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.

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The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT–β-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3–5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT–β-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.

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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.