Terminal Differentiation Of Megakaryocytes Research Articles

Actin Bundling Protein L-Plastin Regulates Megakaryocyte Membrane Rigidity and Platelet Spreading

An adequate number of normally functioning platelets is required for normal hemostasis, while thrombocytopenia and platelet dysfunction are common causes of pathologic bleeding. Platelets are released into the blood from proplatelets (PPs) that extend from megakaryocyte (MK) intracellular invaginated membrane system (IMS) that is continuous with the plasma membrane. Tubulin provides the motor for PP extension, and actin reorganization is believed to be critical for MK migration and PP branching. However, the role for actin in PP formation (PPF) is less well understood. The initiation of PPF requires podosomes - actin-based structures with an F-actin core surrounded by a ring of adhesion molecules (e.g., integrins) and many actin-binding proteins. We have been intrigued by how podosomes form and how the IMS is able to penetrate the dense cortical actin cytoskeleton to form PPs. We recently showed that human MKs and platelets express a novel actin bundling protein, L-plastin. L-plastin levels were inversely correlated with platelet number, and its expression was down-regulated during MK maturation. L-plastin knockdown in cultured human CD34+ derived MKs caused increased PP, IMS, and podosome formation. These data indicate L-plastin is a negative regulator of MK platelet production. To assess the in vivo effects of L-plastin, we have investigated platelet production and function in vivo using L-plastin knockout (KO) mice (Lcp1-/-). Compared to wildtype (WT) littermate controls, Lcp1 KO mice showed both significantly increased platelet counts and increased PPF from cultured murine bone marrow MKs. Elegant work in both HUVECs and epithelial cells has shown high membrane proximal F-actin (MPA) prohibits membrane protrusions, and lowering MPA density is required to sensitize areas for membrane protrusion (Bisaria, Science 2020). We hypothesize that normal MK levels of L-plastin inhibits PPF in less mature MKs by F-actin bundling, which raises the MPA. Conversely, lower L-plastin levels result in lower MPA and enabling PP protrusion. Enhanced L-plastin-mediated F-actin bundling is expected to increase membrane stiffness, so we measured MK membrane stiffness at single cell level using a state-of-the-art Biomembrane Force Probe (BFP). MKs from KO mice showed 68% reduction in membrane stiffness as compared to WT controls (p<0.0001) (Fig 1). Ultrastructure of MKs also revealed increased IMS in Lcp1-/- MKs. Preliminary data suggests that L-plastin deficiency also associated with increased MK podosome formation in vitro, which is consistent with our findings in human CD34+ derived MKs. Although L-plastin levels decrease during terminal MK differentiation, they persist at moderate levels in platelets. Platelet integrin activation, granule release and clot retraction also depend on actin reorganization, but L-plastin function in platelets has not been studied. We observed no difference between WT and Lcp1 KO platelets for measures of inside-out integrin signaling (activation-induced JON/A binding or P-selectin expression). However, when plated on fibrinogen and stimulated with thrombin, platelets from KO mice showed significantly increased spreading compared to WT mice (Fig 2). A similar trend was observed for platelet clot retraction. Taken together these results show that L-plastin regulates MK membrane stiffness, which likely inhibits PPF. L-plastin also appears to participate in platelet outside-in, but not inside-out, integrin signaling. On-going studies are determining whether integrin beta3 is the molecular link between these two findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modelling the Progression of a Preleukemic Stage to Overt Leukemia in Children with Down Syndrome

