Regulator Of Megakaryopoiesis Research Articles

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (<10%; micro-MK low) and high rate of micro-MK (>40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

Research Progress on the Mechanism of Tubulin in Megakaryopoiesis and Regulation of Platelet Count--Review

Tubulin affects platelets count through the control of mitosis and the formation of pro-platelets during the maturation of megakaryoblast to platelets. Tubulin is involved in maintaining the integrity of platelet skeleton, and also participates in the change of platelet morphology during platelet activation. Some new anti-tumor drugs targeting cell mitosis are trying to reduce the effect on tubulin in order to reduce the side effect of drugs on platelet formation. In some patients with thrombocytopenia, the variation and polymorphism of the tubulin gene affect the structure of microtubule multimers, which leads to the decrease of platelet formation. This review summarized the latest progresses of tubulin in the regulation of megakaryopoiesis and thrombopoiesis.

PDGF-BB and Its Role in Megakaryopoiesis and Thrombocythemia

Background: Essential Thrombocythemia (ET) is characterized by persistently elevated platelet counts in the context of a normal red cell mass. However, the molecular mechanism of ET is still under investigation. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on megakaryocytes and their ability to mediate hematopoiesis and megakaryopoiesis (Ye et al, Haematologica, 2010). The role of PDGF-BB on megakaryocytic progenitors (CD41+, CD34+ cells) and its mechanisms on ET will be further studied in this project.Methods: ELISA, CFU assay, immunofluorescence microscope, flow cytometry and NOD/SCID mice were used in this study.Results: Bone marrow plasma levels of PDGF-BB in ET patients (n=18) and normal control (n=10) were tested and found an increased PDGF-BB levels in ET patients (2071.2±124.8 pg/ml), compared with normal control (1382.5±128.3pg/ml) (P=0.002). In vitro experiment, PDGF-BB promoted the ex vivo expansion of human hematopoietic stem (CD34 +) and progenitor (CD41 + CD61 +) cells. More significantly, PDGF enhanced the engraftment of human CD45 + cells and their myeloid subsets (CD33 +, CD14 + cells) in NOD/SCID mice. PDGF-BB stimulated megakaryopoiesis via PDGFR and its signalling. It also showed a direct stimulatory effect of PDGF-BB on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF-BB on the regulation of megakaryopoiesis. PDGF-BB also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like TPO, significantly promoted platelet recovery and the formation of CFU-MK in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the CHRF apoptotic model, PDGF-BB had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF-BB activated the p -Akt , p-Jak2 and p-Stat3 expression, while addition of imatinib mesylate reduced p-Akt, p-Jak2 and p-Stat3 expression in CHRF cells.Conclusion: Our findings suggested that PDGF-BB is likely to be mediated via PDGF receptors with subsequent activation of the Akt and Jak2/ Stat3 pathways in megakaryopoiesis. These studies provide a possible explanation that PDGF-BB and its signaling may be involved in the molecular mechanism of essential thrombocythemia. DisclosuresNo relevant conflicts of interest to declare.

Regulation of megakaryopoiesis by bone marrow macrophage polarization.

Regulation of megakaryopoiesis by bone marrow macrophage polarization.

The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

Megakaryopoiesis and Platelet-Innate Immune Cell Interactions Are Developmentally Regulated

Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis. The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program. Disclosures Palis: Rubius Therapeutics: Consultancy.

Recent advances in mechanisms of megakaryopoiesis and platelet formation

Platelets are specialized cells, which are functioned to prevent bleeding and minimize blood vessel injury, and are also the predominating factor in the processes of hemostasis and thrombosis, while implicate in other processes including inflammation, innate immunity, angiogenesis and tumor metastasis. Among current mechanisms of platelet formation, platelets are produced by megakaryocytes, which are themselves generated by a process of controlled differentiation and maturation of bone marrow hematopoietic stem cells (HSC) and progenitor cells. Megakaryocytes release platelets by extending long, branching proplatelets, into sinusoidal blood vessels. Bone marrow cavity and extracellular matrix composition together with cytokines as thrombopoietin (TPO) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet formation. Understanding the mechanisms of megakaryocyte differentiation and platelet formation can provide a solid theoretical basis for the treatment of platelet-related diseases. This review summarizes the current scientific research progress in the mechanisms of megakaryopoiesis and thrombopoiesis. Key words: Megakaryocytes; Cell differentiation; Thrombopoiesis; Transcription factors; Apoptosis; Megakaryopoiesis

