Megakaryocyte Development Research Articles

Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function

The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors. In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCγ2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1- and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, αIIbβ3, and GPIbα. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function.

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis

AbstractPrimary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1low mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1low mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1low phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1low mice and may represent additional targets for therapeutic intervention in PMF.

The human NPM1 mutation A perturbs megakaryopoiesis in a conditional mouse model

AbstractThe NPM1 mutation is the most frequent genetic alteration thus far identified in acute myeloid leukemia (AML). Despite progress in the clinical and biological characterization of NPM1-mutated AML, the role of NPM1 mutation in leukemogenesis in vivo has not been fully elucidated. We report a novel mouse model that conditionally expresses the most common human NPM1 mutation (type A) in the hematopoietic compartment. In Npm1-TCTG/WT;Cre+ mice, the NPM1 mutant localized in the cytoplasm (NPMc+) of bone marrow (BM) cells. The mutant mice developed no AML after 1.5-year follow-up. However, NPMc+ expression determined a significant platelet count reduction and an expansion of the megakaryocytic compartment in the BM and spleen. Serum thrombopoietin levels overlapped in mutant vs control mice, and BM cells from Npm1-TCTG/WT;Cre+ mice formed more megakaryocytic colonies in vitro. Moreover, we demonstrated the up-regulation of microRNAs (miRNAs; miR-10a, miR-10b, and miR-20a) inhibiting megakaryocytic differentiation along with increased expression of HOXB genes. Notably, these findings mimic those of human NPM1-mutated AML, which also exhibits a similar miRNA profile and expansion of the megakaryocytic compartment. Our mouse model provides evidence that the NPM1 mutant affects megakaryocytic development, further expanding our knowledge of the role of NPM1 mutant in leukemogenesis.

Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, proliferation and terminal differentiation of megakaryocytic progenitors (MK-p) and maturation of megakaryocytes (MKs) to produce functional platelets. This complex process occurs in specialized niches in the bone marrow where MKs align adjacent to vascular endothelial cells, form proplatelet projections and release platelets into the circulation. Thrombopoietin (THPO, TPO) is the primary growth factor for the MK lineage and necessary at all stages of development. THPO is constitutively produced in the liver, and binds to MPL (c-Mpl) receptor on platelets and MKs. This activates a cascade of signalling molecules, which induce transcription factors to drive MK development and thrombopoiesis. Decreased turnover rate and platelet number result in increased levels of free THPO, which induces a concentration-dependent compensatory response of marrow-MKs to enhance platelet production. Newly developed thrombopoietic agents operating via MPL receptor facilitate platelet production in thrombocytopenic states, primarily immune thrombocytopenia. Other drugs are available for attenuating malignant thrombocytosis. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and the innovative drugs and therapeutic modalities to stimulate or decrease thrombopoiesis.

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 effects of GATA-1 and NF-E2 deficiency on bone biomechanical, biochemical, and mineral properties.

Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.

INVOLVEMENT OF BONE MARROW MEGAKARYOCYTES IN THE FORMATION OF THE BONE MARROW-BLOOD BARRIER IN TRANSGENIC MICE

Mature cell release takes place through the hypothe tical barrier- the bone marrow-blood barrier. It co nsists of blood vessels and cells that surround them. Such ce lls are, among others, megakaryocytes. They are involved in the release of platelets or other cellular componen ts of blood. The amount and the development of megakaryocytes and thus the maturity of barrier dep ends on many factors. One of them is IL-6, which gene is located on the short arm of chromosome 7 (7p15-p21). The study was performed on wild-type mice (C57B4/6J) and on mice lacking the gene encoding IL-6 (IL-6 KO-C57B4/6J IL-6tm1 Kopf-/-). Bone marrow of all animals in both groups has been collected, fixed and staine d. Megakaryocytes were identified in each microscopic specimen using immunohistochemical reaction with the CD61 antibody. Then all specimens were subjected to histomorphometric analysis. Statistically significa nt differences in the total number and size of maga kariocytes was noted, which affects bone marrow-blood barrier functions. This could be used in the conventional t reatment of cancer, respiratory diseases and coagulation dis orders and in the treatment using nanoparticles.

