title: The human polynucleotide kinase/phosphatase (hPNKP) inhibitor A12B4C3 radiosensitizes human myeloid leukemia cells to Auger electron-emitting anti-CD123 ¹¹¹In-NLS-7G3 radioimmunoconjugates.
abstract: BACKGROUND
  Leukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131⁻ phenotype uniquely displayed by LSCs.
  
  
  METHODS
  The surviving fraction (SF) of CD123⁺/CD131⁻ AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33-266 nmols/L; 0.74MBq/μg) or to γ-radiation (0.25-5Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) or with γ-radiation (0.25-2Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.
  
  
  RESULTS
  Binding of (111)In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131⁻ epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25-0.5Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6Gy to AML-5 cells.
  
  
  CONCLUSIONS
  We conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Arman
authors.initials: A
authors.lastname: Zereshkian
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada; Department of Nuclear Medicine and Radiobiology, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Jeffrey V
authors.initials: JV
authors.lastname: Leyton
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Dane
authors.initials: D
authors.lastname: Bergstrom
authors.affiliation: Department of Oncology, University of Alberta, Edmonton, AB.
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada; Department of Medical Imaging, University of Toronto, Toronto, ON, Canada; Toronto General Research Institute, University Health Network, Toronto, ON, Canada. Electronic address: raymond.reilly@utoronto.ca.
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002999:Clone Cells / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects; D056831:Coordination Complexes / Q000737:chemistry / Q000276:immunology*; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors*; D004583:Electrons; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000494:pharmacology*; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology*; D042541:Intracellular Space / Q000187:drug effects / Q000528:radiation effects; D007951:Leukemia, Myeloid / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D011758:Pyrroles / Q000737:chemistry / Q000494:pharmacology*; D011838:Radiation-Sensitizing Agents / Q000737:chemistry / Q000494:pharmacology; D011874:Radiometry
chemical_list: C562074:111In-nuclear translocation sequence-7G3; C547167:2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo(3,4-b)pyridine-5,7(2H,4aH)-dione; D000911:Antibodies, Monoclonal; D056831:Coordination Complexes; D004791:Enzyme Inhibitors; D018796:Immunoconjugates; D053650:Interleukin-3 Receptor alpha Subunit; D011758:Pyrroles; D011838:Radiation-Sensitizing Agents; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D045643:DNA Repair Enzymes
keywords: Acute myeloid leukemia; Auger electrons; CD123; Indium-111; Leukemia stem cells; Radioimmunotherapy