Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent.

APO866 is a small molecule drug that specifically inhibits nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Although, the antitumor activity of APO866 on various types of cancer models has been reported, information regarding mechanisms by which APO866 exerts its cytotoxic effects is not well defined. Here we show that APO866 induces a strong, time-dependent increase in highly reactive ROS, nitric oxide, cytosolic/mitochondrial superoxide anions and hydrogen peroxide. We provide evidence that APO866-mediated ROS production is modulated by PARP1 and triggers cell death through mitochondria depolarization and ATP loss. Genetic or pharmacologic inhibition of PARP1 prevented hydrogen peroxide accumulation, caspase activation, mitochondria depolarization, ATP loss and abrogates APO866-induced cell death, suggesting that the integrity of PARP1 status is required for cell death. Conversely, PARP1 activating drugs enhanced the anti-leukemia activity of APO866 Collectively, our studies show that APO866 induces ROS/RNS productions, which mediate its anti-leukemia effect. These results support testing new combinatorial strategies to enhance the antitumor activities of APO866.

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis.

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.