Initial Testing of VS-4718, a novel inhibitor of Focal Adhesion Kinase (FAK), against pediatric tumor models by the Pediatric Preclinical Testing Program

Abstract

VS-4718 was tested against the PPTP’s in vitro cell line panel and showed a median relative IC₅₀ of 1.22 µM. VS-4718 was tested in vivo against the PPTP xenograft models using a dose of 50 mg/kg administered by the oral route twice daily for 21 days. VS-4718 induced significant differences in EFS distribution compared to control in 18 of 36 of the evaluable solid tumor xenografts and in 0 of 8 acute lymphoblastic leukemia (ALL) xenografts, but no xenograft lines showed tumor regression. Future plans include further evaluating the role of FAK inhibition in combination with ABL kinase inhibitors for Ph-positive ALL.

INTRODUCTION

Focal Adhesion Kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that is encoded by PTK2 (chromosome 8q24.3). FAK is involved in many cellular processes through its role in integrin and growth factor signaling, with activation of FAK inducing recruitment of several SH2-domain and SH3-domain containing proteins that mediate signaling to downstream pathways ¹. A role for FAK in cancer has been suspected since it was described as a protein with increased tyrosine phosphorylation in viral Src transformed chicken embryo cells ^2–4. Subsequent research has provided evidence for a role of FAK in promotion of cancer through effects on cell survival, angiogenesis, migration, proliferation, epithelial-mesenchymal transformation, invasion, and stem cell maintenance ⁵.

Several small molecule inhibitors of FAK have entered clinical development, including defactinib (VS-6063, PF-04554878), VS-4718, and GSK2256098 ⁵. VS-4718 (previously named PND-1186) is a selective, reversible inhibitor that has an IC₅₀ of 1.5 nM using an in vitro kinase assay and a cellular IC₅₀ of approximately 100 nM for FAK inhibition as measured by effects on phosphorylation at Tyr-397 ⁶. VS-4718 induced apoptosis in cells cultured in suspension but not in cells grown under adherent conditions ⁶.

Here we report the in vitro and in vivo activity of VS-4718 against PPTP cell lines and xenograft models.

MATERIALS AND METHODS

In vitro testing

In vitro testing was performed using DIMSCAN and cells were incubated in the presence of VS-4718 for 96 hours at concentrations from 1.0 nM to 10 µM and analyzed as previously described ^7,8

In vivo tumor growth inhibition studies

Procedures for propagation of xenografts in CB17SC scid^−/− for solid tumors, BALB/c nu/nu mice for glioma models, ⁹, and leukemia models (NOD)/scid^−/− mice have been described previously ¹⁰. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. An in-depth description of the analysis methods is included in Supplemental Appendix S1. Statistical methods for analysis have been described previously ⁹.

Drugs and Formulation

VS-4718 was provided to the Pediatric Preclinical Testing Program by Verastem Inc., through the Cancer Therapy Evaluation Program (NCI). VS-4718 was formulated in 0.5% carboxymethylcellulose/0.1% Tween 80, and administered P.O. twice daily for 21 consecutive days at 50 mg/kg/dose, a dose and schedule shown to be synergistic when administered with dasatinib in the ALL-4 leukemia model ¹¹. VS-4718 was provided in coded vials for blinded testing.

RESULTS

VS-4718 in vitro testing

VS-4718 was tested against the PPTP’s in vitro cell line panel at concentrations ranging from 1.0 nM to 10 µM using the PPTP’s standard 96-hour exposure period. The median relative IC₅₀ (rIC₅₀) was 1.22 µM, with a range from 0.25 µM (CHLA-9, Ewing sarcoma) to 3.53 µM [COG-LL-317, acute lymphoblastic leukemia (ALL)]. The median Ymin (T/C%) was 4%, with a range from 0% to 29%.

A metric used to compare the relative responsiveness of the PPTP cell lines to VS-4718 is the ratio of the median rIC₅₀ of the entire panel to that of each cell line (Table 1). Overall, there was not a large variation in rIC₅₀ by histotype. There is a trend for the Ewing cell lines (TC-71 through CHLA-258) to be more sensitive than the non-Ewing cell lines (0.85 µM versus 1.44 µM, p=0.05). The median Relative I/O% value for the PPTP cell line panel was -56%, indicating a cytotoxic effect at the highest concentrations tested for most of the cell lines.

