Megakaryocyte Gene Expression Research Articles

Pro-inflammatory megakaryocyte gene expression in murine models of breast cancer.

Despite abundant research demonstrating that platelets can promote tumor cell metastasis, whether primary tumors affect platelet-producing megakaryocytes remains understudied. In this study, we used a spontaneous murine model of breast cancer to show that tumor burden reduced megakaryocyte number and size and disrupted polyploidization. Single-cell RNA sequencing demonstrated that megakaryocytes from tumor-bearing mice exhibit a pro-inflammatory phenotype, epitomized by increased Ctsg, Lcn2, S100a8, and S100a9 transcripts. Protein S100A8/A9 and lipocalin-2 levels were also increased in platelets, suggesting that tumor-induced alterations to megakaryocytes are passed on to their platelet progeny, which promoted in vitro tumor cell invasion and tumor cell lung colonization to a greater extent than platelets from wild-type animals. Our study is the first to demonstrate breast cancer–induced alterations in megakaryocytes, leading to qualitative changes in platelet content that may feedback to promote tumor metastasis.

Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells.

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

Gene and protein expression in human megakaryocytes derived from induced pluripotent stem cells

BackgroundThere is interest in deriving megakaryocytes (MKs) from pluripotent stem cells (iPSC) for biological studies. We previously found that genomic structural integrity and genotype concordance is maintained in iPSC‐derived MKs. ObjectiveTo establish a comprehensive dataset of genes and proteins expressed in iPSC‐derived MKs. MethodsiPSCs were reprogrammed from peripheral blood mononuclear cells (MNCs) and MKs were derived from the iPSCs in 194 healthy European American and African American subjects. mRNA was isolated and gene expression measured by RNA sequencing. Protein expression was measured in 62 of the subjects using mass spectrometry. Results and ConclusionsMKs expressed genes and proteins known to be important in MK and platelet function and demonstrated good agreement with previous studies in human MKs derived from CD34+ progenitor cells. The percent of cells expressing the MK markers CD41 and CD42a was consistent in biological replicates, but variable across subjects, suggesting that unidentified subject‐specific factors determine differentiation of MKs from iPSCs. Gene and protein sets important in platelet function were associated with increasing expression of CD41/42a, while those related to more basic cellular functions were associated with lower CD41/42a expression. There was differential gene expression by the sex and race (but not age) of the subject. Numerous genes and proteins were highly expressed in MKs but not known to play a role in MK or platelet function; these represent excellent candidates for future study of hematopoiesis, platelet formation, and/or platelet function.

FLI1 Induces Megakaryopoiesis Gene Expression Through WAS/WIP-Dependent and Independent Mechanisms; Implications for Wiskott-Aldrich Syndrome.

Wiskott–Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the WAS gene, which together with its homolog, N-WASP, regulates actin cytoskeleton remodeling and cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of WAS and its binding partner WIP. Depletion of either WAS or WIP in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of WAS/WIP also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott–Aldrich syndrome.

SON inhibits megakaryocytic differentiation via repressing RUNX1 and the megakaryocytic gene expression program in acute megakaryoblastic leukemia.

A high incidence of acute megakaryoblastic leukemia (AMKL) in Down syndrome patients implies that chromosome 21 genes have a pivotal role in AMKL development, but the functional contribution of individual genes remains elusive. Here, we report that SON, a chromosome 21-encoded DNA- and RNA-binding protein, inhibits megakaryocytic differentiation by suppressing RUNX1 and the megakaryocytic gene expression program. As megakaryocytic progenitors differentiate, SON expression is drastically reduced, with mature megakaryocytes having the lowest levels. In contrast, AMKL cells express an aberrantly high level of SON, and knockdown of SON induced the onset of megakaryocytic differentiation in AMKL cell lines. Genome-wide transcriptome analyses revealed that SON knockdown turns on the expression of pro-megakaryocytic genes while reducing erythroid gene expression. Mechanistically, SON represses RUNX1 expression by directly binding to the proximal promoter and two enhancer regions, the known +23 kb enhancer and the novel +139 kb enhancer, at the RUNX1 locus to suppress H3K4 methylation. In addition, SON represses the expression of the AP-1 complex subunits JUN, JUNB and FOSB which are required for late megakaryocytic gene expression. Our findings define SON as a negative regulator of RUNX1 and megakaryocytic differentiation, implicating SON overexpression in impaired differentiation during AMKL development.

