Megakaryocyte-erythrocyte Progenitor Cells Research Articles

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-β.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

LSD1 Inhibition with IMG7289 (bomedemstat) Rebalances Megakaryopoiesis and Erythropoiesis in a Patient with MPN/MDS with Refractory Anemia with Ringed Sideroblast and Marked Thrombocytosis with JAK2 and SF3B1 Mutations

Lysine-specific demethylase-1 (LSD1) is a histone H3 K4 demethylase that plays a critical role in hematopoietic progenitor cell differentiation, in particular, the production of megakaryocytes and erythrocytes from their common classical bi-potent progenitor (MEP). LSD1 is recruited to chromatin by the transcription factor GFI1B to license the maturation of megakaryocyte-erythrocyte progenitor cells (MEPs). Anemia is a common and serious complication of primary myelofibrosis (PMF), which is often refractory to conventional therapy. We report here the effect of IMG-7289 (bomedemstat), an LSD1 inhibitor, on skewing the fate of MEPs favoring the production of red cells over platelets and producing a complete hematologic remission in a JAK2V617F-positive PMF patient who had developed a refractory anemia with ringed sideroblasts. A 63 year old woman presented in 2016 with a 1 year history of with fatigue significantly compromising her lifestyle. She denied fever, night sweats, or weight loss. The physical examination was unremarkable with no palpable splenomegaly. Laboratory studies showed a hemoglobin (Hb) of 11.3 g/dL, a total white blood cell count (WBC) of 20.3k/µL with leukoerythroblastosis with 1% circulating blast cells, and a platelet count of 1,192k/µL. A bone marrow biopsy (BMB) showed a hypercellular marrow with increased dysmorphic megakaryocytes but without dyserythropoiesis. There were no ringed sideroblasts with Perls stain, and silver staining showed grade 2 reticulin fibrosis. The karyotype was del(7)(q11.2) and genotyping identified a JAK2V716F allele (VAF not known). The patient was considered to have PMF. Initial treatment consisted of hydroxyurea but without any improvement in her symptoms. Financial constraints initially prevented access to ruxolitinib but the patient was eventually started on 15 mg BID in January 2018. She tolerated therapy well but discontinued therapy a year later owing to no improvement in her fatigue and opted for observation alone. Over the next year, her hemoglobin fell to the range of 9.0-9.5g/dl and she developed drenching night sweats. A BMB in August 2019 showed a hypercellular marrow with increased dysplastic megakaryocytes but now with 76% ringed sideroblasts. Reticulin stain showed grade 2-3 fibrosis. No other dysplastic features were observed. Additional genotyping was declined by insurance and the patient was considered to have an MPN/MDS overlap syndrome. Accordingly, the patient was enrolled in clinical trial IMG-7289-CTP-102 (NCT03136185), a study of the LSD1 IMG-7289. On entry, her Hb was 9.5 gm/dL with an absolute reticulocyte count of 129K/µL. The WBC was 22.7k/µL with 69% neutrophils and 11% monocytes. The platelet count was 1,585k/µL with a mean platelet volume (MPV) of 8.1 fL. Genotyping showed a JAK2V617F VAF of 27%, a new SF3B1K700E mutation with a VAF of 26%, and a DNMT3A mutation with a VAF of 95%. Spleen volume by imaging was 283.3 cm³. The starting IMG-7289 dose was 40 mg po QD. At week 12, the Hb rose to 13.8 g/dL while the absolute reticulocyte count fell to 40K/µL. The platelet count was 271k/µL with the MPV increasing to 10.5 fL. The WBC was 5.2k/µL with 58% neutrophils and 18% monocytes. The mutant JAK2 allele burden had dropped to 14%. The spleen volume was 223 cm³. The IMG-7289 dose was reduced to 35 mg QD after complaints of fatigue. At Week 24, Hb was 11.7 g/dL, WBC was 12.1k/µL and platelets were 743k/µL. The total symptom score (MPN SAF TSS) was reduced by 31% from baseline. The expected pharmacodynamic effects of LSD1 inhibition on hematopoiesis were evident in this patient: monocytosis with a decrease in neutrophils and a marked reduction in the platelet count. Most striking was the concurrent improvement in the patient's anemia which was associated with a persistent reticulocytosis present at the start of treatment but with a significant increase in red blood cells in the setting of a SF3B1 mutation. MEP fate decisions hinge on the balance between KLF1 and FLI1 with the former favoring an erythroid fate, the later, megakaryocytes. The transcription factor complex of FLI1, GATA1, FOG1 which recruits the LSD1-containing NuRD complex is essential for megakaryocyte function; IMG-7289 likely disrupts that complex favoring erythropoiesis over megakaryopoiesis. The inhibition of LSD1 may, therefore, in the setting of thrombocytosis and anemia, rebalance the fate of MEPs to ameliorate both abnormalities. Disclosures Yacoub: Novartis: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Roche: Other: Support of parent study and funding of editorial support. Rienhoff:Imago BioSciences: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Lysophosphatidic acid receptors 2 and 3 regulate erythropoiesis at different hematopoietic stages

