[Daporinad Potently Induces Cell Death in Ph+ Acute Lymphoblastic Leukemia SUP-B15 Cells Independent of the Intrinsic Apoptotic Pathway].
To investigate whether daporinad (APO866), a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, can potently kill Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) cells via an intrinsic apoptosis-independent pathway.
Ph+ ALL SUP-B15 cells were treated with varying concentrations of APO866. After treatment, cell viability was measured via Deep Blue assay, while apoptosis and cell death were determined by Annexin V/7-AAD double staining. Cleavage of Caspase-3 and PARP1 was detected by Western blot, and rescue experiments with the pan-caspase inhibitor Z-VAD-FMK were performed to rule out apoptotic involvement. To determine whether APO866-induced cell killing depends on nicotinamide adenine dinucleotide (NAD+) depletion, the NAD+ precursor nicotinic acid (NA) was supplemented and NAD+consuming enzyme CD38 was knocked down. Furthermore, TP53 knockout and BAX/BAK double-knockout SUP-B15 cell models were used to verify that the cytotoxic effect of APO866 is independent of the intrinsic apoptotic pathway.
Both Deep Blue and Annexin V/7-AAD staining assays showed that APO866 effectively kills SUP-B15 cells. This cytotoxic effect could not be abrogated by the pan-caspase inhibitor Z-VAD-FMK, and no cleavage of Caspase-3 and PARP1 was detected during the process of cell killing. Intracellular NAD+ levels were markedly decreased following APO866 treatment, and supplementation with NA or knockout of CD38 partially reversed such cytotoxicity. Neither TP53 knockout nor BAX/BAK double knockout impaired the killing efficiency of APO866 against SUP-B15 cells.
APO866 could potently induce SUP-B15 cell death by depleting intracellular NAD+ levels, a process that lacks canonical features of the intrinsic apoptotic pathway. Moreover, its cytotoxicity persists even with deficiencies in key regulators of this pathway, indicating a mechanism of action that is independent of intrinsic apoptosis.
Ph+ ALL SUP-B15 cells were treated with varying concentrations of APO866. After treatment, cell viability was measured via Deep Blue assay, while apoptosis and cell death were determined by Annexin V/7-AAD double staining. Cleavage of Caspase-3 and PARP1 was detected by Western blot, and rescue experiments with the pan-caspase inhibitor Z-VAD-FMK were performed to rule out apoptotic involvement. To determine whether APO866-induced cell killing depends on nicotinamide adenine dinucleotide (NAD+) depletion, the NAD+ precursor nicotinic acid (NA) was supplemented and NAD+consuming enzyme CD38 was knocked down. Furthermore, TP53 knockout and BAX/BAK double-knockout SUP-B15 cell models were used to verify that the cytotoxic effect of APO866 is independent of the intrinsic apoptotic pathway.
Both Deep Blue and Annexin V/7-AAD staining assays showed that APO866 effectively kills SUP-B15 cells. This cytotoxic effect could not be abrogated by the pan-caspase inhibitor Z-VAD-FMK, and no cleavage of Caspase-3 and PARP1 was detected during the process of cell killing. Intracellular NAD+ levels were markedly decreased following APO866 treatment, and supplementation with NA or knockout of CD38 partially reversed such cytotoxicity. Neither TP53 knockout nor BAX/BAK double knockout impaired the killing efficiency of APO866 against SUP-B15 cells.
APO866 could potently induce SUP-B15 cell death by depleting intracellular NAD+ levels, a process that lacks canonical features of the intrinsic apoptotic pathway. Moreover, its cytotoxicity persists even with deficiencies in key regulators of this pathway, indicating a mechanism of action that is independent of intrinsic apoptosis.