Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

Sugitani, Norie; Vendetti, Frank P.; Cipriano, Andrew J; Pandya, Pinakin; Deppas, Joshua J.; Moiseeva, Tatiana N.; Schamus-Haynes, Sandra; Wang, Yiyang; Palmer, Drake; Osmanbeyoglu, Hatice U.; Bostwick, Anna; Snyder, Nathaniel W.; Gong, Yi-Nan; Aird, Katherine M.; Delgoffe, Greg M.; Beumer, Jan H.; Bakkenist, Christopher J.

doi:10.1016/j.celrep.2022.111371

Cited by 12 publications

(22 citation statements)

References 59 publications

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“…On average, ATRi QDx3 plus RT further decreased Ki67 + CD8 + T cells in TILs compared with ATRi QDx3 alone, but this difference was not statistically significant ( Figure 2B ). Consistent with these findings, we recently showed that ATRi directly kills rapidly proliferating, but not naive, CD8 + T cells ex vivo ( 36 ).…”

Section: Resultssupporting

confidence: 71%

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy

Vendetti¹,

Pandya²,

Clump³

et al. 2023

JCI Insight

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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.

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Section: Resultssupporting

confidence: 71%

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy

Vendetti¹,

Pandya²,

Clump³

et al. 2023

JCI Insight

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“…We hypothesized that dU and rN, concentrated in active replicons by ATRiinduced origin firing, may generate multiple damaged sites whose repair generates cytoplasmic nucleic acid fragments that induce IFN-1. Consistent with this premise, we recently showed that ATRi-induced dU incorporation and IFN-1 are reversed by low doses of thymidine, which implicates UNG-and SMUG1-initiated BER in the mechanism underlying ATRi-induced IFN-1 (10). cGAS and RNA Polymerase III (Pol III) are pattern recognition receptors (PRRs) that identify dsDNA in the cytoplasm and initiate signaling pathways that induce IFN-1.…”

Section: Introductionmentioning

confidence: 75%

“…We show that unrepaired dU in DNA blocks innate immune responses and potentiates responses to checkpoint inhibitors in mouse models of cancer. This would suggest that patients with low tumor UNG levels may respond to antimetabolites combined with checkpoint inhibitors, and patients with high tumor UNG levels may respond to UNG inhibitors ATR kinase inhibitors (ATRi) induce origin firing, inhibit deoxycytidine kinase, and cause the degradation of ribonucleotide reductase and this disrupts both nucleoside salvage and nucleoside biosynthesis in undamaged cells (10,12). ATRi therefore increase DNA synthesis and decrease dNTP concentrations and thus dU contamination.…”

Section: Discussionmentioning

confidence: 99%

“…We suggest that both paradigms underlie our observations. First, ATRi induce origin firing and the unlimited incorporation of dU at replication forks, unlimited because ATR kinase-dependent cell cycle checkpoints are inhibited (10)(11)(12). Nevertheless, ATRi treatment is associated with the accumulation of ssDNA gaps at replication forks, presumably because ATRi-induced origin firing depletes dNTP pools causing polymerase stalling.…”

Section: Discussionmentioning

confidence: 99%

“…ATR is an essential DNA damage response kinase activated at stalled and collapsed replication forks (7). ATR kinase signaling stabilizes stalled forks, reduces the rate of DNA replication, and increases nucleotide biosynthesis, thereby limiting the incorporation of dU by polymerases in cells treated with antimetabolites (8)(9)(10). ATR kinase inhibitors (ATRi) induce origin firing, inhibit deoxycytidine kinase, and induce the degradation of ribonucleotide reductase, thereby inhibiting nucleoside biosynthesis and nucleoside salvage, respectively, in otherwise unperturbed cells (11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

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

Pandya,

Vendetti,

El-Ghoubaira

et al. 2024

Preprint

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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-γin vitro, and potentiates responses to anti-PD-L1 in resistant tumorsin 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.STATEMENT OF SIGNIFICANCEAntimetabolites disrupt nucleotide pools and increase dU incorporation by DNA polymerases. We show that unrepaired dU potentiates responses to checkpoint inhibitors in mouse models of cancer. Patients with low tumor UNG may respond to antimetabolites combined with checkpoint inhibitors, and patients with high tumor UNG may respond to UNG inhibitors combined with checkpoint inhibitors.

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Advancements and challenges of R-loops in cancers: Biological insights and future directions

Li,

Shao,

et al. 2025

Cancer Letters

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Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

Cited by 12 publications

References 59 publications

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy

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

Advancements and challenges of R-loops in cancers: Biological insights and future directions

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