Abstract 3675: HER2 missense mutations in breast cancer cells do not alter HER2 internalization or sensitivity to T-DM1
Authors: Akamatsu Hiroaki , Mino Rosa , Mettlen Marcel , Ye Dan , Sudhan Dhivya R. , Lin Albert , Marin Arnaldo , Servetto Alberto , Lee Kyung-min , Chatterjee Sumanta , Schmid Sandra L. , Arteaga Carlos L. , Hanker Ariella B. 
Published: September 2020

Abstract
Background: Activating mutations in ERBB2 (HER2) are found in ~2% of solid tumors, including ~5% of metastatic breast cancers. The spectrum of HER2 mutations varies according to tumor type, with kinase domain (KD) missense mutations being most common in breast cancer, while exon 20 insertions are most common in non-small cell lung cancer (NSCLC). Breast cancer cells engineered with activating HER2 mutations show elevated HER2 phosphorylation and enhanced oncogenic growth. Activating mutations of other receptor tyrosine kinases such as EGFR affect receptor internalization, but whether HER2 mutations alter receptor internalization is not yet known. In addition, clinical responses to HER2 antibody-drug conjugates (ADCs) like T-DM1 have been noted in HER2-mutant NSCLC, raising the possibility that HER2 mutations alter receptor internalization, thus enhancing ADC sensitivity. Therefore, we aimed to 1) determine whether mutant HER2 receptor internalization is altered, and 2) determine whether altered internalization contributes to HER2 ADC sensitivity in HER2-mutant breast cancer.
Methods: HER2 subcellular localization was investigated by immunofluorescence (IF) in MCF7 cells where HER2L755S, HER2V777L, or HER2L869R (or HER2WT) have been ‘knocked-in' using AAV-mediated recombination. We used ELISA-based endocytosis assays to quantify the internalization efficiency of trastuzumab-labeled HER2 in cell lines with WT and mutant HER2. Cell viability upon treatment with T-DM1 was tested using conventional 2D and 3D growth assays.
Results: Partial colocalization of HER2 with clathrin, measured by IF, was observed in MCF7 cells, which was not affected by HER2 mutations. In the endocytosis assay, trastuzumab was inefficiently internalized over a period of 4 hours. In MCF7 or MCF10A breast epithelial cells, introduction of HER2L755S, HER2V777L, or HER2L869R did not significantly affect internalization following trastuzumab labeling, or after stimulation with EGF or neuregulin. In line with these results, MCF7 cells expressing HER2L755S, HER2V777L, or HER2L869R were not sensitive to T-DM1 in 2D cell viability assays or 3D Matrigel assays. In contrast, HER2 internalization was significantly more efficient in BT474 cells with wild-type HER2 amplification, which was accompanied by exquisite sensitivity of BT474 cells to T-DM1 both in vitro and in vivo. HER2 endocytic efficiency was significantly increased in MCF10A cells ectopically expressing HER2S310F, a mutation in the extracellular domain, or HER2A775&gt;YVMA, a common insertion mutation in NSCLC.
Conclusion: Breast cancer cells expressing HER2 KD missense mutations do not exhibit enhanced HER2 internalization and are not sensitive to T-DM1. However, cells with the HER2S310F mutation and the HER2A775&gt;YVMA insertion showed enhanced HER2 endocytosis, consistent with clinical responses to T-DM1 in NSCLC patients with these mutations.
Citation Format: Hiroaki Akamatsu, Rosa Mino, Marcel Mettlen, Dan Ye, Dhivya R. Sudhan, Albert Lin, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Sumanta Chatterjee, Sandra L. Schmid, Carlos L. Arteaga, Ariella B. Hanker. HER2 missense mutations in breast cancer cells do not alter HER2 internalization or sensitivity to T-DM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3675.