Validating the WD Repeat Protein CDC40 as a Potential Therapeutic Target for Lung Cancer

Lung cancer is the second most diagnosed and most lethal cancer globally. Presently, there is a need for novel targets for the development of improved therapeutics. Data mining of whole genome genetic dependency screens suggested that the pre-mRNA splicing factor Cell Division Cycle 40 (CDC40) is essential for lung cancer cell survival. In this study, I characterized the effects of CDC40 knockdown in a panel of lung cancer cells. I show that CDC40 knockdown induced cell cycle defects resulting in strong growth inhibition and activation of late-stage apoptotic markers in all lung cancer cells tested. To further understand the cellular consequences of CDC40 suppression, I investigated global transcriptional and splicing changes and found that CDC40 knockdown resulted in a perturbation of splicing and translation-related genes in addition to a higher number of transcripts with intron retention. I explored one specific intron retention event in the mRNA transcript of the cell cycle regulatory protein CDCA5, a potential prognostic marker for cancer, and showed that CDC40 knockdown induced retention of its first intron leading to an increase in the unspliced CDCA5 transcript, and subsequent decrease in CDCA5 protein expression. Finally, I explored the direct protein interactions of CDC40 in lung cancer lines and found spliceosome components as the predominant binding partners for CDC40, further highlighting the major role of CDC40 in splicing. CDC40-CDC5 interaction, identified in the current thesis, was investigated using peptide displacement assay for drug development purpose. So far, no hits have been confirmed from screens. In addition, no disruption was observed even with quadruple mutations within the putative binding pocket on CDC40. This implies that it may be difficult to disrupt key protein-protein interactions using small molecules within this very large complex. In summary, I was able to demonstrate that CDC40 is important for lung cancer cell survival and has potential as a novel therapeutic target for lung cancer treatment. I confirmed that CDC40 is involved in the human pre-mRNA splicing process as it is an important component of the spliceosome like its yeast counterpart. Furthermore, I characterized several CDC40 interactors to look for pockets for future therapeutic intervention.

Department dc:contributor.department

Pharmacology

Year dc:date.issued

2023

Author dc:creator

• Hu, Die

Advisors dc:contributor.advisor

• Al-awar, Rima RA
• Vedadi, Masoud MV

• CDC40
• Drug discovery
• Protein-protein interaction
• shRNA knockdown
• Splicing
• WD Repeat protein

Handle dc:identifier.uri

http://hdl.handle.net/1807/128164

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/128164