Abrogating the G2/M checkpoint with PROTACs to enhance DNA damaging therapies

Buckley-Benbow, Lauryn and Allinson, Sarah and Fielding, Andrew and Parsons, Jason L. and Gadd, Morgan (2024) Abrogating the G2/M checkpoint with PROTACs to enhance DNA damaging therapies. PhD thesis, Lancaster University.

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Abstract

Head and neck squamous cell carcinoma (HNSCC) incidence is expected to increase by 30% by 2030. Whilst rare, the five-year survival rate for HNSCC patients is only 50%, with tumours often resistant to genotoxic treatments. Survival data shows that the current treatment modality is ineffective, thus, it is critical to find targets for combinatorial treatment, to increase the efficacy of common tumour therapies against HNSCC. HNSCC tumours often have a dysfunctional G1/S checkpoint due to p53 mutations. Abrogating the G2/M checkpoint, via targeting of Wee1 (a serine/threonine kinase involved at the G2/M checkpoint), in this context would promote tumour cell death via synthetic lethality after radiotherapy or chemotherapy treatment. Small molecule inhibitors (SMIs) are often used to target proteins clinically and AZD1775, a Wee1 inhibitor (Wee1i), has shown promise in clinical trials. Although potent, the Wee1i has many off-target effects, therefore our group developed Wee1 PROteolysis TArgeting Chimeras (PROTACs) to overcome selectivity issues. PROTACs are heterobifunctional molecules that contain an E3 ubiquitin ligase recruiting domain and a target protein domain. These ligands are connected via a linker and this molecule facilitates the formation of a productive ternary complex to polyubiquitylate the target protein and target it for degradation by the ubiquitin proteasome system (UPS). Here, characterisation of Wee1 PROTACs in HNSCC with varying p53-status demonstrated that cereblon (CRBN)- and Von Hippel-Lindau (VHL)-based Wee1 PROTACs can successfully and rapidly degrade Wee1 and reduce the phosphorylation of its substrate, pCDK1 (Tyr15). Monotherapy use of Wee1 PROTACs caused a loss of cell viability and the most potent compounds were used in lung and kidney cancer cell lines to investigate the bearing of E3 ligase expression on efficacy of the molecules. This work found that the ability for PROTACs to degrade their protein of interest was not dependent on absolute levels of E3 ligase present. Clonogenic survival assays, cell viability assays and apoptosis detection flow cytometry were performed to evaluate the cytotoxicity of the combination of Wee1 inhibition or degradation with genotoxic agents. p53-deficient cell lines displayed more radiation- and cisplatin-induced cell death as a result of sensitization to the genotoxins from Wee1-targeting treatments. In addition, use of Wee1-targeting compounds alone induced apoptosis in the absence of a genotoxic agent. HNSCC cell lines showed replication stress and irregularities in DNA content, indicative of chromosome missegregation or improper mitosis, when treated with Wee1-targeting compounds as single and combinatorial strategies. These observations provide insight that Wee1 PROTACs have potential to be used in place of SMIs and has provided a good foundation for future work to investigate if increased specificity is an advantageous trait for cancer treatment.