Csf1r Is a Downstream Target of C/EBPβ in Ly6C¯ Monocytes

Abstract Currently, monocytes are classified into at least two subsets. Classical monocytes, also known as inflammatory monocytes (a Ly6C+ subset in mice and a CD14+ CD16− subset in human), are involved in innate immune responses. On the other hand, patrolling monocytes (a Ly6C− subset in mice and a CD14− CD16+ subset in human) have been recently identified. Ly6C− monocytes are found attached on the luminal side of endothelium and scavenge microparticles. Developmentally, Ly6C+ and Ly6C− monocytes share common monocyte progenitors (cMoPs), or Ly6C− monocytes might be converted from Ly6C+ monocytes. Although involvement of Ly6C− monocytes in various kinds of diseases has been reported, molecular mechanisms which regulate the homeostasis of Ly6C− monocytes are largely unknown. CCAAT/Enhancer Binding Protein β (C/EBPβ) is a leucine zipper type transcription factor. We and others have previously shown that C/EBPβ is required for stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Satake et al. J Immunol, 2012, Hayashi et al. Leukemia 2013). However, its roles in steady state hematopoiesis remain relatively unknown. We have recently found that peripheral blood monocytes are significantly reduced in Cebpb−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year in this meeting, we have reported that Cebpb mRNA is highly upregulated during differentiation from myeloid progenitors or Ly6C+ monocytes to Ly6C− monocytes, and that Ly6C− monocytes are almost completely absent in Cebpb−/− mice due to enhanced cell death [Abstract #224]. Here, we further investigated the molecular mechanisms underlying C/EBPβ-dependent survival of Ly6C− monocytes. In this study, we focused on the regulation of Csf1r (also known as M-CSF receptor). Csf1r is an essential molecule for the development and survival of monocytes. To determine the developmental stages at which Csf1r plays critical roles, we measured the expressions of Csf1r mRNA in hematopoietic stem/progenitor cells and monocyte subsets obtained from wild-type (WT) mice. Csf1r mRNA was expressed at at low levels in hematopoietic stem/progenitors including macrophage dendritic precursors (MDPs) and cMoPs. Csf1r mRNA started to be upregulated in Ly6C+ monocytes, followed by a drastic increase in Ly6C− monocytes. These expression patterns were quite similar to those of Cebpb, suggesting the close relationship between Csf1r and C/EBPβ. Interestingly, such drastic increase of Csf1r mRNA in Ly6C− monocytes was blunted in Cebpb−/− mice, and protein levels of Csf1r in Cebpb−/− Ly6C− monocytes were significantly lower than those in WT Ly6C− monocytes. In order to evaluate the effect of C/EBPβ overexpression on Csf1r expression, EML cells, a mouse hematopoietic stem cell line, were engineered to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significantly increased levels of Csf1r mRNA and protein when compared to nuclear translocation of ER alone. Previous reports have demonstrated that a combination of a promoter sequence and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) is enough to recapitulate the endogenous Csf1r expression and that these elements contained consensus binding sites for C/EBP transcription factors. Then, we hypothesized that C/EBPβ binds to these sites, activates transcription of Csf1r gene and promotes survival of Ly6C- monocytes. To evaluate this hypothesis, we utilized an expression vector, in which green fluorescent protein (GFP) is driven by a combination of the Csf1r promoter and FIRE sequences (Csf1r-EGFP-FIRE) (a kind gift from Drs Clare P and David A Hume, University of Edinburgh). When a C/EBPβexpression vector was co-transfected with the vector containing Csf1r-EGFP-FIRE into HEK293 cells, the frequencies of GFP positive cells were significantly higher when compared to a control vector (C/EBPβ vs control; 4.6±0.6 vs 1.6±1.0, p=0.01), suggesting that C/EBPβ regulates Csf1r expression through these elements. We are currently evaluating the significance of C/EBP consensus binding sites in the promoter and the enhancer. ChIP PCR is also in progress to further verify our hypothesis. Collectively, these results suggest that Csf1r is a critical downstream target of C/EBPβ in Ly6C- monocytes. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Essential Roles of C/EBPβ in Survival of Ly6C– monocytes

Blood ◽

10.1182/blood.v124.21.224.224 ◽

2014 ◽

Vol 124 (21) ◽

pp. 224-224

Author(s):

Akihiro Tamura ◽

Hideyo Hirai ◽

Asumi Yokota ◽

Atsushi Sato ◽

Hisayuki Yao ◽

...