Involvement of TGF 1 in autocrine regulation of proplatelet formation in healthy subjects and patients with primary myelofibrosis

Megakaryocytes release platelets into the bloodstream by elongating proplatelets. In this study, we showed that human megakaryocytes constitutively release Transforming Growth Factor β1 and express its receptors. Importantly, Transforming Growth Factor β1 downstream signaling, through SMAD2/3 phosphorylation, was shown to be active in megakaryocytes extending proplatelets, indicating a type of autocrine stimulation on megakaryocyte development. Furthermore, inactivation of Transforming Growth Factor β1 signaling, by the receptor inhibitors SB431542 and Stemolecule ALK5 inhibitor, determined a significant decrease in proplatelet formation. Recent studies indicated a crucial role of Transforming Growth Factor β1 in the pathogenesis of primary myelofibrosis. We demonstrated that primary myelofibrosis-derived megakaryocytes expressed increased levels of bioactive Transforming Growth Factor β1; however, higher levels of released Transforming Growth Factor β1 did not lead to enhanced activation of downstream pathways. Overall, these data propose Transforming Growth Factor β1 as a new element in the autocrine regulation of proplatelet formation in vitro. Despite the increase in Transforming Growth Factor β1 this mechanism seems to be preserved in primary myelofibrosis.

Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases

The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations.

High Doses of Romiplostim Induce Proliferation and Reduce Proplatelet Formation by Human Megakaryocytes

BackgroundRomiplostim (AMG531) is a Thrombopoietin (TPO) receptor agonist with no homology with the endogenous TPO that has been used to treat patients affected by immune thrombocytopenia (ITP). Despite the use of TPO mimetics in the clinical practice, the mechanisms underlying their impact on megakaryocyte function is still unknown.Methodology/Principal FindingsIn this project we took advantage of an in vitro human model, that we have established in our laboratory for long time to study megakaryocyte development from human cord blood-derived progenitor cells, and we demonstrated that increasing doses of AMG531 (100 to 2000 ng/mL) determine a progressive increase of megakaryocyte proliferation with a parallel decrease in megakaryocyte ploidy and capacity of extending proplatelets. Most importantly, these differences in megakaryocyte function seemed to be correlated to modulation of AKT phosphorylation.Conclusions/SignificanceOverall our results shed new light on the mechanisms and on the relevance of dosage related to AMG531 impact on megakaryocyte function.

Expression of plasma membrane receptor genes during megakaryocyte development

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function.

Thrombotic events in models GATA-1 low myelofibrosis characterized by altered localization of P-selectin during megakaryocyte development

Patients with primary myelofibrosis (PMF) have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions.This interactions are mediated by P-selectin and even though the megakaryocytes(Mk)of MF patients express normal levels of P-selectin,it remains abnormally localized to the DMS rather than being assembled into the a-granules in platelets.Mice carrying the hypomorphic Gata1low mutation express the same Mk abnormalities presented by PMF patients,including abnormal P-selectin localization to the DMS and develop with age myelofibrosis,that closely resembles human PMF.The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and,in this case,the role played by P-selectin in the development of the trait.To this aim,Gata1low mice were crossed with P-selnull mice according to standard genetic protocols and Gata1lowP-selWT,Gata1lowP-selnull and Gata1WTP-selnull or Gata1WTP-selWT littermates obtained.Platelet count,hematocrit as well as platelet microparticle levels were determined on all the different mutants.It was shown that platelet counts are reduced in Gata1low mice.Moreover,platelet microparticles are reduced in Gata1low mice and P-selectin positive platelet microparticles were not found.The presence of thrombosis was determined by immunohistological staining of organs.Gata1low mice with or without the P-selectin null trait had a prolonged bleeding time and thrombosis was seen adult and old Gata1low mice,but the Gata1low/P-selnull mice were rescued.Thus,presence of the P-selectin null trait rescued Gata1low mice from the thrombotic phenotype,but did not change level of platelet microparticles.All these data indicate that abnormal localization of P-selectin,induced by the Gata1low mutation,and thus, increased pathological interactions with leucocytes,is responsible for the increased presence of thrombosis seen in these mice.

Use of zinc-finger nucleases to knock out theWASgene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

SUMMARYMutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.

