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  • American Society of Hematology  (7)
  • 2010-2014  (7)
  • Medicine  (7)
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  • American Society of Hematology  (7)
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  • 2010-2014  (7)
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  • Medicine  (7)
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  • 1
    Online Resource
    Online Resource
    Cell-Intrinsic and Cell-Extrinsic Involvement Of C/EBPβ In The Regulation Of Hematopoietic Stem Cells
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1202-1202
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1202-1202
    Abstract: Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, in emergency granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPβ is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem/progenitor cells (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPβ is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and leukemic stem cells exhaustion in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPβ plays important roles in normal hematopoietic stem cells (HSCs). Here we investigated the cell-intrinsic and -extrinsic function of C/EBPβ in the regulation of HSCs by analyzing C/EBPβ knockout (KO) mice. At steady state, no obvious defects have been reported in hematopoiesis of C/EBPβ KO mice. Accordingly, the frequencies of long-term and short-term HSCs and various kinds of progenitor cells in bone marrows (BM) of C/EBPβ KO mice were identical to those in BM of wild type (WT) mice. To examine the functional consequences of C/EBPβ deletion, competitive repopulation assay was performed. In brief, 5x105 BM cells from WT or C/EBPβ KO mice (CD45.2+) and the same number of competitor CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the chimerisms of CD45.2+ cells in the peripheral blood of the recipient mice were monitored monthly. The chimerisms of C/EBPβ KO cells were significantly lower than that of WT cell at 1 month after transplantation and the differences were maintained thereafter (Figure A). In order to elucidate the reason for the difference, homing ability of C/EBPβ KO cells were assessed. Lineage depleted CD45.2+ WT or C/EBPβ KO BM cells together with the equal number of lineage negative CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the frequencies of CD45.2+ cells were analyzed 16 hours after transplantation. The frequencies of CD45.2+ WT and C/EBPβ KO donor cells in the recipient BMs were identical and the data indicated that the differences in the chimerisms after primary BM transplantation were due to the difference in the initial expansion of transplanted cells after equivalent levels of homing. To see the roles of C/EBPβ in hematopoiesis under stressed conditions, CD45.1+ mice were transplanted with CD45.2+ WT or C/EBPβ KO BM cells with equal numbers of CD45.1+ BM cells and these mice were administered with 150mg/kg 5-fluorouracil (5-FU) once a month and the chimerisms of peripheral blood were monitored every time before the next 5-FU administration. In consistent with the results mentioned above, the frequencies of CD45.2+ C/EBPβ KO cells were significantly lower than those of CD45.2+ WT cells 1 month after transplantation. After repetitive administration of 5-FU, however, the chimerisms of CD45.2+ C/EBPβ KO cells gradually caught up with those of CD45.2+ WT cells, suggesting that C/EBPβ is involved in the exhaustion of HSCs under stressed conditions (Figure B). To explore the functions of C/EBPβ in hematopoietic microenvironments, 1x106 CD45.1+ BM cells from WT mice were transplanted into irradiated (5Gy or 7Gy) WT or C/EBPβ KO mice (CD45.2+). All the WT recipient mice survived after 5Gy or 7Gy irradiation (4/4 and 4/4, respectively). In contrast, only 2/4 and 1/4 C/EBPβ KO recipient mice survived after 5Gy or 7Gy irradiation, respectively. We are currently trying to identify the cells expressing C/EBPβ in BM microenvironments and investigating the mechanisms for the higher sensitivity of C/EBPβ KO mice to irradiation. In summary, these data suggested that C/EBPβ is required for initial expansion of hematopoietic stem/progenitor cells at the expense of HSCs under stressed conditions, while it is dispensable for maintenance of HSCs at steady state. We are now investigating the cellular and molecular targets of C/EBPβ in HSC regulation and would like to elucidate the cell-intrinsic and cell-extrinsic mechanisms in regulation of the homeostasis of hematopoietic system by C/EBPβ. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1202.1202
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
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  • 2
    Online Resource
    Online Resource
    Expression of C/EBPβ in Bone Marrow Mesenchymal Stem Cells Is Mandatory for Early-Stage B Cell Lymphopoiesis
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 3457-3457