Keyword(s):

Peripheral Blood ◽

Leucine Zipper ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Cell Types ◽

Hematopoietic Stem ◽

Retroviral Transduction ◽

Inflammatory Monocytes ◽

And Function ◽

Myeloid Progenitors

Abstract Accumulating evidences have shown that mouse monocytes can be divided into two subsets, based on the expression of a surface marker Ly6C. Although distinct functions of Ly6C+ monocytes (also called classical or inflammatory monocytes) and Ly6C– monocytes (also known as patrolling monocytes) have been gradually uncovered, molecular mechanisms which govern development of these monocytes remain largely unknown. We have previously reported the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, for ‘emergency’ granulopoiesis (Nat Immunol, 2006, J Immunol, 2012, Leukemia 2013). C/EBPβ is also known to play roles in the differentiation and function of macrophages. However, involvement of C/EBPβ in monocyte development has not been fully investigated. The aim of this study is to elucidate the roles of C/EBPβ in monopoiesis. First, we measured C/EBPβ mRNA expression in purified hematopoietic stem cells, myeloid progenitors and monocyte subsets, and found that monocytes, especially Ly6C– monocytes, expressed C/EBPβ mRNA at extremely higher level than any of other cell types examined. When we analyzed peripheral blood, the frequencies of total monocytes (CD11b+ CD115+ cells) in C/EBPβ–/– mice was significantly lower than those in wild type (WT) mice (4.24±2.71% in WT mice vs. 0.72±0.50% in C/EBPβ–/– mice, p<0.001). Of note, Ly6C– monocytes were almost absent in peripheral blood of C/EBPβ–/– mice (0.67±0.57% in WT mice vs. 0.017±0.021% in C/EBPβ–/– mice). In order to clarify whether the defects in C/EBPβ–/– monopoiesis were cell-intrinsic or cell-extrinsic, we generated mixed bone marrow (BM) chimeras by reconstituting lethally irradiated mice (CD45.1+) with BM cells from WT (CD45.1+) mice together with the equal number of BM cells from either WT or C/EBPβ–/– (CD45.2+) mice. Six weeks after reconstitution, we confirmed that C/EBPβ–/– BM-derived Ly6C– monocytes were absent in peripheral blood of the recipient mice, suggesting that monopoiesis in C/EBPβ–/– mice is impaired in a cell-intrinsic manner. A recent report revealed that MX1-Cre transgenic system can be used for monocyte specific deletion of genes of interest, as MX1 is highly expressed by monocytes (Hashimoto D et al. Immunity 2013). In MX1-Cre+ C/EBPβfloxed/floxed mice, the number of monocytes were decreased to the level similar to C/EBPβ–/– mice, suggesting that C/EBPβ is specifically required in monocytes rather than other progenitors during monopoiesis. As cell cycle status of myeloid progenitors and monocytes did not differ between WT mice and C/EBPβ–/– mice, we evaluated apoptosis by flow cytometry. The frequencies of late apoptotic/dead cells within Ly6C– monocytes in peripheral blood of C/EBPβ–/– mice were significantly higher than those in peripheral blood of WT mice (5.84±2.90% in WT mice vs. 50.4±22.4% in C/EBPβ–/– mice, p<0.001). These enhanced apoptosis of C/EBPβ–/– Ly6C– monocyte was partially reversed by retroviral transduction of Bcl2 gene. Previous reports have shown that Nr4a1, CX3CR1 and S1PR5 are required for survival or BM egress of Ly6C– monocytes. We found that mRNA expressions of these factors are severely reduced in C/EBPβ–/– Ly6C– monocytes. These results suggested that C/EBPβ maintains survival of Ly6C– monocytes through direct or indirect association of these molecules. Collectively, our data strongly indicate that C/EBPβ is essential for survival of Ly6C– monocytes. We are currently investigating the molecular mechanisms involved in the enhanced apoptosis of of Ly6C– monocytes in C/EBPβ–/– mice. Disclosures No relevant conflicts of interest to declare.

