Identification of Pre-Gmps and Pre-Meps: Two Novel Types of Committed Progenitor Cells in Bone Marrow

Abstract Elucidating the stepwise differentiation processes that leads from multipotent hematopoietic stem cells to mature effector cells is critical for understanding both normal and neoplastic hematopoiesis. Early studies suggested that common myeloid progenitors (CMPs) are oligo-lineage hematopoietic progenitors that produce all lineages of myeloid cells, including granulocytes, monocytes, erythrocytes and megakaryocytes. CMPs do so by first giving rise to megakaryocyte-erythroid progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs), two types of bi-lineage progenitors. However, this concept was challenged by several recent studies where single cell techniques demonstrated that CMPs, GMPs and MEPs are highly heterogenic. The existence of lineage-restricted subsets within the CMP population leads to questions about whether erythroid and megakaryocytic lineage commitment is actually initiated at the multipotent progenitor or CMP stage. During the past 15 years, several lineage-restricted subsets of progenitors have been separated out from CMP population, including monocyte-dendritic progenitors, megakaryocyte progenitors, and erythroid progenitors based on expression of CD115/Flt3, CD41/CD150, and CD105/CD150, respectively. However, the remaining CMP population is still highly heterogenic. Thus, further separation of functional subsets within the CMP compartment is required. By screening cell surface markers that can further separate CMPs, we have identified CD27 as a reliable marker to separate all megakaryocyte/erythrocyte-committed progenitors from granulocyte/monocyte-committed progenitors. In addition, we found that CD62L is only expressed on granulocyte/monocyte-committed progenitors. CD27 and CD62L co-staining can separate CMP into CD27 +CD62L +, CD27 +CD62L - and CD27 -CD62L - subsets. Biology and morphology study showed that CD27 +CD62L - cells are closely associated with GMPs, whereas CD27 -CD62L - cells are closely associated with MEPs. In vitro culture and in vivo transplantation functional studies demonstrated that 1) CD27 +CD62L + cells are pre-GMPs that give rise to FcGRII/III + GMPs and only produce granulocytes and monocytes; 2) CD27 -CD62L - cells are pre-MEPs that give rise to MEPs and primarily produce erythrocytes and megakaryocytes with minimal contribution to granulocytes and monocytes; 3) CD27 +CD62L - subset enriches cells with genuine CMP potential capable of producing GMPs, MEPs, and subsequent progeny. Taken together, we have identified two novel populations of committed progenitors that serve as intermediates between CMP-GMP and CMP-MEP commitment pathways. Identification of pre-GMPs and pre-MEPs fills in the gap between CMPs-GMPs and CMPs-MEPs, supporting the hierarchal relationship of myeloid lineage differentiation. Disclosures No relevant conflicts of interest to declare.

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Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽

10.1182/blood.v130.suppl_1.81.81 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 81-81

Author(s):

Silvana Di Giandomenico ◽

Pouneh Kermani ◽

Nicole Molle ◽

Mia Yabut ◽

Fabienne Brenet ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Progenitor Cells ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Effector Cells ◽

Erythroid Precursors ◽

Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.2899.2899 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2899-2899

Author(s):

Jodi Murakami ◽

Baohui Xu ◽

Christopher B. Franco ◽

Xingbin Hu ◽

Stephen J. Galli ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Adhesion ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Effector Cells ◽

Cell Cycle Status

Abstract α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche. Disclosures No relevant conflicts of interest to declare.

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Alteration of HSC Functions in Thalassemia

Blood ◽

10.1182/blood.v126.23.4752.4752 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4752-4752 ◽

Cited By ~ 2

Author(s):

Annamaria Aprile ◽

Maria Rosa Lidonnici ◽

Alessandro Gulino ◽

Claudio Tripodo ◽

Giacomo Mandelli ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Pathological Condition ◽

