single-cell sequencing of BRCA2 depleted HeLa cells treated with PARP and ATR inhibitors

Poly(ADP-ribose)-polymerase (PARP) inhibitors are selectively cytotoxic in homologous recombination (HR)-defective cancer cells, for instance due to BRCA1/2 or mutations. However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knock-out cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors to accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Combined, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.

聚腺苷二磷酸核糖聚合酶（Poly(ADP-ribose)-polymerase, PARP）抑制剂在同源重组（homologous recombination, HR）缺陷的癌细胞中具有选择性细胞毒性，例如因BRCA1/2基因突变或其他突变所致。然而，并非所有HR缺陷型肿瘤都能有效响应PARP抑制剂治疗，且易产生获得性耐药。因此，阐明PARP抑制剂诱导细胞毒性的机制，并开发联合策略以增强HR缺陷型肿瘤中PARP抑制剂的疗效，具有重要意义。本研究发现，在BRCA2敲低及BRCA2基因敲除的癌细胞系模型中，抑制ATR激酶可迫使细胞提前进入有丝分裂，从而增强奥拉帕利（olaparib）介导的PARP抑制剂细胞毒性效应。单分子DNA纤维分析结果显示，ATR抑制并不会加剧复制叉降解。相反，我们观察到ATR抑制剂可加速细胞进入有丝分裂，进而导致染色质桥与滞后染色体的形成。此外，通过全基因组单细胞测序，我们证实ATR抑制可增强经奥拉帕利处理的BRCA2敲低细胞的基因组不稳定性。通过抑制细胞周期蛋白依赖性激酶1（CDK1）以延缓有丝分裂进程，可减轻ATR抑制所引发的有丝分裂异常及基因组不稳定性，这进一步印证了ATR在DNA损伤情境下协调细胞周期正常时序的关键作用。此外，我们发现奥拉帕利处理会导致BRCA2缺陷细胞中微核数量增加，并伴随cGAS/STING通路相关的炎症应答。ATR抑制还可进一步增加经奥拉帕利处理的BRCA2缺陷癌细胞中cGAS阳性微核的数量，以及细胞因子的产生水平。综上，本研究证实ATR抑制可诱导细胞提前进入有丝分裂，并与PARP抑制剂协同增强HR缺陷癌细胞的细胞毒性，这一过程涉及增强的基因组不稳定性及炎症信号通路激活。