The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity. Overall design: To investigate the biomerker for CHK1i sensitivity in NSCLC, we established H1299 and MCF7 cell lines in which each target gene has been knocked down by shRNA.

检查点激酶1（Checkpoint kinase 1, CHK1）在复制应激（Replication stress, RS）条件下对细胞存活至关重要。CHK1抑制剂（CHK1i）联合化疗方案在临床前研究中已展现出可观前景，但在临床试验中却表现为疗效有限且毒性显著。为探寻可克服上述局限的联合治疗策略，我们在非小细胞肺癌（Non-small cell lung cancer, NSCLC）细胞系中开展了无偏高通量筛选，鉴定出硫氧还蛋白1（Thioredoxin1, Trx1）——哺乳动物抗氧化系统的核心组分——作为CHK1i敏感性的决定因素。我们阐明了核糖核苷酸还原酶（Ribonucleotide reductase, RNR）大亚基RRM1的氧化还原循环作用，以及脱氧核苷酸池耗竭在该Trx1介导的CHK1i敏感性中的功能。进一步研究发现，获批用于类风湿关节炎治疗的硫氧还蛋白还原酶（TrxR）抑制剂金诺芬，可通过干扰脱氧核苷酸池与CHK1i产生协同相互作用。综上，本研究证实了一种依托Trx系统与哺乳动物RNR活性间氧化还原调控关联的非小细胞肺癌药理学联合治疗方案。整体实验设计：为探究非小细胞肺癌中CHK1i敏感性的生物标志物，我们构建了H1299与MCF7细胞系，各靶基因均通过短发夹RNA（short hairpin RNA, shRNA）实现敲低。