Blood Cell Development Research Articles

Circulatory Glycan-Modifying Enzymes As Systemic Regulators In Blood Cell Development

Regulating blood cell production is crucial to meeting physiologic demands and to maintaining hematopoietic equilibrium. Elucidating the systemic factors and how they contribute to the interaction of hematopoietic progenitors with their bone marrow microenvironments is crucial to the mechanistic understanding of hematopoietic regulation and development of novel therapeutic strategies. Glycosyltransferases are enzymes that normally reside within the intracellular secretory apparatus to assemble glycans on nascent proteins and lipids in transit, but they are also present in abundance in the extracellular spaces, especially in systemic circulation. One such blood-borne enzyme is the ST6Gal-1 sialyltransferase that mediates the attachment of sialic acids in a6-linkages to glycoproteins. By virtue of their presence on cell surfaces, sialyl-glycans, which are conserved structures in mammals, occupy the critical interphase between cells and their external environment. We hypothesize that remodeling of these cell surface glycans can drastically modulate the communication of systemic cues among marrow hematopoietic cells. A mouse model genetically modified for low circulating ST6Gal-1 has increased numbers of hematopoietic progenitors, increased ability to produce inflammatory cells upon acute challenge, and faster recovery from cyclophosphamide-induced myelosuppression. In ex vivo cultures, addition of ST6Gal-1 strikingly delays expansion and differentiation of murine LSK (Linneg sca-1pos cKitpos) and human CD34pos cells. We developed a subcutaneously implantable B16-F10 model genetically modified to overexpress circulatory ST6Gal-1 (B16st6gal1). Mice bearing B16st6gal1 had >10-fold elevation in circulating ST6Gal-1 levels, accompanied by >50% reduction in marrow granulocyte and B cell populations. B16st6gal1 bearing mice also had 2-fold increase in the number of Linneg cells, suggesting a blockade in development of progenitors into end-effector cells. Murine LSK cell surfaces are decorated with a6-sialic acids but they do not endogenously express ST6Gal-1. Bone chimeras show that the a6-sialic acids on LSK surfaces require the distally synthesized ST6Gal-1 enzyme in circulation. Taken together, our data indicates the glycan-modifying enzyme, the ST6Gal-1 in circulation, is a potent systemic regulator of hematopoiesis by remodeling of cell surface glycan architecture of marrow hematopoietic stem and progenitors. (Funded by NIH Program of Excellence in Glycosciences award P01HL107146 and NIH R01AI56082) Disclosures:No relevant conflicts of interest to declare.

Epo-Induced Erythroid Maturation Is Dependent On Plcγ1 Signalling

Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis.Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44high 21% vs. Plcγ1 shRNA CD44high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p<0.001) - when compared to control cells. Flow cytometry analysis of the colonies revealed a higher amount of immature populations (CD44high, KIT+) in PLCγ1-deficient cells as compared to controls whereas the content of TER119+ cells, reflecting more mature erythroid cells, was higher in controls.To elucidate on the mechanism of Plcγ1-mediated regulation of erythroid development, we performed global gene expression analysis in I/11 cells at various time points of differentiation after knockdown of Plcγ1. Several of the genes that change expression in the absence of Plcγ1 can be classified as transcription/co-transcription factors, epigenetic regulators, metabolic factors or adaptor molecules involved in intracellular signaling. Thus, Plcγ1-deficient cells showed up-regulation of the transcription factor RUNX1 and the adaptor molecule GRAP2 over time compared to controls whereas the epigenetic regulator H2AFY2 was significantly decreased. Stimulated by our observation that profound changes in global gene expression also included the epigenetic machinery (H2afy2), we speculated whether Plcγ1 signalling also modifies the global epigenetic landscape of I/11 pro-erythroblasts. Therefore, we performed genome-wide DNA methylome analysis in I/11 cells upon Plcγ1 knockdown using MCIP-seq (methyl-CpG immunoprecipitation combined with next-generation sequencing). The observed methylation changes were by far dominated by an apparent hypomethylation of differentially methylated regions (DMRs) in Plcγ1 knockdown cells as compared to control cells. In line with this, gene ontology analysis of DMRs revealed a highly significant enrichment of biological terms associated with developmental processes and cell differentiation.Taken together, our findings provide evidence for an essential role of Plcγ1 in regulating erythroid differentiation through alteration of the transcriptomic and epigenetic landscape. Disclosures:No relevant conflicts of interest to declare.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures:No relevant conflicts of interest to declare.

