title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells.
abstract: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL.
  
  
  
  Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing.
  
  
  
  Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright"stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the β5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+induced apoptosis and proteasome inhibition independent of CT-like activity inhibition.
  
  
  
  We provide evidence that DSF/Cu2+overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.
authors.affiliation: Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ, USA.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Bista
authors.affiliation: Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ, USA.|Department of Child Health, University of Arizona College of Medicine-Phoenix, Biosciences Partnership Building (BSPB), 5th floor, 475 N 5th Street, Phoenix, AZ, 85004, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Lee
authors.affiliation: Department of Child Health, University of Arizona College of Medicine-Phoenix, Biosciences Partnership Building (BSPB), 5th floor, 475 N 5th Street, Phoenix, AZ, 85004, USA.|Department of Biology and Biochemistry, University of Bath, Bath, UK.
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Pepper
authors.affiliation: Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ, USA.|Department of Child Health, University of Arizona College of Medicine-Phoenix, Biosciences Partnership Building (BSPB), 5th floor, 475 N 5th Street, Phoenix, AZ, 85004, USA.
authors.forename: David O
authors.initials: DO
authors.lastname: Azorsa
authors.affiliation: Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ, USA.|Department of Child Health, University of Arizona College of Medicine-Phoenix, Biosciences Partnership Building (BSPB), 5th floor, 475 N 5th Street, Phoenix, AZ, 85004, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Arceci
authors.affiliation: Institute of Molecular Medicine at Phoenix Children's Hospital, Phoenix, AZ, USA. ealeem@email.arizona.edu.|Department of Child Health, University of Arizona College of Medicine-Phoenix, Biosciences Partnership Building (BSPB), 5th floor, 475 N 5th Street, Phoenix, AZ, 85004, USA. ealeem@email.arizona.edu.|Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt. ealeem@email.arizona.edu.
authors.forename: Eiman
authors.initials: E
authors.lastname: Aleem
mesh_terms: D000293:Adolescent; D000328:Adult; D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000069286:Bortezomib / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D003561:Cytarabine / Q000008:administration & dosage / Q000494:pharmacology; D004221:Disulfiram / Q000494:pharmacology*; D004314:Down Syndrome / Q000150:complications*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005260:Female; D006801:Humans; D007223:Infant; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000209:etiology / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics*; D055815:Young Adult
chemical_list: D003561:Cytarabine; D000069286:Bortezomib; D000444:Aldehyde Dehydrogenase; C088874:PSMB5 protein, human; D046988:Proteasome Endopeptidase Complex; D004221:Disulfiram
keywords: ALDH; Bortezomib; Chemoresistance; Cytarabine; Disulfiram; Down syndrome-associated AML; Relapsed acute myeloid leukemia