High‐throughput sequencing approaches for diagnosing hereditary bleeding and platelet disorders

Hereditary bleeding and platelet disorders (BPDs) are characterized by marked genetic heterogeneity, far greater than previously appreciated. The list of genes involved in the regulation of megakaryopoiesis, platelet formation, platelet function and bleeding has been growing rapidly since the introduction of high-throughput sequencing (HTS) approaches in research. Thanks to the gradual adoption of HTS in diagnostic practice, these discoveries are improving the diagnostic yield for BPD patients, who may or may not present with bleeding problems and often have other clinical symptoms unrelated to the blood system. However, it was previously found that screening for all known etiologies gives a diagnostic yield of over 90% when the phenotype closely matches a known BPD but drops to 10% when the phenotype is indicative of a novel disorder. Thus, further research is needed to identify currently unknown etiologies for BPDs. Novel genes are likely to be found to be implicated in BPDs. New modes of inheritance, including digenic inheritance, are likely to play a role in some cases. Additionally, identifying and interpreting pathogenic variants outside exons is a looming challenge that can only be tackled with an improved understanding of the regulatory landscape of relevant cell types and with the transition from targeted sequencing to whole-genome sequencing in the clinic.

PDGF/PDGFR May be Involved in the Physiopathologic Mechanism of Essential Thrombocythemia

Our previous studies have demonstrated that PDGF (Platelet-derived growth factor) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis (Yang et al, Thromb Haemastasis, 1997; Ye et al, Haematologica, 2010). Essential Thrombocythemia (ET) is characterized by persistently elevated platelet counts in the context of a normal red cell mass. However, the physiopathologic mechanism of ET is still under investigation. Here, we tested the bone marrow plasma levels of PDGF-BB in essential thrombocythemia patients (n=16) and normal control (n=8), and found an increased PDGF-BB levels in ET patients (2070.92±123.98 pg/ml), compared with normal control (1381.85±128.37pg/ml) (P=0.002). Furthermore, we have demonstrated the presence of functional PDGF receptors (PDGFR) in human megakaryocytes, and their ability to mediate a mitogenic response by bone marrow colony-forming unit-megakaryocyte (CFU-MK) formation assay (n=6). PDGF-BB stimulated in vitro megakaryopoiesis via PDGFR. It also showed a direct stimulatory effect of PDGF-BB on c-Fos expressions in megakaryocytic cells, CHRF. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow CFU-MK in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. We also demonstrated that PDGF activated the p- Akt, p-Jak2 and p-Stat3 expression, while addition of imatinib mesylate reduced p-Akt, p-Jak2 and p-Stat3 expression in CHRF cells. Our findings suggested that the PDGF-initiated megakaryopoiesis is likely to be mediated via PDGF receptors with subsequent activation of the Akt and Jak2/ Stat3 pathways. These studies provide a possible explanation that PDGF/PDGFR may be involved in the physiopathologic mechanism of essential thrombocythemia. DisclosuresYang:National Natural Science Foundation of China(81270580): Research Funding.

