Recent approvals of polymeric adenosine diphosphate ribose (poly(ADP-ribose) polymerase inhibitors (PARPi) for BRCA-mutant metastatic castration resistant prostate cancer necessitate an understanding of the factors that shape sensitivity and resistance. Reversion mutations that restore homologous recombination (HR) repair are detected in ~50 to 80% of BRCA-mutant patients who respond but subsequently relapse, but there is currently little insight into why only ~50% of BRCA-mutant patients display upfront resistance. To address this question, we performed a genome-wide CRISPR screen to identify genomic determinants of PARPi resistance in murine Brca2 Δ/Δ prostate organoids genetically engineered in a manner that precludes the development of reversion mutations. Remarkably, we recovered multiple independent single guide RNAs (sgRNAs) targeting three different members ( Cdt1, Cdc6, and Dbf4 ) of the DNA prereplication complex (pre-RC), each of which independently conferred resistance to olaparib and the next-generation PARP-1 selective inhibitor AZD5305. Moreover, sensitivity to PARP inhibition was restored in Brca2 Δ/Δ , Cdc6-depleted prostate cells by knockdown of geminin, a negative regulator of Cdt1, further implicating the critical role of a functional pre-RC complex in PARPi sensitivity. Furthermore, ~50% of CRPC tumors have copy number loss of pre-RC complex genes, particularly CDT1 . Mechanistically, prostate cells with impaired pre-RC activity displayed rapid resolution of olaparib-induced DNA damage as well as protection from replication fork degradation caused by Brca2 loss, providing insight into how Brca2-mutant cancer cells can escape cell death from replication stress induced by PARP inhibition in the absence of HR repair. Of note, a pharmacologic inhibitor that targets the CDT1/geminin complex (AF615) restored sensitivity to AZD5305, providing a potential translational avenue to enhance sensitivity to PARP inhibition.

中文翻译：

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BRCA2 逆转突变 – 通过 DNA 前复制复合物功能受损对 PARP 抑制产生非依赖性耐药

最近批准聚合物二磷酸腺苷核糖（聚 （ADP-核糖） 聚合酶抑制剂 （PARPi） 用于 BRCA 突变的转移性去势抵抗性前列腺癌，需要了解影响敏感性和耐药性的因素。在 ~50% 至 80% 有反应但随后复发的 BRCA 突变患者中检测到恢复同源重组 （HR） 修复的逆转突变，但目前对为什么只有 ~50% 的 BRCA 突变患者表现出前期耐药知之甚少。为了解决这个问题，我们进行了全基因组 CRISPR 筛选，以确定小鼠 Brca2 Δ/Δ 前列腺类器官中 PARPi 耐药的基因组决定因素，这些类器官以排除逆转突变发展的方式进行基因工程改造。值得注意的是，我们回收了多个独立的单向导 RNA （sgRNA），靶向 DNA 复制前复合物 （pre-RC） 的三个不同成员 （Cdt1、Cdc6 和 Dbf4），每个成员都独立赋予对奥拉帕尼和下一代 PARP-1 选择性抑制剂 AZD5305 的耐药性。此外，通过敲低 Cdt1 的负调节因子 geminin，在 Brca2 Δ/Δ 、 Cdc6 耗尽的前列腺细胞中恢复了对 PARP 抑制的敏感性，进一步暗示了功能性前 RC 复合物在 PARPi 敏感性中的关键作用。此外，~50% 的 CRPC 肿瘤具有 pre-RC 复合基因的拷贝数丢失，尤其是 CDT1。从机制上讲，RC 前活性受损的前列腺细胞显示出奥拉帕尼诱导的 DNA 损伤的快速消退，以及对 Brca2 丢失引起的复制叉降解的保护，从而深入了解 Brca2 突变癌细胞如何在没有 HR 修复的情况下逃避 PARP 抑制诱导的复制应激引起的细胞死亡。 值得注意的是，靶向 CDT1/geminin 复合物 （AF615） 的药物抑制剂恢复了对 AZD5305 的敏感性，为增强对 PARP 抑制的敏感性提供了潜在的转化途径。