Targeting USP7 to treat colorectal cancer

Abstract

Ubiquitination is a post-translational modification important for cellular homeostasis. Its dysregulation has been linked to various cancers including colorectal cancer (CRC). Ubiquitination involves E3 ubiquitin ligases adding ubiquitin to target proteins and deubiquitinase enzymes (DUBs) reversing this process. Ubiquitin-specific protease 7 (USP7) is one of the most well studied DUBs with target proteins including MDM2, EZH2, and FOXO4; involved in DNA damage, immune signalling, epigenetic regulation and apoptosis. Much research has focused on the role of USP7 in regulation of the p53-MDM2 axis, and inhibiting USP7 has been explored as a way of re-activating p53 in cancer. In this thesis, we explored the p53-dependent and -independent effects of a novel USP7 inhibitor, AD04, in CRC models and how it impacts response to 5-Fluorouracil (5FU) chemotherapy. Using a panel of p53 CRISPR-knockout isogenic CRC models, we identified modest p53-dependent effects of AD04 on cell viability which correlated with increased p53 stabilisation and its transcriptional targets p21 and MDM2. Co-treatment with AD04 and 5FU induced a significant reduction in cell viability compared to treatment with AD04 or 5FU alone. Notably, this synergy was independent of p53 status. We identified two independent mechanisms of synergy, in a p53 WT setting the synergy was dependent on caspase-8, but this was not the case in p53 mutant. We employed transcriptomic approaches to further characterise these effects. RNA-sequencing revealed TNFa signalling via NFΚB as a consistent pathway altered in response to both USP7 inhibition and its combination with 5FU.We further employed complex co-culture models to develop more clinically relevant models. Again, we found that AD04 enhances the effects of 5FU, independent of p53. Moreover, co-cultures with activated T-cells further enhanced the effects of AD04/5FU in these co-cultures. Altogether, these results reveal a novel synergy between USP7 inhibition and 5FU that is not dependent on p53.

Thesis embargoed until 31 July 2030.

Date of Award	Jul 2025
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Almac Discovery Ltd
Supervisor	Simon McDade (Supervisor) & Daniel Longley (Supervisor)