Abrogating the G2/M checkpoint with PROTACs to enhance DNA damaging therapies
Creators: name: Buckley-Benbow, Lauryn
Publisher: name: Lancaster University
Year: 2024
Subjects: PROTAC
Cancer
Wee1
DNA damage response

description: 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.