MiR-494-3p Overexpression Leads to SOCS6 Downregulation and Supports Megakaryocytopoiesis in Primary Myelofibrosis CD34+ Hematopoietic Stem/Progenitor Cells

Abstract Primary Myelofibrosis (PMF) belongs to the Philadelphia negative Myeloproliferative Neoplasms (MPNs) and is characterized by hematopoietic stem-cell derived clonal myeloproliferation, involving especially megakaryocyte (MK) lineage, bone marrow fibrosis and extramedullary hematopoiesis. Recent studies have suggested that alterations in miRNAs expression could play a critical role in MPN's pathogenesis. In order to shed some light on this issue, we have previously performed the integrative analysis (IA) of gene and miRNA expression profiles of PMF CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patients compared with 31 healthy donors (R. Norfo et al., Blood, 2014). IA identified miR-494-3p as one of the most upregulated miRNAs in PMF CD34+ cells associated to the highest number of downregulated predicted targets (eighty-six, Fig. 1). In order to study the role of miR-494-3p in hematopoietic commitment and differentiation, and to elucidate its possible involvement in PMF pathogenesis, we performed miRNA overexpression experiments in cord blood (CB) CD34+ cells through miRNA mimic electroporation. The data showed that miR-494-3p promotes MK differentiation of HSPCs. Indeed, the fraction of cells expressing MK surface antigen CD41 was steadily increased in miR-494-3p overexpressing samples compared with controls (75.4±0.3% vs 53.2±3.5% at day 8, 82.6±1.3% vs 60.4±4.3% at day 10 of culture, p<0.05), as well as the percentage of cells expressing the late MK antigen CD42b was similarly increased. Furthermore, the percentage of MK colonies was increased in collagen-based clonogenic assay upon miR-494-3p overexpression compared to control (44.8±4.1% vs 24.1±2.1%, p<0.05). Next, to better characterize the molecular mechanisms underlying megakaryocytopoiesis stimulation by miR-494-3p, we profiled CB CD34+ cells overexpressing this miRNA using the Affymetrix HG-U219 microarray platform. Gene Expression profile analysis allowed the identification of 134 differentially expressed genes between cells overexpressing miR-494-3p and controls. In particular, we highlighted the presence of 13 genes downregulated both upon miRNA overexpression and in PMF CD34+ cells. Among them, Suppressor of Cytokine Signaling 6 (SOCS6) turned out to be the miR-494-3p predicted target associated to the most favorable prediction scores according to TargetScan, microRNA.org and miRDB prediction algorithms. Furthermore, 3' UTR luciferase reporter assays, performed in K562 cell line, proved the predicted interaction between miR-494-3p and SOCS6 3'UTR. Subsequently, we studied the role of SOCS6 in HSPCs differentiation by inhibiting its expression in CB CD34+ cells through small interfering RNAs. SOCS6 silencing stimulated megakaryocytopoiesis in CB CD34+ cells, as demonstrated by the expansion of CD41+ and CD42b+ cell fractions in SOCS6 silenced samples compared with controls (52.8±7.0% vs 37.7±4.5% at day 8, 66.9±7.2% vs 50.7±7.2% at day 10 for CD41+ cells, p<0.05). Moreover, MK colonies were increased upon SOCS6 silencing in collagen-based clonogenic assays (62.4±7.7% vs 51.3±6.5%, p<0.05) and morphological analysis further supported these results. Finally, in order to study the possible contribution of miR-494-3p overexpression to PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells by means of miR-494-3p antagomiR. As a result, miR-494-3p silencing led to SOCS6 upregulation and impaired MK differentiation in PMF HSPCs as demonstrated by the decrease in CD41+ cell fraction in silenced samples compared with controls (28.6±7.1% vs 39.2±7.7% at day 12 of culture, p<0.05) and by the reduction of MK colonies in collagen-based clonogenic assay (44.4±3.6% vs 54.7±2.5%, p<0.05). The values are reported as mean±S.E.M from 3 independent experiments. Taken together, our results showed that miR-494-3p overexpression promotes megakaryocytopoiesis in HSPCs. Moreover, we demonstrated for the first time that SOCS6 is a direct target of miR-494-3p. Since SOCS6 downregulation promotes MK differentiation of HSPCs, SOCS6 could be considered, at least in part, responsible for the biological effects observed after miR-494-3p overexpression. As miR-494-3p and SOCS6 showed the same expression trend in PMF CD34+ cells, our results could suggest that miR-494-3p/SOCS6 axis is involved in the induction of MK hyperplasia typically observed in PMF patients. Figure Figure. Disclosures Vannucchi: Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

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MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

Blood ◽

10.1182/blood.v116.21.3871.3871 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3871-3871

Author(s):

Li-Sheng Wang ◽

Ling LI ◽

Liang Li ◽

Keh-Dong Shiang ◽

Min Li ◽

...

