CD62L expression level determines the cell fate of 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.

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Balance of MafB and PU.1 specifies alternative macrophage or dendritic cell fate

Blood ◽

10.1182/blood-2004-04-1448 ◽

2005 ◽

Vol 105 (7) ◽

pp. 2707-2716 ◽

Cited By ~ 110

Author(s):

Youssef Bakri ◽

Sandrine Sarrazin ◽

Ulrich P. Mayer ◽

Silke Tillmanns ◽

Claus Nerlov ◽

...

Keyword(s):

Cell Fate ◽

Transcriptional Activity ◽

Blood Monocytes ◽

Myeloid Dendritic Cells ◽

Macrophage Differentiation ◽

Myeloid Progenitor ◽

Pathological Conditions ◽

Normal Human ◽

Expression And Activity ◽

Myeloid Progenitors

AbstractMacrophages and myeloid dendritic cells (DCs) represent alternative differentiation options of bone marrow progenitors and blood monocytes. This choice profoundly influences the immune response under normal and pathological conditions, but the underlying transcriptional events remain unresolved. Here, we show that experimental activation of the transcription factors PU.1 and MafB in transformed chicken myeloid progenitors triggered alternative DC or macrophage fate, respectively. PU.1 activation also was instructive for DC fate in the absence of cytokines in human HL-60 cell-derived myeloid progenitor and monocyte clones. Differentiation of normal human monocytes to DCs led to a rapid increase of PU.1 to high levels that preceded phenotypic changes, but no MafB expression, whereas monocyte-derived macrophages expressed MafB and only moderate levels of PU.1. DCs inducing levels of PU.1 inhibited MafB expression in monocytes, which appeared to be required for DC specification, since constitutive MafB expression inhibited DC differentiation. Consistent with this, PU.1 directly bound to MafB, inhibited its transcriptional activity in macrophages, and repressed its ability to induce macrophage differentiation in chicken myeloid progenitors. We propose that high PU.1 activity favors DCs at the expense of macrophage fate by inhibiting expression and activity of the macrophage factor MafB.

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Expression level is a key determinant of E2F1-mediated cell fate

Cell Death and Differentiation ◽

10.1038/cdd.2017.12 ◽

2017 ◽

Vol 24 (4) ◽

pp. 626-637 ◽

Cited By ~ 19

Author(s):

Igor Shats ◽

Michael Deng ◽

Adam Davidovich ◽

Carolyn Zhang ◽

Jungeun S Kwon ◽

...

Keyword(s):

Cell Fate ◽

Expression Level

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BCOR Regulates Cell Fate Transition, Myeloid Differentiation and Leukaemogenesis

Blood ◽

10.1182/blood-2018-99-117893 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3907-3907 ◽

Cited By ~ 1

Author(s):

Lev M Kats ◽

Madison J Kelly ◽

Gareth Gregory ◽

Ricky W Johnstone ◽

Stephin J Vervoort

Keyword(s):

Stem Cell ◽

Cell Fate ◽

Target Genes ◽

Conflicts Of Interest ◽

Haematopoietic Stem Cell ◽

Myeloid Differentiation ◽

Transcriptional Networks ◽

Cell Fate Decisions ◽

Myeloid Progenitors

Abstract Stem cell self-renewal and lineage specification are highly dynamic and tightly controlled processes that are essential for normal haematopoiesis and are dysregulated in cancer. The X-linked BCL6 Corepressor (BCOR) gene encodes a protein that is widely expressed across adult human tissues and is a component of a non-canonical Polycomb repressive complex 1 (PRC1). The BCOR gene is recurrently mutated in various malignant and non-malignant blood disorders, and we and others have recently provided experimental evidence that BCOR has cell-context dependent functions in regulating the proliferation, differentiation and survival of haematopoietic cells. To comprehensively examine the role of BCOR in haematopoiesis in vivo we used a conditional mouse model that mimics the truncating mutations observed in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Using stem and progenitor populations isolated ex vivo we comprehensively analysed the role of BCOR in regulating gene expression, modifying chromatin and altering genome architecture. We demonstrate that BCOR has a pivotal role in down-regulating haematopoietic stem cell (HSC) associated transcriptional networks during the transition from multi-potent stem cells to lineage-committed myeloid progenitors. Inactivation of Bcor in HSCs results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Mechanistically, Bcor regulates a subset of PRC1-target genes including key HSC super-enhancer-linked transcription factors that are normally down-regulated during myeloid differentiation. We used CRISPR/Cas9 to explore the function of Bcor target genes and identified those that are necessary for the proliferation of Bcor mutant leukaemic cells. This study provides a comprehensive mechanistic understanding of how BCOR regulates cell fate decisions and contributes to the development of leukaemia. Disclosures No relevant conflicts of interest to declare.

