herein
includes (and describes) embodiments that are directed to that value or
parameter per se. For
example, description referring to "about X" includes description of "X".
It is understood that aspect and embodiments of the invention described herein
include
"consisting" and/or "consisting essentially of" aspects and embodiments. As
used herein, the
singular form "a", "an", and "the" includes plural references unless indicated
otherwise.
An "individual," "subject," or "patient" is a vertebrate. In certain
embodiments, the vertebrate is
a mammal. Mammals include, but are not limited to, farm animals (such as
cows), sport animals,
pets (such as cats, dogs, and horses), primates, mice and rats. In certain
embodiments, a mammal
is a human.
The term "sample," or "test sample" as used herein, 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. For example, the phrase "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. In
one embodiment, the
definition encompasses blood and other liquid samples of biological origin and
tissue samples
such as a biopsy specimen or tissue cultures or cells derived therefrom. The
source of the tissue
sample 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,
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mucus, tumor lysates, and tissue culture medium, tissue extracts such as
homogenized tissue,
tumor tissue, cellular extracts, and combinations thereof.
The term "sample," or "test sample" includes biological samples that have been
manipulated in
any way after their procurement, such as by treatment with reagents,
solubilization, or
enrichment for certain components, such as proteins or polynucleotides, or
embedding in a semi-
solid or solid matrix for sectioning purposes. For the purposes herein 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. In one embodiment, the sample is a clinical sample. In
another
embodiment, the sample is used in a diagnostic assay. In some embodiments, the
sample is
obtained from a primary or metastatic tumor. Tissue biopsy is often used to
obtain a
representative piece of tumor tissue. Alternatively, tumor cells can be
obtained indirectly in the
form of tissues or fluids that are known or thought to contain the tumor cells
of interest; for
instance, skin samples.
By "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. Optionally,
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. In one embodiment, a reference sample, reference cell,
reference tissue,
control sample, control cell, or control tissue is obtained from a healthy
and/or non-diseased part
of the body (e.g., tissue or cells) of the same subject or individual. For
example, healthy and/or
non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g.,
cells or tissue adjacent to
a tumor). In some embodiments, the reference sample is non-cancerous skin
cells. In another
embodiment, a reference sample is obtained from an untreated tissue and/or
cell of the body of
the same subject or individual. In some embodiments, the reference sample is
non-cancerous
skin cells of the body of the same subject or individual. In yet another
embodiment, a reference
sample, reference cell, reference tissue, control sample, control cell, or
control tissue is obtained
from a healthy and/or non-diseased part of the body (e.g., tissues or cells)
of an individual who is
not the subject or individual. In some embodiments, the reference sample is
non-cancerous skin
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cells of an individual who is not the subject or individual. In even another
embodiment, 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.
In certain embodiments, 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.
In certain embodiments, a reference sample includes all types of biological
samples as defined
above under the term "sample" that is obtained from one or more individuals
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.
In certain embodiments, 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.
For the purposes herein 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. In certain
embodiments,
expression/amount of a gene or biomarker in a first sample is increased as
compared to
expression/amount in a second sample. In certain embodiments,
expression/amount of a gene or
biomarker in a first sample is decreased as compared to expression/amount in a
second sample.
In certain embodiments, the second sample is reference sample.
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In certain embodiments, 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. In
certain
embodiments, 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, 10x, 25x, 50x, 75x, or 100x the
expression
level/amount of the respective gene or biomarker in the reference sample.
In certain embodiments, 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. In certain
embodiments, 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, 0.1x,
0.05x, or 0.01x 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.
In certain embodiments, by "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.
The word "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.
The term "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
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component. Also included within the definition are, for example, polypeptides
containing one or
more analogs of an amino acid (including, for example, unnatural amino acids,
etc.), as well as
other modifications known in the art. The term "polypeptide" as used herein
specifically
encompasses a "protein". The terms "polypeptide" and "protein" as used herein
specifically
encompass antibodies.
An "isolated" nucleic acid molecule is a nucleic acid molecule that is
identified and separated
from at least one 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.
However, 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. Generally, a "template," as used herein, is a polynucleotide that
contains the target
nucleotide sequence. In some instances, 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. Thus, 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. In certain embodiments, 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
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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. Such
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. Ordinarily, a variant will have at least
about 80% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, and even
more preferably at least about 95% amino acid sequence identity with the
native sequence
polypeptide.
The term "benefit" is used in the broadest sense and refers to any desirable
effect and
specifically includes clinical benefit as defined herein.
Clinical benefit can be measured by 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;
decrease of auto-immune response, which may, but does not have to, result in
the regression or
ablation of the disease lesion; relief, to some extent, of one or more
symptoms associated with
the disorder; increase in the length of disease-free presentation following
treatment, e.g.,
progression-free survival; increased overall survival; higher response rate;
and/or decreased
mortality at a given point of time following treatment.
Methods of the invention
The present invention provides methods for treating melanoma in an individual
comprising
contacting the melanoma with a therapeutically effective amount of a PAK1
inhibitor. In some
embodiments, the method comprises administering to the individual a
therapeutically effective
about of a PAK1 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
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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
PAK1. In some embodiments, the invention provides methods of treating a Stage
II melanoma
in an individual by contacting the melanoma with an inhibitor of PAK1. In some
embodiments,
the invention provides methods of treating Stage III melanoma in an individual
by contacting the
melanoma with an inhibitor of PAK1. In some embodiments, the invention
provides methods of
treating Stage IV melanoma in an individual by contacting the melanoma with an
inhibitor of
PAK1. In some embodiments, the invention provides methods of treating
metastatic melanoma
in an individual by contacting the melanoma with an inhibitor of PAK1. In some
embodiments,
the invention provides methods of treating recurrent melanoma in an individual
by contacting the
melanoma with an inhibitor of PAK1. In some embodiments, the method comprises
administering to the individual a therapeutically effective amount of the PAK1
inhibitor. In
some embodiments, the PAK1 inhibitor is a small molecule inhibitor of PAK1. In
some
embodiments, the individual is a mammal. In some embodiments the individual is
a human.
In some aspects, 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 PAK1 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 PAK1 inhibitor. BRAF is a
member of the Raf
kinase family of serine/threonine-specific protein kinases. BRAF plays a role
in regulating the
MAP kinase/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). For example, 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). In some embodiments the invention
provides
methods of treating melanoma in an individual wherein the melanoma is a wild-
type BRAF
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melanoma. In some embodiments the invention provides methods of treating
melanoma in an
individual wherein the melanoma is a wild-type BRAF melanoma and the melanoma
overexpresses PAK1 compared to non-cancerous cells. In some embodiments the
invention
provides methods of treating melanoma in an individual wherein the melanoma
comprises a
wild-type BRAF and PAK1 is amplified in the melanoma. In some embodiments, the
melanoma
is a wild-type BRAF melanoma wherein PAK1 is overexpressed in the melanoma
compared to
non-cancerous cells and PAK1 is amplified in the melanoma. In some embodiments
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 PAK1 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 PAK1 is amplified in the melanoma. In some
embodiments 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. In
some
embodiments, the individual in a mammal. In some embodiments, the individual
is a human.
