Note: Descriptions are shown in the official language in which they were submitted.
CA 02817133 2013-05-07
WO 2012/065935 -1-
PCT/EP2011/070008
METHODS OF TREATING TUMORS
The present invention discloses a method of treating cancerous tumors which
amplify or over-
express PAK1 by contacting the tumor with a PAK1 inhibitor in combination with
a second anti-
proliferative agent.
Protein kinases are a family of enzymes that catalyze phosphorylation of the
hydroxyl groups of
specific tyrosine, serine, or threonine residues in proteins. Typically, such
phosphorylation can
dramatically change the function of the protein and thus protein kinases can
be pivotal in the
regulation of a wide variety of cellular process, including metabolism, cell
proliferation, cell
differentiation, and cell survival. The mechanism of these cellular processes
provides a basis for
targeting protein kinases to treat disease conditions resulting from or
involving disorder of these
cellular processes. Examples of such diseases include, but are not limited to,
cancer and diabetes.
Protein kinases can be broken into two types, protein tyrosine kinases (PTKs)
and serine-
threonine kinases (STKs). Both PTKs and STKs can be receptor protein kinases
or non-receptor
protein kinases. PAK is a family of non-receptor STKs. The p21-activated
protein kinase (PAK)
family of serine/threonine protein kinases plays important roles in
cytoskeletal organization,
cellular morphogenesis, cellular processes and cell survival (Daniels et 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).
The present invention relates to methods of treating tumors or hyper-
proliferative conditions
wherein the tumor cells or hyper-proliferating cells over-express or amplify
PAK1 by treating
the patient or contacting the tumor with a PAK1 inhibitor and a second anti-
hyper-proliferative
or anti-tumor agent selected from an inhibitor of EGFR, the Raf/MEK/ERK
pathway, Src, Akt or
an inhibitor of apoptosis proteins
CA 02817133 2013-05-07
WO 2012/065935 - 2 -
PCT/EP2011/070008
Figure 1 - Analysis of PAK1 genomic amplification and functional role in human
breast tumors.
(A) Genomic Identification of Significant Targets in Cancer (GISTIC) analysis
of 11q13 copy
number gains. Points are proportionately spaced and arranged in genome order.
Vertical line
represents chromosome location of the PAK1 gene. GISTIC Q-value for DNA gain
are defined
by the multiple-testing-corrected probability of gain frequency and mean copy
gain occurring by
chance displayed as the negative logio of the Q-value for each SNP array probe
set. (B) PAK1
DNA copy and mRNA expression dot plot depicts the relationship of DNA copy
number to the
226507 at Affymetrix MAS 5.0 signal for 51 tumor samples. The Pearson
correlation statistic
(0.75) is shown for the plot. The solid line represents the best-fit line
through these points. (C)
Increasing proportion of Annexin V¨positive cells following knockdown of PAK1
expression is
shown for 3 breast cancer cell lines with focal PAK1 genomic amplification,
MDA-MB-175,
HCC1500 and MDA-MB-134 IV. Cells were harvested 3-5 days following transient
transfection
of pooled siRNA oligonucleotides. (D) Fluorescence-activated cell sorting
analysis for Annexin
V/PI. MDA-MB-175 cells were cultured in the presence or absence of IPA-3 for
48 h. Annexin
V/PI staining was then done to assess apoptosis/necrosis. Annexin V labeling
(bottom right
quadrants) represents the population undergoing early apoptosis. Annexin V and
PI double
labeling (top right quadrants) represent cells that have already died by
apoptosis. Live cells are
represented in the bottom left quadrants. Percentages of cells are shown for
each quadrant.
Figure 2 - PAK1 is highly expressed in human lung tumors and plays a critical
role in
proliferation of squamous NSCLC cell lines. (A) Analysis of PAK1 mRNA
expression in laser-
capture microdissected lung tissues. Data for Affymetrix probe 226507 at are
plotted as the
mean (horizontal line), middle 50% of data (box), and 95% confidence interval
(lines). Pair-wise
comparisons were performed by Student's t-test. Relative to normal tissues
(n=9), PAK1
expression was significantly greater in squamous NSCLC (n=16; **, p=0.0005)
and
adenocarcinoma NSCLC (n=29; *, p=0.008). The difference in PAK1 mRNA
expression
between squamous and adenocarcinoma NSCLC was not significant in this panel of
lung tumors
(p=0.1). (B) Proliferation of a panel of squamous NSCLC cell lines was
measured by
[3H]thymidine uptake assay. EBC-1, NCI-H520, KNS-62, SK-MES-1 and NCI-H441
cells were
transfected with either a non-targeting control siRNA oligonucleotide (black
columns) or a pool
of siRNA oligonucleotides against PAK1 and PAK2 (white columns). The extent of
proliferation
under each condition was plotted as a percentage of the normalized non-
targeting control value
for each cell line and data is shown as the mean SD.
Figure 3 - (A) Accumulation of cells in G1 phase of the cell cycle is evident
following PAK1
knockdown. NCI-H520.X1 cells were treated with 200 ng/mL Dox for 4 days and
analyzed by
CA 02817133 2013-05-07
3
WO 2012/065935 - -
PCT/EP2011/070008
propidium iodide staining and flow cytometry. (B) NCI-H520.X1 cells were serum
starved for
24 hours and cell cycle re-entry was monitored by harvesting cell lysates at
the indicated time
points following growth in 10% serum-containing media. Cell lysates were
analyzed by
immunoblotting using antibodies against PAK1, p27KiPl, E2F1 and actin. (C) The
percentages of
cells with nuclear accumulation of p27'"' are indicated (2000 total cells per
condition). Columns
represent mean SD. *, p<0.05. **, p<0.0001.
Figure 4 - PAK1 is required for growth of established NCI-H520.X1 and EBC-1
squamous
NSCLC tumors. (A) NCI-H520.X1 cells expressing inducible shRNAs against LacZ,
PAK1,
PAK2 or PAK1+PAK2 were implanted in the flank of athymic mice as described in
Materials
and Methods. Treatment in each experiment was initiated when tumor size ranged
from 200 to
250 mm3. Administration of 1 mg/mL doxycycline via drinking water resulted in
inhibition of
tumor growth for mice bearing shPAK1 and shPAK1+2 NCI-H520.X1 cells. Induction
of PAK2-
or LacZ-specific shRNAs did not affect tumor growth kinetics. No animal weight
loss was
observed. Data consist of 10 mice per treatment group and errors bars
represent the standard
error. Individual mice were removed from data plotting when tumors reached
volume end point
of 2000 mm3: shLacZ control n=5; shLacZ+Dox n=3; shPAK1 control n=2; shPAK2
control
n=5; shPAK2+Dox n=2; shPAK1+2 control n=5. (B) EBC-1 tumors expressing shLacZ
or
shPAK1 were allowed to grow to 200-250 mm3 before groups of mice with tumors
of equivalent
size were administered doxycycline to inhibit PAK1. Data consist of 10 mice
per treatment
group and errors bars represent the standard error.