Myeloid leukemia of Down syndrome (ML-DS) is a tractable human model of acute myeloid leukemia. A preleukemia phase, transient abnormal myelopoiesis (TAM) and silent TAM, occurs in 28% of neonates with Down Syndrome (Roberts et al. Blood 2013). TAM is caused by trisomy 21 and acquired mutations in GATA1 that result in a N-terminal truncated protein, GATA1s, in hematopoietic stem and progenitor cells (HSPCs) of fetal origin. ML-DS evolves from TAM by acquisition of additional genetic lesions. The nature of these lesions and the mechanism of transformation are incompletely understood.We performed exome sequencing and targeted resequencing of 141 ML-DS and 111 TAM patients to characterize the evolving mutational landscape from TAM to ML-DS. On average 1.6 acquired mutations were detected in ML-DS (in addition to GATA1 mutations), significantly more than in TAM (0.4 mutations per sample). Additional anticipated loss-of-function mutations acquired in ML-DS mainly affected cohesin components including CTCF (43% of patients), PRC2 components (13%), KANSL1 and other epigenetic regulators (14%). Conversely, anticipated gain-of-function mutations were most prevalent in signaling pathways, e.g. JAK kinases, MPL, KIT and RAS family members (40%). Importantly, we detected a novel recurrent hotspot mutation in 4% of patients (6/141 cases) in CSF2RB encoding the IL3-, IL5-, GM-CSF-receptor common beta chain. To test if the A455D/T variant in the CSF2RB transmembrane domain is a putative oncogenic driver, we ectopically expressed CSF2RBA455D in TF1 cells. Cells expressing CSF2RBA455D exhibited cytokine independent growth and STAT5 autonomous phosphorylation. In a CD34+-HSPC megakaryocytic differentiation assay, CSF2RBA455D blocked terminal megakaryocytic differentiation whilst increasing proliferation by 30-fold (P=0.046). Moreover, the median survival of NSG mice transplanted with CSF2RBA455DTF1 cells was shortened by 30 days compared to wild type TF1 cells (23 days compared to 53 days, P=0.0097).To experimentally test the potential of loss-of-function mutations to transform TAM to ML-DS, we performed an in vivo murine isogenic transplantation screen using Gata1s expressing fetal hematopoietic cells from Cas9-knockin mice. We tested variants in 22 genes, recurrently detected in ML-DS, with a pool of prevalidated gRNAs. This resulted in short latency (n=18 mice; median survival 36 days) and high penetrance (100%) leukemia. Leukemia was not detected in mice infected with control gRNAs. Leukemias had a typical ML-DS megakaryoblastic phenotype (CD117+ and CD41a+). Amplicon sequencing revealed on average 2.9 gRNAs per leukemia and high representation (61% of all leukemias) of gRNAs directed to the tumor suppressor Trp53, which was alone sufficient to induce leukemia with 100% penetrance. When excluding the Trp53 gRNA from pools, leukemic cells from moribund mice contained gRNAs against negative regulators of the RAS and JAK-STAT signaling cascade, such as Nf1, Cbl and Sh2b3 (70% of the mice), Ezh2, Asxl1, Kdm6a,Bcor and other epigenetic modifiers (85%) or Ctcf (15%), closely resembling the mutational landscape of ML-DS. In contrast to ML-DS, gRNAs targeting cohesion components, such as Rad21 and Stag2, were not present in any of the leukemias.In summary, we performed the largest genetic analysis of transforming events in ML-DS that cooperate with trisomy 21 and GATA1s and uncovered a previously undescribed activating mutation in CSR2B. We experimentally validated many of the loss-of-function mutations in a novel murine fetal leukemia assay for ML-DS. The field is now well-placed to study mechanisms of oncogenic cooperativity and identify novel therapeutic approaches for this leukemia. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Abstract 1482: SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL)

Abstract Individuals with Down syndrome (DS) are highly predisposed to develop hematopoietic malignancies, especially acute megakaryoblastic leukemia (DS-AMKL). Generally, genes located on chromosome 21 tend to be increased in Down syndrome patients, but how these individual genes contribute to development of DS-AMKL is still unclear. SON, a gene located on chromosome 21, is upregulated in Down syndrome patients, and it has been reported that DS-AMKL cells have higher level of SON expression. The SON protein functions as a splicing factor and a transcription regulator in leukemia but the role of SON upregulation in DS-AMKL pathogenesis is still not fully elucidated. In addition, runt-related transcription factor 1 (RUNX1) is also located on chromosome 21, but its expression level is downregulated in DS-AMKL patients despite the increased gene dosage. We show that SON represses RUNX1 expression and blocks terminal megakaryocytic differentiation in DS-AMKL. Knockdown of SON in DS-AMKL cell lines leads to morphologic changes and increased cell adhesion. Moreover, cell surface marker analyses demonstrate that the depletion of SON induces increased CD41+ CD61+ populations as well as polyploidy cells, indicating megakaryocytic differentiation. Consistent with these results, SON deletion causes upregulation of RUNX1, which is a critical factor in the megakaryocytic differentiation. Furthermore, our data show that knockdown of SON increases several megakaryocytic transcription factors and decreases erythropoietic transcription factors, facilitating terminal megakaryocytic differentiation of leukemic blasts. Mechanistically, SON binds to the proximal promoter region as well as two intronic enhancer regions upstream of the proximal promoter at the RUNX1 gene locus, and suppresses transcription of RUNX1 by lowering the level of H3K4me3. Taken together, we propose that the increased genomic dosage of SON in Down syndrome impairs terminal differentiation of megakaryo-erythroblasts into mature megakaryocytes through binding to the RUNX1 promoter/enhancer to repress its transcription, which contributes to the higher incidence of AMKL in Down syndrome patients. Our results reveal a novel connection of SON to DS-AMKL pathogenesis and provide clinical importance of SON upregulation in DS. These findings suggest that SON could be a promising therapeutic target for DS-AMKL patients. Citation Format: Lana Vukadin, Eun young Park, Jung Hyun Kim, Erin Eun Young Ahn. SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1482.

