TIMELESS haploinsufficiency induces a PARG-dependency that is rescued by nucleoside supplementation, thus mimicking intrinsic PARG inhibitor sensitivity of ovarian cancer cells

A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the oncogenic vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analyzed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork-restart ability, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential, in both a TIMELESShaploinsufficient model and intrinsically sensitive cells. Importantly nucleoside supplementation restores PARG inhibitor resistance without reverting PAR chain accumulation, indicating that sensitivity correlates with PAR chain intolerance. We show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency, and, using label-free, quantitative proteomics, we show that PKMYT1, a negative regulator of CDK1, is overexpressed in PARG inhibitor-sensitive cells. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances, in turn applying selective pressure on mitotic entry controls to maintain genome integrity.

部分癌细胞对靶向PARG（多聚ADP核糖水解酶，poly(ADP-ribose) glycohydrolase）的抑制剂具有内在敏感性，PARG是一种可降解聚ADP核糖（PAR）链的酶。该敏感性伴随持续性DNA复制应激，且可通过抑制复制体加速因子TIMELESS来诱导。然而，介导内在敏感性的致癌脆弱性本质仍未明确。为阐明PARG活性依赖性机制，本研究分析了TIMELESS模型系统及内在敏感的卵巢癌细胞。研究结果显示，在TIMELESS单倍体不足模型与内在敏感细胞中，核苷补充疗法可挽救所有与PARG抑制剂敏感性相关的表型，包括复制体速度、复制叉重启能力、S期完成与有丝分裂进入、增殖动力学及集落形成能力。值得注意的是，核苷补充疗法可恢复PARG抑制剂耐药性，且不会逆转PAR链积累，这表明敏感性与PAR链不耐受性相关。本研究还发现，维持脱氧核苷三磷酸（dNTP）稳态所需的胸苷酸合酶受抑制后可诱导PARG依赖性；通过无标记定量蛋白质组学分析，证实CDK1的负调控因子PKMYT1在PARG抑制剂敏感细胞中存在过表达。综上，上述结果表明，PARG抑制剂敏感性反映了细胞在核苷酸失衡状态下无法调控复制体速度和/或维持解旋酶-聚合酶偶联，进而对有丝分裂进入调控机制施加选择压力以维持基因组完整性。