Abstract 1776: Loss of estrogen receptor alters drug responsiveness and supports a basal-like phenotype in endocrine-resistant HER2+/ER+ breast cancer
Authors: Bahnassy Shaymaa , Stires Hillary , Jin Lu , Tam Stanley , Dunn Yasmin J. , Kohrn Brendan F. , Loeb Lawrence A. , Balachandran Manasi , Podar Mircea , McCoy Matthew D. , Beckman Robert A. , Riggins Rebecca B. 
Published: June 2022

Abstract
At least 50% of human epidermal growth factor 2 (HER2) enriched breast cancer (BC) is estrogen receptor positive (ER+). In clinical trials, patients with HER2+ BC expressing ER respond poorly to neoadjuvant anti-HER2 therapy versus those lacking ER expression. Additionally, combining anti-HER2 with endocrine therapy (ET), like aromatase inhibitor (AI), does not significantly improve pathological complete response in HER2+/ER+ patients. In advanced or de novo HER2+/ER+ metastatic BC (MBC), treatment with ET plus anti-HER2 yields the highest 5-year overall survival. Unfortunately, in the real-world setting outside of clinical trials, just 23% of patients receive this combination, and nearly 40% receive ET alone. The clinical challenge associated with treating advanced HER2+/ER+ BC demands a better understanding of ER signaling and downstream pathways in HER2-driven BC. Our goal is to identify alternative targeted therapies and/or tailor therapeutic combinatorial strategies to treat and/or delay progression of HER2+/ER+ BC. In this study, we established two long-term estrogen (E2) deprived (LTED) HER2+/ER+ cell line models from BT-474 (BT) and MDA-MB-361 (MM) to mimic endocrine therapy resistance (ETR) to AI, and characterized the response of these cell lines to anti-HER2 and other ETs. We also performed whole-genome sequencing (WGS) and single-cell RNA sequencing (scRNAseq) to identify differences between parental and derived ETR-LTEDs. The ER expression was retained in BT-LTEDs but lost in MM-LTEDs as compared to parental cells. Additionally, ER transcriptional activity was confirmed by increased expression of ER-target genes upon E2 stimulation only in BT-LTEDs. However, both LTED variants were less responsive to fulvestrant and showed upregulation of pro-survival AKT signaling. Focusing on the now ER- MM-LTED model, WGS and targeted duplex sequencing identified a significant increase in exonic missense mutations, notably C&gt;T and C&gt;A. Several of the mutated genes encode for transcription and chromatin regulatory factors. scRNAseq analysis showed that MM-LTEDs shift from a luminal- to more basal-like phenotype, with enrichment of genes that regulate immune response and cell motility. Thus, loss of ER expression in MM-LTEDs on the mRNA and protein levels may explain observed intrinsic resistance to fulvestrant and gain of the basal-like phenotype. In summary, we report that our ETR BT- and MM-LTED models capture distinct ER-associated phenotypes of HER2+/ER+ BC. Additional studies are necessary to understand the functional significance of these missense mutations on the development of drug resistance in HER2+/ER+ BC. Ongoing studies are testing pharmacological inhibition of key upregulated genes associated with the basal phenotype to potentially delay disease progression and provide better treatments for ETR HER2+/ER+ BC.
Citation Format: Shaymaa Bahnassy, Hillary Stires, Lu Jin, Stanley Tam, Yasmin J. Dunn, Brendan F. Kohrn, Lawrence A. Loeb, Manasi Balachandran, Mircea Podar, Matthew D. McCoy, Robert A. Beckman, Rebecca B. Riggins. Loss of estrogen receptor alters drug responsiveness and supports a basal-like phenotype in endocrine-resistant HER2+/ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1776.