Normal Megakaryocyte Maturation Research Articles

S847 DISTINCT AND COMMON DEREGULATED PATHWAYS IN RUNX1, GATA1 AND GFI1B ASSOCIATED BLEEDING DISORDERS

Background:Inherited mutations in the transcription factors (TF) Runt‐related transcription factor‐1 (RUNX1), Growth factor independence 1B (GFI1B) and GATA Binding Protein1 (GATA1) cause megakaryocyte and platelet dysfunction resulting in bleeding disorders. Several platelet abnormalities overlap (e.g. platelet α‐granule paucity) suggesting commonly affected pathways.Aims:The functional effects of many pathogenic mutations remain largely unknown. To further the understanding of the pathobiology of TF mutated bleeding disorders, we identified the affected pathways and proteins important during megakaryopoiesis.Methods:We identified variants in RUNX1 (TD2‐6, Q154fs, G165S), GFI1B (Q287∗, T174N) and GATA1 (R216Q) by next generation sequencing of DNA from cases with thrombocytopenia/pathy and bleedings. According to AMCG guidelines all but the RUNX1 G165S and GFI1B T174N mutations were classified as pathogenic. To identify (commonly) affected biological pathways downstream of mutated TFs, we analyzed platelets from these cases and five controls with label free quantification (LFQ) mass spectrometry.Results:Proteomes from controls were highly similar and clearly distinct from all cases with bleedings. Besides, the non‐pathogenic variants in RUNX1 (G165S) and GFI1B (T174N) clustered together but separate from controls and cases with pathogenic mutations. Pathogenic RUNX1, GFI1B and GATA1 proteomes were also clearly distinct from each other (P < 0.001, between 220 and 520 proteins significantly differentially expressed). For cases with pathogenic TF mutations, gene ontology (GO) pathway analysis detected significantly downregulated platelet a‐granule, ‐ degranulation and ‐ activation pathways. Platelet α‐granule proteins such as platelet factor 4V1, SERPINE2 and F5, but also the structural MYL9 protein were all significantly downregulated. In addition, all pathogenic TF patient samples had negative enrichment for coagulation proteins, compared to control samples. Remarkably, in all TF affected platelets we observed a negative enrichment for JAK‐STAT signaling (AKT1, JAK2, JAK3), normally required for proper megakaryocyte differentiation. To address this finding in more detail, we determined the proteomes of normal megakaryocytes at different stages of differentiation derived from primary immature CD34 positive cells. TF mutated platelet proteomes clearly resembled immature rather than mature megakaryocytes, in line with defective megakaryocyte maturation. For example, in affected platelets an increase in oxidative phosphorylation proteins (ATP6V1A, NDUFS3, NDUFA2) was observed, that are downregulated upon normal megakaryocyte maturation. Many other affected pathways, including the ubiquitin‐proteasome pathway were deregulated in affected platelets.Summary/Conclusion:Pathogenic TF mutations have dominant distinctive effects on platelet proteome composition. Platelets from cases without a proven TF mutation have similar affected proteomes distinct from controls and cases with pathogenic TF mutations. Thus, the studied TF mutations have a dramatic effect on the platelet proteome, in line with the plethora of abnormalities in megakaryocytes and platelets. Identifying which pathways and proteins act during megakaryopoiesis and which are affected by TF mutations allows identification of biological pathways previously unknown to be fundamental to platelet development.

CBF\u03b2-MYH11 interferes with megakaryocyte differentiation via modulating a gene program that includes GATA2 and KLF1

The inv(16) acute myeloid leukemia-associated CBFβ-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFβ-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFβ-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFβ-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.

Imetelstat, a telomerase inhibitor, differentially affects normal and malignant megakaryopoiesis.

Imetelstat (GRN163L) is a specific telomerase inhibitor that has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report, we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy Distribution and Platelet Generation

