Genome-wide screen for modulators of cell death induced by the NEDD8-activating enzyme inhibitor MLN4924

The NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of ubiquitin E3 ligases termed the cullin-RING ligases (CRLs) through conjugation of the cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrate proteins targets them for degradation by the proteasome. In this way, NAE regulates the stability of proteins required for cancer cell growth and survival. MLN4924 is an investigational small molecule that is a potent and selective inhibitor of NAE in Phase I clinical trials. In order to investigate the primary genetic determinants that confer sensitivity of cells to NAE inhibition, we have performed a genome-wide synthetic lethal RNAi screen using MLN4924 in the A375 melanoma cell line. We have also investigated the biological consequences of NAE inhibition by studying the regulation of protein and transcript levels in MLN4924-treated A375 cells using large-scale quantitative proteomics and gene expression profiling, respectively. The RNAi screen has identified 123 genes whose down-regulation modulates MLN4924-induced cell death, and approximately one-third of these interfere with components of the NEDD8 pathway itself, the cell cycle and apoptotic machinery, and DNA damage-response pathways. Of these genes, 99 were subsequently assessed using high throughput FACS analysis for their contribution to the major phenotype induced by MLN4924. The results emphasize replication, p53, BRCA1/BRCA2, and transcription-coupled repair as being particularly important for MLN4924-induced cell death. In addition, the interactions of 80 genes with roles in cell cycle and DNA damage repair were explored with proteasome inhibitors and 12 DNA damaging agents, demonstrating that MLN4924 induces DNA damage by a distinct mechanism.