Mediator Kinase Inhibition Triggers a Phenotypic Switch from Neonatal to Adult-like Megakaryopoiesis

Several recent studies have demonstrated substantial biological differences between cord blood (CB)- and adult peripheral blood (PB)-derived megakaryocytes (MKs). Specifically, neonatal (CB) progenitors proliferate at a much higher rate than adult (PB) progenitors, and generate 10-fold more MKs per progenitor when cultured with thrombopoietin (Tpo). The highly proliferative neonatal MKs undergo cytoplasmic maturation without polyploidization, which ultimately generates large numbers of small, low ploidy, but fully mature MKs. Adult MKs, in contrast, undergo successive rounds of endomitosis to reach much higher ploidy levels, and maturation is coupled with polyploidization, so that MKs with the highest ploidy levels are also the most mature. The molecular mechanisms underlying these developmental differences are just beginning to be elucidated. Here, we investigated the effects of cortistatin A (CA), a highly specific small molecule inhibitor of cyclin-dependent kinase 8 (CDK8) and its paralog CDK19, on megakaryopoiesis. CDK8 and CDK19, together with CCNC (Cyclin C), MED12/MED12L and MED13/MED13L, form "CDK modules" which can associate with Mediator, a 26-subunit complex that acts as a bridge between transcription factors and the transcriptional machinery to coordinate gene expression. The Mediator complex has been implicated in developmental disorders and cancer. CB-derived MK progenitors treated with CA from day 7 to day 14 of culture exhibited a dose-dependent reduction in proliferation (6.2±1.7 vs. 24.9±2.2 fold expansion in treated vs. control cultures; p=0.003), accompanied by an increase in ploidy levels to those comparable to adult PB-derived MKs (34±6% vs. 8.9±0.9% MKs with ploidy ≥8N in treated vs. control cultures; p=0.014). MK maturation, evaluated by CD42b surface expression level, also increased with advancing ploidy in CA-treated MKs, in a manner similar to that observed in adult MKs, and CA-treated mature MKs were capable of pro-platelet formation in vitro. These changes were not observed when undifferentiated CB-derived CD34+ or CD41-negative cells were treated, indicating that the effect was specific to committed MK progenitors. CA treatment induced the expected decrease in STAT-1 phosphorylation at Serine 727, a specific site of CDK8-mediated phosphorylation, confirming effective CDK8 inhibition in treated MKs. Next, we used microarray to evaluate the gene expression profile of CB-derived MKs following CA treatment for 4 and 8 hours vs. untreated cells. These studies revealed significant CA-induced changes in the MK gene expression profile. By Gene Set Enrichment Analysis (GSEA), CA treatment significantly upregulated genes that were downregulated in CB- vs. PB-derived MKs. Furthermore, genes upregulated by CA in CB-derived MKs were also upregulated in the megakaryoblastic cell line SET2 (Pelish, Liau et al., Nature, in press), suggesting that the gene expression program affected in SET2 cells is likely the same one affected in neonatal MKs. At the protein level, we observed time- and dose-dependent increases in RUNX-1 in CA-treated vs. control MKs. In summary, this study demonstrated that treatment with CA induced a phenotypic switch from neonatal to adult-like megakaryopoiesis, accompanied by changes in the MK gene expression profile. These findings indicate a novel role for Mediator kinases in the regulation of megakaryopoiesis, and potentially on the developmental differences between neonatal and adult MKs. These studies open the door to a better understanding of and to potential novel therapies for a number of developmental stage-specific megakaryocyte and platelet disorders, which exclusively or more severely affect neonates and infants, including the thrombocytopenia/absent radius (TAR) syndrome and the transient myeloproliferative disorder associated with trisomy 21 and GATA1s mutations. DisclosuresNo relevant conflicts of interest to declare.

The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis

Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years, the molecular mechanism accounting for this activity is still unclear. To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions, using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. We identified 328 annotated genes differentially regulated by anagrelide, including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3, whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4, a known TRIB3 inducer. Notably, immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α, which is an upstream regulator of ATF4, and increased ATF4 protein levels. Furthermore, salubrinal, an inhibitor of eIF2α dephosphorylation, increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis.

