poly(ADP-ribose) Glycohydrolase Inhibitors Research Articles

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.

DNAR-01. PAR-MEDIATED DNA DAMAGE SIGNALING IN IDH MUTANT GLIOMAS: COMBINING PARG INHIBITION AND RADIATION FOR SELECTIVE CYTOTOXICITY

Abstract Isocitrate dehydrogenase (IDH) 1 or 2 mutant gliomas have unique metabolic characteristics and DNA damage response. Radiotherapy and chemotherapy have been proven to be effective treatments for IDH mutant gliomas. Poly (ADP-ribose) (PAR) mediates DNA damage response and is regulated by opposing enzymes: poly (ADP-ribose) polymerases (PARPs) synthesize PAR, while poly (ADP-ribose) glycohydrolase (PARG) breaks it down. Combining PARG inhibitor (PARGi) and Temozolomide (TMZ) in IDH mutant cells leads to imbalanced NAD sequestration into PAR, causing metabolic lethality. This study characterizes the combination of PARGi and radiation. PARGi monotherapy showed no difference between IDH mutant and wild-type cells. However, we found that combining PARGi with radiation significantly increased cytotoxicity in IDH mutant glioma models, similar to PARPi and radiation. (Wang et al, ACi.Adv. 2020) Our findings showed that unlike TMZ+PARGi, PARGi and radiation did not consume and deplete NAD. Cytotoxicity was partially reversed by IDH inhibitor, highlighting the contribution of PAR-mediated DNA damage signaling in IDH mutant contexts. This convergence of vulnerabilities in IDH mutant cells identifies PAR-mediated DNA repair mechanisms that operate independently of NAD metabolism. IDH mutant enzyme generates 2 hydroxyglutarate (2HG), which is critical early in tumor development. However, the effect of 2HG on radiation sensitivity varies across cellular contexts. In this study, we found that IDH1 mutation and 2HG production enhance radiosensitivity in glioma cells both in vivo and in in vitro models, facilitated by PAR-dependent mechanisms. This augmentation of radiosensitivity is associated with S-phase blockage of the cell cycle, unveiling a vulnerability in the replication process of these cancer cells. In summary, this study demonstrates that the combination of PARGi and radiation regulates DNA repair pathways, augmenting cytotoxicity specifically in IDH mutant gliomas. Targeting PAR during radiation holds promise for treating IDH mutant gliomas, tailoring therapy to maximize durability while minimizing impact on normal cells.

Poly(ADP-Ribose) Glycohydrolase (PARG) Removes Repressive Poly-ADP Ribose Marks from DNA Polymerase Theta (PolΘ) to Stimulate DNA Double-Strand Breaks Repair in Myeloid Malignancies

