Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells.
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.
Zoppoli
Gabriele
G
Department of Internal Medicine, University of Genoa, Genoa, Italy.
Cea
Michele
M
Soncini
Debora
D
Fruscione
Floriana
F
Rudner
Justine
J
Moran
Eva
E
Caffa
Irene
I
Bedognetti
Davide
D
Motta
Giulia
G
Ghio
Riccardo
R
Ferrando
Fabio
F
Ballestrero
Alberto
A
Parodi
Silvio
S
Belka
Claus
C
Patrone
Franco
F
Bruzzone
Santina
S
Nencioni
Alessio
A
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
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