Specific Inhibitor Of Nicotinamide Phosphoribosyltransferase Research Articles

Nicotinamide phosphoribosyltransferase regulates the cell differentiation and mineralization in cultured odontoblasts.

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC-23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.

Subcellular Characterization of Nicotinamide Adenine Dinucleotide Biosynthesis in Metastatic Melanoma by Using Organelle-Specific Biosensors.

Aim: Nicotinamide adenine dinucleotide (NAD+) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described genetically encoded NAD+ biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM). Results: FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD+-producing enzyme in MM cells, was used to monitor NAD+ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD+ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD+ biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Innovation and Conclusions: Throughout this work, we validated the use of genetically encoded NAD+ biosensors to characterize subcellular distribution of NAD+ production routes in MM. The chance of real-time monitoring of NAD+ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD+ pool manipulation in cancer and metabolic diseases.

Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level.

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD+) and a reduced form (NADH). NAD + plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD + and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD +/NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD + salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two‐photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)‐induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT‐mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.

Abstract 3515: Down the rabbit hole with structural effects of FK866 in primary CLL B cells

Abstract FK866 (Daporinad or APO866) is a specific inhibitor of NAMPT (nicotinamide phosphoribosyltransferase), a rate limiting enzyme involved in NAD generation. It is a prospective cancer treatment utilized in several clinical trials and has generated much interest. It has been previously demonstrated that FK866 (10nM) induces cell death in CLL patient derived B-cells (CLL-B cells) by early depletion of NAD (1 day after treatment) followed by a subsequent decrease in cellular ATP levels (2 days after treatment). Upon further study we observed that CLL-B cells have structural changes after treatment with FK866 for either 6 or 18-20 hours via electron microscopy. There is a presence of bound structures as well as nuclear invaginations. These structures are only observed in cells that were treated with FK866 and not with any other drugs used (fludarabine, bendamustine, chlorambucil) nor with the DMSO or untreated controls. We stained f-actin (a cytoskeletal protein) with FITC labelled phalloidin in CLL-B cells with and without FK866 treatment and then viewed with confocal microscopy to exclude the chance that these structures were artifacts. Changes in the f-actin were only observed in the FK866 treated cells. Phalloidin staining was performed at 6 and 18 hours. We observed holes in the f-actin framework that passed through the entire cell and in some instances, went through the nuclei too. A comparison of the phalloidin staining was performed in non-CLL based cell lines that were known to be sensitive to treatment with FK866 though structural differences had not been observed at the same time points (6 and 18-20 hours). Two Ewing's sarcoma cell lines and two small cell lung cancer cell lines were used. All four cell lines showed no changes in the f-actin. Labeled dextran beads were observed via confocal microscopy to be taken up more readily by the FK866 treated CLL -B cells as compared to the vehicle control. This would imply that the treated cells are actively removing nutrients from the environment. A lysosome stain, LysoTracker, was employed to determine if there was any involvement of lysosomes in this phenomenon. No change was observed between the untreated, vehicle control and the FK866 treated CLL patient derived B-cells. We were curious about the mitochondrial dynamics in the FK866 treated cells so they were stained with MitoTracker. We were able to detect mitochondria in the cytoplasm of the cells in all treatments but the FK866 treated cells displayed a more distinct punctate pattern. Interestingly, we also observed the mitochondria were appearing at or near the holes in the cytoskeleton. As the FK866 treated cells are deprived of their NAD, the CLL cells may be undergoing macropinocytosis, an mTOR mediated phenomenon used by cells to extract nutrients from the environment. We have shown that FK866 causes an increase in 4EBP1 phosphorylation compared to controls. In conclusion, FK866 may mediate macropinocytosis in CLL cells in an mTOR dependent manner. Citation Format: Cheryl Peltier, Eileen McMillan-Ward, Elizabeth Henson, Nabanita Chatterje, Donna Hewitt, Danielle Desautels, Matthieu Bourrier, Cornelia Mutz, Iris Gehrke, Eric Bouchard, Heinrich Kovar, Shantanu Banerji, Spencer Gibson, Sara Israels, Versha Banerji. Down the rabbit hole with structural effects of FK866 in primary CLL B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3515.