Figure 5 - PAK1 inhibition decreases NF-KB pathway activation and combines
with IAP
antagonists to promote apoptosis of NSCLC cells. (A) EBC-1-shPAK1 and -shLacZ
cells were
treated with BV6 IAP antagonist and 300 ng/mL doxycycline (Dox). The highest
concentration
of BV6 was 20 M and 2-fold serial dilutions were assessed in a 10-point
dilution curve. Cells
were pre-incubated in the presence of Dox for 3 days prior to addition of BV6
for an additional 3
days. Cell cultures were then analyzed by a CellTiterGlo viability assay. Data
points were
performed in quadruplicate. (B) Fluorescence-activated cell sorting analysis
for Annexin V and
propidium iodide (PI) staining. EBC-1-shPAK1 cells were cultured in the
presence or absence of
300 ng/mL doxcycline (Dox) for 72 h and 5 jtM BV6 for an additional 24 h.
Annexin V/PI
staining and fluorescence-activated cell sorting (FACS) analysis was then done
to assess
apoptosis/necrosis.
Figure 6 - (A)Down-regulation of XIAP expression potentiates the proapoptotic
activity of PF-
3758309 PAK small molecule inhibitor (PAK SMI; p ( 0.0001, Dunnett's t-test).
Cells were
CA 02817133 2013-05-07
4
WO 2012/065935 - -
PCT/EP2011/070008
transfected with non-targeting control (NTC) or XIAP-specific siRNA
oligonucleotides for 48 h
prior to treatment with DMSO or PAK SMI as indicated for an additional 72 h.
Cell viability was
determined via Cell Titer Glo assay and results represent mean standard
deviation from three
experiments. (B)Combined antagonism of XIAP and PAK1 promotes efficient
cleavage of
PARP and caspase-3. XIAP siRNA oligonucleotides were transfected for 72 h
prior to treatment
with DMSO or 5 mM PAK SMI.
Figurre 7 - (A) Combinatorial accumulation of cleaved PARP and caspase-3 by
PAK1 and IAP
antagonism. Cells were incubated with Dox and 5 M for indicated time points,
lysed and then
used for Western blot analysis (B) Dual PAK1 and IAP inhibition results in a
synergistic
decrease in viability of SK-MES-1 (squamous subtype) and NCI-H441
(adenocarcinoma
subtype) NSCLC cells. Cellular ATP consumption was determined via Cell Titer
Glo assay
following transient siRNA-mediated inhibition of PAK1 and 5 jtM BV6 treatment
as indicated.
Inhibition of cell viability was significantly greater for PAK1 siRNA and BV6
combination than
for single agents (p ( 0.0001, Student's t-test).
Figure 8 - PAK1 inhibition induces cleavage of caspases and poly ADP ribose
polymerase
(PARP) in breast cancer cells with focal genomic amplification of PAK1. (A)
HCC-1500 cells
were transiently transfected with individual or pooled siRNA oligonucleotides
(100 nM) to
induce PAK1 knockdown. Apoptosis induction was monitored by harvesting cell
lysates after 48
h and immunoblotting using antibodies against cleaved caspase-3, cleaved
caspase-7 and cleaved
PARP. (B) Ablation of PAK1 protein expression in MDA-MB-134 IV cells was also
associated
with a decrease in MEK1 (5er298) and ERK1/2 (Thr202/Tyr204) phosphorylation.
Total
proteins and I3-actin were used as controls.
Figure 9 - Combined PAK1 and IAP inhibition results in apoptosis of squamous
NSCLC cells.
(A) Percentages of Annexin V¨positive cells are shown for each treatment
condition. (B)
Cellular apoptosis markers were increased following genetic ablation of PAK1
and IAP
antagonist treatment for indicated times. Cell lysates were analyzed by
immunoblotting. PARP
cleavage and caspase-3/6/7/9 activation was dramatically elevated by combined
Dox and BV6
treatment.
Figure 10 - Combination of ATP-competitive pan-PAK inhibitor PF-3758309 (B.W.
Murray et al.
Proc. Nat. Acad. Sci. USA 2010 107(20):9446-9471 and IAP small molecule
antagonist results in
apoptosis of squamous NSCLC cells. (A) Catalytic inhibition of PAK1 via PF-
3758309
treatment was tested with BV6 for in vitro combination efficacy in EBC-1 cells
using a 4-day
CA 02817133 2013-05-07
WO 2012/065935 - -
PCT/EP2011/070008
CellTiterGlo viability assay. Calcusyn, a program utilizing the Chou and
Talalay (Chou TC,
Talalay P. Quantitative analysis of dose-effect relationships: the combined
effects of multiple
drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984; 22:27-55) method of
calculating synergy,
was used to calculate the combination index and, thus, determine the level of
synergy. Strong
5 synergy, as indicated by combination index (CI) values 0.3, was observed
(CI = 0.113). (B)
Combination of 5 M PAK inhibitor (PAKi) and 5 jtM BV6 for the indicated times
resulted in
dramatic induction of cellular apoptotic markers.
Figure 11 - Combinatorial effect of PAK1, PAK2, MEK and PI3K inhibition on
tumor cell
viability. CellTiter-Glog (CTG) assays of cellular viability were performed
following PAK1
and PAK2 siRNA transfection and compound treatment for 3 days, as indicated.
GDC-0623 is a
potent and highly selective inhibitor of MEK1 and MEK2. GDC-0941 is a potent
inhibitor of
Class I PI3K isoforms with biochemical IC50 values of 3-75 nM for the four
Class I isoforms of
PI3K. (A) Viability of SKMES-1 (KRASN85K mutation) lung cancer cells treated
with PAK1
and PAK2 siRNA oligonucleotides, 0.2 M GDC-0623 and 0.5 M GDC-0941. (B)
Viability of
Calu-6 (KRASQ61K mutation) lung cancer cells treated with PAK1 and PAK2 siRNA
oligonucleotides, 0.2 M GDC-0623 and 0.4 M GDC-0941. (C) Viability of Cal-
120 (basal
subtype) breast cancer cells treated with PAK1 and PAK2 siRNA
oligonucleotides, 2 M GDC-
0623 and 2.5 M GDC-0941.
Figure 12 - Combinatorial regulation of apoptotic and proliferation biomarkers
following
combined PAK1, PAK2, MEK and PI3K inhibition in NSCLC cells. SKMES-1 (KRASN85K
mutant) NSCLC cells were treated with PAK1 and PAK2 siRNA oligonucleotides,
0.4 M
GDC-0623 and 1 M GDC-0941 for 24 hours. The accumulation of cleaved caspase-3
and poly
ADP ribose polymerase (PARP), and decrease of cyclin D1 protein, was enhanced
by
combination of PAK knockdown with inhibitors of the MEK and PI3K pathways.
The phrase "a" or "an" entity as used herein refers to one or more of that
entity; for example, a
compound refers to one or more compounds or at least one compound. As such,
the terms "a"
(or "an"), "one or more", and "at least one" can be used interchangeably
herein.
The words "comprise," "comprising," "include," "including," and "includes"
when used in this
specification and claims are intended to specify the presence of stated
features, integers,
components, or steps, but they do not preclude the presence or addition of one
or more other
features, integers, components, steps, or groups thereof
CA 02817133 2013-05-07
WO 2012/065935 - 6 -
PCT/EP2011/070008
The terms "treat" and "treatment" refer to both therapeutic treatment wherein
the object is to
prevent or slow down (lessen) an undesired physiological change or disorder,
such as the growth,
development or spread of cancer. For purposes of this invention, beneficial or
desired clinical
results include, but are not limited to, alleviation of symptoms, diminishment
of extent of
disease, stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression,
amelioration or palliation of the disease state, and remission (whether
partial or total), whether
detectable or undetectable. "Treatment" can also mean prolonging survival as
compared to
expected survival if not receiving treatment. Those in need of treatment
include those already
with the condition or disorder as well as those prone to have the condition or
disorder or those in
which the condition or disorder is to be prevented.
