Abstract 999: ESR1 mutations activate and confer hormone resistance via distinct mechanisms
Authors: Toy Weiyi , Carlson Kathryn E. , Martin Teresa A. , Mayne Christopher G. , Fanning Sean W. , Razavi Pedram , Baselga José , Shen Yang , Greene Geoffrey , Katzenellenbogen Benita , Katzenellenbogen John , Chandarlapaty Sarat 
Published: July 2017

Mutations in the ligand-binding domain (LBD) of ESR1 have been identified in the tumors and plasma of hormone-resistant, metastatic ER+ breast cancer patients. The most prevalent alterations are Y537S and D538G, and both mechanistic and clinical investigations have focused on the implications of these 2 mutations. Through large-scale clinical sequencing efforts, we have examined the coding region of ESR1 from over 1000 cases of metastatic breast cancer and found a diversity of ESR1 mutations. These have included a number of never before reported alterations including mutations in the DNA binding domain and dimerization interface. The biochemical and biologic impact of many of these mutations is unknown. In this study, we have examined the impact of these alterations on ER conformation, transcriptional activity, breast cancer growth, and drug sensitivity. Using transcriptional assays, we found several classes of mutation including: (1) mutations that weakly promoted ligand-independent activity, (2) mutations that led to ligand-independent activity comparable to estradiol stimulation, and (3) mutations that impaired transcriptional activity. Among mutations that promoted some level of ligand-independent activation, we examined whether they had similar effects on receptor conformation in vitro. First, we characterized a subset using a FRET-based co-activator recruitment assay and found that, unlike Y537S and D538G, several mutants (e.g. E380Q and S463P) were unable to recruit SRC in the absence of estradiol. In keeping with this observation, trypsin digestion assays also revealed differences in the local structure within helix 11 between these mutants. To further characterize the molecular basis for these differences, we conducted molecular dynamic (MD) modeling of mutants and compared these with the MD and crystal structure models of apo Y537S and D538G mutants. These studies further revealed differences in overall receptor conformation including localization of H12. From a therapeutic point of view, we examined the effect of different mutants on sensitivity to various ER antagonists. Once again, we observed important differences between mutants, with several mutants of class 2 exhibiting reduced sensitivity to SERDs compared to mutants of class 1. Nevertheless, it appeared that all mutants could be effectively antagonized by more potent SERDs, implying a continued ability of the mutant ERs to become distorted into the antagonist conformation. Taken together, the data reveal distinct conformational restrictions on ER activity that can be relieved by different ESR1 mutations. These data also imply the value of more broad coverage of ER in clinical sequencing efforts in order to effectively capture all potential resistance alleles.

Citation Format: Weiyi Toy, Kathryn E. Carlson, Teresa A. Martin, Christopher G. Mayne, Sean W. Fanning, Pedram Razavi, José Baselga, Yang Shen, Geoffrey Greene, Benita Katzenellenbogen, John Katzenellenbogen, Sarat Chandarlapaty. ESR1 mutations activate and confer hormone resistance via distinct mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2017-999