To overcome the major challenge of variants of unknown significance in precision oncology, high-throughput functional genomics is essential. Only a comprehensive characterization of the vast landscape of cancer mutations enables optimized clinical treatment targeting molecular aberrations. Aberrant activation of fibroblast growth factor receptors (FGFRs) is a common oncogenic driver across multiple cancer types, with selective FGFR inhibitors (FGFRis) already approved for clinical use. However, the therapeutic potential of these inhibitors is constrained by the unknown characteristics of point mutations in FGFR1, FGFR2, FGFR3 and FGFR4, which can drive signaling and/or confer resistance. Here, we established a saturation mutagenesis scanning platform to assess all 11520 possible single nucleotide variants in the kinase domains of FGFR1-4 through pooled positive selection screens.

We generated lentiviral libraries of all possible 11520 SNVs in the kinase domains of FGFR1-4 and tested their impact on FGFR activation as well as their capacity to mediate resistance to the FDA-approved FGFR inhibitors pemigatinib and futibatinib in positive selection screens. Selected activating and resistance mutations were individually cloned and their impact validated in a second cell line model.

We classified 474 activating mutations and 738 resistance-conferring mutations to pemigatinib and/or futibatinib – many of which clustered within specific structural elements. Merging these two datasets we identified 301 targetable FGFR point mutations analogous to a strong PS3/BS3 evidence level. Notably, mutations at the same codon or the same mutations in different FGFRs could exhibit strikingly diverse effects, highlighting the necessity for a saturation mutagenesis approach. The screens also identified loss-of-function mutations and FGFR2-specific activating nonsense mutations. Importantly, the functional screens detected 97% of acquired resistance mutations in a clinical trial. To enhance experimental robustness and universality, the screening protocol was also established using an urothelial cancer cell line - a common FGFR-altered cancer entity. This approach revealed a significant overlap in identified resistance mutations, contributing to a more comprehensive understanding of the utility of model cell systems in saturation mutagenesis screening.

In summary, we present a thorough and clinically valuable catalog of all druggable point mutations within the FGFR kinase domains which is readily accessible for clinical decision making to select the optimal cancer therapy and expanding the potential application of the approved inhibitors.