PARP 抑制剂 (PARPi) 对同源重组修复 (HRR) 缺陷 (HRD) 癌症有效。正在探索使 HRR 熟练 (HRP) 肿瘤对 PARPi 与其他药物的组合（重新）敏感。我们的目的是确定细胞周期检查点激酶 ATR、CHK1 和 WEE1 抑制剂对 PARPi 敏感的机制。使用一组 HRD 和 HRP 细胞（包括匹配的 BRCA1 或 2 突变体和校正对）以及卵巢癌腹水细胞。 Rucaparib (PARPi) 诱导复制应激 (RS) 和 HRR（分别对 γH2AX 和 RAD51 灶进行免疫荧光显微镜检查）、细胞周期变化（流式细胞术）、ATR、CHK1 和 WEE1 的激活（分别对 pCHK1S345、pCHK1S296 和 pCDK1Y15 进行蛋白质印迹） ）和细胞毒性（集落形成测定），然后研究 ATR（VE-821，1μM）、CHK1（PF-477736，50nM）和 WEE1（MK-1775）抑制剂对所有这些参数的影响, 100 nM)。Rucaparib 诱导 RS（3 至 10 倍）、S 期积累（2 倍）和 ATR、CHK1 和 WEE1 激活（高达 3 倍），以及 VE-821、PF-477736 和 MK- 1775 抑制了它们的靶点并消除了 rucaparib 诱导的 HRP 和 HRD 细胞的细胞周期变化。 Rucaparib 仅在 HRP 细胞中激活 HRR，并且对 HRD 细胞的细胞毒性更强 (60-1,000 倍)。 VE-821、PF-477736 和 MK-1775 阻断 HRR 并使 HRP 敏感，但不会阻断 HRD 细胞和原发性卵巢腹水对 rucaparib 的影响。我们的数据表明，ATR、CHK1 和 WEE1 抑制剂不是通过废除细胞周期检查点来发挥作用，而是导致HRD 表型，从而通过 PARPi“诱导合成致死率”。© 2024。作者。

PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1.A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM).Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib.Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence "induced synthetic lethality" with PARPi.© 2024. The Author(s).