Abstract P4-02-09: The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer
Authors: Emch Michael J , Aspros Kirsten GM , Bruinsma Elizabeth S , Kalari Krishna R , Jones Calley J , Simone Brandon W , Goetz Matthew P , Hawse John R 
Published: March 2022

Abstract
Background:. Poor patient outcomes in triple negative breast cancer (TNBC) largely stem from a lack of understanding of therapeutic vulnerabilities and an insufficient armamentarium of effective drugs. The standard of care for most early stage TNBC patients continues to be (neo)adjuvant chemotherapy and radiation. Identifying additional therapeutic options for this subset of patients represents a major unmet need. We and others demonstrated that estrogen receptor beta (ERβ) is expressed in about 20% of TN tumors. Prior research has shown that ligand-mediated activation of ERβ decreases proliferation, invasion, and migration in TNBC cell lines. In vivo, ERβ suppresses the growth of cell line xenograft models and prevents the development of metastatic lesions in a ligand dependent manner. Mechanistically, we found that ERβ repurposes EZH2 to suppress oncogenic NFκB signaling and that pharmacologic inhibition of EZH2 diminishes ERβ function and its anti-cancer effects. This research has led to an ongoing clinical trial (NCT03941730) assessing the efficacy of estradiol in ERβ+ TNBC patients with chemorefractory disease. As with all therapies, patients will exhibit de novo and acquired resistance and therefore we sought to understand the mechanisms of resistance to ERβ targeted therapies. Methods:. Using multiple models of ERβ positive TNBC, we developed ERβ resistant cell lines through chronic exposure to estradiol and the ERβ specific agonist LY500307 over a period of 8 months. We employed RNA sequencing to characterize the transcriptomic changes which occurred following ERβ resistance. We profiled XIST expression across multiple publicly available datasets, including TCGA, Metabric, BEAUTY, and GTEx. XIST expression was also modulated using CRISPR/Cas9 to assess subsequent effects on TNBC cell biology, ERβ function, and response to ERβ targeted therapies. Results:. Our resistant cell line models of ERβ positive TNBC maintained ERβ expression, but were no longer growth inhibited by ERβ agonists. RNAseq revealed substantial differences comparing the transcriptome of resistant versus sensitive cell lines, of which the most increased transcript in the resistant setting was the lncRNA XIST. XIST is best known for its role in X-chromosome inactivation through recruitment and association with the PRC2 complex, and thus EZH2. However, little is known about its functions in breast cancer. We therefore assessed the expression levels of XIST in breast tumors and cell lines. XIST expression was highly variable in breast cancer, was found in a proportion of all breast cancer sub-types and did not correlate with ERβ status. However, XIST expression was up-regulated in ERβ expressing cell lines following long term estrogen treatment. No effects on XIST expression were identified in multiple ERα positive models following estrogen, SERM/SERD treatment, or estrogen deprivation. Furthermore, upregulation of XIST was not associated with resistance to Paclitaxel or Doxorubicin, suggesting XIST is not a part of a broad resistance phenotype. Strikingly, CRISPR mediated knockout of XIST in ERβ resistant cells completely re-sensitized cells to ERβ targeted therapies suggesting that XIST expression is a critical component of ERβ resistance. Conclusions:. ERβ represents a relevant therapeutic target that is being tested in clinical trials. Using multiple in vitro models, we provide evidence that XIST expression is sufficient to induce resistance to. ERβ targeted therapies in TNBC and may therefore represent a relevant biomarker for patient stratification. Further strategies to suppress XIST expression may elicit anti-cancer effects on their own and may resensitize a sub-set of ERβ resistant tumors to ERβ agonists.
Citation Format: Michael J Emch, Kirsten GM Aspros, Elizabeth S Bruinsma, Krishna R Kalari, Calley J Jones, Brandon W Simone, Matthew P Goetz, John R Hawse. The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-09.