Role of promoter element in c-mpl gene expression induced by TPO

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

Dysregulation of PSTPIP2 Due to Loss of GATA-1 Contributes to Aberrant Megakaryopoiesis

AbstractAbstract 854GATA-1-deficiency causes dysregulation of multiple genes and leads to aberrant megakaryopoiesis. How the dysregulated genes may contribute to abnormal megakaryocyte development and differentiation is largely unknown. In this study, we focused on Pstpip2, a gene that was upregulated in GATA-1low and GATA-1s mutant megakaryocytes. A strong GATA-1 binding site in the intron 1 of Pstpip2 gene was previously identified by GATA-1 ChIP-Seq studies. These observations strongly suggest that Pstpip2 is a GATA-1 target gene. By using ChIP-PCR we showed that GATA-1 indeed bound to Pstpip2 intron 1 region in megakaryocytes. We also observed that Pstpip2 was expressed in myeloid cells and hematopoietic progenitors, but not lymphocytes. Noticeably, two cell lines with GATA-1 deficiencies, G1ME and CMK, which approximate the MEP and megakaryoblast stages, respectively, showed moderate expression of Pstpip2. Moreover, K562 cells displayed significant upregulation of PSTPIP2 and concomitant downregulation of GATA-1 during TPA-induced megakaryocytic differentiation. To study its function in megakaryocytic differentiation, we modulated PSTPIP2 expression by overexpression or shRNA knockdown. Ectopic expression of PSTPIP2 in K562 or CMK cells impaired TPA-induced megakaryocytic differentiation as evidenced by decreased CD41 expression. In contrast, downregulation of PSTPIP2 by shRNA promoted CD41 expression and increased polyploidy. These findings suggest that failure to suppress PSTPIP2 due to loss of GATA-1 may contribute to abnormal megakaryopoiesis.To probe the mechanism by which PSTPIP2 inhibits megakaryocytic differentiation, we tested whether the interaction with its partner PEST-PTP or phosphorylation of key tyrosine residues in the protein by Src kinase is required. We created two mutants, W232A and Y323/333/F, which block its interaction with PEST-PTP or prevent tyrosine phosphorylation, respectively. Similar to WT PSTPIP2, both mutants retained the ability to inhibit megakaryocytic differentiation suggesting that inhibition of megakaryocytic differentiation does not depend on the PEST-PTP interaction or Src kinase phosphorylation. Separately, we have noted that PSTPIP2 also interacts with Fyn, Grb2, and Shc, suggesting that PSTPIP2 may affect signal transduction. Indeed, we found that ERK phosphorylation was downregulated by PSTPIP2. Since THPO/MPL signaling via JAK/STAT is the physiological pathway in megakaryocyte maturation rather than BCR-ABL signaling seen in K562 cells, we next analyzed the effect of PSTPIP2 on MPL signaling in G1ME cells. Consistent with our findings in K562 and CMK cells, both WT and mutant PSTPIP2 inhibited CD41 expression. Interestingly, ectopic expression of WT PSTPIP2 also caused cell cycle arrest at S phase in G1ME cells. PSTPIP2 also suppressed Erk and Shc phosphorylation. Considering the important role of MAPK/ERK signaling in megakaryocyte development, we believe that PSTPIP2 acts as a negative regulator of THPO-MPL signaling to suppress TPO-stimulated MAPK/ERK activation and subsequently inhibit megakaryopoiesis. Finally, we confirmed the function of PSTPIP2 in megakaryocyte differentiation using mouse bone marrow progenitor cells. Ectopic expression of PSTPIP2 significantly downregulated CD41 expression, reduced CFU-GM and CFU-Mk numbers, and reduced polyploidy, while knockdown of PSTPIP2 exhibited the opposite effects. Taken together, we have identified a new negative regulator of THPO-MPL signaling that is inhibited by GATA-1 in normal megakaryocyte development. Upregulation of PSTPIP2 may contribute to abnormal megakaryopoiesis observed in GATA-1-deficient megakaryocytes. Disclosures:No relevant conflicts of interest to declare.

Platelet Production Occurs Independently of Both the Intrinsic and Extrinsic Apoptosis Pathways

