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.

CHK1 inhibitor induced PARylation by targeting PARG causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer.

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation. We observed elevated Poly-ADP-ribosylation (PAR) of proteins (PARylation) and subsequent decrease in cellular NAD+ levels in OC cells treated with the CHK1 inhibitor prexasertib, indicating activation of NAD+ dependent DNA repair enzymes poly-ADP-ribose polymerases (PARP1/2). While multiple PARP inhibitors are in clinical use in treating OC, tumor resistance to these drugs is highly imminent. We reasoned that inhibition of dePARylation by targeting Poly (ADP-ribose) glycohydrolase (PARG) would disrupt metabolic and DNA repair crosstalk to overcome chemoresistance. Although PARG inhibition (PARGi) trapped PARylation of the proteins and activated CHK1, it did not cause any significant OC cell death. However, OC cells deficient in CHK1 were hypersensitive to PARGi, suggesting a role for metabolic and DNA repair crosstalk in protection of OC cells. Correspondingly, OC cells treated with a combination of CHK1 and PARG inhibitors exhibited excessive replication stress-mediated DNA lesions, cell cycle dysregulation, and mitotic catastrophe compared to individual drugs. Interestingly, increased PARylation observed in combination treatment resulted in depletion of NAD+ levels. These decreased NAD+ levels were also paralleled with reduced aldehyde dehydrogenase (ALDH) activity, which requires NAD+ to maintain cancer stem cells. Furthermore, prexasertib and PARGi combinations exhibited synergistic cell death in OC cells, including an isogenic chemoresistant cell line and 3D organoid models of primary patient-derived OC cell lines. Collectively, our data highlight a novel crosstalk between metabolism and DNA repair involving replication stress and NAD+-dependent PARylation, and suggest a novel combination therapy of CHK1 and PARG inhibitors to overcome chemoresistance in OC.

Oncometabolite 2-hydroxyglutarate suppresses basal protein levels of DNA polymerase beta that enhances alkylating agent and PARG inhibition induced cytotoxicity.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it's estimated that up to 75 % of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD+-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polß), a bifunctional DNA polymerase containing both DNA synthesis and 5'-deoxyribose-phosphate lyase (5'dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD+ metabolism, but still produce sufficient nuclear NAD+ for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polß protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.

Protein lipoylation: mitochondria, cuproptosis, and beyond.

Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions. This review focuses on the mechanisms of lipoylation and its significant impact on cell metabolism and cuproptosis, emphasizing the key genes involved and their implications for human diseases. It offers valuable insights into targeting dysregulated cellular metabolism for therapeutic purposes.

Inspiring basic and applied research in genome integrity mechanisms: Dedication to Samuel H. Wilson.

This Special Issue (SI) of Environmental and Molecular Mutagenesis (EMM), entitled "Inspiring Basic and Applied Research in Genome Integrity Mechanisms," is to update the community on recent findings and advances on genome integrity mechanisms with emphasis on their importance for basic and environmental health sciences. This SI includes two research articles, one brief research communication, and four reviews that highlight cutting edge research findings and perspectives, from both established leaders and junior trainees, on DNA repair mechanisms. In particular, the authors provided an updated understanding on several distinct enzymes (e.g., DNA polymerase beta, DNA polymerase theta, DNA glycosylase NEIL2) and the associated molecular mechanisms in base excision repair, nucleotide excision repair, and microhomology-mediated end joining of double-strand breaks. In addition, genome-wide sequencing analysis or site-specific mutational signature analysis of DNA lesions from environmental mutagens (e.g., UV light and aflatoxin) provide further characterization and sequence context impact of DNA damage and mutations. This SI is dedicated to the legacy of Dr. Samuel H. Wilson from the U.S. National Institute of Environmental Health Sciences at the National Institutes of Health.

TRIP12 governs DNA Polymerase ß involvement in DNA damage response and repair.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase ß (Polß), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polß. Required for Polß foci formation, TRIP12 regulates Polß involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Polß affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Polß-directed BER, optimizes DNA repair pathway choice at complex lesion sites.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polß) revealed that loss of Polß leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models. This technology was exploited to investigate roles for Polß in V(D)J recombination (variable-diversity-joining rearrangement), DNA demethylation, gene complementation, SPO11-induced DNA double-strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin-induced DNA damage, and cancer onset. The revolution in knock-in (KI) mouse models was made possible by CRISPR/cas9-mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polß. Such KI mouse models have helped uncover the importance of key Polß active site residues or specific Polß enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polß protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polß protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polß.

Impact of polß/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway.

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) ß to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polß and LIGIIIα. Yet, the impact of the polß mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polß colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polß by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polß variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polß/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.

Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage.

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transfer of successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been recognized, which identify distinct parts of the PAR chain. Proteins encoding PBDs play an essential role in conveying the PAR-mediated signal through their interaction with PAR chains, which mediates many cellular functions, including the DDR. The WWE domain identifies the iso-ADP-ribose moiety of the PAR chain. We recently described the WWE domain of RNF146 as a robust genetically encoded probe, when fused to EGFP, for detection of PAR in live cells. Here, we evaluated other PBD candidates as molecular PAR probes in live cells, including several other WWE domains and an engineered macrodomain. In addition, we demonstrate unique PAR dynamics when tracked by different PAR binding domains, a finding that that can be exploited for modulation of the PAR-dependent DNA damage response.