In some aspects, the invention provides methods of treatment of melanoma in an
individual by
contacting the melanoma with a therapeutically effective amount of PAK1
inhibitor. In some
aspects, the invention provides methods of treatment of melanoma in an
individual by
administering to the individual a therapeutically effective amount of PAK1
inhibitor. PAKs
participate in a number of pathways that are commonly deregulated in human
cancer cells.
PAK1 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 RAF1 by
phosphorylating them on
serine 298 and serine 338, respectively. The increase of Ras-induced
transformation by PAK1
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). 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.
PAK1 phosphorylation of RAF1 induces RAF1 translocation to mitochondria, where
it
phosphorylates the pro-apoptotic protein BCL2-antagonist of cell death (BAD).
PAK1, PAK2,
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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
al., ibid). However, the relevant pathways downstream of PAK1 in human tumor
cells remain
only partially understood.
PAK1 is widely expressed in a variety of normal tissues; however, expression
is significantly
increased in ovarian, breast and bladder cancer. (S. Balasenthil et al., J.
Biol. Chem. 2004
279:4743; M. Ito et al., J. Urol. 2007 178:1073; P. Schraml et al., Am. J.
Pathol. 2003 163:985).
In luminal breast cancer, genomic amplification of PAK1 is associated with
resistance to
tamoxifen therapy, possibly occurring as a result of direct phosphorylation
and ligand-
independent transactivation of estrogen receptor by PAK1 (S. K. Rayala et al.,
Cancer Res.
2006. 66:1694-1701).
In some aspects, the invention provides methods of treating melanoma in an
individual by
contacting the melanoma with a therapeutically effective amount of a PAK1
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 PAK1 inhibitor. In
some embodiments,
the PAK1 gene is amplified in the melanoma. In some embodiments, the copy
number of the
PAK1 in the melanoma is about any of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or greater
than 5Ø Methods of
determining the copy number of the PAK1 gene in a melanoma are known in the
art. For
example, the copy number of the PAK1 gene may be determined by using SNP
arrays such as
the Affymetrix 500K SNP array analysis. In some embodiments, the invention
provides methods
of treating melanoma in an individual wherein the copy number of PAK1 in the
melanoma is
greater than about 2.5. In some embodiments, the invention provides methods of
determining
the copy number of PAK1 in a melanoma subsequent to treatment with a PAK1
inhibitor. In
some embodiments, the copy number of PAK1 in a melanoma is compared to the
copy number
of PAK1 in non-cancerous cells; for example, non-cancerous skin cells. In some
embodiments,
PAK1 is amplified in the melanoma and the melanoma overexpresses PAK1. In some
embodiments, PAK1 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.
In some aspects, the invention provides methods of treating melanoma in an
individual by
contacting the melanoma with a therapeutically effective amount of a PAK1
inhibitor wherein
PAK1 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 PAK1 inhibitor wherein PAK1 is overexpressed in the melanoma.
Methods to
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determine expression of PAK1 are known in the art. Examples of methods to
determine
expression levels of PAK1 in a melanoma include, but are not limited to
immunohistochemistry,
reverse-phase protein array (RPPA), quantitative PCR, immunoassays, and the
like. Levels of
PAK1 expression can be compared to other tumors and cells by using the Gene
Expression
Omnibus (GEO) database.
In some embodiments, the invention provides methods for treating melanoma in
an individual by
contacting the melanoma with a PAK1 inhibitor wherein PAK1 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 PAK1 inhibitor wherein PAK1 is overexpressed in the melanoma. In
some
embodiments, expression of PAK1 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. In some
embodiments, expression of PAK1 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 PAK1 in non-cancerous cells. In some
embodiments, the
melanoma overexpresses PAK1 compared to non-cancerous cells and the melanoma
is a wild-
type BRAF melanoma. In some embodiments, the melanoma overexpresses PAK1
compared to
non-cancerous cells and PAK1 is amplified in the melanoma. In some
embodiments, the
melanoma overexpresses PAK1 compared to non-cancerous cells and the melanoma
is a wild-
type BRAF melanoma amd PAK1 is amplified in the melanoma. In some embodiments
the
individual is a mammal. In some embodiments, the individual is a human.
In some aspects, 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
PAK1 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 PAK1 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 PAK1
inhibitor. Activation of
CRAF may be measured using phospho-CRAF(Ser338) antibodies. In some
embodiments, the
melanoma is a wild-type BRAF melanoma. In some embodiments, PAK1 is over
expressed in
the melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma
wherein
PAK1 is overexpressed in the melanoma. In some embodiments, the melanoma is a
wild-type
BRAF melanoma wherein PAK1 is amplified in the melanoma. In some embodiments,
the
melanoma is a wild-type BRAF melanoma wherein PAK1 is overexpressed in the
melanoma and
PAK1 is amplified in the melanoma. In some embodiments, PAK1 is overexpressed
in the
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melanoma and PAK1 is amplified in the melanoma. In some embodiments, the
individual is a
mammal. In some embodiments, the individual is a human.
In some aspects, 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
PAK1 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 PAK1 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 PAK1
inhibitor. Activation of
MEK may be measured using phospho-MEK1/1(Ser217/Ser221) antibodies. In some
embodiments, the melanoma is a wild-type BRAF melanoma. In some embodiments,
PAK1 is
over expressed in the melanoma. In some embodiments, the melanoma is a wild-
type BRAF
melanoma wherein PAK1 is overexpressed in the melanoma. In some embodiments,
the
melanoma is a wild-type BRAF melanoma wherein PAK1 is amplified in the
melanoma. In
some embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
overexpressed in the melanoma and PAK1 is amplified in the melanoma. In some
embodiments,
PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma.
In some
embodiments, the individual is a mammal. In some embodiments, the individual
is a human.
Inhibitors of PAK1
Provided herein are inhibitors of PAK1 (e.g. PAK1 antagonists) useful in the
methods described
herein. In some embodiments, the PAK1 inhibitor is a small molecule, a nucleic
acid, a
polypeptide or an antibody. Examples of PAK inhibitors are provided in WO
2007/072153, and
WO 2010/07184 both of which are incorporated herein by reference.
Small molecules
Provided herein are small molecules for use as PAK1 inhibitors for the
treatment of melanoma.
