title: Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: Division of Hematology, Department of Medicine, Stanford University, Stanford, California. jacqueline_garcia@dfci.harvard.edu.
authors.forename: Jacqueline S
authors.initials: JS
authors.lastname: Garcia
authors.affiliation: Stanford Cancer Institute, Stanford University, Stanford, California.
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.affiliation: Division of Hematology, Department of Medicine, Stanford University, Stanford, California. Stanford Cancer Institute, Stanford University, Stanford, California.
authors.forename: Bruno C
authors.initials: BC
authors.lastname: Medeiros
authors.affiliation: Division of Hematology, Department of Medicine, Stanford University, Stanford, California. Stanford Cancer Institute, Stanford University, Stanford, California.
authors.forename: Beverly S
authors.initials: BS
authors.lastname: Mitchell
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001896:Boron Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D016130:Immunophenotyping; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D000090243:Nucleophosmin; D010084:Oxidation-Reduction / Q000187:drug effects; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001896:Boron Compounds; D056572:Histone Deacetylase Inhibitors; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D017382:Reactive Oxygen Species; D013481:Superoxides; D000090243:Nucleophosmin; C548400:ixazomib; D005978:Glutathione; D005998:Glycine