Platelet-Derived Growth Factor Has a Potent Effect on MK Proliferation, F-Actin Reorganization and Proplatelet Formation Via PDGFR and p-Akt or p-ERK1/2 Pathways

Platelet-derived growth factor (PDGF) may intimate the existence of an autocrine and/or paracrine loop constructed by megakaryocytes (MKs) and their granular constituents in the regulation of megakaryopoiesis and thrombopoiesis. Our study demonstrated that PDGF has a promoting effect on thrombopoiesis in an irradiated-mice model (Ye et al, Haematologica, 2010). PDGF may enhance megakaryopoiesis and platelet production via PDGFR and Erk/Akt pathways. PDGF receptors were identified on platelets and megakaryocytic cells (MKs) by ELISA, flow cytometry and immunocytochemical staining. MK proliferation and platelet formation were studied by CFU-MK and pro-platelet assay. Both PDGFR subtype were identified on platelets and MKs; Levels of PDGF beta-receptors exceeded that of alpha-receptors. PDGF-BB significantly stimulated MKs proliferation; this mitogenic effect was partially neutralized by antibodies directed against the PDGF beta-receptors. Western blot also determind that these receptors mediate PDGF-BB-inducible expression of c-fos. In the MKs apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on MKs. We demonstrated that PDGF activated the Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression, suggesting that the PDGF-initiated protective effect is likely to be mediated via PDGF receptors with subsequent activation of the Akt pathway. PDGF also promoted proplatelet formation in MKs and this effect was reduced by PDGFR antibodies. In PDGF-BB (50ng/ml) and TPO (50 ng/ml) groups had more proplatelet bearing MKs; in 200 MKs, proplatelet bearing MKs in PDGF-BB treatment group was (15 % ± 3.2), and in TPO group was (18 % ± 2.5), which were significantly more than the control group (7 % ± 2.6) (n=5). Whether PDGF has effects on cytoskeleton reorganization of MKs, and whether the effect can be reduced by Erk1/2 inhibitor PD98059, MKs were staining with F-actin specific binder rhodamine-phalloidin. We observed that polymerized actin level is lower in control group refer to PDGF-BB group and distributed throughout the cytoplasm with occasionally few aggregation. In contrast, polymerization actin level was higher in PDGF-BB group. Adding PD98059, the fluorescence intensity was reduced (n=5). Our data demonstrated that treated with PDGF-BB for 30 minutes Erk1/2 was activated in MKs. This study suggests that PDGF has a potent effect on megakarpoiesis and platelet formation. This effect is likely mediated via PDGF beta-receptors with subsequent activation of p-ERK1/2 or p-Akt, which leads to MKs proliferation, F-actin reorganization and proplatelet formation. DisclosuresNo relevant conflicts of interest to declare.

Platelets Regulate Thrombopoietin Production: The Missing Link

Humans produce and remove ~1011 platelets daily, and the rate of production can rise sharply under conditions of platelet destruction. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low or arterial occlusion and organ damage if counts are high. Thrombopoietin (TPO), the primary regulator of megakaryopoiesis and platelet production, was first isolated and cloned in 1994. This discovery was a major milestone in medicine and led to the availability of TPO mimetics for clinical care. Hepatocytes are the primary cells responsible for the production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. In one model, circulating TPO levels are maintained solely by its uptake and metabolism by high-affinity c-Mpl receptors on platelets and megakaryocytes. In another model, circulating platelet levels are sensed, resulting in appropriately regulated levels of TPO mRNA expression in liver and bone marrow. Despite these discussions, no physiological ligand-receptor pair capable of regulating steady state TPO production has yet been identified and the mechanisms through which platelets regulate TPO production remain poorly understood. Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. The hepatic Ashwell-Morell receptor (AMR) can bind and remove platelets with reduced α2,3-linked sialic acid (desialylated platelets), such as after cold storage, during sepsis, or in St3gal4-/- mice lacking the ST3Gal-IV sialyltransferase. The AMR is a transmembrane heteroligomeric glycoprotein complex composed of ASGPR1 (also called CLEC4H1, HL-1) and ASGPR2 (CLEC4H2, HL-2) subunits, which are highly conserved among mammalian species. However, since its discovery four decades ago, the regulatory role of the hepatic AMR remains unclear. Injection of desialylated platelets into rabbits stimulates platelet production, suggesting a possible link between desialylated platelet consumption and liver TPO secretion. We report that sialic acid loss determines platelet circulatory lifespan by triggering platelet removal by the hepatic AMR. Binding of desialylated platelets to the AMR induces hepatic TPO gene transcription and translation, thereby regulating platelet production. This conclusion is based on the following results. First, desialylated platelet uptake by the AMR regulates hepatic TPO mRNA expression in situ, as evidenced by increased (~50%) hepatic TPO mRNA levels in St3gal4-/- mice and reduced (~50%) hepatic TPO mRNA levels in Asgr2-/- mice lacking the AMR ASGPR2 subunit, relative to controls. Second, injection of endogenously (isolated from St3gal4-/- and Asgr2-/- mice) and exogenously (sialidase-treated) desialylated mouse platelets into wild-type mice stimulates hepatic TPO mRNA expression by ~50% after 12h and subsequently increases plasma TPO levels and blood platelet counts. In contrast, desialylated platelets injected into Asgr2-/- mice have no effect on hepatic TPO mRNA content, plasma TPO levels and blood platelet counts. Third, incubation of desialylated human platelets with the hepatoma cell line HepG2 increases HepG2 TPO mRNA content by approximately three-fold after six hours in vitro. Recognition of this previously unrecognized physiological feedback mechanism illuminates a novel platelet-dependent regulatory mechanism for hepatic TPO production. Disclosures No relevant conflicts of interest to declare.