Table 1.

In vitro activity for VS-4718

Cell Line	Histotype	rIC50 (µM)	Panel rIC50/ Line rIC50	Ymin	Relative In/Out% (Observed Ymin)1
RD	Rhabdomyosarcoma	1.22	1.00	21%	16%
Rh41	Rhabdomyosarcoma	0.53	2.31	2%	−93%
Rh18	Rhabdomyosarcoma	1.63	0.75	22%	−50%
Rh30	Rhabdomyosarcoma	0.71	1.72	3%	−83%
BT-12	Rhabdoid	3.13	0.39	24%	17%
CHLA-266	Rhabdoid	1.16	1.05	29%	3%
TC-71	Ewing sarcoma	1.05	1.16	2%	1%
CHLA-9	Ewing sarcoma	0.25	4.92	1%	−60%
CHLA-10	Ewing sarcoma	1.03	1.19	5%	−17%
CHLA-258	Ewing sarcoma	0.66	1.84	4%	−90%
SJ-GBM2	Glioblastoma	1.68	0.73	21%	13%
NB-1643	Neuroblastoma	0.54	2.27	6%	−72%
NB-EBc1	Neuroblastoma	1.57	0.78	10%	−56%
CHLA-90	Neuroblastoma	0.64	1.92	26%	−7%
CHLA-136	Neuroblastoma	1.06	1.15	15%	−46%
NALM-6	ALL	2.67	0.46	2%	−24%
COG-LL-317	ALL	3.53	0.35	0%	−94%
RS4;11	ALL	1.23	1.00	6%	−62%
MOLT-4	ALL	2.65	0.46	1%	−88%
CCRF-CEM (1)	ALL	2.55	0.48	1%	−82%
Kasumi-1	AML	1.44	0.84	3%	−90%
Karpas-299	ALCL	0.37	3.32	2%	−73%
Ramos-RA1	NHL	2.83	0.43	1%	0%
Median		1.22	1.00	4%	−56%
Minimum		0.25	0.35	0%	−94%
Maximum		3.53	4.92	29%	17%

¹The Relative In/Out (I/O)% values compare the relative difference in final cell number compared with the starting cell number for treated cells and for control cells calculated as follows: (Observed Y_min−Y₀)/(100−Y₀) if Observed Y_min>Y₀; and (Observed Y_min−Y₀)/(Y₀) if Observed Y_min<Predicted Y_min). Y₀ is an estimate of the starting cell number derived from determinations of the doubling time for each cell line. Relative I/O% values range between 100% (no treatment effect) to -100% (complete cytotoxic effect), with a Relative I/O% value of 0% being observed for a completely effective cytostatic agent.

VS-4718 in vivo testing

VS-4718 was well tolerated, with only a 1.2% mortality rate (5/427) in the treated groups, the same as that observed for control animals. Each of 44 tested xenograft models were considered evaluable for efficacy. Complete details of testing are provided in Supplemental Table 2. VS-4718 induced significant differences in EFS distribution compared to control in 18 of 36 (50%) of the evaluable solid tumor xenografts and in 0 of 8 (0%) of the evaluable ALL xenografts, including the Ph+ ALL xenograft, ALL-4 (Table 2). Significant differences in EFS distribution were most commonly observed for the osteosarcoma panel (6 of 6), the rhabdomyosarcoma panel (4 of 6), and the neuroblastoma panel (4 of 6). VS-4718 did not induce tumor growth inhibition meeting criteria for intermediate EFS T/C(>2) activity in either the solid tumor or ALL xenografts. Only 3 models met criteria for PD2 responses (one Ewing, rhabdomyosarcoma, and neuroblastoma xenograft each). Objective responses were not observed for the solid tumor or ALL xenografts.

Table 2.

