Abstract 1222: Validating the WD repeat protein CDC40 as a potential therapeutic target for lung cancer

Abstract

Abstract Lung cancer is the most lethal cancer globally, accounting for 1.8 million deaths in 2018. Presently, there is a need for novel targets for the development of improved therapeutics. Data mining through genetic screens suggested that Cell Division Cycle 40 (CDC40) is essential for lung cancer cell survival. CDC40 is a WD repeat (WDR) protein that acts as a scaffold mediating protein-protein interactions (PPIs) within the spliceosome complex, and is expected to participate in pre-mRNA splicing pathways. Splicing is a fundamental cellular process for transcriptome and proteome diversity that could support carcinogenesis when hijacked. The therapeutic modulation of cancer-related splicing patterns may thus be an effective strategy for lung cancer treatment, although the precise role of CDC40 in splicing and consequences of CDC40 inhibition remain unknown. With previous success in developing small molecule inhibitors for WDR proteins, our interest now extends to CDC40.Hypothesis: Inhibition of CDC40 will disrupt pre-mRNA splicing and cell cycle control mechanisms in lung cancer cells leading to cell cycle arrest and apoptosis.Through a doxycycline-inducible lentivirus system, several short hairpin RNAs (shRNAs) were introduced into lung cancer cell lines. With CDC40 knockdown (KD), the dependency of 6 lung cancer lines to CDC40 was validated, characterized by significant growth suppression, cell cycle arrest and apoptosis phenotypes. Kinetically, CDC40 mRNA and protein KD were observed as soon as 24h after shRNA induction, followed by cell cycle arrest. This in turn led to Caspase 3/7 activation and an anti-proliferative phenotype. The above phenotypes were rescued upon CDC40 overexpression.RNA-sequencing of a shCDC40-induced lung cancer line revealed the downregulation of genes from cell proliferation and cell structure-related pathways, upregulation of genes from transcription and splicing pathways, as well as a significant increase in intron retention events. RT-qPCR confirmed the downregulation of genes involved in proliferation and glycolipid synthesis, and upregulation of genes involved in splicing. These results indicate that CDC40 plays a role in RNA splicing, and suggest that alterations in splicing through CDC40 inhibition could lead to lung cancer cell growth suppression.Preliminary in silico analysis of CDC40 PPIs identified the top central pocket of the CDC40 WD domain as the most therapeutically tractable site. This region is theorized to interact with CDC5, another spliceosome component. Further investigation of pharmacological (chemical probes) and genetic (point mutation) interventions of CDC40-CDC5 interactions will improve understanding of the role of CDC40 within the spliceosome and the effects of CDC40 PPIs on splicing.Our results demonstrate that CDC40 is essential for lung cancer cell survival, and points to its further investigation as a therapeutic target for lung cancer. Citation Format: Die Hu, Brigitte L. Thériault, Vida Talebian, Julie Owen, Richard Marcellus, Rima Al-awar. Validating the WD repeat protein CDC40 as a potential therapeutic target for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1222.