The phrase "therapeutically effective amount" means an amount of a compound of
the present
invention that (i) treats the particular disease, condition, or disorder, (ii)
attenuates, ameliorates,
or eliminates one or more symptoms of the particular disease, condition, or
disorder, or (iii)
prevents or delays the onset of one or more symptoms of the particular
disease, condition, or
disorder described herein. In the case of cancer, the therapeutically
effective amount of the drug
may reduce the number of cancer cells; reduce the 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, to some extent, tumor
growth; and/or
relieve to some extent one or more of the symptoms associated with the cancer.
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 can be measured, for example, by assessing the
time to disease
progression (TTP) and/or determining the response rate (RR).
The term "synergistic" as used herein refers to a therapeutic combination
which is more effective
than the additive effects of the two or more single agents. A determination of
a synergistic
interaction between a PAK1 inhibitor and a second anti-hyperproliferative
agent may be based
on the results obtained from the assays described herein. The combinations
provided by this
invention have been evaluated in several assay systems, and the data can be
analyzed utilizing a
standard program for quantifying synergism, additivism, and antagonism among
anticancer
agents. The program preferably utilized is that described by Chou and Talalay,
in "New
Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter
2.
Combination Index values less than 0.8 indicates synergy, values greater than
1.2 indicate
antagonism and values between 0.8 to 1.2 indicate additive effects. The
combination therapy
may provide "synergy" and prove "synergistic", i.e., the effect achieved when
the active
ingredients used together is greater than the sum of the effects that results
from using the
CA 02817133 2013-05-07
7
WO 2012/065935 - -
PCT/EP2011/070008
compounds separately. A synergistic effect may be attained when the active
ingredients are: (1)
co-formulated and administered or delivered simultaneously in a combined, unit
dosage
formulation; (2) delivered by alternation or in parallel as separate
formulations; or (3) by some
other regimen. When delivered in alternation therapy, a synergistic effect may
be attained when
the compounds are administered or delivered sequentially, e.g., by different
injections in separate
syringes. In general, during alternation therapy, an effective dosage of each
active ingredient is
administered sequentially, i.e., serially, whereas in combination therapy,
effective dosages of two
or more active ingredients are administered together.
The terms "cancer" and "cancerous" refer to or describe the physiological
condition in mammals
that is typically characterized by unregulated cell growth. A "tumor"
comprises one or more
cancerous cells. 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 squamous cell cancer (e.g., epithelial squamous cell cancer),
lung cancer
including small-cell lung cancer, non-small cell lung cancer ("NSCLC"),
adenocarcinoma of the
lung and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer, 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, as well as head and neck cancer.
The term "carcinoma" refers to an invasive malignant tumor consisting of
transformed epithelial
cells. The term "squamous cell carcinoma" (SCC) refers to subset of carcinomas
that effect
squamous epithelial cells that may occur in many different organs, including
the skin, lips,
mouth, esophagus, urinary bladder, prostate, lungs, vagina, and cervix. It is
a malignant tumor of
squamous epithelium.
A "chemotherapeutic agent" is a biological (large molecule) or chemical (small
molecule)
compound useful in the treatment of cancer, regardless of mechanism of action.
Classes of
chemotherapeutic agents include, but are not limited to: alkylating agents,
antimetabolites,
spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase
inhibitors,
proteins, antibodies, photosensitizers, and kinase inhibitors.
Chemotherapeutic agents include
compounds used in "targeted therapy" and non-targeted conventional
chemotherapy.
CA 02817133 2013-05-07
WO 2012/065935 - 8 -
PCT/EP2011/070008
Examples of chemotherapeutic agents include erlotinib (TARCEVA , Genentech/OSI
Pharm.),
bortezomib (VELCADE , Millennium Pharm.), fulvestrant (FASLODEX ,
AstraZeneca),
sunitib (SUTENT , Pfizer/Sugen), letrozole (FEMARA , Novartis), imatinib
mesylate
(GLEEVEC , Novartis), finasunate (VATALANIB , Novartis), oxaliplatin (ELOXATIN
,
Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE ,
Wyeth),
Lapatinib (TYKERB , G5K572016, Glaxo Smith Kline), Lonafamib (SCH 66336),
sorafenib
(NEXAVAR , Bayer Labs), gefitinib (IRESSA , AstraZeneca), AG1478, alkylating
agents such
as thiotepa and CYTOXAN cyclosphosphamide; 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); a camptothecin (including the
synthetic analog
topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin
synthetic analogs); cryptophycins (particularly cryptophycin 1 and
cryptophycin 8); dolastatin;
duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1);
eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as
chlorambucil,
chlomaphazine, 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 ylI and calicheamicin w1I (Angew Chem.
Intl. Ed. Engl.
1994 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as
clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related chromoprotein
enediyne
antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins,
cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,
dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAIVIYCIN
(doxorubicin),
morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin
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 and 5-
fluorouracil (5-FU); folic acid
analogs 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,
CA 02817133 2013-05-07
9
WO 2012/065935 - -
PCT/EP2011/070008
mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic
acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic
acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine;
diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins;
mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet;
pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK
polysaccharide complex
(JHS Natural Products); razoxane; rhizoxin; sizofuran; spirogermanium;
tenuazonic acid;
triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A,
roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;
mitobronitol;
mitolactol; pipobroman; gacytosine; arabino side ("Ara-C"); cyclophosphamide;
thiotepa;
taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton,
N.J.),
ABRAXANE (Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel
(American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE
(docetaxel, doxetaxel;
Sanofi-Aventis); chloranmbucil; GEMZAR (gemcitabine); 6-thioguanine;
mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine;
etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine; NAVELBINE (vinorelbine); novantrone;
teniposide;
edatrexate; daunomycin; aminopterin; capecitabine (XELODA ); ibandronate; CPT-
11;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMF0); retinoids
such as retinoic
acid; and pharmaceutically acceptable salts, acids and derivatives of any of
the above.
Also included in the definition of "chemotherapeutic agent" are: (i) anti-
hormonal agents that act
to regulate or inhibit hormone action on tumors such as anti-estrogens and
selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen (including
NOLVADEX ;
tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,
keoxifene,
LY117018, onapristone, and FARESTON (toremifine citrate); (ii) aromatase
inhibitors that
inhibit the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such
as, for example, 4(5)-imidazoles, aminoglutethimide, IVIEGASE (megestrol
acetate),
AROMASIN (exemestane; Pfizer), formestanie, fadrozole, RIVISOR (vorozole),
FEMARA
(letrozole; Novartis), and ARIMIDEX (anastrozole; AstraZeneca); (iii) anti-
androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-
dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v)
lipid kinase inhibitors;
(vi) antisense oligonucleotides, particularly those which inhibit expression
of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for example, PKC-
alpha, Ralf and H-
Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYIVIE )
and HER2
CA 02817133 2013-05-07
WO 2012/065935 - 10 -
PCT/EP2011/070008
expression inhibitors; (viii) vaccines such as gene therapy vaccines, for
example,
ALLOVECTIN , LEUVECTIN , and VAXID , PROLEUKIN , rIL-2; a topoisomerase 1
inhibitor such as LURTOTECAN , ABARELIX rmRH; (ix) anti-angiogenic agents
such as
bevacizumab (AVASTIN ), Genentech); and (x) pharmaceutically acceptable salts,
acids and
derivatives of any of the above.