Summary of in Vivo Activity of VS-4718

Line	Tumor Type	Median Time to Event	P- value	EFS T/C1	Median RTV/CD45 at End of Study	Tumor Volume T/C2	Median Group Response	EFS Activity
BT-29	Rhabdoid	28.2	<0.001	1.5	>4	0.64	PD1	Low
KT-16	Rhabdoid	15.4	0.796	1.4	>4	0.82	PD1	Low
KT-14	Rhabdoid	20.7	0.378	1.3	>4	0.80	PD1	Low
KT-10	Wilms	18.6	0.089	1.4	>4	0.74	PD1	Low
KT-11	Wilms	15.2	0.367	1.1	>4	0.84	PD1	Low
KT-13	Wilms	12.0	0.598	0.9	>4	1.18	PD1	Low
SK-NEP-1	Ewing	13.5	0.110	1.4	>4	0.66	PD1	Low
EW5	Ewing	10.7	<0.001	1.9	>4	1.36	PD2	Low
EW8	Ewing	18.6	0.925	1.3	>4	0.84	PD1	Low
TC-71	Ewing	10.6	0.197	1.1	>4	0.81	PD1	Low
CHLA258	Ewing	8.0	0.351	0.8	>4	0.99	PD1	Low
Rh10	Alveolar RMS	24.1	0.351	1.2	>4	0.67	PD1	Low
Rh28	Alveolar RMS	19.2	0.015	0.7	>4	1.29	PD1	Low
Rh30	Alveolar RMS	15.1	0.013	1.5	>4	0.57	PD1	Low
Rh30R	Alveolar RMS	19.9	<0.001	1.6	>4	0.48	PD2	Low
Rh41	Alveolar RMS	12.4	0.010	1.4	>4	0.66	PD1	Low
Rh18	Embryonal RMS	16.7	0.009	1.5	>4	0.65	PD1	Low
BT-28	Medulloblastoma	16.5	0.002	1.3	>4	0.73	PD1	Low
BT-50	Medulloblastoma	33.0	0.123	1.1	>4	1.05	PD1	Low
BT-36	Ependymoma	27.0	0.074	0.8	>4	1.50	PD1	Low
GBM2	Glioblastoma	24.6	0.636	1.3	>4	0.83	PD1	Low
BT-39	Glioblastoma	6.9	0.517	0.9	>4	1.14	PD1	Low
D645	Glioblastoma	6.7	0.470	1.0	>4	1.04	PD1	Low
D456	Glioblastoma	8.1	0.004	1.4	>4	0.50	PD1	Low
NB-SD	Neuroblastoma	10.1	0.048	1.3	>4	0.87	PD1	Low
NB-1771	Neuroblastoma	8.7	0.138	1.2	>4	0.88	PD1	Low
NB-1691	Neuroblastoma	10.6	0.013	1.4	>4	0.77	PD1	Low
NB-EBc1	Neuroblastoma	4.9	0.162	1.1	>4	0.90	PD1	Low
CHLA-79	Neuroblastoma	12.1	0.002	1.5	>4	0.73	PD2	Low
NB-1643	Neuroblastoma	6.7	0.020	1.1	>4	0.82	PD1	Low
OS-1	Osteosarcoma	> EP	<0.001	> 1.3	3.1	0.58	NE	NE
OS-2	Osteosarcoma	21.9	<0.001	1.2	>4	0.69	PD1	Low
OS-17	Osteosarcoma	17.0	0.024	1.3	>4	0.75	PD1	Low
OS-9	Osteosarcoma	30.3	<0.001	1.4	>4	0.54	PD1	Low
OS-33	Osteosarcoma	28.4	<0.001	1.4	>4	0.50	PD1	Low
OS-31	Osteosarcoma	20.9	<0.001	1.4	>4	0.71	PD1	Low
ALL-2	ALL B-precursor	15.5	0.940	0.9	>25	.	PD1	Low
ALL-4	ALL B-precursor	4.9	0.834	1.1	>25	.	PD1	Low
ALL-7	ALL B-precursor	7.0	0.258	0.9	>25	.	PD1	Low
ALL-8	T-cell ALL	10.0	0.718	1.0	>25	.	PD1	Low
ALL-17	ALL B-precursor	9.7	0.452	0.9	>25	.	PD1	Low
ALL-19	ALL B-precursor	5.7	0.331	0.9	>25	.	PD1	Low
ALL-31	T-cell ALL	9.0	0.598	1.0	>25	.	PD1	Low
MLL-7	Infant ALL3	9.0	1.000	1.0	>25	.	PD1	Low

