Megakaryocytic Differentiation Research Articles

Role of platelet endothelial aggregation receptor 1 polymorphisms in idiopathic thrombocytopenic purpura: Is there an association?

Background: Genetic risk factors are implicated in the etiology and pathogenesis of immune thrombocytopenic purpura (ITP). Platelet endothelial aggregation receptor 1 (PEAR1) plays an important role in regulating megakaryopoiesis and thrombopoiesis. rs12041331 and rs12566888 single-nucleotide polymorphisms of PEAR1 are associated with megakaryocyte differentiation and platelet function. Materials and Methods: To conduct this study, 68 peripheral blood samples of patients with ITP (56 acute and 12 chronic) were collected. The amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used to detection of rs12041331 and rs12566888 PEAR1 polymorphisms. Results: Statistically significant differences were not seen between rs12041331 and rs12566888 genotypes in acute and chronic groups (P = 0.778, P = 0.844). The frequency of rs12041331 AG/AA genotypes and the rs12566888 GT genotype was more in acute ITP patients; on the other hand, the rs12566888 TT genotype was more in the chronic group. The highest platelet counts and platelet distribution width (PDW) were related to the rs12041331 AG allele. GT and TT of rs12566888 had more PDW and platelet count, respectively. Mean platelet volume values between alleles of both the polymorphisms were constant and did not differ much. In general, no statistically significant differences were observed between genotypes of polymorphisms and platelet parameters. Conclusions: There was no association between rs12041331 and rs12566888 with platelet parameters in ITP patients and the severity of this disease. Further investigation with a larger size is recommended.

Aurka loss in CD19+ B cells promotes megakaryocytopoiesis via IL-6/STAT3 signaling-mediated thrombopoietin production.

Rationale: Aurora kinase A (Aurora-A), which is required for mitosis, is a therapeutic target in various tumors. Targeting Aurora-A led to an increase in the differentiation and polyploidization of megakaryocytes both in vivo and in vitro. However, the mechanisms involved in controlling megakaryocyte differentiation have not been fully elucidated.Methods: Conditional Aurka knockout mice were generated. B cell development, platelet development and function were subsequently examined. Proplatelet formation, in vivo response to mTPO, post-transfusion experiment, colony assay, immunofluorescence staining and quantification, and ChIP assay were conducted to gain insights into the mechanisms of Aurka loss in megakaryocytopoiesis.Results: Loss of Aurka in CD19+ B cells impaired B cell development in association with an increase in the number of platelets in peripheral blood (PB). Surprisingly, thrombopoietin (TPO) production and IL-6 were elevated in the plasma in parallel with an increase in the number of differentiated megakaryocytes in the bone marrow (BM) of Aurkaf/f;Cd19Cre/+ mice. Interestingly, compared with that of the Aurkaf/f mice, a higher number of CD19+ B cells close to megakaryocytes was observed in the BM of the Aurkaf/f;Cd19Cre/+ mice. Moreover, Aurka loss in CD19+ B cells induced signal transducer and activator of transcription-3 (STAT3) activation. Inhibition of STAT3 reduced the Tpo mRNA levels. ChIP assays revealed that STAT3 bound to the TPO promoter. Additionally, STAT3-mediated TPO transcription was an autocrine effect provoked by IL-6, at least partially.Conclusions: Deletion of Aurka in CD19+ B cells led to an increase in IL-6 production, promoting STAT3 activation, which in turn contributed to TPO transcription and megakaryocytopoiesis.

Forming megakaryocytes from murine induced pluripotent stem cells by the inducible overexpression of supporting factors

BackgroundPlatelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. ObjectivesTo optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). MethodsTo avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. ResultsOverexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. ConclusionWe demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production. [Display omitted]

Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

1616. Mechanism of Thrombocytopenia Induced by Oxazolidinones Antibiotics (Linezolid, Tedizolid): Demonstration of Impairment of Megakaryocyte Differentiation From Human Hematopoietic Stem Cells associated with Mitochondrial Toxicity