Download Full-text

C/EBPβ Is Required for Survival of Ly6C- Monocytes after Committment to Monocyte Lineage through Upregulation of Csf1r

Blood ◽

10.1182/blood.v128.22.1325.1325 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1325-1325

Author(s):

Akihiro Tamura ◽

Hideyo Hirai ◽

Asumi Yokota ◽

Naoka Kamio ◽

Atsushi Sato ◽

...

Keyword(s):

Binding Sites ◽

Research Funding ◽

Leucine Zipper ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Regulatory Element ◽

Regulatory Elements ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Reporter Activity

Abstract Monopoiesis is the process in which hematopoietic stem cells (HSCs) continuously give rise to monocytes. Accumulating evidence has identified cellular constituents of monopoiesis. Common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), macrophage-dendritic cell precursors (MDPs) and common monocyte progenitors (cMoPs) are the intermediates during the differentiation of HSCs into mature monocytes. In mice, CD11b+ CD115+ monocytes are further divided into two subsets based on the expression of Ly6C. Classical monocytes express Ly6C on their surface. By contrast, Ly6C− patrolling monocytes have been recently identified, and the molecular mechanisms which regulate the development and homeostasis of Ly6C−monocytes still remain elusive. C/EBPβ is a leucine zipper transcription factor which regulates stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Hayashi et al. Leukemia 2013). We have recently found that peripheral blood (PB) monocytes are significantly reduced in steady-state Cebpb−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year at this meeting, we have reported that cell death of Ly6C− monocytes was accelerated through reduced expression of Csf1r (encoding a receptor for M-CSF) in Cebpb−/− mice. Here in this study, we determined the precise developmental stage where C/EBPβ is mandatory for survival of Ly6C− monocytes, and investigated the mechanism of Csf1r regulation by C/EBPβ. A recent publication demonstrated that Mx1 is preferentially expressed by monocytes and a Mx1 promoter-mediated conditional system targets monocytes without inoculation of polyI:C (Hashimoto et al. Immunity, 2013), suggesting that Mx1-Cre Cebpbf/f mouse is ideal to evaluate the monocyte-specific requirement for C/EBPβ. We confirmed that upregulation of Cebpb mRNA during monopoiesis was significantly impaired after cMoP stage in Mx1-Cre+Cebpbf/f mice. In order to exclude the possible involvement of Cebpβ deficient microenvironment, bone marrow (BM) cells of Mx1-Cre+Cebpβf/f mice (CD45.2+) were transplanted into lethally irradiated CD45.1+ wild type mice. The frequencies of Ly6C− monocytes in the recipients of Mx1-Cre+Cebpbf/f BM cells were significantly reduced when compared to mice that received Mx1-Cre−Cebpbf/f BM cells (Figure). These results strongly suggest that C/EBPβ is specifically required after commitment to monocytes. In order to investigate the molecular mechanisms involved in the regulation of Csf1r by C/EBPβ, we utilized a combination of a promoter and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) for reporter assay (Pridans et al. Mol Ther Methods Clin Dev, 2014). These regulatory elements contain at least 2 consensus binding sites for C/EBPβ (one in the promoter and the other in the enhancer). C/EBPβ significantly enhanced the reporter activity of the regulatory elements in a dose-dependent manner, and introduction of mutations into either of the consensus binding sites abrogated the reporter activity. Next, we engineered EML cells, a mouse HSC line, to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significant increase of Csf1r mRNA and protein. Using these cells, we performed chromatin immunoprecipitation PCR. Upon treatment with tamoxifen, significant enrichment of C/EBPβ at the promoter region and the FIRE region was observed. These data indicated that C/EBPβ regulates Csf1r through direct binding to these regulatory elements. Collectively, these results demonstrate that C/EBPβ supports survival of Ly6C− monocytes after commitment to monocyte lineage through direct regulation of Csf1r, which is critical for survival and differentiation of monocytes. Figure Figure. Disclosures Hirai: Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

Download Full-text

C/EBPβ Isoforms Regulate Proliferation and Differentiation of Regenerating Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood-2019-128026 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3713-3713

Author(s):

Atsushi Sato ◽

Asumi Yokota ◽

Yoshihiro Hayashi ◽

Naoka Kamio ◽

Satoshi Sagai ◽

...

