Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer

Purpose Studies have identified several estrogen receptor alpha (ER alpha) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in similar to 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ER alpha, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens. Methods ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ER alpha via the ubiquitin-proteasome pathway. Results Wild-type MCF-7 and ER alpha LBD mutant cells were treated with ERD-148 (10 pM-1 mu M) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1 nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1 nM ERD-148 for 24 h exhibited reduced ER alpha protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24 h of ERD-148 treatment at 10 nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the similar to IC90 observed in the ER-positive cells. Conclusion ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ER alpha with comparable potency to Fulvestrant with marginal non-specific toxicity.