BackgroundLinezolid causes thrombocytopenia, which limits its use. In cell culture and in tissues from treated patients, linezolid impairs mitochondrial protein synthesis (due to structural similarities and common binding sites between bacterial and mitochondrial ribosomes). Recent studies have shown that mitochondria act as a key relay in the process leading from activation of the thrombopoietin receptor to megakaryocytes differentiation.MethodsValidated ex-vivo human model of hematopoietic stem cells (HSC) differentiation for (i) measuring megakaryocytes, granulocyte-monocytes, and burst-forming unit-erythroids colony formation; (ii) differentiation into megakaryocytes (conversion of CD34+ into CD41+/CD42+ cells; morphology) and proplatelets formation, (iii) mitochondrial toxicity (electron microscopy; cytochrome c-oxidase activity [partly encoded by the mitochondrial genome]).ResultsWe show that linezolid (and the recently approved tedizolid), both at concentrations corresponding to their human serum concentrations) inhibit the maturation of HSC into fully differentiated megakaryocytes (CD41 and CD42-positive cells) and the formation of proplatelets. Optic and Electron microscopy) showed an impairment of the formation of typical megakaryocytes (lack of large polylobulated nuclei and of intracellular demarcation membrane system [required for platelet formation]), together with disappearance of the internal structure of mitochondria. Biochemical studies showed a complete suppression of the activity of cytochrome c-oxidase (a key enzyme of the mitochondrial respiratory chain).ConclusionOur study provides for the first time insights in the mechanism of thrombocytopenia induced by linezolid and tedizolid, identifying mitochondria as their target and showing that the drugs will impair the differentiation of hematopoietic stem cells into mature platelets-releasing megakaryocytes. It illustrates how mitochondria dysfunction may play a key role in toxicology and diseases, while paving the way for rational approaches for the design and screening of less toxic derivatives for the benefit of future patients.Disclosures Paul M. Tulkens, MD, PhD, Bayer (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Menarini (Speaker’s Bureau)Merck (Advisor or Review Panel member, Speaker’s Bureau)Trius (now part of Merck) (Advisor or Review Panel member, Research Grant or Support) Françoise Van Bambeke, PharmD, PhD, Bayer (Speaker’s Bureau)

Gene Network Analysis Reveals Association between Somatic Loss of Y and Genes Involved in Hemostasis

Background and Hypothesis: 
 Somatic loss of the Y chromosome (LOY) is associated with diagnoses of various cancers, Alzheimer’s disease, and cardiovascular events. In the case of cardiovascular disorders, no mechanistic link to LOY has been elucidated. To explore this relationship, we utilized Weighted Gene Co-Expression Analysis (WGCNA), a powerful technique that can reveal associations between modules of co-expressed genes and clinical traits. We hypothesized WGCNA would reveal a module that illuminates the relationship between LOY and cardiovascular disease. 
 
 Methods: 
 Information about 403 male participants was obtained from Alzheimer’s Disease Neuroimaging Initiative (ADNI), including Genome Wide Association Study (GWAS), microarray, and patient demographic data. GWAS data was obtained from each participant within one year of collecting gene expression data. Using GWAS data for each participant, a median log R ratio was calculated for probes in the male specific region of the Y (MSY), which showed detectable LOY in 16 participants. Microarray data was used for WGCNA. Genes with correlated expression levels were organized into modules which were tested for associations with age, Alzheimer’s disease status, APOE-e4 allele status, and LOY. Ensembl IDs were used to investigate Gene Ontology (GO) enrichment in the GO database. 
 
 Results: 
 One module was associated with LOY (p=0.001). GO enrichment revealed significant representation of 47 GO terms within this module. Highly enriched GO terms included “platelet aggregation” (FDR = 8.05 x 10-6), “blood coagulation, fibrin clot formation” (FDR= 0.0328), “regulation of megakaryocyte differentiation” (FDR=3.01 x 10-5), and other terms related to the vascular response to injury. 
 
 Potential Impact: 
 WGNCA revealed a module associated with LOY that is related to vascular injury response. While LOY is associated with cardiovascular events, more work is needed to explore its association with biomarkers for cardiovascular disease. Our findings warrant more investigation into the relationship between LOY and cardiovascular dysfunction in males.

