AbstractThe emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFR^T790M-negative but EGFR^G724S-positive subclones and osimertinib resistance. We demonstrate that EGFR^G724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFR^G724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFR^G724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFR^G724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFR^G724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors.