poly(ADP-ribose) Glycohydrolase Inhibitors Research Articles

Abstract 546: PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach

Abstract Colorectal carcinomas (CRC) and pancreatic ductal adenocarcinomas (PDAC) are the 2nd and 3rd leading causes of cancer-related deaths in the U.S. respectively. Recent work from our lab highlighted Poly ADP-Ribose Glycohydrolase (PARG) as a target in PDAC, and PARG inhibition (PARGi) sensitized PDAC cells to the DNA damaging agents, 5-FU and oxaliplatin. Other labs have shown that PARG inhibition causes replication stress in cancer cells. CRC and PDAC cells, while cellularly different, share a high frequency of KRAS and P53 inactivating mutations. High levels of replication stress (RS) has also been well documented in these tumor types. Targeting DNA Damage Response Kinases (DDRKs) in combination with chemotherapy (mostly DNA damage inducers like 5-FU, oxaliplatin, etc.) has gained recent interest in solid tumors but overlapping toxicities and tolerability have been issues clinically. Accordingly, we hypothesized that combined inhibition of PARG and DDRKs could yield synergistic effects in tumors with high replication stress while alleviating adverse events seen in other combination strategies. We determined in drug sensitivity assays that PARGi did not synergize with either the CHK1 inhibitor prexasertib or the ATR inhibitor VE-821 in our cell lines but did synergize with Wee1i (AZD1775) in both PDAC and CRC cell lines (Comb. Analysis, p<.05). Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for Panc1, MiaPaCa-2, CFPAC1 (PDAC cells), HCT116 (CRC cells) and SW480 (CRC cells) cell lines with the combination of PARGi and Wee1i as compared to single-agent controls (p<.001). To validate the targeted synergy between AZD1775 and PARGi, we utilized genetic models of PARG and Wee1 silencing, including CRISPR PARG knockout cell lines, siRNA, and shRNA knockdown models. Genetic knockdown of Wee1 and PARG in PDAC and CRC cell lines yielded strikingly similar results in colony formation assays, with 2 and 12-fold changes respectively compared to Wee1i alone (P-value < .0001). HCT116 CRISPR PARG knockouts yielded a 5-fold difference in Wee1i sensitivity in colony formation assays. Mechanistically, western blot analysis demonstrated PARGi and Wee1i led to >2-fold increases in PARylation, yH2AX, and cleaved-caspase 3 as compared to single-agent controls. In conclusion, combined PARG - Wee1 inhibition is a novel targeting strategy in PDAC and CRC tumors that results in decreased cell viability. We are currently testing the efficacy of AZD1775 in PARG CRISPR-knockout tumors in in vivo xenograft models. Ongoing studies are focused on assessing the effect of Wee1i + PARGi on chromosomal aberrations, disruption in replication fork dynamics via DNA fiber labeling, and cell cycle dependencies. Citation Format: Lebaron C. Agostini, Aditi Jain, Michael Pishvaian, Charles Yeo, Jonathan Brody. PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 546.

Androgen Receptor and Poly(ADP-ribose) Glycohydrolase Inhibition Increases Efficiency of Androgen Ablation in Prostate Cancer Cells

There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.

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.

Structural insights into the active site of poly(ADP-ribose) glycohydrolase using docking modes of 6-hydroxy-3H-xanthen-3-one derivative inhibitors

Poly(ADP-ribose) glycohydrolase (PARG) plays an essential role in poly(ADP-ribose) (PAR) turnover, and thereby regulating DNA transactions, such as DNA repair, replication, transcription and recombination. Here, we examined the inhibitory activities of 6-hydroxy-3H-xanthene-3-one (HXO) derivatives and analyzed their binding modes in the active site of PARG by in silico docking study. Among the derivatives, Rose Bengal was found to be the most potent inhibitor of PARG and its halogen groups were revealed to cooperatively potentiate the inhibitory activity. Importantly, the binding mode of Rose Bengal occupied the active site of PARG revealed the presence of unique “Sandwich” residues of Asn869 and Tyr792, which enable the inhibitor to bind tightly with the active pocket. This sandwich interaction could stabilize the π-π interactions of HXO scaffold with Phe902 and Tyr795. In addition, to increase the binding affinity, the iodine and chlorine atoms of this inhibitor could contribute to the inducing of favorable disorders, which promote an entropy boost on the active site of PARG for structural plasticity, and making the stable configuration of HXO scaffold in the active site, respectively, as judged by the analysis of binding free energy. These results provide new insights into the active site of PARG and an additional opportunity for designing selective PARG inhibitors.

Selective small molecule PARG inhibitor causes replication fork stalling and cancer cell death

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.

Abstract NT-117: INHIBITION OF PARG, SENSITIZES OVARIAN CANCER CELLS TO PARP INHIBITORS AND DNA DAMAGING AGENTS

Abstract BACKGROUND: Poly(ADP-ribose) glycohydrolase (PARG) is responsible poly(ADP-ribose) (PAR) catabolism, which is synthesized by poly(ADP-ribose) polymerases (PARPs) at the site of DNA single-strand breaks (SSB). Faulty PAR formation or disintegration inhibits SSB repair. PARP inhibitors (PARPi) exploit the synthetic lethality between SSB repair and homologous recombination (HR) double strand break (DSB) repair. The most effective PARPi traps PARP at the SSB site, causing a double strand break during DNA synthesis that the cell cannot repair. The trapping of PARP can also be achieved through PARG inhibition. Due to the high rate of HR defects in epithelial ovarian cancer, the aim of this study is to evaluate the effect of a PARG inhibitor (PARGi) on epithelial ovarian cancer cell lines, alone and in combination with PARPi or cisplatin. METHODS: PARG protein levels were assessed in 74, unselected, snap-frozen, tumors kept in our bio-bank by western blotting and immuno-histochemistry (IHC), as well as in the TCGA database. PARG was knocked down in two cell lines resistant to PARP inhibition, OVCAR3 and SKOV3. The knockdowns were subjected to cell migration and cell proliferation assays, as well as single cell analysis. BRCA1 mutated and BRCA1 wild-type cell lines were exposed to clinically relevant doses of PARGi, PARPi, or cisplatin, separately or in various combination. Therapeutic efficacy was assessed using colony formation assay. Western Blotting was used to detect the levels of PARG and PARP proteins in the cell lines. RESULTS: PARG mRNA was expressed in 30% of tumors in TCGA. PARG protein was also detected in 30% of the biobank tumors analyzed. PARG knockdowns exhibited reduced rates of cellular proliferation and significant G2/M cell cycle arrest. In PARG over expressed ovarian cancer cell lines, PARG inhibitor similarly reduced cell migration (OVCAR3 and SKOV3: 75% reduction, SNU-251: 48% reduction). CONCLUSION: PARG inhibition appears to be a viable, complementary strategy to PARP inhibition in HR-deficient cancers. The effect of BRCA1 on the efficacy of PARG inhibition, as well as various treatment combinations, are currently under investigation. Citation Format: Emad Matanes, Tahira Baloch , David Octeau, Roy Kessous, Liron Kogan, Ido Laskov, Walter Gotlieb, Amber Yasmeen. INHIBITION OF PARG, SENSITIZES OVARIAN CANCER CELLS TO PARP INHIBITORS AND DNA DAMAGING AGENTS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-117.

Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) exhibit single agent therapeutic activity and sensitize glioblastoma cells to ionizing radiation

Abstract Objective The lack of an effective therapy for glioblastoma (GBM) largely results from the intrinsic resistance of GBM cells. The radiosensitizing activity of inhibitors of poly(ADP-ribose) polymerases (PARPs) highlights the important role of poly(ADP-ribose) (PAR) in the DNA damage response. In contrast to PARPs, inhibition of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for degrading PAR chains, has shown single agent therapeutic activity in non-glioma cancer cells. This work aims to validate the therapeutic potential of PARG inhibitors (PARGi) in GBM. Results Baseline PAR levels were found to vary between different primary and commercial GBM cells, with PARylation increasing upon exposure of cells to ionizing radiation (IR), as expected. Target engagement of a novel PARGi, PDD00017273, was confirmed by the accumulation of nuclear PAR in treated cells. Inhibitor specificity was demonstrated using an inactive control compound and by combining PARGi with the PARP inhibitor olaparib, which blocked the effect. Single agent treatment with PARGi reduced the clonogenic survival of GBM cells in a concentration-dependent manner. Importantly, PARGi also sensitized GBM cells to IR (sensitizer enhancement ratios, SER, ≥ 1.40) Conclusion In contrast to PARP inhibitors, novel PARGi exhibit single agent activity against a panel of GBM cell lines, and also show robust radiosensitizing activity. PARGi therefore have therapeutic potential in this cancer of unmet need.

Poly (ADP) Ribose Glycohydrolase Can Be Effectively Targeted in Pancreatic Cancer.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair-deficient cells compared with homologous repair-proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. SIGNIFICANCE: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells.

Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.

DNA Replication Vulnerabilities Render Ovarian Cancer Cells Sensitive to Poly(ADP-Ribose) Glycohydrolase Inhibitors

SummaryInhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%–20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.

Oncogenic activity of poly (ADP-ribose) glycohydrolase

Poly (ADP-ribosylation), known as PARylation, is a post-translational modification catalyzed by poly (ADP-ribose) polymerases (PARP) and primarily removed by the enzyme poly (ADP-ribose) glycohydrolase (PARG). While the aberrant removal of post-translation modifications including phosphorylation and methylation has known tumorigenic effects, deregulation of PARylation has not been widely studied. Increased hydrolysis of PARylation chains facilitates cancer growth through enhancing estrogen receptor (ER)-driven proliferation, but oncogenic transformation has not been linked to increased PARG expression. In this study, we find that elevated PARG levels are associated with a poor prognosis in breast cancers, especially in HER2-positive and triple-negative subtypes. Using both in vitro and in vivo models, we demonstrate that heightened expression of catalytically active PARG facilitates cell transformation and invasion of normal mammary epithelial cells. Catalytically inactive PARG mutants did not recapitulate these phenotypes. Consistent with clinical data showing elevated PARG predicts poor outcomes in HER2+ patients, we observed that PARG acts in synergy with HER2 to promote neoplastic growth of immortalized mammary cells. In contrast, PARG depletion significantly impairs the growth and metastasis of triple-negative breast tumors. Mechanistically, we find that PARG interacts with SMAD2/3 and significantly decreases their PARylation in non-transformed cells, leading to enhanced expression of SMAD target genes. Further linking SMAD-mediated transcription to the oncogenicity of PARG, we show that PARG-mediated anchorage-independent growth and invasion are dependent, at least in part, on SMAD expression. Overall, our study underscores the oncogenic impact of aberrant protein PARylation and highlights the therapeutic potential of PARG inhibition in breast cancer.

Cell-Active Small Molecule Inhibitors of the DNA-Damage Repair Enzyme Poly(ADP-ribose) Glycohydrolase (PARG): Discovery and Optimization of Orally Bioavailable Quinazolinedione Sulfonamides

DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure-activity relationships of these novel inhibitors.

Abstract A72: PARP vs PARG: Exploring Poly (ADP-ribose) glycohydrolase inhibitors in the context of high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer is characterized by TP53 mutation and rampant genomic instability, in part due to DNA damage repair defects. This latter feature opens up opportunities for synthetic-lethality-based chemotherapy, exemplified by the ability of PARP inhibitors to selective kill cells harboring homologous recombination defects. To further explore the synthetic lethality concept, we are characterizing a first-in-class inhibitor of PARG, a glycohyrolase that counterbalances PARP activity. In particular, we are asking a number of specific questions: Will PARG inhibitors be effective in the context of HGSOC? If so, via what mechanism, and will they offer something different to PARP inhibition? Can we also start to devise predictive biomarkers and rational combination strategies? To address these questions, we treated an exemplar panel of six HGSOC cell lines with selective PARG and PARP inhibitors and assayed proliferation, cell cycle dynamics, and survival. This approach identified one line that is exquisitely sensitive to PARG inhibition but resistant to the PARP inhibitor. Conversely, we identified one line exquisitely sensitive to the PARP inhibitor but resistant to PARG inhibition. The remaining four lines were relatively resistant to both. Sensitivity to the PARG inhibitor was accompanied by pan-nuclear γH2AX staining and a G2 cell cycle block. To define the mechanism underlying PARG inhibitor sensitivity, we performed an RNAi library screen. In brief, a resistant cell line was transfected with siRNAs targeting known PARP synthetic lethal genes and the induction of γH2AX measured. We identified a number of genes involved in DNA replication and replication stress. These insights have allowed us to begin exploring rational drug combinations to determine if we can sensitize additional HGSOC cell lines to PARG inhibition. To translate our findings beyond cell lines, we are testing the PARG inhibitor against a panel of ex vivo cultures generated from patient biopsies collected in Manchester. To do this, we have set up a biopsy pipeline that delivers ascites and surgical specimens from both chemotherapy-naive and treated patients direct to the laboratory. Cells are disaggregated, isolated by centrifugation, then plated into OCMI media and cultured under low-oxygen conditions. Selective trypisinization and/or magnetic bead separation yields tumor and stromal populations that are validated by flow cytometry, immunofluorescence microscopy, and TP53 genotyping. Importantly, this protocol routinely produces proliferative populations of tumor cells suitable for drug sensitivity profiling. Our latest results testing the PARG inhibitor will be presented. Citation Format: Stephen S. Taylor, Anthony Tighe, Nisha Pillay, Nourdine Bah, Louisa Nelson, Dominic James, Andrew Clamp, Richard Edmondson, Gordon Jayson. PARP vs PARG: Exploring Poly (ADP-ribose) glycohydrolase inhibitors in the context of high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A72.

Abstract 1973: Targeting PARG in pancreatic cancer: Implications for synthetic lethal therapeutic strategies

Abstract Metastatic pancreatic ductal adenocarcinoma (PDA) has an average survival of less than one year. There is a pressing need to identify patient subgroups for treatment with novel targeted agents and the necessity to combat increasing incidence of therapeutic resistance. In a previous study, we identified that poly(ADP) ribose glycohydrolase (PARG) is a critical player in mediating resistance to PARP inhibitor (PARPi); therefore targeting PARG is a strategy to enhance PARPi therapy in PDA and can be optimized to benefit patients with or without homologous repair (HR) deficiencies. We developed and characterized multiple PARG inhibition models in both DNA- repair proficient (MIA PaCa-2) and deficient (Hs766t) PDA lines; doxycycline-inducible shPARG knockdown, CRISPR- mediated PARG knockout and small molecule inhibition via a series of potent first-in-class, cell-active PARG inhibitors (PARGi). Our data show that PARG inhibition is synthetic lethal with DNA damage repair deficiency in PDA cells. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality: DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-). We have also shown that PARG inhibition enhances PARPi sensitivity through increased accumulation of DNA damage, apoptosis and persistence of detrimental PARylation. Moreover, PARP1 was trapped on the chromatin in response to both PARPi treatment as well as DNA damaging agents such as oxaliplatin. Complementary xenograft experiments were performed wherein MIA.shPARG cells were injected in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 100mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Similar results were obtained when DOX-fed mice with MIA.shPARG cells were treated with olaparib at 50mg/kg. Furthermore, in an attempt to mimic and break long-term in vivo PARPi resistance, doxycycline-mediated PARG inhibition was induced in the olaparib treatment arm on day 56 (with established tumors, and exposed to olaparib for 3weeks i.e. 15 injections). This resulted in a significant decrease in tumor volume when compared to control untreated arm (46% decrease, p-value 0.0025) and the olaparib only treatment arm (25% decrease, p-value 0.0124). We are currently validating these results in a DDR-deficient HST.shPARG cell line, as well as with CRISPR knockouts of PARG. Together these studies validate PARG as a therapeutically relevant and “druggable” target in both HR-proficient and deficient PDA cells, and lays the groundwork to optimize PARPi-based as well as other DNA targeted therapies in the treatment of PDA. Citation Format: Saswati N. Chand, AnnJosette Ramirez, Aditi Jain, Avinoam Nevler, Cinthya Yabar-Lowder, Joseph A. Cozzitorto, Dominic I. James, Allan Jordan, Kate M. Smith, Ian Waddell, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. Targeting PARG in pancreatic cancer: Implications for synthetic lethal therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1973.

Abstract 1943: PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition

Abstract Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models. Citation Format: Leenus Martin, Tzuling Cheng, Dominic I. James, Habiba Begum, Kate M. Smith, Allan Jordan, Ian Waddell, Kedar Vaidya, Marcus Fischer, Bing Yao, Jason Drummond, Leah Cleary, Ruben Martinez, James Sutton, Nandini Ravindran, James Joseph, Eleni Venetsanakos, Michael Dillon, Jeffrey H. Hager, Lisa D. Belmont. PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1943.

Abstract LB-A13: Targeting poly-ADP ribose glycohydrolase: pursuing a synthetic lethal therapeutic strategy to treat all pancreatic cancers