Abstract 385The small GTPase, RhoA orchestrates actin cytoskeletal dynamics, which plays a role in platelet development and function. After platelet activation, RhoA rearranges the cytoskeleton by facilitating shape change, granule release, and clot retraction. In addition, RhoA is involved in platelet development. It does this by presumably regulating cytokinesis during megakaryocyte-erythroid progenitor cell expansion and megakaryopoiesis. RhoA also regulates thrombopoiesis by coordinating end bifurcation of the proplatelet extensions to amplify preplatelet numbers and the regulation of platelet release. Previously, most studies utilized pharmacological Rho inhibitors, such as C3 exotoxin. This could potentially obfuscate the data because of the incomplete knockdown of RhoA, the non-specific disruption of closely related Rho family members, or the long incubation times that could alter platelet biology. Therefore, we developed a transgenic mouse model that knocked out RhoA only in megakaryocytes and in platelets by using a CRE-LOX strategy to further investigate the role of RhoA in platelet biology. First, mice were generated that had loxP sites flanking the 3rd exon of RhoA (RhoAfl/fl). These mice were then crossed with mice expressing CRE recombinase driven by the platelet factor 4 promoter (PF4 CRE+), thus limiting CRE expression only in megakaryocytes and in platelets. The offspring, RhoAfl/fl PF4CRE+ mice were phenotypically normal, and had normal complete blood counts, except for macrothrombocytopenia. Their platelet counts were 25 ±3% of that observed in their littermate controls, (RhoAfl/fl PF4CRE−) and their platelet size was 130 ±10% of their littermate controls. The lack of RhoA only disrupted aggregation, granule release, and clot retraction when stimulated at the lowest dosage of agonists. To determine the causes of macrothrombocytopenia in RhoAfl/fl PF4CRE+ mice, histological examination of the spleen showed that 26.0±13.3% of the megakaryocytes had pyknotic nuclei as compared to 1.0±0.5% of the controls. In the bone marrow, apoptosis was present in 6.8±3.3% of the RhoA null megakaryocytes, but only in 1.2 ±0.3% of the control megakaryocytes. Furthermore, flow cytometry revealed that megakaryocyte counts in the bone marrow were 51.5 ±4.2% lower than in that of the controls. To determine if lacking RhoA impairs normal megakaryocyte maturation, we measured DNA ploidy using propidium iodide and flow cytometry. In megakaryocytes derived from adult bone marrow or from cultured fetal livers (E13.5, 8 days culture), the RhoAfl/fl PF4CRE+ cells had higher ploidy, a lower number of 2N cells, and an increased number of 16N cells than megakaryocytes derived from control animals. Together these data show that loss of RhoA causes deficiency of megakaryocytes probably due to increased apoptosis, and also causes aberrant maturation of the surviving megakaryocyte. To analyze whether RhoA was also required for thrombopoiesis, cultured RhoA-null megakaryocytes derived from fetal livers were infused into recipient mice. The megakaryocytes lacking RhoA more rapidly release platelets during the first 3 hours post infusion than controls. However, unlike control platelets, the Rho-null platelets were essentially gone within 24 hours. We analyzed whether the increased release of knockout platelets could be due to the up-regulation of proplatelet generation since the deletion of RhoA might impair myosin activity and impair cortical tension. However, micropipette aspiration analysis, which mimics the shear forces found in the bone marrow sinusoid capillaries, showed that the RhoA-null megakaryocytes were less compliant than the controls, primarily due to their large size. These data suggest that the higher ploidy and larger sizes of RhoA knockout megakaryocytes causes them to lodge in the pulmonary capillary bed more quickly, and to rapidly release defective macrothrombocytes. In contrast, the smaller (and thereby more compliant) wild type-cell megakaryocytes fragmented more slowly into platelets through proplatelet extension. Together, our findings demonstrate that RhoA is essential for normal megakaryocyte survival, maturation, and thrombopoiesis. Disclosures:No relevant conflicts of interest to declare.

Serum response factor is an essential transcription factor in megakaryocytic maturation

AbstractSerum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

Megakaryocyte proliferation and ploidy regulated by the cytoplasmic tail of glycoprotein Ibα

AbstractWe have investigated the ability of glycoprotein (GP) Ibα, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3ξ and to influence megakaryocytopoiesis. Using a Gp1ba–/– mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibα allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3ξ. The observed phenotypes illustrate an involvement for GP Ibα in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibα/14-3-3ξ/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibα cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.

Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome

AbstractAn absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an α-subunit of GP Ib (GP Ibα) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the α-subunit of the human interleukin-4 receptor (IL-4Rα). The IL-4Rα/GP Ibα fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the β-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome–producing animals devoid of mouse GP Ibα but expressing the IL-4Rα/GP Ibα fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Rα/GP Ibα subunit interacts with filamin-1 and 14-3-3ζ, known binding proteins to the GP Ibα cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibα extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibα subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.