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis

AbstractMutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Analysis of Clonal Evolution/Heterogeneity of MDS By Simultaneous Detection of Both Mutation and Gene Expression By Single Cell Sequencing

Recent advances in single-cell sequencing (sc-Seq) technologies have enabled high-throughput transcriptome analysis in thousands of cells to understand the heterogeneity among cancer populations in terms of genome-wide gene expression. However, its application to the analysis of clonal evolution of cancer populations is largely limited by the lack of an efficient sc-Seq platform that allows for accurate detection of gene mutations at the same time with transcriptome analysis. The major challenge here is a frequent allele dropout of just two copies per single cell, which results in an inaccurate genotype assignment for many cells, preventing identification of relevant genotype-phenotype correlations. To overcome this, we developed a novel sc-Seq platform (scMutSeq) that allows for precise determination of both genotype and genome-wide gene expression simultaneously with negligible allele dropouts, on the basis of the Fluidigm C1 Single-Cell mRNA Seq HT system and applied it to the analysis of clonal evolution and intratumor heterogeneity of myelodysplastic syndromes (MDS) characterized by frequent clonal evolution to acute amyloid leukemia (AML).We first evaluated the performance of our plat form using an AML-derived cell line with heterozygous SF3B1K700E mutation, HNT-34, for which efficiency of the detection of both wild-type and mutant allele, together with global gene expression, was evaluated. Among 400 cells subjected to scMutSeq analysis, a total of 125 passed QC, in which cell viability was assessed in terms of expression of mitochondrial genes. Global gene expression and heterozygous SF3B1mutation were successfully detected in all the QC-confirmed cells with none of the cells showing the wild-type allele or homozygous SF3B1mutation, where evaluable transcript reads (unique molecular identifier >=1) were obtained for a median of 2,753 genes, designated as nGene. The performance was also tested for flow-sorted hematopoietic stem/progenitor cells (HSPCs) (Lin−CD34+) from an MDS patient positive for the SF3B1K700E mutation. Gene expression was successfully analyzed all the QC-confirmed cells (n=81) with a median nGene of 1,953. No substantial allele dropouts were suspected, because none of the cells genotyped had homozygous SF3B1mutation.We then applied scMutSeq to the analysis of TP53-mutated AML/MDS with complex karyotype, including del(5q) and del(7q), for which longitudinal samples were obtained for the assessment of clonal evolution. scMutSeq successfully analyzed the mutation status of TP53and global gene expression profiles at a single-cell level, where copy number abnormalities were also evaluated on the basis of gene expression. We identified two discrete clones in the HSPC fraction, carrying both del(5q) and del(7q) and del(5q) alone, respectively, even though the analysis of bulk DNA had failed to detect the latter clone, indicating that a minor clone having a distinct genotype came under detection with scMutSeq. Moreover, the HSPCs with both del(5q) and del(7q) showed aberrant expression of erythroid and megakaryocytic genes, increased expression of inflammatory signals and decreased expression of cell cycle-related genes, exhibiting a clear genotype phenotype correlation. Subsequent analysis of samples at later time points further disclosed evolution of clones having discrete del(5q) deletions and expression, revealing a complexity of clonal evolution in MDS.Next, to investigate the early process of MDS development, we analyzed clonal hematopoiesis found in a minor fraction (1.2-12%) of bone marrow samples from three elder individuals having hip replacement surgery, in which DNMT3A(n=1) (R882H) and TET2(n=2) (D905fs and Q1540fs) mutations had been detected by ddPCR or targeted deep sequencing, respectively. scMutSeq analysis of the HSPCs from these individuals revealed that mutant HSPCs showed distinct gene expression profiles, depending on the type of CHIP mutations.To summarize, our single-cell sequencing platform enables to detect both genetic and transcriptional heterogeneities, providing a powerful clue to understand clonal evolution and intratumor heterogeneity of MDS. DisclosuresNakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Yoda:Chordia Therapeutics Inc.: Research Funding.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

