Macrodomains: Structure, Function, Evolution, and Catalytic Activities.

Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair.

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

PARP and PARG inhibitors in cancer treatment.

Oxidative and replication stress underlie genomic instability of cancer cells. Amplifying genomic instability through radiotherapy and chemotherapy has been a powerful but nonselective means of killing cancer cells. Precision medicine has revolutionized cancer therapy by putting forth the concept of selective targeting of cancer cells. Poly(ADP-ribose) polymerase (PARP) inhibitors represent a successful example of precision medicine as the first drugs targeting DNA damage response to have entered the clinic. PARP inhibitors act through synthetic lethality with mutations in DNA repair genes and were approved for the treatment of BRCA mutated ovarian and breast cancer. PARP inhibitors destabilize replication forks through PARP DNA entrapment and induce cell death through replication stress-induced mitotic catastrophe. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) exploit and exacerbate replication deficiencies of cancer cells and may complement PARP inhibitors in targeting a broad range of cancer types with different sources of genomic instability. Here I provide an overview of the molecular mechanisms and cellular consequences of PARP and PARG inhibition. I highlight clinical performance of four PARP inhibitors used in cancer therapy (olaparib, rucaparib, niraparib, and talazoparib) and discuss the predictive biomarkers of inhibitor sensitivity, mechanisms of resistance as well as the means of overcoming them through combination therapy.

(ADP-ribosyl)hydrolases: structure, function, and biology.

ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.

O-GlcNAc has crosstalk with ADP-ribosylation via PARG.

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, a prevalent protein post-translational modification (PTM) that occurs intracellularly, has been shown to crosstalk with phosphorylation and ubiquitination. However, it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that the poly(ADP-ribosyl)ation "eraser", ADP-ribose glycohydrolase (PARG), is O-GlcNAcylated at Ser26, which is in close proximity to its nuclear localization signal. O-GlcNAcylation of PARG promotes nuclear localization and chromatin association. Upon DNA damage, O-GlcNAcylation augments the recruitment of PARG to DNA damage sites and interacting with proliferating cell nuclear antigen (PCNA). In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances the poly(ADP-ribosyl)ation of DNA damage-binding protein 1 (DDB1) and attenuates its auto-ubiquitination, thereby stabilizing DDB1 and allowing it to degrade its downstream targets, such as c-Myc. We further demonstrated that PARG-S26A, the O-GlcNAc-deficient mutant, promoted HCC in mouse xenograft models. Our findings thus reveal that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs.

ADP-ribose contributions to genome stability and PARP enzyme trapping on sites of DNA damage; paradigm shifts for a coming-of-age modification.

ADP-ribose is a versatile modification that plays a critical role in diverse cellular processes. The addition of this modification is catalyzed by ADP-ribosyltransferases, among which notable poly(ADP-ribose) polymerase (PARP) enzymes are intimately involved in the maintenance of genome integrity. The role of ADP-ribose modifications during DNA damage repair is of significant interest for the proper development of PARP inhibitors targeted toward the treatment of diseases caused by genomic instability. More specifically, inhibitors promoting PARP persistence on DNA lesions, termed PARP "trapping," is considered a desirable characteristic. In this review, we discuss key classes of proteins involved in ADP-ribose signaling (writers, readers, and erasers) with a focus on those involved in the maintenance of genome integrity. An overview of factors that modulate PARP1 and PARP2 persistence at sites of DNA lesions is also discussed. Finally, we clarify aspects of the PARP trapping model in light of recent studies that characterize the kinetics of PARP1 and PARP2 recruitment at sites of lesions. These findings suggest that PARP trapping could be considered as the continuous recruitment of PARP molecules to sites of lesions, rather than the physical stalling of molecules. Recent studies and novel research tools have elevated the level of understanding of ADP-ribosylation, marking a coming-of-age for this interesting modification.

Deletion of DWORF does not affect cardiac function in aging and in PLN-R14del cardiomyopathy.

