PAK4 Allosteric Modulators (PAMs) Repress the Wnt/β-catenin Signaling Pathway and Tumor Growth
William Senapedis, Scott Donovan, Gali Golan, Dilara McCauley, Joel Ellis, Marsha Crochiere, Trinayan Kashyap, Boris Klebanov, Sharon Shacham, Yosef Landesman and Erkan Baloglu
Karyopharm Therapeutics, Newton, MA, USA
0 .8
In vitro MTS IC50 curves from
>150 solid and heme cancer
cell lines
Mutational analysis of cancer
genes from CCLE compared
to PAM sensitivity
40
50 mg/kg 150 mg/kg
KPT-9274 KPT-9274
Migration
Cyclin D1
Phos-LRP6
LRP6
Dvl2
Phos-S675
β-catenin
Wnt
4EBP1
Caspase-3
Caspase-8
Apoptosis
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b
Vehicle
50 mg/kg
KPT-9274
75 mg/kg
KPT-9274
Phos-Pak4
Pak4
Vinculin
β-Actin
GEFH1
Migration
Phos-Cofilin
Cofilin
Cyclin D1
50 mg/kg
QDx5
Phos-LRP6
LRP6
5 mg/kg
QDx5
Phos-4EBP1
Figure 6: PAMs show anti-tumor activity in colon cancer. (a) Mice were inoculated on the hind flank with tumor
cells. Mice began treatment when average Colo-205 tumor volume reach ~100 mm3 per group (n = 10 animals/dose
group). Mice were treated either with vehicle or oral PAM (KPT-9274) twice daily (5 days/week) without major toxicity
(minimal or no side effects or weight loss). (b) Tumors were collected 2 hours post-dose after 3 weeks of dosing with
oral KPT-9274. Frozen tumors were grounded up and lysed in a Tissue Lyser (Qiagen) using the T-PER buffer
(Pierce). (c) Colo-205 tumors were collected and formalin fixed 2 hours post-dose. Cellular biomarkers of PAK4 and
Wnt/β-catenin signaling were dramatically changed after treatment with oral KPT-9274. There was a reduction in
proliferation (Ki67) and an increase in apoptosis (Apoptag).
Proliferation
4EBP1
10 mg/kg
QDx5
Apoptosis
Caspase-8
PAK4
GEF-H1
β-catenin
Cyclin D1
Ki67
Apoptag
Figure 7: PAMs show anti-tumor activity in Prostate cancer in nude rats. Nude rats were inoculated on the hind
flank with PC3 tumors. (a) Rats began treatment when average tumor volume reach ~100 mm3 (n = 8/group). Rats
were treated either with vehicle or oral PAM (KPT-9274) at 5 and 10 mg/kg daily (5 days/week) with dose
escalations as marked. Tumor regression occurred after escalating to 50 and 75 mg/kg BIDx5 with stable disease
achieved with 10 and 50 mg/kg QDx5. (b) Tumors were collected 2 hours post-dose at the end of the study. Frozen
tumors were grounded up and lysed. (c) IHC staining of formalin fixed tumors from (b).
3
M e a n T u m o r V o lu m e ( m m )
V e h ic le (n = 1 2 )
K P T -9 2 7 4 (1 0 0 m g /k g B ID x 5 ; n = 1 1 )
4000
3000
2000
1000
0
10
20
30
D a y s o f T re a tm e n t
Vehicle
Pak4
100 mg/kg
KPT-9274
40
d
H&E
0
5
10
D a y s o f T re a tm e n t
PAK4
GEF-H1
Paxillin
15
Vinculin
Migration
Paxillin
Vinculin
Cyclin D1
Phos-LRP6
LRP6
Cyclin D1
Wnt
Dvl2
β-catenin
Phos-4EBP1
Proliferation
4EBP1
Bcl-2
Apoptosis
Caspase-8
Dvl2
Ki67
Apoptag
KPT-9274 Vehicle
0
Figure 5: PAMs show anti-tumor activity in MCL. Mice were inoculated on the hind flank with tumor cells. Mice began treatment
when average Z-138 tumor volume reach (a) ~300 or (b) 1500 mm3 per group (n = 8-12 animals/dose group). Mice were treated
either with vehicle or oral PAM (KPT-9274) twice daily (5 days/week). (a) Tumor regression(“cures”) occurred after ~2 weeks of
dosing. After stopping treatment on Day 21, 8 out of 8 animals remained tumor free for more than 2 weeks before euthanasia. (b)
Large tumors regressed to near full elimination after ~2 weeks of dosing. (c) Tumors were collected 2 hours post-dose of 100 mg/kg
oral KPT-9274 administered for ~1 week. Frozen tumors were grounded up and lysed in a Tissue Lyser (Qiagen) using the T-PER
buffer (Pierce). (d) Formalin fixed tumors from (c) were processed and stained for immuno-histochemistry. Cellular biomarkers of
PAK4 and Wnt/β-catenin signaling were dramatically changed after treatment with oral KPT-9274. There was a reduction in
proliferation (Ki67) and an increase in apoptosis (Apoptag).