Nicotinamide phosphoribosyltransferase regulates cocaine reward through Sirtuin 1

Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis in mammals, converts nicotinamide into nicotinamide mononucleotide (NMN). NMN is subsequently converted to NAD, a component that is critical for cell energy metabolism and survival. Sirtuin 1 (SIRT1), an NAD-dependent histone deacetylase, plays an important role in mediating memory and synaptic plasticity. Here, we found that NAMPT was significantly upregulated in the ventral tegmental area (VTA) of cocaine-conditioned mice. Intraperitoneal or intra-VTA injection of FK866, a specific inhibitor of NAMPT, significantly attenuated cocaine reward. However, such effects were clearly repressed by intra-VTA expression of NAMPT or supplementation with NMN. Using 1H-nuclear magnetic resonance metabolomic analysis, we found that the content of NAD and NMN were increased in the VTA of cocaine-conditioned mice; moreover, the expression of SIRT1 was also upregulated. Interestingly, the inhibitory effect of FK866 on cocaine reward was significantly weakened in Sirt1 midbrain conditional knockout mice. Our results suggest that NAMPT-mediated NAD biosynthesis may modify cocaine behavioral effects through SIRT1. Moreover, our findings reveal that the interplay between NAD biosynthesis and SIRT1 regulation may comprise a novel regulatory pathway that responds to chronic cocaine stimuli.

Preclinical efficacy of the novel competitive NAMPT inhibitor STF-118804 in pancreatic cancer.

NAD salvage is one of the pathways used to generate NAD in mammals. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in this pathway, uses nicotinamide (NAM) to generate nicotinamide mononucleotide (NMN). NMN is one of the main precursors of NAD synthesis in cells. Our previous study showed the importance of NAMPT in maintaining NAD levels in pancreatic ductal adenocarcinoma cells (PDAC), and that the NAMPT inhibitor FK866 decreased pancreatic cancer growth. We now tested the effect of STF-118804, a new highly specific NAMPT inhibitor, in models of pancreatic ductal adenocarcinoma. STF-118804 reduced viability and growth of different PDAC lines, as well as the formation of colonies in soft agar. In addition, STF-118804 decreased glucose uptake, lactate excretion, and ATP levels, resulting in metabolic collapse. STF-118804 treatment activated AMPK and inhibited of mTOR pathways in these cells. This effect was significantly potentiated by pharmacological AMPK activation and mTOR inhibition. Exogenous NMN blocked both the activation of the AMPK pathway and the decrease in cell viability. Panc-1 cells expressing GFP-luciferase were orthotopically implanted on mice pancreas to test the in vivo effectiveness of STF-118804. Both STF-118804 and FK866 reduced tumor size after 21 days of treatment. Combinations of STF-118804 with chemotherapeutic agents such as paclitaxel, gemcitabine, and etoposide showed an additive effect in decreasing cell viability and growth. In conclusion, our preclinical study shows that the NAMPT inhibitor STF-118804 reduced the growth of PDAC in vitro and in vivo and had an additive effect in combination with main current chemotherapeutic drugs.

A pancreatic ductal adenocarcinoma subpopulation is sensitive to FK866, an inhibitor of NAMPT

