MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating 
enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a 
mechanism-based inhibitor, with enzyme inhibition occurring through the 
formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models 
of cancer, we identified treatment-emergent heterozygous mutations in the 
adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the 
primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants 
revealed slower rates of adduct formation and reduced adduct affinity for the 
mutant enzymes. A compound with tighter binding properties was able to potently 
inhibit mutant enzymes in cells. These data provide rationales for patient 
selection and the development of next-generation NAE inhibitors designed to 
overcome treatment-emergent NAEβ mutations.