Keyword(s):

Protein Expression ◽

Myeloid Cells ◽

Erythroid Differentiation ◽

Luciferase Reporter ◽

Regulatory Function ◽

Control Vector ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Direct Target

Abstract Abstract 3871 Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures: No relevant conflicts of interest to declare.

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A Novel TWIST2-ID2 Axis That Modulates The Growth and Imatinib Sensitivity Of CML CD34+ Cells

Blood ◽

10.1182/blood.v122.21.511.511 ◽

2013 ◽

Vol 122 (21) ◽

pp. 511-511

Author(s):

Yun Zhao ◽

Wenjuan Ma ◽

Xiuyan Zhang ◽

Jiangxia Cui ◽

Ivan Sloma ◽

...

Keyword(s):

Dna Binding ◽

Progenitor Cells ◽

Lentiviral Vector ◽

Critical Role ◽

K562 Cells ◽

Luciferase Reporter ◽

Control Group ◽

Wild Type ◽

Mesenchymal Transition ◽

Cd34 Cells

Abstract TWIST is a basic helix-loop-helix transcription factor that specifies Drosophila mesoderm development. In mammals there are 2 members, TWIST1 and TWIST2. TWIST2 is a regulator of osteoblasts and muscle development and plays a critical role in the epithelial-mesenchymal transition process, as well as in cancer initiation and metastasis. Twist2-deficient mice develop a myeloproliferative disease. These findings led us to query a potential role of TWIST2 in normal and leukemic (CML) human hematopoietic cells. RT-PCR and immuno-fluorescence analysis of CD34+ bone marrow (BM) cells obtained from healthy donors demonstrated their expression of TWIST2 transcripts and protein. Lentiviral vector-mediated knockdown of TWIST2 with 2 independent shRNA sequences enhanced the erythroid and granulopoietic colony-forming activity of transduced normal BM cells ∼2-fold compared with control transduced cells (n=3, p<0.05). Interestingly, ChIP studies showed that TWIST2 can bind directly to the DNA promoter for ID2 in CD34-enriched BM cells and knockdown of TWIST2 reduced ID2 expression by 50%. In lin-CD34+ cells from 14 chronic myeloid leukemia (CML) patients, we found both TWIST2 and ID2 transcripts to be 5 and 6 fold lower than those from 6 healthy BM donors (p<0.05), with similar findings for TWIST2 and ID2 protein in the same cells. BCR-ABL1-transduced Baf/3 cells also showed a reduction in Twist2 expression. Conversely, TWIST2 expression became elevated when K562 cells were treated with Imatinib mesylate (IM). We then generated a lentiviral vector encoding TWIST2 which proved capable of inhibiting the growth of K562 and MEG-01 cells as well as CFC production from CML CD34+ cells (n=11, p<0.05). Overexpression of TWIST2 in MEG-01 cells also reduced their tumorigenic ability in subcutaneously injected nude mice (0/8 for TWIST2 group, 7/8 for control group). In addition, increased TWIST2 sensitized the IM response of K562 cells and IM-resistant CD34+ cells from CML patients (2 in chronic phase and 2 in blast crisis). Correspondingly, knockdown of TWIST2 in K562 cells enhanced their cloning efficiency by 15% and made them IM-resistant. To obtain further insight into these biological effects of TWIST2, we generated several TWIST2 mutant cDNAs, including ones with a N-terminal truncation (ΔN), a C-terminal truncation (ΔC), a F86P dimerization mutant and a b- DNA binding mutant. Analysis of the effects of these mutants when overexpressed in CML cells and cell lines showed TWIST2 dimer formation was critical for the effects obtained with wild-type TWIST2, whereas the DNA binding domain could modulate these effects but was not essential, and the N-terminal and C-terminal domains were dispensable. We also found that overexpression of TWIST2 enhanced ID2 expression in CML CD34+ cells (n=3), as well as K562 and MEG-01 cells, and ChIP analyses confirmed the binding of TWIST2 to ID2 promoter DNA from K562 and MEG-01 cells. Using ID2 promoter-driven luciferase reporter and a mutant derivative (with only the E-box sequence altered), we found that TWIST2 could activate the wild-type promoter but not the mutated one in both K562 and MEG-01 cells. Finally, we co-transduced CML cells from 3 patients with TWIST2 and shRNA against ID2 and found that this reversed the suppressed production of CFC obtained with TWIST2 alone. Similarly in K562 cells this treatment partially restored their growth rate and IM resistance. Taken together, we report a novel TWIST2-ID2 regulatory axis in normal hematopoietic progenitor cells, which can also modulate the growth and IM response of CML progenitor cells. These findings provide a baseline for the future development of more effective therapy of CML. Disclosures: No relevant conflicts of interest to declare.