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3457-3457
    Abstract: Abstract 3457 The interplay between hematopoietic cells and bone marrow microenvironment organized by mesenchymal stem cells is important for the maintenance of hematopoiesis. With respect to B cell lymphopoiesis, several constituents of bone marrow microenvironment specific for B cells (“B cell niche”) have been identified, including CXCL12/stromal cell-derived factor-1 (SDF-1)-abundant reticular cells as cellular factors, and CXCL12/SDF-1, interleukin (IL)-7, stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt3-L), and nuclear factor kappa-B ligand (RANKL), as essential humoral factors. However, the precise mechanism through which mesenchymal stem cells in the bone marrow microenvironment support B cell lymphopoiesis, especially the role of transcription factors, remains unknown. We show that the mesenchymal stem cells lacking a transcription factor, CCAAT enhancer binding protein (C/EBP) b, are functionally abnormal, which contribute to the impairment of B cell lymphopoiesis in C/EBPb knockout mice. In C/EBPb knockout mice, the number of B cells, in particular, B220+CD43+ precursor B cells, was significantly decreased in bone marrow compared with that in wild-type littermates (Figure 1A and 1B). As shown in Fig. 1A, the percentage of total B220+ B cells was decreased at 19.1 ± 7.1% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 26.5 ± 7.3%: *P 〈 0.05). The percentage of B220+CD43+ precursor B cells was also decreased at 5.2 ± 1.5% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 7.4 ± 1.6%: **P 〈 0.01). Intriguingly, in vivo bone marrow transplantation experiments demonstrated that the bone marrow cells derived from C/EBPb knockout mice were engrafted in lethally-irradiated (10 Gy) wild-type mice with equivalently B cell recovery compared to the bone marrow cells from normal wild-type mice. Conversely, when normal wild-type c-kit+ Sca-1+ lineages− hematopoietic stem cells (KSL cells) were co-cultured with C/EBPb deficient mesenchymal stem cells in vitro (KO), they showed impaired B cell differentiation compared to the co-culture with normal wild-type mesenchymal stem cells (WT, Figure 1C). Mechanistically, the CXCL12/SDF-1 production by C/EBPb deficient mesenchymal stem cells was reduced compared with that by wild-type mesenchymal stem cells (KO, n = 5, 4.47 ± 1.16 ng/mL; WT, n = 5, 9.90 ± 1.93 ng/mL; **P 〈 0.01). These results suggest a possibility that abnormal C/EBPƒÀ deficient mesenchymal stem cells in bone marrow microenvironment contribute to impaired B cell lymphopoiesis in C/EBPb knockout mice. We further found that C/EBPb deficient mesenchymal stem cells displayed several functional abnormalities. First, calcium accumulation was significantly reduced in 4 week osteogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells compared to cultures of wild-type mesenchymal stem cells. This occurred along with the down-regulated expression of the principal osteogenic master molecule runt-related transcription factor 2 (Runx2). Second, lipid deposition was significantly reduced in 1 week adipogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells. The expression of adipogenic markers, including peroxisome proliferator-activated receptor b (PPARb) was significantly reduced in adipogenic cultures of C/EBPb-deficient mesenchymal stem cells compared with cultures of wild-type mesenchymal stem cells Finally, the number of colony-forming unit fibroblast (CFU-F) was higher in the bone marrow of C/EBPb knockout mice than in that of wild-type mice. Collectively, C/EBPb-deficient mesenchymal stem cells have aberrant multi-differentiation capability and increased proliferation activity compared with wild-type mesenchymal stem cells, further supporting that C/EBPb-deficient mesenchymal stem cells were functionally abnormal. Altogether, this work demonstrates that impaired B cell lymphopoiesis in C/EBPb knockout mice is attributed to abnormal mesenchymal stem cells in bone marrow microenvironment, at least in a steady-state, an effect that is due in part to the impaired CXCL12/SDF-1 production. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.3457.3457
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    Online Resource
    Online Resource
    C/EBPβ Promotes Initial Expansion and Exhaustion of Hematopoietic Stem and Progenitor Cells in Response to Hematopoietic Stresses
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 5126-5126
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 5126-5126
    Abstract: Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions. At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state. To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p 〈 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation. In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation. Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress. From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.5126.5126
