Characterization Of Molecular, Phenotypic, And Drug Sensitivity Changes Due To Estrogen Receptor Alpha Mutations In Endometrial Cancer.

Estrogen signaling is essential for normal uterine function and has been shown to play critical roles in the development of endometrial cancer. Mutations in the ligand binding domain (LBD) of estrogen receptor alpha (ER) have been identified in primary endometrial cancer and hormone therapy resistant breast cancer. In endometrial cancer, ER mutations are associated with worse outcomes and obesity independent disease. However, additional molecular and phenotypic characterizations of these mutations are needed in the context of endometrial cancer. Utilizing a CRISPR/Cas9 tagging strategy to model the more common Y537S and D538G LBD mutations while epitope tagging ER’s endogenous locus in Ishikawa cells, we studied the regulatory and phenotypic consequences of mutant ER in endometrial cancer and have identified potential targeted therapies for the treatment of this disease. RNA sequencing (RNA-seq) experiments uncovered expression changes caused by ER mutations, including both estradiol-regulated genes and mutation-specific, estradiol-independent genes. We found mutation-specific changes in gene expression were not recapitulated with prolonged estrogen exposure in wildtype cells, suggesting the mutant ER transcriptome is distinct from constitutive ER activity. The unique mutant ER expression program was related to phenotypic changes in ER mutant endometrial cancer cells, including increased proliferation specifically in vivo and increased migration in vitro relative to wildtype cells, consistent with more aggressive endometrial tumors. Based on the transcriptional and phenotypic alterations caused by mutant ER, we explored gene regulation based drugs for the potential treatment of mutant ER endometrial tumors. We found that two small molecules, cyclin dependent kinase 9 (CDK9) inhibitor Alvocidib and steroid receptor coactivator 3 (SRC-3) inhibitor SI-2, decreased mutant ER’s ability to promote proliferation and migration. In conclusion, mutant ER confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells. This activity has led us to identify two drugs that show efficacy in treating mutant ER endometrial cancer cells, however further study is needed to ascertain whether they can be utilized as novel therapeutic targets in this disease. Citation Format: Zannel Blanchard, Jeffery M. Vahrenkamp, Spencer Arnesen, Kristofer C. Berrett, Jason Gertz. Characterization of molecular, phenotypic, and drug sensitivity changes due to estrogen receptor alpha mutations in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO012.