DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8 + T cell–dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1–2). Short-course ATRi (days 1–3) plus RT caused expansion of tumor antigen–specific, effector CD8 + T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1–9) prevented the expansion of tumor antigen–specific, effector CD8 + T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti–PD-L1. Our data argue that ATRi cessation is essential to allow CD8 + T cell responses to both RT and immune checkpoint inhibitors.

ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. However, little is known about the role of ATR in the cell cycle and the impact of DDR inhibitors in immune cells in the absence of DNA damage. We previously showed that the ATR inhibitor AZD6738 (ATRi) combines with radiation to generate delayed, CD8+ T cell-dependent antitumor responses in mouse models of cancer. Here, we show that ATRi induces untimely CDK1 activity during S phase in CD8+ T cells and this induces origin firing and simultaneous degradation of dNTP synthesis and salvage enzymes. These pleiotropic effects of ATRi in proliferating CD8+ T cells induce deoxyuridine contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and death. Remarkably, thymidine significantly rescues ATRi-induced CD8+ T cell death. Our data identifies critical considerations for the design of clinical ATRi regimens with genotoxic chemo- and radiation and immunotherapies.

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