Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD+ / SIRT1 Mediated Deacetylation of FOXO3a in Myeloid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1352-1352
Author(s):  
Yannick Lippka ◽  
Basant Kumar Thakur ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Nature Medicine ◽  
Mrna Levels ◽  
Leukemic Transformation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Myeloid Progenitor ◽  
Damage Repair ◽  
Gene Assay ◽  
Myeloid Progenitor Cells

Abstract Abstract 1352 Poster Board I-374 Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 10/l. G-CSF treatment increases blood neutrophil numbers in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. Recently we found that NAMPT, a protein involved in biosynthesis of NAD+ was significantly increased in CN patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD+ levels correlated with the elevated levels of SIRT1, an enzyme involved in deacetylation of several histone and non -histone proteins (e.g. FOXO3a) by utilising NAD+. In search of the downstream factors regulated by G-CSF/ NAMPT /SIRT1 pathway, we found elevated levels of FOXO3a protein in CN patients treated with G-CSF compared to healthy controls or patients with other types of neutropenia. Therefore, we were interested whether NAMPT/ NAD+ -dependent activated SIRT1 affects FOXO3a levels. Indeed we observed that overexpression of NAMPT leads to the upregulation of FOXO3a protein in 293T cells and endogenous FOXO3a interacts with endogenous SIRT1 in both 293T cells and promyelocytic cell line NB4. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. As acetylation of FOXO3a is considered to be important for its tumor suppressor function we asked if inhibition of NAMPT using FK866 increases the acetylation of FOXO3a. Indeed we show that acetylation level of FOXO3a is enhanced when 293T and NB4 cells were treated with FK866. Furthermore, increased acetylation of FOXO3a correlates with the decreased interaction between FOXO3a and SIRT1 in 293T cells after treatment with FK866. GADD45, a protein involved in DNA damage repair, is a well known target of FOXO3a. We found that in CD33+ myeloid progenitor cells from G-CSF treated CN patients with high NAMPT / SIRT1 / FOXO3a levels also had low GADD45 mRNA levels. We performed reporter gene assay using luciferase construct containing wild type GADD45 promoter and observed that SIRT1 enhances the FOXO3a mediated downregulation of GADD45 promoter and interestingly NAD+ further augments this effect. Taken together we hypothesized that NAMPT/NAD+ activated SIRT1 mediates deacetylation and activation of FOXO3a protein, leading to downregulation of target genes (e.g. GADD45) with tumor suppression functions which might possibly be involved in the leukemic transformation in CN patients. Disclosures No relevant conflicts of interest to declare.

Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD+ / SIRT1 Mediated Deacetylation of p53 in Myeloid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3589-3589
Author(s):  
Basant Kumar Thakur ◽  
Yannick Lippka ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
P53 Protein ◽  
Nature Medicine ◽  
Leukemic Transformation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Downstream Target ◽  
P21 Promoter

Abstract Abstract 3589 Poster Board III-526 Recently we found that NAMPT, a protein involved in biosynthesis of NAD+ was significantly increased in Severe Congenital Neutropenia (CN) patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD+ levels correlated with the elevated levels of SIRT1, a NAD+-dependent deacetylase involved in the deacetylation of histone and non-histone proteins. CN is considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. SIRT1 mediated deacetylation of p53 at the lysine 382 has been shown to attenuate the transcriptional activity of p53. We asked if inactivation of p53 by NAMPT/ NAD+ / SIRT1 dependent deacetylation plays a role in the leukemic transformation in patients suffering from CN. Here we can demonstrate that presence of NAMPT or NAD+ enhances the SIRT1 mediated deacetylation of acetylated lysine peptide in vitro. Further in 293T cells overexpression of NAMPT induces the SIRT1 mediated deacetylation of p53. Using the promyelocytic cell line NB4 we show that endogenous p53 interacts with SIRT1 and the presence of NAMPT leads to decreased acetylation of p53 at the lysine 382. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. Acetylation of p53 is considered to be important for its tumor suppressor function and we asked if inhibition of NAMPT using FK866 increases the acetylation of p53. Indeed we demonstrate that the acetylation level of p53 is enhanced when NB4 cells were treated with FK866 and increased acetylation of p53 correlates with the decreased interaction between p53 and SIRT1. Treatment of NB4 with NAMPT further leads to the decreased expression of p53 target gene cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein. p21 is a well known target of p53 and is involved in cell cycle arrest after the cell undergoes stress. Using p21 promoter-luciferase construct we show that SIRT1 inhibits the p53 mediated activation of p21 promoter and this inhibition was further enhanced in the presence of NAMPT or NAD+. Taken together our working hypothesis is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of p53 protein thereby leading to p53 inactivation and downregulation of downstream target genes with tumor suppression functions (e.g. p21) which might possibly resulting in the leukemic transformation in CN patients. Disclosures: No relevant conflicts of interest to declare.

HoxA10 Regulates Myeloid Progenitor Proliferation by Activating Transcription of the Gene Encoding Transforming Growth Factor Beta 2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1572-1572
Author(s):  
Chirag Shah ◽  
Hao Wang ◽  
Elizabeth A. Eklund
Keyword(s):  
Progenitor Cells ◽  
Transforming Growth Factor Beta ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Growth Factor Beta ◽  
Differentiation Block

Abstract Abstract 1572 HoxA10 is a homeodomain transcription factor which functions as a myeloid leukemia promoter. Correlative clinical studies found that increased expression of a group of HoxA proteins, including HoxA10, in acute myeloid leukemia (AML) was associated with poor prognosis. In murine models, overexpression of HoxA10 in the bone marrow was associated with development of a myeloproliferative disease which progressed to AML with time. These results suggested that HoxA10-overexpression dysregulated cell proliferation and/or survival, and predisposed to acquisition of additional mutations which led to differentiation block and AML. Additional investigations, we and others demonstrated that HoxA10 overexpression in murine hematopoietic stem cells (HSC) expanded the granulocyte/monocyte progenitor (GMP) population in vitro and in vivo. Despite this information about the impact of HoxA10 overexpression on myeloid leukemogenesis, the mechanisms by which HoxA10 exerts this effect are largely unknown. To investigate such mechanisms, we have been identifying HoxA10 target genes. In previous studies, we identified a number of HoxA10 target genes that encode phagocyte effector proteins. HoxA10 represses transcription of these gene in myeloid progenitors, and decreased HoxA10 repression activity contributes to phenotypic differentiation as myelopoiesis proceeds. This provided a potential mechanism for HoxA10 involvement in differentiation block, but not progenitor survival or expansion. We used a chromatin immuno-precipitation based approach to identify additional HoxA10 target genes involved in these activities. Previously, we reported that HoxA10 activated the DUSP4 gene in myeloid progenitor cells. This gene encodes Mitogen Activated Protein Kinase Phosphatase 2 (Mkp2) which inhibits Jnk-induced apoptosis in myeloid progenitor cells. This provided a mechanism for increased cell survival in HoxA10-overexpressing cells. In the current studies, we identified TGFB2 as a HoxA10 target gene. This gene encodes Transforming Growth Factor Beta 2 (TgfB2) a member of the TgfB super family of cytokines. Similar to TgfB1 and 3, TgfB2 interacts with TgfB-receptors I and II. However, unlike these more classical family members, TgfB2 induces proliferation of hematopoietic stem and progenitor cells. We found that HoxA10 activated the TGFB2 promoter via tandem cis elements in the proximal promoter. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing GMP and leukemia cells in vitro. Increased proliferation in HoxA10-overexpressing cells involved activation of the MAP kinase pathway in a TgfB2 dependent manner. These studies identify autocrine production of pro-proliferative cytokines as a novel mechanism for the function of Hox proteins. These findings have implications for ex vivo expansion of HSC and myeloid progenitors for tissue engineering. These result also have implications for therapeutic approaches to poor prognosis AML characterized by increased Hox expression. Disclosures: No relevant conflicts of interest to declare.