Myeloid malignant cells (AML, MPN, CML) expressing oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1, respectively] accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We reported before that formaldehyde generated by altered serine/one-carbon cycle metabolism in malignant cells harboring OTKs contributed to accumulation of toxic DNA-protein crosslinks (DPCs) which were converted to highly lethal DNA double-strand breaks (DSBs). To counteract the toxicity of DSBs, OTKs enhanced the expression of DNA polymerase theta (PolΘ, encoded by POLQ gene), a unique DNA helicase-DNA polymerase fusion protein that promotes error-prone repair of DSBs by a mechanism referred to as PolΘ-mediated DNA end-joining (TMEJ). PolΘ plays an essential role in the initiation and maintenance of hematological malignancies harboring OTKs. Although the cellular activities of PolΘ have been widely studied, little is known about how PolΘ is regulated at the molecular level. For example, whether post-translational modifications of PolΘ are important for its TMEJ activity and regulation is unknown. Poly(ADP-ribose) polymerase 1 (PARP1) dependent poly-(ADP)-ribosylation (PARylation) of proteins is one of the major post-translational modification events involved in the DNA damage response (DDR). PARP1 - the founding member of the poly(ADP-ribose) polymerase family -transfers adenosine diphosphate (ADP)-ribose from nicotinamide adenine dinucleotide (NAD +) to substrate proteins enabling their PARylation. PARylation is tightly controlled by the glycohydrolase activity of poly(ADP-ribose) glycohydrolase (PARG). Thus, interplay between PARP1 and PARG is thought to regulate the PARylation status of relevant DDR proteins and multiple studies demonstrated that both PARP1 and PARG contribute to DDR. Here, we investigated whether the interplay of PARP1 and PARG is important for the regulation of TMEJ and the specific activities of PolΘ in myeloid malignancies harboring OTKs. We found that upon DNA damage OTKs exerted temporal effect on PolΘ, PARP1 and PARG detection in chromatin fractions. While both PolΘ and PARG displayed continuous time-dependent accumulation during 120 min. after irradiation, PARP1 levels sharply increased at 20 min. and dissipated at 120 min. We find that PARP1 binds to and directly PARylates PolΘ in vitro and in cells. However, PARylated PolΘ is unable to perform TMEJ in vitro due to its inability to bind DNA despite its PARylation dependent recruitment to DNA. Hence, PARG is needed to reactivate PolΘ DNA binding and its TMEJ activity by removing repressive PAR marks on PolΘ. In support of this, cellular studies show that PARG is essential for TMEJ and support a two-step mechanism of a PARP1-PARG regulatory axis of PolΘ and TMEJ. In the first step, the rate of PARP1-PolΘ spatiotemporal recruitment to DNA damage foci supports a rapid step whereby PARP1 PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage in an inactive state. The rate of PARG spatiotemporal recruitment supports a second step whereby subsequent recruitment of PARG corresponds to dissipation of PARP1 and PAR at DNA damage sites, dePARylation of PolΘ, and activation of TMEJ. These studies elucidate an unprecedented mechanism of PARP1-PARG activation of TMEJ and reveal the molecular basis by which PARP1 and PARG inhibition suppresses TMEJ. In conclusion, using intracellular and biochemical approaches we show here that both PARP1 and PARG are required for TMEJ despite their counteracting enzymatic activities. We also pinpointed a unique dynamic spatiotemporal interplay between PolΘ, PARP1 and PARG to regulate TMEJ activity. During the initial stage, PARP1 activated by DNA damage PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage. PARG removal of PAR from PolΘ reactivated its interaction with DNA resulting in robust TMEJ activity in vitro and in cells. Thus, DNA/chromatin binding and TMEJ activity of PolΘ were restored upon PARG-mediated removal of PAR repressive marks on PolΘ. This process seems to play a critical role in protecting OTK-positive hematopoietic malignant cells from genotoxic effect of formaldehyde generated by altered serine/one-carbon cycle metabolism.

Abstract 2667: PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells

Abstract Ovarian cancer (OC) is one of the leading causes of cancer-related deaths in women in the United States. PARP inhibitors showed promising clinical responses; however, most of the patients eventually relapse and succumb to the resistant disease. Currently, it lacks effective therapies to prevent and treat OC, which warrants novel drugs and combination therapies to improve patients' prognoses. Epithelial OCs are heavily dependent on ATR-CHK1-mediated DNA damage checkpoint signaling for survival, which is also regulated by hedgehog/GLI1 (Hh) signaling in cancer cells. However, both Hh/GLI1 and CHK1 inhibitors failed to show promising results in clinical trials as single agents. Poly(ADP-ribose) glycohydrolase (PARG) is the critical enzyme that removes Poly-ADP ribosylated (PARylated) proteins upon replication stress. We hypothesized that inhibition of PARG could amplify CHK1 inhibitor-induced replication stress by trapping PARylated proteins on the chromatin and compromising DNA repair and causing a metabolic and mitotic catastrophe. We evaluated CHK1 inhibitor prexasertib in combination with PARG inhibitor (PARGi) to target metabolic and DNA repair crosstalk to effectively kill OC cells and to overcome resistance. Our studies demonstrate that prexasertib treatment induces PARylation by inducing replication stress. Similarly, PARG inhibition by (PDD00017273) fails to remove PARylation and activates checkpoint kinase 1 (CHK1) by phosphorylating at S296, S317, and S345 in OC cells. Based on these observations, we hypothesized that a combination of CHK1 and PARG inhibition would augment oncogenic replication stress in OC cells and cause increased DNA damage and cell death. Interestingly, our evaluation of prexasertib and PARGi combination treatment by clonogenic survival and MTT assays showed synergistic killing in a panel of OC cells, including in isogenic chemoresistant models as well as 3D organoid models of primary OC cells compared to individual drugs. As predicted, combined treatment increased DNA double-strand breaks as demonstrated by COMET assays, and increased γH2AX foci, pRPA32 foci, perturbed S/G2 phase arrest, and nuclear disintegration relative to individual-drug treatment. Furthermore, significant depletion of NAD+ levels was seen in PARGi-treated OC cells compared to untreated cells. Together, these results indicate that this combination treatment cause persistence of heavy PARylation at DNA damage sites abrogates cell cycle checkpoint mechanisms, exhausts cellular NAD+ levels, and inhibits DNA repair leading to the metabolic and mitotic collapse of cancer cells and synergistic lethality in OC cells. Currently, prexasertib is under clinical trials and our studies will provide novel mechanistic insight into the therapeutic potential of this combination and provides preclinical evidence to develop further this combination therapy in treating OC. Citation Format: Ganesh Acharya, Chinnadurai Mani, Mark B. Reedy, Komaraiah Palle. PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2667.

