Chronic treatment with ATR and CHK1 inhibitors does not substantially increase the mutational burden of human cells

DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs.To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.

DNA复制压力（replication stress, RS）指多种阻碍基因组精确且持续复制的条件，可频繁导致复制叉减慢甚至停滞。复制压力水平升高会引发基因组不稳定、复制灾难，最终导致细胞死亡。复制压力在癌细胞中尤为高发，而通过化疗药物将其加剧至不可承受的水平，仍是癌症治疗的核心策略之一。
DNA复制压力带来的不良后果，通常可通过ATR与CHK1这两种检查点激酶得以防范：它们能够稳定受胁迫的复制叉、抑制复制起始位点激活，并在复制受扰时促进细胞周期阻滞。目前已开发出针对这两种激酶的特异性抑制剂，多项体外癌症实验已证实其可增强复制压力与细胞死亡效应。当前正在开展的临床试验正探索这类抑制剂单药或联合一线化疗药物对多种癌症的治疗效果。
尽管这类抑制剂有望成为抗癌药物库中的有力补充，但目前我们仍缺乏这类新药全基因组层面的潜在致突变性研究数据。为探究这一问题，我们对敲除TP53的人类癌细胞进行了长期慢性处理，分别使用ATR抑制剂（ATRi：AZD6738/塞拉塞替布）与CHK1抑制剂（CHK1i：MK8776/SCH-900776）。
抑制ATR或CHK1并未显著提升细胞的突变负荷，也未产生特异性突变特征。实际上，尽管处理过程中诱导了复制相关的DNA断裂，但未观察到碱基替换、短插入/缺失以及大规模染色体重排的数量出现显著变化。有趣的是，抑制ATR确实会导致紧密相邻突变的轻微增加——这一特征此前被认为与跨损伤合成DNA聚合酶有关。
上述结果表明，当作为单药使用时，ATRi与CHK1i在体外环境中并未表现出显著的致突变效应。