The phospholamban (PLN) pathogenic gene variant p.Arg14del causes cardiomyopathy, which is characterized by perinuclear PLN protein clustering and can lead to severe heart failure (HF). Elevated expression of dwarf open reading frame (DWORF), a protein counteracting the function of PLN in the sarcoplasmic reticulum (SR), can delay disease progression in a PLN-R14del mouse model. Here, we evaluated whether deletion of DWORF (DWORF-/-) would have an opposite effect and accelerate age-dependent disease progression in wild-type (WT) mice and mice with a pathogenic PLN-R14del allele (R14Δ/+). We show that DWORF-/- mice maintained a normal left ventricular ejection fraction (LVEF) during aging and no difference with WT control mice could be observed up to 20 mo of age. R14Δ/+ mice maintained a normal cardiac function until 12 mo of age, but at 18 mo of age, LVEF was significantly reduced as compared with WT mice. Absence of DWORF did neither accelerate the R14Δ/+-induced reduction in LVEF nor enhance the increases in gene expression of markers related to cardiac remodeling and fibrosis and did not exacerbate cardiac fibrosis caused by the R14Δ/+ mutation. Together, these results demonstrate that absence of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, our data indicate that DWORF appears to be dispensable for cardiac function during aging.NEW & NOTEWORTHY Although DWORF overexpression significantly delayed heart failure development and strongly prolonged life span in PLN-R14del mice, the current study shows that deletion of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, DWORF appears to be dispensable for cardiac function during aging. Changes in DWORF gene expression are therefore unlikely to contribute to the clinical heterogeneity observed in patients with PLN-R14del cardiomyopathy.

Dysfunction of poly (ADP-ribose) glycohydrolase suppresses osteoclast differentiation in RANKL-stimulated RAW264 cells.

Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.

Long-term reliability of the phospholamban (PLN) p.(Arg14del) risk model in predicting major ventricular arrhythmia: a landmark study.

AIMS: Recently, a genetic variant-specific prediction model for phospholamban (PLN) p.(Arg14del)-positive individuals was developed to predict individual major ventricular arrhythmia (VA) risk to support decision-making for primary prevention implantable cardioverter defibrillator (ICD) implantation. This model predicts major VA risk from baseline data, but iterative evaluation of major VA risk may be warranted considering that the risk factors for major VA are progressive. Our aim is to evaluate the diagnostic performance of the PLN p.(Arg14del) risk model at 3-year follow-up. METHODS AND RESULTS: We performed a landmark analysis 3 years after presentation and selected only patients with no prior major VA. Data were collected of 268 PLN p.(Arg14del)-positive subjects, aged 43.5 ± 16.3 years, 38.9% male. After the 3 years landmark, subjects had a mean follow-up of 4.0 years (± 3.5 years) and 28 (10%) subjects experienced major VA with an annual event rate of 2.6% [95% confidence interval (CI) 1.6-3.6], defined as sustained VA, appropriate ICD intervention, or (aborted) sudden cardiac death. The PLN p.(Arg14del) risk score yielded good discrimination in the 3 years landmark cohort with a C-statistic of 0.83 (95% CI 0.79-0.87) and calibration slope of 0.97. CONCLUSION: The PLN p.(Arg14del) risk model has sustained good model performance up to 3 years follow-up in PLN p.(Arg14del)-positive subjects with no history of major VA. It may therefore be used to support decision-making for primary prevention ICD implantation not merely at presentation but also up to at least 3 years of follow-up.

Arg4810Lys mutation in RNF213 among Eastern Indian non-MMD ischemic stroke patients: a genotype-phenotype correlation.