Cytosolic carboxypeptidase CCP6 is required for megakaryopoiesis by modulating Mad2 polyglutamylation.

Bone marrow progenitor cells develop into mature megakaryocytes (MKs) to produce platelets for hemostasis and other physiological functions. However, the molecular mechanisms underlying megakaryopoiesis are not completely defined. We show that cytosolic carboxypeptidase (CCP) 6 deficiency in mice causes enlarged spleens and increased platelet counts with underdeveloped MKs and dysfunctional platelets. The prominent phenotypes of CCP6 deficiency are different from those of CCP1-deficient mice. We found that CCP6 and tubulin tyrosine ligase-like family (TTLL) members TTLL4 and TTLL6 are highly expressed in MKs. We identify Mad2 (mitotic arrest deficient 2) as a novel substrate for CCP6 and not CCP1. Mad2 can be polyglutamylated by TTLL4 and TTLL6 to modulate the maturation of MKs. CCP6 deficiency causes hyperglutamylation of Mad2 to promote activation of Aurora B, leading to suppression of MK maturation. We reveal that Mad2 polyglutamylation plays a critical role in the regulation of megakaryopoiesis.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

Cholinergic drugs inhibit in vitro megakaryopoiesis via the alpha7-nicotinic acetylcholine receptor

Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca2+ channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.

Novel treatments in the management of immune thrombocytopenic purpura (ITP)

ITP is generally considered an antibody mediated condition with platelet destruction characterised by an abnormally low platelet count. More recently it has also been shown that insufficient or inadequate platelet production also plays a role. Less than 10% with chronic ITP experience spontaneous remission and need long-term management. Conventional first line management is with corticosteroids or intravenous immune globulins (including anti-D) followed by a variety of agents including cytotoxic and immune suppressive agents for refractory and relapsed patients. Splenectomy may also be considered but one-third fail to respond. However, up to 50% of the mortality seen in ITP is due to infection secondary to immune suppression and treatment is generally unsatisfactory. In view of the recognised suboptimal platelet production there has been a search for treatments that enhance platelet production. The identification of thrombopoietin, the key regulator of megakaryopoiesis, in 1994, led to its evaluation in ITP. The first studies were discontinued because of the development of cross-reacting antibodies, leading to severe thrombocytopenia. Nevertheless, this paved the way for the development of the next generation of agents, including the peptibody, romiplostim and the small-molecule TPO receptor agonist, eltrombopag. Both showed promising results in early studies, confirmed in Phase III clinical trials, and are now being evaluated for their place in the treatment pathway.

Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation

AbstractA major determinant in platelet production is the megakaryocyte (MK) size that is regulated both by ploidization and the increase in cytoplasmic volume at the end of maturation. Here we investigated the involvement of the mammalian target of rapamycin (mTOR) pathway in the regulation of megakaryopoiesis. We show that phosphorylation of mTOR, p70S6K1, and 4E-BP1 was diminished in thrombopoietin-cultured human MKs after rapamycin treatment. Rapamycin induced an inhibition in the G1/S transition and a decrease in the mean MK ploidy via a diminution of p21 and cyclin D3 occurring at a transcriptional level. Both cycling (2N/4N) and polyploid (8N/16N) MKs were reduced in size, with a size reduction slightly more pronounced in mature polyploid MKs than in immature ones. Rapamycin also induced a delay in the expression of MK markers and prevented the generation of proplatelet MKs. Additional experiments performed in vitro with MKs from mutant mice showed that the decrease in mean ploidy level and the delay in MK differentiation in the presence of rapamycin were less pronounced in CdknIa (p21)–/– MKs than in CdknIa (p21)+/+ MKs. These findings indicate that the mTOR pathway plays an important role during megakaryopoiesis by regulating ploidy, cell size, and maturation, in part by regulating p21 and cyclin D3.

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias.

Megakaryocyte-Active Chemokines: Dysregulation in the SDF-1a/CXCR4 Axis in Patients with Essential Thrombocythemia.

Chemokines belong to a large family of molecules that are implicated in the localization and production of blood cells. Some of them, such as Interleukin 8 (IL-8) and GRO-a, participate in the regulation of megakaryopoiesis, mostly by exerting an inhibitory action. Recently, the involvement of stromal derived factor 1 (SDF-1) in synergizing the stimulatory effect of thrombopoietin on megakaryopoiesis and its participation in megakaryocyte transendothelial migration has been described. The aim of the present study was the evaluation of the plasma levels of IL-8, GRO- a and SDF-1 in patients with essential thrombocythemia (ET), a myeloproliferative disorder characterized by megakaryocytic hyperproliferation with increased circulating platelet count. Besides, the corresponding chemokine receptors were assayed on platelet membrane from ET patients. A cohort of 27 patients diagnosed according to the Polycitemia Vera Study Group were enrolled in the study (mean age, 45; 21 women). Twenty-seven normal subjects matched by sex and age were taken as the control group. The Ethic Committee from IDIM A. Lanari approved the study and all patients and normal controls signed the informed consent. Plasma levels of the chemokines were measured by ELISA technique (R&D Systems) according to the manufacturer. Expression levels of IL-8 receptors (CXCR1 and CXCR2), GRO-a receptors (CXCR2) and SDF-1 receptors (CXCR4) on platelet membrane were evaluated by flow cytometry using specific MoAbs and the corresponding isotype controls (B-D Pharmingen). Flow cytometry results were expressed as relative fluorescence intensity (RFI, the relationship between the mean fluorescence intensity from the specific antibody and the isotype control). Results were expressed as median and range. Statistic analysis was carried out using Mann-Whitney Wilcoxon rank sum test and Wilcoxon signed rank test. Plasma levels of the chemokines measured in 19 ET patients were similar to that found in normal controls, IL-8 2.5, pg/ml (0.8–28.2) and 2.8 pg/ml (1.1–16.5), GRO-a, 30.0 pg/ml (7.4–463.1) and 23.9 pg/ml (9.6–148.0), SDF-1, 1895.0 pg/ml (1246.0–2719.0) and 1915.0 pg/ml (822–2424.0), respectively. The expression levels of CXCR4 receptor was found diminished in platelets from ET patients, RIF 16.94 (1.3–31.3) compared to normal controls, 27.4 (2.4–58.4); p=0.0059, n=10. However, the expression of CXCR1 and CXCR2 in platelets from ET patients was normal. In conclusion, although plasma levels of the chemoquines IL-8, GRO-a and SDF-1 were normal in these patients, the decreased level of CXCR4 on platelet membrane suggests a dysregulation in the SDF-1a/CXCR4 axis in patients with essential thrombocythemia.