Abstract 2962: Enhanced HER2 internalization by clathrin-dependent endocytosis in non-small cell lung cancer positive for <i>HER2</i> mutations

Abstract Introduction: Mutations in the gene for human epidermal growth factor 2 (HER2) result in constitutive phosphorylation of the protein and are driver alterations in cancer. HER2 mutations are present at a frequency of 1% to 4% in non-small cell lung cancer (NSCLC), with exon-20 insertion mutations such as A775_G776insYVMA and G776delinsVC being among the most common of these latter mutations. Although HER2-targeted ADCs have shown marked efficacy for HER2 mutation-positive NSCLC, the detailed molecular mechanisms underlying such efficacy are not well understood. Methods: The intracellular trafficking and dimerization patterns of HER2 were examined in cells expressing wild-type (WT) or mutant forms of the protein with the use of live cell imaging and an in situ proximity ligation assay, respectively. Proteins that preferentially associate with mutant HER2 were identified by liquid chromatography and tandem mass spectrometry to shed light on the pathway of HER2 trafficking in HER2-mutated cells. Results: HER2 internalization was enhanced in NSCLC cells expressing mutant forms of HER2 compared with those expressing HER2(WT). Homodimers of HER2(WT) were localized mainly at the cell surface, whereas those of mutant HER2 were present mostly in the cytoplasm. Although siRNA interference of EGFR or HER3 effectively suppressed the HER2 internalization in cells stably expressing WT forms of HER2, such internalization was not affected in the cell lines expressing mutant forms of HER2. The enhanced internalization of mutant HER2 was mediated by clathrin-dependent endocytosis, as was reflected by increased binding of the ubiquitin ligase c-Cbl to mutant HER2 and its consequent ubiquitination. Zongertinib, a HER2-specific tyrosine kinase inhibitor, attenuated HER2 phosphorylation, the binding of c-Cbl to HER2, and HER2 internalization in HER2-mutated cells. Conclusions: Upregulation of HER2 phosphorylation promotes internalization of mutant forms of HER2 mediated by clathrin-dependent endocytosis, which may contribute to the efficacy of HER2-targeted ADCs in NSCLC positive for HER2 mutations. Citation Format: Atsushi Shimauchi, Eiji Iwama, Ritsu Ibusuki, Hirono Tsutsumi, Daisuke Shibahara, Kohei Otsubo, Yasuto Yoneshima, Kentaro Tanaka, Hiroyuki Inoue, Isamu Okamoto. Enhanced HER2 internalization by clathrin-dependent endocytosis in non-small cell lung cancer positive for HER2 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 2962.