Dual targeting of FGFR3 and ERBB3 enhances the efficacy of FGFR inhibitors in FGFR3 fusion-driven bladder cancer

Abstract

Background: Mutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10-20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents.

Methods: Acquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis.

Results: Long term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation.

Conclusions: We demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

Keywords: Acquired resistance; Bladder cancer; EGFR; ERBB2; ERBB3; FGFR3; Targeted therapy.

Fig. 1

Sensitivity of SW780 (A-D) and RT4 (E–H) parental (PAR) and FGFR inhibitor resistant cell lines (SW780-RS, SW780-RD, RT4-RS, RT4-RD) to BGJ398 (A, E), erdafitinib (B, F), TAS-120 (C, G) and trametinib (D, H). Cells were treated with BGJ398, erdafitinib, TAS-120 or trametinib for 96 h and cell viability determined using the Cell Titre-Glo assay. Values shown are mean ± SEM of a representative experiment performed in triplicate

Fig. 2

Activation of pERBB receptors in FGFR inhibitor-resistant bladder cancer cell lines. A SW780 and RT4 parental (PAR) and Resistant (RS) lines were grown in fresh medium for 24 h without exposure to BGJ398, and cell lysates hybridised to phospho-RTK arrays. Hybridisation signals at the corners serve as controls. B Western blot analysis confirming the increase in pERBB3 in SW780 and RT4 parental (PAR) and FGFR inhibitor-resistant (RS) cell lines. Data shown are from a representative experiment

Fig. 3

Effect of combinatorial treatment with an FGFR and pan-ERBB inhibitor on (A, D) cell viability and (B, E) cell cycle kinetics, and (C, F) apoptosis in bladder cancer cell lines with acquired resistance to FGFR inhibitors. A, D FGFR-inhibitor resistant (A) SW780-RS and (D) RT4-RS cell lines were treated with a range of concentrations of BGJ398 alone and in combination with the pan-ERBB inhibitor, AZD8931, for 72 h and cell viability assessed using the Cell-Titer Glo assay. Plots shown are the BLISS synergy analysis, which shows synergistic growth inhibition across a range of concentrations. B, E FGFR inhibitor-resistant (B) SW780-RS and (E) RT4-RS cell lines were treated with BGJ398 alone and in combination with the pan-ERBB inhibitor, AZD8931, for 24 h and changes in cell cycle distribution determined by propidium iodide staining and FACS analysis. C, F Assessment of the effect of combination treatment with BGJ398 and AZD8931 on apoptosis by propidium iodide staining and FACS analysis in the same samples analysed in panels B and D. Values shown are mean ± SEM of a representative experiment performed in triplicate. *P < 0.05 and ***P < 0.0005, t test

Fig. 4

Effect of short-term treatment with BGJ398 on pERK and ERBB family receptors. A SW780 and (B) RT4 parental cells were treated with BGJ398 for 4–72 h and changes in pERK and ERBB family receptors was determined by western blot. Data shown are from a representative experiment

Fig. 5

Effect of combinatorial treatment of parental FGFR3 fusion harboring bladder cell lines with an FGFR and pan-ERBB inhibitor on (A, E) cell signaling, (B, F) cell viability, (C, G) cell cycle kinetics and (D, H) apoptosis. A, E Parental SW780 and RT4 cells cells were treated with BGJ398 (0.1 µM) or AZD8931 (1 µM), alone or in combination for 72 h and changes in pERBB3 and pERK determined by western blot. B, F Parental SW780 and RT4 cells were treated with a range of concentrations of BGJ398 or AZD8931 alone or in combination for 72 h and cell viability determined using Cell-Titer Glo assays. Plots shown are the BLISS synergy analysis from a representative experiment, which shows synergistic growth inhibition across a range of concentrations. C, G FGFR inhibitor-resistant (C) SW780-RS and (G) RT4-RS cell lines were treated with BGJ398 alone and in combination with the pan-ERBB inhibitor, AZD8931, for 24 h and changes in cell cycle distribution determined by propidium iodide staining and FACS analysis. D, H Assessment of the effect of combination treatment with BGJ398 and AZD8931 on apoptosis by propidium iodide staining and FACS analysis in the same samples analysed in panels C and G. Values shown are mean ± SEM of a representative experiment performed in triplicate. *P < 0.05 and ***P < 0.0005, t test

Fig. 6

Effect of combinatorial treatment of parental FGFR3 fusion harboring bladder cell lines with (A, C) erdafitinib and AZD8931, or (B, D) TAS-120 and AZD8931 on cell viability. A, B Parental SW780 and (C, D) RT4 cells were treated with a range of concentrations of BGJ398 or AZD8931 alone or in combination for 72 h and cell viability determined using Cell-Titer Glo assays. Plots shown are the BLISS synergy analysis from a representative experiment, which shows synergistic growth inhibition across a range of concentrations