Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (∼10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib and afatinib). The basis of this primary resistance is poorly understood.  We study a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients; and find that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered ATP-binding pocket and the inserted residues form a wedge at the end of the C-helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro; and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C-helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology and their response to EGFR TKIs.

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