High PRMT1 Expression Promotes the Progression of Acute Megakaryocytic Leukemia Via Controlling Metabolic Pathways

Metabolic reprogramming is needed not only to accommodate but also to drive leukemia progression. Yet very little is known on genetic factors other than IDH1 mutations, which can drive leukemogenesis via metabolic reprogramming. Here, we will present data to suggest that protein arginine methyltransferases 1 (PRMT1) is a driver for acute megakaryocytic leukemia via reprogramming metabolism. PRMT1 is highly expressed in megakaryocyte-erythrocyte progenitor cells and downregulated during the terminal differentiation of megakaryocytes. Constitutively high expression of PRMT1 in acute megakaryoblastic leukemia (AMKL) blocks megakaryocyte differentiation. PRMT1 upregulates RBM15 protein level via methylation-dependent ubiquitylation pathway (Zheng et al. Elife, 2015). In this presentation, we discovered that metabolic stress such as hypoxia downregulates PRMT1 protein level. Thus, metabolic stress is the upstream signal for the PRMT1-RBM15 pathway. We have identified that RBM15 specifically binds to 3'UTR of mRNAs of genes involved in metabolic pathways. Using RNA-immunoprecipitation with anti-RBM15 antibody and real-time PCR assays, we validated that RBM15 binds to mRNAs of genes involved in fatty acid oxidation and glycolysis. We transduced PRMT1 into an RBM15-MKL1 expressing cell line 6133. Overexpression of PRMT1 renders 6133 cells to grow in a cytokine-independent manner with increased mitochondria biogenesis, which in turn produces higher concentration of ATP in our metabolomic analysis. Based on the analysis of metabolomics data and RBM15-target genes, we conclude that PRMT1 promotes the usage of glucose as bioenergy via oxidative phosphorylation and inhibits fatty acid oxidation. Given that acetyl-coA is higher in PRMT1 expressing 6133 cells, we asked whether histone acetylation is upregulated in PRMT1 overexpressed 6133 cells. Indeed, we found higher histone acetylation level in PRMT1 highly expressed cells. We also found that propionylated histone is reduced, which is consistent with reduced fatty acid oxidation. Propionyl-CoA molecules are produced from fatty acids with odd carbon numbers. Thus PRMT1-mediated metabolic reprogramming changes epigenetic programming during leukemia progression. Intriguing, we also found PRMT1 overexpression enhances histone H3S10 phosphorylation via methylation-dependent ubiquitylation of DUSP4. DUSP4 promotes polyploidy during megakaryocyte differentiation. Thus PRMT1 caused profound epigenetic changes to promote leukemogenesis. In this vein, we established mouse AMKL models by bone marrow transplantation of 6133 cells as well as human AMKL patient samples respectively. Using this mouse model, we tested PRMT1 inhibitors, acetyltransferase inhibitors as well as other metabolic inhibitors. Treating cells with PRMT1 inhibitors as well as metabolic inhibitors promote MK differentiation of AMKL leukemia cells. Metabolomics analysis of cells recovered from mouse models will be discussed in the presentation. In summary, our data demonstrated that PRMT1 is a major sensor for metabolic stress and that PRMT1 in turn reprograms metabolic pathways to bring epigenetic changes in leukemogenesis. Therefore, targeting PRMT1 and downstream PRMT1-regulated metabolic pathways will offer new avenues in treating acute megakaryocytic leukemia and other hematological malignancies with defective megakaryocyte differentiation. DisclosuresNo relevant conflicts of interest to declare.