Treating pancreatic cancer is extremely challenging due to multiple factors, including chemoresistance and poor disease prognosis. Chemoresistance can be explained by: the presence of a dense stromal barrier leading to a lower vascularized condition, therefore limiting drug delivery; the huge intra-tumoral heterogeneity; and the status of epithelial-to-mesenchymal transition. These factors are highly variable between patients making it difficult to predict responses to chemotherapy. Nicotinamide phosphoribosyl transferase (NAMPT) is the main enzyme responsible for recycling cytosolic NAD+ in hypoxic conditions. FK866 is a noncompetitive specific inhibitor of NAMPT, which has proven anti-tumoral effects, although a clinical advantage has still not been demonstrated. Here, we tested the effect of FK866 on pancreatic cancer-derived primary cell cultures (PCCs), both alone and in combination with three different drugs typically used against this cancer: gemcitabine, 5-Fluorouracil (5FU) and oxaliplatin. The aims of this study were to evaluate the benefit of drug combinations, define groups of sensitivity, and identify a potential biomarker for predicting treatment sensitivity. We performed cell viability tests in the presence of either FK866 alone or in combination with the drugs above-mentioned. We confirmed both inter- and intra-tumoral heterogeneity. Interestingly, only the in vitro effect of gemcitabine was influenced by the addition of FK866. We also found that NAMPT mRNA expression levels can predict the sensitivity of cells to FK866. Overall, our results suggest that patients with tumors sensitive to FK866 can be identified using NAMPT mRNA levels as a biomarker and could therefore benefit from a co-treatment of gemcitabine plus FK866.

NAMPT protects against 6-hydroxydopamine-induced neurotoxicity in PC12 cells through modulating SIRT1 activity.

Parkinson's disease (PD) is the second most common progressive neurodegenerative movement disorder. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate‑limiting step in the nicotinamide adenine dinucleotide (NAD+) biosynthetic pathway in mammals, is a substrate for NAD+‑dependent enzymes, such as sirtuin1 (SIRT1), and contributes to cell fate decisions. However, the role of NAMPT in PD has remained to be fully elucidated. In the present study, PC12 cells were treated with the neurotoxin 6-hydroxydopamine (6‑OHDA) to establish an invitro model of PD, following which an obvious inhibitory effect on the levels of NAMPT and NAD+ as well as the NAD+/NADH ratio was detected. In addition, pre‑incubation with FK866, a highly specific NAMPT inhibitor, enhanced the inhibitory effects of 6‑OHDA on the viability of PC12, while pre‑incubation with nicotinamide mononucleotide (NMN), am enzymatic product of NAMPT, had the opposite effect. Furthermore, it was revealed that NMN markedly attenuated 6‑OHDA‑induced decreases in superoxide dismutase activity and glutathione levels, as well as 6‑OHDA‑induced increases in malondialdehyde and lactate dehydrogenase in PC12 cells. Furthermore, 6‑OHDA significantly reduced SIRT1 activity in PC12 cells, which was inhibited by NMN. The pharmacological activator resveratrol also significantly inhibited 6‑OHDA‑mediated decreases in PC12 cell viability while reversing 6‑OHDA‑induced decreases in SIRT1 levels. The results of the present study suggested that NMT protected against 6‑OHDA‑induced decreases in PC12 cell viability, and that SIRT1 activation had a role in this process. Treatment with NMN to activate SIRT1 may represent a novel therapeutic strategy for treating PD.

SIRT2 Plays Essential Role in Early Hematopoiesis through Deacetylation of LMO2 Protein