Also included in the definition of "chemotherapeutic agent" are therapeutic
antibodies such as
alemtuzumab (Campath), bevacizumab (AVASTIN , Genentech); cetuximab (ERBITUX ,
Imclone); panitumumab (VECTIBIX , Amgen), rituximab (RITUXAN ,
Genentech/Biogen
Idec), pertuzumab (OMNITARG , 2C4, Genentech), trastuzumab (HERCEPTIN ,
Genentech)
and tositumomab (Bexxar, Corixia.
Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic
agents in
combination with the PI3K inhibitors of the invention include: alemtuzumab,
apolizumab,
aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine,
cantuzumab
mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,
daclizumab,
eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab,
gemtuzumab
ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab,
matuzumab,
mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab,
numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab,
pecfusituzumab,
pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab,
reslizumab,
resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab,
tacatuzumab
tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,
trastuzumab,
tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and
visilizumab.
The following abbreviations are used in the present application: DCIS (ductal
carcinoma in situ),
SCLC (small cell lung cancer), NSCLC (non-small cell lung cancer); SCC
(squamous cell
carcinoma).
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 l<13(NFK12.) signalling
pathways, which all
have been associated with oncogenesis. PAKs activate MEKand 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.
CA 02817133 2013-05-07
WO 2012/065935 - 11 -
PCT/EP2011/070008
(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,
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., supra) 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., i
Biol. Chem. 2004
279:4743; M. Ito et al., 1 Urol. 2007 178:1073; P. Schraml et al., Am. i
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) PAK1 is an attractive target for developing therapeutic
agents effective for
use in treatment of hyperproliferative disorders. (R Kumar et al. supra)
Amplification Experiments - PAK1 genomic copy number and gene expression were
determined
for a large panel of breast, lung and head-and-neck tumors. PAK1 genomic
amplification was
prevalent in luminal breast cancer and PAK1 protein expression was associated
with lymph node
invasion and metastasis.
Several genomic regions with copy number gains have been identified in breast
cancer via
comparative genomic hybridization approaches (J. Climent et al., Biochem. Cell
Biol. 2007
85:497-508; E.H. van Beers and P. M. Nederlof, Breast Cancer Res. 2006 8:210)
An assay of 51
breast tumors for DNA copy number changes using high-resolution single
nucleotide
polymorphism (SNP) arrays and analyzed data using the Genomic Identification
of Significant
Targets in Cancer (GISTIC) method (P. M. Haverty et al., Genes Chromosomes
Cancer 2008
47:530-542. 21; R. Beroukhim et al., Proc. Natl. Acad. Sci. USA 2007 104:20007-
20012.). A
CA 02817133 2013-05-07
WO 2012/065935 - 12 -
PCT/EP2011/070008
chromosome 11 region of amplification is shown in Figure 1A. Two distinct
GISTIC peaks
were observed at 69 and 76 Mb, suggesting that the 11q13.5 region contains 2
independent
amplicons. The 69-Mb peak corresponds to amplification of CCND1, a very well
described
genomic alteration in breast cancer. (C. Dickson, et al., Cancer Lett. 1995
90:43-50) The plateau
of the 76-Mb peak contains the PAK1 gene (shown as a dotted line). The
frequency of PAK1
amplification was 17% (copy number > 2.5) in this tumor panel and copy number
gain was well
correlated with mRNA expression (Pearson correlation = 0.75; Figure 1B).
Similar results were
also obtained in a larger panel (n=165) of breast tumors that were also
analyzed for genomic
amplification by high-resolution SNP arrays. PAK1 gene amplification was
prevalent and mean
DNA copy number was greatest in luminal, hormone receptor-positive tumors (7.7
mean copy
number) and least in basal breast tumors (2.8 mean copy number). (Z. Kan et
al., Nature 2010
466:869-873) These experiments suggest that PAK1 could be a tumor-promoting
"driver" gene
in the 76-Mb amplicon of chromosome 11. PAK1 expression was absent in normal
breast
epithelial cells, but was detected in the malignant cells of 39% of primary
breast
adenocarcinomas.
PAK1 protein expression level and subcellular localization were ascertained
via
immunohistochemical (IHC) staining of tissue microarrays. Robust and selective
IHC reactivity
of PAK1 antibody was confirmed in cancer cell lines with immunoblot analysis
of protein
extracts from these cells performed in parallel. PAK1 protein expression data
for 226 primary
breast cancers, 15 DCIS, 32 breast cancer lymph node metastases, 97 NSCLC, 27
SCLC and 130
head and neck squamous cell carcinomas are summarized in Table 1. PAK1
staining intensity
varied among tumor tissues, ranging from no or low staining to very strong
staining in the either
the cytoplasm and/or nucleus.
RNA was purified from 88 primary breast cancer specimens and cytoplasmic PAK1
IHC
staining was correlated with increased mRNA expression. These data show that
PAK1
expression is broadly up-regulated in breast cancer and that high expression
is correlated with
disease aggressiveness.
Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous
non-small cell
lung and selective PAK1 inhibition was correlated with delayed cell cycle
progression in vitro
and in vivo.
Expression of PAK1 protein was analyzed on tissue microarrays of 27 SCLCs and
97 NSCLCs,
the latter being comprised of 30 adenocarcinomas and 67 SCCs. 43/67 (64%)
squamous NSCLC
CA 02817133 2013-05-07
-13 -
WO 2012/065935
PCT/EP2011/070008
samples were positive for PAK1 expression and 52% of all cases showed staining
of moderate
(2+) or strong (3+) intensity in the malignant cells. Nuclear localization of
PAK1 was also
evident in a significant proportion of squamous NSCLC tumors (17/67; 25%). In
contrast to
squamous cell carcinoma, NSCLC adenocarcinoma (p=0.0008) and SCLC (p=0.003)
tumors
expressed only weak to moderate levels of PAK1 in the cytoplasm only. Adjacent
normal lung
tissue did not express appreciable levels of PAK1. Elevated PAK1 expression
was also
prevalent (79/130; 61%) in head-and-neck tumors, an additional indication of
squamous cell
carcinoma.
