Induction of Apoptosis and Autophagy By Nampt Inhibition in Adult T-Cell Leukemia/Lymphoma and Leukemic Cell Lines

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus type I (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes is one of the poorest among hematological malignancies. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide (NAD⁺)-dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels and novel small-molecule SIRT1 inhibitors are highly effective against ATL cells.^1,2 Nicotinamide phosphoribosyltransferase (Nampt) also known as pre-B-cell colony-enhancing factor 1 or visfatin is a rate-limiting enzyme in NAD⁺ biosynthesis, and it regulates intracellular ATP levels in mammalian cells. Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma) is sensitive to low concentrations of FK866, Nampt inhibitor, as measured in cytotoxicity and clonogenic assays.³Here, we assessed how Nampt is regulated in ATL cells and leukemic cell lines.

Results: We observed that ATL patients had significantly higher SIRT1 and Nampt protein levels than healthy controls. FK866 induced significant growth inhibition and apoptosis (Annexin V⁺ cells and TUNEL) in leukemia/lymphoma cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60). FK866 showed potent activities with GI₅₀values of 0.63, 3.7, 1.0, and 3,4 nM for S1T, MT-2, Jurkat, and HL60 cells, respectively. FK866 also activated caspase activity (caspase-3, 8, and 9) with DNA fragmentation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. Interestingly, FK866 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Autophagy detection was also performed using the CytoID Autophagy detection kit. Autophagy levels are increased in the presence of STF-62247 pre-treated with bafilomycin A1, a specific inhibitor of vacuolar proton ATPase, whose inhibition is known to block the fusion of autophagosomes with lysosomes for 2 h. Thus, FK866 simultaneously caused apoptosis and autophagy.

Conclusion:These results suggest that Nampt inhibitor is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.

1. Kozako T, Aikawa A, Shoji T, et al. High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells. Int J Cancer. 2012;131:2044-2055.

2. Kozako T, Suzuki T, Yoshimitsu M, et al. Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep. 2015;5:11345.

3. Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood. 2009;113:3276-3286.