Fli1 Expression Level Affects Megakaryopoiesis and Thrombopoiesis: More Is Not Always a Bad Thing

Friend leukemia integration 1 (FLI1) is a critical transcription factor responsible for terminal megakaryocyte differentiation. This transcription factor is amongst the genes missing in the inherited disorder Jacobsen syndrome, resulting from a hemizygous deletion on chromosome 11q. The deletion causes dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Also, FLI1 mutations in its DNA-binding domain region results in thrombocytopenia in affected patients. We examined induced pluripotent stem cell- (iPSC) derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated and if varied levels of Fli1 expression affected megakaryopoiesis and thrombopoiesis. Beginning with a normal control (WT) iPSC line, genome editing was performed to generate three lines with: 1) one copy of FLI1 disrupted (FLI1+/-), 2) hemizygous transgene expression of Fli1 in the adeno-associated virus site 1 (AAVS1) safe harbor locus using a megakaryocyte-specific GP1balpha promoter (WT-overexpressing line, WT-OE), and 3) homozygous transgene expression in AAVS1 (WT-OE2). Additionally, we established an iPSC line from a PTSx patient and edited this line for a similar hemizygous transgene expression of FLI1 (PTSx-OE). Data described here are summarized in the tables below. We confirm our genome editing strategies by examining mRNA and protein levels of iMegs and found WT-OE and WT-OE2 iMegs have ~3X higher mRNA and ~7X higher protein levels than WT iMegs. FLI1+/- and PTSx iMegs both have lower mRNA and ~0.5X the protein levels of WT iMegs, while PTSx-OE iMegs had comparable levels of mRNA and protein as WT-OE iMegs. Megacult colony assays showed WT-OE and WT-OE2 yielded more CFU-Megs than WT HPCs (p²0.01, p²0.05, respectively). PTSx and FLI1+/- had much less CFU-Megs compared to PTSx-OE and WT (p²0.0001). After growth in liquid culture, WT-OE and -OE2 lines had an increase in iMeg numbers (p=0.29, p²0.01) while FLI1+/- and PTSx iMeg numbers were 43% and 10% that of the WT control (p²0.01, p²0.001). PTSx-OE iMeg numbers were comparable to WT. Surface marker CD41 and CD42b levels were increased compared to WT iMegs in the WT-OE and -OE2 iMegs (p=0.25, p²0.05) and less on FLI1+/- and PTSx iMegs (p²0.01, p²0.05). PTSx-OE iMegs were normal compared to WT, but p²0.05 vs. PTSx. Infused CD41+CD42b+ iMegs into NSG mice showed a trend toward same yield of platelets in the OE lines and lower yield in FLI1+/- and PTSx lines compared to WT. However, when calculations were made from HPCs rather than iMegs infused, the FLI1+/- and PTSx iMegs generated significantly less number of released platelets (p²0.05, p²0.01). The half-life of WT-OE and -OE2 released platelets were increased at 4 and 10 hours compared to WT, whereas FLI1+/- and PTSx released platelets have lower half-lives of 2 hours and 0.5 hours, respectively. These decreased platelet half-lives are due to the majority of platelets being defective and cleared at a higher rate. This was corrected to WT in the PTSx-OE-released platelets. Both in vitro iMegs and in vivo released platelets within the murine blood were assessed for function. Analysis of activation by thrombin was performed via FACS for PAC-1 binding and cell surface P-selectin levels, which are both indicators of platelet activation. Initial studies show no difference between WT and the WT-OE and WT-OE2 lines, but FLI1+/- platelet activation was slightly impaired. In summary, we show that studies of iMegs with decreased Fli1 levels replicate many of the clinical features previously described: less iMegs, lower CD41 and CD42b density with less platelets released while having shorter half-lives. On the other hand, increased Fli1 levels resulted in higher number of iMegs with more surface antigen density and released platelets with increased half-lives. Based on overexpression studies of other transcription factors during megakaryopoiesis, such as GATA1, the expectation would have been that the excess of Fli1 in the OE lines would have also resulted in defects in megakaryopoiesis and thrombopoiesis, but instead improvements were seen. The basis for this difference when overexpressing different transcriptional factors needs further analysis. That high Fli1 levels enhance iMeg yield with maintained numbers and function of released platelets may be of value for generating platelets for clinical use from in vitro grown Megs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Megakaryopoiesis and ex vivo differentiation of stem cells into megakaryocytes and platelets