TABLE 1. Combination of PAK1 inhibition with ay small molecule panel
Single Combo
g Fold
agent PAKi- Mechanism of action and/or
approved
Compound change
EC50 EC50 use
in EC50
(mM) (mM)
Antagonist of inhibitor of apoptosis
BV6 20 0.35 57.54
(IAP) proteins
erlotinibl 20 1.56 5.02
Epidermal growth factor receptor
1
(EGFR) inhibitor for NSCLC
EGFR kinase inhibitor
gefitinib 2 20 3.3 12.8
Antagonist of IAP proteins
G-416 20 1.67 12
Inhibitor of MAPK/ERK kinase-1/2
U0126 19.46 2.3 8.45
(MEK1/2)
lapatinib 3 6.31 0.86 7.34
EGFR/HER2 inhibitor used for HER2-
positive breast cancer
dasatinib4 8.29 1.53 5.43 Dual BCR/ABL and Src family
kinase
inhibitor for CML
altretamine5 20 3.94 5.07 Alkylating chemotherapy used for
refractory ovarian cancer
oxaliplatin6 10.5 2.48 4.23 Platinum-based chemotherapy used
for
colorectal cancer
ZD64747 20 6.88 2.91 VEGF and EGF inhibitor in
clinical
development
Akt-1/2 kinase inhibitor
Akt inhibitor VIII 3.21 2.03 1.58
Trade Names: 1. Tarceva, 2. Iressa, 3. Tykerb, 4. Sprycel, 5. Hexalen,
6.Eloxatin, 7. Vandetanib
8. PAK1 knockdown shRNA and compound.
In head and neck tumors elevated PAK1 expression was also prevalent (79/130;
61%) in head-
and-neck tumors, another indication of squamous cell carcinoma (Table 1).
Anti-tumor efficacy of PAK1 inhibition in preclinical tumor models of squamous
NSCLC -
Inhibition of PAK1 expression using shRNA in NCI-H520.X1 and EBC-1 xenograft
models
resulted significant inhibition of tumor growth (Figure 4A and B). Following
tumor
establishment (200-250 mm3), animals were administered Dox in sucrose drinking
water and
CA 02817133 2013-05-07
WO 2012/065935 - 14 -
PCT/EP2011/070008
tumor growth was monitored for 21-24 days. For NCI-H520.X1 tumor-bearing
animals,
inhibition of PAK1, but not PAK2, significantly impaired tumor growth relative
to control
shLacZ mice as measured on the final day of dosing (Dunnett's t-test,
p<0.0001; Figure 4A).
Combined knockdown of PAK1 and PAK2 resulted in inhibition of tumor growth
that was
comparable to that of PAK1 inhibition alone (63.7% and 59.7%, respectively).
Equivalent
results were obtained using EBC-1 tumor xenografts and 66.8% inhibition of
tumor growth was
observed following in vivo knockdown of PAK1 (Figure 4B). Lastly, tumor
progression data for
both xenograft models were analyzed for the time required for tumor size to
double from the
onset of treatment. By this metric, PAK1 inhibition also resulted in a
significant anti-tumor
effect compared to the other cohorts (p<0.0001). Dox treatment was well
tolerated and no
animals exhibited any appreciable body weight loss. Analysis of xenograft
tumors by
immunohistochemistry revealed a substantial decrease in Ki-67 positive tumor
cells in Dox-
treated tumors expressing shPAK1 compared to shLacZ controls. The proportion
of Ki-67
positive nuclei was quantified and the anti-proliferative effect of PAK1
knockdown in vivo was
shown to be statistically significant (p<0.01; NCI-H520.X1 shLacZ 69 6%; NCI-
H520.X1
shPAK1 50 4%; EBC-1 shLacZ 91 3%; EBC-1 shPAK1 75 11%). PAK1 and PAK2
levels
were reduced by greater than 80% in Dox-treated tumors and PAK1 knockdown was
not
associated with decreased AKT activation as has been suggested. (T.C.
Hallstrom and J.R.
Nevins, Cell Cycle 2009 8:532-535.)
Analysis of cell lines with PAK1 genomic copy number gain revealed a
dependence on PAK1
expression and activity for cell survival. Inhibition of PAK1 catalytic
activity using IPA-3, an
allosteric inhibitor that prevents PAK1-3 activation by Rho family GTPases (J.
Viaud, J. and J.R.
Peterson, Mol. Cancer Ther. 2009 8:2559-2565), resulted in a pronounced
induction of apoptosis
as determined by fluorescence-activated cell sorting analysis for Annexin-
V/propidium iodide
staining of dying cells (7-fold increase; Figure 1D). This phenotype was
evident within 24-48 h
and was confirmed using selective, siRNA-mediated knockdown of PAK1 expression
(2-6-fold
increase in Annexin-V incorporation; Figure 1C). Cell death induced by PAK1
inhibition was
also associated with caspase activation, PARP cleavage and attenuated
phosphorylation of
MEK1-S298 and ERK1/2 (Figure 8). Hence, the strong induction of cell death
resulting from
PAK1 inhibition in breast cancer cells with PAK1 amplification suggests that
this kinase
contributes to the oncogenic phenotype, at least in part, by suppressing tumor
cell apoptosis.
The dependence on PAK1 suggests it may be an "Achilles' heel" for a
subpopulation of breast
cancer provides evidence of oncogene addiction (I.B. Weinstein and A. Joe,
Cancer Res. 2008
68:3077-3080) and a rationale for PAK1-directed therapy in this disease
indication.
CA 02817133 2013-05-07
WO 2012/065935 - 15 -
PCT/EP2011/070008
The aberrant cytoplasmic expression of PAK1 in greater than 50% of squamous
non-small cell
lung cancers and in head and neck squamous cell carcinoma further suggest they
also may be
dependent on PAK1 expression for continued growth and survival.
At present additional known genetic aberrations in squamous NSCLC include p53,
p1614a,
PTEN and LKB1 loss-of-function via mutation or methylation, and activating
mutations or
amplification of protein kinases, such as EGFR, MET, HER2 and PIK3CA. (R.S.
Herbst et al.,
N. Engl. J. Med. 2008 359:1367-1380) Thus inhibition of PAK1 enzymatic
activity or scaffold
function might combine synergistically with therapeutic agents that target
these critical growth
and survival pathways to increase anti-tumor efficacy and tumor cell death in
tumor cells that
) over-amplify or over-express PAK1. Such tumor cells include, but are not
limited to DCIS,
squamous NSCLC and head and neck SCC.
Inhibitors of PAK kinases have been described. (D. Bouzida et al W02006072831
published
7/13/2006; C. Guo et al., W02007023382 published 3/1/2007, L. Dong et al.,
W02007072153
published 6/30/2007; D. Campbell et al., W02010/071846 published 6/24/2010; K.
Daly et al.,
U520090275570 published 11/5/2009)
Combination of shRNA induced PAK1 knockdown with molecularly targeted
therapeutics
induces apoptosis of NSCLC cells - Small molecule library was screened to
identify potent
synergistic interactions between PAK1 antagonists and other anti-hyper-
proliferative agents
(Table 1).
A cellular viability screen was performed using EBC-1-shPAK1 isogenic cells
and a panel of
200 small molecule compounds that included Food and Drug Administration (FDA)
approved
oncology drugs, signaling pathway inhibitors and DNA damaging agents. Among
the tested
compounds, antagonists of inhibitor of apoptosis proteins (IAP; 12 and 57-
fold), epidermal
growth factor receptor (EGFR; 2.9, 7.4, 12.8 and 15-fold), MAPK/ERK kinase-1/2
(MEK1/2;
8.5-fold) and Src family kinases (5.4-fold) displayed dramatically enhanced
efficacy in
combination with PF-3758309 (Table 1). None of these agents demonstrated a
profound single
agent effect (EC50 > 6 M) on the growth and survival of EBC-1 cells in the
absence of PAK1
inhibition. Thus, PAK1 inhibition can greatly augment the efficacy of several
classes of well-
characterized molecularly targeted therapeutics.
Inhibitors of MEK kinase (S. Price, Expert Opin. Ther. Patents 2008 18(6):603-
626; E. M.