Functional Consequences Of RPS29 Germline Mutations In Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risks of cancer. Approximately half of DBA cases have a germline mutation or deletion in a ribosomal protein gene (i.e., RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26), or in GATA1, an X-linked hematopoietic transcription factor. The inheritance of mutated ribosomal protein genes is autosomal dominant (AD), but de novo germline mutations may also occur. We previously reported a novel nonsynonymous ribosomal protein S29 (RPS29) mutation, I31F, which segregated with DBA in a large multi-case family (NCI-193). We now present functional data that link this RPS29 mutation to the DBA phenotype and report an additional large multi-case DBA family (NCI-38) with another novel RPS29 mutation.Individuals in this study are participants in the IRB-approved longitudinal cohort IBMFS study at the NCI. We have identified a second large family with DBA, NCI-38, in which the male proband had steroid-responsive anemia as a child, elevated erythrocyte adenosine deaminase (eADA = 1.26 IU/g Hb, normal <0.96), and has been in remission for approximately 20 years. Two unaffected male half-siblings have no symptoms of DBA, and 1 female half-sibling has only a borderline elevated eADA (1.00 IU/g Hb). The proband's mother is an asymptomatic obligate carrier because her full brother and two half-siblings have DBA or an unspecified bone marrow failure; her eADA is normal (0.8 IU/g Hb). We performed whole-exome sequencing using Nimblegen v2 capture arrays and paired-end sequencing on the Illumina HiSeqTM. After applying quality control filters, removing variants present in publicly available databases (1000Genomes, ESP, Kaviar, and dbSNP), and applying an AD inheritance model, we identified an I50T mutation in RPS29 that tracked with AD inheritance in this family (NCI-38). The sibling with elevated eADA and the mother also carried this RPS29 mutation while the two asymptomatic healthy siblings were mutation negative. I50T, like I31F, occurs in a very highly evolutionarily conserved region of the RPS29 protein and in silico prediction programs (i.e., SIFT, Polyphen 2, Condel, MutationTaster) predict this variant to be deleterious.The original DBA family, NCI-193, has AD DBA due to the heterozygous I31F mutation in RPS29. We used quantitative rtPCR to determine RPS29 expression in the proband with the I31F mutation compared with a healthy individual with wildtype RPS29. The proband demonstrated haploinsufficiency of RPS29 mRNA, with levels consistent with approximately 50% expression compared with wildtype RPS29 values. Zebrafish are ideal for modeling ribosomal protein knockdown because hematopoietic regulation is conserved with mammals, they are amenable to high throughput in vivo genetics, and ribosomal proteins can be knocked down in the embryo with morpholino technology. The rps29-/- mutant zebrafish displays many aspects of the DBA phenotype including significant defects in red blood cell development, shown by a decrease in hemoglobin levels. We used this as a model to determine if the RPS29 mutation identified in our DBA cases could rescue the hemoglobin defect. The rps29-/- zebrafish embryos were injected with 50pg of either wildtype or the mutated (I31F) human RPS29 RNA. The wildtype human RPS29 RNA rescued the hemoglobin phenotype (increased hemoglobin levels) of the rps29-/- zebrafish embryos, whereas the mutated human RPS29 RNA could not (no increase in hemoglobin levels; P <0.01).In summary, germline mutations in RPS29 can cause AD DBA. RPS29 is an essential protein in the 40s small subunit of the ribosome, important for ribosomal RNA processing and ribosome biogenesis. Using functional assays, we show for the first time that the mutant RPS29 in our DBA cases results in haploinsufficiency of RPS29. The patient-associated I31F RPS29 mutation failed to rescue the defective hematopoiesis in the rps29-/- mutant zebrafish DBA model and provides further evidence that RPS29 is a DBA-associated gene. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Effect of birth weight on the association between necrotising enterocolitis and red blood cell transfusions in ≤1500 g infants

