NAMPT/PBEF Enzymatic Activity Is Indispensable for Myeloma Cell Growth and Osteoclast Activity

Abstract 886

Clinical MRI and PET-CT analyses implicated hypermetabolic activity of multiple myeloma (MM) cells in focal lesions with tumor burden, metastasis and survival. Osteoclasts induced by MM are also naturally hypermetabolic suggesting that targeting cellular metabolism may contribute to controlling MM and associated bone disease. The coenzyme nicotinamide adenine dinucleotide (NAD) not only is essential for proper cellular metabolism but also affects activity of NAD-dependent enzymes, such as PARP1 and sirtuins. Nicotinamide phosphoribosyl transferase (NAMPT/PBEF/Visfatin) is a rate-limiting enzyme in NAD biosynthesis from nicotinamide and also functions extracellularly. APO866 (TopoTarget, Switzerland), an inhibitor of NAMPT, depletes cellular NAD particularly in cancer cells. The aims of the study were to evaluate expression of NAMPT (PBEF1) in clinical and experimental MM samples using global gene expression profiling (GEP) and investigate the effect of APO866 on MM cell growth and osteoclastogenesis. Our molecular classification data revealed elevated expression of PBEF1 in the PR, MAF and LB subtypes of newly diagnosed MM patients (n=796) and in MM cell lines (n=41). Based on our GEP70 risk model, PBEF1 expression was higher in high risk of the CD-1, HY, and MS subtypes relative to their low risk counterparts, and in focal lesions at relapse than at diagnosis. PBEF1 expression was 2.5 fold higher in myelomatous versus nonmyelomatous human bones from SCID-hu mice (p<0.0001). In primary MM cell-osteoclast cocultures (n=8) PBEF1 expression was upregulated by >3 folds in cocultured MM cells or osteoclasts compared to baseline MM cells (p<0.02) or control osteoclasts (p<0.04). APO866 dose dependently inhibited growth of 6 MM cell lines at low nanomolar concentrations (IC50 at 0.1–5 nM, 72 hrs) and similarly inhibited growth of MM cells in coculture with stromal cells or osteoclasts. NAD depletion by APO866 (5 nM) in 3 MM cell lines was consistently associated with reduced enzymatic activities of PARP1 by 73 ± 5% (p<0.001) and of SIRT1 by 49 ± 3% (p<0.005). In vivo, SCID-rab mice engrafted with our DAS MM cell line (MF subtype) were intraperitoneally treated with saline or APO866 (10 mice/group, 20 mg/kg, twice weekly) for 3 weeks. At experiment's end circulating human Ig levels in control and APO866 groups were 43 ± 15 μg/ml and 1 ± 0.6 μg/ml, respectively (p<0.01). APO866 prevented bone loss as assessed by X-rays and measurement of bone mineral density (p<0.05). Histological examination confirmed high infiltration of MM cells in myelomatous bones of control hosts while in APO866-treated hosts the implanted bones reduced numbers of osteoclasts by 53% (p<0.0007). In vitro, at concentration of ≥0.1 nM, APO866 significantly suppressed osteoclast differentiation and bone resorption activity of mature osteoclasts on dentine slices (p<0.007). We conclude that NAMPT/PBEF/Visfatin is a potential target in certain molecularly classified and high risk MM patients, and that MM cells and osteoclasts are highly sensitive to NAD depletion.