Bok Promotes Erythropoiesis in a Mouse Model of Myelodysplastic Syndrome

Abstract BCL-2 Ovarian Killer (BOK) is a pro-apoptotic member of the BCL-2 family of proteins best characterized for its putative ability to induce apoptosis in response to Endoplasmic Reticulum (ER) stress, when stabilized from ER-associated degradation (ERAD). Although ER stress appropriately activates the unfolded protein response (UPR) in BOK-disrupted cells, as measured by PERK and eIF2-alpha phosphorylation, downstream effector signaling, including ATF4 and CHOP, is defective. A functional role for BOK as a tumor suppressor is suggested by its genetic location in one of the 20 most frequent, focally deleted chromosomal regions across all human cancers. To evaluate the consequences of BOK loss in the pathogenesis of myelodysplasia (MDS) and Acute Myeloid Leukemia (AML), we used the Nup98-HoxD13 (NHD13) transgenic mouse model of MDS/AML. In this model, both overexpression of anti-apoptotic BCL-2 and deletion of pro-apoptotic PUMA rescue cytopenias, but surprisingly delay progression to AML. In contrast, AML developed in 36.7% of NHD13 mice lacking BOK between the age of 8 and 13 months with a similar overall survival to the NHD13 mice. However, the loss of BOK exacerbated the anemia of the NHD13 mice, which raised a potential connection between BOK and the regulation of erythropoiesis in cells experiencing stress from the NHD13 translocation. NHD13 mice deficient for BOK exhibited significantly lower hemoglobin (Hb), lower mean cell hemoglobin concentration (MCHC) and higher mean cell volume (MCV) than NHD13 mice, whereas other lineages were unaffected. Mouse colony forming unit assays revealed there is a decreased amount of erythroid progenitor stem cells (BFU-E) in the bone marrow of NHD13-transgenic/BOK-deficient mice, which hinted at a diminished ability to produce RBCs in the absence of BOK. Isolation of various stages of erythroid progenitors in the bone marrow by CD44/TER119 FACS sorting revealed that both NHD13 and NHD13-transgenic/BOK-deficient mice have an increase in proerythroblasts relative to more mature red blood cells. Preliminary RT-QPCR analysis shows decreased expression of UPR components in the RBC progenitors of both BOK-deficient and NHD13-transgenic/BOK-deficient mice. Interestingly, CHOP is not only a component of the UPR, but also an erythropoietin target gene necessary for erythroid differentiation. These results suggest that in addition to its pro-apoptotic function, BOK may have other regulatory roles within the cell, and specifically a role in regulating erythropoiesis when certain RBC progenitors experience ER stress. Disclosures Katz: Gene-in-Cell: Equity Ownership.

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Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes

Blood ◽

10.1182/blood.v59.3.646.646 ◽

1982 ◽

Vol 59 (3) ◽

pp. 646-651 ◽

Cited By ~ 8

Author(s):

D Brookoff ◽

L Maggio-Price ◽

S Bernstein ◽

L Weiss

Keyword(s):

Bone Marrow ◽

Membrane Protein ◽

Stromal Cells ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Plasma Clot ◽

Dark Cells ◽

Erythroid Precursors ◽

The Status ◽

Deficient Mice

Abstract In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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Selective Expression of the Receptor Tyrosine Kinase, HTK, on Human Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v89.8.2757 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2757-2765 ◽

Cited By ~ 32

Author(s):

Tomohisa Inada ◽

Atsushi Iwama ◽

Seiji Sakano ◽

Mitsuharu Ohno ◽

Ken-ichi Sawada ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Bone Marrow Cells ◽

Regulatory System ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Marrow Cells

Abstract HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34low, and glycophorin A−/low. Assays of progenitors showed that HTK+c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK−c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

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RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v118.21.610.610 ◽

2011 ◽

Vol 118 (21) ◽

pp. 610-610 ◽

Cited By ~ 2

Author(s):

