Abstract 3877: Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer

Abstract The estrogen receptor-positive (ER+) subtype accounts for 70% of breast cancer, and endocrine therapies that target ER are effective in the early-stage clinical setting. However, endocrine resistance is ubiquitous in late-stage metastatic breast cancer (MBC). The chief mechanism is the acquisition of ER mutations (ERmuts) in 25% of endocrine-resistant tumors; ERmuts are strongly resistant to aromatase inhibitor (AI) endocrine therapy. We previously identified four novel acquired activating mutations in human epidermal growth factor receptor 2 (HER2) by whole exome sequencing (WES) of paired ER+ MBC in a clinical cohort. These HER2 mutations (HER2muts) were enriched in multiple resistant tumors, and like ERmuts, were resistant to AIs. In contrast to ERmuts, these HER2muts also conferred complete resistance to all other classes of endocrine therapy, as well as cross-resistance to combination therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors; the latter is standard-of-care in the refractory setting in ER+ MBC. At the same time, these HER2muts had variable responses to HER2-targeting therapies used in HER2+ breast cancer. Thus, HER2muts represent only the second validated mechanism of clinical endocrine resistance in ER+ MBC, accounting for ~8% of cases, and that is therapeutically intractable. Systematic phenotypic characterization of HER2muts in ER+ MBC is a prerequisite for effective targeting of this class of resistance. We used cBioPortal to identify >20 novel HER2 variants of unknown significance (VUS) that were frequent pan-cancer, in ER+ breast tumors, or annotated in a domain-restricted HER2 saturation mutagenesis screen, and that had not been previously characterized in ER+ MBC. We also identified a novel HER2 VUS by WES in an ER+/HER2+ tumor with resistance to HER2 therapy, from a clinical cohort of breast cancer patients. We phenotypically characterized all VUS through clinically comprehensive targeted therapy viability assays, and downstream activation assays. In sum, we identified seven novel activating HER2muts, spanning domains, with clinically important phenotypes in ER+ MBC. We found these HER2muts also conferred complete resistance to all classes of endocrine therapies, and cross-resistance to CDK4/6 inhibition. While novel HER2muts had variable responses to HER2-targeting therapies, they could be endocrine re-sensitized by combination with an irreversible tyrosine kinase inhibitor. Lastly, we report that constitutive HER2 homodimerization via reduction-sensitive disulfide bridges that occur as a consequence of mutations in the extracellular and juxtamembrane domains is a more common mechanism of HER2 hyperactivation than previously appreciated. This finding has unique functional consequences, and provides important structure-function insight into HER2 biology, beyond its immediate clinical ramifications in ER+ MBC. Citation Format: Nelson Vicente Guevara, Zirui Zhu, Esha Jain, Jennifer Kavran, Selime Arslan, Nikhil Wagle, Utthara Nayar. Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3877.