Nicotinamide phosphoribosyltransferase (NAMPT) is known as a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) and supplies NAD+ to sirtuins (SIRTs) that require NAD+ to activate their protein deacetylase functions. SIRTs are members of the NAD+-dependent class III histone deacetylase family. We already reported that NAMPT-NAD+-SIRT1 pathway is essential for granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis (Skokowa J, et al. Nat Med. 2009).In the present study, we analyzed the mechanism of NAMPT-NAD+ -SIRTs-triggered hematopoiesis and myelopoiesis using human induced pluripotent stem (iPS) cells model. We applied a serum- and feeder-free monolayer hematopoietic differentiation system of iPS cells by sequential incubation of iPS cells with combinations of mesoderm- and hematopoiesis-inducing cytokines. We found that addition of NAMPT to granulopoiesis-inducing iPS cells culture medium resulted in a marked increase of CD15+ and CD16+ myeloid and granulocytic cell numbers. These results are compatible with our published data in healthy donor derived CD34+ cells. To evaluate at which stage of hematopoietic differentiation NAMPT plays a role, we cultured iPS cells in the presence or absence of the specific inhibitor of NAMPT, FK866. Interestingly, we found marked reduction of VEGFR2+ CD34+ early hematopoietic progenitor cells at day 6 of differentiation after inhibition of NAMPT. These data argue for the requirement of NAMPT and its downstream effectors at early stages of hematopoietic differentiation. To evaluate which of the SIRTs is essential in this process, we tested early hematopoietic differentiation of iPS cells in the presence of specific inhibitors of several SIRTs. We found that inhibition of SIRT2 using its specific inhibitor AC93253 significantly suppressed differentiation into VEGFR2+ CD34+ early hematopoietic progenitor fraction. Furthermore, the emergence of CD43+ hematopoietic progenitor cells at day 13 was completely suppressed after inhibition of SIRT2. These results indicate that NAMPT-NAD+-SIRT2 signaling is needed for early hematopoiesis. We further evaluated downstream targets of NAMPT-NAD+-SIRT2. qRT-PCR analysis of well-known early hematopoietic genes (GATA2, RUNX1, LMO2, TAL1) revealed that mRNA expression of these factors was not down-regulated after NAMPT or SIRT2 inhibition. We therefore tested whether hematopoietic functions of these proteins might be affected by post-transcriptional modifications via NAMPT-NAD+-SIRT2-triggered deacetylation. We tested protein-protein interaction between SIRT2 and these candidate proteins in differentiated iPS cells on day 6 of culture using Duolink technique, which allows identification of protein complexes in single cells. We found that RUNX1, LMO2 and TAL1 proteins interact with SIRT2 protein. To identify if these proteins could be deacetylated by SIRT2, we compared the signals from total- and de-/acetylated- candidate proteins in differentiated iPS cells on day6 treated or not with SIRT2 inhibitor using Duolink-FACS, Duolink technique in combination with flow cytometry, as a readout method. This analysis revealed that only LMO2 is deacetylated by SIRT2. LMO2 is known as an essential transcriptional regulator in early hematopoiesis. It is a component of TAL1 complex, which consist of LMO2, TAL1, LDB1, E47 and GATA1/2 proteins and has transcriptional activity as a complex on hematopoiesis-specific target genes as KIT, GATA1, GYPA, KLF1 and KDR. Interestingly, mRNA expression levels of all these genes were markedly downregulated after inhibition of NAMPT or SIRT2, which was in line with severely reduced numbers of early hematopoietic progenitors.Overall, these results indicate that NAMPT, known to be essential for myeloid differentiation, induces early stages of hematopoietic differentiation by SIRT2-triggered deacetylation of LMO2. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the NAMPT-Triggered Deacetylation of HCLS1 Protein: A New Therapeutic Option in Chronic Myeloid Leukemia