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MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1220710110 ◽

2013 ◽

Vol 110 (33) ◽

pp. 13410-13415 ◽

Cited By ~ 15

Author(s):

K. Okuyama ◽

T. Ikawa ◽

B. Gentner ◽

K. Hozumi ◽

R. Harnprasopwat ◽

...

Keyword(s):

B Cell ◽

Cell Fate ◽

Transcriptional Factors ◽

Potential Alternative ◽

Myeloid Progenitors

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Balance of Transcription Factors Downstream of Notch Signaling Determines the Fate of Myeloid Progenitors toward Differentiation to Mast Cells or Immortalization without Differentiation.

Blood ◽

10.1182/blood.v108.11.676.676 ◽

2006 ◽

Vol 108 (11) ◽

pp. 676-676

Author(s):

Mamiko Sakata-Yanagimoto ◽

Fumio Nakahara ◽

Etsuko Yamaguchi-Nakagami ◽

Keiki Kumano ◽

Toshiki Saito ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Cell Differentiation ◽

Mast Cells ◽

Notch Signaling ◽

Cell Fate ◽

Myeloid Cell ◽

Cell Generation ◽

Notch Activation ◽

Myeloid Progenitors

Abstract Notch signaling represents one of the fundamental communication channels in various types of cells. While Notch activation has been shown to inhibit myeloid differentiation in a subset of hematopoietic progenitors, the role of Notch signaling in mast cell differentiation is not clear. When common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) purified from mouse bone marrow cells were stimulated with Delta1-Fc, a soluble form of Notch ligand, in the presence of stem cell factor, IL-3, IL-6, and thrombopoietin, granulocyte and macrophage differentiation, which is observed at day 7 of culture in the absence of Delta1-Fc, was markedly inhibited. Instead, Lin-c-Kit+FcεR+ mast cells dominated in the culture. Delta1-Fc did not increase mast cell generation from either CMPs or GMPs of the bone marrow of pI:pC-treated Mx-Cre x Notch2 flox/flox (N2-MxcKO) mice, in contrast to littermate Notch2 flox/flox mice treated with pI:pC, which suggests that Notch2 is responsible for the Delta1-Fc-augmented mast cell generation from CMPs and GMPs in culture. Retroviral transfer of constitutive active form of Notch2 (aN2) into CMPs and GMPs resulted in the complete loss of granulocyte-macrophage colony-forming cells and the emergence of basophilic granules-containing blast like cells, indicating the cell fate instruction. Real-time PCR analysis revealed that Delta1-Fc stimulation and aN2 introduction up-regulated the expression of Hes1, a transcriptional suppressor that is known to be a direct target of Notch activation in several cell types, within 12 h. Moreover, among GATA genes, Delta1-Fc stimulation and aN2 introduction resulted in increase of GATA3 mRNA, while expression levels of GATA1 and GATA2, which have been suggested to play a role in regulating mast cell differentiation, were unchanged. Next, we retrovirally expressed Hes1 and/or a GATA gene into CMPs and GMPs to see if the same effects were observed. Mast cells were increased only when both genes were expressed. On the other hand, when Hes1 alone was transduced, we observed rapid growth and immortalization of these cells without differentiation. C/EBPa, which is known to be suppressed in mast cell differentiation and upregulated in myeloid cell differentiation, was down-regulated within 48 h from the initiation of Hes1 retroviral transduction, suggesting that C/EBPa is a downstream target of Hes1 in this myeloid cell fate determination. Theses results indicate that, at the downstream of Notch activation, there are a C/EBPa down-regulation pathway that is Hes1-dependent and a GATA3 up-regulation pathway. Balanced regulation of these pathways should play a physiological role in myeloid and mast cell differentiation, while imbalance between these two pathways might provide a new model of myeloid transformation.