Abstract 6093: IDE161, a potential first-in-class clinical candidate PARG inhibitor, selectively targets homologous-recombination-deficient and PARP inhibitor resistant breast and ovarian tumors

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) were the first clinically approved drugs designed to exploit synthetic lethality. Despite initial responses, patients harboring homologous recombination (HR) alterations that receive PARPi develop treatment resistance, which has generated a need for additional therapies targeting homologous recombination deficient (HRD) tumors. Poly (ADP-Ribose) glycohydrolase (PARG) plays a key role in the resolution of PARP1/2-dependent DNA damage repair through hydrolysis of Poly (ADP-ribose) (PAR) chains and consequent release of DNA repair protein complexes from chromatin. In culture, the accumulation of PAR chains in the absence of a functional PARG enzyme causes delayed repair of DNA breaks resulting in increased sensitivity to alkylating agents and ionizing radiation. Notably, some HRD cell models have shown differential sensitivity to PARGi vs PARPi, suggesting that PARGi may overcome some PARPi resistance mechanisms. The ability of PARGi to exacerbate replication deficiencies nominates it as a potential therapeutic target for a broad range of cancer types with genomic instability. IDE161 is an orally bioavailable small molecule inhibitor of PARG. Biochemical and cellular assays demonstrate that IDE161 is a potent inhibitor of PAR chain hydrolysis and has anti-proliferative activity in breast and ovarian HRD cancer cell lines with both inherent and acquired PARPi resistance. IDE161 anti-proliferative effects were associated with induction of mitotic arrest and activation of the DNA damage response pathway, suggesting the position of PARG in the DNA repair cycle remains essential in the context of some PARPi resistance mechanisms. Furthermore, profiling of IDE161 across a panel of 264 molecularly characterized cancer cell lines indicated that PARGi may show benefit beyond HRD and in indications other than ovarian and breast. Nonclinical studies in cell line and patient derived xenograft models of breast, ovarian, and gastric tumor types harboring defects in the HR pathway demonstrated anti-tumor activity in response to IDE161. Consistent with in vitro findings, we observed IDE161 antitumor activity in PARPi resistant xenograft models. Moreover, studies in cell lines, tumors and tissues revealed that dose and time-dependent accumulation of PAR chains serves as a robust proximal pharmacodynamic biomarker indicative of PARG target engagement. IDE161 is a novel targeted therapy that exploits the synthetic lethal relationship between PARG and genomic instability, thus leading to selective anti-proliferative effects in tumors harboring defects in the HR pathway. Citation Format: Monah Abed, Diana Muñoz, Vidya Seshadri, Steve Federowicz, Arjun A. Rao, Deepthi Bhupathi, Marya Liimatta, Rita Ousterhout, Firoz Jaipuri, Claire Neilan, Mark Lackner, Mike White, Zineb Mounir. IDE161, a potential first-in-class clinical candidate PARG inhibitor, selectively targets homologous-recombination-deficient and PARP inhibitor resistant breast and ovarian tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6093.

