Download FAK Inhibitor VS-4718 Attenuates Breast Cancer Stem Cell Function

FAK Inhibitor VS-4718 Attenuates Breast Cancer Stem Cell
Function in vitro and in vivo
P6-11-09
Vihren N. Kolev, Quentin G. Wright, Christian M. Vidal, Irina M. Shapiro, Mahesh V. Padval,
Mitchell Keegan, Qunli Xu and Jonathan A. Pachter
Verastem, Inc., Cambridge, MA
RESULTS
ABSTRACT
Fig 3: VS-4718 preferentially reduces CSCs in xenograft tumors in vivo
Control
Fig 1: FAK is important for the self renewal of CSCs in vitro
Aldefluor FACS
VS4718
Re-implantation in limiting dilutions
100 mg/kg, ip, bid x 9
Primary tumorsphere
A.
Secondary tumorspheres
200
600
C o lo n y # p e r 1 0 k c e lls
C o lo n y # p e r 1 0 k c e lls
800
P = 0.05
*
400
200
150
100
P = 0.015
0
B.
Fig 2: FAK inhibitor VS-4718 reduces the proportion of CSCs in Aldefluor and
Hoechst dye exclusion assays
A.
125
100
Control
VS-4718
VS-4718
1 .0
0 .5
0 .0
V S -4 7 1 8
Tumor Initiation
75
500 k
50k
5k
Tumor Ini.
Probability
50
25
0
10
100
Vehicle
6/6
5/6
1/6
VS-4718
5/6
2/6
0/6
1:28,000
1:230,000
Vehicle
VS-4718
1000
Tumor bearing mice were treated with VS-4718 at the indicated schedules. Tumors from Sum159 xenograft model
were dissociated and subject to tumorsphere assays (A) and Aldefluor FACS analysis (B). Frozen sections of harvested
tumors were also prepared and subject to ALDH1 immunofluorescence analysis (C). Cells from dissociated MDA-MB231 tumors were xenografted in limiting dilutions in SHrN mice (D).
200
Fig 4: Potent in vivo antitumor activity of VS-4718
100
V e h ic le , B ID x 2 8 d a y s
800
V S -4 7 1 8 2 5 m g /k g , B ID x 2 8 d a y
0
DM SO
D EA B
10 n M
30 n M
100 n M
Paclitaxel
Control
1 mM VS-4718
30 n M
100 n M
300 n M
Cisplatin
50 μM Verapamil
T u m o r W e ig h t (m g )
(% o f C o n tr o l)
A ld e f lu o r - P o s it iv e C S C s
Control
1 .5
D.
300
B.
VS-4718
C.
C o n tro l
VS-4718, nM
FAK has been implicated in the self-renewal of cancer stem cells (CSC) and breast
cancer development
A ld e f lu o r - P o s it iv e C S C s ( % )
Control
MDA-MB-231 cells were treated with VS-4718 in tumorsphere formation assay. VS-4718 inhibited sphere forming
efficiency of MDA-MB-231 breast cancer cells in a dose-dependent manner.
*
50
0
INTRODUCTION
 Inactivation of FAK or b1 integrin compromised mammary CSC self renewal (Taddei, Nature Cell Biol 2008)
 In the MMTV-PyMT model, targeted deletion of FAK in mouse mammary epithelium reduced the number & self
renewal capability of cancer stem/progenitor cells & impaired tumor growth (Luo, Cancer Res 2009)
 FAK amplification correlates with poor survival of breast cancer patients (Pylayeva, JCI 2009)
 Integrin b1 – FAK signaling is critical for proliferation of micro-metastatic breast cancer cells in the lung (Shibue
& Weinberg, PNAS 2009)
Liberase
ALDH1 staining
Aldefluor-Positive CSCs
(% of Control)
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates signal transduction by
integrins as well as growth factor receptors. FAK has been implicated in multiple steps of
carcinogenesis including tumor initiation, growth and metastasis. Amplification and overexpression
of FAK have been observed in aggressive human cancers including breast cancer. We have now
observed that VS-4718, a selective FAK kinase inhibitor, exhibits preferential inhibitory effects on
breast cancer stem cells.
VS-4718 is a potent and selective FAK kinase inhibitor that blocks fibronectin-stimulated FAK
autophosphorylation at Tyr397 at low nanomolar concentrations. To determine if FAK plays a role
in the biology of breast cancer stem cells, VS-4718 was evaluated in a multitude of cancer stem cell
assays both in vitro and in vivo. Treatment of SUM159 triple negative breast cancer cells in vitro
with FAK shRNA inhibits tumorsphere formation, and therefore indicates a role of FAK in breast
cancer stem cell renewal. Similarly, pre-treatment of SUM159 cells with VS-4718 in matrigel
attenuated secondary tumorsphere formation. Furthermore, VS-4718 reduced the side population
(SP) and the percentage of Aldefluor+ cancer stem cells in SUM159 breast cancer cells in vitro. In
direct contrast, standard-of-care agents such as paclitaxel and cisplatin increased the percentage of
Aldefluor+ cancer stem cells under equivalent conditions.
The effect of VS-4718 on cancer stem cells in vivo was evaluated in SUM159 and MDA-MB-231
human triple negative breast cancer xenograft models. Following systemic administration, VS4718 induced significant reduction of cancer stem cells in tumors as assessed by a decrease in
Aldefluor+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors.
In summary, our results indicate the importance of FAK in the self-renewal of breast cancer stem
cells in vitro and in vivo, and support the clinical development of the selective FAK inhibitor VS4718 to target cancer stem cells for the treatment of triple negative breast cancer.
Tumorspheres
V S -4 7 1 8 1 0 0 m g /k g , B ID x 2 8 d a y
600
P a c lita x e l 1 5 m g /k g , IP Q D x 5 d a y
400
200
CSCs
13%
0
0
5
10
15
20
25
30
S tu d y D a y
ICR-scid mice bearing MDA-MB-231 breast xenograft tumors were treated with VS-4718 and paclitaxel at the
indicated doses and schedules.
VS-4718 is a potent and selective FAK kinase inhibitor
SUMMARY
1. VS-4718 is a potent and selective FAK kinase inhibitor
MDA-MB-231 cells were treated with VS-4718, pacliatxel or cisplatin for 4 days in 3D matrigel. Cells extracted from
matrigel were plated on tissue culture plates and subjected to Aldefluor assay. The percent of Aldefluor-positive cells
normalized to control is shown (A). SUM159 cells were treated with VS-4718 or Verapamil, a PGP inhibitor, for 4 days
before Hoechst dye exclusion assay was carried out (B).
2. FAK inhibitor VS-4718 preferentially reduces cancer stem cells in vitro
and in vivo as assessed with multiple CSC assays
3. VS-4718 effectively inhibits breast cancer tumor growth in vivo
4. Our results demonstrate the importance of FAK in the self-renewal of
cancer stem cells and support the clinical development of a FAK inhibitor
to achieve more durable clinical responses for cancer patients
Cellular pFAK EC50 = 4 nM
Verastem, Inc. | 215 First Street | Suite 440 | Cambridge | Massachusetts | 02142