Abstract 314: Low-dose NAMPT inhibition by FK866 initiates autophagy to counteract cellular energy crisis, which is overridden by apoptosis at higher drug concentrations

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of mature, but immune-incompetent monoclonal B-lymphocytes. Dependent on external stimuli, these CLL-cells can either be quiescent or active, with the latter being suggested to be critical for disease aggressiveness, drug-resistance and relapse. The active state of the malignant cells is associated with abnormal cellular metabolism. Nicotinamide adenine dinucleotide (NAD) is a critical factor in cellular metabolism by acting as coenzyme in ATP-synthesizing mitochondrial electron transport chain reactions. Nicotinamide phoysphorybosyltransferase NAMPT is the rate-limiting enzyme for nicotinamide-salvaging to NAD. NAMPT has been demonstrated to be over-expressed in several cancer entities. Also in CLL-cells increased NAMPT expression was found in tissue-associated CLL-cells by gene expression profiling. FK866 is a NAMPT-inhibitor and has been demonstrated to selectively reduce cancer cell viability mainly in solid tumors. FK866 has also been demonstrated to be effective in several hematological cell lines where it leads to ATP-reduction and delayed induction of cell death. While some studies proposed apoptosis-induction, others showed caspase-independent autophagic cell death. Most available data about the antitumor effect of FK866 is derived from studies in cell lines. Further, inconsistencies about sensitivity of different cell lines to FK866 in the literature strongly warrant studies in primary cells. In this study we address the impact of FK866-mediated NAMPT-inhibition on cellular energy content, cell viability and involvement of apoptotic and autophagic signaling in primary CLL-cells. FK866 potently reduced cellular NAD-content at low concentration of 10 nM as early as day one. This was followed by reduction in cellular ATP-content at day two and subsequent viability reduction at day three as assessed by flow cytometric measure of annexin V/PI staining. FK866 at 1 nM reduced cellular NAD-content slightly at day one with no significant further downregulation up to day four. At this drug concentration we neither saw significant ATP-downregulation, nor viability reduction up to day four. In contrast, at 1 nM FK866 we saw protection from spontaneous apoptosis by flow cytometry, stabilization of caspases 9 and 3, upregulation of antiapoptotic proteins XIAP and Mcl1 going along with increased phosphorylation of mTOR and AMPK. We conclude that downregulation of NAD by FK866 results in a cellu