Blood agencies worldwide face the same recurring problem of enrolling healthy donors to produce platelet concentrates (PC) to meet the fluctuating demand. Research in stem cell biology has now made possible the production of culture‐derived platelets (CDP) raising the possibility of producing donor‐independent platelets. The aim of this paper was to first review known key concepts about megakaryopoiesis and second to describe efforts done in our laboratory and that of others for the establishment of culture processes for the production of megakaryocytes and platelets from cord blood haematopoietic stem and progenitor cells (HSPC). Recent progresses made with induced pluripotent stem cells (iPSCs) are also presented. Expansion of HSPCs and megakaryocytes can be accomplished by combining thrombopoietin (TPO) and a wide array of other cytokines. However, expansion of HSPCs ex vivo is associated with a rapid loss of megakaryocyte differentiation potential. Optimization of cytokine combinations and concentrations can improve yields and reduce consumption of growth factors. Moreover, the development of inducible megakaryocytic cell lines that can be triggered to undergo terminal megakaryocytic differentiation offers an independent solution to some of the problems associated with megakaryocyte production ex vivo. Functional characterization of platelets produced ex vivo has so far revealed that they form a heterogeneous population with some culture‐derived platelets (CDP) showing normal platelet properties and functions. In summary, functional CDPs can be produced in HSPC‐ and iPSC‐based cultures. Remaining challenges that need to be addressed before culture‐derived PCs can be used to complement blood‐derived PCs are also discussed.

Aurora A Kinase Is Required For Hematopoiesis and Couples Polyploidization With Terminal Differentiation In Megakaryocytes Through Phosphorylation Of NF-E2

We have recently shown that small molecule inhibitors of Aurora A kinase (AURKA) including dimethyfasudil and MLN827 induce polyploidization and differentiation of normal and malignant megakaryocytes, as evidenced by increased DNA content and upregulation of the cell surface markers CD41 and CD42. Furthermore, our pre-clinical studies demonstrate that MLN8237 shows potent anti-leukemia activity and anti-myelofibrotic activity in MPNs by promoting polyploidization and terminal differentiation of abnormal megakaryocytes. To determine the mechanism by which AURKA inhibitors ameliorate the leukemic and myelofibrotic phenotypes, we have examined the functional requirement for Aurka in adult hematopoiesis. To circumvent the embryonic lethality of the germline knockout and study the requirement for AURKA in adult hematopoiesis, we induced deletion in Aurka floxed mice with Mx1-Cre. Complete loss of AURKA caused a rapid and profound defect in hematopoiesis, with the mice developing pancytopenia and marked hypocellularity of the bone marrow. Notably, we observe an increase in the proportion of CD41 and CD42-positive megakaryocytes in contrast to the deficiency of myeloid and lymphoid cells in the bone marrow. To determine whether the observed defects are cell autonomous, we transplanted Aurkafl/fl, Mx1-Cre bone marrow cells to lethally irradiated recipients, confirmed engraftment, and induced deletion with pIpC. Upon AURKA deletion, the transplanted mice develop an identical phenotype to the one observed in the conditional knockout mice, demonstrating a cell autonomous requirement for AURKA during hematopoiesis. Moreover, in competitive transplantation experiments, Aurka-/- cells are selectively lost and fail to contribute to hematopoiesis. Mechanistically, loss of AURKA led to a significant degree of apoptosis in hematopoietic cells, likely due to mitotic catastrophe resulting from impaired chromosome segregation. To bypass the requirement for AURKA in progenitor cells and examine AURKA function in the megakaryocyte lineage, we deleted AURKA in megakaryocytes ex vivo by infecting Aurka floxed bone marrow with MSCV-Cre. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased ploidy of the megakaryocyte fraction. Together these results are consistent with a selective differentiation effect of AURKA deficiency on megakaryocytes.To investigate whether AURKA modulates differentiation of megakaryocytes through interactions with lineage specific transcription factors, we performed co-immunoprecipitation experiments between AURKA and megakaryocyte transcription factors containing consensus AURKA phosphorylation sites in primary megakaryocytes. Results confirmed a robust interaction between AURKA and p45 NF-E2. Additionally, we found by an in vitro kinase assay that AURKA phosphorylates p45 NF-E2 on the S170 residue, as the S170A mutant of NF-E2 cannot be phosphorylated by AURKA. To explore the functional importance of NF-E2 phosphorylation at S170, we created a phosphomimetic mutant, S170D, and assayed its ability to induce megakaryocyte differentiation in primary cultures. While overexpression of wild-type NF-E2 significantly increased the expression of the megakaryocyte differentiation markers CD41 and CD42, overexpression of the S170D mutant was markedly less effective in promoting megakaryocyte differentiation, indicating that the loss of phosphorylation promotes NF-E2 activity and that NF-E2 activity may be suppressed by AURKA phosphorylation. Finally, to determine if AURKA inhibitors promote megakaryocyte differentiation by allowing activation of NF-E2, we knocked-down down NF-E2 in megakaryocytic cell lines and subsequently treated with diMF or MLN8237. Strikingly, cells with NF-E2 knocked-down displayed significantly less CD41 and CD42 expression in response to AURKA inhibitor treatment compared to control knockdowns. This experiment demonstrates that NF-E2 is required for the differentiation effect caused by AURKA inhibition. Taken together, our data show that Aurora A kinase is required for adult hematopoiesis and that Aurora A regulates NF-E2 function during megakaryocyte differentiation. Our results also reveal that inhibition of AURKA kinase activity couples polyploidization and terminal differentiation of megakaryocytes. Disclosures:Crispino: Sanofi: Research Funding.