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
    Online Resource
    Online Resource
    Mesenchymal Stem Cells Skewed Their Phenotype Toward the Osteogenic Lineage Support the Hematopoietic Cell Differentiation
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 3458-3458
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3458-3458
    Abstract: Abstract 3458 The interaction between hematopoietic cells and bone marrow microenvironment is important for the regulation of hematopoiesis. Recent studies have identified the specific bone marrow microenvironment for the hematopoietic stem cells, the “endosteal niche” and the “vascular niche”, in which mesenchymal stem cells and their progenies including osteoblasts are demonstrated to be major cellular constituents. We found that osteogenesis-induced mesenchymal stem cells by the combinations of ascorbic acid, dexamethasone, and potassium dihydrogen phosphate have unique capabilities of both expanding CD34+ hematopoietic progenitor cells and of differentiating CD34+ hematopoietic progenitor cells into mature cells. The osteogenesis-induction was achieved by treating human MSCs with ascorbic acid, dexamethasone, and potassium dihydrogen phosphate (osteogenesis-inducing cocktails: OICS). When human MSCs were osteogenesis-induced by OICS for 3 weeks, MSCs differentiated into mature osteoblasts with abundant calcium accumulation assessed by Alizarin red S staining. However, when human MSCs were treated with OICS for short periods, they did not show apparent calcium accumulation but expressed early-stage osteogenic marker, osterix, and maintain differentiation capability to adipocytes. Intriguingly, purified CD34+ hematopoietic cells (5.0×103 cells/dish) were expanded to the number of 12.6±1.01×104 (#3 of the figure) in 10 day co-cultures with the osterix positive osteogenesis-induced cells in StemSpan Medium (StemCell Technologies) supplemented with 100ng/mL SCF, 100ng/mL Flt-3 ligand, 50ng/mL TPO, and 20ng/mL IL-3. As a control, CD34+ hematopoietic cells was expanded to the number of 6.2±0.4×104 (#2 of the figure) in co-cultures of unstimulated MSCs. Moreover, although the most of the hematopoietic cells expanded on unstimulated MSCs showed an immature blast-like morphology, the hematopoietic cells expanded in the co-cultures with OICS-stimulated MSCs showed a tendency to differentiate into the mature hematopoietic cells, which was supported by the expression of glycophorin-A and CD14 on the hematopoietic cells by FACS analysis. When purified CD34+ hematopoietic cells were co-cultured with OICS-stimulated MSCs in the transwell, the number of expanded CD34+ hematopoietic cells was decreased to 21.7% (#4 of the figure). In contrast, there was no apparent difference in the expression of differentiation markers in the expanded hematopoietic cells between the co-cultures in the presence and in the absence of transwell. Therefore, cell-cell interactions through surface membrane molecules were involved in CD34+ hematopoietic cells expansion mediated by OICS-stimulated MSCs, and soluble factors were mainly involved in the enhancement of hematopoietic differentiation. Real-time PCR analysis showed that the expression of CXCL12 and LIF was reduced in OICS-stimulated MSCs. Given that osteogenic stimulation of MSCs by OICS enhances the expansion and differentiation of CD34+ hematopoietic cells in vitro, we tested the possibility of in vivo administration of OICS to mice receiving bone marrow transplantation after myeloablative conditioning for obtaining quick hematopoietic recover. Lethally irradiated (9Gy) C57BL/6 mice were injected with OICS on day 1–7 after receiving total bone marrow transplantation. The number of leukocytes was decreased to bottom level around 7 days after transplantation in both OICS-treated and non-treated (control) mice. However, the number of leukocyte showed a rapid increase in OICS-treated mice compared with that in control mice. These results suggested that short-term osteogenic stimulation supports the hematopoietic recovery in vivo, probably in part, through acting on MSCs in bone marrow microenvironment. In conclusion, osteogenesis-induced, osterix positive mesenchymal stem cells have unique capabilities to enhance both expansion of CD34+ hematopoietic cells through surface membrane molecules, and differentiation of CD34+ hematopoietic cells into mature cells through soluble factors. This work suggests a possibility that “pharmacological stimulation” of MSCs could modify the bone marrow microenvironment through enhancement of biological potency of MSCs. Further studies are needed whether this strategy may be applied in the clinical settings. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.3458.3458
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 5
    Online Resource
    Online Resource
    C/EBPβ-Mediated Expansion of Hematopoietic Stem/Progenitors Precedes ‘Emergency’ Granulopoiesis Induced by Candidemia.
