Investigating small molecule approaches to the pharmacological inhibition of CtIP function

  • Hugh Osborne

Student thesis: Phd

Abstract

In recent decades, cancer research has increasingly focussed on precision medicine; treatments that are selectively lethal to a given cancer due to a genetic abnormality, leading to potent therapies with fewer dose-limiting adverse reactions. An emerging precision medicine approach is the targeting of DNA repair factors, such as poly(ADP-ribose) polymerase (PARP) inhibition. PARP function is critical to multiple DNA repair pathways, and the maintenance of DNA replication fork stability. PARP inhibitors have made a considerable impact in the clinic, most notably in the treatment of patients suffering from BRCA1/2-mutant breast and ovarian cancers. However, the overall utility of this treatment modality has been hamstrung by the development of acquired drug resistance, in addition to the intrinsic insensitivity of many cancers to PARP inhibition. Therefore, there is a pressing need to develop DNA repair inhibitors that are effective in a distinct cohort of patients and/or potentiate the effects of PARP inhibitors, like olaparib, in recalcitrant cancers. In this regard, CtIP, is an intriguing DNA repair factor to target. It is an essential mediator of DNA end resection, a nucleolytic process in which the opposing ends of a DNA double-strand break (DSB) are degraded, allowing downstream repair to take place. Due to its prominence in multiple DSB repair pathways and replication fork stability, CtIP protein depletion sensitises cells to PARP inhibition. However, its disordered protein structure makes it challenging to target directly. Nonetheless, the post-translational modification (PTM) of CtIP is often essential to its recruitment and activity. In this thesis, the pharmacological inhibition of CtIP function is explored via two key PTMs: its N-terminal ubiquitylation and C-terminal sumoylation. Following its chemical synthesis, a published UBE2D inhibitor (BdS) was evaluated as a CtIP antagonist. UBE2Ds represent a ubiquitin-conjugating enzyme family that mediate CtIP ubiquitylation, a PTM important for its recruitment to DNA damage sites. It was found in this work that while BdS is unlikely to represent a selective UBE2D inhibitor, it potentiates olaparib efficacy in selected cell lines through a cooperative induction of replication catastrophe, as indicated by increased RPA consumption followed by pan-nuclear γH2AX staining. Subsequently, a CBX4/7 inhibitor, UNC3866 was explored to evaluate its antagonism of the CBX4-mediated sumoylation of CtIP, which increases CtIP-dependent DNA repair efficiency. UNC3866 reduced the efficiency of CtIP-dependent DNA repair and selectively sensitised a high-grade serous ovarian cancer cell line with dysregulated DSB repair to ionising radiation, but failed to synergise with olaparib which revealed key mechanistic insights into CBX4/7 inhibition. Taken together, CBX4/7 inhibition via UNC3866 might serve as a platform for the development of bona fide CtIP function antagonists, while UBE2D inhibition remains a potential avenue for further research.
Date of Award31 Dec 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorChristine Schmidt (Supervisor), Stephen Taylor (Supervisor) & Igor Larrosa (Supervisor)

Keywords

  • SUMO
  • Ovarian cancer
  • DNA repair
  • Ubiquitin-like protein
  • CtIP
  • Ubiquitin

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