Abstract 7025: Mechanisms of zongertinib resistance in HER2-mutant non-small cell lung cancer and potential strategies to overcome resistance.

Abstract Activating mutations in HER2 are found in approximately 2-4% of non-small cell lung cancer (NSCLC). Zongertinib is currently the only FDA-approved tyrosine kinase inhibitor (TKI) for patients with HER2-mutant NSCLC. However, the mechanisms underlying acquired resistance remain unclear, and understanding zongertinib-resistance mechanisms is essential for developing subsequent effective therapeutic strategies in HER2-mutant NSCLC. To identify candidate genomic alterations in HER2 mediating zongertinib resistance, we analyzed HER mutation profiles in clinical samples from NSCLC patients after progression on zongertinib, reviewed previously reported resistance mutations from other HER2 TKIs, and employed the LentiMutate scanning mutagenesis system. Candidate HER2 resistance mutations identified through these approaches were then transduced into Ba/F3 cells and the effect on drug sensitivity was assessed. We detected secondary HER2 mutations including S783C, C805S, and T862A in post-zongertinib clinical samples in combination with the originally observed activating HER2 mutations or NRG1 fusions. Our LentiMutate analysis identified HER2 alterations including C805S, T862A, S783P as well as T798I and L726F as being enriched in zongertinib resistant cells. We next engineered Ba/F3 cells to express HER2 activating mutations (e.g. Y772dupYVMA) alone or in combination with potential secondary HER2 resistance mutations including S783C, T798I, C805S, or T862A, and observed that expression of these mutations rendered cells resistant to zongertinib in vitro. However, S783C and C805S mutations did not impact in vitro sensitivity to the HER2 TKI sevabertinib. Structural analysis revealed that T798I acts as a gatekeeper mutation, whereas S783C and T862A disrupt hydrogen bonds between the drug and HER2, and C805S disrupts a covalent bond, thereby weakening zongertinib-HER2 binding. Next, we implanted mice with tumor cells expressing HER2 activating mutations alone or in combination with S783C or C805S and evaluated the anti-tumor activity of zongertinib and sevabertinib. Consistent with our in vitro findings, tumors bearing C805S or S783C secondary mutations were resistant to zongertinib but sensitive to sevabertinib. Our findings identify novel HER2 mutations that mediate zongertinib resistance and indicate that sevabertinib may be effective against a subset of these genomic alterations. Citation Format: Yuji Shibata, Monique B. Nilsson, Ximeng Liu, Li (Lily) Cai, Hong Jiang, Linghzi Hong, Alvaro Guimaraes Paula, Hibiki Udagawa, Jacqulyne Ponville Robichaux, Junqin He, Xiaoxing Yu, John V. Heymach. Mechanisms of zongertinib resistance in HER2-mutant non-small cell lung cancer and potential strategies to overcome resistance [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 7025.