PROJECT SUMMARY While there have been tremendous advances in the treatment of estrogen receptor-positive (ER+) breast cancer, late-stage ER+ metastatic breast cancer (MBC) has very poor prognosis. The chief reason for this is tumor endocrine resistance, wherein tumors do not respond to ER-targeting therapies. Endocrine resistance is the major reason for breast cancer mortality, and a primary challenge in the treatment of ER+ MBC. The most well known mechanism of endocrine resistance is hotspot activating mutations in ER that lead to ligand independence. These hotspot mutations are effectively inhibited by the newest class of endocrine therapy, novel oral selective ER degraders (SERDs), and serve as a biomarker for SERD responsive tumors. We found a novel non-hotspot ER variant of unknown significance, and double hotspot mutations (in cis, on the same ER molecule) from clinical whole-exome sequencing of tumors with acquired resistance to ER inhibition. These single and double mutations are frequent, with the latter present in ~5% of resistant tumors. Without preclinical characterization, these variants represent an unknown entity in terms of biology and therapy response. In pilot studies, we found they confer strong resistance to select ER and CDK4/6-targeting therapies, and that structure modeling suggests they are predicted to alter ER binding to its ligand, inhibitors, and/or transcriptional co-factors. Robust preclinical studies in well-established models of breast cancer are required before these findings can be translated to the clinic. In this application, we propose comprehensive preclinical characterization of these prevalent novel ER variants through two specific aims: 1) assess oncogenic potential and downstream signaling by novel ER variants, specifically to determine how they dysregulate downstream ER transactivation, through transcriptomic, epigenomic, and proteomic profiling, reporter assays, and structure modeling, and 2) profile their response to targeted therapies in cell line, patient-derived organoid, and animal xenograft models. At the end of the grant term, we expect to a) validate novel ER variants as potential biomarkers that may predict resistance to ER and CDK4/6 inhibitors, particularly the novel SERDs, b) identify the structural basis by which these novel variants, and particularly, double hotspot mutations dysregulate ER, and c) identify potential targetable nodes for tumors that acquire these novel ER mutations. Our results would help design clinical trials to overcome resistance, and that may avoid unnecessary additional burden of toxicity in heavily pre-treated patients. Developing biomarkers that predict response to oral SERDs will also enable the stratification of patients whose tumors express these novel ER variants onto alternate therapeutic regimens without delay (precision medicine). These studies have strong potential to reduce the mortality associated with breast cancer, and are thus, well-aligned with the m...