Small molecules are preferably organic molecules other than binding
polypeptides or antibodies
as defined herein that bind to PAK1 or interfere with PAK1 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
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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. WO
00/00823 and WO 00/39585). 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, enamines, sulfonamides,
epoxides,
aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides,
or the like.
Small molecule inhibitors of PAK kinases have been described (see
W02006072831,
W02007023382, W02007072153, W02010/071846, U520090275570).
CONHRI
R3
HilAC
NL0 Z X
I />¨Ar
/
N Il Y
RI R2 R2
(I) (II)
A series of PAK1 selective inhibitors elaborated on the 2-aminopyrido[2,3-
d]pyrimidin-7(8H)-
one (I) scaffold have been disclosed by Afraxis, Inc. in a series of patent
applications
(W02009086204, W02010071846, W02011044535, W02011156646,
W02011156786,W02011156640, W02011156780,W02011156775, W02011044264)
AstraZeneca has disclosed bicyclic heterocyclic PAK1 inhibitors of formula II
(see
W02006106326).
N¨NH
H ¨R1
ii Me me
c.... HNAl
N I- RiA
i= `_ = CONHR2 N; I N¨CONHR-2 1 N
µ µ
I .*L
S
R1OCNH 1210CNH R2 N Ni.....)s,
Iii
(III) (IV) (V) NH2
Pfizer has disclosed PAK inhibitors elaborated on 1H-thieno[3,2-c]pyrazole
(III), 3-amino-
tetrahydropyrrolo[3,4-c]pyrazole (IV) and N4-(1H-pyrazol-3-yl)pyrimidine-2,4-
diamine (V) (see
WO 2004007504, WO 2007023382, W02007072153, and W02006072831).
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Me me
r)s.i....H 0
Ni I N4 Ph
ç..1
NH c.
HN¨s.
NMe2
I
NN
I (VI)
Me
PF-3758309 (VI) is a potent ATP-competitive inhibitor of PAK1, 4, 5 and 6 that
has been in
clinical testing. (B. W. Murray et al., Proc. Natl. Acad. Sci USA 2010
107(20):9446; Rosen L
et al. Phase 1, dose escalation, safety, pharmacokinetic and pharmacodynamic
study of single
agent PF-03758309, an oral PAK inhibitor, in patients with advanced solid
tumors [abstract]. In:
Proceedings of the AACR-NCI-EORTC International Conference on Molecular
Targets and
Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA):
AACR; Mol
Cancer Ther 2011;10(11 Suppl):Abstract nr A177.
N--NH
Al--R7
HN
R6,,.._ (VII)
.N 0 Rib
I
* X
R5 NL N A
1
Ra
A series of N2-bicyclic indolyl, indazolyl and benzimidazolyl derivatives of
N4-(1H-pyrazol-3-
yl)pyrimidine-2,4-diamines (VII) (U.S. Ser. No.: 61/527,453 filed 08/25/2011)
and aza-indolyl,
indazolyl and benzimidazolyl derivatives thereof (U.S. Ser. No. 61/579,227,
filed 12/22/2011)
have been disclosed and those references are incorporated by reference in
their entirety. (A =
indolyl, indazolyl and benzimidazolyl or aza derivatives thereof).
Nucleic acids
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). In some
embodiments, the polynucleotide targets PAK1 expression (e.g. targets PAK1
mRNA).
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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 PAK1.
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 PAK1 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 PAK1
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, e.g., the
5' untranslated
sequence up to and including the AUG initiation codon, should work most
efficiently at
inhibiting translation. However, 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. Thus, oligonucleotides
complementary to
either the 5'- or 3'-non-translated, non-coding regions of the PAK1 gene,
could be used in an
antisense approach to inhibit translation of endogenous PAK1 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 PAK1 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.
In one embodiment, the PAK1 antisense nucleic acid of the invention is
produced intracellularly
by transcription from an exogenous sequence. For example, a vector or a
portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the PAK1 gene. Such
a vector would
contain a sequence encoding the PAK1 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 PAK1, or
fragments thereof, can be by any promoter known in the art to act in
vertebrate, preferably
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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 (siRNAs) can also function as PAK1 inhibitors for use in
the treatment of
melanoma. PAK1 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 PAK1 is specifically inhibited. Methods for
selecting an
appropriate dsRNA or dsRNA-encoding vector are well known in the art (Tuschi,
T et al (1999)
Genes Dev. 13(24):3191-3197; Elbashir, SM et al., (2001) Nature 411:494-498;
Hannon, GF
(2002) Nature 418:244-251; McManus MT and Sharp, PA (2002) Nature Reviews
Genetics
3:737-747; Bremmelkamp, TR et al. (2002) Science 296:550-553,US Patents Nos.
6,573,099 and
6,506,559 and International Patent Publications W001/36646, WO 99/32619 and WO
01/68836.
Examples of PAK1 siRNA oligonucleotide sequences include, but are not limited
to 1)
GAAGAGAGGTTCAGCTAAA, 2) GGAGAAATTACGAAGCATA, 3)
ACCCAAACATTGTGAATTA, 4) GGTTTATGATTAAGGGTTT, all obtained from
Dharmacon, Inc.
Polyp eptides
The invention provides polypeptide inhibitors of PAK1 activity for the
treatment of melanoma in
an individual. For example, binding polypeptides are polypeptides that bind,
preferably and
specifically to PAK1 as described herein. In some embodiments, the binding
polypeptides are
PAK1 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 PAK1, as described herein. Binding polypeptides may be
identified without
undue experimentation using well known techniques. In this regard, it is noted
that techniques
for screening polypeptide libraries for binding polypeptides that are capable
of specifically
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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.
U.S.A., 81:3998-
4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985);
Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol.
Meth., 102:259-274
(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al.
(1990) Proc. Natl.
Acad. Sci. USA, 87:6378; Lowman, H.B. et al. (1991) Biochemistry, 30:10832;
Clackson, T. et
al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol.,
222:581; Kang, A.S. et al.
(1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current
Opin. Biotechnol.,
2:668).
In this regard, 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. (1990) Proc.
Natl. Acad. Sci. USA,
87:6378) or protein (Lowman, H.B. et al. (1991) Biochemistry, 30:10832;
Clackson, T. et al.
(1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581;
Kang, A.S. et al.
(1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used
for screening
millions of polypeptides or oligopeptides for ones with specific binding
properties (Smith, G. P.
(1991) Current Opin. Biotechnol., 2:668). 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. U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143.
Although most phage display methods have used filamentous phage, lambdoid
phage display
systems (WO 95/34683; U.S. 5,627,024), T4 phage display systems (Ren et al.,
Gene, 215: 439
(1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998); Jiang et al.,
Infection &
Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311 (1997);
Ren, Protein
Sci., 5: 1833 (1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage
display systems
(Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. 5,766,905)
are also
known.