ContextReports evaluating a possible association between necrotising enterocolitis (NEC) and blood transfusion have been predominantly case–control studies. As the possible associations of disease with any variable on which cases and...

HIV-1 infection of hematopoietic progenitor cells in vivo in humanized mice

AbstractHIV infection has been associated with defective hematopoiesis since the earliest days of the HIV/AIDS epidemic. Generation of all hematopoietic lineages suffers in the face of infection. The mechanisms by which HIV impairs normal blood cell development remain unclear, and direct infection of intermediate hematopoietic progenitors has not been established as a source of HIV-associated hematopoietic pathology. Here, we demonstrate infection of multiple subsets of highly purified intermediate hematopoietic progenitors by wild-type HIV both in vitro and in vivo. Although direct infection is clearly cytotoxic, we find that some infected progenitors can survive and harbor proviral DNA. We report intermediate hematopoietic progenitors to be a novel target of infection and their permissivity to infection increases with development. Further, the nonobese diabetic severe combined immunodeficiency common γ chain knockout-bone marrow-liver-thymus humanized mouse provides a unique model for studying the impact of HIV infection on bone marrow–based human hematopoiesis.

A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

Transcriptional networks orchestrate complex developmental processes, and such networks are commonly instigated by master regulators for development. By now, considerable progress has been made in elucidating GATA factor-dependent genetic networks that control red blood cell development. Here we reported that GATA-1 and GATA-2 co-regulated the expression of two microRNA genes, microRNA-27a and microRNA-24, with critical roles in regulating erythroid differentiation. In general, GATA-2 occupied the miR-27a∼24 promoter and repressed their transcription in immature erythroid progenitor cells. As erythropoiesis proceeded, GATA-1 directly activated miR-27a∼24 transcription, and this involved a GATA-1-mediated displacement of GATA-2 from chromatin, a process termed ‘GATA switch’. Furthermore, the mature miR-27a and miR-24 cooperatively inhibited GATA-2 translation and favoured the occupancy switch from GATA-2 to GATA-1, thus completing a positive feedback loop to promote erythroid maturation. In line with the essential role of GATA factors, ectopic expression of miR-27a or miR-24 promoted erythropoiesis in human primary CD34+ haematopoietic progenitor cells and mice, whereas attenuated miR-27 or miR-24 level led to impaired erythroid phenotypes in haematopoietic progenitor cells and zebrafish. Taken together, these data integrated micro RNA expression and function into GATA factor coordinated networks and provided mechanistic insight into a regulatory circuit that comprised GATA1/2 switch and miR-27a/24 in erythropoiesis.

Establishing a hematopoietic genetic network through locus-specific integration of chromatin regulators

