Acute Myeloid Leukemia Articles and Brief Reports The role of sirtuin 2 activation by nicotinamide phosphoribosyltransferase in the aberrant proliferation and survival of myeloid leukemia cells Lan Dan, 1,4 Olga Klimenkova, 1 Maxim Klimiankou, 1 Jan-Henning Klusman, 2 Marry M. van den Heuvel-Eibrink, 3 Dirk Reinhardt, 2 Karl Welte, 1 and Julia Skokowa 1 1Department of Molecular Hematopoiesis, Children’s Hospital, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Hematology and Oncology, Children’s Hospital, Hannover Medical School, Hannover, Germany; and 3Department of Pediatric Oncology and Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands; 4Department of Pediatrics, The First Affiliated Hospital of GuangXi Medical University, NanNing, China ABSTRACT Acknowledgments: we thank Background A. Gigina, A. Müller Brechlin Inhibitors of nicotinamide phosphoribosyltransferase have recently been validated as therapeu - and M. Reuter for their excellent tic targets in leukemia, but the mechanism of leukemogenic transformation downstream of this technical assistance. enzyme is unclear. Manuscript received on Design and Methods September 14, 2011. Revised version arrived on November 21, Here, we evaluated whether nicotinamide phosphoribosyltransferase’s effects on aberrant pro - 2011. Manuscript accepted liferation and survival of myeloid leukemic cells are dependent on sirtuin and delineated the on December 19, 2011. downstream signaling pathways operating during this process. Correspondence: Results Karl Welte, Department of We identified significant upregulation of sirtuin 2 and nicotinamide phosphoribosyltransferase Molecular Hematopoiesis, Hannover levels in primary acute myeloid leukemia blasts compared to in hematopoietic progenitor cells Medical School, Carl-Neuberg from healthy individuals. Importantly, specific inhibition of nicotinamide phosphoribosyltrans - Str. 1, 30625 Hannover, ferase or sirtuin 2 significantly reduced proliferation and induced apoptosis in human acute Germany. myeloid leukemia cell lines and primary blasts. Intriguingly, we found that protein kinase E-mail: B/AKT could be deacetylated by nicotinamide phosphoribosyltransferase and sirtuin 2. The [email protected] anti-leukemic effects of the inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 were accompanied by acetylation of protein kinase B/AKT with subsequent inhibition by de- Skokowa Julia, Department of phosphorylation. This leads to activation of glycogen synthase kinase-3 via diminished phos - Molecular Hematopoiesis, Hannover β Medical School, Carl-Neuberg-Str. phorylation and, ultimately, inactivation of β-catenin by phosphorylation. 1, 30625 Hannover, Germany. Conclusions E-mail: [email protected] Our results provide strong evidence that nicotinamide phosphoribosyltransferase and sirtuin 2 participate in the aberrant proliferation and survival of leukemic cells, and suggest that the pro - The online version of this article tein kinase B/AKT/ glycogen synthase kinase-3 β/β-catenin pathway is a target for inhibition of has a Supplementary Appendix. nicotinamide phosphoribosyltransferase or sirtuin 2 and, thereby, leukemia cell proliferation. Key words: Nampt, SIRT2, Akt, GSK3, acute myeloid leukemia , acetylation. Citation: Dan L, Klimenkova O, Klimiankou M, Klusman J-H, van den Heuvel-Eibrink MM, Reinhardt D, Welte K, and Skokowa J. The role of sirtuin 2 activation by nicotinamide phosphoribo - syltransferase in the aberrant proliferation and survival of myeloid leukemia cells. Haematologica 2012;97(4):551-559. doi:10.3324/haematol.2011.055236 ©2012 Ferrata Storti Foundation. This is an open-access paper. haematologica | 2012; 97(4) 551 l. Dan et al. Introduction B-cell neoplasia and multiple myeloma. 27-32 Here, we aimed to evaluate the involvement of NAMPT Recently, we demonstrated that nicotinamide phospho - and SIRT2 in the aberrant proliferation and survival of ribosyltransferase (NAMPT) is an essential enzyme medi - leukemic cells, and to ascertain whether the ator of granulocyte colony-stimulating factor (G-CSF)- AKT/GSK3 β/β-catenin signaling pathway plays a role in triggered granulopoiesis. 1 NAMPT is the rate-limiting mediating this process. enzyme in the biosynthesis of NAD + and supplies NAD + to sirtuins, which require NAD + to activate their protein deacetylase functions. 2 In vitro stimulation of CD34 + cells Design and Methods with NAMPT leads to granulocytic differentiation via SIRT1-C/EBP (CCAAT/enhancer binding protein)-depen - Patients and control subjects dent activation of autocrine G-CSF synthesis and G-CSF Primary blasts from 11 patients with AML and CD34 + bone receptor expression in myeloid cells. 