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 2336-2336
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 2336-2336
    Abstract: Abstract 2336 Granulopoiesis, the process of granulocyte production in the bone marrow (BM), is tightly regulated to meet host demands during both 'steady state' and 'emergency' situations such as infections. The transcription factor CCAAT/Enhancer Binding Protein β (C/EBPβ) plays critical roles in emergency granulopoiesis (Hirai et al. Nat Immunology, 2006). However, the precise developmental stages in which C/EBPβ is required are unknown. In this study, we investigated the roles of C/EBPβ in the proliferation and differentiation of prospectively identified intermediates between hematopoietic stem cells and mature granulocytes in mouse BM. In order to analyze the mouse BM cells undergoing granulopoiesis, novel flow cytometric method was developed. Mouse BM cells retaining the ability to give rise to granulocytes were dissected into five distinct subpopulations (#1–#5) according to their levels of c-kit and Ly-6G expression. Upon infection of Candida albicans (4 × 106 CFU/20 g body weight/mouse) on day 1, C/EBPβ was upregulated at the protein level but not at mRNA level in all the granulopoietic subpopulations, suggesting the importance of the transcription factor in □emergency' granulopoiesis. Then, the role of C/EBPβ was further assessed by analyzing C/EBPβ knockout (KO) mice. At steady state, the distribution of granulopoietic cells in BM of C/EBPβ KO mice at □esteady state' was identical to that of wild type (WT) mice. In contrast, the rapid increase in immature subpopulations #1 and #2 observed in WT mice at 1 day post-infection was significantly attenuated in C/EBPβ KO mice. The levels of mRNA expression for granule proteins (cathepsin G, myeloperoxidase, elastase 2, proteinase 3, lactoferrin and MMP9) within each subpopulation from WT and C/EBPβ KO mice were identical at both steady state and during infection. When the cell cycle status of these models was evaluated using in vivo BrdU labeling experiments, incorporation of BrdU in subpopulation #1 and #2 in C/EBPβ KO mice was always slightly lower than in WT mice, but the differences were not statistically significant. These findings suggest that C/EBPβ is required for efficient proliferation of early granulocytic precursors but not directly involved in the differentiation/maturation process. To elucidate the roles of C/EBPβ in the proliferation of the early granulopoietic subpopulations, the hematopoietic stem cells (HSCs) and myeloid progenitor compartments were analyzed in WT and C/EBPβ KO mice. The frequency and number of c-kit+ Sca-1+ lineage markers− HSC were identical between WT mice and C/EBPβ KO mice during the steady state, and were not significantly affected on day 1 post-infection. Induction of candidemia increased the frequency and number of granulocyte-macrophage progenitors (GMP) in WT mice, and these increases were significantly attenuated in C/EBPβ KO mice. Upon induction of candidemia, the frequency of BrdU-positive cells in the HSC and common myeloid progenitors (CMP) populations from WT mice increased significantly; however, an increase of BrdU-positive cells was observed only within the HSC compartment in C/EBPβ KO mice, and at a lower level than that in WT mice Taken together, these data suggest that the proliferation of early granulocytic precursors is tightly coupled to differentiation/maturation and that C/EBPβ is involved in the efficient amplification of early granulocyte precursors including HSC and myeloid progenitors during candidemia-induced 'emergency' granulopoiesis. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.2336.2336
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
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    Cytokine-STATs Signalings Upregulate C/EBPβ In BCR-ABL+ Leukemic Cells Independently From BCR-ABL/JAK-STAT Pathway
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1468-1468
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1468-1468