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PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis

Blood ◽

10.1182/blood.v124.21.4335.4335 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4335-4335

Author(s):

Fan LIU ◽

Guoyan Cheng ◽

Fabiana Perna ◽

Xu Haiming ◽

Pierre-Jacques Hamard ◽

...

Keyword(s):

Cell Biology ◽

Conflicts Of Interest ◽

Critical Role ◽

Conditional Knockout ◽

Major Type ◽

Cytokine Signaling ◽

Methyltransferase Activity ◽

Hematopoietic Stem ◽

Arginine Residues

Abstract Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology. Disclosures No relevant conflicts of interest to declare.

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MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.

Blood ◽

10.1182/blood.v108.11.1334.1334 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1334-1334

Author(s):

Robert W. Georgantas ◽

Richard Hildreth ◽

Jonathan Alder ◽

Carlo M. Croce ◽

George A. Calin ◽

...

Keyword(s):

Protein Expression ◽

Expression Profiles ◽

In Vivo Studies ◽

Luciferase Reporter ◽

Hematopoietic Differentiation ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.

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A Novel Role of Complement Components C1q and C5a in Hematopoietic Stem/Progenitor Cell Migration.

Blood ◽

10.1182/blood.v108.11.4169.4169 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4169-4169 ◽

Cited By ~ 2

Author(s):

Ali Jalili ◽

Leah Marquez-Curtis ◽

Jencet Montano ◽

Neeta Shirvaikar ◽

Mariusz Z. Ratajczak ◽

...

Keyword(s):

Progenitor Cells ◽

Mrna Level ◽

Classical Pathway ◽

Immune Functions ◽

Complement Protein ◽

Complement Components ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

C5ar Expression

Abstract The complement protein C1q primarily provides a recognition and activation signal that triggers the classical pathway of complement, but it also has multiple immune functions including acting as a chemoattractant for neutrophils, eosinophils, mast and monocyte-derived dendritic cells. Moreover, hematopoietic stem cells have recently been shown to express the C1q receptor, C1qRp. C5a also strongly chemoattracts monocyte/macrophages to inflammatory sites. Recently we showed that G-CSF mobilization activates complement by a classical IgM-dependent pathway (Blood2005;106:1976a). In this study we examined the possible roles of C1q and C5a in hematopoietic stem/progenitor cells (HSPC) migration. We found that C5aR was expressed at the mRNA level on mobilized peripheral blood (mPB) and cord blood (CB) CD34+ cells but at the protein level only on mPB CD34+ cells. Flow cytometry revealed high C1qRp expression on both mPB and CB CD34+ cells. When the expression of C5aR and C1qRp was examined on the CB CD34+ cells expanded towards myeloid, megakaryocytic and erythroid lineages (at days 0, 3, 6, 11 and 14 of expansion), we found that C5aR expression increased with cell maturation (days 3–14) in both myeloid and megakaryocytic progenitors. In contrast, C1qRp was highly expressed on day 0, stayed constant in myeloid and megakaryocytic cells (days 3–11), and was down-regulated by day 14 in megakaryocytic cells. C1qRp was down-regulated in erythroid precursors during their maturation. We also found that C5a but not C1q is a chemoattractant for mPB CD34+ cells. In chemotaxis assays towards an SDF-1 gradient, C5a primed chemotactic responses of both mPB and CB CD34+ cells to a low (10 ng/mL) gradient of SDF-1 (up to 80% of their response to a high (200 ng/mL) SDF-1 gradient); C1q primed the chemotactic responses of both types of CD34+ cells (up to 100% of the response to a high SDF-1 gradient), and the priming effect of C1q on SDF-1-induced chemotaxis of expanded myeloid and megakaryocytic precursors decreased, consistent with down-regulation of C1qRp on these cells by day 14. Hence these results indicate that HSPC and progenitor cells express functional C5a and C1q receptors and that both the C5a-C5aR and C1q-C1qRp axes sensitize the responses of these cells to SDF-1 and thus could play a role in HSPC homing/mobilization to bone marrow. Further studies in animal models are needed to elucidate the roles of C5a and C1q in HSPC trafficking.