Hyperglycemia enhances arsenic-induced platelet and megakaryocyte activation

ObjectiveLow to moderate inorganic arsenic (iAs) exposure is independently associated with cardiovascular disease (CVD), particularly for patients with diabetes mellitus (DM). The mechanism of increased CVD risk from iAs exposure in DM has not been adequately characterized. We evaluated whether increasing concentrations of glucose enhance the effects of iAs on platelet and megakaryocyte activity, key steps in atherothrombosis.MethodsHealthy donor whole blood was prepared in a standard fashion and incubated with sodium arsenite in a range from 0 to 10 µM. iAs-induced platelet activation was assessed by platelet receptor CD62P (P-selectin) expression and monocyte-platelet and leukocyte-platelet aggregation (MPA and LPA, respectively) in the presence of increasing sodium arsenite and glucose concentrations. Megakaryocyte (Meg-01) cell adhesion and gene expression was assessed after incubation with or without iAs and increasing concentrations of d-glucose.ResultsPlatelet activity markers increased significantly with 10 vs. 0 µM iAs (P < 0.05 for all) and with higher d-glucose concentrations. Platelet activity increased significantly following co incubation of 1 and 5 µM iAs concentrations with hyperglycemic d-glucose (P < 0.01 for both) but not after incubation with euglycemic d-glucose. Megakaryocyte adhesion was more pronounced after co incubation with iAs and hyperglycemic than euglycemic d-glucose, while gene expression increased significantly to iAs only after co incubation with hyperglycemic d-glucose.ConclusionWe demonstrate that glucose concentrations common in DM potentiate the effect of inorganic arsenic exposure on markers of platelet and megakaryocyte activity. Our results support recent observational cohort data that DM enhances the vasculotoxic effects of arsenic exposure, and suggest that activation of the platelet-megakaryocyte hemostatic axis is a pathway through which inorganic arsenic confers atherothrombotic risk, particularly for patients with DM.

Increased megakaryocytic proliferation, pro-platelet deposition and expression of fibrosis-associated factors in children with chronic myeloid leukaemia with bone marrow fibrosis.

Paediatric chronic myeloid leukaemia (ped-CML) is rare and ped-CML with fibre accumulation in the bone marrow (MF) is thought to be even rarer. In adults (ad-CML), fibrosis represents an adverse prognostic factor. So far, the pro-fibrotic changes in the bone marrow microenvironment have not been investigated in detail in ped-CML. From a total of 66 ped-CML in chronic phase, biopsies were analysable and 10 had MF1/2 (MF1, n=8/10; MF2, n=2/10). We randomly selected 16 ped-CML and 16 ad-CML cases with and without fibrosis (each n=8) as well as 18 non-neoplastic controls. Bone marrow samples were analysed with a real-time PCR-based assay (including 127 genes for paediatric cases) and by immunohistochemistry. We found increased expression of megakaryocytic genes in ped-CML. The number of megakaryocytes and pro-platelets are increased in CML patients, but the most significant increase was noted for ped-CML-MF1/2. Anti-fibrotic MMP9 expression was lower in children than in adults. Cell mobilisation-related CXCL12 was decreased in young and adult patients with CML but not the corresponding receptor CXCR4. In summary, fibre accumulation in ped-CML-MF1/2 is associated with increased megakaryocytic proliferation and increased interstitial pro-platelet deposition. Deregulated expression of matrix-modulating factors shifts the bone marrow microenvironment towards fibrosis.

