Afatinib Overcomes Osimertinib Resistance via Egfr V804F Mutation in a Syngeneic Egfr ‐Mutant Lung Cancer Mouse Model

Osimertinib is the standard first‐line treatment for advanced EGFR ‐mutant non‐small cell lung cancer. However, acquired resistance invariably develops, and identifying novel resistance pathways is clinically important as a substantial portion remains undefined. We used an immunocompetent syngeneic lung cancer mouse model harboring an Egfr exon 19 deletion (mDEL tumors) to establish acquired resistance. Osimertinib‐resistant tumors were generated in vivo using a drug‐holiday/re‐challenge protocol. The resistance mechanism was identified using Sanger sequencing, receptor tyrosine kinase arrays, and western blotting. Egfr V804F knock‐in cells were generated using CRISPR/Cas9, and their sensitivity to osimertinib and afatinib was assessed in vitro and in vivo. We established two distinct osimertinib‐resistant tumor lines in a syngeneic lung cancer mouse model (mDEL OsiR #1/#3). The analysis revealed an on‐target secondary Egfr V804F mutation (corresponding to human EGFR V802F) in both tumor lines. Importantly, Egfr V804F knock‐in cells demonstrated significant osimertinib resistance, with a 5.7–10.5‐fold increase in in vitro IC 50 (mean, 8.2‐fold), but remained highly sensitive to afatinib. Furthermore, afatinib effectively overcame osimertinib resistance in the V804F knock‐in cell‐derived mouse model. Consistently, afatinib treatment resulted in marked tumor shrinkage and suppression of EGFR signaling in the established mDEL OsiR #1/#3 in vivo. These findings establish secondary Egfr V804F/ EGFR V802F as an on‐target osimertinib resistance mechanism, providing a preclinical rationale for evaluating afatinib in biomarker‐selected patients harboring this alteration.