INTRODUCTION: RNF213 mutations have been reported mostly in moyamoya disease (MMD) with varying frequencies across different ethnicities. However, its prevalence in non-MMD adult-onset ischemic stroke is still not well explored. AIMS AND OBJECTIVES: This present study thus aims to screen the most common RNF213 variant (Arg4810Lys, among East Asians) in the Eastern Indian non-MMD ischemic stroke patients and correlate it with long-term progression and prognosis of the patients. The subjects were analyzed for this variant using PCR-RFLP and confirmed using Sanger sequencing method. RESULT AND CONCLUSION: We have identified Arg4810Lys variant among eleven young-onset familial ischemic stroke patients in heterozygous manner. A positive correlation of the variant with positive family history (P = 0.001), earlier age at onset (P = 0.002), and history of recurrent stroke (P = 0.015) was observed. However, the carriers showed better cognitive performances in memory (P = 0.042) and executive function (P = 0.004). Therefore, we can conclude that Arg4810Lys/RNF213 - a pathogenic variant for young-onset familial ischemic stroke with higher incidence of recurrent events unlike in MMD cases, have no additional impact on cognition among Eastern Indians.

PARG Promotes Esophagus Cancer Cell Metastasis by Activation of the Wnt/ß-Catenin Pathway.

Esophagus cancer (EC) is a highly malignant and metastatic cancer. Poly(ADP-ribose) glycohydrolase (PARG), a DNA replication and repair regulator, inhibits cancer cell replication defects. This study aimed to explore the role of PARG in EC. The biological behaviors were analyzed using MTT assay, Transwell assay, scratch test, cell adhesion assay, and western blot. PARG expression was detected using quantitative PCR and immunohistochemical assay. The regulation of the Wnt/ß-catenin pathway was assessed using western blot. The results showed that PARG was highly expressed in EC tissues and cells. Knockdown of PARG suppressed cell viability, invasion, migration, adhesion, and epithelial-mesenchymal transition. Conversely, overexpression of PARG promoted the biological behaviors mentioned above. Moreover, overexpression of PARG promoted the activation of the Wnt/ß-catenin pathway rather than the STAT and Notch pathways. XAV939, the Wnt/ß-catenin pathway inhibitor, partly abolished the biological behaviors mediated by PARG overexpression. In conclusion, PARG promoted the malignant advancement of EC via activating the Wnt/ß-catenin pathway. These findings suggested that PARG might be a new therapeutic target for EC.

PARG Protein Regulation Roles in Drosophila Longevity Control.

Aging, marked by a gradual decline in physiological function and heightened vulnerability to age-related diseases, remains a complex biological process with multifaceted regulatory mechanisms. Our study elucidates the critical role of poly(ADP-ribose) glycohydrolase (PARG), responsible for catabolizing poly(ADP-ribose) (pADPr) in the aging process by modulating the expression of age-related genes in Drosophila melanogaster. Specifically, we uncover the regulatory function of the uncharacterized PARG C-terminal domain in controlling PARG activity. Flies lacking this domain exhibit a significantly reduced lifespan compared to wild-type counterparts. Furthermore, we observe progressive dysregulation of age-related gene expression during aging, accelerated in the absence of PARG activity, culminating in a premature aging phenotype. Our findings reveal the critical involvement of the pADPr pathway as a key player in the aging process, highlighting its potential as a therapeutic target for mitigating age-related effects.

The First Korean Case with Cardiac, Facial, and Digital Anomalies with Developmental Delay Caused by De Novo TRAF7 p.Arg655Gln Variant.

TRAF7-related disorders represent some of the rarest inherited disorders, exhibiting clinical features that overlap with cardiac, facial, and digital anomalies with developmental delay (CAFDADD) syndrome, as well as blepharophimosis-mental retardation syndrome (BMRS). A 36-year-old male, presenting with total blindness, blepharophimosis, and intellectual disability, was admitted for the assessment of resting dyspnea several months previously. He had a history of being diagnosed with obstructive sleep apnea (OSA). Transesophageal and transthoracic echocardiography unveiled right ventricular dilatation without significant pulmonary hypertension, bicuspid aortic valve with aortic root aneurysm, and aortic regurgitation in the proband. Sanger sequencing identified a de novo TRAF7 variant (c.1964G>A; p.Arg655Gln). Subsequently, aortic root replacement using the Bentall procedure was performed. However, despite the surgery, he continued to experience dyspnea. Upon re-evaluating OSA with polysomnography, it was discovered that continuous positive airway pressure support alleviated his symptoms. The underlying cause of his symptoms was attributed to OSA, likely exacerbated by the vertebral anomaly and short neck associated with CAFDADD syndrome. Clinicians should be attentive to the symptoms associated with OSA as it is a potentially serious medical condition in patients with TRAF7 variants.