Abstract This study lays the groundwork to target poly-ADP ribose glycohydrolase (PARG), a DNA repair enzyme that counteracts PARP-1 activity and is upregulated in response to PARP inhibitor (PARPi) treatment. The goal of this study is to optimize PARPi-based therapies for all pancreatic cancer patients, irrespective of their DNA repair status (i.e., homologous repair (HR) deficiency). Metastatic pancreatic ductal adenocarcinoma (PDA) is a lethal disease with an average survival of less than one year, which highlights the need for novel treatment regimens. Though PARPi therapy offers a personalized strategy in a subset of tumors with HR defects, initially responsive patients typically become resistant to therapy. Therefore, identifying and combating PARPi resistance mechanisms is a rising concern. We have previously shown that PARG is a critical player in mediating resistance; therefore we evaluated PARG as a candidate target in PDA. Importantly, we demonstrated that the pro-oncogenic protein, HuR (ELAVL1), upregulates PARG specifically in tumor microenvironment niches (e.g., drug exposure), thus providing a unique, cancer-specific therapeutic window for targeting PARG. We evaluated a series of potent first-in-class, cell-active small molecule PARG inhibitors; PDDX-01, PDDX02 and PDDX-04 in pre-clinical models of PDA. These PARGi effectively inhibited PARG activity, caused persistence of PAR polymers, increased accumulation of γH2AX foci and inhibited short and long-term growth of PDA cell lines. Similar to PARPi agents, PARG inhibition was also shown to be synthetic lethal; DNA damage repair (DDR)- deficient cell lines were more sensitive than the DDR- proficient PDA cell lines. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality; DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-) modulation. Using chromatin tethering assays, these PARGis effectively trapped PARP1 on DNA, alone as well as combination with the PARPi olaparib, and a DNA damaging agent oxaliplatin. We also developed and characterized a PARG inhibition model in vivo in a DNA- repair proficient PDA line, MIA PaCa2. The doxycycline-inducible shPARG knockdown was validated for PARG inhibition, PAR accumulation and increased PARPi sensitivity in vitro. Complementing xenograft experiments were performed wherein 3 million cells were injected per flank in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 50mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Taken together, these studies help define and break a potent PARPi resistance mechanism and also validate pursuing PARG as a valuable “druggable” target in both HR-proficient and deficient PDA cells. Citation Format: Saswati N. Chand, AnnJosette Ramirez, Aditi Jain, Avinoam Nevlar, Cinthya Yabar-Lowder, Joseph A. Cozzitorto, Dominic I. James, Ian D. Waddell, Charles J. Yeo, Michael J. Pishvaian, Jordan M. Winter, Jonathan R. Brody. Targeting poly-ADP ribose glycohydrolase: pursuing a synthetic lethal therapeutic strategy to treat all pancreatic cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A13.

Radiosensitization with an inhibitor of poly(ADP-ribose) glycohydrolase: A comparison with the PARP1/2/3 inhibitor olaparib

Upon DNA binding the poly(ADP-ribose) polymerase family of enzymes (PARPs) add multiple ADP-ribose subunits to themselves and other acceptor proteins. Inhibitors of PARPs have become an exciting and real prospect for monotherapy and as sensitizers to ionising radiation (IR). The action of PARPs are reversed by poly(ADP-ribose) glycohydrolase (PARG). Until recently studies of PARG have been limited by the lack of an inhibitor. Here, a first in class, specific, and cell permeable PARG inhibitor, PDD00017273, is shown to radiosensitize. Further, PDD00017273 is compared with the PARP1/2/3 inhibitor olaparib. Both olaparib and PDD00017273 altered the repair of IR-induced DNA damage, resulting in delayed resolution of RAD51 foci compared with control cells. However, only PARG inhibition induced a rapid increase in IR-induced activation of PRKDC (DNA-PK) and perturbed mitotic progression. This suggests that PARG has additional functions in the cell compared with inhibition of PARP1/2/3, likely via reversal of tankyrase activity and/or that inhibiting the removal of poly(ADP-ribose) (PAR) has a different consequence to inhibiting PAR addition. Overall, our data are consistent with previous genetic findings, reveal new insights into the function of PAR metabolism following IR and demonstrate for the first time the therapeutic potential of PARG inhibitors as radiosensitizing agents.

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.

First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib.

The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).

PARG Inhibitors and Functional PARG Inhibition Models.

Poly(ADP-ribose) polymerases (PARPs) family proteins catalyze poly(ADP-ribosylation) (PARylation) by conjugating ADP-ribose residues repeatedly on amino acid residues using nicotinamide adenine dinucleotide as a substrate. The inhibitors of PARP widely block DNA repair processes and are currently examined in clinical trials of cancer therapy. Poly(ADP-ribose) glycohydrolase (PARG) is the main nuclear enzyme, which digests poly(ADP-ribose) into ADP-ribose. PARG inhibitor could also be considered as a chemotherapeutic agent for cancer, because of its involvement in DNA repair. Various PARG inhibitors with IC50 value of micromolar to submicromolar range have been reported. However, for most of these chemicals, the specificity of inhibition has not been fully evaluated. PARG functional inhibition models in various organisms have been developed. Here, inducible PARG knockdown system was developed in HeLa cells and the cell line will be useful for identifying the synthetic lethal genes or affecting genes for PARG inhibitor treatment and also for functional elucidation of PARP superfamily molecules.