MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating 
enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via 
rereplication in most cell lines. This distinct mechanism of DNA damage may 
affect its ability to combine with standard-of-care agents and may affect the 
clinical development of MLN4924. As such, we studied its interaction with other 
DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, 
and gemcitabine demonstrated synergy in combination with MLN4924 in vitro. The 
combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse 
xenograft model. Importantly, depletion of genes within the ataxia 
telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin 
modification, and transcription-coupled repair reduced the synergy between 
mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA 
strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest 
that mitomycin C causes stalled replication forks, which when combined with 
rereplication induced by MLN4924 results in frequent replication fork 
collisions, leading to cell death. This study provides a straightforward 
approach to understand the mechanism of synergy, which may provide useful 
information for the clinical development of these combinations.