PROJECT SUMMARY/ABSTRACT Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies manifest intrinsic or acquired therapy resistance. One-third of these patients present with metastatic tumors containing ERα Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. Previous research suggests that ERα and the progesterone receptor (PR) engage in complex interactions involving reciprocal regulation of ERα and PR transcription factor activity known as ERα/PR crosstalk. Thus, there may be therapeutic value in targeting both nuclear receptors simultaneously in ERα/PR-positive breast cancers. However, preliminary data suggests that ERα/PR crosstalk is altered in the context of ERα Y537S, likely contributing to therapy resistance. Although the constitutive activity associated with ERα Y537S and the efficacy of combined ERα/PR therapies have independently been assessed, there has yet to be characterization of the effects of ERα Y537S on ERα/PR crosstalk or the specific effects of ERα/PR-targeted therapies on ERα/PR crosstalk in the context of ERα Y537S. The objective of this proposal is to determine the effects of the most commonly occurring, treatment-resistant ERα Y537S mutation on ERα/PR crosstalk and resultant transcriptional activity, and to elucidate how this unique interaction leads to endocrine therapy resistance in ERα- positive breast cancer. Previous research identified a synergistic effect of combined ERα/PR antagonism in ERα WT cancer cells, where combined treatment results in tumor regression in vivo. Conversely, preliminary data suggests that treatment of ERα Y537S tumors with combined ERα and PR antagonists results in significantly increased tumor proliferation in vivo. As previous research has highlighted a significant alteration in gene expression associated with ERα Y537S, it is likely that changes to both ERα- and PR-driven gene expression drive the altered response to ERα and PR antagonists. My central hypothesis is that ERα Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. This hypothesis will be assessed with the following Specific Aims: 1. Determine how the activating ERα Y537S point mutation affects ERα transcriptional activity and alters ERα/PR crosstalk. 2. Assess the efficacy of various selective estrogen and progesterone receptor modulators (SERMs and SPRMs) in treating patient-derived models of ERα Y537S tumors. Understanding the role of ERα Y537S in altering gene expression and reducing the response to SERM and SPRM treatment will provide understanding as to why these tumors are resistant to standard-of-care treatment and will additionally suggest alternative targets for treatment.