Strategies for the Management of Patients with Pancreatic Cancer with PARP Inhibitors.

A subset of patients with pancreatic adenocarcinomas (PDAC) harbor mutations that are exploitable in the context of DNA-damage response and repair (DDR) inhibitory strategies. Between 8-18% of PDACs harbor specific mutations in the DDR pathway such as BRCA1/2 mutations, and a higher prevalence exists in high-risk populations (e.g., Ashkenazi Jews). Herein, we will review the current trials and data on the treatment of PDAC patients who harbor such mutations and who appear sensitive to platinum and/or poly ADP ribose polymerase inhibitor (PARPi) based therapies due to a concept known as synthetic lethality. Although this current best-in-class precision treatment shows clinical promise, the specter of resistance limits the extent of therapeutic responses. We therefore also evaluate promising pre-clinical and clinical approaches in the pipeline that may either work with existing therapies to break resistance or work separately with combination therapies against this subset of PDACs.

The role of poly (ADP-ribose) glycohydrolase in phosphatase and tensin homolog deficiency endometrial cancer.

AIM: To explore the relationship between poly(ADP-ribose) glycohydrolase (PARG) and the occurrence, development, and prognosis of endometrial carcinoma (EC), and investigate whether the PARG inhibitor PDD0017273 could increase the sensitivity of EC cells to cisplatin. METHODS: The expression of PARG, phosphatase and tensin homolog (PTEN), and p53 in normal endometrial tissues (NE), endometrial hyperplasia without atypia (EH), atypical endometrial hyperplasia (AH), and EC was detected by immunohistochemistry. AN3CA EC cells with PTEN deficiency were treated with different cisplatin and PDD0017273, alone or in combination. Cell proliferation was detected by MTT method, apoptosis was detected by flow cytometry, and the expression of PARG in EC cells after treatment with different drugs was detected by western blot and immunohistochemistry. RESULTS: Expression of PARG in NE, EH, AH, and EC increased gradually. In addition, compared with low PARG expression in PTEN-positive EC, patients who had high PARG expression in PTEN-negative EC had more advanced clinical stages (r = -0.399, p = 0.032) and shorter overall survival time (p = 0.037). A dose of 40 µM PDD0017273 effectively inhibited PARG expression, increased the sensitivity of AN3CA cells to cisplatin. CONCLUSIONS: The findings suggest that PARG overexpression is a promising immunohistochemical marker to predict the occurrence and prognosis of EC. Moreover, PARG inhibition produced antitumor effects and increased the sensitivity of EC cells with PTEN deficiency to cisplatin.

Synthesis and Degradation of Poly(ADP-ribose) in Zebrafish Brain Exposed to Aluminum.

Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD+. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.

Disrupting Poly(ADP-ribosyl)ating Pathway Creates Premalignant Conditions in Mammalian Liver.

Hepatocellular carcinoma (HCC) is a major global health concern, representing one of the leading causes of cancer-related deaths. Despite various treatment options, the prognosis for HCC patients remains poor, emphasizing the need for a deeper understanding of the factors contributing to HCC development. This study investigates the role of poly(ADP-ribosyl)ation in hepatocyte maturation and its impact on hepatobiliary carcinogenesis. A conditional Parg knockout mouse model was employed, utilizing Cre recombinase under the albumin promoter to target Parg depletion specifically in hepatocytes. The disruption of the poly(ADP-ribosyl)ating pathway in hepatocytes affects the early postnatal liver development. The inability of hepatocytes to finish the late maturation step that occurs early after birth causes intensive apoptosis and acute inflammation, resulting in hypertrophic liver tissue with enlarged hepatocytes. Regeneration nodes with proliferative hepatocytes eventually replace the liver tissue and successfully fulfill the liver function. However, early developmental changes predispose these types of liver to develop pathologies, including with a malignant nature, later in life. In a chemically induced liver cancer model, Parg-depleted livers displayed a higher tendency for hepatocellular carcinoma development. This study underscores the critical role of the poly(ADP-ribosyl)ating pathway in hepatocyte maturation and highlights its involvement in liver pathologies and hepatobiliary carcinogenesis. Understanding these processes may provide valuable insights into liver biology and liver-related diseases, including cancer.