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Homozygous Mutations in PHRF1 and PPARG in a Patient with Congenital Neutropenia and Profound Monocytosis: Understanding the Pathway Beyond Monocyte Versus Granulocyte Cell-Fate Choice

Blood ◽

10.1182/blood.v126.23.648.648 ◽

2015 ◽

Vol 126 (23) ◽

pp. 648-648

Author(s):

Houra Loghmani-khouzani ◽

Maksim Klimiankou ◽

Siarhei Kandabarau ◽

Cornelia Zeidler ◽

Karl Welte ◽

...

Keyword(s):

Bone Marrow ◽

Cell Fate ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Myeloid Cells ◽

Healthy Individuals ◽

Congenital Neutropenia ◽

Maturation Arrest ◽

Functional Connections ◽

Myeloid Progenitors

Myelopoiesis is a strictly regulated process of monocytes and granulocytes production, originating from common myeloid progenitors. Studies on patients with inherited severe congenital neutropenia (CN) could help to better elucidate myelopoiesis regulation. In CN, maturation arrest of granulocytic precursors at the promyelocytic stage leads to a shift towards monopoiesis and an ineffective granulopoiesis. Hereby we report on a particular CN patient with a typical "maturation arrest" of granulopoiesis at the stage of promyelocytes, very low levels of neutrophils but extremely high levels of monocytes in the peripheral blood and bone marrow. Upon G-CSF treatment (5μg/kg/day) this patient produces high levels of monocytes (up to 24.8x103/μL, more than 50 % of total leukocytes) and only up to 1x103/μl neutrophils. Sanger sequencing of DNA from this patient revealed no mutations in CN-related genes such as ELANE, HAX1 and G6PC3. Therefore we performed whole genome sequencing (Complete Genomics. Inc, Mountain View, CA) of DNA from blood of this patient and his mother to discover causative gene mutations. We identified a homozygous deletion in PHRF1 (PHD and Ring Finger Domain-Containing Protein 1) (p.R1015-G1019, NP_065952.2; rs144630030) and a homozygous missense mutation in PPARG (Peroxisome proliferator-activated receptor gamma) (p.P12A, NP_0569553.2; rs1801282). Both mutations are heterozygote in the patient's mother. Population frequency for heterozygote allele of these two variations was reported to be 13% and 7%, respectively, but no homozygote variants were reported till date. PHRF1 functions as an essential component of the TGF-ß tumour suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF (TG-Interacting Factor) and a consequent retinoic acid signalling activation in haematopoiesis and monopoiesis. PPARG interacts with Retinoid X Receptors (RXR) and controls the expansion of macrophages. In order to evaluate the functional role of the detected mutations on disturbed G-CSF-triggered myelopoiesis in reference CN patient, CD33+ bone marrow myeloid progenitor cells of two healthy controls and this patient were treated with G-CSF in vitro and mRNA expression profiles were analysed in an Affymetrix Microarray platform, followed by Ingenuity Pathway Analysis (IPA). We found, that 'TREM1 signalling' was among the top three pathways with most significant differences (p<9x10-7) between this CN patient and healthy individuals. 'Granulocyte adhesion and diapedesis' and 'LXR/RXR pathway' were the next two significantly affected gene sets (p<8x10-6 and p<1.3x10-5, respectively). TREM1 (Triggering Receptor Expressed on Myeloid cells 1) is a chemokine receptor that is expressed by neutrophils and monocytes, however the ligand that activates this receptor is yet unknown. TREM-1 is involved in neutrophil apoptosis and is known to positively regulate monopoiesis by activation of M-CSF synthesis. Intriguingly, M-CSF was 4.2-fold upregulated in myeloid cells of patient, in comparison to healthy individuals. Other known components of TREM 1 signalling were also among the top 10 differentially expressed genes identified by IPA: HSD11B1 (+20 fold), ATP1B2 (+14 fold) and THBS1 (-10 fold). Functional connections between PPARG and TREM1 is known. PPARG mutation could lead to TREM1 signalling activation that consequently lead to M-CSF over-expression (+4.2 fold). In addition to the activation of TREM1 signalling, deletion in the PHFR1 gene could be the causative effect of marked upregulation of ALDH1A2 (+17 fold), which also could lead to an increase in M-CSF levels and in a retinoid acid signalling activation ultimately leading to increased monocyte production. Together, PPARG and PHRF1 mutations could hyper-activate the secretion of M-CSF by myeloid progenitors leading to a strong shift towards monopoiesis upon G-CSF treatment. Disclosures No relevant conflicts of interest to declare.