Wallace et al., Curr. Topics Med. Chem. 2005 5(2):215), Akt (C. Lindsley,
Curr. Top. Med.
CA 02817133 2013-05-07
WO 2012/065935 - 16 -
PCT/EP2011/070008
Chem. 2010 10:458-477; S.E. Ghayad and P.A. Cohen, Rec. Pat. Anti-Cancer Drug
Discov.
2010 5:29-57), Src (X.Cao et al., Mini-Rev. Med. Chem. 2008 8:1053-1063) and
Inibitor of
Apoptosis Proteins (IAP) (D. Vucic and W.J. Fairbrother, Clin. Cancer Res.
2007 13(20)5995;
A.D. Schimmer and S. Dalili, Hematology 2005 215) have been reviewed.
The prosurvival activity of IAP proteins is antagonized by the second
mitochondrial activator of
caspases (SMAC) (C. Du et al., Cell 2000 102:33-42; A.M. Verhagen, et al.,.
Cell 2000 102:43-
53) and a number of antagonists have been described that mimic SMAC amino-
terminal peptides
to disrupt the association of IAP with SMAC and activated caspase-9 ( K. Zobel
et al., ACS
Chem. Biol. 2006 1:525-533; E. Varfolomeev et al., Cell 2007 131:669-681). In
particular, BV6
(C. Ndubaku et al., Future Med. Chem. 2009 1(8):1509) represents one such
class of small
molecule antagonist that binds to baculovirus IAP repeat (BIR) domains and
promotes rapid
auto-ubiquitination and proteasomal degradation of c-IAP1 and cIAP-2 (Zobel
supra).
Consistent with the small molecule screening data, strong combinatorial
activity was confirmed
for dual inhibition of PAK1 and IAP in EBC-1 cells (Figure 5A). This dramatic
increase in BV6
potency on EBC-1-shPAK1 cells when co-treated with Dox (EC50 = 4.1 x 10-704)
relative to
controls (EC50 = 3.0 x 101 M) translated into a strong induction of cellular
apoptosis as
determined by Annexin-V flow cytometry assay (Figure 5B) and immunoblotting
for cleaved
caspases-3, 6, 7 and 9 (Figure 9). Importantly, evidence of enhanced cell
killing was also
observed using pharmacological inhibitors of PAK (IPA-3, PF-3758309) or Racl
(N5C23766)
catalytic activity (Figure 7A). To further explore this apparent synergy we
investigated possible
molecular mechanisms of PAK1 and IAP inhibition on the induction of apoptosis.
Combined
PAK1 and IAP inhibition did not involve either altered kinetics of IAP protein
degradation
induced by BV6 or increased autocrine signaling by TNFa. Lastly, additional
NSCLC cell lines,
including those that are only minimally responsive to the activity of either
single agent, were
examined for anti-tumor efficacy resulting from combined inhibition of PAK1
and IAP.
Transient siRNA-mediated knockdown of PAK1 expression followed by BV6
treatment resulted
in a significant reduction of SK-MES-1 and NCI-H441 cell viability (Figure
7B).
Combined inhibition of PAK1 and PAK2 with inhibitors of MEK (GDC-0623) and
PI3K (GDC-
0941; A.J. Folkes et al., J. Med. Chem. 2008 57:5522-5532) pathways was also
examined.
Combinatorial efficacy, as determined by reduced cellular viability (Figure
11) and induction of
apoptotic biomarkers (Figure 12), was observed following inhibition of PAK,
MEK and PI3K
signaling. Taken together, these studies provide strong preclinical support
that breast and
NSCLC may provide several opportunities for rational combination therapies
with PAK1
CA 02817133 2013-05-07
WO 2012/065935 - 17 -
PCT/EP2011/070008
inhibitors. In one embodiment of the present invention there is provided a
method of treating
tumors comprising contacting the tumor with a PAK 1 inhibitor and a second
anti-
hyperproliferative compound.
In another embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with a PAK 1 inhibitor and an inhibitor or
antagonist of EGFR,
the Raf/MEK/ERK pathway, Src, PI3K/AKT/mTOR pathway or inhibitor of apoptosis
proteins.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with a
PAK 1 inhibitor and a second anti-hyperproliferative compound.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of PAK1 comprising contacting the
tumor with a
PAK 1 inhibitor and an inhibitor or antagonist of EGFR, the Raf/MEK/ERK
pathway, Src,
PI3K/AKT/mTOR pathway or inhibitor of apoptosis proteins.
In one embodiment of the present invention there is provided a method of
treating tumors
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor comprising contacting the tumor with a PAK 1 inhibitor and a
second anti-
hyperproliferative compound.
In another embodiment of the present invention there is provided a method of
treating tumors
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor comprising contacting the tumor with a PAK 1 inhibitor and an
inhibitor or
antagonist of EGFR, the Raf/MEK/ERK pathway, Src, Akt or a inhibitor of
apoptosis proteins.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor that exhibits elevated levels of a PAK1 comprising contacting
the tumor with a
PAK 1 inhibitor and a second anti-hyperproliferative compound.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor that exhibits elevated levels of a PAK1 comprising contacting
the tumor with a
PAK 1 inhibitor and an inhibitor or antagonist of EGFR, the Raf/MEK/ERK
pathway, Src, Akt
or inhibitor of apoptosis proteins.
CA 02817133 2013-05-07
WO 2012/065935 - 18 -
PCT/EP2011/070008
N--NH
S N
HN Me
r (I)
HN
N Me (s) Ph
NMe2
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I (PF-3758309)
and a second
anti-hyperproliferative compound.
In another embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an
inhibitor or antagonist
of EGFR, the Raf/MEK/ERK pathway, Src, Akt or inhibitor of apoptosis proteins.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and a second anti-hyperproliferative compound.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of PAK1 comprising contacting the
tumor with the
compound of formula I and an inhibitor or antagonist of EGFR, the Raf/MEK/ERK
pathway,
Src, Akt or inhibitor of apoptosis proteins.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor that exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and a second anti-hyperproliferative compound.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor is breast tumor, a squamous non-small cell lung tumor or a
squamous head
and neck tumor that exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an inhibitor or antagonist of EGFR, the Raf/MEK/ERK
pathway,
Src, Akt or inhibitor of apoptosis proteins.
CA 02817133 2013-05-07
WO 2012/065935 - 19 -
PCT/EP2011/070008
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an
inhibitor of inhibitor of
apoptosis proteins.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an inhibitor of inhibitor of apoptosis proteins.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and BV6 or
G24416.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and BV6 or G24416.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an EGFR
inhibitor
antagonist.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an EGFR inhibitor or antagonist.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and erlotinib,
gefitinib or
lapatinib.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and erlotinib, gefitinib or lapatinib.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an
inhibitor of the
Ras/Raf/MEK/Erk signaling cascade.
CA 02817133 2013-05-07
WO 2012/065935 - 20 -
PCT/EP2011/070008
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an inhibitor of the Ras/Raf/MEK/Erk signaling
cascade.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an
inhibitor of Akt kinase.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an inhibitor of Akt kinase.
In one embodiment of the present invention there is provided a method of
treating tumors
comprising contacting the tumor with the compound of formula I and an
inhibitor of Src kinase.