Molecular Pathways: Induction of Polyploidy as a Novel Differentiation Therapy for Leukemia

Differentiation therapy has emerged as a powerful way to target specific hematologic malignancies. One of the best examples is the use of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL), which has significantly improved the outcome for patients with this specific form of acute myeloid leukemia (AML). In considering how differentiation therapy could be used in other forms of AML, we predicted that compounds that induce terminal differentiation of megakaryocytes would be effective therapies for the megakaryocytic form of AML, named acute megakaryocytic leukemia (AMKL). We also speculated that such agents would reduce the burden of abnormal hematopoietic cells in primary myelofibrosis and alter the differentiation of megakaryocytes in myelodysplastic syndromes. Using a high-throughput chemical screening approach, we identified small molecules that promoted many features of terminal megakaryocyte differentiation, including the induction of polyploidization, the process by which cells accumulate DNA to 32N or greater. As the induction of polyploidization is an irreversible process, cells that enter this form of the cell cycle do not divide again. Thus, this would be an effective way to reduce the tumor burden. Clinical studies with polyploidy inducers, such as aurora kinase A inhibitors, are under way for a wide variety of malignancies, whereas trials specifically for AMKL and PMF are in development. This novel form of differentiation therapy may be clinically available in the not-too-distant future. Clin Cancer Res; 19(22); 6084-8. ©2013 AACR.

C1galt1-deficient mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes

AbstractC1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galtflox allele (Mx1-C1). Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIbα messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIbα protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of α- and β-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIbα in cells lacking O-glycan might be caused by increased proteolysis.

