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. STATEMENT OF SIGNIFICANCE: Antimetabolites 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.

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

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-ß, 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.

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

ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. ATR kinase inhibitors (ATRi's) combine with radiation to generate CD8+ T cell-dependent responses in mouse models of cancer. We show that ATRi's induce cyclin-dependent kinase 1 (CDK1)-dependent origin firing across active replicons in CD8+ T cells activated ex vivo while simultaneously decreasing the activity of rate-limiting enzymes for nucleotide biosynthesis. These pleiotropic effects of ATRi induce deoxyuridine (dU) contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and interferon-α/ß (IFN-α/ß). Remarkably, thymidine rescues ATRi-induced dU contamination and partially rescues death and IFN-α/ß expression in proliferating CD8+ T cells. Thymidine also partially rescues ATRi-induced cancer cell death. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell-dependent anti-tumor responses in mouse models. We conclude that ATR is essential to limit dU contamination in genomic DNA and IFN-α/ß expression.

p53 Promotes Cytokine Expression in Melanoma to Regulate Drug Resistance and Migration.

The transcription factor p53 is frequently lost during tumor development in solid tumors; however, most melanomas retain a wild type p53 protein. The presence of wild type p53 in melanoma has fueled speculation that p53 may play a neutral or pro-tumorigenic role in this disease. Here we show that p53 is functional in human melanoma cell lines, and that loss of p53 results in a general reduction in basal NF-kB regulated cytokine production. The reduced cytokine expression triggered by p53 loss is broad and includes key inflammatory chemokines, such as CXCL1, CXCL8, and the IL6 class cytokine LIF, resulting in a reduced ability to induce chemotactic-dependent migration of tumor cells and immune cells and increased sensitivity to BRAF inhibition. Taken together, this result indicates that wild type p53 regulates cytokine expression and induces cytokine-dependent phenotype on melanoma.

PICOT binding to chromatin-associated EED negatively regulates cyclin D2 expression by increasing H3K27me3 at the CCND2 gene promoter.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Grx3; Glrx3)) is a ubiquitous protein that can interact with the embryonic ectoderm development (EED) protein via each of its two C-terminal PICOT/Grx homology domains. Since EED is a Polycomb-Group protein and a core component of the polycomb repressive complex 2 (PRC2), we tested the involvement of PICOT in the regulation of PRC2-mediated H3 lysine 27 trimethylation (H3K27me3), transcription and translation of selected PRC2 target genes. A fraction of the cellular PICOT protein was found in the nuclei of leukemia cell lines, where it was associated with the chromatin. In addition, PICOT coimmunoprecipitated with chromatin-residing EED derived from Jurkat and COS-7 cell nuclei. PICOT knockdown led to a reduced H3K27me3 mark and a decrease in EED and EZH2 at the CCND2 gene promoter. In agreement, PICOT-deficient T cells exhibited a significant increase in CCND2 mRNA and protein expression. Since elevated expression levels of PICOT were reported in several different tumors and correlated in the current studies with decreased transcription and translation of the CCND2 gene, we tested whether this opposite correlation exists in human cancers. Data from the Cancer Genome Atlas (TCGA) database indicated statistically significant negative correlation between PICOT and CCND2 in eight different human tumors where the highest correlation was in lung (p = 8.67E-10) and pancreatic (p = 1.06E-5) adenocarcinoma. Furthermore, high expression of PICOT and low expression of CCND2 correlated with poor patient survival in five different types of human tumors. The results suggest that PICOT binding to chromatin-associated EED modulates the H3K27me3 level at the CCND2 gene promoter which may be one of the potential mechanisms for regulation of cyclin D2 expression in tumors. These findings also indicate that a low PICOT/CCND2 expression ratio might serve as a good predictor of patient survival in selected human cancers.

PICOT (GLRX3) is a positive regulator of stress-induced DNA-damage response.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Glrx3)) is a ubiquitously expressed protein that possesses an N-terminal monothiol thioredoxin (Trx) domain and two C-terminal tandem copies of a monothiol Glrx domain. It has an overall highly conserved amino acid sequence and is encoded by a unique gene, both in humans and mice, without having other functional gene homologs in the entire genome. Despite being discovered almost two decades ago, the biological function of PICOT remains largely ill-defined and its ramifications are underestimated considering the fact that PICOT-deficiency in mice results in embryonic lethality. Since classical Glrxs are important regulators of the cellular redox homeostasis, we tested whether PICOT participate in the stress-induced DNA-damage response, focusing on nuclear proteins that function as integral components of the DNA repair machinery. Using wild type versus PICOT-deficient (PICOT-KD) Jurkat T cells we found that the anti-oxidant mechanism in PICOT-deficient cells is impaired, and that these cells respond to genotoxic drugs, such as etoposide and camptothecin, by increased caspase-3 activity, a reduced survival and a slower and diminished phosphorylation of the histone protein, H2AX. Nevertheless, the effect of PICOT on the drug-induced phosphorylation of H2AX was independent of the cellular levels of reactive oxygen species. PICOT-deficient cells also demonstrated reduced and slower γH2AX foci formation in response to radiation. Furthermore, immunofluorescence staining using PICOT- and γH2AX-specific Abs followed by confocal microscopy demonstrated partial localization of PICOT at the γH2AX-containing foci at the site of the DNA double strand breaks. In addition, PICOT knockdown resulted in inhibition of phosphorylation of ATR, Chk1 and Chk2 kinases, which play an essential role in the DNA-damage response and serve as upstream regulators of γH2AX. The present data suggest that PICOT protects cells from DNA damage-inducing agents by operating as an upstream positive regulator of ATR-dependent signaling pathways. By promoting the activity of ATR, PICOT indirectly regulates the phosphorylation and activation of Chk1, Chk2, and γH2AX, which are critical components of the DNA damage repair mechanism and thereby attenuate the stress- and replication-induced genome instability.

PICOT binding to the polycomb group protein, EED, alters H3K27 methylation at the MYT1 PRC2 target gene.

PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.