Potassium Voltage-Gated Channel Subfamily H Member 1 (KCNH1) Missense Mutation Causing Epileptic Encephalopathy And Autistic Behaviour.

The phenotypically similar genetic diseases Zimmermann Laband syndrome (ZLS) and Temple-Baraitser syndrome (TMBTS) cause neurodevelopmental problems. Mutations in the gene coding for potassium voltage-gated channel, primarily KCNH1, cause these symptoms. An uncommon mutation in KCNH1 (p.Arg357Trp) present on Exon 7, reported to replace arginine with tryptophan at codon 357 of the KCNH1 protein c.1069C>T, caused pharma coresistantseizures and autistic behaviour in a 2.7-year-old boy. This mutation causes problems with protein modelling and has yet to be documented in any genetic databases around the world. This mutation was overlapped with GPHN gene, c.828+1G>A, in our patient, causing GPHN related spectrum disorder (autosomal dominant) along with molybdenum cofactor deficiency (autosomal recessive) leading to a neuropsychiatric presentation including autistic behaviour, making diagnosis and management even more complicated.

PARG inhibition limits HCC progression and potentiates the efficacy of immune checkpoint therapy.

BACKGROUND & AIMS: Although the treatment of hepatocellular carcinoma (HCC) has been revolutionized by the advent of effective systemic therapies, the prognosis of patients with HCC remains dismal. Herein, we examined the pathophysiological role of PARG and assessed the utility of targeting dePARylation for HCC therapy. METHODS: The oncogenic function of PARG was evaluated in 2 orthotopic xenograft models and a Pargflox/flox mice model. The therapeutic efficacy of PARG inhibitors in combination with an anti-PD-1 antibody were assessed in murine orthotopic models. Microarray analysis was used to evaluate the pathological relevance of the PARG/DDB1/c-Myc/MMR axis. RESULTS: High PARG expression was strongly associated with poor HCC prognosis. Hepatocyte-specific PARG deletion significantly impaired liver tumorigenesis. PARG promoted HCC growth and metastasis through DDB1-dependent modulation of c-Myc. Specifically, PARG dePARylated DDB1 and consequently promoted DDB1 autoubiquitination, thus stabilizing the c-Myc protein in HCC cells. PARG downregulation attenuated c-Myc-induced MMR expression and PARG deficiency was correlated with a favorable prognosis in patients with HCC treated with anti-PD-1-based immunotherapy. In addition, PARG inhibitors could act in synergy with anti-PD-1 antibodies in orthotopic mouse models. CONCLUSIONS: PARG can act as an oncogene in HCC by modulating PARG/DDB1/c-Myc signaling and could be used as a biomarker to identify patients with HCC who may benefit from anti-PD-1 treatment. Our findings suggest that co-inhibition of PARG and PD-1 is an effective novel combination strategy for patients with HCC. LAY SUMMARY: The increase in deaths due to hepatocellular carcinoma (HCC) is a growing concern, with the mechanisms responsible for HCC development still incompletely understood. Herein, we identify a novel mechanism by which the protein PARG contributes to HCC development. Inhibition of PARG increased the efficacy of anti-PD-1 therapy (a type of immunotherapy) in HCC. These findings support the future clinical development of PARG inhibitors, potentially in combination with anti-PD-1 inhibitors.