Aurora A Kinase Is Required for Hematopoiesis

Abstract 1201In acute megakaryocytic leukemia (AMKL), there is a failure of megakaryocytes to differentiate, become polyploid and stop dividing. We used an integrated screening approach that included chemical, proteomic and genetic screens to identify small molecules and their targets that control polyploidization and differentiation of normal and malignant megakaryocytes. We identified several small molecule inducers of polyploidy and used siRNA and proteomic target ID approaches to determine the cellular targets of the lead small molecule dimethylfasudil (diMF). Aurora Kinase A (AURKA) was identified as one of the top targets of diMF. AURKA is an attractive target in AMKL for several reasons. First, AURKA is overexpressed in AMKL cells. Second, at the biologically effective doses used in our cell-based assays, AURKA inhibition was selective for the megakaryocyte lineage. Third, AURKA inhibition by diMF or the selective AURKA inhibitor MLN8237 increased MK polyploidy, induced features of differentiation, blocked proliferation of AMKL blasts, and improved survival in an AMKL mouse model.AURKA is important in mitotic spindle assembly, mitosis, chromosomal alignment and segregation. Moreover, it is required for embryonic development, as Aurka−/− embryos fail to grow beyond the blastocyst stage. However, the extent to which AURKA is necessary for steady state hematopoiesis in adults is unknown. To investigate the necessity of AURKA in hematopoiesis, we utilized a conditionally targeted strain of mice (Aurkaflox/flox). To delete AURKA in megakaryocytes ex vivo, Aurkaflox/flox bone marrow cells were expanded, transduced with a retrovirus expressing Cre and GFP, and then cultured in the presence of THPO for 72 hours. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased DNA content. Assays for apoptosis by Annexin V staining of Aurkaflox/flox cells infected with Cre also showed increased apoptosis in AURKA-deleted cells at 24 and 48 hours.To delete AURKA in vivo, we crossed Aurkaflox/flox mice to MX1-Cre mice and injected wild-type, heterozygous and homozygous floxed mice expressing MX1-Cre with pIpC every other day for six days. We found that deletion of AURKA in hematopoietic progenitors leads to pancytopenia, profound bone marrow defects and death within two weeks. Colony formation assays showed significantly decreased myeloid, erythroid and megakaryocyte colony formation with AURKA deficiency. Bone marrow histology displayed markedly hypocellular marrow, but curiously, flow cytometry revealed a significant increase in the percentage of CD41 and CD42 positive cells. This observation suggests that AURKA normally acts to restrain terminal differentiation of megakaryocytes and is consistent with the CD41 and CD42 inducing ability of AURKA inhibitors.To confirm that AURKA is the key target of our recently identified polyploidy inducers, we assayed the effects of diMF and MLN8237 on Aurka+/+, Aurka+/− and Aurka−/− megakaryocytes. 300 nM diMF and 100 nM MLN8237, concentrations that strongly induce polyploidy, did not increase MK polyploidization in Aurka−/− MKs. diMF and MLN8237 treatment increased polyploidy in Aurka+/− MKs with no significant difference in comparison to Aurka+/+ MKs. We also assayed the ability of wild-type or the T217D mutant of AURKA, which is resistant to inhibition by MLN8237, to reduce the induction of polyploidy caused by diMF and MLN8237 upon overexpression. CMK cells were infected with viruses harboring wild-type or T217D AURKA, treated with DMSO, 3 μM diMF or 30 nM MLN8237 for 72 hours, and then evaluated for DNA content. The increase in polyploidization induced by both compounds was significantly decreased in cells overexpressing the T217D mutant of AURKA. With overexpression of the wild-type AURKA, there was a trend towards reduction in polyploidy, but more variable effects and no significant difference. Thus, AURKA T217D overexpression reduced the ability of diMF and MLN8237 to induce polyploidization, consistent with our conclusion that diMF targets AURKA.Together, our data support a role of AURKA in megakaryocyte polyploidization and differentiation and show that AURKA is required for steady state hematopoiesis. The results also show that AURKA is the key target of diMF in the induction of polyploidization of megakaryocytes and support the development of Aurora A kinase inhibitors in clinical trials for AMKL. Disclosures:No relevant conflicts of interest to declare.

Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

ObjectivePlatelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease.Methods and ResultsWe therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen.ConclusionsOur data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.

Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation

During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine‐1‐phosphate receptor S1P 4

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine‐1‐phosphate receptor S1P ₄

GATA-2 Controls Proliferation of Megakaryocyte Progenitors and Its Expression Contributes to Human DS-AMKL.