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 982-982
Author(s):  
Tohru Fujiwara ◽  
Haruka Saitoh ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Wide Range ◽  
The Impact ◽  
Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

c-Maf Induces Monocytic Differentiation and Apoptosis in Bipotent Myeloid Progenitors

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1578-1589 ◽  
Author(s):  
Shrikanth P. Hegde ◽  
JingFeng Zhao ◽  
Richard A. Ashmun ◽  
Linda H. Shapiro
Keyword(s):  
Target Genes ◽  
Cell Model ◽  
Myeloid Cell ◽  
Model Systems ◽  
Monocytic Differentiation ◽  
Developmentally Regulated ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Direct Binding ◽  
Myeloid Progenitors

Abstract The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated Myb:Maf status seen in differentiating cells can be recapitulated by overexpression of c-Maf in myeloid cell lines, we inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of c-Maf protein led to marked increases in Myb:Maf complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of c-Myb regulated targets, such as Bcl-2 and CD13/APN, coupled with the activation of as yet undefined differentiation-promoting genes.

Use of CALM-AF10 and NUP98-HOXD13 fusions to Identify Oncogenic Pathways That Collaborate with HOXA9 Overexpression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2499-2499
Author(s):  
Rachel L Novak ◽  
David P Harper ◽  
David L. Caudell ◽  
Sarah H Beachy ◽  
Yang Jo Chung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Insertional Mutagenesis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Insertion Site ◽  
Cell Number ◽  
Incomplete Penetrance ◽  
Leukemic Transformation

Abstract Abstract 2499 Acute myeloid leukemias (AMLs) are thought to result from a series of complementary mutations that affect several vital pathways, including differentiation, self-renewal, apoptosis, and proliferation. Several mutations associated with AML (MLL fusions, NUP98 fusions, CALM-AF10 fusion, and NPM1 mutations) are thought to impair hematopoietic differentiation by dysregulation of target genes; one of the most consistently dysregulated target genes is HOXA9. Mice that express a CALM-AF10 (CA10) or NUP98-HOXD13 (NHD13) fusion gene overexpress HOXA9 and develop AML with a delayed onset and incomplete penetrance, suggesting the need for complementary mutations. We have used three techniques including candidate gene resequencing, retroviral insertional mutagenesis (RIM), and gene expression profiling to compare and contrast the complementary mutations that occur in these mice. The frequency of Ras pathway mutations is similar for both CA10 (20%) and NHD13 (32%) leukemic mice, and previously reported RIM studies identified common integration sites near Mn1 and Evi1 in both models, suggesting that dysregulation of similar collaborative pathways can lead to leukemia in both models. However, there were significant differences between these models. The aforementioned RIM studies identified Zeb2 as the most frequent insertion site for CA10 leukemias, suggesting that Zeb2 collaborated with CA10, whereas no Zeb2 insertions were found in the NHD13 RIM study. Another distinction between the NHD13 and CA10 leukemias is the expression of the HOXA9 co-regulatory factor Meis1. Meis1 is markedly upregulated in bone marrow from clinically healthy, pre-leukemic CA10 mice and CA10 leukemic tissues compared to wildtype bone marrow. Conversely, Meis1 expression was decreased in bone marrow from clinically healthy NHD13 mice compared to wildtype, and only occasionally overexpressed in NHD13 leukemias. Interestingly, RIM analysis of NHD13 mice identified Meis1 as the most frequent insertion site, suggesting that Meis1 overexpression can collaborate with NHD13 during leukemic transformation. Flt3, which is frequently mutated in patients with AML, has been shown to be transcriptionally activated by Meis1. Of note, Flt3 is consistently overexpressed in CA10 leukemias but is overexpressed only in those NHD13 leukemias that have upregulated Meis1. Intriguingly, the frequency of Flt3 activating mutations in CA10 leukemias (which overexpress Flt3) was 25%, but none were found in the NHD13 leukemias (which do not overexpress Flt3). Lastly, using gene expression arrays, N-myc was identified as one of the most differentially overexpressed mRNAs, suggesting that it may cooperate with CA10 during leukemic transformation. Furthermore, N-myc has recently been shown to promote AML in mice, and our preliminary experiments suggest that N-myc and CA10 collaborate and lead to increased cell number and replating potential in bone marrow colony formation assays. In conclusion, we have taken several genetic approaches to identify key similarities and differences in the biology of two leukemic models (CA10 and NHD13), both of which overexpress HOXA9. Disclosures: No relevant conflicts of interest to declare.

AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 265-273 ◽  
Author(s):  
Chunaram Choudhary ◽  
Joachim Schwäble ◽  
Christian Brandts ◽  
Lara Tickenbrock ◽  
Bülent Sargin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Activating Mutations ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Kinase Domain Mutations

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein α (c/EBPα) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPα and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.

scholarly journals Interferon Consensus Sequence Binding Protein (ICSBP) Decreases β-Catenin Activity in Myeloid Cells by Repressing GAS2 Transcription

2010 ◽  
Vol 30 (19) ◽  
pp. 4575-4594 ◽  
Author(s):  
Weiqi Huang ◽  
Wei Zhou ◽  
Gurveen Saberwal ◽  
Iwona Konieczna ◽  
Elizabeth Horvath ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Target Genes ◽  
Binding Protein ◽  
Blast Crisis ◽  
Consensus Sequence ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Histone Deacetylase 3 ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

ABSTRACT The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and β-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases β-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases β-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased β-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in β-catenin activity in Bcr/abl-positive (Bcr/abl+) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences β-catenin activity in myeloid leukemia.

Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3164-3173 ◽  
Author(s):  
Masao Mizuki ◽  
Joachim Schwäble ◽  
Claudia Steur ◽  
Chunaram Choudhary ◽  
Shuchi Agrawal ◽  
...  
Keyword(s):  
Target Genes ◽  
Wild Type ◽  
Transcriptional Program ◽  
Serine Threonine Kinase ◽  
Myeloid Progenitor ◽  
Genome Wide ◽  
Flt3 Mutations ◽  
Myeloid Progenitor Cells ◽  
And Function ◽  
Microarray Expression

Abstract The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT– and FLT3-ITD–expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD–expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPα, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3–independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.

Cyclin A Is a Target Gene of Activated CREB Downstream of GM-CSF Signaling That Regulates Normal and Malignant Myelopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2181-2181
Author(s):  
Deepa B. Shankar ◽  
Kentaro Kinjo ◽  
Jerry Cheng ◽  
Samuel Esparza ◽  
Noah Federman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Target Genes ◽  
Leukemia Cell ◽  
Cyclin A ◽  
Gm Csf ◽  
Myeloid Progenitor ◽  
Leukemia Cell Lines ◽  
Myeloid Progenitor Cells ◽  
Blast Cells

Abstract Granulocyte-Macrophage Colony-Stimulating Factor stimulates the proliferation and survival of myeloid progenitor cells in vitro and in vivo. Primary AML blast cells express high affinity GM-CSF receptors. We previously reported that the basic leucine zipper transcription factor, CREB (cAMP Responsive Element Binding Protein), is phosphorylated at serine 133 in response to GM-CSF stimulation through a MEKK- and pp90RSK-dependent pathway. CREB protein and mRNA levels are elevated in the bone marrow of over 60% of AML patients at diagnosis. Furthermore, CREB is activated in primary AML blast cells that overexpress CREB. CREB overexpression in myeloid leukemia cell lines results in increased cell proliferation and survival, and decreased myeloid cell differentiation. Cells overexpressing CREB have increased numbers of cells in S phase in Brdu incorporation experiments in comparison to control cells. In addition, myeloid progenitor cells from CREB transgenic mice proliferate more and are growth-factor independent compared to cells from littermate controls. In colony assays, progenitor cells from CREB overexpressing mice are immortalized and acquire a blast-like phenotype. To understand the downstream pathways that regulate CREB-induced proliferation and survival in normal myeloid and leukemic cells, we examined the expression of two known CREB target genes, BCL-2 and Cyclin A, in CREB overexpressing myeloid leukemia cell lines. Cyclin A, but not BCL-2, was upregulated in cells overexpressing CREB compared to control cells. Cyclin A regulates G1 to S transition through activation of the cyclin-dependent kinase, CDK2. Myeloid progenitor cells from transgenic mice that overexpress CREB also overexpressed Cyclin A. We next investigated Cyclin A expression in primary blast cells from AML patients. Cyclin A protein levels were elevated in bone marrow from AML patients in which CREB was overexpressed but not in normal bone marrow. CREB has been shown to directly bind the CRE in the cyclin A promoter and increase transcription of cyclin A upon activation in response to growth factor stimulation. To study the possibility of cyclin A upregulation downstream of CREB, we examined transcriptional activation of Cyclin A in CREB overexpressing cells. A luciferase reporter construct containing the cyclin A promoter with the CRE was transfected into NFS60 cells that overexpressed CREB. A 4-fold increase in relative luciferase activity was observed in CREB overexpressing cells compared to control cells (p<0.05). Microarray analysis was performed with RNA from CREB overexpressing and non-overexpressing cells. In addition to cyclin A, several other target genes such as the pim-1 oncogene were found to be upregulated in CREB overexpressing cells. Our results suggest that aberrant cell cycle progression and increased proliferation from overexpression and activation of CREB downstream of GM-CSF signaling is a consequence of critical target genes that control myelopoiesis.