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Centrosome Aurora A gradient ensures single polarity axis in C. elegans embryos

Biochemical Society Transactions ◽

10.1042/bst20200298 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1243-1253 ◽

Cited By ~ 1

Author(s):

Sukriti Kapoor ◽

Sachin Kotak

Keyword(s):

Symmetry Breaking ◽

Cell Fate ◽

Cell Cortex ◽

Axis Formation ◽

Aurora A Kinase ◽

Aurora A ◽

C Elegans ◽

Cell Embryo ◽

Polarity Establishment

Cellular asymmetries are vital for generating cell fate diversity during development and in stem cells. In the newly fertilized Caenorhabditis elegans embryo, centrosomes are responsible for polarity establishment, i.e. anterior–posterior body axis formation. The signal for polarity originates from the centrosomes and is transmitted to the cell cortex, where it disassembles the actomyosin network. This event leads to symmetry breaking and the establishment of distinct domains of evolutionarily conserved PAR proteins. However, the identity of an essential component that localizes to the centrosomes and promotes symmetry breaking was unknown. Recent work has uncovered that the loss of Aurora A kinase (AIR-1 in C. elegans and hereafter referred to as Aurora A) in the one-cell embryo disrupts stereotypical actomyosin-based cortical flows that occur at the time of polarity establishment. This misregulation of actomyosin flow dynamics results in the occurrence of two polarity axes. Notably, the role of Aurora A in ensuring a single polarity axis is independent of its well-established function in centrosome maturation. The mechanism by which Aurora A directs symmetry breaking is likely through direct regulation of Rho-dependent contractility. In this mini-review, we will discuss the unconventional role of Aurora A kinase in polarity establishment in C. elegans embryos and propose a refined model of centrosome-dependent symmetry breaking.

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Centromere assembly and non-random sister chromatid segregation in stem cells

Essays in Biochemistry ◽

10.1042/ebc20190066 ◽

2020 ◽

Vol 64 (2) ◽

pp. 223-232 ◽

Cited By ~ 1

Author(s):

Ben L. Carty ◽

Elaine M. Dunleavy

Keyword(s):

Stem Cells ◽

Cell Division ◽

Cell Fate ◽

Sister Chromatid ◽

Asymmetric Cell Division ◽

Protein A ◽

Germline Stem Cells ◽

Sister Chromatids ◽

Centromere Protein A ◽

Oocyte Meiosis

Abstract Asymmetric cell division (ACD) produces daughter cells with separate distinct cell fates and is critical for the development and regulation of multicellular organisms. Epigenetic mechanisms are key players in cell fate determination. Centromeres, epigenetically specified loci defined by the presence of the histone H3-variant, centromere protein A (CENP-A), are essential for chromosome segregation at cell division. ACDs in stem cells and in oocyte meiosis have been proposed to be reliant on centromere integrity for the regulation of the non-random segregation of chromosomes. It has recently been shown that CENP-A is asymmetrically distributed between the centromeres of sister chromatids in male and female Drosophila germline stem cells (GSCs), with more CENP-A on sister chromatids to be segregated to the GSC. This imbalance in centromere strength correlates with the temporal and asymmetric assembly of the mitotic spindle and potentially orientates the cell to allow for biased sister chromatid retention in stem cells. In this essay, we discuss the recent evidence for asymmetric sister centromeres in stem cells. Thereafter, we discuss mechanistic avenues to establish this sister centromere asymmetry and how it ultimately might influence cell fate.

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