Abstract 1117: Acquired resistance to ATP-competitive and irreversible FGFR inhibitors (FGFRi's): A library-based approach

Abstract

Abstract Background: The emergence of secondary FGFR kinase domain (KD) mutations with FGFRi's leads to drug resistance and disease progression. The frequency of resistance mutations with individual FGFRi's is unknown. Using an unbiased library-based approach, we examined the development of acquired resistance with the irreversible FGFRi futibatinib and the ATP-competitive FGFRi's erdafitinib (ERD) and pemigatinib (PEM). Methods: Mutant (mt) libraries were generated with random mutagenesis in two FGFR2 KD regions: #1 (amino acids [aa] 540-569), and #2 (aa 640-669). Library were transfected as TEL-fusions into Ba/F3 cells, resulting in FGFR2-dependent cell growth. Transfected cells were treated with FGFRi's at concentrations suppressing parental cell line growth. Surviving drug-resistant clones were counted, isolated, and sequenced to identify the KD mutation. The inhibitory activity of FGFRi's against resistant clones was assessed using phospho-FGFR2 ELISA. Results: Treatment of the library-transfected Ba/F3-TEL-FGFR2 KD cells with PEM, ERD, and futibatinib resulted in an average (n=2) of 93, 27, and 13 resistant clones in region #1 and 28, 21 and 1 resistant in region #2, respectively. Sequencing (table) identified, among others, the following aa mutations: N549, E565, and K641 (regulatory triad), V564 (gatekeeper region) and K659 (activation loop), also seen in patients who developed resistance to Debio1347, infigratinib, and PEM. Among FGFR mts shown (table), futibatinib inhibited 8/10, and ERD, 5/10 mts at IC50 values comparable to wild type (wt) FGFR; however, PEM activity against all FGFR mts was attenuated by ≥5-fold vs wt. Conclusions: Resistance mutations identified with this unbiased library-based in vitro approach were consistent with those observed in the clinic. Acquired drug resistance mutations were less frequent with futibatinib than with ATP-competitive FGFRi's. Futibatinib demonstrated the most robust inhibition of drug resistant mts. FGFR mutationKD regionFutibatinib IC50 (nM)Erdafitinib IC50 (nM)Pemigatinib IC50 (nM)Wild-type (parent)-2.12.51.5N549DRegulatory triad4.225.5153.2N549KRegulatory triad16.732.0246.2V562L-6.32.97.7V564IGatekeeper8.32.762.4V564LGatekeeper91.956.5502.3E565ARegulatory triad6.82.911.8E565GRegulatory triad3.93.09.0K641IRegulatory triad3.855.829.6K641RRegulatory triad4.73.210.3K659MActivation loop9.546.633.8 Citation Format: Hiroshi Sootome, Suzuko Kato, Masanori Kato, Hiroshi Hirai. Acquired resistance to ATP-competitive and irreversible FGFR inhibitors (FGFRi's): A library-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1117.