Existence of a differentiation blockage at the stage of a megakaryocyte precursor in the thrombocytopenia and absent radii (TAR) syndrome

The thrombocytopenia and absent radii (TAR) syndrome is a rare disease associating bilateral radial agenesis and congenital thrombocytopenia. Here, we investigated in vitro megakaryocyte (MK) differentiation and expression of c-mpl in 6 patients. Using blood or marrow CD34(+) cells, the colony-forming unit (CFU)-MK number was markedly reduced. CD34(+) cells were also cultured in liquid medium in the presence of a combination of 3 cytokines (stem cell factor, interleukin-3, and interleukin-6) or megakaryocyte growth and development factor (PEG-rHuMGDF) with or without SCF. In the presence of PEG-rHuMGDF, the majority of mature megakaryocytes (CD41 high, CD42 high) underwent apoptosis. This phenomenon was also observed in cultures stimulated by three cytokines. However, this last combination of cytokines allowed a more complete terminal MK differentiation. Surprisingly, a homogeneous population of CD34(-)CD41(+)CD42(-) cells accumulated during the cultures. This population was unable to differentiate along the myeloid pathways. This result suggests that a fraction of MK cells is unable to differentiate in the TAR syndrome. We subsequently investigated whether this could be related to an abnormality in c-mpl. No mutation or rearrangement in the c-mpl gene was found by Southern blots or by sequencing of the c-mpl coding region and its promoter in any of the patients. Using Western blot analysis, a decreased level of Mpl was found in patient platelets. A decreased level of c-mpl messenger RNA in TAR platelets was also detected with a lower c-mpl-P to c-mpl-K ratio in comparison to adult platelets. Altogether, these results demonstrate that the thrombocytopenia of the TAR syndrome is associated with a dysmegakaryocytopoiesis characterized by cells blocked at an early stage of differentiation. (Blood. 2000;95:1633-1641)

Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21

AbstractPatients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.

Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition.

Numerous recurrent genetic mutations are known to occur in acute myeloid leukemia (AML). Among these common mutations, Fms-like tyrosine kinase 3 remains as one of the most frequently mutated genes in AML. We observed apparent marrow expansion of megakaryocytes in three out of six patients with Flt3-mutated AML following treatment with a recently FDA-approved Flt3 inhibitor, gilteritinib which possesses activity against internal tandem duplication and tyrosine kinase domain Flt3 mutations and also inhibits tyrosine kinase AXL. To assess whether biopsy findings can be attributed to promotion of megakaryocytic (Mk) differentiation with gilteritinib, we devised a cellular assay by overexpressing double mutated Flt3-ITDY591F/Y919F in chronic myeloid leukemia cell line K562 to study Mk differentiation in the presence of Flt3 and AXL inhibitors with non-mutually exclusive mechanisms. These experiments demonstrated the lack of direct effect Flt3 inhibitors gilteritinib and quizartinib on megakaryocytic differentiation at either transcriptional or phenotypic levels, and highlighted antileukemic effects of AXL receptor tyrosine kinase inhibitor and its potential role in megakaryocytic development.

On the Quest for In Vitro Platelet Production by Re-Tailoring the Concepts of Megakaryocyte Differentiation.

The demand of platelet transfusions is steadily growing worldwide, inter-donor variation, donor dependency, or storability/viability being the main contributing factors to the current global, donor-dependent platelet concentrate shortage concern. In vitro platelet production has been proposed as a plausible alternative to cover, at least partially, the increasing demand. However, in practice, such a logical production strategy does not lack complexity, and hence, efforts are focused internationally on developing large scale industrial methods and technologies to provide efficient, viable, and functional platelet production. This would allow obtaining not only sufficient numbers of platelets but also functional ones fit for all clinical purposes and civil scenarios. In this review, we cover the evolution around the in vitro culture and differentiation of megakaryocytes into platelets, the progress made thus far to bring the culture concept from basic research towards good manufacturing practices certified production, and subsequent clinical trial studies. However, little is known about how these in vitro products should be stored or whether any safety measure should be implemented (e.g., pathogen reduction technology), as well as their quality assessment (how to isolate platelets from the rest of the culture cells, debris, microvesicles, or what their molecular and functional profile is). Importantly, we highlight how the scientific community has overcome the old dogmas and how the new perspectives influence the future of platelet-based therapy for transfusion purposes.

Two Sequential and Independent Pathways of Erythromyeloid Progenitor Commitment in Their Niche of Emergence

The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocyte/granulocytes, mast cells and macrophages, the latter in a Myb-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from bipotent progenitors and is absent in Myb-deficient embryos. We identified Csf2rb as a novel marker of these Myb-dependent megakaryocyte-erythroid bipotent progenitors that colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes.

