Biochemical and structural basis for differential inhibitor sensitivity of EGFR with distinct exon 19 mutations

Tyrosine kinase inhibitors (TKIs) are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). TKI responses vary across tumors driven by the heterogeneous group of exon 19 deletions and mutations, but the molecular basis for these differences is not understood. Using purified TKDs, we compared kinetic properties of several exon 19 variants. Although unaltered for the second generation TKI afatinib, sensitivity varied significantly for both the first and third generation TKIs erlotinib and osimertinib. The most sensitive variants showed reduced ATP-binding affinity, whereas those associated with primary resistance retained wild type ATP-binding characteristics (and low K M, ATP ). Through crystallographic and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies, we identify possible origins for the altered ATP-binding affinity underlying TKI sensitivity and resistance, and propose a basis for classifying uncommon exon 19 variants that may have predictive clinical value. Although small molecule tyrosine kinase inhibitors are effective in lung cancer driven by mutated EGFR, some receptor variants fail to respond. Here, the authors identify structural features of an important set of EGFR variants with reduced inhibitor sensitivity, guiding future inhibitor selection.