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

A single-cell multiomics approach to study tumor-driven perturbations during hematopoiesis in mice

Abstract Cancer is associated with concomitant myeloid cell responses that are characterized by an expansion of tumor associated myeloid cells, including myeloid derived suppressor cells, tumor associated macrophages and neutrophils. The prevailing paradigm is that cancer interferes with normal hematopoiesis by skewing the generation of myeloid cells with tumor promoting functions and promoting extramedullary hematopoiesis in organs such as the spleen. Advances in single cell multiomic technologies now enable the analysis of high dimensional protein and mRNA expression from thousands of cells simultaneously. Using the power of the BD Rhapsody™ Single-Cell Analysis System, we have carefully investigated melanoma associated extramedullary hematopoiesis in mice. To do this, we isolated 4 hematopoietic stem and progenitor cell populations simultaneously, including Lin− Sca-1+ c-Kit+, common myeloid progenitor, granulocyte-macrophage progenitor, and megakaryocyte-erythrocyte progenitor cells from the spleen and bone marrow of melanoma bearing mice using the BD FACSMelody™ cell sorter in combination with a multiplex BD® AbSeq Oligos panel for surface protein expression and a single-cell whole transcriptome analysis. Using advanced analysis plugins in SeqGeq™ software, we were able to delineate the developmental trajectories of these hematopoietic stem and progenitor cells in this systemic immuno-suppressive myeloid environment. These advanced technologies are critical to uncover tumor mediated abnormalities in hematopoiesis. Disclaimers: For Research Use Only. Class 1 Laser Product. BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice.

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. [Display omitted] DisclosuresNakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Red blood cell-derived extracellular vesicles communicate with megakaryocyte–erythrocyte progenitor cells in bone marrow

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3−ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

Deregulated Genes in Hematopoietic Stem Cells Isolated from Spleen of Patients with Myelofibrosis

An important issue in myeloproliferative diseases research is to test the hypothesis that in the neo-angiogenesis, which is observed in the spleen and bone marrow of patients, involves endothelial cells derived from the neoplastic clone. In this study we analyzed the gene expression profile of 138 genes in CD34+ hematopoietic cells from the spleen and peripheral blood of patients with myelofibrosis (MF) and healthy individuals (HI). These genes have been selected among those that characterize the neoangiogenic gene signature or belonging to deregulated expression pathways in MF, and are listed in Table1.By using quantitative reverse transcription-PCR (RT-qPCR), we measured the expression levels of 141 targets and 5 reference genes in CD34+ cells isolated by immunomagnetic method from the spleen and peripheral blood of 4 patients and 3 HI. For calculation of the statistical significance of the differences we used the t-test for unpaired samples. A p<0.05 was considered to be statistically significant. All data analyses were performed with GenEx (version 6.1, MultiD).To characterize the differentially expressed genes obtained in each comparison, the lists of genes were analyzed through several tools, i.e. String, Enrichr and GSEA analysis. The comparison between spleen CD34+ cells and circulating CD34+ cells reveal a group of common genes in both patients and heathy donors. Those genes are significantly overexpressed in spleen. Some of the common genes are involved in the regulation of cell migration and in the development of blood vessels were significantly overexpressed in splenic CD34+ cells compared to circulating CD34+ cells. However, we observed that many of the genes implicated in the development of the vessels were under-expressed in CD34+ cells isolated from the spleen of patients compared to those of HI. The most significant upregulated genes in CD34+ splenic cells from patients included transcripts (GATA1, HBB, TAL1, GATA2, PTGS1) belonging to the molecular signature of CD34+ cells isolated from the bone marrow (BM) of patients with chronic myeloid leukemia (Diaz-Blanco E. et al., Leukemia 2007). Another group of upregulated genes (GATA1, TAL1, ITGB3, GATA2, PF4) are also identified in a study designed to characterize the genes essential to the development of megakaryocytes (Tenedini et al. Blood 2004). These two groups of genes are part of an expression pattern characteristic for immature stem cells as well as megakaryocyte-erythrocyte progenitor cells. Another group of genes overexpressed in patients spleens (CD34, ANGPT1, PF4, GATA2, PTGS1), which includes some of the above mentioned genes, has been observed in a comparison between circulating CD34+ cells from patients with myelofibrosis, and CD34 + cells isolated from the bone marrow of HI (Guglielmelli P. et al. Stem Cells 2007). Among the underexpressed genes in the spleen of patients we mention CXCR4, in keeping with previous observations. Interestingly, among the aforementioned genes, GATA1 and GATA2 genes are overexpressed in granulocytes of patients with MPNs regardless of the JAK2/CALR mutational status whereas PTGS1 is overexpressed in the JAK2-V617F homozygous and CALR-mutated granulocytes (Rampal et al. Blood 2014).We focused our attention on PTGS1 because its well-known activity in regulating angiogenesis, which can be inhibited in vitro by treatment with aspirin (Tsujii, M. et al. Cell 1998). To explain the mechanism of action of low-doses aspirin in this context, it has been proposed a model that involves permanent inactivation of PTGS1 in platelets. Indeed, PTGS1 is the only cyclooxygenase isoenzyme present in platelets. Moreover, Dixon and colleagues (Dixon, D.A. et al. JCI 2006), demonstrated that activated platelets induced the expression of PTGS2 in monocytes. It is believed that the synthesis of prostaglandin E2 by PTGS2 in tissues, is linked to an increment of angiogenesis and cell proliferation, and to a reduction of apoptosis. According to this concept, our data point toward a model in which, in the spleen of patients with MF, an altered hematopoietic stem cell differentiation could induce an inflammation-mediated angiogenesis through the overexpression of PTGS1 and a consequent induction of PTGS2 in cells of the splenic microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High PRMT1 Expression Promotes the Progression of Acute Megakaryocytic Leukemia Via Controlling Metabolic Pathways