Additional improvements enhance the ability of display systems to screen
peptide libraries for
binding to selected target molecules and to display functional proteins with
the potential of
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screening these proteins for desired properties. Combinatorial reaction
devices for phage display
reactions have been developed (WO 98/14277) and phage display libraries have
been used to
analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and
properties of
constrained helical peptides (WO 98/20036). 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. The use of Staphlylococcus 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.
Methods of generating peptide libraries and screening these libraries are also
disclosed in U.S.
Patent Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434,
5,734,018,
5,698,426, 5,763,192, and 5,723,323.
Antibodies
In some embodiments of the invention, the PAK1 inhibitor for the treatment of
melanoma in an
individual is an isolated antibodies that bind to PAK1. In some embodiments,
the antibody is
humanized. In a further aspect of the invention, an anti-PAK1 antibody or an
antibody that
inhibits PAK1 function. In some embodiments the antibody is a monoclonal
antibody, including
a chimeric, humanized or human antibody. In some embodiments, the antibody is
an antibody
fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In
another embodiment, the
antibody is a full length antibody, e.g., an intact IgGl" antibody or other
antibody class or
isotype as defined herein.
In certain embodiments, amino acid sequence variants of the antibodies and/or
the binding
polypeptides provided herein are contemplated. For example, it may be
desirable to improve the
binding affinity and/or other biological properties of the antibody and/or
binding polypeptide.
Amino acid sequence variants of an antibody and/or binding 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
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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.
In certain embodiments, 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.,
retained/improved antigen binding, decreased immunogenicity, or improved ADCC
or CDC.
One type of substitutional variant involves substituting one or more
hypervariable region
residues of a parent antibody (e.g., a humanized or human antibody).
Generally, the 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 (e.g., substitutions) 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. Affinity maturation by constructing
and reselecting from
secondary libraries has been described, e.g., in Hoogenboom et al. in Methods
in Molecular
Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In
some embodiments
of 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
randomized. 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.
In certain embodiments, 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
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antigen. For example, conservative alterations (e.g., conservative
substitutions as provided
herein) that do not substantially reduce binding affinity may be made in HVRs.
Such alterations
may be outside of HVR "hotspots" or SDRs. In certain embodiments of the
variant VH and VL
sequences provided above, each HVR either is unaltered, or contains no more
than one, two or
three amino acid substitutions.
Combination therapy
The PAK1 inhibitors of the methods described herein can be used either alone
or in combination
with other agents in a therapy for the treatment of melanoma. For instance, a
PAK1 inhibitor
described herein may be co-administered with at least one additional
therapeutic agent including
another PAK1 inhibitor. In certain embodiments, an additional therapeutic
agent is a
chemotherapeutic agent. In some embodiments, the additional therapeutic agent
may be
Aldesleukin, Dacarbazine, DTIC-Dome (Dacarbazine), Ipilimumab, Proleukin
(Aldesleukin),
Vemurafenib, Yervoy (Ipilimumab), and/or Zelboraf (Vemurafenib). The example
of the use of
PAK1 inhibitors in combination therapies is provided by PCT/EP2011/070008
filed November
14, 2011.
Such 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 PAK1 inhibitor can occur
prior to,
simultaneously, and/or following, administration of the additional therapeutic
agent and/or
adjuvant. In some embodiments, PAK1 inhibitors are used for the treatment of
melanoma in an
individual in combination with radiation therapy. In some embodiments, PAK1
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.
In some embodiments of the invention, the individual has been previously
treated for melanoma,
for example, using an anti-cancer therapy. In one example, the anti-cancer
therapy is surgery. In
another embodiment, the subject can be further treated with an additional anti-
cancer therapy
before, during (e.g., simultaneously), or after administration of the PAK1
inhibitor. Examples of
anti-cancer therapies include, without limitation, surgery, radiation therapy
(radiotherapy),
biotherapy, immunotherapy, chemotherapy, or a combination of these therapies.
Route of administration
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.,
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injection or infusion by subcutaneous, intravenous, intraperitoneal,
intramuscular, intraarterial,
intralesional or intraarticular routes, topical administration, inhalation or
by sustained release or
extended-release means. In some embodiments, the invention provides for
methods for the
treatment of melanoma in an individual with a PAK1 inhibitor wherein the
PAKlinhibitor is
administered intravenously to the individual. In other embodiments, 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.
Pharmaceutical compositions
For the methods of the invention, therapeutic formulations of the invention
are prepared for
storage by mixing the PAK1 inhibitor having the desired degree of purity with
optional
physiologically acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are 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 sorbitol; salt-forming
counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as
TWEENTm, PLURONICSTh4 or polyethylene glycol (PEG).
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-
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particles and nanocapsules) or in macroemulsions. Such techniques are
disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
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.
Examples of 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 y ethyl-L-glutamate, non-degradable ethylene-
vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT Th4
(injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-
D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and
lactic acid-
glycolic acid enable release of molecules for over 100 days, certain hydrogels
release proteins
for shorter time periods. 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.
In some aspects, the invention provides a composition comprising a PAK1
inhibitor for use in
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. In some
embodiments, 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. In some
embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
amplified in the
melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma
wherein
PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma.
In some
embodiments, PAK1 is overexpressed in the melanoma and PAK1 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 PAK1
compared to
non-cancerous cells and/or PAK1 is amplified in the melanoma. In some
embodiments, the
invention provides a composition comprising PAK1 inhibitor for use in the
treatment of
melanoma in a mammal. In some embodiments, the invention provides a
composition
comprising PAK1 inhibitor for use in the treatment of melanoma in a human.
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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. In
some
embodiments, 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. In some
embodiments, the melanoma is a wild-type BRAF melanoma wherein PAK1 is
amplified in the
melanoma. In some embodiments, the melanoma is a wild-type BRAF melanoma
wherein
PAK1 is overexpressed in the melanoma and PAK1 is amplified in the melanoma.
In some
embodiments, PAK1 is overexpressed in the melanoma and PAK1 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 PAK1
compared to
non-cancerous cells and/or PAK1 is amplified in the melanoma. In some
embodiments, the
invention provides a use for a PAK1 inhibitor in the manufacture of a
medicament for the
treatment of melanoma in a mammal. In some embodiments, the invention provides
a use for a
PAK1 inhibitor in the manufacture of a medicament for the treatment of
melanoma in a human.
Kits
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. In some embodiments, the kit
further comprises another
therapeutic agent.
The 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
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The invention provides methods to identify human melanoma patients suitable
for treatment with
a PAK1 inhibitor by determining the presence of one or more melanoma
biomarkers. In some
embodiments, the melanoma biomarker is overexpression of PAK1 in the melanoma,
amplification of PAK1 in the melanoma, and/or the presence of wild-type BRAF
in the
melanoma. In some embodiments the overexpression of PAK1 is determined by
comparison to
non-cancerous tissue; for example non-cancerous skin tissue. In some
embodiments, the
biomarkers are detected in a test sample obtained from the individual. In some
embodiments, the
presence of the biomarker is determines by comparison of a test sample with a
reference sample.
