PURPOSE: This study was performed to determine whether the investigational 
proteasome inhibitor ixazomib demonstrated selective antineoplastic activity 
against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 
gene and to gain a better understanding of its mechanisms of action.
EXPERIMENTAL DESIGN: The cytotoxic effects of ixazomib treatment were analyzed 
in human acute myelogenous leukemia (AML) cell lines and primary AML samples 
expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative 
stress in mediating cytotoxic activity were determined using flow cytometry, 
enzyme-based assays, and Western blots.
RESULTS: Apoptosis induced by ixazomib was abrogated by knockdown of 
NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by 
NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation 
and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells 
expressing NPMc(+)had significantly reduced levels of intracellular glutathione 
and NADPH associated with reduced antioxidant responses to drug treatment. 
Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic 
effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in 
the peripheral blood. Efficacy was associated with superoxide generation, 
reduced glutathione levels, and reduced mRNA and protein expression of 
antioxidant effectors in responding cells.
CONCLUSIONS: In this study, a direct association was observed between 
NPMc(+)expression in AML, reduced antioxidant responses, and enhanced 
sensitivity to an oral proteasome inhibitor that induces oxidative stress. These 
data suggest that intracellular determinants of antioxidant responses may be 
good predictors of therapeutic response to ixazomib.