Rajasekhar NVS Suragani ◽

Aaron Mulivor ◽

R. Scott Pearsall ◽

Ravindra Kumar

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Supportive Care ◽

Red Blood Cells ◽

Blood Cells ◽

Significant Proportion ◽

Erythroid Differentiation ◽

Wild Type ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Abstract Abstract 610 Myelodysplatic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Patients develop peripheral blood cytopenias; however, the bone marrow shows increased proliferation and apoptosis. In addition to bone marrow apoptosis, a failure of differentiation contributes to reduced terminally differentiated blood cells. A significant proportion of patients with MDS will develop anemia that are refractory to treatment with recombinant human erythropoietin (EPO) and must rely on transfusions as supportive care. The use of blood transfusions as supportive care is associated with iron overload and significant morbidity. Therefore, alternative therapies to treat anemia in MDS patients are needed. Members of the TGFβ super family of signaling molecules have been implicated in erythropoiesis and represent alternative, EPO-independent targets for the treatment of anemia. ACE-536 is a soluble receptor fusion protein consisting of a modified Activin Receptor Type IIB extracellular domain linked to a human Fc domain. ACE-536 acts as a ligand trap to modulate the activity of TGFβ ligands and promote erythroid differentiation in an EPO independent manner. Subcutaneous administration of ACE-536 to C57BL/6 mice resulted in significant increases in hematocrit, hemoglobin and red blood cells compared to vehicle treated controls within four days. These effects were observed with concurrent treatment of an EPO neutralizing antibody, indicating that EPO is not directly responsible for the initial RBC response of ACE-536. BFU-E or CFU-E colony formation assays from bone marrow or spleen of mice 48 hours after ACE-536 were normal, indicating no effect on the erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by FACS analysis following 72 hours after RAP-536 (murine version of ACE-536) treatment revealed a decrease in basophilic erythroblasts and an increase in late stage poly-, ortho-chromatophilic and reticulocytes in bone marrow and spleen compared to vehicle treated mice. The data demonstrate that while EPO treatment increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. The efficacy of ACE-536 has been demonstrated in various animal models of acute and chronic anemia. In this study we investigated the effect of ACE-536 on anemia in mouse model of MDS. The NUP98-HOXD13 (NHD13) transgenic mouse carries a common translocation found in MDS patients. NHD13 mice develop anemia, neutropenia and lymphopenia at 4–7 months of age, with normal or hypercellular bone marrow. Starting at 4 months of age, mice were treated with RAP-536 (murine homolog of ACE-536) at 10 mg/kg or vehicle control twice per week for 8 months. Wild-type littermate controls were also dosed on the same schedule. As expected, at study baseline (mice 4 months of age), NHD13 mice had reduced RBC, Hb and HCT compared to wild-type littermates. The progression of anemia over the study period was reduced by treatment with RAP-536 compared to vehicle (HCT: −8.3% v. −22%, RBC: −13% v. −30%). Based on blood smear analyses, there was no indication of increased leukemic cells with ACE-536 treatment. Our data demonstrate that RAP-536 can increase hematology parameters through enhancing maturation of terminally differentiated red blood cells and can serve as a therapeutic molecule for the treatment of anemia. As anemia contributes significantly to the morbidity of patients with MDS, a mouse model was used to test the therapeutic efficacy of ACE-536 in this disease. We have shown that systemic administration of RAP-536 to MDS mice promotes increases in red blood cell mass without enhanced progression to AML. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. Disclosures: Suragani: Acceleron Pharma Inc: Employment. Mulivor:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment. Kumar:Acceleron Pharma Inc: Employment.

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Specific Domains of Fibronectin Mediate Adhesion and Migration of Early Murine Erythroid Progenitors

Blood ◽

10.1182/blood.v90.1.138 ◽

1997 ◽

Vol 90 (1) ◽

pp. 138-147 ◽

Cited By ~ 19

Author(s):

Kristin L. Goltry ◽

Vikram P. Patel

Keyword(s):

Bone Marrow ◽

Erythroid Differentiation ◽

Heparin Binding ◽

Erythroid Cells ◽

Cell Binding ◽

Erythroid Progenitors ◽

Adhesive Properties ◽

Erythroid Progenitor ◽

And Migration ◽

Carboxy Terminal

Abstract The binding of late stage erythroid cells to fibronectin (FN) has been well characterized and is believed to be critical for the terminal stages of erythroid differentiation, but the adhesive properties of more primitive murine erythroid progenitors and the role of these interactions during earlier stages of erythropoiesis has not been determined. Using chymotryptic fragments and inhibitory probes, we have tested the ability of each of the major cell binding domains of FN; the RGDS sequence, the CS-1 sequence, and the carboxy-terminal heparin-binding domain (HBD), to promote adhesion of primitive burst-forming unit-erythroid (BFU-E), mature BFU-E, and colony-forming unit-erythroid (CFU-E). We found that only 10% to 15% of BFU-E bound to FN or to the RGDS sequence in contrast to 75% to 85% of CFU-E. Approximately 50% to 70% of BFU-E and 60% to 80% of CFU-E bound to the carboxy-terminal HBD and to the CS-1 sequence. The binding of BFU-E and CFU-E to the RGDS and CS-1 sites was blocked by β1 integrin antibodies. These results suggest that binding to FN determinants is developmentally regulated during early erythroid differentiation. Erythroid progenitor migration within the bone marrow is thought to be important for the eventual release of reticulocytes into the circulation. A correlation between FN binding and the migratory capacity of erythroid cells has been suggested, although the ability of FN to promote migration of erythroid progenitors has not been directly measured. We measured migration of CFU-E on fragments of FN containing each cell binding region. CS-1–containing fragments, in addition to promoting adhesion of both BFU-E and CFU-E, supported the highest levels of CFU-E migration (11-fold above background). Migration was sixfold above background on intact FN and only threefold above background on RGDS-containing fragments. Fragments containing HBD alone, although they promoted adhesion of CFU-E, failed to support significant levels of migration. These results show that specific domains of FN possess distinct adhesion- and migration-promoting properties for murine erythroid progenitors. Regulation of the adhesive properties during erythroid differentiation may alter the ability of progenitors to migrate in the bone marrow and thus play an important role in normal murine erythroid differentiation.