Keyword(s):

Cell Cycle ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Flow Cytometric Analysis ◽

Myeloid Cells ◽

Stress Conditions ◽

Hematopoietic Stem ◽

N Terminus ◽

Factor C ◽

The Impact

Under stress or regenerative conditions, HSCs rapidly enter into cell cycle and are reprogrammed toward myeloid-biased hematopoiesis to meet the increasing demand of myeloid cells. We have previously shown that the transcription factor C/EBPβ plays critical roles at the level of HSPCs under stress conditions (Nat Immunol 2006, J Immunol 2012, Leukemia 2013 and Blood Adv 2019). However, underlying molecular mechanisms of action remain largely unknown. In this study, we have investigated the detailed function of C/EBPβ in regulation of HSPCs. We first evaluated the impact of C/EBPβ on the cell cycle status of LT-HSCs. To exclude the cell-extrinsic contribution of C/EBPβ, CD45.2+ BM cells from WT or Cebpb-/- mice were transplanted into lethally irradiated CD45.1+ WT mice, and these "BM-replaced" recipients were subjected to the following experiments. At steady state, the cell cycle statuses and the numbers of HSPCs did not significantly differ between the recipients of WT cells and those of Cebpb-/- cells. Immediately after 5-FU treatment, WT LT-HSCs entered the cell cycle, as revealed by the decreased percentage of cells in G0 phase and the increased percentage of cells in S/G2M phase. All these parameters of cell cycle acceleration were observed prior to the nadir of LT-HSCs induced by 5-FU and were significantly attenuated in Cebpb-/- LT-HSCs. Next, we assessed the numbers of LT-HSCs, KSL cells, and KL cells after 5-FU treatment. Following the nadir, the recovery of LT-HSCs preceded that of KSL and KL cells, suggesting the differentiation of LT-HSCs to KSL and KL cells. In the recipients of Cebpb-/- cells, the recovery of KSL and KL cells was delayed significantly. Collectively, cell cycle acceleration and subsequent differentiation of LT-HSCs under stress conditions were impaired in the absence of Cebpb. The Cebpb is a single exon gene, and three isoforms, namely, LAP*, LAP and LIP which lacks N-terminus, are translated from its unique mRNA. Due to their structural difference, they should have distinct functions. Here, we focused on expression and functions of these isoforms in regenerating HSPCs. To monitor expression of these isoforms in small numbers of HSCs, we devised a novel intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies. An antibody against the C-terminus of C/EBPβ recognized all three isoforms, while an antibody against the N-terminus of C/EBPβ only recognized LAP* and LAP. Thus, simultaneous staining with both antibodies should enable us to distinguish cells that dominantly expressed LIP (C-term+ N-term-) from those that expressed all three isoforms (C-term+ N-term+). Using this method, we monitored the expression patterns of these isoforms in LT-HSCs after 5-FU treatment. LT-HSCs initially became C-term single positive in response to 5-FU and subsequently changed to C- and N-term double positive, suggesting that LIP was upregulated prior to LAP/LAP* under stress conditions. These results suggest that phase-specific upregulation of LIP and LAP/LAP* is strongly associated with phase-specific functions of C/EBPβ in cell cycle activation and differentiation, respectively. Indeed, when EML cells, a mouse HSC line, were retrovirally transduced with LIP, the transduced cells were more proliferative and actively cycling than those transduced with the control vector, whereas proliferation and cell cycle were markedly suppressed in LAP*- and LAP-expressing EML cells. LIP-expressing cells remained undifferentiated, while LAP*- and LAP-expressing cells rapidly differentiated into CD11b+ myeloid cells and eventually stopped proliferating. In summary, our findings clearly suggest that sequential upregulation of C/EBPβ isoforms is critical for the regulation of HSCs under stress conditions. LIP amplifies the "reservoir" of HSPCs by accelerating the proliferation of HSCs during the early phase of regeneration, while LAP*/LAP induce their myeloid differentiation at a later phase. These findings should facilitate our understanding of the pathophysiology of infection, inflammation, and regenerating hematopoiesis in response to myeloablative chemotherapies or hematopoietic stem cell transplantation, all of which increase the hematopoietic demands. Disclosures Hirai: Kyowa Kirin: Research Funding.