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The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells

Blood ◽

10.1182/blood-2010-04-281329 ◽

2011 ◽

Vol 117 (6) ◽

pp. 1840-1850 ◽

Cited By ~ 38

Author(s):

Kam Tong Leung ◽

Kathy Yuen Yee Chan ◽

Pak Cheung Ng ◽

Tze Kin Lau ◽

Wui Man Chiu ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Critical Role ◽

Janus Kinase ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Short Term Exposure ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract The stromal cell–derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34+ cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34+CD38−/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34+ cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1–mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34+ cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34+CD9− cells displayed lower bone marrow homing capacity compared with that of total CD34+ cells. CD9 expression on homed CD34+ cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

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c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression

Blood ◽

10.1182/blood-2009-08-238311 ◽

2010 ◽

Vol 116 (22) ◽

pp. e99-e110 ◽

Cited By ~ 74

Author(s):

Elisa Bianchi ◽

Roberta Zini ◽

Simona Salati ◽

Elena Tenedini ◽

Ruggiero Norfo ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Luciferase Reporter ◽

Myb Transcription Factor ◽

Promoter Regions ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb–silenced CD34+ cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

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A Possible Role Of The Tetraspanin CD9 In Bone Marrow Retention and Engraftment Of Human CD34+ Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v122.21.3237.3237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3237-3237 ◽

Cited By ~ 5

Author(s):

Kam Tong Leung ◽

Karen Li ◽

Kam Sze Kent Tsang ◽

Kathy Yuen Yee Chan ◽

Pak Cheung Ng ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Motility ◽

Progenitor Cells ◽

Neutralizing Antibody ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cxcr4 Axis

Abstract The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing, engraftment and retention of hematopoietic stem/progenitor cells. We previously demonstrated that expression of CD9 is a downstream signal of the SDF-1/CXCR4 axis, and that CD9 regulates short-term (20 hours) homing of cord blood (CB) CD34+ cells in the NOD/SCID mouse xenotransplantation model (Leung et al, Blood, 2011). Here, we provided further evidence that pretreatment of CB CD34+ cells with a CD9-neutralizing antibody significantly reduced their long-term (6 weeks) engraftment, as indicated by the presence of human CD45+ cells, in the recipient bone marrow and spleen by 70.9% (P = .0089) and 87.8% (P = .0179), respectively (n = 6). However, CD9 blockade did not bias specific lineage commitment, including the CD14+ monocytic, CD33+ myeloid, CD19+ B-lymphoid and CD34+ stem/progenitor cells (n = 4). We also observed an increase of the CD34+CD9+ subsets in the bone marrow (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4). These data indicate that CD9 possesses important functions in regulating stem cell engraftment and its expression level on CD34+ cells is up-regulated in the target hematopoietic organs. Analysis of paired bone marrow (BM) and peripheral blood (PB) samples from healthy donors revealed a higher CD9 expression in BM-resident CD34+ cells (57.3% ± 8.1% CD9+ cells in BM vs. 29.3% ± 5.8% in PB; n = 5, P = 0.0478). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (33.8% ± 3.0% CD9+ cells, n = 24), when compared with those in BM (56.4% ± 4.9% CD9+ cells, n = 8, P = 0.0025). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.55-fold increase, n = 4, P = 0.0103), concomitant with a 75.2% reduction in the CD34+CXCR4+ subsets (P = 0.0118). Treatment of NOD/SCID chimeric mice with G-CSF increased the frequency of circulating CD45+ cells (3.4-fold) and CD34+ cells (3.3-fold), and substantially decreased the CD34+CD9+ subsets in the BM from 75.8% to 30.8%. Importantly, the decline in CD9 levels during G-CSF mobilization was also observed in the CD34+CD38-/low primitive stem cell subpopulation. Interestingly, in vitro treatment of BM CD34+ cells with G-CSF did not affect CD9 expression (n = 3), suggesting that a signaling intermediate is required for G-CSF-mediated CD9 down-regulation in vivo. Transwell migration assay revealed a significant enrichment of CD9- cells that were migrated towards a SDF-1 gradient (n = 4 for BM CD34+ cells, P = 0.0074; n = 7 for CB CD34+ cells, P = 0.0258), implicating that CD9 might negatively regulate stem cell motility. In contrast, pretreatment with the CD9-neutralizing antibody inhibited adhesion of CD34+ cells to the osteoblastic cell line Saos-2 by 33.5% (n = 2). Our results collectively suggest a previously unrecognized role of CD9 in stem cell retention by dual regulation of cell motility and adhesion, and reveal a dynamic regulation of CD9 expression in the BM microenvironment, which might represent an important event in controlling stem cell homing and mobilization. Disclosures: No relevant conflicts of interest to declare.