GATA1 Activity Governed by Configurations of cis-Acting Elements

The transcription factor GATA1 regulates the expression of essential erythroid and megakaryocytic differentiation genes through binding to the DNA consensus sequence WGATAR. The GATA1 protein has four functional domains, including two centrally located zinc-finger domains and two transactivation domains at the N- and C-termini. These functional domains play characteristic roles in the elaborate regulation of diversified GATA1 target genes, each of which exhibits a unique expression profile. Three types of GATA1-related hematological malignancies have been reported. One is a structural mutation in the GATA1 gene, resulting in the production of a short form of GATA1 that lacks the N-terminal transactivation domain and is found in Down syndrome-related acute megakaryocytic leukemia. The other two are cis-acting regulatory mutations affecting expression of the Gata1 gene, which have been shown to cause acute erythroblastic leukemia and myelofibrosis in mice. Therefore, imbalanced gene regulation caused by qualitative and quantitative changes in GATA1 is thought to be involved in specific hematological disease pathogenesis. In the present review, we discuss recent advances in understanding the mechanisms of differential transcriptional regulation by GATA1 during erythroid differentiation, with special reference to the binding kinetics of GATA1 at conformation-specific binding sites.

Modulation of miR-185-5p expression by EBV-miR-BART6 contributes to developmental differences in ABCG4 gene expression in human megakaryocytes

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by low platelet count and bleeding, and is usually triggered by viral infections. We previously reported that 14 viral microRNAs of megakaryocytes cultured with serum from patients with ITP, including ebv-miR-BART6, are up-regulated. Previous research has reported that ebv-miR-BART6 down-regulated the expression of miR-185-5p. We therefore predicted that the ABCG4 gene, which is highly expressed in megakaryocyte progenitor cells, is a direct target of miR-185-5p. We hypothesized that ebv-miR-BART6 may play a role in development and differentiation of megakaryocytes. First, we verified the negative regulation of ABCG4 by miR-185-5p through luciferase assay analysis. Second, after transfection of ebv-miR-BART6 into megakaryocytes developing from normal cord blood mononuclear cells (MNCs), we found that the level of miR-185-5p in the ebv-miR-BART6 group was reduced to almost a third of that in the control groups, accompanied by up-regulation of ABCG4 at both the mRNA and protein levels. Meanwhile, proliferation of megakaryocytes was significantly repressed in the ebv-miR-BART6 group compared with the blank and negative control groups (14.89%±3.13%, 34.15%±2.42% and 30.96%±4.37%, respectively; P<0.001). Our results further revealed that ebv-miR-BART6 inhibited megakaryocyte colony unit formation, decreased CD41 expression and inhibited megakaryocyte polyploidization. These data suggest a new paradigm to explain the mechanisms underlying ITP, involving the regulation of megakaryocytopoiesis by viral microRNAs through the intronic hsa-microRNA.

Regulation of Erythro-Megakaryocytic Lineage Divergence By the Transcriptional Repressor Gfi1b and Its Rgs Targets