Maturation of Megakaryoblastic Cells Is Accompanied by Upregulation of GSα-L Subtype and Increased cAMP Accumulation

In platelets and megakaryoblasts Gs, the trimeric G-protein that stimulates adenylyl cyclase, is present in a short, 45 kDa, and a long, 52 kDa isoform termed G(s)alpha-S and G(s)alpha-L, respectively. To assess the relative contribution of these isoforms in the cellular synthesis of cAMP, the ratio G(s)alpha-S/G(s)alpha-L was changed in the megakaryoblastic cell line DAMI by inducing cell maturation with recombinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cytometric analysis confirmed that this treatment induced a moderate (TPO) and extensive (PMA) increase in nuclear ploidy and expression of the glycoproteins-IIIa and -Ib. Northern blot analysis revealed downregulation of total Gs-mRNA after treatment of DAMI-cells with TPO and PMA. Western blot analysis showed significant (P < 0.05) upregulation of Gs-L with respective amounts of 27 +/- 4% of total Gs in untreated cells, 35 +/- 1% in TPO- and 41 +/- 3% in PMA-treated DAMI cells (n = 3-4). DAMI cells contained 6 +/- 1 pmol cAMP/10(6) cells, which was not changed by treatment with TPO or PMA. In untreated cells this level increased to 70 +/- 9 pmol cAMP/10(6) cells after 10 min stimulation with 1 micromol/l of the stable prostacyclin analog iloprost. The same stimulation with iloprost resulted in 165 +/- 32 pmol cAMP/10(6) in TPO-treated cells and in 588 +/- 100 pmol cAMP/10(6) in cells treated with PMA. Thus, a shift from G(s)alpha-S to G(s)alpha-L during megakaryoblast maturation strongly potentiates the production of cAMP. A similar shift may occur during normal megakaryocyte maturation and may explain the extreme sensitivity to prostacyclin of platelets, which contain G(s)alpha-S and G(s)alpha-L in approximately equal amounts.

7 Megakaryocytes and platelets in α-granule disorders

This chapter summarizes research data contributing to current understanding of disorders affecting alpha-granules of megakaryocytes and platelets. Diagnostic features of the gray platelet syndrome are well defined. Combined evidence suggests a defect, specific to the megakaryocyte cell lineage, causing a cytoskeletal abnormality and defective targeting of endogenously synthesized proteins to the alpha-granule. The abnormalities linked by signal transduction pathways. von Willebrand disease and afibrinogenaemia are disorders which highlight the functional importance of platelet storage pools of von Willebrand factor and fibrinogen, essential ligands in the process of adhesion and aggregation. The abnormality in the factor V Quebec disorder leads to a degradation of most proteins contained within the alpha-granule. The familial platelet disorder Paris-Trousseau thrombocytopenia is the only alpha-granule disorder associated with a cytogenetic abnormality, and it presents a useful model for exploring the genetic influence on regulation of thrombopoiesis. Study of these syndromes has elucidated aspects of the physiology of normal megakaryocyte maturation and platelet formation, including storage organelle biosynthesis.

Effect of diet-induced hypercholesterolemia on proteoglycan metabolism in guinea pig megakaryocytes and platelets.

Proteoglycan metabolism was evaluated in megakaryocytes and platelets from guinea pigs fed a 1% cholesterol diet for 3 or 7 weeks. The animals were injected with a single dose of [35S]sulfate at the end of the feeding period, and megakaryocytes and platelets were isolated after 3 hours and then daily for 4 days thereafter. Proteoglycans were extracted from the cells of each animal and analyzed by ion-exchange chromatography, gel filtration, and electrophoresis. The maximal labeling of platelets occurred 2 days after [35S]sulfate injection as compared with 3 days in controls. A proteoglycan that eluted at Kav 0.2 from the Sepharose CL-6B column appeared 1 day after labeling. Additional proteoglycans of Kav 0.15 appeared at subsequent time points. The labeling profile for cholesterol-fed animals was unchanged from 2-4 days, unlike profiles from controls, which had exhibited a gradual increase in mean proteoglycan size. Thus, the progressive change in size of proteoglycans synthesized during normal megakaryocyte maturation was altered. The mean chain length of the proteoglycan-associated glycosaminoglycans from cholesterol-fed animals was increased relative to that of controls. In conjunction with the twofold increase in mean megakaryocyte size induced by cholesterol feeding in guinea pigs, the changes in proteoglycan synthesis suggest a state of stimulated megakaryocytopoiesis.