In one embodiment, the invention provides methods to identify human melanoma
patients
suitable for treatment with a PAK1 inhibitor by determining the expression of
PAK1 in the
melanoma wherein overexpression of PAK1 in the melanoma compared to non-
cancerous cells
indicated that the patient is suitable for treatment with a PAK1 inhibitor. In
some embodiments,
overexpression of PAK1 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 PAK1 inhibitor. In some embodiments,
overexpression of
PAK1 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 PAK1
in non-cancerous
cells indicates that the patient is suitable for treatment with a PAK1
inhibitor. Methods to
determine expression of PAK1 are known in the art. Examples of methods to
determine
expression levels of PAK1 in a melanoma include, but are not limited to
immunohistochemistry,
reverse-phase protein array (RPPA), quantitative PCR, immunoassays, and the
like. Levels of
PAK1 expression can be compared to other tumors and cells by using the Gene
Expression
Omnibus (GEO) database.
In another embodiment, the invention provides methods to identify human
melanoma patients
suitable for treatment with a PAK1 inhibitor by detecting the amplification of
PAK1 in the
melanoma wherein amplification of the PAK1 gene in the melanoma indicates that
the patient is
suitable for treatment with a PAK1 inhibitor. In some embodiments, a copy
number of PAK1 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
PAK1 inhibitor.
Methods to determine amplification of a gene are known in the art. For
example, the copy
number of the PAK1 gene may be determined by using SNP arrays such as the
Affymetrix 500K
SNP array analysis.
In another embodiment, the invention provides methods to identify human
melanoma patients
suitable for treatment with a PAK1 inhibitor by detecting the genotype of BRAF
in the
melanoma wherein wild-type BRAF in the melanoma indicates that the patient is
suitable for
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treatment with a PAK1 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
PAK1 in the
melanoma, amplification of PAK1 in the melanoma and/or the presence of wild-
type BRAF in
the melanoma; the method comprising administering to the patient a
therapeutically effective
amount of a PAK1 inhibitor. In some embodiments, the patient is a human
patient. In some
embodiments of the above embodiment, at least one of the biomarkers is
overexpression of
PAK1 wherein PAK1 is overexpressed by about any of 10%, 20%, 30%, 40%, 50%,
60%, 70%,
80%, 90%, 100% or greater in the melanoma compared to non-cancerous cells. In
some
embodiments, expression of PAK1 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 PAK1 in non-cancerous cells. Methods to
determine expression
of PAK1 are known in the art. Examples of methods to determine expression
levels of PAK1 in
a melanoma include, but are not limited to immunohistochemistry, reverse-phase
protein array
(RPPA), quantitative PCR, immunoassays, and the like. Levels of PAK1
expression can be
compared to other tumors and cells by using the Gene Expression Omnibus (GEO)
database.
In some embodiments of the above embodiment, at least one of the biomarkers is
amplification
of PAK1 in the melanoma wherein a copy number of PAK1 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.
Methods to determine amplification of a gene are known in the art. For
example, the copy
number of the PAK1 gene may be determined by using SNP arrays such as the
Affymetrix 500K
SNP array analysis.
In some embodiments of the above embodiment, 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 PAK1
inhibitor.
The invention provides methods of adjusting treatment of melanoma in a patient
undergoing
treatment with a PAK1 inhibitor wherein the expression of PAK1 in the melanoma
is
determined. In some embodiments, the melanoma is a wild-type BRAF melanoma. In
some
embodiments, the overexpression of PAK1 in the melanoma indicates that
treatment with the
PAK1 inhibitor may continue. In some embodiments, the expression of PAK1 in a
melanoma in
a patient undergoing treatment with PAK1 is monitored over time. In some
embodiments, the
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expression of PAK1 in the melanoma is monitored at least daily, at least
weekly, at least
monthly. In some embodiments, the expression of PAK1 in a melanoma in a
patient undergoing
treatment with a PAK1 inhibitor is monitored over time. If PAK1 expression
increases over the
course of treatment with the PAK1 inhibitor, the amount of PAK1 inhibitor
administered to the
patient is increased or remains the same. In some embodiments, the amount of
PAK1 inhibitor
administered to the patient is increased until the level of PAK1 expression
decreases or is no
longer detected. If PAK1 expression decreases over the course of treatment
with the PAK1
inhibitor, the amount of PAK1 inhibitor administered to the patient is
decreased or remains the
same. In some embodiments, the expression of PAK1 expression in a melanoma of
a patient
undergoing treatment with a PAK1 inhibitor is monitored over time where
treatment with the
PAK1 inhibitor is continued until PAK1 expression in the melanoma is no longer
detected.
Exemplary embodiments
In some embodiments the invention provides methods for treating a melanoma in
an individual
comprising contacting the melanoma with a therapeutically effective amount of
a PAK1
inhibitor. In further embodiments, the melanoma is a wild-type BRAF melanoma.
In yet further
embodiments, PAK1 is overexpressed in the tumor compared to non-cancerous skin
cells. In
further embodiments of any of the above embodiments, PAK1 is amplified in the
tumor. In
further embodiments, the copy number of the PAK1 in the tumor is greater than
about 2.5.
In further embodiments of any of the above embodiments, the inhibitor is a
small molecule, a
nucleic acid, or a polypeptide. In some embodiments, the small molecule is PF-
3758309. In
some embodiments, the small molecule is a compound of formula I.
N--NH
HN
IR6..._ (VII)
. - N Ri a R11)
i
R5 N X N A .
1
Ra
In further embodiments, the small molecule is a compound of formula I and A is
4-indolyl, 5-
indolyl, 4-indazolyl, 5-indazolyl, 4-benzimidazoly1 or 5-benzimidiazoly1; Ra,
Rla and Rib are
independently hydrogen or Ci_3 alkyl; R5 is hydrogen or Ci_6 alkyl; R6 is
hydrogen, halogen or
Ci_6 alkyl; and, R7 is cycloalkyl optionally substituted by fluorine.
In further embodiments of any of the above embodiments, the individual is a
human.
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In further embodiments of any of the above embodiments, the PAK1 inhibitor is
used in
combination with a therapeutic agent.
The invention provides the use of a PAK1 inhibitor for the treatment of
melanoma in an
individual. In some embodiments of the use, the melanoma is a wild-type BRAF
melanoma.
The invention provides compositions comprising a PAK1 inhibitor for use in the
treatment of
melanoma. In some embodiments of the composition, the melanoma is a wild-type
BRAF
melanoma. In some embodiments, the composition further comprises a
pharmaceutically
acceptable excipient.