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Integrative Analysis Of mRNA/miRNA Expression Profiles Identified JARID2 As a Shared Target Of Deregulated Mirnas In Primary Myelofibrosis

Blood ◽

10.1182/blood.v122.21.1600.1600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1600-1600

Author(s):

Roberta Zini ◽

Ruggiero Norfo ◽

Valentina Pennucci ◽

Elisa Bianchi ◽

Simona Salati ◽

...

Keyword(s):

Gene Expression ◽

Mirna Expression ◽

Expression Profiles ◽

Primary Myelofibrosis ◽

Integrative Analysis ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Potential Contribution ◽

Cd34 Cells ◽

Mirna Expression Profiles

Abstract Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients [A.M. Vannucchi et al., Leukemia, 2013]. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported [P. Guglielmelli et al., Exp Hematol, 2007]. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Among 726 differentially expressed genes (DEG) we found that several putative cancer markers (WT1, ANGPT1) and several genes related to PMF progression, i.e. involved in megakaryocyte (MK) differentiation (NFE2, CD9), and fibrosis development (DLK1, LEPR1), were significantly more expressed in PMF samples than in the normal counterpart. Similarly, as regards the miEP, among 74 human differentially expressed miRNAs (DEM) in PMF compared to controls we found the upregulation of several miRNAs associated with hematological malignancies or known as oncomiRs (i.e. hsa-miR-155-5p [S. Jiang et al., Cancer Res, 2010], miRNAs belonging to the miR-17-92 cluster [L. Venturini et al., Blood, 2007]), and other aberrantly expressed miRNAs never described in hematopoiesis (i.e. hsa-miR-335-5p). Then, in order to construct regulatory networks of the functional human miRNA-target interactions, we performed an integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the miRNA expression profile with computational predicted targets and with the gene expression data. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. Of note, IA identified an interaction network (see Figure) in which the upregulated oncomirs miR-155-5p [R.M. O'Connel et al., J Exp Med, 2008], miR29a-3p [Y.C. Han et al., J Exp Med, 2010] and miR-19b-3p [K.J. Mavrakis et al., Nat Cell Biol, 2010] could explain the downregulation of targets whose lower expression was already described as involved in myeloproliferative phenotypes, such as NR4A3, CDC42, HMGB3. Additionally, IA disclosed the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of chronic myeloid malignancies, as a shared target of several upregulated miRNAs in PMF samples (i.e. miR-155-5p, miR-152-3p). Noteworthy, these miRNA-mRNA interactions were functionally confirmed by 3' UTR luciferase reporter assays. Next, in order to characterize the role of JARID2 in PMF pathogenesis, we performed RNAi-mediated gene silencing experiments on CD34+ cells of healthy donor. Interestingly, inhibition of JARID2 expression produces in silenced cells a significant increase of CD41 expression when compared with control (28.6±3.1% vs 15.3±1.8% at day 8, 52.6±7.6% vs 35.4±4.9% at day 12 of serum free liquid culture) and a remarkable increase in CFU-MK colonies (59.6±6.5% vs 39.8±5.9%). The values are reported as mean ± 2S.E.M from five independent experiments. Moreover, morphological analysis after May-Grunwald-Giemsa staining showed that JARID2 silencing induces in normal CD34+ cells a considerable enrichment in MK precursors at different stages of maturation. This study allowed the identification of different networks possibly involved in PMF onset, highlighting the potential contribution of miRNAs to PMF pathogenesis. Furthermore, for the first time, we demonstrated that the JARID2 downregulation in CD34+ cells might contribute to the abnormal megakaryopoiesis typical of PMF. Disclosures: Rambaldi: Novartis: Honoraria; Sanofi: Honoraria; Italfarmaco: Honoraria.

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Necdin, a p53 target gene, regulates the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells

Blood ◽

10.1182/blood-2011-11-393983 ◽

2012 ◽

Vol 120 (8) ◽

pp. 1601-1612 ◽

Cited By ~ 34

Author(s):

Takashi Asai ◽

Yan Liu ◽

Silvana Di Giandomenico ◽

Narae Bae ◽

Delphine Ndiaye-Lobry ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Progenitor Cells ◽

Target Gene ◽

Critical Role ◽

Genotoxic Stress ◽

Dependent Manner ◽

Hematopoietic Stem ◽

P53 Target Gene

Abstract We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle–dependent and cell-cycle–independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.

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