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. 2012 Apr;97(4):551-9.
doi: 10.3324/haematol.2011.055236. Epub 2011 Dec 29.

The role of sirtuin 2 activation by nicotinamide phosphoribosyltransferase in the aberrant proliferation and survival of myeloid leukemia cells

Lan Dan  1 , Olga Klimenkova, Maxim Klimiankou, Jan-Henning Klusman, Marry M van den Heuvel-Eibrink, Dirk Reinhardt, Karl Welte, Julia Skokowa
Affiliations

The role of sirtuin 2 activation by nicotinamide phosphoribosyltransferase in the aberrant proliferation and survival of myeloid leukemia cells

Lan Dan et al. Haematologica. 2012 Apr.

Abstract

Background: Inhibitors of nicotinamide phosphoribosyltransferase have recently been validated as therapeutic _targets in leukemia, but the mechanism of leukemogenic transformation downstream of this enzyme is unclear.

Design and methods: Here, we evaluated whether nicotinamide phosphoribosyltransferase's effects on aberrant proliferation and survival of myeloid leukemic cells are dependent on sirtuin and delineated the downstream signaling pathways operating during this process.

Results: We identified significant upregulation of sirtuin 2 and nicotinamide phosphoribosyltransferase levels in primary acute myeloid leukemia blasts compared to in hematopoietic progenitor cells from healthy individuals. Importantly, specific inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 significantly reduced proliferation and induced apoptosis in human acute myeloid leukemia cell lines and primary blasts. Intriguingly, we found that protein kinase B/AKT could be deacetylated by nicotinamide phosphoribosyltransferase and sirtuin 2. The 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 dephosphorylation. This leads to activation of glycogen synthase kinase-3 β via diminished phosphorylation and, ultimately, inactivation of β-catenin by phosphorylation.

Conclusions: 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 protein kinase B/AKT/ glycogen synthase kinase-3 β/β-catenin pathway is a _target for inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 and, thereby, leukemia cell proliferation.

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Figures

Figure 1.
Figure 1.
(A) Model of the effects of NAMPT. NAMPT is the rate-limiting enzyme for converting nicotinamide (NA) into NAD+, which, in turn, activates sirtuins, protein deacetylases, with further transcriptional regulation by deacetylation. (B-D) NAMPT (B), SIRT2 (C) and SIRT1 (D) mRNA expression levels in CD34+ cells of healthy individuals (n = 6) and AML patients (n = 11) were measured by quantitative reverse transcriptase polymerase chain reaction, normalized to β-actin levels and reported as arbitrary units (AU). Data represent means ± SD of triplicate measurements (*P<0.05); (E,F) representative DUOLINK images of NAMPT (E) and SIRT2 (F) protein expression in AML blasts (n = 6) and in CD34+ cells from healthy individual (n = 6); (G-I) intensity of mRNA expression levels of SIRT2 in AML patients, measured by Affymetrix Human Genome U133 Plus 2.0 Arrays (*P<0.05): (G) 33 high-risk (HR) AML patients and 18 standard-risk (SR) AML patients; (H) 17 AML patients with CBF AML – t(8;21) and inv(16) – and 35 patients with other types of AML; (I) four AML M1 patients, nine AML M2 patients, 19 AML M4 patients including 11 with AML M4eo, and 13 AML M5 patients.
Figure 2.
Figure 2.
(A, B) HL60 cells were cultured with 1 nM, 10 nM, or 100 nM of FK866 for 96 h or 10 nM, 100 nM or 1000nM of AC93253 for 48 h, or with DMSO as a control. SIRT2 activity was measured using a CycLex® SIRT2 Deacetylase Fluorometric Assay Kit; data represent means ± SD of triplicate measurements derived from three experiments (*P<0.05); (C,E) HL60 cells, NB4 cells, or primary blasts of three AML patients were cultured with 1 nM, 10 nM, or 100 nM of FK866 for 96 h (C) or 10 nM, 100 nM or 1000 nM of AC93253 for 48 h (E), or with DMSO for the respective times as a control; apoptosis was assessed by the Caspase-GLO 3/7 Assay; the ratio of apoptotic to viable cells is shown; data represent means ± SD of triplicate measurements and are derived from three experiments (*P<0.05; **P<0.01); (D,F) bone marrow CD34+ cells were cultured in ex-vivo supplemented medium in the presence of 10 nM of FK866 for 96 h, or 100 nM of AC93253 for 48 h or with DMSO for the respective times; appoptosis was assessed as described above.
Figure 3.
Figure 3.
(A, B) HL60 and NB4 cells were labeled with BrdU for 20 min, cell cycle profile (A) and percentage of BrdU+ cells (B) were assessed by the BrdU Flow Kit and FACS analysis; data are means ± SD of duplicate measurements and are derived from three experiments (* P<0.05).
Figure 4.
Figure 4.
(A, B) HL60 and NB4 cells were cultured with or 10 nM of FK866 for 96 h or 100 nM of AC93253 for 48 h, or with DMSO as a control, (A) representative western blot images of total- and phospho-AKT (Ser473) (left part) as well as densitometric analysis of phospho- to total-AKT (Ser473) protein levels of three experiments (right part); data represent means ± SD (*P<0.05); (B) representative images of acetylated-AKT protein, as measured by the DUOLINK technique; left panel, Cy3 dots of acetylated-AKT staining; middle panel, DAPI stained nuclei; right panel, DAPI/Cy3 overlay; (C) effects of AKT on GSK3β: activated (phosphorylated) AKT phosphorylates and by this inhibits GSK3β; (D) HL60 cells were cultured with 10 nM of FK866 for 96 h or 100 nM of AC93253 for 48 h, or with DMSO as a control. Representative western blot images of total- and phospho-GSK3β (Ser9) (left part) as well as densitometric analysis of phospho- to total-GSK3β (Ser9) proteins levels (right part) of three experiments; data represent means ± SD (*P<0.05); (E) representative DUOLINK images of phospho-GSK3β (Ser9) protein expression in HL60 cells treated with 10 nM of FK866 for 96 h or 100 nM of AC93253 for 48 h, or with DMSO as a control.
Figure 5.
Figure 5.
(A) Nuclear localization of β-catenin protein in AML blasts, in comparison to that in CD34+ cells from healthy individuals. Representative images of immunofluorescence staining with β-catenin antibody and ToPro-3 (nuclei); (B) representative images of inactive phospho-β-catenin (Ser33/37) protein expression in HL60 cells treated with 10 nM of FK866, 100 nM of AC93253 or DMSO as a control; (C) representative images of inactive phospho-β-catenin (Ser33/37) protein expression in AML blasts treated or not with 100 nM of AC03253, or DMSO as control; (D) downregulation of mRNA levels of Wnt/β-catenin _target genes in AML blasts after treatment with 10 nM of FK866 or 100 nM of AC93253 or DMSO as control; assessed by quantitative reverse transcriptase polymerase chain reaction analysis, data represent mean ± SD of triplicate measurements (*P<0.05) and are derived from three experiments; (E) elevated levels of NAMPT are involved in aberrant proliferation of AML blasts by sequential activation of SIRT2, further elevated by deacetylation, phosphorylation and activation of AKT with subsequent phosphorylation/deactivation of GSK3β. This leads to activation and nuclear accumulation of the proto-oncogene, β-catenin. Nuclear β-catenin binds its co-factors LEF-1/TCF transcription factors and by this activates _target genes (e.g. c-myc, cyclin D1 and survivin) involved in cell proliferation as well as in the regulation of cell cycle and apoptosis, which may contribute to aberrant proliferation of AML blasts.
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