Deep Mutational Scanning Reveals EGFR Mutations Conferring Resistance to the 4th-generation EGFR tyrosine kinase inhibitor BLU-945

Abstract

IntroductionOsimertinib, a covalent third-generation EGFR tyrosine kinase inhibitor (TKI) is the first-line standard of care for EGFR L858R and ex19del lung adenocarcinoma; however, tumors frequently acquire resistance through second-site mutations. Fourth-generation inhibitors designed to overcome common second-site resistance liabilities are in clinical development. MethodsWe performed deep mutational scanning (DMS) of the EGFR kinase domain in the context of an EGFR L858R driver mutation by transducing Ba/F3 cells with a saturation library of [~]17,000 EGFR L858R kinase domain variants. Ba/F3 cells expressing the DMS library were exposed to either osimertinib or BLU-945 to select for escape mutations. ResultsL718X mutations were enriched across all conditions as well as mutations private to BLU-945 treated samples including K714R, K716T, L718V, T725M, K728E, K754E/N, N771S/T, T783I, Q791L/K, G863S, S895N, K929I, and M971L. In silico pairwise comparisons of resistance profiles between each single agent condition suggested that combination treatment with osimertinib and BLU-945 would effectively suppress orthogonal resistance mechanisms, apart from L718X. A secondary DMS screen with osimertinib and BLU-945 in combination exclusively enriched for L718X mutations. L718X mutations were present in two patients treated with BLU-945 at our institution. One patient with both EGFR L858R and L718Q mutations prior to treatment was noted to have early progression. A second patient with EGFR L858R, T790M, and C797S at the time of enrollment acquired an L718V mutation at progression. ConclusionsThis study underscores the utility of comprehensive resistance profiles of single compounds, which can be used to predict the emergence of clinical resistance mutations and to devise combination treatments designed to suppress clonal escape.