G-CSFR-triggered phosphorylation and activation of the hematopoietic-specific lyn-substrate 1 (HCLS1) protein is important for the granulocytic differentiation of hematopoietic stem cells (HSCs) (Skokowa J. et al., Nat Med 2012). G-CSF also activates protein deacetylation via Nicotinamide phosphoribosyltransferase (NAMPT)/NAD+/sirtuin 1 (SIRT1) pathway (Skokowa J. et al., Nat Med 2009). SIRT1 is NAD+-dependent protein deacetylase playing an important role in the regulation of gene transcription. Interestingly, having strong pro-differentiation effects on myeloid progenitors, both HCLS1 and NAMPT are markedly hyper-activated in myeloid leukemias, inducing proliferation and survival of undifferentiated blasts.Knowing this, we investigated whether HCLS1 could be activated by NAMPT/NAD+/SIRT1 pathway via deacetylation in HSCs. Indeed, we found that HCLS1 is deacetylated in HSCs by NAMPT/SIRT1 and that G-CSF treatment of these cells induced deacetylation and phosphorylation of HCLS1 via NAMPT/SIRT1. Moreover, we found that deacetylation of HCLS1 is important for myeloid differentiation and proliferation of HSCs.We further demonstrated that HCLS1 protein was activated by deacetylation in blasts of chronic myeloid leukemia (CML) patients, as compared to HSCs of healthy individuals. This was in line with elevated mRNA and protein levels of NAMPT and HCLS1. We further investigated, whether inhibition of NAMPT-triggered deacetylation of HCLS1 has any effect on the survivial and differentiation of CML blasts in vitro. We found that treatment of CML blasts with the specific NAMPT inhibitor FK866 resulted in increased acetylation of HCLS1. In addition, treatment of CML blasts with the FK866 in a dose of 10nM resulted in marked inhibition of proliferation and elevated cell death, which was consistent between different patients and was comparable to the effects of Imatinib treatment. In CFU assay in the presence of FK866 (10nM) no CFU colonies were grown. Even at a dose of 3nM FK866 drastically suppressed total colony counts. This inhibitory effect was further amplified by combination of FK866 with Imatinib. Addition of 10nM of FK866 to CFU culture medium was not toxic for CD34+ cells of healthy donors.Taken together, we demonstrated that NAMPT and HCLS1 have dose-dependent effects on hematopoietic cells and that NAMPT activates HCLS1 functions by SIRT1-triggered deacetylation. Inhibition of NAMPT in CML blasts markedly reduced their survival, proliferation and differentiation. In conclusion, we suggest a possible therapeutic application of NAMPT inhibition in the treatment of CML patients via deactivation of HCLS1 function by acetylation. This therapy could be applied in combination with Imatinib, or in Imatinib-resistant CML patients. DisclosuresNo relevant conflicts of interest to declare.

Nicotinamidephosphoribosyltransferase (NAMPT) Induces Ubiquitination of LEF-1 Transcription Factor By Deacetylation

We and others demonstrated, that transcription factor lymphoid enhancer-binding factor 1 (LEF-1) is crucial for neutrophil granulocytopoiesis and lymphopoiesis. LEF-1 expression is severely diminished in myeloid progenitors of severe congenital neutropenia (CN) patients, leading to "maturation arrest" of granulopoiesis.In the present study we evaluated the mechanisms of LEF-1 regulation. Previously we identified elevated levels of the enzyme Nicotinamide phosphoribosyltransferase (NAMPT) and its target Sirtuin 1 (SIRT1) in myeloid cells of CN patients. SIRT1 is NAD+-dependent protein deacetylase and in this study we analyzed whether LEF-1 could be regulated by NAMPT/SIRT1-triggered deacetylation. We identified lysine residue in LEF-1 protein, which could be de-/acetylated, generated specific rabbit polyclonal antibody recognizing this acetyl-Lys and confirmed NAMPT/SIRT1-triggered deacetylation of LEF-1 protein on this lysine in T-cell leukemia cell line Jurkat and in AML cell line LW/SO.We first studied the effects of LEF-1 deacetylation by NAMPT/SIRT1 on its expression and functions. We found markedly diminished levels of total LEF-1 protein as well as its nuclear translocation in Jurkat and LW/SO cells treated with NAMPT. At the same time, inhibition of NAMPT using specific NAMPT inhibitor FK866 resulted in elevated mRNA and protein levels of LEF-1 and its target genes survivin, c-myc and cyclin D1.We also performed reporter gene assay using WT or acetyl-Lys LEF-1 mutant and TOP promoter construct, containing six LEF-1/TCFs binding sites. Acetyl-Lys LEF-1 mutant was not able to activate TOP promoter, as compared to its strong activation by WT LEF-1. Moreover, co-transfection with NAMPT- or SIRT1-expression constructs also showed inhibitory effects on LEF-1-triggered activation of TOP promoter. These experiments clearly demonstrated that NAMPT/SIRT1-triggered deacetylation of the LEF-1 protein inhibited its expression and functions.We further evaluated the mechanism of NAMPT-triggered reduction of LEF-1 expression. In silico analysis of LEF-1 protein using CPLM software revealed that lysine residue in LEF-1 protein, which is de-/acetylated, could be also ubiquitinated. We therefore studied the effects of deacetylation on the ubiquitination of LEF-1 protein in Jurkat cells. We evaluated the effects of 26S proteasome inhibitor, bortezomib, on LEF-1 protein levels after treatment with NAMPT. Indeed, upon treatment with bortezomib the level of LEF-1 protein was restored. This confirmed, that deacetylation of LEF-1 by NAMPT led to degradation of LEF-1 by ubiquitination. We assumed, that in CN patients, hyperactivation of NAMPT/SIRT1 pathway might lead to downregulation of LEF-1 by deacetylation and ubiquitination. Indeed, we observed decreased G-CSF-triggered granulocytic differentiation and proliferation of CD34+ cells from healthy individuals transduced with LEF-1 acetyl-lysine mutant, as compared to cells transduced with WT LEF-1. Moreover, treatment of CD34+ cells of CN patients with bortezomib resulted in restoration of defective LEF-1 expression ultimately leading to normalization of granulocytic differentiation.In conclusion, NAMPT/SIRT1-triggered deacetylation of LEF-1 resulted in its degradation by ubiquitination. DisclosuresNo relevant conflicts of interest to declare.

