title: A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells.
abstract: Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand.
authors.forename: Stephen M F
authors.initials: SM
authors.lastname: Jamieson
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand.
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
authors.forename: Donya Moradi
authors.initials: DM
authors.lastname: Manesh
authors.affiliation: Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
authors.forename: Jad
authors.initials: J
authors.lastname: El-Hoss
authors.affiliation: Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
authors.forename: Duohui
authors.initials: D
authors.lastname: Jing
authors.affiliation: Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Mackenzie
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Guise
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand.
authors.forename: Annika
authors.initials: A
authors.lastname: Foehrenbacher
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Pullen
authors.affiliation: Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
authors.forename: Juliana
authors.initials: J
authors.lastname: Benito
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Smaill
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand.
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.affiliation: Comprehensive Cancer Centre Ulm, Institute for Experimental Tumor Research, University of Ulm, Germany.
authors.forename: Medhanie A
authors.initials: MA
authors.lastname: Mulaw
authors.affiliation: Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
authors.forename: Marina
authors.initials: M
authors.lastname: Konopleva
authors.affiliation: Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand.
authors.forename: Stefan K
authors.initials: SK
authors.lastname: Bohlander
authors.affiliation: Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Lock
authors.affiliation: Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand. Electronic address: wr.wilson@auckland.ac.nz.
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000332:Aerobiosis; D000074425:Aldo-Keto Reductase Family 1 Member C3; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D001853:Bone Marrow / Q000201:enzymology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D045325:HCT116 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007962:Leukocytes / Q000201:enzymology; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism; D009588:Nitrogen Mustard Compounds / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors; D014508:Urea / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D009025:Morpholines; D009588:Nitrogen Mustard Compounds; C524170:PR-104A; D011355:Prodrugs; C587797:SN 34037; C533769:coumberone; D014508:Urea; D015096:3-Hydroxysteroid Dehydrogenases; D006911:Hydroxyprostaglandin Dehydrogenases; C487341:AKR1C3 protein, human; D000074425:Aldo-Keto Reductase Family 1 Member C3
keywords: Aldo-keto reductase 1C3; Fluorogenic assays; Leukaemia; PR-104; SN34037