The establishment and maintenance of cell type-specific transcriptional programs require an ensemble of broadly expressed chromatin remodeling and modifying enzymes. Many questions remain unanswered regarding the contributions of these enzymes to specialized genetic networks that control critical processes, such as lineage commitment and cellular differentiation. We have been addressing this problem in the context of erythrocyte development driven by the transcription factor GATA-1 and its coregulator Friend of GATA-1 (FOG-1). As certain GATA-1 target genes have little to no FOG-1 requirement for expression, presumably additional coregulators can mediate GATA-1 function. Using a genetic complementation assay and RNA interference in GATA-1-null cells, we demonstrate a vital link between GATA-1 and the histone H4 lysine 20 methyltransferase PR-Set7/SetD8 (SetD8). GATA-1 selectively induced H4 monomethylated lysine 20 at repressed, but not activated, loci, and endogenous SetD8 mediated GATA-1-dependent repression of a cohort of its target genes. GATA-1 used different combinations of SetD8, FOG-1, and the FOG-1-interacting nucleosome remodeling and deacetylase complex component Mi2β to repress distinct target genes. Implicating SetD8 as a context-dependent GATA-1 corepressor expands the repertoire of coregulators mediating establishment/maintenance of the erythroid cell genetic network, and provides a biological framework for dissecting the cell type-specific functions of this important coregulator. We propose a coregulator matrix model in which distinct combinations of chromatin regulators are required at different GATA-1 target genes, and the unique attributes of the target loci mandate these combinations.

Implication of IRF4 Aberrant Gene Expression in the Acute Leukemias of Childhood

The most frequent targets of genetic alterations in human leukemias are transcription factor genes with essential functions in normal blood cell development. The Interferon Regulatory Factor 4 (IRF4) gene encodes a transcription factor important for key developmental stages of hematopoiesis, with known oncogenic implications in multiple myeloma, adult leukemias and lymphomas. Very few studies have reported an association of IRF4 with childhood malignancy, whereas high transcript levels have been observed in the more mature immunophenotype of ALL. Our aim was to investigate the expression levels of IRF4 in the diagnostic samples of pediatric leukemias and compare them to those of healthy controls, in order to determine aberrant gene expression and whether it extends to leukemic subtypes other than the relatively mature ALL subpopulation. Quantitative real-time RT-PCR methodology was used to investigate IRF4 expression in 58 children with acute leukemias, 4 leukemic cell lines and 20 healthy children. We show that aberrant IRF4 gene expression is implicated in a variety of leukemic subtypes; higher transcript levels appear in the more immature B-common ALL subtype and in T-cell than in B-cell leukemias, with the highest expression levels appearing in the AML group. Interestingly, we show that childhood leukemia, irrespective of subtype or cell maturation stage, is characterised by a minimum of approximately twice the amount of IRF4 gene expression encountered in healthy children. A statistically significant correlation also appeared to exist between high IRF4 expression and relapse. Our results show that ectopic expression of IRF4 follows the reverse expression pattern of what is encountered in normal B-cell development and that there might be a dose-dependency of childhood leukemia for aberrantly expressed IRF4, a characteristic that could be explored therapeutically. It is also suggested that high IRF4 expression might be used as an additional prognostic marker of relapse at diagnosis.

Potential Pathological and Functional Links Between Long Noncoding RNAs and Hematopoiesis

Differential abundance and activity of long noncoding RNAs (lncRNAs) are recognized as the hallmark features in various diseases. We highlight the lncRNAs that play a functional role in the development of blood cells. Many lncRNAs and the protein complexes within which they interact have been implicated in various types of cancers. Multiple lncRNAs participate in normal and may be implicated in malignant hematopoiesis associated with blood cell cancers, such as leukemia, by regulating gene expression through such mechanisms as redirecting chromatin remodeling complexes and activating epigenetic silencing, either of which can inactivate tumor suppressor genes or activate oncogenes. Because of their potential importance in cancers of the blood, lncRNAs may be useful as diagnostic and prognostic markers, and it may be possible to develop lncRNA-mediated therapy.

Transcriptional hierarchies regulating early blood cell development

Hematopoiesis represents one of the paradigmatic systems for studying stem cell biology, but our understanding of how the hematopoietic system develops during embryogenesis is still incomplete. While many lessons have been learned from studying the mouse embryo, embryonic stem cells have come to the fore as an alternative and more tractable model to recapitulate hematopoietic development. Here we review what is known about the embryonic origin of blood from these complementary systems and how transcription factor networks regulate the emergence of hematopoietic tissue from the mesoderm. Furthermore, we have performed an integrated analysis of genome-wide microarray and ChIP-seq data sets from mouse embryos and embryonic stem (ES) cell lines deficient in key regulators and demonstrate how this type of analysis can be used to reconstruct regulatory hierarchies that both confirm existing regulatory linkages and suggest additional interactions.

