Resistance to Selective FGFR Inhibitors in FGFR-Driven Urothelial Cancer

Abstract

Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients (FGFR3 N540K, V553L/M, V555L/M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, and L608V). Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K-mTOR pathway (n = 4 TSC1/2, n = 4 PIK3CA, n = 1 TSC1 and PIK3CA, n = 1 NF2, n = 1 PTEN). In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib-gefitinib combination was able to overcome bypass resistance mediated by EGFR activation.

Significance: In the largest study on the topic thus far, we detected a high frequency of FGFR kinase domain mutations responsible for resistance to FGFR inhibitors in urothelial cancer. Off-target resistance mechanisms involved primarily the PI3K-mTOR pathway. Our findings provide preclinical evidence sustaining combinatorial treatment strategies to overcome bypass resistance. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

Figure 1.

Mutational landscape of urothelial cancers included in MATCH-R or MOSCATO studies, with WES ± RNA-seq performed. CNV, copy-number variation; INDEL, insertion/deletion; mut/Mb, mutations per megabase; SNP, single-nucleotide polymorphism; TMB, tumor mutation burden.

Figure 2.

Overview of patients with urothelial cancer progressing on FGFR inhibitors, treatment outcomes, and postprogression sample availability. Granular view at the individual patient level. BOR, best objective response; CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease. ^aPatient MR560 received erdafitinib combined with a PD-1 inhibitor. ^bPatient CTC1845 was treated with a sequence of erdafitinib–futibatinib (with intervening immunotherapy between the two FGFR inhibitors), and the ctDNA sample was collected at progression on futibatinib. ^cPatient CTC615 received pazopanib before futibatinib.

Figure 3.

FGFR kinase domain mutations detected at progression on FGFR inhibitors in urothelial cancer. A, Heat map representing individual patient data among the cohort of FGFR3-driven urothelial cancers. B–G, Molecular and radiologic findings. Mutations and fusions in ctDNA are reported as variant allele frequencies (VAF). Mutations in the FGFR tyrosine kinase domain are reported in red. ampl, amplification. ^aPatient MR560 received erdafitinib combined with a PD-1 inhibitor. ^bPatient CTC1845 was treated with a sequence of erdafitinib–futibatinib (with intervening immunotherapy between the two FGFR inhibitors) before the ctDNA sample was collected at progression on futibatinib.

Figure 4.

Functional characterization of FGFR2/3 kinase domain mutations in Ba/F3 cell models. A, IC₅₀ values of eight selective FGFR inhibitors in viability assays against FGFR3::TACC3 with a WT kinase domain or harboring mutations found in postprogression patient samples. IC₅₀ values (nmol/L) are reported as means of ≥3 independent datasets (see Supplementary Table S3). B, Immunoblot analysis confirming the ability of erdafitinib to abrogate intracellular signaling in FGFR3::TACC3 WT Ba/F3 cells, while resistance was observed in N540K, V553M, V555L, V555M, and L608V mutants. C, Higher concentrations of pemigatinib (100–300 nmol/L) were required to abrogate FGFR3 signaling in FGFR3::TACC3 V553L Ba/F3 compared with erdafitinib (10–30 nmol/L). D, Exposing FGFR3::TACC3 WT, N540K, and V555L Ba/F3 cells to erdafinitib corroborated the findings of resistance to erdafinitib, and confirmed the role of “molecular brake disruption” of the N540K mutant. E and F, Functional characterization of FGFR2 L551F mutation in Ba/F3 cells harboring the FGFR2::BICC1 transcript. A representative study of a cell viability assay is reported at the top (E), and IC₅₀ values (nmol/L) are reported at the bottom as means of ≥3 independent datasets (F).

Figure 5.

Synergistic effect of combinatorial treatments in patient-derived models of FGFR-driven urothelial cancer. A, In an MR86 PDX, combined erdafitinib–pictilisib (PI3K inhibitor) administration had synergistic effects leading to prolonged inhibition of tumor growth. B, Protein extracts from the MR15 cell line subjected to a phospho-RTK array revealed EGFR activation. C and D, Concomitant EGFR and FGFR inhibition with gefitinib and erdafitinib was synergic both in viability assays (C) and in immunoblot analyses (D). E, The in vivo pharmacologic experiment in the corresponding PDX confirmed the efficiency of the combined treatment strategy.