AbstractAbstract 389The survival of megakaryocytes and platelets is regulated by the intrinsic apoptosis pathway. Both cell types express Bak and Bax, the essential mediators of intrinsic apoptosis, which must be kept in check for cellular viability to be maintained. In platelets, Bak and Bax are restrained by the pro-survival protein Bcl-xL. Mutations that reduce the pro-survival activity of Bcl-xL cause dose-dependent cell-intrinsic reductions in circulating platelet life span in mice. Accordingly, pharmacological blockade of Bcl-xL with the BH3 mimetic drugs ABT-737 or ABT-263 (Navitoclax) triggers platelet death and thrombocytopenia in mice, dogs and humans. In mice, loss of Bak and Bax almost doubles platelet life span, and renders platelets refractory to the effects of ABT-737. In megakaryocytes, we and others have recently demonstrated that Bcl-xLand its pro-survival relative Mcl-1 are essential for restraint of the intrinsic apoptosis pathway. Their loss triggers Bak/Bax-mediated death. Conversely, ablation of Bak and Bax can protect megakaryocytes from acute apoptotic insults, such as treatment with carboplatin. Combined with the fact that platelet production is normal in the absence of Bak and Bax, these studies have brought into question the long-standing theory that megakaryocytes deliberately undergo apoptosis in order to shed platelets. However, whilst it is clear that the intrinsic apoptosis pathway is not required for thrombopoiesis, the role of the extrinsic pathway—the other major route to apoptotic cell death—has not been established.In the current study we examined the functionality of, and physiological requirement for, the extrinsic apoptosis pathway in megakaryocytes and platelets. The extrinsic pathway is triggered when members of the tumor necrosis factor (TNF) superfamily such as Fas ligand (FasL) bind to cell surface death receptors (e.g. Fas). This induces receptor multimerization, recruitment of death domain adaptor proteins (e.g. FADD) and subsequent activation of Caspase-8, which is the essential mediator of extrinsic pathway-mediated cell death. We found that both megakaryocytes and platelets express critical components of the pathway, including FADD, Caspase-8 and Bid. Megakaryocytes, but not platelets, also expressed the death receptor Fas. Mature fetal liver-derived megakaryocytes treated with soluble FasL exhibited activation of Caspase-8 and the effector Caspases-3/7. This was accompanied by mitochondrial damage and a failure of pro-platelet formation.To establish the requirement for the extrinsic pathway in megakaryocyte development and platelet production, we conditionally deleted Caspase-8 from the megakaryocyte lineage. Platelet counts and platelet life span in Casp8Pf4Δ/Pf4Δ mice were indistinguishable from those of wild-type littermates. Megakaryocyte numbers, morphology, ploidy and in vitro pro-platelet formation capacity were also normal. Caspase-8-deficient megakaryocytes were resistant to FasL treatment. Casp8Pf4Δ/Pf4Δ animals responded to experimentally-induced thrombocytopenia in a manner similar to wild-type. Collectively, these data indicate that the extrinsic apoptosis pathway is dispensable for the generation and survival of platelets. To examine any potential redundancy between the extrinsic and intrinsic apoptosis pathways, we generated Bak−/−BaxPf4Δ/Pf4ΔCasp8Pf4Δ/Pf4Δ triple knockout mice. The ability of these animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers and morphology were normal. Thus, platelet shedding by megakaryocytes does not require the intrinsic or extrinsic apoptosis pathways. Together with recent work demonstrating that the apoptotic initiator caspase, Caspase-9, is also dispensable for platelet production, we conclude that platelet biogenesis is not an apoptotic process. Disclosures:No relevant conflicts of interest to declare.

Increased TGF-β1 expression Cooperates with the Gata1low Mutation in Determining the Gata1low Phenotype in CD1 Mice

