Investigating the interplay between TIMELESS and PARG inhibitor sensitivity

Abstract

Cancer treatment has been revolutionised by synthetic lethality, which describes redundant genes that are lethal when both disrupted. For instance, inhibitors of poly(ADP-ribose) polymerase (PARP) have shown impressive efficacy in the clinic treating women with homologous recombination (HR)-deficient ovarian cancer. However, ~50% of ovarian cancers are HR-proficient meaning there is a pressing need for additional therapies. PARG, the glycohydrolase that counterbalances PARP1/2 activity, is a potential target for HR-proficient ovarian cancer. However, a better understanding of the molecular determinants of PARG inhibitor (PARGi)-sensitivity is required to identify predictive biomarkers required for clinical translation. Intrinsic PARGi-sensitivity of ovarian cancer cells is accompanied by persistent DNA replication stress and replication catastrophe, indicating an underlying DNA replication vulnerability. Indeed, depletion of the pivotal DNA replication protein, TIMELESS confers PARGi-sensitivity. This thesis explores the molecular determinants of PARGi-sensitivity by focussing on the TIMELESS synthetic lethal relationship. Previous work used CRISPR/Cas9-mutagenesis to identify a four amino acid deletion in TIMELESS which results in PARGi-sensitivity. It follows, this work set out to address how the mutation affects TIMELESS, and DNA replication, to increase reliance on PARG activity. To determine the direction of the thesis work, firstly the TIMELESS-PARG synthetic lethal relationship was explored in additional model systems and the TIMELESS mutant cell line was re-isolated. Characterisation of the mutation found it is a separation-of-function-mutation that sustains DNA replication but renders cells critically reliant on PARG activity to maintain replisome progression. PARGi-treatment of TIMELESS mutant cells induces markers of DNA replication stress including replication fork asymmetry, RPA foci, CHK1 and Kap1 phosphorylation. Pan-nuclear gamma-H2AX staining and a pre-mitotic cell block are also displayed by PARGi-treated TIMELESS mutant cells, indicating replication catastrophe. The TIMELESS mutation results in a hyper-PARylated state with slowed replisome progression that is exacerbated by PARGi-treatment. Restricting the PARylation of the mutant TIMELESS protein, by mutating a key PAR-acceptor site, increased replisome speed and restored resistance to PARGi. These observations support a model whereby TIMELESS regulates replisome progression by integrating the opposing forces of PARP and PARG. Hyper-PARylation, from the TIMELESS mutation and PARGi-treatment, dissociate TIMELESS from chromatin. Potentially, the model could be based on TIMELESS’ role in coordinating helicase and polymerase activities to promote fork progression; increased TIMELESS-chromatin dissociation hence reduces fork progression in a PAR-dependent manner. Consistently, PARP inhibition protects cells from the replication stress effects of PARGi. Taken together, these results propose a novel mechanism of regulating replication fork progression in response to replication stress.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen Taylor (Supervisor)