Erythroid development in the mammalian embryo

Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation

AbstractThe evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell–based, chemically-defined, serum and feeder cell–free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice–compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

Delineating the mixed lineage leukemia gene expression network in hematopoietic stem cells

Hematopoiesis is maintained and sustained throughout life by rare hematopoietic stem cells (HSCs). The hematopoietic system is organized in a hierarchy with the primitive HSCs at the top, generating mature progeny through a series of intermediate and progressively restricted progenitor cells. Normal blood cell development is a tightly controlled process, regulated by transcription factors and epigenetic regulators internally and by cytokines and cellular interactions in the bone marrow microenvironment. Perturbation of the critical molecular regulators is associated often with malignancy of the hematopoietic system, such as leukemia. A key transcriptional regulator in HSCs, the mixed lineage leukemia (MLL) gene, is commonly mutated in both myeloid and lymphoid leukemia (1). MLL is the mammalian ortholog of Drosophila melanogaster Trithorax and widely expressed in multiple organs and tissues including the hematopoietic system and neuronal and vascular systems (2⇓–4). It belongs to a family of histone methyltransferases, where the MLL protein specifically methylates histone H3 on lysine 4 (H3K4), a mark associated with active transcription. MLL is included in a larger multiprotein complex containing protein with chromatin modification and remodeling functions (5). The MLL gene is frequently rearranged through chromosomal translocations, most notably in >70% of cases of infant leukemia and in 10% of all acute myelogeneous leukemia (AML) and acute lymphoblastic leukemia (ALL) (5, 6). The presence of MLL rearrangement in leukemia is generally associated with a poor prognosis. The chromosomal translocations result in MLL fusion proteins that lead to aberrant target gene expression. …

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.

The cell-mediated immunity of Drosophila melanogaster: Hemocyte lineages, immune compartments, microanatomy and regulation

In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom.

The lasting influence of LSD1 in the blood

An enzyme called LSD1 that controls the development of blood cells by manipulating gene expression in progenitor cells could be a therapeutic target for leukemia.

Abstract 761: Importance of protein interactions in mediating AF9/MLLT3 function.

Abstract AF9/MLLT3, located on chromosome band 9p22, was first identified as a fusion partner of the Mixed Lineage Leukemia (MLL) gene in acute myeloid leukemia. The AF9 protein has been shown to play an important role in erythrocyte/ megakaryocyte development by regulating the expression of genes encoding hematopoietic transcription factors. Moreover, in hematopoietic precursor cells, expression of a chimeric protein comprised of the C-terminus of AF9 fused to the N-terminus of MLL leads to leukemic transformation. Thus AF9 contributes both to normal as well as neoplastic blood cell development. Our lab and others have shown the direct or indirect association of the C-terminus of AF9 with several distinct proteins including activators and repressors of transcription (AF4, pTEFb, DOT1L, CBX8, RING1b, and BCoR), thus suggesting that the function of AF9 can be substantially changed depending upon the nature of molecules that specifically associate with it. We hypothesize that AF9 is an important component of several distinct macromolecular complexes, each of which has a unique function. Specifically, DOT1L is a chromatin modifying enzyme that methylates histone H3 at lysine 79. CBX8 is component of the repressive Polycomb PRC1 complexes. Here, we report that the association of AF9 with CBX8 competes with DOT1L for AF9 binding and alters expression of genes regulated by DOT1L. In this context, CBX8 enhances rather than represses gene expression. Citation Format: Bhavna Malik, Charles S. Hemenway. Importance of protein interactions in mediating AF9/MLLT3 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 761. doi:10.1158/1538-7445.AM2013-761 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time.