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

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

Regulation of platelet numbers and sizes by signaling pathways

Either the glycoprotein (GP) Ib deficiency or hyper-function in humans can cause macrothrombocytopenia, the molecular mechanisms of which remain unclear. Herein, the investigations for disease pathogenesis were performed in the human induced pluripotent stem cell (hiPSC) model. The hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD) were generated using CRISPR/Cas9. The GP1BA-null hiPSCs were previously derived from a Bernard–Soulier syndrome (BSS) patient. After full megakaryocyte differentiation in culture, both hiPSC mutations showed large proplatelet tips under fluorescence microscopy and yielded fewer but larger platelets compared with those of wild-type cells. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to wild-type hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and wild-type hiPSCs with a MAPK inhibitor. In conclusion, the genetically modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.

8-Hydroxydaidzein, an Isoflavone from Fermented Soybean, Induces Autophagy, Apoptosis, Differentiation, and Degradation of Oncoprotein BCR-ABL in K562 Cells.

8-Hydroxydaidzein (8-OHD, 7,8,4′-trihydoxyisoflavone) is a hydroxylated derivative of daidzein isolated from fermented soybean products. The aim of this study is to investigate the anti-proliferative effects and the underlying mechanisms of 8-OHD in K562 human chronic myeloid leukemia (CML) cells. We found that 8-OHD induced reactive oxygen species (ROS) overproduction and cell cycle arrest at the S phase by upregulating p21Cip1 and downregulating cyclin D2 (CCND2) and cyclin-dependent kinase 6 (CDK6) expression. 8-OHD also induced autophagy, caspase-7-dependent apoptosis, and the degradation of BCR-ABL oncoprotein. 8-OHD promoted Early Growth Response 1 (EGR1)-mediated megakaryocytic differentiation as an increased expression of marker genes, CD61 and CD42b, and the formation of multi-lobulated nuclei in enlarged K562 cells. A microarray-based transcriptome analysis revealed a total of 3174 differentially expressed genes (DEGs) after 8-OHD (100 μM) treatment for 48 h. Bioinformatics analysis of DEGs showed that hemopoiesis, cell cycle regulation, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducers and activators of transcription (JAK-STAT)-mediated apoptosis/anti-apoptosis networks were significantly regulated by 8-OHD. Western blot analysis confirmed that 8-OHD significantly induced the activation of MAPK and NF-κB signaling pathways, both of which may be responsible, at least in part, for the stimulation of apoptosis, autophagy, and differentiation in K562 cells. This is the first report on the anti-CML effects of 8-OHD and the combination of experimental and in silico analyses could provide a better understanding for the development of 8-OHD on CML therapy.

Modeling Acute Megakaryoblastic Leukemia of Down Syndrome Using Induced Pluripotent Stem Cells

Introduction Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2). How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs). Methods Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1sMPLW515K/W515Kclones were selected as well as a revertant Dis21 GATA1sMPLW515K/W515Kclone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/-iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes. Results GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts. RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1sMPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility. Conclusion Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development. Disclosures No relevant conflicts of interest to declare.

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

Conditional CRISPR-Mediated Deletion of Lyn Kinase Promotes Megakaryocyte Differentiation, Platelet Production and Function