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

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. 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. 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. 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.

DNAR-03. TARGETING POLY (ADP-RIBOSE) SIGNALING TO INDUCE LETHAL DNA DAMAGE IN IDH MUTANT GLIOMA

Abstract Isocitrate dehydrogenase (IDH) 1 or 2 mutant gliomas have unique metabolic characteristics and DNA damage response. Radiotherapy and chemotherapy has been proven to be an effective treatment for IDH mutant gliomas. A key mediator of DNA damage response, Poly(ADP-ribose) (PAR) is regulated by opposing enzymes, poly(ADP-ribose) polymerases (PARPs) that synthesizes PAR through enzymatic polymerization of nicotinamide adenine dinucleotide (NAD), and poly(ADP-ribose) glycohydrolase (PARG) that breaks down and recycles PAR. We recently demonstrated that combining PARG inhibitor (PARGi) and Temozolomide (TMZ) in IDH mutant cells drove an imbalanced sequestration of NAD into PAR, resulting in metabolic lethality through NAD depletion. Here, we sought to characterize the effect of combining PARGi and radiation. Isogenic pairs of IDH1-mutant glioma cells, alongside multiple IDH mutant patient-derived glioma cells, were treated with TMZ, radiation, PARPi, PARGi, and combination thereof. In contrast to PARPi which can have a “trapping” effect, PARGi monotherapy displayed no significant difference between IDH mutant and wild-type isogenic cells. However, we found a substantial augmentation of radiation cytoxicity when combined with PARGi, that was genotype-specific in IDH mutant glioma models, mirroring that seen with PARPi and radiation (Wang et al, Science Advances 2020). Unlike TMZ+PARGi, NAD was not depleted with PARGi and radiation. Conversely, the cytotoxic effect was partially reversed with either PARPi or IDH inhibitor, indicative of a key contribution of PAR mediated DNA damage signaling in the IDH mutant context. Therefore, these results support a model of converging vulnerabilities in IDH mutant cells, with PAR-mediated DNA repair mechanisms independent from NAD metabolism.In summary, this study demonstrates that the combination of PARGi and radiation regulates DNA repair pathways with cytoxicity specific to IDH mutant gliomas. Targeting PAR can potentially be a promising treatment for clinical translation in IDH mutant gliomas, minimizing the radiation effect to surrounding normal cells.

Genomic and biological aspects of resistance to selective poly(ADP-ribose) glycohydrolase inhibitor PDD00017273 in human colorectal cancer cells.

Poly(ADP-ribose) glycohydrolase (PARG) is a key enzyme in poly(ADP-ribose) (PAR) metabolism and a potential anticancer target. Many drug candidates have been developed to inhibit its enzymatic activity. Additionally, PDD00017273 is an effective and selective inhibitor of PARG at the first cellular level. Using human colorectal cancer (CRC) HCT116 cells, we investigated the molecular mechanisms and tumor biological aspects of the resistance to PDD00017273. HCT116RPDD , a variant of the human CRC cell line HCT116, exhibits resistance to the PARG inhibitor PDD00017273. HCT116RPDD cells contained specific mutations of PARG and PARP1, namely, PARG mutation Glu352Gln and PARP1 mutation Lys134Asn, as revealed by exome sequencing. Notably, the levels of PARG protein were comparable between HCT116RPDD and HCT116. In contrast, the PARP1 protein levels in HCT116RPDD were significantly lower than those in HCT116. Consequently, the levels of intracellular poly(ADP-ribosyl)ation were elevated in HCT116RPDD compared to HCT116. Interestingly, HCT116RPDD cells did not exhibit cross-resistance to COH34, an additional PARG inhibitor. Our findings suggest that the mutated PARG acquires PDD00017273 resistance due to structural modifications. In addition, our findings indicate that PDD00017273 resistance induces mutation and PARP downregulation. These discoveries collectively provide a better understanding of the anticancer candidate PARG inhibitors in terms of resistance mechanisms and anticancer strategies.

Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens.

