title: Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells.
abstract: OBJECTIVE
  The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells.
  
  
  METHODS
  Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively.
  
  
  RESULTS
  APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation.
  
  
  CONCLUSIONS
  Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.
authors.affiliation: Department of Internal Medicine, University of Genoa, Genoa, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Zoppoli
authors.forename: Michele
authors.initials: M
authors.lastname: Cea
authors.forename: Debora
authors.initials: D
authors.lastname: Soncini
authors.forename: Floriana
authors.initials: F
authors.lastname: Fruscione
authors.forename: Justine
authors.initials: J
authors.lastname: Rudner
authors.forename: Eva
authors.initials: E
authors.lastname: Moran
authors.forename: Irene
authors.initials: I
authors.lastname: Caffa
authors.forename: Davide
authors.initials: D
authors.lastname: Bedognetti
authors.forename: Giulia
authors.initials: G
authors.lastname: Motta
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ghio
authors.forename: Fabio
authors.initials: F
authors.lastname: Ferrando
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
authors.forename: Silvio
authors.initials: S
authors.lastname: Parodi
authors.forename: Claus
authors.initials: C
authors.lastname: Belka
authors.forename: Franco
authors.initials: F
authors.lastname: Patrone
authors.forename: Santina
authors.initials: S
authors.lastname: Bruzzone
authors.forename: Alessio
authors.initials: A
authors.lastname: Nencioni
mesh_terms: D000178:Acrylamides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000378:metabolism; D004357:Drug Synergism; D005260:Female; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D007938:Leukemia / Q000378:metabolism / Q000473:pathology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008875:Middle Aged; D009243:NAD / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D014404:Tubulin / Q000378:metabolism
chemical_list: D000178:Acrylamides; D016207:Cytokines; C480543:N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide; D010880:Piperidines; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; D014404:Tubulin; D009243:NAD; D000255:Adenosine Triphosphate; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human; D053148:Caspase 3