The invention provides the use of a PAK1 inhibitor in the manufacture of a
medicament for the
treatment of melanoma. In some embodiments of the use, the melanoma is a wild-
type BRAF
melanoma.
The invention provides kits comprising a PAK1 inhibitor for use in treating
melanoma
comprising PAK1 inhibitor and directions for use in the treatment of melanoma.
In some
embodiments of the kit, 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 PAK1
inhibitor.
The invention provides methods of inhibiting MEK signaling in a melanoma tumor
comprising
contacting the melanoma with a therapeutically effective amount of a PAK1
inhibitor.
The invention provides methods of identifying a human melanoma patient
suitable for treatment
with a PAK1 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
PAK1 inhibitor.
The invention provides methods of identifying a human melanoma patient
suitable for treatment
with a PAK1 inhibitor comprising determining the expression of PAK1 in the
melanoma,
wherein overexpression of PAK1 in the melanoma compared to non-cancerous skin
cells
indicates that the patient is suitable for treatment with a PAK1 inhibitor. In
some embodiments
of the method, the overexpression of PAK1 in the melanoma is 2.5-fold greater
than the
expression of PAK1 in the non-cancerous skin cellsThe invention provides
methods for treating
a human melanoma patient with a PAK1 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 PAK1 inhibitor;
and (b) administering to
the selected patient a therapeutically effective amount of a PAK1 inhibitor.
The invention provides methods for treating a human melanoma patient with a
PAK1 inhibitor
comprising: (a) selecting a patient based on the PAK1 expression level of the
melanoma,
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wherein a overexpression of PAK1 in the melanoma indicates that the patient is
suitable for
treatment with a PAK1 inhibitor; and (b) administering to the selected patient
a therapeutically
effective amount of a PAK1 inhibitor. In some embodiments of the methods, the
overexpression
of PAK1 in the melanoma is 2.5-fold greater than the expression of PAK1 in the
non-cancerous
skin cells.
The invention provides methods for treating a human melanoma patient
comprising
administering to the selected individual a therapeutically effective amount of
a PAK1 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
administering to the patient a therapeutically effective amount of a PAK1
inhibitor, wherein the
melanoma had been determined to overexpress PAK1 compared to non-cancerous
skin cells. In
some embodiments of the methods, the overexpression of PAK1 in the melanoma is
2.5-fold
greater than the expression of PAK1 in the non-cancerous skin cells.
The invention provides methods of adjusting treatment of melanoma in a patient
undergoing
treatment with a PAK1 inhibitor, said method comprising assessing the PAK1
expression in the
melanoma, wherein overexpression of PAK1 in the melanoma indicates that
treatment of the
individual is adjusted until PAK1 overexpression is no longer detected.
All of the features disclosed in this specification may be combined in any
combination. Each
feature disclosed in this specification may be replaced by an alternative
feature serving the same,
equivalent, or similar purpose. Thus, unless expressly stated otherwise, each
feature disclosed is
only an example of a generic series of equivalent or similar features.
Further details of the invention are illustrated by the following non-limiting
Examples. The
disclosures of all references in the specification are expressly incorporated
herein by reference.
EXAMPLES
The examples below are intended to be purely exemplary of the invention and
should therefore
not be considered to limit the invention in any way. The following examples
and detailed
description are offered by way of illustration and not by way of limitation.
Example 1: Elevated PAK1 protein expression and genomic amplification in
melanoma.
To determine the possible extent of PAK1 dysregulation in human melanoma,
primary tumor
tissue from 87 melanoma patients was assayed for DNA copy number changes using
high-
resolution single nucleotide polymorphism (SNP) arrays. Affymetrix 500K SNP
array analysis,
genomic DNA preparation, chip processing and data analysis were performed as
published
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previously (Harvey PM, et al., (2008) Genes Chromosomes Cancer, 47(6):530-542)
to measure
copy gains of 1 1q13, the region of chromosome 11 that harbors the PAK1 gene,
in the sampled
melanoma tissue. To collect expression array data for matched tumor samples,
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 1B). Dysregulated PAK1
expression was more
frequent than would be predicted by genomic amplification alone, thereby
suggesting that
additional transcriptional or regulatory mechanisms increase PAK1 expression
in this indication
(Reddy SD, et al, (2008) Cancer Res, 68(20):8195-8200 and de la Torre-Ubieta
L, et al., (2010)
Genes Dev, 24(8):799-813). Elevated expression of PAK1 in melanoma compared to
normal
skin tissues was also demonstrated using gene expression data deposited in the
Gene Expression
Omnibus database (G5E4587). Interestingly, PAK1 gene amplification was
preferentially
observed in tumors that lacked activating mutations in the BRAF oncogene at
22% versus 0% for
BRAF wild-type or mutant, respectively (p = 0.005, two-sided t-test; Figure
1B). The levels of
PAK1 mRNA expression differed between wild-type and BRAF(V600E) or BRAF(V600M)
genotypes (p = 0.006 and p=0.125, respectively; Figure 1B). Taken together,
this suggests that
PAK1 could be a tumor-promoting "driver" gene in a subset of BRAF wild-type
melanomas.
To further evaluate the extent of PAK1 dysregulation in human melanomas, 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. Four cancers were stage
pT1, 17 were
stage pT2, 28 were stage pT3, 35 were stage pT4 and 8 cases could not be
accurately staged.
Tissue microarrays (TMAs) were assembled as described previously (Bubendorf L,
et al., (2001)
J Pathol, 195(1):72-79). Approval was obtained for the use of all human tissue
from the local
research ethics committee (CO2.216). 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. The
highest intensity score among replicate cores was used as the score for each
patient. The same
pathologist scored all cases, blind to the clinical data. The chi-squared test
was used to evaluate
associations between categorical variables. Robust and selective IHC
reactivity of PAK1
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antibody was previously demonstrated ( Ong CC, et al., (2011) PNAS,
108(17):7177-7182). In
malignant melanoma, 46 of 92 (50%) primary tumor samples were positive for
PAK1 expression
and 26% of all cases showed staining of moderate (2+) or strong (3+) intensity
in the malignant
cells (Figure 1C, panels III and IV; Table 1). Nuclear localization of PAK1
was only evident in a
very small proportion of melanomas. Identical results were seen with an
alkaline phosphatase
label and fast red chromogen in place of a horseradish peroxidase label and
brown
diaminobenzidine. PAK1 was weakly expressed in basal keratinocytes in normal
skin, and
lymphocytes and presumed Langerhans cells were positive for PAK1 expression
(Figure 1C,
panel IV). Together, these data show that PAK1 DNA copy number, mRNA and
protein
expression are broadly upregulated in human melanoma.