Simple SummaryPoly(ADP-ribose) (PAR) functions in genome repair, cell metabolism, and cancer treatment; however, in vivo molecular tools to assess PAR levels are not readily available. Here, we describe two methods to measure PAR with high sensitivity in real time in vivo using the PAR binding domain (termed WWE) of Ring Finger Protein 146 (RNF146). In response to genotoxic stress, the WWE-EGFP fusion protein, termed LivePAR, displays nuclear enrichment enabling quantitative and visual detection of PAR formation. Additionally, a split luciferase assay using the same WWE domain facilitates rapid PAR quantitation in a plate format and provides a tool for chemotherapeutic candidate compound screening. Both tools can be used to identify genetic determinants of PAR formation and help in identifying compounds that alter PAR formation or degradation.Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds.

Inhibition of Poly (ADP-Ribose) Glycohydrolase Accelerates Osteoblast Differentiation in Preosteoblastic MC3T3-E1 Cells.

Poly ADP-ribosylation (PARylation) is a post-translational modification catalyzed by poly (ADP-ribose) polymerase (PARP) family proteins such as PARP1. Although PARylation regulates important biological phenomena such as DNA repair, chromatin regulation, and cell death, little is known about the relationship between osteoblast differentiation and the PARylation cycle involving PARP1 and the poly (ADP-ribose)-degrading enzyme poly (ADP-ribose) glycohydrolase (PARG). Here, we examined the effects of PARP inhibitor olaparib, an approved anti-cancer agent, and PARG inhibitor PDD00017273 on osteoblast differentiation. Olaparib decreased alkaline phosphatase (ALP) activity and suppressed mineralized nodule formation evaluated by Alizarin Red S staining in preosteoblastic MC3T3-E1 cells, while PDD00017273 promoted ALP activity and mineralization. Furthermore, PDD00017273 up-regulated the mRNA expression levels of osteocalcin and bone sialoprotein, as osteoblast differentiation markers, and osterix as transcription inducers for osteoblast differentiation, whereas olaparib down-regulated the expression of these genes. These findings suggest that PARG inhibition by PDD00017273 accelerates osteoblast differentiation in MC3T3-E1 cells. Thus, PARG inhibitor administration could provide therapeutic benefits for metabolic bone diseases such as osteoporosis.

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

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. 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. 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. 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. 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.

Replication catastrophe is responsible for intrinsic PAR glycohydrolase inhibitor-sensitivity in patient-derived ovarian cancer models

BackgroundPatients with ovarian cancer often present at advanced stage and, following initial treatment success, develop recurrent drug-resistant disease. PARP inhibitors (PARPi) are yielding unprecedented survival benefits for women with BRCA-deficient disease. However, options remain limited for disease that is platinum-resistant and/or has inherent or acquired PARPi-resistance. PARG, the PAR glycohydrolase that counterbalances PARP activity, is an emerging target with potential to selectively kill tumour cells harbouring oncogene-induced DNA replication and metabolic vulnerabilities. Clinical development of PARG inhibitors (PARGi) will however require predictive biomarkers, in turn requiring an understanding of their mode of action. Furthermore, differential sensitivity to PARPi is key for expanding treatment options available for patients.MethodsA panel of 10 ovarian cancer cell lines and a living biobank of patient-derived ovarian cancer models (OCMs) were screened for PARGi-sensitivity using short- and long-term growth assays. PARGi-sensitivity was characterized using established markers for DNA replication stress, namely replication fibre asymmetry, RPA foci, KAP1 and Chk1 phosphorylation, and pan-nuclear γH2AX, indicating DNA replication catastrophe. Finally, gene expression in sensitive and resistant cells was also examined using NanoString or RNAseq.ResultsPARGi sensitivity was identified in both ovarian cancer cell lines and patient-derived OCMs, with sensitivity accompanied by markers of persistent replication stress, and a pre-mitotic cell cycle block. Moreover, DNA replication genes are down-regulated in PARGi-sensitive cell lines consistent with an inherent DNA replication vulnerability. However, DNA replication gene expression did not predict PARGi-sensitivity in OCMs. The subset of patient-derived OCMs that are sensitive to single-agent PARG inhibition, includes models that are PARPi- and/or platinum-resistant, indicating that PARG inhibitors may represent an alternative treatment strategy for women with otherwise limited therapeutic options.ConclusionsWe discover that a subset of ovarian cancers are intrinsically sensitive to pharmacological PARG blockade, including drug-resistant disease, underpinned by a common mechanism of replication catastrophe. We explore the use of a transcript-based biomarker, and provide insight into the design of future clinical trials of PARGi in patients with ovarian cancer. However, our results highlight the complexity of developing a predictive biomarker for PARGi sensitivity.

NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

PARP1-modulated chromatin remodeling is a new target for cancer treatment.

Cancer progression requires certain tumorigenic mutations in genes encoding for different cellular and nuclear proteins. Altered expressions of these mutated genes are mediated by post-translational modifications and chromatin remodeling. Chromatin remodeling is mainly regulated by the chromatin remodeling enzyme complexes and histone modifications. Upon DNA damage, Poly-(ADP-ribose) Polymerase1 (PARP1) plays a very important role in the induction of chromatin modifications and activation of DNA repair pathways to repair the DNA lesion. It has been targeted to develop different anti-cancer therapeutic interventions and PARP inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for clinical use. But it has been found that the cancer cells often develop resistance to these PARP inhibitors and chromatin remodeling helps in enhancing this process. Hence, it may be beneficial to target PARP1-mediated chromatin remodeling, which may allow to reverse the drug resistance. In the current review, we have discussed the role of chromatin remodeling in DNA repair, how PARP1 regulates modifications of chromatin dynamics, and the role of chromatin modifications in cancer. It has also been discussed how the PARP1-mediated chromatin remodeling can be targeted by PARP inhibitors alone or in combination with other chemotherapeutic agents to establish novel anti-cancer therapeutics. We have also considered the use of PARG inhibitors that may enhance the action of PARP inhibitors to target different types of cancers.

Abstract 2043: Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies

Abstract Background:The enzyme Poly(ADP-ribose) glycohydrolase (PARG) plays an important role during the DNA damage repair process through hydrolysis of poly(ADP-ribose)(PAR) chains and accounts for approximately 90% of dePARylation activity within the cell. PAR chain synthesis is achieved by poly(ADP-ribose) polymerases (PARPs), which use donor NAD+ molecules to link repeating ADP-ribose units. The resulting PAR chains serve as a platform to recruit DNA repair proteins that are critical for single-strand break (SSB) repair. Following DNA repair PARG hydrolyzes the linkages to break down PAR chains, completing the cycle. Recent studies also found that dePARylation is necessary for the translocation of DNA repair proteins such as XRCC1 to SSB suggesting a more direct role for PARG in DNA damage repair. PARP inhibitors (PARPi), demonstrate synthetic lethality in cells with impaired homologous recombination (HR)-mediated DNA repair. Clinical studies have shown that responses to PARPi are often accompanied by high rates of resistance, generating a need for additional therapies for patients with HR-deficient tumors. The ability of PARG inhibition (PARGi) to exacerbate replication deficiencies makes it a promising therapeutic target for a broad range of cancer types with genomic instability. We therefore sought out to evaluate the effects of PARGi in HR-deficient ovarian and breast cancer models. Methods: To understand the effects of PARGi in cell growth and survival, a panel of HR-deficient cell lines and cell line derived xenografts (CDX) were exposed to PARGi alone or in combination with DNA damage response inhibitors (DDRi). In these studies, PAR chain accumulation was assessed as a marker of target engagement and PARPi was used to assess on target effects in vitro. Results: Using cellular proliferation assays and xenograft models, we find that PARGi increases the cellular levels of PAR and significantly decreases the viability of HR-deficient cancer cell lines. Furthermore, inhibition of cell proliferation by PARGi is antagonized by PARPi, which is consistent with an on-target cellular mechanism of action (MOA). In conclusion, PARGi induces significant accumulation of PAR chains and decreases cell proliferation both in vitro and in vivo in HR-deficient tumor cells. Ongoing exploratory studies will further delineate the molecular underpinning of the relationship between HR-deficiency and PARGi. Citation Format: Monah Abed, Diana Munoz, Firoz Jaipuri, Nandini Ravindran, Vidya Seshadri, Marya Liimatta, Claire Neilan, Xin Linghu, An Nguyen, Marie-Claire Wagle, Zineb Mounir. Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2043.

Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors

Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono(ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly(ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.

DNA replication stress and emerging prospects for PARG inhibitors in ovarian cancer therapy

Poly (ADP-ribosyl)ation has central functions in maintaining genome stability, including facilitating DNA replication and repair. In cancer cells these processes are frequently disrupted, and thus interfering with poly (ADP-ribosyl)ation can exacerbate inherent genome instability and induce selective cytotoxicity. Indeed, inhibitors of poly (ADP-ribose) polymerase (PARP) are having a major clinical impact in treating women with BRCA-mutant ovarian cancer, based on a defect in homologous recombination. However, only around half of ovarian cancers harbour defects in homologous recombination, and most sensitive tumours eventually acquire PARP inhibitor resistance with treatment. Thus, there is a pressing need to develop alternative treatment strategies to target tumours with both inherent and acquired resistance to PARP inhibition. Several novel inhibitors of poly (ADP-ribose)glycohydrolase (PARG) have been described, with promising anti-cancer activity in vitro that is distinct from PARP inhibitors. Here we discuss, the role of poly (ADP-ribosyl)ation in genome stability, and the potential for PARG inhibitors as a complementary strategy to PARP inhibitors in the treatment of ovarian cancer.

An efficient chemical screening method for structure-based inhibitors to nucleic acid enzymes targeting the DNA repair-replication interface and SARS CoV-2.

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.

TAMI-20. POLY(ADP-RIBOSE) GLYCOHYDROLASE INHIBITION SEQUESTERS NAD+ TO POTENTIATE THE METABOLIC LETHALITY OF ALKYLATING CHEMOTHERAPY IN IDH MUTANT TUMOR CELLS

Abstract Mutations in IDH1 or IDH2 characterize the majority of diffuse infiltrative gliomas of younger adulthood. DNA alkylator chemotherapy has proven to be an effective treatment for IDH mutant glioma, yet recurrences remain frequent and improved treatments are needed. Upon exposure of cancer cells to DNA alkylating agents, poly(ADP-ribose) polymerases (PARPs) catalyze the transient oligomerization of nicotinamide adenine dinucleotide (NAD+) into poly-ADP-ribose (PAR) chain-assemblies, which stimulate the DNA damage response. Here, we find that, in IDH mutant glioma cells, alkylator-induced cytotoxicity is dramatically augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme poly(ADP-ribose) glycohydrolase (PARG). Mechanistically, we show this augmentation is driven by a metabolic state pairing amplified alkylator-induced DNA damage with catastrophic depletion of NAD+. IDH mutant cancer cells have low basal levels of NAD+, rendering them vulnerable to prolonged NAD+ depletion. Either of two clinically-utilized monofunctional alkylators, procarbazine (PCZ) or temozolomide (TMZ), when exposed to multiple IDH mutant lines, activated PARP conversion of cellular NAD+ monomers into polymerized PAR. Subsequent PAR breakdown and recycling of NAD+ monomers was then halted by concomitant PARG inactivation, amplifying PARylation-mediated DNA damage signaling and critically depleting free NAD+, resulting in profound cytotoxicity. This effect was partially reversed by NAD+ rescue supplementation, confirming the key contribution of metabolic stress. Combined alkylator treatment and PARG inhibition in vivo maximized both NAD+ depletion and cytotoxicity in an endogenous IDH mutant cancer model. Thus, alkyating DNA damaging agents expose a metabolic liability in IDH mutant cancers, and subsequent targeted blockade of PAR breakdown and NAD+ metabolite recovery can exploit this vulnerability to provide genotype-specific therapeutic benefit.

Inhibition of PARG sensitizes ovarian cancer cells to PARP inhibitors and DNA damaging agents

Objective: Poly(ADP-ribose) glycohydrolase (PARG) is responsible for PAR catabolism. The aim of this study was to evaluate the effect of a PARG inhibitor on epithelial ovarian cancer cell lines, alone and in combination with a poly(ADP-ribose) polymerase inhibitor (PARPi) and/or cisplatin.