GATA2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Mutations in GATA2 are associated with blast crisis phase of chronic myelogenous leukemia (CML) while overexpression of GATA2 is a hallmark of Down syndrome acute megakaryoblastic leukemia (DS-AMKL), a malignancy that is defined by the combination of trisomic chromosome 21 and a GATA1 mutation. Here, we show thatGATA2 is essential for megakaryocyte development from both wild-type and GATA-1mutant hematopoietic progenitors. Furthermore, we reveal that GATA2 is indispensable for cell cycle progression and that its expression cooperates with activated JAK3 to promote TPO-independent expansion of megakaryocytes. Genome-wide microarray analysis revealed that GATA2 regulates a wide set of genes including cell cycle regulators and lineage-specific genes that are essential for terminal differentiation of megakaryocytes. Of note, a subset of genes within the GATA2 signature is contained within human DS-AMKL patient specimens. These observations implicate overexpression of GATA2 in the aberrant gene expression and proliferation of AMKL. Taken together, our data demonstrate thatGATA2 is a critical regulator of normal and malignant megakaryopoiesis.

MafG-Null Mice Show Delayed Leukocyte Engraftment Following Whole Bone Marrow Transplant.

Megakaryocytes, which give rise to platelets, constitute a small population of cells. However, molecular aspects of megakaryocyte development are poorly understood. MafG is the small subunit of the transcription factor, NF-E2, which plays a major role in terminal megakaryocyte differentiation and platelet release. We sought to determine the effect of impaired platelet release due to targeted ablation of the mafG gene following a hematopoietic stem cell transplant on engraftment kinetics. We performed three types transplants:C57Bl/6 Ly5.1 murine bone marrow (mafGWT/WT bm) into lethally irradiated mafG null hosts expressing green fluorescent protein, (GFP),mafG-null/GFP bm into lethally irradiated C57BL/6 mafGWT/WThosts, andmafGWT/WT GFP bm into lethally irradiated C57BL/6 mafGWT/WT hosts.We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight, and megakaryocyte size and concentration on day 5, 7, 14, and 28 post transplant. We found:Platelet engraftment kinetics differed when mafG null animals served as donors or recipients. Specifically, platelets reached their lowest point at day 7 when mafGWT/WT or mafG null bm was transplanted into wildtype hosts. When mafGWT/WT bm was transplanted into mafG null hosts, platelets reached a low point on day 5.MafG null hosts transplant with mafGWT/WT bm had 5-fold lower circulating platelet levels than mafGWT/WT hosts at day 5.When mafGWT/WT served as donors, circulating platelets began to increase after day 7, while animals receiving mafG null donor cells had platelet counts that continued to diminish.Splenic hematopoiesis was markedly decreased when mafGWT/WT bm was transplanted into mafG null hosts. In contrast, when mafG null bm was transplanted into mafGWT/WT hosts, splenic hematopoiesis was increased 2.5 fold on day 28 compared to animals receiving mafGWT/WTdonor cells.Mortality rate was much higher in mafG null hosts.Leukocyte engraftment was delayed when mafGWT/WT bm was transplanted into mafG null hosts.These results suggest that host factors determine the timing of the platelet nadir following bone marrow transplant. Furthermore, the mafG protein, contributes in previously unrecognized ways to splenic hematopoiesis and successful engraftment, possibly through its function in promoting megakaryocyte development and platelet release.

RUNX1 suppression induces megakaryocytic differentiation of UT-7/GM cells

The transcription factor RUNX1 plays a crucial role in hematopoiesis. RUNX1 regulates both differentiation and proliferation of hematopoietic cells. Several reports have shown that RUNX1 participates in megakaryopoiesis, which is a process that leads to formation of platelets. However, to date, the mechanisms by which this occurs have not been fully elucidated. In the present study, we investigated whether siRNA-mediated depletion of RUNX1 affected megakaryopoiesis of UT-7/GM cells. The depletion of RUNX1 in UT-7/GM cells resulted in up-regulation of the expression of megakaryocytic markers and polyploidization, while cell proliferation was down-regulated. Furthermore, the overexpression of RUNX1 decreased the activity of megakaryocytic gene promoters. These results suggest that RUNX1 down-regulates terminal differentiation of megakaryocytes and promotes proliferation of megakaryocytic progenitors.

GATA1-mediated megakaryocyte differentiation and growth control can be uncoupled and mapped to different domains in GATA1.

The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

Megakaryocyte apoptosis: sorting out the signals

British Journal of Pharmacology (2005) 145, 271–273. doi:10.1038/sj.bjp.0706202