A comprehensive genetic analysis of metastatic ER+ breast cancer tumors identified mutations in the ligand binding domain (LBD) of ESR1 in 14 out of 80 cases. These mutations, including p.Tyr537Ser/Asn and p.Asp538Gly, were associated with hormone resistance. Molecular dynamics simulations suggest that these mutations promote the agonist conformation of the estrogen receptor (ER), leading to increased ER activity and reduced response to ER antagonists. These findings indicate that LBD mutations in ER may play a key role in clinical resistance to hormonal therapy. The study also showed that these mutations are enriched in tumors that have relapsed while on hormonal therapy, suggesting they may contribute to acquired resistance. Functional studies demonstrated that these mutations promote ER-dependent transcription and proliferation in the absence of hormone, and that they are less sensitive to ER antagonists. The mutations were found to be located within the LBD/activation function-2 (AF-2) domain of the receptor, with most occurring at amino acids 537 and 538 of helix 12. The study also showed that these mutations can promote hormone-independent ER activity and that they may be resistant to ER-targeted therapies. Molecular dynamics simulations further revealed that these mutations stabilize the agonist conformation of ER, which may contribute to their functional activity. The study highlights the importance of understanding the mechanisms of acquired resistance to hormonal therapy and suggests that more potent ER antagonists may be beneficial for patients with these mutations. The findings have important implications for the development of more effective therapies for hormone-resistant breast cancer.A comprehensive genetic analysis of metastatic ER+ breast cancer tumors identified mutations in the ligand binding domain (LBD) of ESR1 in 14 out of 80 cases. These mutations, including p.Tyr537Ser/Asn and p.Asp538Gly, were associated with hormone resistance. Molecular dynamics simulations suggest that these mutations promote the agonist conformation of the estrogen receptor (ER), leading to increased ER activity and reduced response to ER antagonists. These findings indicate that LBD mutations in ER may play a key role in clinical resistance to hormonal therapy. The study also showed that these mutations are enriched in tumors that have relapsed while on hormonal therapy, suggesting they may contribute to acquired resistance. Functional studies demonstrated that these mutations promote ER-dependent transcription and proliferation in the absence of hormone, and that they are less sensitive to ER antagonists. The mutations were found to be located within the LBD/activation function-2 (AF-2) domain of the receptor, with most occurring at amino acids 537 and 538 of helix 12. The study also showed that these mutations can promote hormone-independent ER activity and that they may be resistant to ER-targeted therapies. Molecular dynamics simulations further revealed that these mutations stabilize the agonist conformation of ER, which may contribute to their functional activity. The study highlights the importance of understanding the mechanisms of acquired resistance to hormonal therapy and suggests that more potent ER antagonists may be beneficial for patients with these mutations. The findings have important implications for the development of more effective therapies for hormone-resistant breast cancer.