In another embodiment of the present invention there is provided a method for
treating a tumor
wherein said tumor exhibits elevated levels of a PAK1 comprising contacting
the tumor with the
compound of formula I and an inhibitor of Src kinase.
In another embodiment of the present invention there is provided a method of
treating a patient
suffering from a cancer or a hyperproliferative disorder comprising co-
administering to a patient
in need thereof a PAK 1 inhibitor and a second anti-hyperproliferative agent.
In another embodiment of the present invention there is provided a method of
treating a patient
suffering from a cancer or a hyperproliferative disorder comprising co-
administering to a patient
in need thereof a PAK 1 inhibitor and an inhibitor or antagonist of EGFR, the
Raf/MEK/ERK
pathway, Src, Akt or inhibitor of apoptosis proteins.
In another embodiment, the present invention provides a combination of a PAK1
inhibitor with a
second anti-hyperproliferative compound for the treatment of tumors.
In another embodiment, the present invention provides a co-administration of a
PAK 1 inhibitor
CA 02817133 2013-05-07
WO 2012/065935 - 21 -
PCT/EP2011/070008
In another embodiment, the present invention provides the use of a combination
of a PAK1
inhibitor with a second anti-hyperproliferative compound for the preparation
of a medicament
for the treatment of tumors .
In another embodiment, the present invention provides the use of a PAK 1
inhibitor and a second
anti-hyperproliferative agent for the preparation of a medicament for the
treatment of a cancer
or a hyperproliferative disorder.
The following examples illustrate the biological evaluation of compounds
within the scope of the
invention. These examples which follow are provided to enable those skilled in
the art to more
clearly understand and to practice the present invention. They should not be
considered as
limiting the scope of the invention, but merely as being illustrative and
representative thereof
Example 1
Tissue samples
Formalin-fixed paraffin-embedded tissue blocks and corresponding pathology
reports were
obtained for 97 sequential NSCLC and 27 sequential SCLC, 130 head and neck
SCC, 15 DCIS,
226 primary breast cancers and 32 breast cancer lymph node metastases (John
Radcliffe
Hospital, Oxford, UK). Tissue microarrays (TMAs) were assembled as described
previously (L.
Bubendorf, et al., J. Pathol. 2001 195:72-79.).
For the sequential patients with breast adenocarcinoma, surgery was performed
between 1989
and 1998, and patients were treated with a wide local excision and
postoperative radiotherapy or
mastectomy with or without postoperative radiotherapy. Patients received
adjuvant
chemotherapy and/or adjuvant hormone therapy, or no adjuvant treatment.
Tamoxifen was used
as endocrine therapy for 5 years in estrogen receptor (ER) positive patients.
Patients who were
<50 years of age, with lymph node positive tumors, or ER¨ and/or a primary
tumor >3 cm in
diameter, received adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil
(CMF) for six
cycles, in a three weekly intravenous regimen. Patients >50 years of age with
ER¨, lymph node¨
positive tumors also received CMF. Estrogen receptor (ER) content was
determined using an
enzyme-linked immunosorbent assay technique (Abbott Laboratories, Abbott Park,
IL). Tumors
were considered positive when cytosolic ER levels were >10 fmol/mg of total
cytosolic protein.
HER2 status was assessed with the HercepTest (DAKO, Carpinteria, CA). Receptor
values were
monitored by participation in the EORTC quality control scheme.
The operable NSCLC series comprised surgical resection specimens from 30
adenocarcinomas
and 67 squamous cell carcinomas (surgery was performed from 1984 to 2000).
Clinical and
CA 02817133 2013-05-07
WO 2012/065935 - 22 -
PCT/EP2011/070008
pathological data were available for 75 cancers. Thirty-five cases (47%) were
stage T1 and 40
cases (53%) were stage T2. Fifty-three cases (71%) were stage NO and twenty-
two cases (29%)
were stage N1. Patients did not receive adjuvant chemotherapy and information
regarding
radiotherapy was not available.
The head and neck squamous cell carcinoma series comprised surgical resection
specimens from
11 oropharyngeal cancers, 27 cancers arising in the oral cavity, 17 laryngeal
cancers and 75
hypopharyngeal cancers (definitive surgery was performed from 1995 to 2005).
Nine cancers
were UICC stage 1, 16 were stage 2, 29 were stage 3 and 76 were stage 4. Post-
operatively 108
patients (83%) received radiotherapy.
Example 2
Genome copy number and expression analysis
For the Affymetrix 500K SNP array analysis, genomic DNA preparation, chip
processing and
data analysis were performed as published previously. (P.M. Haverty et al.,
Genes Chromosomes
Cancer 2008 47:530-542) Regions of significant gains or losses were identified
using the
GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm (R.
Beroukhim, et
al., Proc Natl Acad Sci U S A 2007 104:20007-20012). To collect expression
array data for
matched tumor samples, RNA was extracted from frozen tissue from 88 cases of
the primary
breast cancer series and applied to Affymetrix (Santa Clara, CA) HGU133 gene
expression
microarrays. Gene probe intensity data were used to subclassify the tumors
into basal, luminal-
A, luminal-B, Her2 and normal types according to published criteria (C.M.
Perou et al., 2000
Nature 2000 406:747-752). The 226507 at probeset was chosen to represent PAK1
mRNA
expression.
Example 3
Cell culture and viability assays.
Cell lines were acquired from either the Health Science Research Resources
Bank (HSRRB,
Japan) or 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. For
analysis of cell proliferation by thymidine incorporation into DNA, cells were
incubated with 1
Ki/well [3H]thymidine for 18 h and harvested onto Unifilter GF/C plates using
a Filtermate TM
196 harvester (Perkin Elmer, Waltham, MA). MicroScint TM 20 liquid
scintillation cocktail was
added to the dried filter plates that were subsequently sealed and counted in
a TopcountTm
(Perkin Elmer, Waltham, MA). For cell cycle analysis via flow cytometry, cells
at a density of 1
CA 02817133 2013-05-07
WO 2012/065935 - 23 - PCT/EP2011/070008
x 106 were fixed in 70% ice-cold ethanol for 1 hour and then washed with PBS
and incubated in
propidium iodide (PI) solution (0.05 mg/ml RNase solution (Sigma, St. Louis,
MO), 0.05mg/m1
PI (Sigma, St. Louis, MO), in PBS) for 3 hours at 4 C. Cells were immediately
analyzed with a
FacScan flow cytometer (Becton Dickinson, San Jose, CA).
CA 02817133 2013-05-07
WO 2012/065935
PCT/EP2011/070008
24
To ascertain the role of PAK1 in cell survival, the quantity of cytoplasmic
histone-
associated DNA fragments was quantified using the Cell Death Detection ELISA
Plus kit
from Roche (Mannheim, Germany). Alternatively, for cell death analyses via
flow
cytometry, cells were collected by centrifugation and stained with Annexin
V¨FITC and
PI solution (BD Biosciences, San Jose) according to the manufacturer's
instructions.
CA 02817133 2013-05-07
WO 2012/065935
PCT/EP2011/070008
For the pharmarray viability screen using a 200 compound library, EBC1-shPAK1
cells were
cultured in complete growth medium and either untreated or treated with 300
ng/mL doxycycline
for 3 days prior to compound addition. Cells were then replated at appropriate
density in 384-
well plates and treated with 6 concentrations (4-fold serial dilutions from 10
uM) of each
5 compound for 72 hr treatment. Cell viability was assessed via ATP content
using the CellTiter-
Glo Luminescent Assay (Promega, Madison, WI). Cell growth inhibition and EC50
differences
were determined for PAK1 knockdown and wild-type cells.
