Targeting NEDDylation in Colorectal Cancer


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Jennifer Ferris1,Jennifer Ferris2,Caitriona Holohan3,Alexander McIntyre3,Simon McDade3,Daniel B Longley3,Vicky Coyle3
1Centre of Cancer Research and Cell Biology,2Cell Death & Drug Resistance Group, Centre for Cancer Research & Cell Biology, Queen’s University Belfast,3Centre of Cancer Research and Cell Biology, QUB

Abstract

Background

Protein NEDDylation is a process by which the ubiquitin-like protein NEDD8 is tagged onto substrate proteins, with the Cullin-RING E3 ligases (CRLs) being the best characterised substrates. CRLs mediate ubiquitination and degradation of a vast range of cellular proteins. Pevonedistat (MLN4924) specifically inhibits the NEDDylation pathway, thereby preventing ubiquitination and degradation of CRL substrates. Accumulation of such may impede tumour cell proliferation and initiate cell death. Recent studies have shown that Pevonedistat abrogates cell cycle progression and promotes cell death, and it is currently under investigation in early phase clinical trials.

Method

Colorectal cancer (CRC) cell lines with differing p53 status were used: HCT116 p53 wild-type and null; LoVo shScr and shp53. The cell lines were treated with differing concentrations of Pevonedistat to determine its effects on cell viability, apoptosis and cell cycle progression. MTT and clonogenic survival, Annexin V/PI and PI flow cytometry assays were conducted. Protein levels were measured by Western blotting and caspase 3/7 and 8 activity was determined by Caspase Glo assay (Promega).

Results

Pevonedistat induced G2/M cell cycle arrest at low concentrations (10-30 nM) and early timepoints (24h) and cell death at higher concentrations (30-100 nM) and later timepoints (48-72h), independent of p53 status. Pevonedistat treatment induced a significant increase in caspase 3/7 activity at 24 and 48 hours indicative of apoptotic cell death. Pevonedistat also synergized potently with 5-Fluorouracil and Oxaliplatin and this synergy was again independent of p53 status. Work is ongoing to establish the mechanism by which Pevonedistat induces p53-independent cell cycle arrest and apoptosis, with increased expression of the CRL substrate WEE1 currently being investigated.

Conclusion

In this study, we have so far demonstrated that a specific inhibitor of NEDDylation, Pevonedistat, is capable of mediating cytostatic and cytotoxic effects in a p53-independent manner across a panel of CRC cells and synergizes with standard-of-care chemotherapeutics.