Download Full-text

Promoter Methylation and Decreased Expression of Mir-124 In Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.4014.4014 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4014-4014

Author(s):

Yuesheng Meng ◽

Qiao Xia ◽

Jun Hu

Keyword(s):

Myelodysplastic Syndromes ◽

Promoter Methylation ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Quantitative Polymerase Chain Reaction ◽

Methylation Status ◽

Polymerase Chain Reaction Analysis ◽

Promoter Regions ◽

Hematopoietic Stem

Abstract Abstract 4014 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders of hematopoietic stem/progenitor cells. Although demethylating agents such azacytidine and decitabine have been widely used to treat MDS, the underlying molecular mechanisms remain obscure. Abnormalities of microRNAs (miRNA) have been recently associated with hematological malignancies including MDS. The miR-124 was initially demonstrated to modulate neurogenesis. It was recently shown that EVI1-induced methylation and silencing of miR-124 were present in murine MDS cells. In the retrospective study we evaluated methylation status and expression levels of miR-124 in fifteen MDS patients (subtypes included RCUD, RCMD, RAEB-1, RAEB-2 and CMML). Genomic DNA samples were modified with bisulfite and methylation at three promoter regions of miR-124 was examined with methylation-specific real time quantitative polymerase chain reaction analysis (MQPCR). In general, we observed an increased methylation levels of miR-124 in MDS patients than that in normal bone marrow (NBM, P<0.01). In accordance with this, marked depression of miR-124 was seen in six patients when compared with NBM (more than 2 times lower), as determined with quantitative reverse-transcriptive PCR assay. Moreover, there were higher degrees of promoter methylation in cases with depressed miR-124 than that in remaining cases. A negative correlation between the expression and methylation levels was statistically significant (R= -0.498, P<0.01). The change of miR-124 was not directly related to short-term clinical response or prognosis, possibly due to limited size of the sample. However, the miR-124 amount returned to basal levels in two cases (RCMD and CMML subtypes respectively) after low-dose decitabine therapy and DNA methylation of all three loci disappeared. Continued work is underway to accumulate more cases and make long-term clinical follow-up. In conclusion, this primary work suggested a possible role of the methylation-mediated silencing of miR-124 in the pathogenesis or disease progression of MDS. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽

10.1182/blood.v118.21.3461.3461 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3461-3461

Author(s):

Beiyan Zhou

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Fusion Genes ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Effect of Lenalidomide Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients

Blood ◽

10.1182/blood.v118.21.4635.4635 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4635-4635

Author(s):

Matilde Y Follo ◽

Cristina Clissa ◽

Sara Mongiorgi ◽

Francesca Chiarini ◽

Michele Baccarani ◽

...

Keyword(s):

Supportive Care ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Best Supportive Care ◽

