Targeting NAD⁺ Salvage Pathway Induces Autophagy in Multiple Myeloma Cells

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme crucially involved in several cellular functions, including energy metabolism, reactive oxygen species scavenging, DNA repair, and gene expression. Intracellular NAD⁺ stores are continuously replenished through pathways whose activity depends on the tissue and availability of substrates. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD⁺ salvage pathway from nicotinamide. During neoplastic transformation, Nampt is upregulated to compensate for increased metabolic demands. It promotes myeloid and lymphoid differentiation and increases specific cytokine production (TNF- α, IL-6 and VEGF). Importantly, cancer and leukaemia cells appear to be more sensitive to Nampt inhibitor drugs than normal cells. The reasons for this selectivity are not fully understood, but may include aberrant metabolic demands and increased reliance on NAD⁺-dependent enzymes. Promising results obtained with Nampt inhibitors (such as FK866) in preclinical cancer models suggest that Nampt activity represents an innovative therapeutic target for novel anticancer agents. Methods: A panel of eighteen different MM cell lines, both sensitive and resistant to conventional and novel anti-myeloma drugs, as well as patient MM cells were used in the study. The mechanism of action of FK866 was investigated by Annexin-V/propidium iodide staining, thymidine incorporation, Western-blotting, and with lentivirus-mediated shRNAs. For the autophagy assay, EGFP-LC3⁺ cells were treated with FK866 and the number of GFP-LC3 punctae was analyzed and quantified by fluorescence microscopy and flow cytometry, respectivley. Intracellular NAD⁺ content was measured using a biochemical assay. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assay. Results: To study the role of Nampt in MM cells, we performed a protein analysis of this enzyme in eighteen MM cell lines. Nampt is constitutively activated in all cell lines tested. Moreover, patient MM cells highly express this enzyme whereas normal cells lack this protein. Indeed, the Nampt inhibitor FK866 decreased MM cell line viability in a dose and time dependent manner, with an IC50 ranging from 3–30nM. Similar results were observed in patient MM cells. Importantly, FK866 did not inhibit viability of normal peripheral blood mononuclear cells. Tritiated thymidine uptake assay confirmed the antiproliferative effects of FK866 in MM cell lines and patient cells. To examine the mechansim of action, we showed that intracellular NAD⁺ levels decreased with FK866 treatment at 24 and 48 hours. Furthermore, knock-down of Nampt by small interfering RNAs caused significant inhibition of MM cell growth. FK866 triggered anti-MM activity in our models of MM in the bone marrow (BM) microenvironment, confirming its ability to overcome the proliferative advantage conferred by the BM milieu. FK866 treatment also inhibited angiogenesis via suppression of pivotal MM pathways PI3K/AKT and ERK. In further studies to delineate its mechanisms of action, no activation of apoptosis was observed in treated-cells. Instead FK866 treatment resulted in a marked increase in autophagy, evidenced by autophagic vacuoles in the cytoplasm and proteolitic processing of endogenous LC3-I to LC3-II. FK866 inhibited mTOR signaling and triggered increased formation of EGFP-LC3 punctae, confirming involvement of autophagic cell death. Finally, combined FK866 with bortezomib (CI < 0.6), melphalan (CI < 0.9), and dexamethasone (CI < 0.8), induced synergistic cytotoxicity against MM cells. Conclusion: Our data therefore show a pivotal role of Nampt in MM cell growth, survival and drug resistance. The ability of FK866 to inhibit Nampt activity strongly supports its clinical evaluation to improve patient outcome in MM.

Hideshima:Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.