Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising 
approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, 
we show that selective pressure results in HCT116 colorectal carcinoma cells 
with decreased MLN4924 sensitivity and identify a single-nucleotide transition 
that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This 
reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 
activation at physiological ATP concentrations. Expression of UBA3 A171T is 
sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating 
that it is a dominant suppressor of MLN4924-mediated cell death. Our data 
suggest that the on-target potency of MLN4924 selects for a point mutation in 
NAE that overcomes the molecule's inhibitory effects, allowing cancer cell 
survival.