Acetylation of p53 Protein Is Crucial for Its Function In Myeloid Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1480-1480
Author(s):  
Basant Kumar Thakur ◽  
Tino Dittrich ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Tumor Suppressor ◽  
Cell Line ◽  
Acute Myelocytic Leukemia ◽  
Mrna Levels ◽  
Down Regulation ◽  
Leukemic Transformation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Nb4 Cells ◽  
Over Expression ◽  
Cd34 Cells

Abstract Abstract 1480 Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 109/L. G-CSF treatment increases blood neutrophil counts in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop Acute Myelocytic Leukemia (AML) or Myelodysplastic Syndrome (MDS). Surprisingly no mutations in genes, which typically occur in primary AML/MDS patients, were detected in CN patients who developed leukemia. But studies in CN patients reveal a high association of G-CSF receptor mutation and the incidence of leukemia, indicating the dysregulation of certain factors downstream of G-CSF receptor signalling. Recently, we reported that Nicotinamide Phosphoribosyltransferase (NAMPT), a protein involved in biosynthesis of NAD+, was significantly increased in CN patients treated with G-CSF as compared to healthy individuals. Elevated NAMPT/NAD+ levels correlated with increased levels of SIRT1, a NAD+-dependent deacetylase which is involved in the deacetylation of histone and non-histone proteins e.g. p53. The acetylation of tumor-suppressor p53 is considered necessary for its transcriptional activation, while SIRT1-mediated deacetylation of p53 has been shown to attenuate the transcriptional activity of p53. Therefore, we asked if deacetylation-dependent inactivation of p53 might play a role in leukemic transformation in CN patients. In this study we demonstrate that the presence of NAMPT or NAD+ enhances the SIRT1-mediated deacetylation of p53 in both the 293T cell line and the promyelocytic leukemia NB4 cell line. Treatment with exogenous recombinant NAMPT also leads to a decrease in the acetylation levels of endogenous p53 in CD34+ cells. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein is a well-known target of p53 and is involved in cell cycle arrest. We have shown that over-expression of NAMPT leads to down-regulation of p21 mRNA, and specific knockdown of SIRT1 leads to up-regulation of p21 mRNA. The presence of NAMPT also decreases the mRNA levels of p21 in both NB4 and CD34+ cells. The compound FK866 specifically inhibits NAMPT and has recently entered clinical trials as a potential chemotherapeutic agent. In a recent preclinical in vitro study FK866 has been shown to elicit massive cell death in numerous leukemia/lymphoma cell lines, but the underlying molecular mechanism remains unknown. We tested if inhibition of NAMPT using FK866 enhances the tumor-supressing role of p53 by increasing its acetylation levels. We have demonstrated that the treatment of NB4 cells with FK866 increases the acetylation of endogenous p53, and this increased acetylation is in part due to decreased interaction of p53 with SIRT1. In addition, the mRNA levels of p21 down-regulated in CD34+ and NB4 cells on treatment with NAMPT were up-regulated on use of FK866. Knockdown of p53 using specific shRNA against p53 inhibits the expression of p21 and treatment with FK866 under p53 knockdown does not induce the expression of p21 when compared to control cells. In functional studies we show that over-expression of NAMPT leads to increased proliferation of both 293T and NB4 cells. Treatment with FK866 leads to increased death of NB4 cells compared to the cells in which p53 has been silenced, due to the lack of p53 available to be acetylated. Taken together, our conclusion is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of p53 leading to down-regulation of the downstream target gene p21. This inhibition of the tumor-suppressor functions of p53 might be involved in the leukemic transformation seen in CN. Disclosures: No relevant conflicts of interest to declare.

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