Introduction: Appropriate diversification of hematopoietic lineages from multi-potent progenitors is essential for normal development and health. The molecular programs that govern the divergence of erythroid and megakaryocytic lineages remain incompletely defined. Gene targeting experiments have shown the transcriptional repressor Gfi1b (Growth factor independence 1b) to be essential for erythro-megakaryocyte lineage development. Transcriptional repression of Gfi1b target genes is mediated by the cofactors LSD (lysine demethylase) 1 and Rcor (CoREST) 1. To understand the mechanism of Gfi1b action, its target genes were identified by chromatin immunoprecipitation (ChIP on Chip) screens. Three members of the Rgs (Regulator of G protein signaling) family were prominently represented in this target gene pool. In this study we present the role of Rgs18, a GTPase activating protein (GAP), in modulating erythro-megakaryocytic lineage divergence in hematopoietic progenitors. The results presented below demonstrate Rgs18 as a key arbitrator of this process in murine and human contexts.Approach: Following identification of Rgs18 as a potential Gfi1b and LSD1 target, its regulation by these factors was ascertained in erythro-megakaryocytic cells. Subsequently, to interrogate the role of Rgs18 in erythro-megakaryocyte differentiation, cDNA and shRNA mediated manipulations were performed in primary hematopoietic progenitors and cell lines, and the resulting phenotypes were analyzed. Finally, to trace the underlying mechanistic alterations responsible for these phenotypes the status of two branches of the MAPK (mitogen activated protein kinase) pathway and gene expression patterns of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1/Klf1 (Krupple like factor 1) were determined in Rgs18 manipulated cells.Result: Rgs18 expression was found to be low in immature megakaryoblasts in keeping with strong Gfi1b and LSD1 expression, but was reciprocally upregulated in mature megakaryocytes following declining Gfi1b and LSD1 levels in cells and on the rgs18 promoter. In contrast, expression of Gfi1b was strong in immature erythroid cells and increased further in mature cells, while Rgs18 expression which was modest in immature erythroid cells exhibited a reciprocal decline during maturation. Manipulation of Rgs18 expression in murine hematopoietic progenitors and a bipotential human cell line produced divergent outcomes, with expression augmenting megakaryocytic, and potently suppressing erythroid differentiation and vice versa. These phenotypes resulted from differential impact of Rgs18 expression on the P38 and ERK branches of MAPK signaling in the erythroid and megakaryocytic lineages. Repercussions of these signaling changes impacted relative expression of the mutually antagonistic transcription factors Fli1 and Klf1 and were compensated by ectopic Fli1 expression demonstrating activity of this transcription factor downstream of Rgs18.Conclusion: These results identify Rgs18 as a critical downstream effector of Gfi1b and an upstream regulator of MAPK signaling and Klf1/Fli1 gene expression. Sustained Gfi1b expression during erythroid differentiation represses Rgs18 and limits megakaryocytic gene expression. However during progression of megakaryocytic differentiation, declining Gfi1b levels results in robust expression of Rgs18 and lineage progression.Overall, this study provides new perspectives on lineage determination by highlighting multi-tier interactions between transcriptional and signaling networks in orchestrating hematopoietic lineage divergence. These insights could exemplify generic mechanisms exhibited by this large family of signal modulators in mediating lineage diversification in various contexts. DisclosuresNo relevant conflicts of interest to declare.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. DisclosuresNo relevant conflicts of interest to declare.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

AimThe aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα).MethodsThe effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV).ResultsIn DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment.ConclusionsThe present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.

Systems biology of megakaryocytes.

The molecular pathways that regulate megakaryocyte production have historically been identified through multiple candidate gene approaches. Several transcription factors critical for generating megakaryocytes were identified by promoter analysis of megakaryocyte-specific genes, and their biological roles then verified by gene knockout studies; for example, GATA-1, NF-E2, and RUNX1 were identified in this way. In contrast, other transcription factors important for megakaryopoiesis were discovered through a systems approach; for example, c-Myb was found to be critical for the erythroid versus megakaryocyte lineage decision by genome-wide loss-of-function studies. The regulation of the levels of these transcription factors is, for the most part, cell intrinsic, although that assumption has recently been challenged. Epigenetics also impacts megakaryocyte gene expression, mediated by histone acetylation and methylation. Several cytokines have been identified to regulate megakaryocyte survival, proliferation, and differentiation, most prominent of which is thrombopoietin. Upon binding to its receptor, the product of the c-Mpl proto-oncogene, thrombopoietin induces a conformational change that activates a number of secondary messengers that promote cell survival, proliferation, and differentiation, and down-modulate receptor signaling. Among the best studied are the signal transducers and activators of transcription (STAT) proteins; phosphoinositol-3-kinase; mitogen-activated protein kinases; the phosphatases PTEN, SHP1, SHP2, and SHIP1; and the suppressors of cytokine signaling (SOCS) proteins. Additional signals activated by these secondary mediators include mammalian target of rapamycin; β(beta)-catenin; the G proteins Rac1, Rho, and CDC42; several transcription factors, including hypoxia-inducible factor 1α(alpha), the homeobox-containing proteins HOXB4 and HOXA9, and a number of signaling mediators that are reduced, including glycogen synthase kinase 3α(alpha) and the FOXO3 family of forkhead proteins. More recently, systematic interrogation of several aspects of megakaryocyte formation have been conducted, employing genomics, proteomics, and chromatin immunoprecipitation (ChIP) analyses, among others, and have yielded many previously unappreciated signaling mechanisms that regulate megakaryocyte lineage determination, proliferation, and differentiation. This chapter focuses on these pathways in normal and neoplastic megakaryopoiesis, and suggests areas that are ripe for further study.

