Deoxyuridine-rich cytoplasmic DNA antagonizes STING-dependent innate immune responses and sensitizes resistant tumors to anti-PD-L1 therapy

DNA damage and cytoplasmic DNA induce type-1 interferon (IFN-1) and potentiate responses to immune checkpoint inhibitors. Our prior work found that inhibitors of the DNA damage response kinase ATR (ATRi) induce IFN-1 and deoxyuridine (dU) incorporation by DNA polymerases, akin to antimetabolites. Whether and how dU incorporation is required for ATRi-induced IFN-1 signaling is not known. Here, we show that ATRi-dependent IFN-1 responses require uracil DNA glycosylase (UNG)-initiated base excision repair and STING. Quantitative analyses of nine distinct nucleosides reveals that ATRi induce dU incorporation more rapidly in UNG wild-type than knockout cells, and that induction of IFN-1 is associated with futile cycles of repair. While ATRi induce similar numbers of micronuclei in UNG wild-type and knockout cells, dU containing micronuclei and cytoplasmic DNA are increased in knockout cells. Surprisingly, DNA fragments containing dU block STING-dependent induction of IFN-1, MHC-1, and PD-L1. Furthermore, UNG knockout sensitizes cells to IFN-γ in vitro , and potentiates responses to anti-PD-L1 in resistant tumors in vivo . These data demonstrate an unexpected and specific role for dU-rich DNA in suppressing STING-dependent IFN-1 responses, and show that UNG-deficient tumors have a heightened response to immune checkpoint inhibitors.