Example 4
RNA interference and generation of inducible-shRNA cell pools.
10 Short interfering RNA (siRNA) oligonucleotides for PAK1 and PAK2 were
obtained
from Dharmacon RNAi Technologies (Chicago, IL). Short-hairpin RNA
oligonucleotides used in this study are as follows: LacZ shRNA (sense) 5'-CTT
ATA
AGT TCC CTA TCA GTG ATA GAG ATC CCC AAT AAG CGT TGG CAA TTT
ATT CAA GAG ATA AAT TGC CAA CGC TTA TTT TTT TTG GAA-3', LacZ
15 shRNA (antisense) 5'-TTC CAA AAA AAA TAA GCG TTG GCA ATT TAT CTC
TTG AAT AAA TTG CCA ACG CTT ATT GGG GAT CTC TAT CAC TGA TAG
GGA ACT TAT AAG-3', PAK1 shRNA-1 (sense) 5'-GAT CCC CGA AGA GAG GTT
CAG CTA AAT TCA AGA GAT TTA GCT GAA CCT CTC TTC TTT TTT GGA AA-
3', PAK1 shRNA-1 (antisense) 5'-AGC TTT TCC AAA AAA GAA GAG AGG TTC
20 AGC TAA ATC TCT TGA ATT TAG CTG AAC CTC TCT TCG GG-3', PAK2
shRNA-3 (sense) 5'-GAT CCC CCT GCA TAA CCT GAA TGA AAT TCA AGA GAT
TTC ATT CAG GTT ATG CAG TTT TTT GGA AA-3', PAK2 shRNA-3 (antisense) 5'-
AGC TTT TCC AAA AAA CTG CAT AAC CTG AAT GAA ATC TCT TGA ATT
TCA TTC AGG TTA TGC AGG GG-3'. Inducible-shRNA bearing lentivirus constructs
25 were made based on previously described methods (K.P. Hoeflich et al.,
Cancer Res.
2006 66:999-1006; J. Climent et al., Biochem. Cell Biol. 2007 85:497-508.) by
co-
transfecting pHUSH-Lenti-GFP and/or pHUSH-Lenti-dsRed constructs containing a
desired shRNA with plasmids expressing the vesicular stomatitis virus (VSV-G)
envelope glycoprotein and HIV-1 packaging proteins (GAG-POL) in HEK293T cells
using LipofectamineTM (Invitrogen, Carlsbad, CA). Target cells were transduced
with
CA 02817133 2013-05-07
WO 2012/065935
PCT/EP2011/070008
26
these viruses and sterile sorted (top 2-5%) by flow cytometry for presence of
dsRed or
GFP or both. Cells were characterized for doxycycline-inducible protein
knockdown by
western blot analysis.
Example 5
Immunoblotting and immunofluorescence.
Frozen tumors were 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 mIVI
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). Lysates were
subjected to
centrifugation at 16,100 g for 15 minutes and protein concentration was
determined using
the BCA protein assay (Pierce Biotechnology, Rockford, IL). 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 primary antibodies for PAK1, p27, E2F1 (Cell Signaling Technology,
Danvers,
MA) and anti-f3-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies
were
obtained from Pierce Biotechnology (Rockford, IL).
Immunoflourescence imaging was performed using primary antibodies for p27KT1
(Becton Dickinson, San Jose, CA). Secondary antibodies were obtained from
Millipore
Corporation (Billerica, MA). Images were analyzed in Metamorph (version
7.5.3.0, MDS
Analytical; Sunnyvale, CA) using an automated analysis routine. Briefly, a
smoothing
filter was applied to the DAPI channel to even out the nuclear staining
pattern. The
MVVCS application in Metamorph was then used to identify and count DAPI
stained
nuclei, and classify them as positive or negative for p27 in the Cy3 channel.
Example 6
Tumor xenograft models
Cultured NCI-H520.X1 and EBC1 cells were removed from culture, suspended in
Hank's
buffered saline solution (HMS), mixed 1:1 with Matrigel (BD Biosciences, USA),
and
CA 02817133 2013-05-07
WO 2012/065935
PCT/EP2011/070008
27
implanted subcutaneously into the right flank of naïve female NCR nude mice
(Taconic
Farms, Hudson, NY). Mice with tumors of a mean volume of approximately 250 mm3
were grouped into treatment cohorts of 10 mice each. Mice received 5% sucrose
only or
5% sucrose plus 1 mg/ml doxycycline (Clontech, Mountain View, CA) for control
and
knockdown cohorts, respectively. All water bottles were changed 3 times per
week.
Body weights and tumor volume measurements (as obtained by length and width
measurements with calipers) were taken twice per week during the study. 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.
Tumor volumes were calculated by the following formula: Tumor Volume = 0.5 *
(a *
b2), where 'a' is the largest tumor diameter and '13' 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).
Xenograft tissues were fixed for 24 h in 10% neutral buffered formalin and
were then
processed and paraffin embedded. Sections were cut at a thickness of 3 [tm,
and
specimens with sufficient viable tumor (assessed on H&E-stained slides) were
further
evaluated by immunohistochemistry. Anti¨Ki-67 (clone 1VIIB- 1, mouse anti-
human) was
used with the DAKO ARK Kit for detection. Tissues were counterstained with
hematoxylin, dehydrated, and mounted. Antigen retrieval was done with the DAKO
Target Retrieval Kit as per manufacturer's instructions. For quantification of
immunohistochemically Ki-67¨positive cells, images were acquired by the Ariol
SL-50
automated slide-scanning platform (Genetix Ltd.) at x100 final magnification.
Tumor-
specific areas were identified manually for analysis in the Ariol software. A
3,3'-
diaminobenzidine¨specific color range was specified using the hue, saturation,
and
intensity color space to quantify the area of staining and the output was
total Ki-67¨
positive cells in relation to total cell count.
CA 02817133 2013-05-07
WO 2012/065935
PCT/EP2011/070008
28
The features disclosed in the foregoing description, or the following claims,
expressed in
their specific forms or in terms of a means for performing the disclosed
function, or a
method or process for attaining the disclosed result, as appropriate, may,
separately, or in
any combination of such features, be utilized for realizing the invention in
diverse forms
thereof
The foregoing invention has been described in some detail by way of
illustration and
example, for purposes of clarity and understanding. It will be obvious to one
of skill in
the art that changes and modifications may be practiced within the scope of
the appended
claims. Therefore, it is to be understood that the above description is
intended to be
illustrative and not restrictive. The scope of the invention should,
therefore, be
determined not with reference to the above description, but should instead be
determined
with reference to the following appended claims, along with the full scope of
equivalents
to which such claims are entitled.
The patents, published applications, and scientific literature referred to
herein establish
the knowledge of those skilled in the art and are hereby incorporated by
reference in their
entirety to the same extent as if each was specifically and individually
indicated to be
incorporated by reference. Any conflict between any reference cited herein and
the
specific teachings of this specifications shall be resolved in favor of the
latter. Likewise,
any conflict between an art-understood definition of a word or phrase and a
definition of
the word or phrase as specifically taught in this specification shall be
resolved in favor of
the latter.
* * * * * *