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Specific Domains of Fibronectin Mediate Adhesion and Migration of Early Murine Erythroid Progenitors

Blood ◽

10.1182/blood.v90.1.138.138_138_147 ◽

1997 ◽

Vol 90 (1) ◽

pp. 138-147 ◽

Cited By ~ 1

Author(s):

Kristin L. Goltry ◽

Vikram P. Patel

Keyword(s):

Bone Marrow ◽

Erythroid Differentiation ◽

Heparin Binding ◽

Erythroid Cells ◽

Cell Binding ◽

Erythroid Progenitors ◽

Adhesive Properties ◽

Erythroid Progenitor ◽

And Migration ◽

Carboxy Terminal

The binding of late stage erythroid cells to fibronectin (FN) has been well characterized and is believed to be critical for the terminal stages of erythroid differentiation, but the adhesive properties of more primitive murine erythroid progenitors and the role of these interactions during earlier stages of erythropoiesis has not been determined. Using chymotryptic fragments and inhibitory probes, we have tested the ability of each of the major cell binding domains of FN; the RGDS sequence, the CS-1 sequence, and the carboxy-terminal heparin-binding domain (HBD), to promote adhesion of primitive burst-forming unit-erythroid (BFU-E), mature BFU-E, and colony-forming unit-erythroid (CFU-E). We found that only 10% to 15% of BFU-E bound to FN or to the RGDS sequence in contrast to 75% to 85% of CFU-E. Approximately 50% to 70% of BFU-E and 60% to 80% of CFU-E bound to the carboxy-terminal HBD and to the CS-1 sequence. The binding of BFU-E and CFU-E to the RGDS and CS-1 sites was blocked by β1 integrin antibodies. These results suggest that binding to FN determinants is developmentally regulated during early erythroid differentiation. Erythroid progenitor migration within the bone marrow is thought to be important for the eventual release of reticulocytes into the circulation. A correlation between FN binding and the migratory capacity of erythroid cells has been suggested, although the ability of FN to promote migration of erythroid progenitors has not been directly measured. We measured migration of CFU-E on fragments of FN containing each cell binding region. CS-1–containing fragments, in addition to promoting adhesion of both BFU-E and CFU-E, supported the highest levels of CFU-E migration (11-fold above background). Migration was sixfold above background on intact FN and only threefold above background on RGDS-containing fragments. Fragments containing HBD alone, although they promoted adhesion of CFU-E, failed to support significant levels of migration. These results show that specific domains of FN possess distinct adhesion- and migration-promoting properties for murine erythroid progenitors. Regulation of the adhesive properties during erythroid differentiation may alter the ability of progenitors to migrate in the bone marrow and thus play an important role in normal murine erythroid differentiation.

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The Anemic Friend Virus gp55 Envelope Protein Induces Erythroid Differentiation in Fetal Liver Colony-Forming Units-Erythroid

Blood ◽

10.1182/blood.v91.4.1163 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1163-1172 ◽

Cited By ~ 27

Author(s):

Stefan N. Constantinescu ◽

Hong Wu ◽

Xuedong Liu ◽

Wendy Beyer ◽

Amy Fallon ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Liver Cells ◽

Erythropoietin Receptor ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Fetal Liver Cells

Abstract The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR−/− fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.

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Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010535 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3906-3917 ◽

Cited By ~ 2

Author(s):

Aaron M. Wortham ◽

Devorah C. Goldman ◽

Juxing Chen ◽

William H. Fleming ◽

An-Sheng Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Transferrin Receptor ◽

Function Analysis ◽

Hereditary Hemochromatosis ◽

The Body ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Deficient Mice ◽

Transferrin Receptor 2

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit–erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit–erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

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CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation

Blood ◽

10.1182/blood.v124.21.2149.2149 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2149-2149

Author(s):

Qi Cai ◽

Robin Jeannet ◽

Hongjun Liu ◽

ya-Huei Kuo

Keyword(s):

Mouse Model ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Altered Expression

Abstract Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice. To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b. The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation. In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. Disclosures No relevant conflicts of interest to declare.

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Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes

Blood ◽

10.1182/blood.v59.3.646.bloodjournal593646 ◽

1982 ◽

Vol 59 (3) ◽

pp. 646-651 ◽

Cited By ~ 1

Author(s):

D Brookoff ◽

L Maggio-Price ◽

S Bernstein ◽

L Weiss

Keyword(s):

Bone Marrow ◽

Membrane Protein ◽

Stromal Cells ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Plasma Clot ◽

Dark Cells ◽

Erythroid Precursors ◽

The Status ◽

Deficient Mice

In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.

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