Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation

Abstract

Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).

Conflict of interest statement

TGH, RES, YY, SE, TH, GA, JS, and BS are employees of ArQule, Inc. KP and WS have no competing financial interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Mode of FGFR inhibition for ARQ 087.

(A) ARQ 069. (B) ARQ 087. (C) Enzyme kinetic analysis was performed to determine the mode of inhibition of ARQ 087 with FGFR1 and FGFR2. Concentrations of ATP [ATP] and ARQ 087 [I] are indicated on the graphs. The rate plotted is the AlphaScreen™ signal obtained from the plate reader with background correction. The experiments were conducted in triplicates and the means and the standard deviations were plotted. The number of parameter (np), the sum of squares rel (SSrel), Akaike information criterion (AIC), weight of AIC (w(AIC)), Bayesian information criterion (BIC) and the weight of BIC (w(BIC)) for each binding mode were determined by the DynaFit software and are summarized below the plots. The dissociation constant (Ki) of ARQ 087 for FGFR1 and FGFR2 are shown. (D) The effect of ARQ 087 on the activation of FGFR1 and FGFR2 was examined in a continuous autophosphorylation assay.

Fig 2. ARQ 087 inhibits FGFR phosphorylation.

COS-1 cells ectopically expressing FGFR1, FGFR2, FGFR3 or FGFR4 were treated with the indicated concentrations of ARQ 087 for 2 hours followed by stimulation with 100 pM of FGF1/2/7 for 15 minutes. Total and phospho-FGFR was assessed by Western blot analyses. β-Actin was used as a loading control. The EC50 values of individual FGFR family members are shown.

Fig 3. ARQ 087 inhibits the FGFR pathway in cancer cell lines.

(A) NCI-H716, SNU-16, and KATO-III cells were treated with the indicated concentrations of ARQ 087 for 2 hours, and phospho-FGFR was assessed by Western blot analyses. β-Actin was used as a loading control. (B) NCI-H716 and SNU-16 cells were treated with increasing concentrations of ARQ 087 from 0 μM to 3 μM for 2 hours. The quantity of indicated protein was assessed by Western blot analyses. β-Actin was used as a loading control. (C) KG-1 cells were treated with indicated concentrations of ARQ 087 for 2 hours followed by stimulation with a mixture of FGF1/FGF2/FGF7 for 15 minutes. Cell lysates were analyzed by Western blot to determine the expression of phospho-FGFR, phospho-ERK, and β-actin.

Fig 4. ARQ 087 arrests cells in the G1 cell cycle phase and induces apoptosis.

(A) NCI-H716 and SNU-16 cells were treated with 1 μM of ARQ 087 or vehicle for 72 hours. Cell cycle profiles were measured by flow cytometric analyses. (B) SNU-16 cells were treated with ARQ 087 (1 mM) for 0, 24, 48 and 72 hrs. Western blot analysis was performed for XIAP, cleaved-PARP (c-PARP), activated-caspase 3 (a-Caspase 3), and phospho-p53 were assessed by Western blot analyses. β-Actin was used as the loading control.

Fig 5. ARQ 087 inhibits the FGFR pathway in xenograft tumors.

Mice were dosed with 75 mg/kg of ARQ 087, and sacrificed at day 9 after administration Tumor samples were collected 4 hr after last dose. IHC staining of pFGFR, FGFR2, pFRS2-a and pERK were performed from tumor tissues. The intensity of staining was scored by a veterinary pathologist and presented as mean±SEM. Representative photomicrographs are shown.

Fig 6. ARQ 087 activity in tumor growth models.

(A, C, D), Growth is inhibited in BaF3/FGFR2, SNU-16, and NCI-H716, xenograft models, but not inhibited in (B) BaF3/INSR. Results are represented as the mean of tumor volume in mm3 ± SEM of each group (n = 8–10) in function of the treatment period. The tumor growth inhibition (TGI) is indicated on the plots.

Fig 7. Animal weights in BaF3/FGFR2 animals dosed with ARQ 087.

Mice were dosed with 0, 50, 100, and 150 mg/kg of ARQ 087, and sacrificed at 10 days after administration after the start of dosing. Mean weights +/- SEM.