Conclusions
Wnt
Dvl2
β-catenin
50 mg/kg
BIDx5
H&E
500
Cofilin
Vehicle
Dvl2
1000
GEFH1
Phos-Cofilin
a
Cyclin D1
K P T - 9 2 7 4 ( 1 0 0 m g /k g P O B ID ; n = 8 )
β-Actin
PAMs Display In Vivo Efficacy in PC3 Xenograft in Rats
Paxillin
V e h ic le ( n = 8 )
3
c
m
g
m /k
g g
m /k
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m /k
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/
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g
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/
m kg
g
m /k
g g
m /k
g g
m /k
g g
m /k
g g
/k
g
Apoptag
5000
1500
0
-5 0
Vehicle
Proliferation
-2 5
Figure 4: PAMs show anti-tumor activity in vivo. Both solid and hematological xenografts in mice show dose
response to KPT-9274 oral treatment. Mice were inoculated with different cancer cell lines in the hind flank. When
the tumor reached 100 – 200 mm3 vehicle or oral KPT-9274 treatment was begun. Mice were treated with various
amount of PAM at BIDx5 per week for ~4 weeks. The best response was used to calculated growth repression or
regression of tumors when compared to vehicle tumors. The hematological tumors showed high levels of regression
with several or all animals “cured” of tumors. The solid tumors scored high for growth repression.
KPT-9274
Phos-4EBP1
H 5 2 0 (lu n g )
75 mg/kg
BiDx5
β-catenin
β-catenin
C o lo -2 0 5 ( c o lo n )
0
Vehicle
Phos-Cofilin
Cofilin
Z -1 3 8 (M C L )
5
0
7
1 5
0
1 0
5
2 0
5
0
5
0
7
1 5
0
1 0
5
2 0
5
0
GEF-H1
GEFH1
H e p 3 B (liv e r)
25
KPT-9274
Vehicle
M M 1 .S (m y e lo m a )
Vehicle
30
M D A - M B - 2 3 1 ( b re a s t)
PAMs Display In Vivo Efficacy in Z-138 with a Reduction in Biomarkers
b
Vehicle
50
M o lt4 (A L L )
a
M e a n T u m o r V o lu m e ( m m )
75
M D A - M B - 4 6 8 ( b re a s t)
KPT-9274
20
1
C
100
D o s in g (B ID x 5 )
Vehicle
10
M
Orally Bioavailable PAMs Display Broad In Vivo Efficacy
PAK4
KPT-9274
0
2
Figure 3: KPT-9274, PAM clinical candidate, displays broad anti-tumor activity in hematological and solid cancer cells. (a) KPT-9274 inhibits the growth of hematological cancer cell lines in a 72 hour MTS assay (Cell-Titer AQueous One). Of 99 cell lines
tested, 89 cell lines had IC50 values < 500 nM (90%). (b) Z-138 (MCL) cancer cells were treated with various amounts of KPT-9274 (IC50 = ~10 nM) for 72 hours then lysed for western blot analysis. Treatment of cells with KPT-9274 showed decreases in PAK4
protein and signaling along with key downstream effectors of cell cycle (cyclin D1), cell migration (GEF-H1), and Wnt signaling (β-catenin, Dvl2, and LRP6). (c) Z-138 and Colo-205 cells were treated with 1 µM KPT-9274 for 48 hours then total RNA was collected.