Example 2: Negative association between PAK1 over-expression and BRAF mutation
in primary
melanomas
Given the prevalence of oncogenic mutation of BRAF and NRAS in melanoma (Lee
JH, et al.,
(2011) Br J Dermatol, 164(4):776-784), melanoma tissues were genotyped for
known 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.
Genotype
data for BRAF (39 Va1600G1u, 1 Va1600Lys and 46 wild-type) and NRAS (1
Gln61His, 7
Gln6lLys, 1 Gln6lLys + Gln6lArg + Leu59Ala, 1 Gln6lLeu, 19 Gln6lArg, 2
Gln6lArg +
Gln6lLys and 53 wild-type) were available for 86 and 84 tumors, respectively,
and were
consistent with the ranges of mutation frequencies that have been previously
published for
cutaneous melanoma (Lee JH, et al., (2011) Br J Dermatol, 164(4):776-784).
PAK1 IHC
staining was scored blind to clinicopathological details and mutation status
and results are
summarized in Table 1. Notably, PAK1 protein expression was dysregulated
selectively in
BRAF wild-type tumors (19 of 46 were positive for strong IHC staining of PAK1)
compared to
melanomas expressing oncogenic V600E or V600K mutants (4 of 40 tumors with
high IHC
staining). This negative correlation between PAK1 expression and BRAF mutation
was
statistically significant (p < 0.001, Chi-squared 10.702). BRAF and NRAS
mutations were not
mutually exclusive, and presence of tumors with either mutation was also
negatively associated
with PAK1 protein expression (p=0.004, Chi-squared 8.128). 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). There was no significant
association between
PAK1 protein expression and mitotic count (p=0.61 Student's T-test),
pathological tumor (pT)
stage (p=0.14 Chi-squared), Breslow thickness (p=0.85 Student's T-test) or
ulceration (p=0.91
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Chi-squared). Taken together, these results provide evidence that PAK1
dysregulation is
strongly associated with cutaneous melanomas that lack oncogenic mutation of
BRAF and define
a subset of human melanoma for which there is no effective targeted therapy.
Table 1. PAK1 protein overexpression in BRAF wild-type melanoma
MAPK Genotype PAK1 IHC P-value Chi-
activator 0, 1 2, 3 squared
BRAF WT 27 19 0.001 10.702
Mutant 36 4
NRAS WT 37 16 0.45 0.569
Mutant 24 7
Example 3: PAK1 is required for proliferation of BRAF wild-type melanoma cells
Given the genomic and histologic data for elevated PAK1 expression in the
subset of human
melanoma that is wild-type for BRAF, PAK1 expression and the effect of RNAi-
mediated
knockdown of PAK1 was examined in a panel of melanoma cell lines in order to
clarify the
contribution of PAK1 towards tumor cell proliferation. Cell lines were
acquired from the
American Type Culture Collection (ATCC; Manassas, VA) and maintained at 37 C
and 5% CO2
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. Cell lines
were
transfected with commercially available short-interfering RNA (siRNA)
oligonucleotide
duplexes from Dharmacon RNAi Technologies (Chicago, IL) that were previously
characterized
for efficiency and selectivity of PAK1 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 PAK1 protein expression in melanomas
expressing wild-type
versus mutant BRAF was also observed for immortalized cell lines in culture.
Cell viability
analysis demonstrated that 537MEL, MeWo, SK-MEL23 and SK-MEL30 melanoma cells
expressed high levels of PAK1 protein and transient knockdown of PAK1 via a
pool of multiple
PAK1-selective siRNA oligonucleotides resulted in a 1.8- to 4.3-fold reduction
in cell viability
when compared with cells transfected with a non-targeting, negative control
siRNA
oligonucleotide (p <0.0001; Figure 2A). Furthermore, inhibition of PAK1
generally reduced
proliferation of BRAF wild-type melanoma cells relative to BRAFv600E
cells (p < 0.07; n=14),
further supporting a role for PAK1 as a driver of proliferation in this
melanoma subtype (Figure
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2B). To better assess the mechanism by which PAK1 contributes to
proliferation, PAK-
dependent cellular signaling was assessed in 537MEL and SK-MEL23 cells.
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). For 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). MAPK
pathway
activation, as determined by phosphorylation of ERK and MEK, was dramatically
inhibited by
PAK knockdown (Figure 2C). In agreement with this result, cyclin D1 levels
(which are
essential for regulating cyclin-dependent kinases and Gl/S progression) were
also diminished as
a consequence of PAK1 ablation. 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. The sample dilutions were printed in
duplicate on slides
that were then subjected to immunostaining 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
log2
transformation and linear scaling (z-score conversion) to ensure normality and
linearity. RPPA
analysis showed a decreased signaling to MAPK, nuclear factor-KB (NF-KB) 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 (5er338) and MEK1(5er298) (17,
29-31).
Hence, the molecular mechanism by which PAK1 triggers activation of the MAPK
pathway in
BRAF wild-type melanoma cells was investigated. Since phospho-specific
antibodies that are
raised to the 5er217/5er221 activation loop sites on MEK proteins do not
distinguish between
MEK1 and MEK2, the MEK isoforms were immunoprecipitated from cells transfected
with
either control or PAK-selective siRNA oligonucleotides as previously described
(Hatzivassiliou
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G, et al., (2010) Nature, 464(7287):431-435) and MEK activation was detected
via
immunoblotting with phospho-MEK1/2(Ser217/Ser221) antibodies. PAK knockdown
diminished both MEK1 (Figure 3A) and MEK2 (Figure 3B) phosphorylation in
537MEL and
SK-MEL23 cells. Since the 5er298 phosphorylation site on MEK1 is not conserved
in MEK2,
PAK-dependent activation of both MEK isoforms would suggest that upstream
signaling to
CRAF might be a driver of MAPK pathway regulation in BRAF wild-type melanoma
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(5er338) antibodies. Western analysis demonstrated that PAK ablation
reduced
phosphorylation of CRAF on 5er338, a residue critical for full activation of
this kinase (Figure
3C). The dependence of CRAF(5er338) phosphorylation (Figure 3D) and CRAF
effector
signaling (Figure 3E) on PAK catalytic activity was also confirmed using PF-
3758309, an
inhibitor of PAKs that is currently in clinical development (Murray BW, et al,
(2010) PNAS,
107(20): 9446-9451), and IPA-3, an allosteric inhibitor that binds PAK1-3 and
prevents
activation by Rho family GTPases (Deacon SW, et al., (2008) Chemistry &
Biology, 15(4):322-
331).
Additional loss-of-function studies to analyze the role of PAK1 in BRAF wild-
type melanoma
cells were conducted by investigating the contribution of PAK1 to melanoma
cell migration.
