TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion-Positive Intrahepatic Cholangiocarcinoma

Abstract

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983.

Figure 1.. TAS-120 is clinically effective in FGFR2 fusion-positive ICC patients whose tumors acquired resistance to BGJ398 or Debio1347.

A, Radiologic scans of patients 1–4 during the course of FGFR inhibitor therapy. B-D, ddPCR analysis of serial ctDNA samples from patients 1–3. Time periods of therapy with the specific FGFR inhibitors are indicated by shading. MAF: mutant allele frequency. Mutations identified in tumor biopsies taken at the indicated times are presented at the bottom of each graph. CCF: cancer cell fraction.

Figure 2.. FGFR-activated ICC models show FGFR2-dependent growth and MEK/ERK signaling in vivo and in vitro

A. Graph of IC50 data and dose response curves for BGJ398 in biliary tract cancer cell lines that show constitutive FGFR activation (red) or lack FGFR activity (black). p < 0.0002 for IC50 difference. B. Graph of IC50 data and dose response curves for TAS-120 in biliary tract cancer cell lines. p < 0.002 for FGFR-activated versus non-FGFR-activated lines. * denotes IC50 was not reached. C and D, Immunoblot of signaling effects of 50 nM BGJ398 treatment versus vehicle control in ICC13–7 cells (C) and CCLP-1 cells (D). Cells were treated for the indicated times before harvesting. E-G. Fragments of an ICC PDX harboring an FGFR2-KIAA1217 fusion were implanted in NSG mice. Mice were randomized for treatment with TAS-120 (25 mg/kg) or vehicle once tumors reached ~500 mm³. E, Histologic images (H&E staining) and measurement of proliferation (Ki67 staining) of tumors isolated at the indicated times. F, Serial measurement of tumor volumes. G, Immunoblot data showing signaling inhibition upon TAS-120 treatment (samples are from 14 days treatment).

Figure 3.. FGFR inhibitors have distinct activity profiles against secondary FGFR2 kinase mutations in ICC cell lines that correlate with clinical data.

A-D, CCLP-1 cells were engineered by retroviral transduction to express the FGFR2-PHGDH fusion with a wild type kinase domain or harboring the indicated mutations, or empty vector. The fusions contain the FGFR2-IIIb splice isoform [NM_001144913.1], and the amino acids are numbered accordingly. A, Graphs of IC50 measurements upon treatment with the indicated FGFR inhibitors. The measured IC50 is also indicated numerically at the right along with the fold-change in IC50 of each cell line relative to cell lines expressing the WT fusion. Red shading highlight mutants conferring a greater than 10-fold increase in IC50. B, Pooled CCLP-1 cell clones of all FGFR2 fusion variants were treated with BGJ398, Debio1347, or TAS-120 at the indicated concentrations over 14 days. The individual clones were monitored using genomic DNA extracted at 14 days, using a ddPCR assay specific to each mutation. Data are mean ± SEM of triplicate determinants of relative change in clonal abundance compared with the start of treatment and are generated from two independent experiments. C, Clonal pools as in (B) were treated sequentially with 50 nM BGJ398 and 10nM TAS-120 to mimic the treatment course of patients. Cells were monitored at 7 and 14 days. Data are expressed in relative mutant allele frequency compared with the start of treatment. Data are mean ± SD of triplicate determinants of relative change in clonal abundance compared with the start of treatment and are generated from two independent experiments. D, Immunoblot of CCLP-1 cells expressing the different FGFR2-PHGDH alleles following treatment with the indicated inhibitor concentrations.

Figure 4.. Structural modeling of secondary FGFR2 kinase domain mutations with TAS-120.

A, Model showing TAS-120 docked into ATP-binding pocket of wild type FGFR2. Amino acid residues corresponding to mutations conferring resistance to ATP competitive FGFR inhibitors are highlighted. Structural representations were prepared using PyMOL. B, A close-up view of TAS-120 in ATP-binding pocket of wild type FGFR2. The gatekeeper residue (V565) is in close proximity to dimethoxy phenyl group of TAS-120.