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.