Intrinsic PARG inhibitor sensitivity is mimicked by TIMELESS haploinsufficiency and rescued by nucleoside supplementation.

A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analysed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork stalling, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential. Importantly nucleoside supplementation restores PARG inhibitor resistance despite the continued presence of PAR chains, indicating that sensitivity does not correlate with PAR levels. In addition, we show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances.

Spatially resolved phosphoproteomics reveals fibroblast growth factor receptor recycling-driven regulation of autophagy and survival.

Receptor Tyrosine Kinase (RTK) endocytosis-dependent signalling drives cell proliferation and motility during development and adult homeostasis, but is dysregulated in diseases, including cancer. The recruitment of RTK signalling partners during endocytosis, specifically during recycling to the plasma membrane, is still unknown. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b) recycling, we reveal FGFR signalling partners proximal to recycling endosomes by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphorylated peptides enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncover that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the recycling endosomes, leading to autophagy suppression and cell survival. This adds to the growing importance of RTK recycling in orchestrating cell fate and suggests a therapeutically targetable vulnerability in ligand-responsive cancer cells. Integrating SRP with other systems biology approaches provides a powerful tool to spatially resolve cellular signalling.

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.