1 In addition, a specif - marrow cells from six healthy individuals were isolated from bone ic inhibitor of NAMPT has been validated as a therapeutic marrow mononuclear cells by Ficoll-Hypaque gradient centrifuga - target in leukemia, 3 suggesting that different mechanisms tion and were subsequently sorted using MACS beads. We operate downstream of NAMPT in “normal” and leuke - obtained approval for this study from Hannover Medical School’s mogenic myeloid cells. However, the mechanisms down - institutional review board. Informed consent was obtained from stream of NAMPT which are responsible for the aberrant the study participants in accordance with the Declaration of proliferation and apoptosis of leukemic cells have Helsinki. remained elusive. Sirtuins are members of the NAD +-dependent class III Cell lines and culture conditions histone deacetylase family; seven members (SIRT1-7) of NB4 and HL60 AML cell lines were cultured in RPMI-1640 this family have been described in humans. Sirtuins pos - medium with 10% fetal calf serum and 1% penicillin/strepto - sess either histone or protein deacetylase activity, and play mycin. CD34 + cells from healthy individuals and AML blasts were a particularly important role in the response to certain cultured for 4 days in 24-well tissue-culture plates (2 ¥10 5 types of stress and toxicity. Sirtuins are involved in life - cells/well) in X-vivo medium supplemented with 1% heat-inacti - span extension, age-related disorders, obesity, heart dis - vated autologous human serum with 20 ng/mL of interleukin-3, 20 ease, neurological function and cancer. 4 SIRT1, one of the ng/mL of interleukin-6, 20 ng/mL of thrombopoietin, 50 ng/mL of most extensively studied sirtuins, is known to deacetylate, stem cell factor and 50 ng/mL of Flt3 ligand. and thereby inactivate, p53 and FOXO3a. 5 SIRT2, unlike SIRT1, is mostly found in the cytoplasm 2 and shows cell Assessment of cell proliferation, cell cycle and apoptosis cycle-dependent intracellular localization, undergoing Cells (2 ¥10 4/100 mL) were plated in 96-well plates and incubated rapid nucleo-cytoplasmic shifts during G2/M cell-cycle with different concentrations of FK866 or AC 93253 for the indi - progression. This observation together with the demon - cated times. We used the CellTiter-Glo Luminescence Cell stration that overexpression of SIRT2 mediates a delay in Proliferation Assay to assess cell proliferation. To analyze apopto - cellular proliferation 6 suggest that SIRT2 may play a role in sis we used the Caspase-Glo 3/7 Activity Assay and counted cell-cycle regulation. The importance of nucleo-cytoplas - viable cells using trypan blue dye exclusion. Both assays were per - mic shuttling in SIRT2 function is highlighted by the formed according to the manufacturer’s instructions (Promega). observation that abnormal intracellular SIRT2 localization For cell cycle analysis we measured BrdU uptake (20 min) using a may lead to pathological downstream effects, such as BrdU Flow Kit (Pharmingen). abnormal cellular response of leukemic cells to DNA dam - age. 7 SIRT2 also deacetylates α-tubulin, 6,8 suggesting a Quantitative reverse transcriptase polymerase function in cytoskeletal organization. In addition to target - chain reaction analysis ing α-tubulin, SIRT2 is known to specifically target NF- We isolated RNA using a RNeasy Mini Kit, amplified cDNA kB, 9 FOXO transcription factors 10-12 and p53. 13-15 with random hexamer primers and a cDNA synthesis kit (Qiagen). The AKT pathway is frequently activated in acute We measured mRNA expression using a SYBR green qPCR kit. myeloid leukemia (AML). 16-18 However, the mechanisms Target gene mRNA expression was normalized to β-actin and was leading to AKT activation in AML are not completely represented as arbitrary units (AU). Primer sequences are available clear. NAMPT (visfatin) has recently been shown to on request. induce AKT phosphorylation in endothelial cells and in cardiac fibroblasts. 19,20 AKT phosphorylates and thereby Western blot analysis inhibits a serine-threonine kinase glycogen synthase We used mouse anti- β-actin, rabbit anti-AKT, rabbit anti-phos - kinase 3 β (GSK3 β). 21 GSK3 β is a well-known inhibitor of pho-AKT (Ser473) (193H12), rabbit anti-GSK-3 β (27 C10 ), and rab - Wnt signaling. GSK3 β targets the proto-oncogene β- bit anti-phospho-GSK-3 β (Ser9 ) (5 B3 ) ( all from Cell Signaling catenin and promotes its ubiquitination and proteasome- Technology ). Horseradish peroxidase-conjugated anti-mouse or mediated degradation. 22,23 Inactivation of GSK3 β leads to anti-rabbit secondary antibodies (Santa Cruz Biotechnology) were β-catenin accumulation and redistribution to the nucle - used as appropriate. We obtained whole-cell lysates through direct us. 22,23 Nuclear β-catenin interacts with LEF-1/TCF