Altered conformational landscape and dimerization dependency underpins the activation of EGFR by αC-β4 loop insertion mutations

Mutational activation of epidermal growth factor receptor (EGFR) in human cancers involves both point mutations and complex mutations (insertions and deletions). In particular, short in-frame insertion mutations within a conserved alpha C-beta 4 loop in the EGFR kinase domain are frequently observed in tumor samples and patients harboring these mutations are insensitive to first-generation EGFR inhibitors. Despite the prevalence and clinical relevance of insertion mutations, the mechanisms by which these mutations regulate EGFR activity and contribute to drug sensitivity are poorly understood. Using cell-based mutation screening, we find that the precise location, length, and sequence of the inserted segment are critical for ligand-independent EGFR activation and downstream signaling. We identify three insertion mutations (N771 P772insN, D770_N771insG, and D770 > GY) that activate EGFR in a unique way by relying more on the "acceptor" interface for kinase activation. Our drug inhibition studies indicate that these activating insertion mutations respond more favorably to osimertinib, a recently Food and Drug Administration-approved EGFR inhibitor for T790M-positive patients with lung cancer. Molecular dynamics simulations and umbrella sampling of WT and mutant EGFR suggest a model in which activating insertion mutations increase catalytic activity by relieving key autoinhibitory interactions associated with alpha C-helix movement and by lowering the transition free energy (Delta G(active-inactive)) between active and inactive states. Our studies also identify a transition state sampled by activating insertion mutations that can be exploited in the design of mutant-selective EGFR inhibitors.