Briefly, WM-266-4 melanoma cells were transfected with non-targeting control
(NTC) or
PAK1/2 siRNA oligonucleotide for 72 h and confluent WM-266-4 melanoma cell
were
subsequently wounded. Images were recorded when wounds were made (dark
shading) and 28 h
after wounding (bright field). Differences in relative wound density were
statistically significant
(p <0.001; n=3) revealing a requirement for PAK1 in melanoma cell migration
(Figure 4).
Taken together, the functional consequences of PAK1 blockade in BRAF wild-type
melanoma
cells encompasses pronounced cytostatic effects via reduced CRAF activation
and subsequent
MAPK pathway signaling.
Example 4: Differential sensitivity of BRAF wild-type melanoma cells to PAK
and BRAF
inhibition
To more closely investigate the activity and cellular mechanism of action of
PAK signaling
within sensitive and insensitive tumor types, small molecule inhibition of PAK
and BRAF were
compared using SK-MEL23 BRAF wild-type and A375 BRAF(V600E) cells. For
analysis of
pathway modulation, cells were treated with 5 [t.M PF-3758309 or with 0.2 [t.M
PLX-4720, a
BRAF inhibitor, for 4 h before cell lysates were analyzed for phosphorylation
of MAPK pathway
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components. Administration of PF-3758309 resulted in profound MAPK pathway
modulation in
SK-MEL23 cells (lane 2), but not A375 cells (lane 5), as determined by
measurement of ERK1/2
and MEK1/2 phosphorylation on kinase loop residues that are critical for
catalytic activity
(Figure 5A). In comparison, analysis of PLX-4720-mediated signaling changes
revealed only
modest inhibition of ERK1/2 and MEK1/2 phosphorylation in SK-MEL23 cells (lane
3),
whereas the same treatment conditions potently inhibited MAPK activation in
BRAF(V600E)
cells (lane 6). As a control, no differences were noted for total ERK1/2 or
MEK1/2 protein levels
in this experiment. Consistent with previous reports, MEK1-Ser298 was
confirmed as a PAK-
specific phosphorylation site but Ser298 phosphorylation was unlinked to MEK
activation loop
phosphorylation in BRAF(V600E) melanoma cells (lane 5). The biological
consequence of
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-PAK1 in BRAF(V600E) cells with only moderate
endogenous
expression of PAK1. Elevated PAK1 signaling in A375 cells resulted in a
significant increase in
CRAF and MEK phosphorylation that was reversible by addition of PF-3758309
(Figure 5B),
suggesting that acquisition of PAK1 overexpression could be another mechanism
to overcome
dependence on oncogenic BRAF in melanoma (Johannessen CM, et al., (2011)
Nature,
468(7326):968-972).
To determine if PAK inhibitors decreased cell viability of wild-type BRAF
melanoma cells, SK-
MEL23 and 537MEL cells were assayed with the CellTiter-Glo Luminescent Assay
(Promega,
Madison, WI) after treatment with PF-3758309 or with (S)-N2-(1-(1H-indo1-5-
yl)ethyl)-N4-(5-
cyclopropyl-1H-pyrazol-3-y1)-6-methylpyrimidine-2,4-diamine (I-007), N2-((1H-
indo1-4-
yl)methyl)-N4-(5-cyclopropyl-1H-pyrazol-3-y1)-6-methylpyrimidine-2,4-diamine
(1-054) and N2-
((4-chloro-1H-benzo[d]imidazol-5-y1)methyl)-N4-(5-cyclopropyl-1H-pyrazol-3-y1)-
N2-methyl-
pyrimidine-2,4-diamine (1-087). Inhibition of PAK1 by treatment with all
tested PAK inhibitors
significantly reduced cell proliferation indicating that inhibition of PAK
signaling is a target for
treatment of BRAF wild-type melanoma (Figure 6A and B).
To extend the in vitro observations, pharmacodynamic modulation by PAK small
molecule
inhibitors was evaluated using tumor xenograft models. Cultured SK-MEL-23,
A2058.X1 and
A375.X1 cells were removed from culture, suspended in Hank's buffered saline
solution
(HBSS), mixed 1:1 with Matrigel (BD Biosciences, USA), and implanted
subcutaneously into
the right flank of naïve female NCR nude (Taconic Farms, Hudson, NY) or Beige
Nude XID
(Harlan Laboratories, CA) mice. Animals with tumors of a mean volume of
approximately 250
mm3 were grouped into treatment cohorts. Tumor volumes were calculated by the
following
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formula: Tumor Volume = 0.5 x (a x b2), where 'a' is the largest tumor
diameter and 'b' is the
perpendicular tumor diameter. Tumor volume results are presented as mean tumor
volumes the
standard error of the mean (SEM). Percent growth inhibition (%INH) at the end
of study (EOS)
was calculated as %INH = 100 [(EOS Vehicle EOS Treatment)/(EOS Vehicle)]. 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, Rancho
Dominguez, CA) and protein extracts 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).
Proteins were
subsequently resolved by 4-12% SDS-PAGE and transferred to nitrocellulose
membranes
(Millipore Corporation, Billerica, MA) for immunoblotting with the indicated
antibodies.
Treatment with PF-3758309 resulted in a substantial decrease in CRAF, MEK1/2
and ERK1/2
phosphorylation in SK-MEL23 tumors (Figure 7A). In A2058.X1 BRAF(V600E)
tumors,
decreased phosphorylation of CRAF(5er338) was not observed following PF-
3758309 dosing.
The effect of PF-3758309 on growth and maintenance of BRAF wild-type tumors
was also
evaluated in an efficacy experiment for 21 days (Figure 7B and Figure 8A-B).
Treatment with
10, 15 and 25 mg/kg PF-3758309 significantly impaired tumor growth (74%, 76%
and 91%
inhibition relative to the control cohort, respectively) relative to the
vehicle cohort as measured
on the final day of dosing (Dunnett's t-test, p<0.0001). In comparison,
minimal anti-tumor
efficacy and inhibition of CRAF phosphorylation were observed for SK-MEL23
tumors treated
with a potent RAF inhibitor in vivo (Figure 9B)(Hoeflich KP, et al., (2009)
Cancer Res,
69(7):3042-3051). In addition, phosphoproteomic analysis of BRAF mutant (A375)
and wild-
type (SK-MEL23) cells treated with PLX-4720 BRAF inhibitor demonstrated that
these cell
subtypes of melanoma exhibit different signaling responses due to BRAF
inhibition (Figure 9A).
Together, the magnitude of MAPK pathway inactivation by PF-3758309 was
correlated with
anti-tumor efficacy in a BRAF wild-type melanoma xenograft model and these
data support the
conclusion that interfering with PAK signaling could have therapeutic efficacy
in this subset of
melanoma (model depicted in Figure 10).