RT-PCR was analyzed by cDNA creation then measured by Taqman probes using Via VII from Invitrogen. KPT-9274 treatment increases the PDn marker TEX29 transcripts in both cells lines and decreases ASB2 in Colo-205. There is also a marked repression of
β-catenin responsive genes such as WNT5A, WNT10A, KREMEN1, and cyclin D1. The overall effects on mRNA expression are more significant in the Colo-205 then at Z138 at 48 hours.
-1 0 0
Ki67
500
Cyclin D1
-7 5
KPT-9274
1000
M
K
N
O
0 .0
Vehicle
3
M e a n T u m o r V o lu m e (m m )
1500
Phos-Pak4
Pak4
β-Actin
Figure 1: PAK4 Allosteric Modulators inhibit cancer cell growth. A novel class of PAK4 inhibitors
display anti-tumor activity in vitro while sparing normal (NIH 3T3). Cells were assessed by MTS assay 72
hours after treatment with various structural analogs of the early lead compound, KPT-6604. The clinical
candidate compound is KPT-9274.
Contact Information: Dr. William Senapedis
e-mail: [email protected] T: +1 617 658 0524
H&E
Dvl2
5
0
7
1 5
0
1 0
5
2 0
5
0
5
0
7
1 5
0
1 0
5
2 0
5
0
5
0
7
1 5
0
1 0
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2 0
5
0
5
0
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1 5
0
1 0
5
2 0
5
0
5
0
7
1 5
0
1 0
5
2 0
5
0
V e h ic le ( n = 1 0 )
Wnt5a/b
5
0
7
1 5
0
1 0
5
2 0
5
0
c
2000
LRP6
β-Catenin
PAMs Display In Vivo Efficacy in Colo-205 with a Reduction in Biomarkers
a
Axin1
0 .2
( % o f V e h ic le )
Figure 2: Bioinformatics analysis predicts
mutations in the Ras and Wnt signaling
pathways influence sensitivity to PAMs. (a)
KPT-8752 inhibits the proliferation of cancer
(blue bars) cell lines in a 72 hour MTS assay
(Cell-Titer AQueous One, Promega). 45
hematological and 109 solid cancer cell lines
were tested. 42 heme and 54 solid cell lines,
respectively, had IC50 < 500 nM to KPT-8752.
On the other hand, KPT-8752 had minimal
effect on normal cells (orange bars). (b)
Bioinformatics revealed that sensitivity to PAMs
was directly correlated with mutations in APC or
N-ras and inversely correlated with mutations in
β-catenin, K-ras, or PI3K. (c) Z138 cells were
treated with KPT-9274 for 48 hours then
collected. PTScan which uses phosphodirected antibodies and mass spectrometry
(Cell Signaling) reveals effects on growth
pathways as well as specific influence on PAK
and Wnt signaling after PAM treatment.
Caveolin-1
Wnt3a
T u m o r G ro w th
M
c
β-actin
0 .4
Conclusion
Wild-type K-Ras, PI3K, or βcatenin correlate with in vitro
sensitivity to PAMs
Mutant
APC
or
N-Ras
correlate
with
in
vitro
sensitivity to PAMs
Solid
85% cell lines
IC50 < 500 nM
(41/48)
Phos-LRP6
S
0 .2
b
Proliferation
0 .6
Data
0 .4
Heme
94% cell lines
IC50 < 500 nM
(48/51)
M
0 .6
Solid
50% cell lines
IC50 < 500nM
(54/109)
GEF-H1
IC5500 ((µM)
M )
IC
Heme
93% cell lines
IC50 < 500nM
(42/45)
D a y s o f T re a tm e n t
Cell
Migration
Phos-Pak4
Pak4
0 .8
0 .0
c
Z-138 in vitro
KPT-9274:
1 .0
0
AntiApoptotic
5
1 .0
K P T - 9 2 7 4 ( 1 5 0 m g /k g P O B ID x 5 )
Cell
Cycle
b
10
5
K P T - 9 2 7 4 ( 1 0 0 m g /k g P O B ID x 5 )
Focal
Adhesion
a
b
10
K P T - 9 2 7 4 ( 5 0 m g /k g P O B ID x 5 )
Survival
Clinical Candidate, KPT-9274, Potently Cytotoxic to Cancer Cells and Inhibits PAK4 and Wnt/β-catenin Signaling In Vitro
KPT-9274
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(m H F
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)
Background: Wnt/β-catenin signaling is a ubiquitous pathway conserved throughout
evolution and plays a role in embryonic and cancer development. However, this pathway
has proven to be difficult to target therapeutically. Another intractable target is oncogenic
Ras which is highly mutated in many cancer types, including pancreatic, colon and lung. An
available target at the intersection of both of these genes is p21-Activated Kinase 4 (PAK4).