NAD+ depletion by APO866 in combination with radiation in a prostate cancer model, results from an in vitro and in vivo study

BackgroundAPO866 is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), inhibition of which reduces cellular NAD+ levels. In this study we addressed the potential of NAD+ depletion as an anti-cancer strategy and assessed the combination with radiation. MethodsThe anticipated radiosensitizing property of APO866 was investigated in prostate cancer cell lines PC3 and LNCaP in vitro and in PC3 xenografts in vivo. ResultsWe show that APO866 treatment leads to NAD+ depletion. Combination experiments with radiation lead to a substantial decrease in clonogenic cell survival in PC3 and LNCaP cells.In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD+ levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Compared with untreated tumors, APO866 and radiation alone resulted in tumor growth delays of 14days and 33days, respectively, whereas the combination showed a significantly increased tumor growth delay of 65days. ConclusionsOur studies show that APO866-induced NAD+ depletion enhances radiation responses in tumor cell survival in prostate cancer. However, the in vitro data do not reveal a solid cellular mechanism to exploit further clinical development at this moment.

Next-Generation NAMPT Inhibitors Identified by Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

Nicotinamide Phosphoribosyltransferase Is Required for the Calorie Restriction-Mediated Improvements in Oxidative Stress, Mitochondrial Biogenesis, and Metabolic Adaptation

Calorie restriction (CR) is one of the most reproducible treatments for weight loss and slowing aging. However, how CR induces these metabolic alterations is not fully understood. In this work, we studied whether nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for nicotinamide adenine dinucleotide biosynthesis, plays a role in CR-induced beneficial metabolic effects using a specific inhibitor of NAMPT (FK866). CR upregulated NAMPT mRNA and protein levels in rat skeletal muscle and white adipose tissue. Inhibition of NAMPT activity by FK866 in rats did not affect the SIRT1 upregulation by CR but suppressed the CR-induced SIRT1 activity and deacetylation of Forkhead box protein O1/peroxisome proliferator-activated receptor γ coactivator-1α. Inhibition of NAMPT activity by FK866 also attenuated the CR-induced SIRT3 activity, evidenced by deacetylation of superoxide dismutase-2. Furthermore, FK866 not only weakened the CR-induced decrease of oxidative stress (dichlorofluorescin signal, superoxide , and malondialdehyde levels), but also greatly attenuated the CR-induced improvements of antioxidative activity (total superoxide dismutase, glutathione, and glutathione/oxidized glutathione ratio) and mitochondrial biogenesis (mRNA levels of nuclear respiratory factor 1, cytochrome c oxidase IV, peroxisome proliferator-activated receptor-γ coactivator-1α, and transcription factor A, mitochondrial and citrate synthase activity). At last, FK866 blocked the CR-induced insulin sensitizing, Akt signaling activation, and endothelial nitric oxide synthase phosphorylation. Collectively, our data provide the first evidence that the CR-induced beneficial effects in oxidative stress, mitochondrial biogenesis, and metabolic adaptation require NAMPT.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity by Small Molecule GMX1778 Regulates Reactive Oxygen Species (ROS)-mediated Cytotoxicity in a p53- and Nicotinic Acid Phosphoribosyltransferase1 (NAPRT1)-dependent Manner