    Abstract: Since residual chronic myelogenous leukemia (CML) stem cells may be a cause of relapse after Imatinib (IM) cessation, targeting these IM-resistant stem cells is mandatory for complete cure of this disease. CCAAT/enhancer binding protein β (C/EBPβ), a leucine zipper transcription factor, promotes both cell cycle progression and differentiation toward granulocytes in hematopoietic stem/progenitor cells under stress conditions such as infection. We have recently reported that C/EBPβ was upregulated in leukemic stem/progenitor cells derived from patients in chronic phase of CML (CP-CML) through STAT5, a major downstream target of BCR-ABL. In CML mouse model, C/EBPβ enhanced exhaustion of CML stem cells through promoting their differentiation (Hayashi Y et al, Leukemia 2013). In spite of the upregulation of C/EBPβ by BCR-ABL, CML stem cells will not be exhausted spontaneously in patients with CP-CMP. Therefore, we hypothesized that quiescent CML stem cells maintain their immature status in the bone marrow niche by suppressing C/EBPβ expression or function induced by BCR-ABL and that induction of C/EBPβ expression in CML stem cells through BCR-ABL-independent pathways may be a novel therapeutic strategy targeting CML stem cells. The aim of this study is to propose a novel therapeutic strategy that can stimulate quiescent CML stem cells with cytokine-STATs signalings and induce their exhaustion through C/EBPβ-mediated differentiation. STATs are family members of molecules which convey signals from various kinds of cytokine receptors to nucleus. In order to investigate whether STAT molecules can induce C/EBPβ expression, we first examined the effects of constitutive active (CA) form of STAT1, STAT3 and STAT5 on C/EBPβ expression in a murine hematopoietic stem cell line, EML cells. Retroviral transduction of CA-STAT5 significantly upregulated C/EBPβ mRNA and protein in EML cells. EML cells begun to differentiate toward CD11b+ myeloid lineage upon introduction of CA-STAT5. CA-STAT1 and CA-STAT3 also upregulated C/EBPβ mRNA when they were retrovirally transduced into EML cells (Figure 1). These results suggest that signaling mediated by various kinds of STATs can upregulate C/EBPβ. Consensus binding sites for STATs were not found in the proximal (∼ 4 kb) promoter region of C/EBPβ and we are currently identifying the cis-regulatory elements responsible for the STATs-dependent activation of C/EBPβ.Figure 1Figure 1. Interferon-α (IFNα) exerts STATs-mediated signaling in hematopoietic stem cells and has been used for therapy of CML. Therefore we investigated the possible involvement of C/EBPβ in efficacy of IFNα in CML treatments. Stimulation of EML cells with 500 U/ml IFNα upregulated C/EBPβ mRNA (Figure 2). Higher levels of phosphorylation of STAT1 than those of STAT3 or STAT5 were observed after stimulation with IFNα, suggesting that STAT1 mediated activation of C/EBPβ was induced by IFNα. As previously reported, C/EBPβ was upregulated in EML cells transduced with BCR-ABL (EMLBCR-ABL). Treatment of EMLBCR-ABL cells with IFNα augmented this effect significantly. IM effectively inhibited phosphorylation of STAT5 and blunted the upregulation of C/EBPβ in EMLBCR-ABL cells. Simultaneous treatment of EMLBCR-ABL cells with IFNα and IM resulted in maintained upregulation of C/EBPβ with increased phosphorylation of STAT1 and decreased phosphorylation of STAT5. These data suggested that IFNα treatment can upregulate C/EBPβ independently of signals mediated by BCR-ABL.Figure 2Figure 2. In conclusion, cytokine-STATs signalings can induce C/EBPβ expression in BCR-ABL+ leukemic cells independently from BCR-ABL/JAK-STAT pathway. Stimulations of dormant CML stem cells with cytokines might be a novel treatment strategy to eliminate these populations, leading to complete cure of CML. We are currently evaluating the in vivo effects of IFNα treatment on CML stem cells in mice models. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1468.1468
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
