PROJECT SUMMARY / ABSTRACT Estrogen receptor-alpha (ER), a member of the nuclear receptor family of transcription factors, drives proliferation of breast cancer cells and is expressed in the majority of breast cancers. Tumors that express ER account for the greatest number of breast cancer associated mortalities. While targeting the ligand- dependent functions of receptor is a successful approach in the clinical management of breast cancer, ER can be activated by other means. This alternative activity of ER can promote ligand-independent properties and our lack of understanding of this activity creates a barrier to the clinical challenge of therapy resistance. We have found that phosphorylation of ER can modulate the regulation of multiple aspects of ER biology in tumor cells, including its fate and transcriptional function, in some cases leading to ligand-independence and resistance to therapy. We recently described a genome-wide DNA binding (i.e. cistrome) analyses of phosphorylated ER, specifically phosphorylation at Ser 118, and found that it has unique properties, including a novel association with Grainyhead-like protein 2 (GRHL2), a developmental transcription factor that has since been recognized to play a role in breast cancer. Based on these novel findings and additional preliminary data, we hypothesize that pS118 and GRHL2 identify selective activities of ER that are necessary for growth of therapy-resistant tumors. Toward addressing this hypothesis, we developed three lines of research. Aim 1 exploits our cistromic analysis of pS118ER and a unique cohort of patient tumor samples to define, for the first time, the pS118ER activity in ER+ therapy-resistant tumors. Aim 2 will test the requirement for pS118ER and GRHL2 in growth and malignant phenotypes in cells bearing ER mutations, a genetic form of therapy resistance. This aim will use newly derived CRISPR edited cell models. Aim 3 will use novel DNA technologies to interrogate mechanisms of pS118ER and GRHL2 interactions on DNA and to develop DNA-targeting selective inhibitors. Collectively, these three aims will employ patient samples, in vitro and in vivo approaches and unique technologies to interrogate how a post- translationally modified variant of ER contributes to the aberrant ER activity in recurrent tumors.