Low Risk ◽

Cyclin D3 ◽

Signalling Pathways ◽

Globin Genes ◽

Downstream Target ◽

Lipid Signalling

Abstract Abstract 4635 Nuclear inositide signalling pathways are involved in the MDS progression to AML. Indeed, in the last few years our group demonstrated not only that MDS cells can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Lenalidomide has proven effectiveness in 70–80% low-risk MDS cases with del(5q), resulting in transfusion-independence with a rise in hemoglobin levels, suppression of the 5q clone, and improvement of bone marrow morphologic features. In particular, in del(5q) MDS, Lenalidomide probably acts by directly suppressing the dysplastic clone, while in non-del(5q) it might enhance an effective erythropoiesis, possibly via activation of the EPO signalling, which in turn is associated with PI-PLCgamma1 pathways. However, the exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still not completely clarified. Interestingly, Lenalidomide might inhibit the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS cells, which display a marked apoptosis and a low proliferation rate. In this study we examined four MDS patients treated with Lenalidomide, and compared them with four low-risk MDS patients (IPSS: Low or Int-1) who only received best supportive care. In our study, all of the patients treated with Lenalidomide were affected by del(5q) low-risk MDS (IPSS: Low or Int-1), with transfusion-dependent anemia, and had only received supportive care before undergoing Lenalidomide treatment. Clinically, all of the patients responded to Lenalidomide: one patient reached Complete Remission, whilst the other three patients showed erythroid Hematologic Improvement. In contrast, all of the patients who were treated only with best supportive care maintained a Stable Disease. As for the molecular effects of Lenalidomide on lipid signalling pathways, we analyzed the expression of critical molecules involved in both cell proliferation and differentiation, that is PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling. Ongoing analyses are also trying to examine the effect of Lenalidomide on Akt phosphorylation and Globin genes, which are specifically associated with erythropoiesis. So far, our results indicate that, in our responder patients, both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Lenalidomide, whereas PI-PLCgamma1 is specifically induced, as compared with both healthy subjects and low-risk MDS patients treated with supportive care. Overall, these findings hint at a specific activation of PI-PLCgamma1 signalling following Lenalidomide treatment, and possibly pave the way to larger investigations aiming to better understand the role of these pathways in the mechanism of action of Lenalidomide in del(5q) MDS. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

BCR/ABL-Mediated Myeloid Expansion Is Promoted by C/EBPβ, a Regulator of Emergency Granulopoiesis,

Blood ◽

10.1182/blood.v118.21.3747.3747 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3747-3747

Author(s):

Yoshihiro Hayashi ◽

Hideyo Hirai ◽

Hisayuki Yao ◽

Satoshi Yoshioka ◽

Sakiko Satake ◽

...

Keyword(s):

Median Survival ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Myeloid Cells ◽

Expression Level ◽

Hematopoietic Stem ◽

Emergency Situations ◽

Proliferation And Differentiation ◽

Myeloid Progenitors

Abstract Abstract 3747 Chronic phase chronic myeloid leukemia (CP-CML) is characterized by the increase of myeloid cells in the peripheral blood (PB) and bone marrow (BM). We have previously shown that the C/EBPβ transcription factor is required for emergency granulopoiesis, increased proliferation and differentiation of granulocytic precursors in emergency situations such as infection (Hirai H et al., Nature Immunol. 2006). Enhanced myelopoiesis is a common feature between emergency situations and CP-CML. However, little is known about the roles of C/EBPβ in the pathogenesis of CP-CML. The aim of this study is to elucidate the regulation and function of C/EBPβ in BCR/ABL-mediated myeloid expansion. We first assessed the expression level of C/EBPβ in hematopoietic stem cells and myeloid progenitors in BM obtained from healthy donors or CP-CML patients. The transcript of C/EBPβ is expressed at significantly higher level in common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in CP-CML BM than those in normal BM. When BCR/ABL was retrovirally transduced into a mouse hematopoietic stem cell line, EML, C/EBPβ expression was significantly upregulated. Treatment of the EML-BCR/ABL cells with imatinib mesylate normalized the expression level of C/EBPβ. These data suggested that C/EBPβ was upregulated in response to the downstream signaling of BCR/ABL. In order to investigate the function of C/EBPβ in BCR/ABL-mediated myeloid expansion, BCR/ABL was retrovirally introduced into BM cells obtained from 5-FU treated C/EBPβ knockout (KO) mice and their properties were compared with those of BCR/ABL-transduced BM cells from wild type (WT) mice. When the transduced cells were cultured in cytokine-free semisolid methylcellulose medium, the number and the size of the colonies of C/EBPβ KO cells were significantly smaller. Flow cytometric analysis of the colony-forming cells revealed that the BCR/ABL-transduced C/EBPβ KO BM cells gave rise to higher frequency of c-kit+ cells and lower CD11b+ cells than BCR/ABL-transduced WT BM cells (%c-kit+ cells=8.2±3.0% vs. 11.3±3.5%, p=0.002, %CD11b+ cells=75.1±2.1% vs. 90.0±4.2%, p=0.003). In addition, BCR/ABL-transduced C/EBPβ KO BM cells revealed higher replating efficiency than BCR/ABL-transduced WT BM cells. To investigate the role of C/EBPβ in leukemogenesis, BCR/ABL-transduced BM cells from C/EBPβ KO mice or WT mice were transplanted into lethally irradiated recipient mice. In mice transplanted with BCR/ABL-transduced C/EBPβ KO cells, the increase of white blood cell count was delayed (Figure) and higher frequency of c-kit+ cells were observed in the BM at day 19 post transplantation (16.0±2.6% vs. 5.5±4.6%, p=0.01). Spleen size of mice transplanted with BCR/ABL-transduced WT cells is much larger than that of BCR/ABL-transduced C/EBPβ KO cells (Figure). The median survival of mice transplanted with BCR/ABL-transduced WT cells was 19 days. In contrast, the median survival of mice transplanted with BCR/ABL-transduced C/EBPβ KO cells was 31 days (p=0.0005). In summary, C/EBPβ is upregulated by BCR/ABL and the absence of C/EBPβ resulted in delayed proliferation and differentiation of myeloid cells both in vitro and in vivo. Our results suggest that C/EBPβ is involved in the BCR/ABL-mediated myeloid expansion in CP-CML and that C/EBPβ can be the novel molecular target for the therapy of CML. We are currently investigating the molecular mechanisms which mediate the upregulation of C/EBPβ and the direct targets of C/EBPβ in CP-CML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

