Abstract HER2 amplification has been identified in 2-5% of patients with EGFR-mutant lung cancer who have acquired resistance to osimertinib; however, its precise mechanisms in mediating resistance remain unclear. This study aims to elucidate how HER2 aberration contributes to acquired resistance to osimertinib through multiple preclinical approaches. EGFR-mutant cell lines stably overexpressing HER2 were generated by lentiviral transduction using PC9 (EGFR exon 19 deletion) and H1975 (EGFR L858R). Although HER2-overexpressing cells displayed comparable short-term (3-day) sensitivity to osimertinib in vitro relative to empty-vector controls, long-term assays (7 days) revealed a remarkable increase in resistant colony formation across both HER2-overexpressing models. Next, xenograft models bearing HER2-overexpressing cells were established in nude mice to evaluate in vivo responses to osimertinib. Continuous oral administration of osimertinib induced initial tumor shrinkage in HER2-overexpressing xenografts; however, these tumors subsequently demonstrated pronounced regrowth, whereas parental tumors exhibited more durable responses. Finally, the combination of a HER2-targeted tyrosine kinase inhibitor and osimertinib synergistically suppressed tumor cell growth in HER2-overexpressing EGFR-mutant cells. In conclusion, HER2 overexpression induced acquired resistance to osimertinib, whereas dual HER2/EGFR blockade overcame this resistance, highlighting this approach as a promising therapeutic strategy for EGFR-mutant lung cancer. Citation Format: Gaku Yamamoto, Yu Tanaka, Miku Tsukuda, Sato Sayaka, Saori Matsui, Tetsuya Sakai, Hiroki Izumi, Eri Sugiyama, Yoshitaka Zenke, Shigeki Umemura, Kiyotaka Yoh, Koichi Goto, Hibiki Udagawa, . HER2 overexpression confers osimertinib resistance in EGFR-mutant lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3297.
Yamamoto et al. (Fri,) studied this question.