Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNF alpha-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNF alpha mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer. (C) 2016 Elsevier Inc. All rights reserved.