PAK4 (1 of 6 PAK proteins) that is downstream of the Ras oncogene and a direct kinase
that stabilizes transcriptional activity of β-catenin. Therefore therapeutically targeting PAK4
could be beneficial in a broad range of cancer types. PAK4 Allosteric Modulators (PAMs)
represent a novel and selective class of compounds that inhibit PAK4 allosterically.
Methods: Flow cytometry and CellTiter AQueous One assay (MTS) were used to
determine compound effects on cell cycle distribution and proliferation. CCLE, COSMIC
and other databases were used for bioinformatics analysis of mutations in cancer genes.
Deep sequencing and qPCR were used to analyze mRNA expression profiles. Proteomic
platforms – KinomeScan, PTScan (Cell Signaling) and immunoblots were used to study
phosphorylation signatures of whole cell proteins and total protein steady state levels.
Results: We identified selective, orally bioavailable small molecules, (PAMs; KPT-6604,
KPT-7523, KPT-8752 and KPT-9274) which demonstrated anti-tumor activity in a variety of
cancer cell lines (IC50 values from 0.005 – 1 µM). Bioinformatics revealed that sensitivity to
PAMs was directly correlated with mutations in APC or N-ras and inversely correlated with
mutations in β-catenin, K-ras, or PI3K. KPT-9274 and other PAMs reduced phosphorylation
and steady state levels of PAK4 protein while reducing Phospho-S675 and total β-catenin,
Wnt5, Phospho- and total LRP6, Dvl2, and Axin1. PAMs also reduced β-catenin
transcriptional activity (i.e. CCND1, WNT5A, and WNT10A). PAMs arrested cancer cell
cycle at the G1 and G2 phases and induced apoptosis through Caspase and PARP
cleavage. KPT-9274 (~100 mg/kg BIDx5 orally) has demonstrated potent anti-tumor activity
against hematological (Z-138, Molt-4, MM1S) and solid (MDA-MB-231, MDA-MB-468,
H520, Hep 3B and Colo-205) xenograft models in mice. PAM-treated xenografts showed
reduction of PAK4, β-catenin and cyclin D1 proteins.
Conclusions: PAK4 represents a novel anti-cancer target at the crossroads of Ras and
Wnt/β-catenin signaling. We have identified selective small molecule PAMs with anti-tumor
activity both in vitro and in vivo. These allosteric modulators induce tumor cell growth arrest
and apoptosis. Bioinformatics helped identify potential predictive markers in the Wnt
signaling pathway while deep sequencing and proteomics revealed possible PDn markers.
Based on the in vitro and in vivo activity, KPT-9274 may be beneficial for the treatment of a
wide variety of cancers and preclinical toxicology studies are ongoing.
Bioinformatics Analysis Suggest PAM In Vitro Sensitivity is Predicted
to be though Ras and Wnt Pathways
IC
(µM)
IC 50
50 ( M )
Abstract
 PAMs
demonstrate anti-tumor activity across a broad range of
hematological and solid malignancies in vitro while sparing normal
cells.
 PAMs
decrease PAK4 protein levels and signaling pathways of treated
 PAMs
decrease activity of Wnt/β-catenin both in vitro and in vivo.
cells.
 PAMs
are orally bioavailable and display anti-tumor activity in
hematological and solid xenograft mouse and rat models with
excellent tolerability.
 KPT-9274
is our clinical candidate with IND expected in 2H2015