Effect Of Age and Gender On Human Platelet mRNA and Micro-RNA Levels

The well-known inter-individual variation in human platelet reactivity is heritable, but there is limited understanding of the responsible genetic mechanisms. Prior work supports an effect of age, gender, race, body mass index (BMI) and other demographic variables on platelet function, but there is little or no data addressing whether these variables are associated with platelet gene expression. Quantitative and qualitative variants of different classes of platelet RNAs are important windows into megakaryocyte and platelet gene expression, and emerging evidence indicates the utility of specific transcripts as disease biomarkers, as vectors for systemically transmitting genetic information in microparticles, as mediators of pharmacogenetic effects, and as potential therapies. However, to date the small sample sizes of platelet transcriptomic studies have not permitted testing for associations between demographic variables and platelet RNA levels. The Platelet RNA And eXpression-1 (PRAX1) study was designed to characterize mRNAs and microRNAs (miRNAs) in highly purified platelets from a group of healthy, non-diabetic subjects. 163 participants (84 whites and 70 blacks) were recruited, and after exclusion due to use of anti-platelet medication (defined as non-responsiveness to arachidonic acid-induced platelet aggregation) or abnormal hematological parameters, 154 subjects were included for RNA profiling and analyses. Leukocyte-depleted platelet RNA was profiled using the Human Gene 1.0 ST Array (Affymetrix) for mRNA and the nCounter (Nanostring) for miRNA. After normalization, mRNAs and miRNAs were defined as commonly expressed if they were above background in over 65% of subjects. This yielded 5813 common mRNAs and 181 common miRNAs. These profiles were validated using a separate cohort with similar demographic characteristics (n=19) and by plotting rank correlations of mRNAs (r = 0.57; p = 2.3x10-311) and miRNAs (r = 0.69; p = 7.3x10-21). We identified 130 mRNAs and 15 miRNAs that were differentially expressed (DE) by age (Q<0.05). These 130 mRNAs were enriched for putative binding sites for these 15 miRNAs (p<0.001). We identified a network of DE miRNAs targeting DE mRNAs, in which the miRNA and mRNA were significantly and inversely correlated by age. Mitochondrial mRNAs were also inversely correlated with age. Second, we identified 54 mRNAs and 9 miRNAs DE by gender (Q<0.05). As expected, the Y-chromosome genes, EIF1AY, TMSB4Y, UTY and DDX3Y were expressed more highly in males (p = 1.22x10-82, p = 9.28x10-70, p = 2.89x10-68 and p = 7.45x10-58, respectively). A network of miRNAs and mRNAs, both DE by gender, was identified in which the miRNAs were predicted to target the mRNAs. Lastly, a single miRNA but no mRNAs were DE by BMI. In summary, levels of platelet mRNAs and miRNAs are strongly associated with age and gender, but for the most part, not with BMI. The inverse relationship between these two DE RNA classes suggests miRNAs may regulate mRNA levels between genders and upon aging. Future association studies between platelet RNAs and either ex vivo platelet function or in vivo platelet-mediated hemostasis and thrombosis must account for age and gender. Disclosures:No relevant conflicts of interest to declare.

Ikaros inhibits megakaryopoiesis through functional interaction with GATA-1 and NOTCH signaling

AbstractThe transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation

AbstractWnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6−/−), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

AbstractAbstract 2867▪▪This icon denotes a clinically relevant abstractPrimary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML.We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease.Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks.We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi.GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks.We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures:Pardanani:Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.