L370F and Y537S ESR1 mutations determine distinct endocrine therapy sensitivities and metabolic vulnerabilities that define new opportunities for synergistic therapeutic combinations

Background. Somatic mutations in the ESR1 gene encoding estrogen receptor α (ERα) are major drivers of resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6in) in ERα+/HER2− advanced breast cancer (ABC). While clinical and preclinical efforts have largely focused on recurrent ERα ligand-binding domain hotspot mutations, the functional and therapeutic relevance of non-hotspot ERα variants remains poorly understood. Whether distinct ERα mutations confer mutation-specific drug sensitivities and actionable vulnerabilities beyond classical endocrine resistance remains largely unknown. Methods. We systematically compared the pharmacological and biological consequences of the non-hotspot L370F ERα mutation and the hotspot Y537S ERα mutation using stable HEK293 models and CRISPR/Cas9-engineered MCF-7 cells. A panel of ET drugs, including SERDs, SERMs, SERCAs, CERANs, and CDK4/6in, was evaluated for effects on cell proliferation, ERα transcriptional activity, receptor abundance, and in vitro binding affinity. Multivariate analyses and an integrated sensitivity score were applied to define mutation-specific drug responses. Metabolic profiling, transcriptomic analysis, and drug combination studies were used to identify mutation-dependent metabolic rewiring and synergistic therapeutic strategies. Results: L370F and Y537S ERα mutations confer distinct pharmacological profiles and non-overlapping vulnerabilities. Both mutations reduce ligand binding and confer resistance to CDK4/6in. However, L370F ERα expressing cells exhibit a predominantly oxidative metabolic phenotype, whereas Y537S ERα expressing cells enhanced glycolytic dependence, and increased susceptibility to ferroptosis. These metabolic states translate into selective sensitivity to oxidative phosphorylation inhibition in L370F ERα expressing cells and to glycolysis inhibition and ferroptosis induction in Y537S ERα expressing cells. Importantly, combining endocrine agents with metabolic inhibitors produces robust but mutation-specific synergistic antiproliferative effects, which vary depending on both the ERα variant and the compounds used. Conclusions: Our findings demonstrate that ERα mutations drive endocrine resistance through distinct, mutation-specific mechanisms that extend beyond receptor signaling to encompass cell-cycle control and metabolic reprogramming. Hotspot and non-hotspot ERα variants define unique therapeutic landscapes with differential sensitivity to endocrine agents, metabolic inhibitors, and drug combinations. These results provide a strong rationale for mutation-informed therapeutic stratification and support the development of precision combination strategies tailored to individual ERα mutations in ABC.