Thomas L Gonzalez1, Molly Hancock2,3, Siqi Sun4, Christina L Gersch2,3, Jose M Larios2,3, Wadie David2,3, Jiantao Hu2,3, Daniel F Hayes2,3, Shaomeng Wang2,3,4,5, James M Rae6,7,8. 1. Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, 48109, USA. 2. The University of Michigan Rogel Cancer Center, Ann Arbor, USA. 3. Department of Internal Medicine, University of Michigan Medical School, 6310 Cancer Center, 1500 E. Medical Center Dr., Ann Arbor, MI, 48109, USA. 4. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, USA. 5. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA. 6. The University of Michigan Rogel Cancer Center, Ann Arbor, USA. jimmyrae@med.umich.edu. 7. Department of Internal Medicine, University of Michigan Medical School, 6310 Cancer Center, 1500 E. Medical Center Dr., Ann Arbor, MI, 48109, USA. jimmyrae@med.umich.edu. 8. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, USA. jimmyrae@med.umich.edu.
Abstract
PURPOSE: Studies have identified several estrogen receptor α (ERα) 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 ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, 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 ERα via the ubiquitin-proteasome pathway. RESULTS: Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM-1 µM) 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 ERα 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 ~ IC90 observed in the ER-positive cells. CONCLUSION: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.
PURPOSE: Studies have identified several estrogen receptor α (ERα) 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 ~ 30% of endocrine resistant metastatic breast cancerpatients. These ESR1 mutations induce the agonist conformation of ERα, 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 ERα via the ubiquitin-proteasome pathway. RESULTS: Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM-1 µM) 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 ERα 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-231breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells. CONCLUSION: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.
Authors: Elizabeth P Darga; Emily M Dolce; Fang Fang; Kelley M Kidwell; Christina L Gersch; Steven Kregel; Dafydd G Thomas; Anoop Gill; Martha E Brown; Steven Gross; Mark Connelly; Michael Holinstat; Erin F Cobain; James M Rae; Daniel F Hayes; Costanza Paoletti Journal: PLoS One Date: 2021-11-15 Impact factor: 3.240