Autologous Transplantation ◽

Beta Thalassemia ◽

Allogeneic Bone ◽

Erythroid Progenitors ◽

Hematopoietic Stem

Abstract Beta-thalassemia represents one of the most globally widespread monogenic disorders and is characterized by significantly reduced or absent synthesis of hemoglobin beta-chains. In its severe form the insufficient production of adult hemoglobin results in altered erythropoiesis, hemolytic anemia, bone marrow (BM) hematopoietic hyperplasia and splenomegaly often associated with extramedullary hematopoiesis, requiring regular blood transfusions and iron chelation treatment. Over the last two decades many progresses were made in the field of allogeneic bone marrow (BM) transplantation to definitively cure beta-thalassemia. In parallel, experimental autologous transplantation protocols were developed to correct the disease by gene therapy also in patients lacking a compatible donor. Both in the allogeneic and autologous setting, thalassemic hematopoietic stem cells (HSCs) and the BM niche represent central elements. Although many aspects of the pathophysiology of thalassemia have been extensively investigated, the HSC and its niche have never been explored. In thalassemia, the BM is a stressed environment, characterized by the compensatory expansion of erythroid progenitors secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations, such as primitive progenitors and/or HSCs, might be affected by such an altered hematopoietic microenvironment is unknown. We investigated the frequency of hematopoietic progenitors in a murine model of severe beta-thalassemia intermedia. Immunophenotypic analyses revealed no differences in MEP, GMP, CMP, LMPP and MPP committed precursor subpopulations, whereas a significantly lower frequency of HSCs (Lin- Sca-1+ c-kit+ CD48- CD150+) was observed in thalassemic mice, as compared to age-matched wild-type controls. Competitive transplantation experiments revealed a disadvantage in the engraftment capacity of thalassemic HSCs, which was substantiated by the preliminary results from in vitro and in vivo cell cycle analyses suggesting an accelerated HSC exhaustion. Analyses of other cellular components, such as BM stroma and differentiated hematopoietic cells, revealed that additional elements are altered in the thalassemic BM microenvironment. The cellular and molecular bases of HSC-niche interaction in this pathological condition are under investigation. Our results uncover a previously ignored defect of HSCs in beta-thalassemia. The investigation of cellular and molecular players that might affect in trans HSC functions in the complexity of this altered microenvironment is ongoing. Disclosures No relevant conflicts of interest to declare.

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Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-142963 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Gordon G. L. Wong ◽

Gabriela Krivdova ◽

Olga I. Gan ◽

Jessica L. McLeod ◽

John E. Dick ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Myeloid Differentiation ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Lineage Differentiation ◽

Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Blood ◽

10.1182/blood.v118.21.917.917 ◽

2011 ◽

Vol 118 (21) ◽

pp. 917-917

Author(s):

Adrienne M. Dorrance ◽

Rachelle Kosoff ◽

Meaghan McGuinness ◽

Chad Harris ◽

Serena De Vita ◽

...

Keyword(s):

Cell Migration ◽

Conflicts Of Interest ◽

Effector Proteins ◽

Proliferating Cells ◽

Hematopoietic Stem ◽

Time Points ◽

Empty Vector ◽

Group A

Abstract Abstract 917 Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures: No relevant conflicts of interest to declare.

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TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽

10.1182/blood.v118.21.4829.4829 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4829-4829

Author(s):

David C Dorn ◽

Wei He ◽

Joan Massague ◽

Malcolm A.S. Moore

Keyword(s):

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Transforming Growth Factor Β ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽

10.1182/blood.v120.21.2332.2332 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2332-2332

Author(s):

Vitalyi Senyuk ◽

Yunyuan Zhang ◽

Yang Liu ◽

Ming Ming ◽

Jianjun Chen ◽

...

Keyword(s):

Cell Transformation ◽

Conflicts Of Interest ◽

Critical Role ◽

Ectopic Expression ◽

Myeloid Differentiation ◽

Dna Hypermethylation ◽

Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Blood ◽

10.1182/blood.v122.21.3669.3669 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3669-3669

Author(s):

Stephan Emmrich ◽

Franziska Schmidt ◽

Ramesh Chandra Pandey ◽

Aliaksandra Maroz ◽

Dirk Reinhardt ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Gene Set Enrichment Analysis ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Lncrna Expression ◽

Fold Reduction ◽

Non Coding Rnas

Abstract Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs. The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis. Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs. Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA). For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays. Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures: No relevant conflicts of interest to declare.

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The Role of STAT5 in HOXA9-Associated Leukemia

Blood ◽

10.1182/blood.v128.22.3919.3919 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3919-3919

Author(s):

Peilin Ma ◽

Yuqing Sun ◽

Jingya Wang ◽

Weihua Song ◽

Tao Xu ◽

...

Keyword(s):

Binding Sites ◽

Myeloid Leukemia ◽

Tandem Duplication ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Constitutive Activation ◽

Oncogenic Mutation

Abstract Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis. Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo. To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. Disclosures No relevant conflicts of interest to declare.

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HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo

Blood ◽

10.1182/blood-2012-03-417022 ◽

2012 ◽

Vol 120 (15) ◽

pp. 3001-3006 ◽

Cited By ~ 19

Author(s):

Andreas Weigert ◽

Benjamin Weichand ◽

Divya Sekar ◽

Weixiao Sha ◽

Christina Hahn ◽

...

Keyword(s):

Bone Marrow ◽

Cell Function ◽

Bone Marrow Cells ◽

Negative Regulator ◽

Low Oxygen ◽

Hematopoietic Stem ◽

Lineage Differentiation ◽

Marrow Cells

Abstract Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L–induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1αfl/fl LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2−/− bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1αfl/fl LysM-Cre and ID2−/−, but not HIF-2αfl/fl LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

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