MLN4924 is a first-in-class experimental cancer drug that inhibits the 
NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and 
stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits 
cancer cell proliferation has not been defined, although it is accompanied by 
DNA rereplication and attendant DNA damage. Here we show that stabilization of 
the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for 
MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 
hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, 
was found to be sufficient to induce DNA rereplication and to activate apoptosis 
and senescence pathways. Cells in S phase were most susceptible, suggesting that 
MLN4924 will be most toxic on highly proliferating cancers. Although 
MLN4924-induced cell senescence seems to be dependent on induction of p53 and 
its downstream effector p21(Waf1), we found that p53(-/-) and p21(-/-) cells 
were even more susceptible than wild-type cells to MLN4924. Our results 
suggested that apoptosis, not senescence, might be more important for the 
antiproliferative effect of MLN4924. Furthermore, our findings show that 
transient exposure to this new investigational drug should be useful for 
controlling p53-negative cancer cells, which often pose significant clinical 
challenge.