Biochemical characterization of EGFR exon 20 insertion variants and their inhibitor sensitivities

Abstract

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer (NSCLC). Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs), the leading class of targeted therapies for NSCLC in clinical use. Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD), to better understand the molecular mechanisms underlying TKI sensitivity and resistance in these variants. From kinetic studies, we found that EGFR insASV and insSVD have lower Km, ATP values compared to the L858R variant, which contributes to their relative resistance to ATP competitive inhibitors. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that BAY-568, BAY-33, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and mutant selective. Kinetic studies with TAK788 showed that these covalent inhibitors have similar reversible binding affinities to wild-type EGFR and exon 20 insertion variants, but different covalent inactivation rates that may underlie their increased potency against insASV and insSVD. Similar experiments with BAY-33, a reversible inhibitor, suggest that mutant selectivity for this compound could be due to different rates of conformational change following reversible binding. Our systematic biochemical and structural analysis suggests that these mutant-selective compounds should be prioritized for clinical development, and in the long run our results should facilitate development of more mutant-selective compounds.