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

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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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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TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽

10.1182/blood.v124.21.2912.2912 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2912-2912

Author(s):

Petros Papadopoulos ◽

Laura Gutierrez ◽

Jeroen Demmers ◽

Dimitris Papageorgiou ◽

Elena Karkoulia ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells ◽

Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

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Bok Promotes Erythropoiesis in a Mouse Model of Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v130.suppl_1.922.922 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 922-922

Author(s):

Seong-Ho Kang ◽

Oscar Perales ◽

Michael Michuad ◽

Samuel G. Katz

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Er Stress ◽

Hemoglobin Concentration ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Mean Cell Volume ◽

The Unfolded Protein Response ◽

Deficient Mice

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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Runx1 promotes murine erythroid progenitor proliferation and inhibits differentiation by preventing Pu.1 downregulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901122116 ◽

2019 ◽

Vol 116 (36) ◽

pp. 17841-17847 ◽

Cited By ~ 2

Author(s):

Michael A. Willcockson ◽

Samuel J. Taylor ◽

Srikanta Ghosh ◽

Sean E. Healton ◽

Justin C. Wheat ◽

...

Keyword(s):

Ectopic Expression ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Chromatin Accessibility ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Down Regulation ◽

Erythroid Progenitor ◽

Terminal Erythroid Differentiation ◽

In Erythroid Cells

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.

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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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Erythroid Development in the Absence of Hemoglobin.

Blood ◽

10.1182/blood.v110.11.1262.1262 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1262-1262

Author(s):

Shan-Run Liu ◽

Sean C. McConnell ◽

Yongliang Huo ◽

Ting-Ting Zhang ◽

Rui Yang ◽

...

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Es Cells ◽

Embryonic Stem ◽

Globin Genes ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Stage Of Development

Abstract The mammalian erythrocyte is a highly specialized blood cell that differentiates via an orderly series of committed progenitors in the bone marrow in a process termed erythropoiesis. Homeostasis of the erythron is carefully maintained by balancing the proliferation and destruction of early and late erythroid progenitors. In mature red blood cells over ninety-five percent of the protein is hemoglobin (Hb). What happens to committed erythroid cells in the absence of hemoglobin? To answer this question we have derived a novel line of embryonic stem (ES) cells from mouse embryos that have all eight adult alpha and beta globin genes knocked out. These “Null” Hb ES cells were injected into wild-type blastocysts to examine their in vivo potential to contribute to the tissues of developing chimeric mice. Examination of the peripheral blood and bone marrow of these chimeras by flow cytometry revealed that the “Null” Hb ES cells were able to produce normal levels of each type of white blood cell analyzed. However, “Null” erythrocytes were absent from the circulation and only early committed progenitors were found in the bone marrow. Very few “Null” erythroid cells matured beyond the proerythoblast to the basophilic erythroblast stage (Ter119low, CD71hi). To study this maturational block in more detail, an erythroid culture system was established by in vitro differentiation of the “Null” Hb ES cells. These pure erythroid progenitor (EP) cultures support and amplify the proerythroblast stage of development. Interestingly, EP cells could be derived from “Null” Hb ES cells demonstrating that Hb is not required for the development of proerythroblasts. “Null” derived EP cells express erythroid lineage markers (EKLF, GATA1, GypA, EpoR, Tal1), but express no adult globins or markers of other hematopoietic lineages (Mpl, GATA3, IL7R, PAX5, CEBPα, CD41b). Upon terminal differentiation most “Null” derived EP cells undergo apoptosis by 48 hours (7AAD−, Annexin V+) and are dead (7AAD+) by 72 hours. These “Null” Hb ES cells provide a novel experimental system to elucidate the role of hemoglobin during erythroid differentiation, maturation, and homeostasis.

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Beta-1 Integrin Controls Homing and Expansion of Erythroid Cells in Stress Erythropoiesis and ß-Thalassemia

Blood ◽

10.1182/blood.v124.21.2661.2661 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2661-2661

Author(s):

Bart Crielaard ◽

Roberta Chessa ◽

Ritama Gupta ◽

Carla Casu ◽

Stefano Rivella

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Cre Recombinase ◽

Blood Parameters ◽

Red Cells ◽

Dramatic Improvement ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Stress Erythropoiesis

Abstract After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β-thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interupting the interaction between macrophages and erythroblasts (macrophage-erythroblast interaction, MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. Targeting MEI, using a number of approaches, caused a significant improvement in blood parameters in β-thalassemia intermedia (BTI) mouse models (Hbbth3/+) and a rapid and dramatic improvement in splenomegaly, an outcome that is relevant for clinical practice. Importantly, MEI is not critical for hematopoiesis under non-stress conditions, and ablation of this interaction in normal mice showed minimal effects on blood parameters. As our initial observations indicate that MEI is essential to support stress erythropoiesis, we investigated adhesion molecules that might activate downstream pathways in erythroblasts that regulate cell proliferation. We also speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta 1 (Itgb1) and also intracellular molecules such as Fak1, Talin1 and Sharpin might play a role in stress erythropoiesis. There is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgb1 and Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgb1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. More specifically, when measuring the content of DNA, we were able to demonstrate that enucleation of erythroblasts was accompanied by a marked loss of Itgb1 expression, indicating that there may be an important role for Itgb1 in erythroblast enucleation, and differentiation in general. Lack of Itgb1 in thalassemic mice prevents erythroid cells from homing to and expanding in the spleen, the major source of chronic stress erythopoiesis in this disorder. In particular, such a role of Itgb1 is supported by our analysis of thalassemic mice in which this molecule was partially depleted by induction of the Cre recombinase. These animals were generated by crossing th3/+ mice with animals in which Itgb1 was floxed and carrying an inducible Cre-recombinase (Mx1-CRE). We utilized the BM of these animals (Hbbth3/+, Itgb1fl/fl, Mx1-CRE) to generate thalassemic animals that expressed the floxed Itgb1 only in hematopietic cells. After serial administration of polyI:C the animals were analyzed for their erythropoiesis in the bone marrow and spleen. Interestingly, all the animals analyzed show chimeric populations of Itgb1 positive and negative erythroid cells in the bone marrow. This indicated that not all the HSCs were successfully depleted of the Itgb1 gene. However, when we investigated Itgb1 in the spleen, we observed only erythroid cells positive for the expression of this adhesion molecule. This last observation strongly suggests that depletion of Itgb1 prevents homing and expansion of erythroid cells in the spleen and drugs that by inhibit Itgb1 could reduce erythroid spleen colonization, splenomegaly and limit erythropoiesis. We are now in the process of identifying compounds that target MEI. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Casu: Merganser Biotech : Research Funding; Isis Pharmaceuticals, Inc.: Research Funding.

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Heterozygous Mutations in DDX41 Cause Erythroid Progenitor Cell Defects

Blood ◽

10.1182/blood-2021-153544 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 148-148

Author(s):

Timothy M Chlon ◽

Emily Stepanchick ◽

Analise Sulentic ◽

Kathleen Hueneman ◽

Daniel Starczynowski

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Progenitor Cells ◽

Liquid Culture ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Colony Assays

Abstract Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

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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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