Blood ◽

10.1182/blood.v120.21.864.864 ◽

2012 ◽

Vol 120 (21) ◽

pp. 864-864

Author(s):

Dewen You ◽

Andrew Volk ◽

Clare Sun ◽

Junping Xin ◽

Geunhyoung Ha ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Phosphatase Activity ◽

Focal Adhesion ◽

Protein Phosphatase ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Critical Role ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Polycytidylic Acid

Abstract Abstract 864 Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten's lipid phosphatase activity. However, the role of Pten's protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied. Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

AK2 Deficiency In Zebrafish Recapitulates Human Reticular Dysgenesis, An Autosomal Recessive Form Of Severe Combined Immunodeficiency

Blood ◽

10.1182/blood.v122.21.2416.2416 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2416-2416

Author(s):

Alberto Rissone ◽

Jaya Jagadeesh ◽

Karen Simon ◽

Kevin Bishop ◽

Raman B. Sood ◽

...

Keyword(s):

Autosomal Recessive ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Severe Combined Immunodeficiency ◽

Combined Immunodeficiency ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Autosomal Recessive Form ◽

Recessive Form ◽

Mutant Lines

Abstract The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP+AMP to 2ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues. Mutations in the AK2 gene are responsible for reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID). RD is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation and it represents less than 2% of total SCID. Affected children succumb to overwhelming infections early in life unless their immune system is successfully restored with allogeneic hematopoietic stem cells transplant (HSCT). The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has never been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). We used the zebrafish model to perform a comprehensive study of the effects of AK2 deficiency using Morpholino oligomers injections and two different kinds of AK2 mutants (a ENU-induced T371C/L124P missense mutant and two null mutant lines generated using zinc-finger nuclease technology). In situ hybridization analyses of AK2-deficient embryos indicated that only erythroid development was affected during primitive hematopoiesis. Conversely, during definitive hematopoiesis, the loss of function of AK2 resulted in abnormalities distributed along all hematopoietic lineages suggesting an impairment of hematopoietic stem cell (HSC) development. Moreover, we observed that the AK2 deficiency induced oxidative stress and consequent apoptosis in both primitive erythroid cells and definitive HSCs. Importantly, antioxidant treatment of AK2 mutant embryos rescued the hematopoietic phenotypes as indicated by the recovered expression of HSC and lymphoid markers (such as c-myb and rag1). Overall, our data indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD and testing of new therapeutic interventions. To date, our mutant lines remain the only animal model of this rare and lethal human disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3764.3764 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3764-3764

Author(s):

Patali S Cheruku ◽

Marina Bousquet ◽

Guoqing Zhang ◽

Guangtao Ge ◽

Wei Ying ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Colony Formation ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text