Abstract 7220: Zongertinib demonstrates potent efficacy against cancer cells harboring HER2 non-tyrosine kinase domain mutations

Abstract Activating mutations in HER2 are found in approximately 2-4% of non-small cell lung cancer (NSCLC). Recurrent activating mutations most commonly occur in the tyrosine kinase domain (TKD) encoded by exons 18-21, with exon 20 insertions being the most frequent, followed by non-TKD mutations in the transmembrane domain (TMD) and juxtamembrane domain (JMD). While tyrosine kinase inhibitors (TKIs) such as the HER2-selective inhibitor zongertinib and the pan-HER inhibitors poziotinib, pyrotinib, and afatinib have been tested primarily in patients harboring TKD mutations, their efficacy against non-TKD mutations has been less well characterized. To systematically evaluate HER2 TKI activity against HER2 non-TKD mutations, we established a panel of Ba/F3 cell lines expressing various HER2 mutations including common TKD mutations (e.g. exon 20 insertions YVMA, GSP, and VC), TMD mutations such as V659E, and JMD mutations such as R678Q and evaluated drug sensitivity by Cell Titer Glo assay. Among HER2 TMD mutations, zongertinib showed potent activity with an average IC50 value of 8.72 nM. For HER2 JMD mutations, zongertinib demonstrated an IC50 value of 0.35 nM, and among HER2 TKD mutations, zongertinib treatment yielded an average IC50 value of 6.14 nM. Moreover, calculation of the relative IC50 value, representing the ratio of IC50 values between HER2 mutations and wild-type EGFR, revealed that zongertinib which had an average log IC50 mutant HER2/wild-type EGFR ratio of -2.31, -3.71, -2.46, for TMD, JMD, and TKD HER2 mutations, respectively, exhibited superior selectivity compared to pyrotinib (TMD -1.00, JMD -4.85, TKD -0.55) and poziotinib (TMD 0.09, JMD -3.13, TKD 0.48). This suggests zongertinib has high efficacy and a potentially lower risk of EGFR inhibition-associated toxicity. In addition, we generated a HER2 TMD mutant NSCLC cell line model by expressing HER2 V659E in HCC4006 cells and knocking out endogenous mutant EGFR. Consistent with our results obtained using Ba/F3 models, HCC4006 HER2 V659E cells were sensitive to HER2 inhibition with zongertinib. Our findings indicate that zongertinib demonstrates comparable or more potent inhibitory activity for HER2 non-TKD mutants (e.g TMD and JMD) as HER2 TKD mutations, providing a potential therapeutic option for patients with these HER2 non-TKD mutant cancers. Citation Format: Yuji Shibata, Monique B. Nilsson, Hibiki Udagawa, Jacqulyne P. Robichaux, Junqin He, Xiaoxing Yu, John V. Heymach. Zongertinib demonstrates potent efficacy against cancer cells harboring HER2 non-tyrosine kinase domain mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7220.