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    C/EBPβ Is Upregulated Through STAT5 and Accelerates Exhaustion of Leukemic Stem Cells During BCR-ABL-Mediated Myeloid Expansion
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 907-907
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 907-907
    Abstract: Abstract 907 Enhanced proliferation and differentiation of myeloid cells are features common to emergency granulopoiesis and chronic phase of chronic myeloid leukemia (CP-CML). We have previously shown that C/EBPβ, a transcription factor regulating emergency granulopoiesis, is upregulated in hematopoietic stem/progenitors in CP-CML and that C/EBPβ promotes BCR-ABL-mediated myeloid cell expansion (ASH annual meeting abstract. 2011; 118: 3747). However, the molecular mechanisms involved in the upregulation of C/EBPβ and the effects of C/EBPβ on the CML stem cells remained to be elucidated. Here we show that STAT5 is involved in the BCR-ABL-mediated C/EBPβ upregulation and that C/EBPβ accelerates the exhaustion of CML stem cells. In order to investigate the regulation of C/EBPβ in CP-CML, BCR-ABL gene was retrovirally introduced into EML cells, a mouse hematopoietic stem cell line, to make EML-BCR-ABL. The expression of C/EBPβ was upregulated at mRNA and protein level in EML-BCR-ABL when compared to EML cells transduced with a control vector. The upregulation of C/EBPβ was significantly repressed by adding imatinib mesylate, suggesting that the downstream signaling pathway of BCR-ABL is directly involved in the process. Neither a PI3K inhibitor nor a MEK inhibitor affected the levels of C/EBPβ in EML-BCR-ABL, but a STAT5 inhibitor reversed the upregulation of C/EBPβ in EML-BCR-ABL. Retroviral transduction of dominant-negative STAT5 mutant also impaired the upregulation of C/EBPβ in EML-BCR-ABL. Transduction of constitutively-active STAT5 mutant into parental EML cells significantly upregulated C/EBPβ expression. These results suggest that BCR-ABL upregulates C/EBPβ expression at least in part through activation of STAT5. To clarify the role of C/EBPβ in the regulation of CML stem cells, BCR-ABL-transduced bone marrow (BM) cells from C/EBPβ knockout (KO) mice or wild type (WT) mice were serially transplanted into irradiated recipient mice. In the first round of transplantation, all the recipient mice developed myeloproliferative status and the mice transplanted with BCR-ABL-transduced C/EBPβ KO BM cells survived longer than the mice transplanted with BCR-ABL-transduced WT BM cells. Interestingly, the frequency of c-kit+ cells within BCR-ABL+ cells was higher in the BM of the mice transplanted with BCR-ABL-transduced C/EBPβ KO cells than in the BM transplanted with BCR-ABL-transduced WT cells (Figure). When 2 × 106 BCR-ABL+ BM cells from primary recipient mice were transplanted into sublethally irradiated secondary recipients, all the mice developed a myeloproliferative status. Transplantation of 1 × 106 BCR-ABL+ BM cells from primary recipient mice, four out of the five recipient mice that transplanted with BCR-ABL-transduced C/EBPβ KO BM cells developed a myeloproliferative status and only one out of the four recipient mice that transplanted with BCR-ABL-transduced WT BM cells developed a myeloproliferative status. Transplantation of less than 0.5 × 106 BCR-ABL+ BM cells engrafted none of the secondary recipient mice. The frequencies of leukemia-initiating cells in BM of primary recipient mice that transplanted with BCR-ABL-transduced WT cells or BCR-ABL-transduced C/EBPβ KO cells were 1 in 1,404,129 and 1 in 683,773, respectively. These findings suggest that C/EBPβ promotes exhaustion of CML stem cells. In conclusion, these results suggest that C/EBPβ is upregulated by BCR-ABL at least in part through STAT5 and that C/EBPβ has the ability to exhaust CML stem cells. We are currently investigating the molecular mechanisms which protect CML stem cells from C/EBPβ-mediated exhaustion for complete eradication of CML stem cells in the future. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.907.907
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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