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

The NAMPT inhibitor FK866 reverts the damage in spinal cord injury

BackgroundEmerging data implicate nicotinamide phosphoribosyl transferase (NAMPT) in the pathogenesis of cancer and inflammation. NAMPT inhibitors have proven beneficial in inflammatory animal models of arthritis and endotoxic shock as well as in autoimmune encephalitis. Given the role of inflammatory responses in spinal cord injury (SCI), the effect of NAMPT inhibitors was examined in this setting.MethodsWe investigated the effects of the NAMPT inhibitor FK866 in an experimental compression model of SCI.ResultsTwenty-four hr following induction of SCI, a significant functional deficit accompanied widespread edema, demyelination, neuron loss and a substantial increase in TNF-α, IL-1β, PAR, NAMPT, Bax, MPO activity, NF-κB activation, astrogliosis and microglial activation was observed. Meanwhile, the expression of neurotrophins BDNF, GDNF, NT3 and anti-apoptotic Bcl-2 decreased significantly. Treatment with FK866 (10 mg/kg), the best known and characterized NAMPT inhibitor, at 1 h and 6 h after SCI rescued motor function, preserved perilesional gray and white matter, restored anti-apoptotic and neurotrophic factors, prevented the activation of neutrophils, microglia and astrocytes and inhibited the elevation of NAMPT, PAR, TNF-α, IL-1β, Bax expression and NF-κB activity.We show for the first time that FK866, a specific inhibitor of NAMPT, administered after SCI, is capable of reducing the secondary inflammatory injury and partly reduce permanent damage. We also show that NAMPT protein levels are increased upon SCI in the perilesional area which can be corrected by administration of FK866.ConclusionsOur findings suggest that the inflammatory component associated to SCI is the primary target of these inhibitors.

Adipocytes as a Source of Increased Circulating Levels of Nicotinamide Phosphoribosyltransferase/Visfatin in Active Acromegaly

Nicotinamide phosphoribosyltransferase (NAMPT)/visfatin is a widely expressed protein with various effects on glucose and lipid metabolism, cell survival, and inflammation. We hypothesized that NAMPT was related to metabolic disturbances in active acromegaly. Body composition, glucose metabolism, and NAMPT levels were measured in 47 patients with active, untreated acromegaly and 24 age-, sex-, and body mass index-matched controls. The in vitro effects of GH/IGF-I on NAMPT expression in human sc adipocytes (SCA), visceral adipocytes, osteoblasts, and hepatocytes were studied. The effects of overnight incubation with the highly specific NAMPT inhibitor FK866 on the GH-stimulated monocyte chemotactic protein-1 and IL-6 expression in mature SCA were evaluated. NAMPT was increased in active acromegaly (P = 0.004) and correlated negatively with limb (arms + legs) fat percentage (% fat, r = -0.32; P = 0.032). After adjusting for age, gender, leptin, and GH, the circulating NAMPT correlated negatively with limb and total body fat percentage (% fat limbs, r = -0.43, P = 0.006; % fat total body, r = -0.36, P = 0.022) and correlated positively with limb and total body lean percentage (% lean limbs, r = 0.31, P = 0.047; % lean total body, r = 0.33, P = 0.034). No correlation between NAMPT and glucose metabolic parameters was found. In vitro studies revealed that GH increased NAMPT expression in adipocytes. The inhibition of NAMPT enzymatic activity attenuated GH-induced monocyte chemotactic protein-1 expression in SCA. NAMPT is increased in active acromegaly and may be an inflammatory mediator that causes monocyte infiltration in adipose tissue.

