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.

中文翻译：

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靶向降解人乳腺癌中激活的雌激素受体α配体结合域突变。

目的 研究已经在内分泌治疗耐药的转移性 ER 阳性乳腺癌中鉴定了几种雌激素受体 α (ERα) 配体结合域 (LBD) 体细胞突变。最常见的突变 Tyr537Ser (Y537S) 和 Asp538Gly (D538G) 在约 30% 的内分泌耐药转移性乳腺癌患者中检测到。这些 ESR1 突变诱导 ERα 的激动剂构象，赋予雌激素非依赖性表型，并促进对抗雌激素药物的耐药性。方法 ER 阳性、雌激素依赖性 MCF-7 细胞被设计为使用 CRISPR 敲入（cY537S 和 cD538G）表达 Y537S 或 D538G 突变体。这些细胞用于筛选使用蛋白水解靶向嵌合体 (PROTAC) 方法合成的几种雌激素受体降解剂 (ERD) 化合物，以通过泛素-蛋白酶体途径诱导 ERα 降解。结果野生型 MCF-7 和 ERα LBD 突变细胞用 ERD-148 (10 pM-1 µM) 处理，并使用 PrestoBlue 细胞活力测定法测定细胞增殖。ERD-148 在 MCF-7、cY537S 和 cD538G 细胞中减弱 ER 依赖性生长，IC50 值分别为 0.8、10.5 和 6.1 nM。蛋白质印迹分析显示，与突变体相比，用 1 nM ERD-148 处理 24 小时的 MCF-7 细胞表现出降低的 ERα 蛋白表达。在 10 nM 的 ERD-148 处理 24 小时后，ER 调节基因 GREB1 在亲本和突变细胞中表现出显着下调。在 ER 阳性细胞中观察到的~IC90 处，ERD-148 未抑制 ER 阴性、不依赖雌激素的 MDA-MB-231 乳腺癌细胞的生长。