AbstractAbstract 3462The Gata1low mutation impairs megakaryocytic and erythroid development. Gata1low mice die at birth with thrombocytopenia and anemia but in the CD1 background, which readily activates spleen hematopoiesis, they recover from anemia at 1 month living up to 2 years but die from fatal anemia within 3-weeks following splenectomy. The increased levels of TGF-β1 expressed by these mice suggests that TGF-β1 may determine some of the traits of their phenotype. This hypothesis was tested by profiling the TGF-β signaling pathway of bone marrow (BM) and spleen of Gata1low mice and by determining the effects of loss of TGF-β1 function on phenotype. Differences (3two-fold) in TGF-β1 signaling gene expression were determined with the Mouse TGF-β RT2Profiler™ PCR array kit (SABiosciences, Frederick, MD, USA) and their biological effects predicted with the David Bioinformatic Database (David Bioinformatics Resources 6.7 NIAID/NIH). Loss of TGF- β1 function was achieved by treatment for 2 months with SB431542, or vehicle as control. During treatment, mice were phlebotomized (50 μL) every 3-days for blood evaluation.Differences in the TGF- β pathway genes abnormally expressed in BM and spleen from Gata1low mice were observed. In BM, 6 genes were up-regulated and 14 down-regulated (Bmp2, Bmp5, Acvrl1, Tgfblil and Igf1 with a p<0.05) with respect to wild-type. In spleen, seven genes were up-regulated (Cdkn1a and Ltbp1 with a p<0.05) and 29 down-regulated (Gdf2 and Nodal with a p<0.05). David analyses identified activation of TGF- β, Hedgehog and p53 pathways both in BM and spleen and predicted biological effects consistent with the Gata1low phenotype. TGF- β signaling alterations predicted increased osteoblast maturation in BM but not in spleen (mice are osteopetrotic), increased apoptosis and G1 arrest in BM and spleen (erythroblast apoptosis increases in both organs) and reduced ubiquitin-mediated proteolysis, a process necessary for erythroid maturation, in BM but not in spleen (erythropoiesis is ineffective in BM). The Hedgehog pathway, required for BMP4 (the stress-erythroid growth factor) expression, was active in both BM and spleen. Also the p53 pathway was activated in both organs but mTOR signaling was activated only in spleen.SB431542–treatment had profound effects on TGF- β expression profile and on the phenotype of Gata1low mice. SB431542- treatment only significantly reduced TGF- β1 mRNA (∼20%) and protein (>80%) levels in BM and spleen. However, numerous TGF- β pathway genes were abnormally expressed in BM and spleen of both vehicle- and SB431542-treated mice. In BM, 48 genes were up-regulated and 2 down-regulated by vehicle-treatment while 46 genes were up-regulated and 2 down-regulated by SB431542-treatment. Thirty-nine of the alterations were common to the two groups and included BMP4 activation, indicating that they were induced by phlebotomy. No gene was abnormally expressed only in vehicle-treated mice but David analyses identified that p53 signaling was normal in SB431542-treated mice, predicting reduced apoptosis and activation of hematopoiesis in their BM. In spleen, 38 genes were up-regulated and 11 down-regulated in vehicle-treated animals while 37 were up-regulated and 6 down-regulated in SB431542-treated animals. Thirty genes were altered in both groups but these common genes did not include BMP4 which was activated in vehicle-treated mice only, suggesting that SB431542-treated mice are unable to activate stress-erythropoiesis in spleen. In addition, Id2 and Stat1 were down-regulated in vehicle-treated animals but expressed at normal levels in SB431542-treated mice, suggesting that these genes may be involved in the activation of splenic erythropoiesis in Gata1low mice. David analyses identified that expression of genes involved in ubiquitin-mediated proteolysis was reduced in spleen from SB431542-treated mice, predicting that inhibition of TGF- β signaling would reduce erythroid maturation in their spleen. Both predictions were confirmed by comparing the phenotype of vehicle- and SB431542-treated mice which indicated that inhibition of TGF-β1 signaling restores hematopoiesis, including megakaryocyte development, in BM and reduces hematopoiesis in spleen. In conclusion, these results suggest that TGF-β1 signaling cooperates with the Gata1low mutation in determining the phenotype of Gata1low mice in the CD1 background. Disclosures:No relevant conflicts of interest to declare.