¹EFS T/C value is defined by the ratio of the median time to event of the treatment group and the median time to event of the respective control group. High activity requires: a) an EFS T/C > 2; b) a significant difference in EFS distributions, and c) a net reduction in median tumor volume for animals in the treated group at the end of treatment as compared to at treatment initiation. Intermediate activity demonstrates criteria a) and b) above, but not having a net reduction in median tumor volume for treated animals at the end of the study. Low activity demonstrates EFS T/C < 2. ³Objective response measures are described in detail in the Supplemental Response Definitions. PD1 = progressive disease with EFS T/C ≤ 1.5, and PD2 = progressive disease with EFS T/C > 1.5.

²Tumor Volume T/C value: Relative tumor volumes (RTV) for control (C) and treatment (T) mice were calculated at day 21 or when all mice in the control and treated groups still had measurable tumor volumes (if less than 21 days). The T/C value is the mean RTV for the treatment group divided by the mean RTV for the control group. High activity = T/C ≤ 0.15; Intermediate activity = T/C ≤ 0.45 but > 0.15; and Low activity = T/C > 0.45.

³KMT2A (MLL) rearranged B-ALL.

DISCUSSION

VS-4718 inhibits FAK phosphorylation at Y397 in cellular assays with an IC₅₀ in the 100 nM range ⁶, and it also reduces the proportion of cancer stem cells (CSCs) in aldefluor and Hoechst dye exclusion assays with an IC₅₀ value of approximately 100 nM ¹². Other work has shown that malignant pleural mesothelioma (MPM) cell lines with mutated NF2 have IC₅₀ values in the 100 nM range, while MPM cell lines with wild-type NF2 have IC₅₀ values greater than 1 µM ¹³. Under the assumption that these results indicate that FAK “on-target” effects are observed at 100 nM concentrations or less, then it is likely that the PPTP in vitro results showing IC₅₀ values generally above 1 µM reflect “off-target” effects of VS-4718.

VS-4718 was well tolerated at the dose and schedule evaluated (50 mg/kg BID×21 days). However, there was limited in vivo activity observed. Significant differences in EFS distribution were noted in approximately one-half of the solid tumor models, but the magnitude of the shift in time to event was small with only 3 xenografts showing EFS T/C ratios greater than 1.5. No ALL models showed significant differences in EFS distribution.

A recent publication highlighted the potential utility for FAK inhibition against ALL that exhibits loss of Ikaros function ¹⁴, a finding that is particularly common in Ph⁺ ALL expressing the BCR-ABL1 fusion gene ¹⁵. Joshi, et al., showed that loss of Ikaros function in early pre-B cells leads to a differentiation block that drives the cells into an adhesive state, promotes self-renewal, and increases the likelihood for malignant transformation ¹⁴. Treatment of Ikaros-defective malignant B-cells with a small molecule FAK inhibitor (VS-6062) led to apoptotic cell death. However, it is unknown how closely leukemias developing from murine pre-B cells null for Ikaros align with the biology of Ph+ ALL in children. The PPTP has two ALL xenograft lines, ALL-4 and ALL-19, for which SNP array data indicate alterations in IKZF1. ALL-4 harbors the BCR-ABL1 translocation while ALL-19 has been identified as harboring a NUP214-ABL1 fusion. However, neither of these lines responded to single agent VS-4718, consistent with previous results¹¹. Churchman, et al., made the important observation that VS-4718 potentiates the activity of dasatinib both in vitro and in vivo against BCR-ABL1 expressing Ph⁺ ALL preclinical models, including ALL-4 ¹¹. A promising line of future research is to further evaluate how VS-4718 can be used to more effectively treat Ph⁺ ALL. An open question is whether there are other childhood cancers for which FAK inhibition can be used to enhance the activity of established anticancer agents.

In summary, the in vivo activity observed for VS-4718 as a single agent against the PPTP preclinical xenograft lines is low. Similarly, the in vitro data are consistent with a limited effect of FAK inhibition alone for the pediatric models studied.

Abbreviations

PPTP

Pediatric Preclinical Testing Program

EFS T/C

Ratio of Event Free Survival Treated/Control

FAK

Focal Adhesion Kinase

(NOD)/scid^−/−

Non-Obese diabetic/severe combined immune deficient