INTRODUCTION EGFR^T790M mostly exists subclonally and is acquired as the most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKIs). Nevertheless, because de novo EGFR^T790M-mutant NSCLC is rare, little is known on acquired resistance mechanisms to third-generation EGFR TKIs.

METHODS Acquired resistance mechanisms were analyzed using tumor and plasma samples before and after third-generation EGFR TKI treatment in four patients with de novo EGFR^T790M-mutant NSCLC. Genetic alterations were analyzed by whole-exome sequencing, targeted sequencing, fluorescence in situ hybridization, and droplet digital PCR. MTOR^L1433S, confirmed for oncogenicity using the Ba/F3 system, was reproduced in H1975 cell lines using CRISPR/Cas9-RNP.

RESULTS Of seven patients with NSCLC with de novo EGFR^T790M/L858R mutation, four (LC1-4) who received third-generation EGFR TKIs acquired resistance after achieving a partial response (median = 27 mo, range: 17-48 mo). Novel MTOR^L1433S and EGFR^C797S/L798I mutations in cis, MET amplification, and EGFR^C797S mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs. The MTOR^L1433S mutation was oncogenic in Ba/F3 models and revealed resistance to osimertinib through AKT signaling activation in NCI-H1975 cells harboring the MTOR^L1433S mutation edited by CRISPR/Cas9 (half-maximal inhibitory concentration, 800 ± 67 nM). Osimertinib in combination with mTOR inhibitors abrogated acquired resistance to osimertinib.

CONCLUSIONS Activation of bypass pathways and the EGFR^C797S or EGFR^C797S/L798I mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs in patients with NSCLC with de novo EGFR^T790M mutation. In addition, MTOR^L1433S- and EGFR^L858R/T790M-mutant NSCLC cells were sensitive to osimertinib plus mTOR inhibitors.