Intrinsic Impairment of Platelet Production in Persistent/Chronic ITP

AbstractAbstract 624Persistent/chronic immune thrombocytopenias (PC-ITP) are acquired thrombocytopenias characterized by a platelet count less than 100 G/L, lasting respectively more than 3 months/one year. It is a diagnosis of exclusion, and other causes of acquired thrombocytopenia such as neoplasms, infections, autoimmune diseases or drugs must have been ruled out. It is commonly admitted that the pathogenesis of ITP associates the presence of antiplatelet antibodies that bind to mature platelets, leading to their elimination by macrophages through Fc-mediated recognition mechanisms mainly in the spleen or liver, or both. These antibodies are also thought to bind to megakaryocytes impairing platelet production. Newly available TPO receptor agonists are highly effective in chronic ITP patients, with a dramatic increase in platelet count, which suggests a crucial role of altered megakaryopoiesis in this disorder. Because antiplatelet antibodies are only found in 20% of PC-ITP, we hypothesized that some of these patients might have an intrinsic defect in megakaryopoiesis rather than an immune-mediated dysmegakaryopoiesis. We thus analyzed in vitro megakaryocyte differentiation and proplatelet formation in 9 PC-ITP patients, 4 acute ITP patients (A-ITP), and 9 healthy controls (CTRL).All PC-ITP patients had ITP criteria regarding last international consensus (Neunert C et al, Blood 2011), with a slowly progressing thrombocytopenia (76 G/L, range: 29–97) lasting more than 3 months (median: 17 months; range: 7–36 months), no anti-platelet antibodies and normal medullar density and number of megakaryocytes. All A-ITP patients also matched ITP criteria. All samples were taken at the time of diagnosis, before any treatment was administered. They were compared with 9 controls including patients undergoing valvular replacement or healthy bone marrow donors with normal blood count. To analyze whether there was a defect of megakaryopoiesis that was cell-intrinsic, we isolated CD34 positive cells from the bone marrow and analyzed in vitro megakaryocytic differentiation with TPO-mimetic romiplostim. Proliferation was measured at days 3, 6 and 10 and compared in the 3 groups by a proliferation coefficient. Membrane maturation was assessed at day 6 and 10 by flow cytometry (FC) after CD41-FITC and CD42-PE staining. Megakaryocytic ploidy was measured at day 10 by FC after propidium iodure and CD41 staining. At day 8, large mature megakaryocytes were isolated after discontinuous HSA density gradient and proplatelets forming megakaryocytes were counted from between days 9 and 13. Late mature megakayocytes (day 12–13) were observed with a confocal microscope to qualitatively analyze proplatelet formation.We did not observe any difference between A-ITP or PC-ITP patients and controls in term of proliferation, ploidy, or expression of surface differentiation markers (CD41, CD42). In contrast, PC-ITP-derived megakaryocytes showed a defect in proplatelet formation, as only 12% of large, mature megakaryocytes were able to form proplatelets in liquid culture at day 11 vs 37% in CTRL (p=0,046) and 39% in A-ITP (p=0,03), 11% at day 12 vs 43,2% in CTRL (p=0,024) and 46% in A-ITP (p=0,0002), and 10% at day 13 vs 44,5% in CTRL (p=0,015) and 46% in A-ITP (p=0,03). Besides, we observed that proplatelet-forming megakaryocytes from ITP patients had less proplatelets per megakaryocyte and less bifurcation per proplatelet.In conclusion, our study shows that megakaryocytes from patients with persistent/chronic ITP have an intrinsic defect in megakaryocyte development that is independent from the medullar environment. This defect affects proplatelet formation and further investigations are now needed to better describe mechanisms underlying proplatelet alteration in this disease. Disclosures:Viallard:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Molecular Triggers of Granule Formation in Megakaryocytes and Platelets

AbstractAbstract SCI-34Platelet secretory granules develop within maturing bone marrow-resident megakaryocytes, where α-granules, δ-granules, and lysosomes are transported to extending proplatelets (1) and undergo further maturation after platelets are released into the circulation. Mature platelets contain 50 to 80 membrane-enclosed α-granules, three to eight dense (δ-) granules, and a few lysosomes. δ-granules store calcium, phosphate, ADP, ATP, and serotonin, which play important roles during platelet activation. α-granules store numerous soluble and membrane-bound proteins, including adhesion molecules, cytokines, chemokines, coagulation and fibrinolytic proteins, immunologic modulators, and an assortment of complement, growth, and pro- and antiangiogenic factors. These play important roles in clotting, angiogenesis, inflammation, wound healing, and bone remodeling, and provide defenses against infections. Insights into megakaryocyte and platelet δ-granule development have come from studying inherited δ-granule deficiencies such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS; MIM214500), for which mouse models also exist. Several genes/proteins linked to the regulation of vesicle trafficking have been implicated in δ-granule formation. These include components of BLOC (biogenesis of lysosome-related organelles complex) protein complexes (BLOC-1, −2, and −3), known vesicle-trafficking proteins (VPS33A and the β3A and δ subunit of AP-3), and the BEACH domain, containing protein LYST. Less is known about α-granule development, in which two inherited disorders result in platelets lacking α-granules: ARC syndrome (Arthrogryposis, Renal dysfunction, and Cholestasis; MIM208085) and gray platelet syndrome (GPS; MIM139090). GPS is characterized by variable thrombocytopenia and large, gray-appearing platelets on blood smears, with α-granules and α-granule proteins markedly decreased or absent. We and others recently determined that GPS is caused by mutations in NBEAL2, encoding a BEACH protein (2, 3, 4). Our work has also shown that the large α-granule-deficient platelets in ARC syndrome can arise due to mutations in VPS33B, encoding the Sec1/Munc18 (SM) protein VPS33B involved in vesicular trafficking (5). SM proteins are known to interact with membrane-associated soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptors (SNAREs) of the syntaxin subfamily. Recently we have also identified VPS16B as a VPS33B-binding protein. A patient with homozygous missense mutations in C14orf133, encoding VPS16B, has ARC syndrome, with platelets lacking α-granules and stored α-granule proteins. Thus VPS16B is also required for megakaryocyte and platelet α-granule formation, and, in contrast to GPS, in which platelets have α-granule membrane proteins such as P-selectin, VPS16 null platelets lack P-selectin. The observation that GPS and ARC platelets lack α-granules but contain δ-granules, while HPS platelets are devoid of δ-granules but contain α-granules, suggests there are distinct pathways for δ-granule and α-granule biogenesis in maturing megakaryocytes. Immunofluorescence microscopy suggests that VPS16B and VPS33B act along the trans-Golgi network/late endosome/α-granule vesicular trafficking pathway during formation of α-granules in megakaryocytes. It is predicted that complexes containing VPS33B and VPS16B facilitate docking and fusion of intracellular vesicles during α-granule formation, while NBEAL2 promotes the maturation of nascent α-granule vesicles. Disclosures:No relevant conflicts of interest to declare.