A Preclinical Study on the Rescue of Normal Tissue by Nicotinic Acid in High-Dose Treatment with APO866, a Specific Nicotinamide Phosphoribosyltransferase Inhibitor

Inhibitor of nicotinamide phosphoribosyltransferase APO866 is a promising cancer drug currently in phase II clinical trials in oncology. Here, we present a strategy for increasing the therapeutic potential of APO866 through the rescue of normal tissues by coadministration of nicotinic acid (Vitamin B(3)). We examined the toxicity profile of APO866 in B6D2F1 mice and the effect of oral administration of nicotinic acid on tissue toxicity. Nicotinic acid (50 mg/kg) protects mice from death and severe toxicity from an APO866 dose (60 mg/kg) four times the monotherapy maximum tolerated dose (15 mg/kg). In a panel of six cancer cell lines, we find that three (including ML-2 cells) are protected by nicotinic acid in vitro, whereas the cytotoxicity of APO866 remains unaffected in the remaining three (including A2780 cells). A selective biomarker for the protection by nicotinic acid was subsequently identified by quantitative RT-PCR. The expression of nicotinic acid phosphoribosyltransferase is low in the cell lines not rescued from APO866 by nicotinic acid compared with protected cell lines. The findings in cell lines translated into xenograft models in which the combination of 50 mg/kg nicotinic acid and 50 mg/kg APO866 in mouse xenografts of A2780 cells increased life span by >3-fold compared with standard treatment of 15 mg/kg, and the effect of APO866 was clearly decreased when using the same treatment paradigm in ML-2 xenografts. In conclusion, the combination of high doses of APO866 with rescue by nicotinic acid may significantly increase the therapeutic potential in a subset of cancers with low expression of nicotinic acid phosphoribosyltransferase.

Visfatin: a new player in mesangial cell physiology and diabetic nephropathy

Visfatin is an adipocytokine that improves insulin resistance and has an antidiabetic effect. However, the role of visfatin in the kidney has not yet been reported. In this experiment, the synthesis and physiological action of visfatin in cultured mesangial cells (MCs) were studied to investigate the role of visfatin in diabetic nephropathy. Visfatin was found synthesized in MCs as well as adipocytes. Visfatin synthesis was markedly increased, not by angiotensin II, but by high glucose stimuli. In addition, visfatin treatment induced a rapid uptake of glucose, peaking at 20 min after visfatin treatment in a dose-dependent manner. A small inhibiting RNA against insulin receptor significantly blocked visfatin-mediated glucose uptake. Visfatin stimuli also enhanced intracellular NAD levels, and treatment with FK866, which is a specific inhibitor of nicotinamide phosphoribosyltransferase (Nampt), significantly inhibited visfatin-induced NAD synthesis and glucose uptake. Visfatin treatment increased glucose transporter-1 (GLUT-1) protein expression in isolated cellular membranes, and pretreatment with cytochalasin B completely inhibited visfatin-induced glucose uptake. Moreover, immunofluorescent microscopy showed the migration of cytosolic GLUT-1 into cellular membranes after visfatin treatment. In accordance with these results, the activation of protein kinase B was detected after visfatin treatment. Furthermore, visfatin treatment dramatically increased the synthesis of profibrotic molecules including transforming growth factor-beta1, plasminogen activator inhibitor-1, and type I collagen, and pretreatment with cytochalasin B completely inhibited visfatin-induced upregulation of profibrotic molecules. These results suggest that visfatin is produced in MCs, which are a novel target for visfatin, and play an important role in the pathogenesis of diabetic nephropathy.