FGFR1-4 kinasedomain saturation mutagenesis

Variants of unknown significance constitute the biggest challenge for precision oncology. Aberrantly activated fibroblast growth factor receptors (FGFRs) are frequently driving tumorigenesis across many entities and approved selective inhibitors (FGFRis) are available. However, it is largely unknown which of the many different point mutations affecting FGFR1, FGFR2, FGFR3 or FGFR4 are druggable, i.e. activating FGFR signaling while not mediating resistance to FGFRis.High throughput functional genomics is essential to characterize the vast number of cancer mutations and tailor the most promising treatment accordingly. Here, we implemented a saturation mutational scanning platform to screen all 11520 possible point mutations in the kinase domains of FGFR1-4. In pooled positive selection screens, we identified 474 activating mutations and 738 resistance-mediating mutations to Pemigatinib and/or Futibatinib - many clustered in particular structural elements. Combining these comprehensive datasets revealed 301 druggable FGFR point mutations analogous to a strong PS3/BS3 evidence level. Notably, mutations in the same codon or the same mutations in different FGFRs could strongly differ in their impact, thus underlining the necessity for a saturation approach to study each mutation individually. The screens also identified loss-of-function mutations and FGFR2-specific activating nonsense mutations. Importantly, the functional screens identified 97% of acquired resistance mutations in a clinical trial.In summary, we provide a comprehensive catalog of all druggable point mutations in the FGFR kinase domains readily available for clinical decision support.