GATA2 Mutations in Pediatric Acute Myeloid Leukemia: A Study of the Japanese Childhood AML Cooperative Study Group.

AbstractAbstract 2536 BackgroundAcute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Although many important molecular markers have already been discovered in AML, no known prognosis-associated cytogenetic aberrations or mutations were detectable in a subset of AML patients. In this regard, recent reports of somatic mutations affecting the C-terminal zinc finger (ZF) 2 of GATA2 are intriguing, because these GATA2 mutations were associated with the progression of chronic myeloid leukemia, whereas hereditary ZF2 of GATA2 mutations predispose to AML and myelodysplastic syndrome. GATA2 mutations were also reported as a predisposing gene of the monocytopenia, mycobacterial infection (MonoMAC) syndrome and the dendritic cell, monoctye, B and NK lymphoid deficiency (DCML) syndrome. GATA2 belongs to a family of zinc finger transcription factors, and is important for hematopoietic stem cell proliferation and normal megakaryocytic development. These findings prompted us to search for possible GATA2 mutations in pediatric AML. MethodsTo explore the frequency and clinical impact of GATA2 mutations, we examined 157 Japanese pediatric AML patients, including 13 with FAB-M3 and 10 with Down syndrome (DS) who were treated on different treatment protocol, by PCR following direct sequencing. As GATA2 mutations thus far reported almost exclusively involved exons 4–6 that encode zinc finger 1 and 2 domain, we confined our analysis to these exons. ResultsGATA2 missense mutations were found in 7 out of 157 patients (4.5%). Notably, All of GATA2 mutations were located in ZF2 in this study, although almost all of GATA2 mutations in adult AML were located in ZF1. Wild type GATA2 were found in 3 of 3 AML patients with GATA2 mutation. The other 4 patients had no history of Mono MAC syndrome, suggesting that these mutations were acquired. The zinc finger region of GATA2 is required for binding to promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA2 and can modify its transactivation capacity. Interestingly, 2 GATA2 mutations were found in FAB-M3 in this study, GATA2 mutations also may be associated with acute promyelocytic leukemia. Clinical and molecular features between patients with and without GATA2 mutations were not significantly different in the clinical parameters (WBC, age, sex, etc.), and the outcome of GATA2 mutation positive patients was not poor when compared to GATA2 mutation negative patients: Two of the 7 patients received allogeneic-stem cell transplantation (Allo-SCT) and one of them died of gastrointestinal hemorrhage after SCT. The other 5 patients who did not receive the SCT were still alive. ConclusionGATA2 is a new predisposition gene for pediatric AML and shows functional changes caused by mutations within a highly conserved threonine repeat located in ZF2. Interestingly, MonoMAC and DCML syndromes were not observed in de novo AML patients with GATA2 mutations. Although further investigation is needed, our results indicated that GATA2 mutations were associated with a favorable outcome in pediatric AML. Most of the patients with GATA2 mutations have been classified into an intermediate risk group in our study, however, their favorable outcome suggests that less aggressive treatment strategy without SCT might be appropriate for AML patients carrying GATA2 mutations. Disclosures:No relevant conflicts of interest to declare.