Activation of p53 by the NEDD8 activating enzyme inhibitor MLN4924 is independent of Cdt1 and involves the cullin-RING ligase CRL4-DTL

NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of E3 ubiquitin ligases termed the cullin-RING ligases (CRLs) through conjugation of cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrates targets them for degradation by the 26S proteasome. NAE thereby regulates the stability of many proteins including those required for cancer cell growth and survival. MLN4924 is an investigational small molecule inhibitor of NAE with 2-3 orders of magnitude selectivity over the related E1’s ubiquitin-activating enzyme (UAE) and sumo-activating enzymes (SAE; Ref. 1). Recently, a genome-wide siRNA screen in the A375 melanoma cell line identified key DNA damage response genes affecting sensitivity of cells to MLN4924, many of which were associated with the p53 pathway which activates a p21-dependent intra-S-phase checkpoint (2). A comparison of the effects of TP53 RNAi in A375 cells to genetic deletion of TP53 in HCT116 cells (HD 104-001 cells, Horizon Discovery Ltd) suggests that p21 induces a cell cycle arrest within 24 h, whereas analysis of the p53-regulated transcriptional signature indicates that proapoptotic proteins are up-regulated at later times. Surprisingly, stabilization of p53 by MLN4924 appears independent of the replication licensing factor Cdt1, ATR activation, or p53 neddylation, but rather involves the E3 ligase complex Cul4A/B-Ddb1-DTL. Furthermore, Nutlin-3induced ubiquitination of p53 could be blocked by MLN4924, suggesting that Mdm2 and DTL may cooperate in the ubiquitination of p53.