The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR–Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.

中文翻译：

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细胞周期蛋白 C 或 CDK8 的缺失通过抑制转录相关的复制应激来提供 ATR 抑制剂抗性


蛋白激酶 ATR 在 DNA 修复、细胞周期检查点参与和 DNA 复制中发挥着关键作用。因此，ATR 抑制剂 (ATRi) 正在临床开发中用于治疗癌症，包括相关激酶 ATM 发生突变的肿瘤。然而，目前仍不清楚哪些功能和途径主导 ATRi 的长期疗效，以及这些功能和途径在临床相关遗传背景之间有何差异。因此，阐明 ATRi 疗效的常见机制和遗传背景特定机制有助于患者分层和预防耐药性。在这里，我们在 ATM 缺陷和成熟的小鼠胚胎干细胞中使用 CRISPR–Cas9 全基因组筛选，以询问 ATRi、ceralasertib 治疗后细胞的适应性。我们确定了增强或抑制 ATRi 功效的因素，其中一部分需要完整的 ATM 信号传导。引人注目的是，ATM 丰富和 ATM 缺陷细胞中两个最强的抗性基因命中编码细胞周期蛋白 C 和 CDK8：RNA 聚合酶 II 介导复合物的 CDK8 激酶模块的成员。我们发现 Cyclin C/CDK8 缺失会减少 S 期 DNA:RNA 杂交体的形成、转录复制应激以及 ATRi 诱导的最终微核形成。总体而言，我们的工作在 ATM 熟练和 ATM 缺陷的细胞中确定了 ATRi 功效的新生物标志物，并强调转录相关的复制应激是 ATRi 诱导的细胞死亡的主要驱动因素。