Summary This proposal studies how drug-induced structural changes to Y537S estrogen receptor alpha (ERα) impact anti- tumoral activities in hormone-resistant breast cancer cells. Breast cancer is the second leading cause of cancer death in the United States. Acquired resistance to hormone therapies is a leading contributor to mortality. In approximately 40% of progressive ER+ patients, prolonged selective pressure by antiestrogenic therapies gives raise to tumors bearing activating somatic ESR1 (the gene for ERα) mutations. These mutations resist inhibition by clinically approved hormone therapies and engage new transcriptional programs that boost metastatic potential. Y537S missense mutation is among the most common and enables the greatest hormone-free transcriptional activities and resistance to antiestrogen. Next generation selective estrogen receptor degraders (SERDs) have been clinically deployed to address this mechanism of drug resistance. However, they show variable activities in Y537S ESR1 breast cancers and possess common side-effects that will limit their long-term use. We recently studied how a panel of 17 selective estrogen receptor modulators (SERMs) and SERDs bind to and affect Y537S ERα activities in breast cancer cells. We identified structurally distinct SERMs and SERDs with improved activities in this setting. While structurally distinct, our x-ray co-crystal structures showed that the most effective molecules engaged the same S537-E380 hydrogen bond to reinforce the therapeutic antagonist conformation. Therefore, we hypothesize that novel ligand-dependent structural interactions will improve therapeutic antagonistic activities in the Y537S ESR1 setting. In this study, we will leverage our library of over 100 diverse SERMs and SERDs to reveal the structural-transcriptional relationships that underlie improved anti- cancer activities Y537S ESR1 breast cancer cells. We will start by studying how our library binds to and affects Y537S ERα structure and anti-cancer activities (Aim 1). This approach will reveal the ligand binding modes and structural interactions that enable potency. Next, we will study how the most effective molecules impact Y537S ERα genomic activities including protein-protein interactions, genome binding, and transcriptional programing (Aim 2). This approach will show whether the efficacies of SERMs and SERDs arise from alterations to Y537S ERα genomic activities. Finally, we will reveal the anti-tumor and tissue-specific activities of the most effective SERMs and SERDs in hormone-resistant ER+ breast cancer in vivo (Aim 3). This approach will reveal whether our in vitro observations correspond to improved anti-cancer activities in patient-relevant tumor models. Overall, these studies will provide detailed structural-transcriptional relationships to improve therapeutic targeting of Y537S ERα in hormone-resistant breast cancer.