Metabolic reprogramming is needed not only to accommodate but also to drive leukemia progression. Yet very little is known on genetic factors other than IDH1 mutations, which can drive leukemogenesis via metabolic reprogramming. Here, we will present data to suggest that protein arginine methyltransferases 1 (PRMT1) is a driver for acute megakaryocytic leukemia via reprogramming metabolism. PRMT1 is highly expressed in megakaryocyte-erythrocyte progenitor cells and downregulated during the terminal differentiation of megakaryocytes. Constitutively high expression of PRMT1 in acute megakaryoblastic leukemia (AMKL) blocks megakaryocyte differentiation. PRMT1 upregulates RBM15 protein level via methylation-dependent ubiquitylation pathway (Zheng et al. Elife, 2015). In this presentation, we discovered that metabolic stress such as hypoxia downregulates PRMT1 protein level. Thus, metabolic stress is the upstream signal for the PRMT1-RBM15 pathway. We have identified that RBM15 specifically binds to 3'UTR of mRNAs of genes involved in metabolic pathways. Using RNA-immunoprecipitation with anti-RBM15 antibody and real-time PCR assays, we validated that RBM15 binds to mRNAs of genes involved in fatty acid oxidation and glycolysis. We transduced PRMT1 into an RBM15-MKL1 expressing cell line 6133. Overexpression of PRMT1 renders 6133 cells to grow in a cytokine-independent manner with increased mitochondria biogenesis, which in turn produces higher concentration of ATP in our metabolomic analysis. Based on the analysis of metabolomics data and RBM15-target genes, we conclude that PRMT1 promotes the usage of glucose as bioenergy via oxidative phosphorylation and inhibits fatty acid oxidation. Given that acetyl-coA is higher in PRMT1 expressing 6133 cells, we asked whether histone acetylation is upregulated in PRMT1 overexpressed 6133 cells. Indeed, we found higher histone acetylation level in PRMT1 highly expressed cells. We also found that propionylated histone is reduced, which is consistent with reduced fatty acid oxidation. Propionyl-CoA molecules are produced from fatty acids with odd carbon numbers. Thus PRMT1-mediated metabolic reprogramming changes epigenetic programming during leukemia progression. Intriguing, we also found PRMT1 overexpression enhances histone H3S10 phosphorylation via methylation-dependent ubiquitylation of DUSP4. DUSP4 promotes polyploidy during megakaryocyte differentiation. Thus PRMT1 caused profound epigenetic changes to promote leukemogenesis. In this vein, we established mouse AMKL models by bone marrow transplantation of 6133 cells as well as human AMKL patient samples respectively. Using this mouse model, we tested PRMT1 inhibitors, acetyltransferase inhibitors as well as other metabolic inhibitors. Treating cells with PRMT1 inhibitors as well as metabolic inhibitors promote MK differentiation of AMKL leukemia cells. Metabolomics analysis of cells recovered from mouse models will be discussed in the presentation. In summary, our data demonstrated that PRMT1 is a major sensor for metabolic stress and that PRMT1 in turn reprograms metabolic pathways to bring epigenetic changes in leukemogenesis. Therefore, targeting PRMT1 and downstream PRMT1-regulated metabolic pathways will offer new avenues in treating acute megakaryocytic leukemia and other hematological malignancies with defective megakaryocyte differentiation. DisclosuresNo relevant conflicts of interest to declare.

Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis.

Intracellular Reactive Oxygen Species Mark and Influence the Megakaryocyte-Erythrocyte Progenitor Fate of Common Myeloid Progenitors

While most studies regarding reactive oxygen species (ROS) focus on their deleterious biological effects, a growing body of evidence indicates the importance of ROS as critical mediators of several signaling pathways, including those involved in hematopoiesis. In this study, we show the critical role of ROS in lineage decision of myeloid progenitors. In megakaryocyte-erythrocyte progenitor cells (MEP), intracellular ROS levels were found to be as low as those in hematopoietic stem cells (HSC). In contrast, remarkably high intracellular ROS levels were observed in granulocyte-monocyte progenitor cells. Intracellular ROS levels in common myeloid progenitors (CMP) were inversely correlated with their MEP differentiation potential. Moreover, gene set enrichment analysis revealed that ROS-low CMP showed gene expression patterns similar to those of MEP, indicating that intracellular ROS levels mark the fate of CMP. In in vitro assays, ROS significantly suppressed the generation of MEP and the formation of megakaryocyte-erythrocyte colonies from CMP. In ROS-high CMP, expression of colony-stimulating factor one receptor (CSF1R) was highly upregulated, and its surface expression correlated with their granulocyte-monocyte differentiation potential. Furthermore, ROS was found to induce the expression of CSF1R mRNA in a leukemia cell line. These data provide novel insights into the relationship between ROS and the hematopoietic differentiation system.

A Critical Role of Reactive Oxygen Species In the Generation of Megakaryocyte-Erythrocyte Progenitor Cells.

AbstractAbstract 1605Reactive oxygen species (ROS) are small molecules containing oxygen with unpaired electron. ROS are always generated as the products of cellular metabolism, and cells have several antioxidant systems to avoid their harmful effect induced by their high chemical reactivity. While studies about biological effects of ROS have been mainly focused on the harmful aspects, a growing body of evidence suggests that ROS are critical mediators of several signaling pathways in cellular homeostasis. In hematopoiesis, for example, it was reported that the generation of intracellular ROS functions as the initiation signal in the development of Drosophila hematopoietic cells and that ROS control self-renewal of hematopoietic stem cells. However, little is known about the functions of ROS in regulating hematopoietic progenitors. In this study, we show a critical role of ROS in lineage decision of myeloid progenitor cells. Firstly, we measured the intracellular ROS level of hematopoietic cells from murine bone marrow by flow cytometry with H2-DCFDA (2‘,7‘-dichlorofluorescin diacetate) staining. It was found that intracellular ROS level of megakaryocyte-erythrocyte progenitor cells (MEP) was kept equal to or lower than that of lineage marker negative, Sca-1 positive, c-Kit positive (KSL) cells. On the other hand, that of granulocyte-monocyte progenitor cells (GMP) was significantly elevated. Additionally, mRNA expression of NADPH oxidase 2 (cytochrome b-245) and NOXA2 (neutrophil cytosolic factor 2), both of which are major components of the membrane-bound oxidase complex generating ROS in cells, was significantly suppressed in MEP and KSL cells, whereas it was up-regulated in GMP. Thus, intracellular ROS level of MEP is kept lowest in hematopoietic cells. Next, we investigated the effect of ROS on the differentiation of myeloid progenitors. In liquid culture assay, loading of ROS with low dose hydrogen peroxide inhibited the differentiation of progenitor cells into MEP, whereas removal of ROS with catalase accelerated the differentiation of those into MEP. Similarly, in the colony-forming assay with semisolid culture medium, we observed that loading of ROS inhibited the formation of megakaryocyte-erythrocyte colonies. To investigate the effect of intrinsic ROS on the colony-forming capacity, we sorted ROS-high and low common myeloid progenitor cells (CMP) individually and cultured them. It was shown that ROS-low CMP had high colony-forming capacity of megakaryocyte-erythrocyte, whereas ROS-high CMP had high colony-forming capacity of granulocyte-monocyte. There is inverse correlation between intracellular ROS level of CMP and colony-forming capacity of megakaryocyte-erythrocyte. In order to confirm that ROS can control the differentiation of CMP into MEP or GMP in vivo, we injected lipopolysaccharide (LPS), which can increase intracellular ROS level of bone marrow cells, into mice. We found that MEP were decreased in LPS-injected mice. Finally, we performed gene expression microarray analysis to compare gene expression profiles between ROS-low and high CMP. In terms of gene expression profiles, ROS-low CMP were similar to MEP and magakaryocyte whereas ROS-high CMP were similar to GMP. In conclusion, these findings suggest that intracellular ROS play a critical role in lineage decision of myeloid progenitor cells, especially in the generation of MEP. In several diseases with anemia or thrombocytopenia, such as myelodysplastic syndrome, Fanconi anemia and autoimmune diseases with chronic inflammation, it was reported that intracellular ROS level of hematopoietic cells was up-regulated. Thus, up-regulated intracellular ROS may be involved in their symptoms via differentiation disorder of MEP. Disclosures:No relevant conflicts of interest to declare.

Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor–related protein 1 (LRP1) on megakaryocytes

AbstractPlatelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor–related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4High). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4High mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.

The Wilms' Tumor Gene WT1 Is Over-Expressed in Immature Leukemia Cells but Not Necessary for Leukemia Development in Mouse Leukemia Models.

The Wilms tumor gene WT1, which is over-expressed in almost all leukemia, is one of the most promising targets for immunotherapy. To clarify which cells express WT1, we generated a knock-in reporter GFP mice (WT1GFP/+) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term hematopoietic stem cells (HSCs) and very few (<1%) of multipotent progenitor cells. WT1 was expressed in 17.4% of megakaryocyte-erythrocyte progenitor cells and 4.1% of common myeloid progenitor cells, while the frequencies of WT1 expressing cells in mature granulocyte or lymphocyte were very low (~1%). In contrast, in murine leukemias induced by TEL/PDGFβR + AML1/ETO or BCR/ABL, WT1 was expressed in 40.5% or 38.9% of c-kit+lin-Sca-1+ (KLS) leukemia cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSC). Furthermore, WT1 was not expressed fetal liver HSC population, mobilized HSCs by cyclophosphamide and granulocyte-colony stimulating factor, or HSCs stimulated with SCF, Flt3L, and IL3 in-vitro. These results suggest that WT1 expression in is not a surrogate marker of cell proliferation. To examine the function of WT1 in leukemogenesis, we used recombinant lentivirus to overexpress WT1. HSCs transduced with either empty or WT1-expressing lentivirus were transplanted into lethally irradiated recipients. The FACS analysis of BM cells at 4 months post transplant showed very low chimerism of WT1-expressing cells compared to control GFP-expressing cells. Moreover, differentiation block of WT1-expressing cells was not observed. These results indicate that WT1 does not induce proliferation or block differentiation in-vivo. Next, we tested whether leukemias could be induced from the WT1-deficient fetal liver hematopoietic cells. All of three mice transplanted with E13.5 WT1-defeicient fetal liver cells transduced with TEL/PDGFβR+AML1/ETO developed leukemia. Similarly, 3 out of 4 mice transplanted with p210BCR/ABL-transduced WT1-deficient fetal liver cells developed leukemia. These results indicate that WT1 is not necessary for leukemia development in two leukemia models examined. Collectively, WT1 is expressed on immature leukemic cells but not normal HSCs, highlighting its potential as a specific target for therapy. However, the exact function of WT1 in leukemogenesis remains unclear from these studies.

Impaired maturation of myeloid progenitors in mice lacking novel Polycomb group protein MBT-1

Polycomb group (PcG) proteins participate in DNA-binding complexes with gene-repressing activity, many of which have been highlighted for their involvement in hematopoiesis. We have identified a putative PcG protein, termed MBT-1, that is associated with Rnf2, an in vivo interactor of PcG proteins. MBT-1 structurally resembles the H-L(3)MBT protein, whose deletion is predicted to be responsible for myeloid hematopoietic malignancies. The human MBT-1 gene is located on chromosome 6q23, a region frequently deleted in leukemia cells, and shows a transient expression spike in response to maturation-inducing stimuli in myeloid leukemia cells. MBT-1(-/-) myeloid progenitor cells exhibit a maturational deficiency but maintain normal proliferative activities. This results in the accumulation of immature myeloid progenitors and hence, a marked decrease of mature myeloid blood cells, causing the MBT-1(-/-) mice to die of anemia during a late embryonic stage. Together, we conclude that MBT-1 specifically regulates the maturational advancement of myeloid progenitor cells during transitions between two developmental stages. We also show that MBT-1 appears to influence myelopoiesis by transiently enhancing p57(KIP2) expression levels.