Abstract

Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924. We also identified roles for the extrinsic (TRAIL-R2/caspase-8) and intrinsic (BAX/BAK) apoptotic pathways in mediating the apoptotic effects of MLN4924 in CRC cells, as well as a role for BID, which modulates a cross-talk between these pathways. Depletion of the anti-apoptotic protein FLIP, which we identify as a novel mediator of resistance to MLN4924, enhanced apoptosis in a p53-, TRAIL-R2/DR5-, and caspase-8-dependent manner. Notably, TRAIL-R2 was involved in potentiating the apoptotic response to MLN4924 in the absence of FLIP, in a ligand-independent manner. Moreoever, when paired with SoC chemotherapies, MLN4924 demonstrated synergy with the irinotecan metabolite SN38. The cell death induced by MLN4924/SN38 combination was dependent on activation of mitochondria through BAX/BAK, but in a p53-independent manner, an important observation given the high frequency of TP53 mutation(s) in advanced CRC. These results uncover mechanisms of cell death induced by MLN4924 and suggest that this second-generation proteostasis-disrupting agent may have its most widespread activity in CRC, in combination with irinotecan-containing treatment regimens.