Abstract:
PARP inhibitors (PARPi) are efficacious in BRCA1-null tumors; however, their utility is limited in tumors with functional BRCA1. We hypothesized that pharmacologically reducing BRCA1 protein levels could enhance PARPi effectiveness in BRCA1 wild-type tumors. To identify BRCA1 downregulating agents, we generated reporter cell lines using CRISPR-mediated editing to tag endogenous BRCA1 protein with HiBiT. These reporter lines enable the sensitive measurement of BRCA1 protein levels by luminescence. Validated reporter cells were used in a pilot screen of epigenetic-modifying probes and a larger screen of more than 6,000 compounds. We identified 7 compounds that could downregulate BRCA1-HiBiT expression and synergize with olaparib. Three compounds, N-acetyl-N-acetoxy chlorobenzenesulfonamide (NANAC), A-443654, and CHIR-124, were validated to reduce BRCA1 protein levels and sensitize breast cancer cells to the toxic effects of olaparib. These results suggest that BRCA1-HiBiT reporter cells hold promise in developing agents to improve the clinical utility of PARPi.
摘要:
PARP抑制剂(PARPi)对BRCA1无效肿瘤有效;然而,它们的效用在具有功能性BRCA1的肿瘤中是有限的。我们假设药理学上降低BRCA1蛋白水平可以增强BRCA1野生型肿瘤中PARPi的有效性。为了识别BRCA1下调因子,我们使用CRISPR介导的编辑方法,用HiBiT标记内源性BRCA1蛋白,产生了报告细胞系.这些报道线使得能够通过发光灵敏地测量BRCA1蛋白水平。验证的报告细胞用于表观遗传修饰探针的先导筛选和超过6,000种化合物的更大的筛选。我们鉴定了7种可以下调BRCA1-HiBiT表达并与奥拉帕尼协同作用的化合物。三种化合物,N-乙酰基-N-乙酰氧基氯苯磺酰胺(NANAC),A-443654和CHIR-124被验证可降低BRCA1蛋白水平并使乳腺癌细胞对奥拉帕尼的毒性作用敏感。这些结果表明,BRCA1-HiBiT报告细胞有望开发药物以改善PARPi的临床应用。