Thrombocytopenia leading to life-threatening excessive bleeding can be caused by defects in megakaryocyte/platelet production, platelet depletion by platelet-specific auto- or allo-antibodies, blood loss following trauma or surgery, or as a result of damage to bone marrow blood stem cells induced by chemo- or radiation therapy. Currently, platelets available for transfusion are entirely dependent on volunteer blood donation, which are often in short supply, carry a risk of infection, and often unable to be antigen-matched for alloimmunized patients. Thus, maintaining an adequate supply of platelets for transfusion is often a challenge. In response to this growing clinical need, a number of laboratories have started differentiating human induced pluripotent stem cells (iPSCs) into megakaryocytes, with the long-term goal of creating a readily available supply of antigen-matched, highly functional platelets. Current methods for generating platelets from iPS cell-derived megakaryocytes have been improved to produce enough platelets in vitro to begin phase I safety trials in humans; however, modifying the iPS cell genome has the potential to further improve both the yield and function of in vitro-generated platelets. The purpose of this investigation, therefore, was to combine recent advances in gene editing technology with iPS cell differentiation techniques to improve the yield and/or functionality of in vitro-derived platelet products. As a start, we sought to examine whether disruption of negative regulators of platelet production and/or activation might allow the generation of increased numbers of platelets with improved hemostatic effectiveness. One such negative regulator of platelet function is the Src family kinase, Lyn. Previous studies using pharmacologic inhibitors and global Lyn knockout mice have shown that that disrupting Lyn activity leads to mild thrombocytosis and megakaryocytosis. The effects of specifically targeting Lyn in human megakaryocytes and platelets, however, are not known. We hypothesized that conditionally disrupting Lyn during the process of iPSC to megakaryocyte differentiation might be an effective way to enhance megakaryocyte differentiation, platelet production, and hemostatic function. Using CRISPR/Cas9 gene editing technology, we generated a temporally controllable, tamoxifen-inducible Lyn-KO iPS cell line system that allowed us to conditionally delete Lyn at any desired stage of the iPSC→hematopoietic stem cell (HSC)→megakaryocyte→platelet differentiation process. We found tamoxifen efficiently disrupted Lyn expression at any of these stages within 24-48 hours after addition of the drug to the cell culture, and also improved the efficiency and yield of the iPSC→megakaryocyte→proplatelet differentiation process. Lyn-deficient human megakaryocytes prepared in this fashion exhibited enhanced reactivity, as reported by increased Erk phosphorylation in response to thrombopoietin stimulation, and by an improved ability to support hemostasis in human blood in vitro, as determined using thromboelastometry (ROTEM). Taken together, these data suggest that temporal disruption of Lyn kinase might represent an effective strategy for the engineering and manufacture of in vitro-derived platelets for both diagnostic and future therapeutic use. Disclosures Newman: NIH: Research Funding; Bloodworks Northwest: Membership on an entity's Board of Directors or advisory committees; Rallybio Corporation: Consultancy.

CD62L Expression Level Dictates the Cell Fate of Myeloid Progenitors in Mice and Humans

Hematopoietic cells are hierarchically differentiated from hematopoietic stem cells through several progenitors. Recent studies showed that each progenitor population has significant heterogeneity and some of the subsets have skewed differentiation potential. However, it has not been elucidated how and when common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) acquire different fates. We hypothesized that progenitor cells with skewed differentiation potential had acquired a part of gene expression profiles of more differentiated cells. By analyzing publicly available single-cell RNA sequencing data of human and murine CMPs and GMPs, we thoroughly explored surface markers with heterogeneous expression patterns and identified CD62L as a candidate to refine the differentiation potential of CMPs and GMPs. First, at CMP level, we clarified that CD62L-low CMPs had genuine CMP potential, whereas CD62L-high CMPs were mostly restricted to GMP potentials in both mice and humans. CD62L expression on CMPs was widely distributed and when divided into low, middle, and high fraction, colony forming-cell assay showed that murine CD62L-high CMPs mostly differentiated into CD11b-positive granulocytes and macrophages (97.4 ± 1.7%), whereas CD62L-low CMPs produced many erythroid and megakaryocytic colonies (38.7 ± 3.3%), and human CMPs had similar tendency. Also, liquid culture assay showed that murine CD62L-low CMPs differentiated into both GMPs (43.3 ± 4.1%) and megakaryocyte-erythrocyte progenitors (MEPs) (20.6 ± 3.7%), while CD62L-high CMPs mainly produced GMPs (82.8 ± 1.7%) and the proportion of MEPs was only 1.3 ± 0.1%. As for in vivo kinetics, we transplanted CD62L-low and high CMPs from GFP-expressing mice into irradiated wild-type mice, thus donor-derived platelets can be detected as GFP-positive. On day 7 after transplantation of each 15,000 cells, GFP-positive platelets from CD62L-low CMPs accounted for 9.8 ± 2.7% of all platelets, meanwhile CD62L-high CMPs accounted for only 0.20 ± 0.14%, which reinforced our hypothesis. Moreover, we performed transcriptome analysis using data of murine and human CMPs and focused on several transcription factors. Gata1, Klf1, Tal1 and Gfi1b are important transcription factors for erythroid and megakaryocytic differentiation, and these expressions were exclusively high in CD62L-low CMPs. On the other hand, Spi1 and Irf8 are important for differentiation into granulocytes and monocytes, and these expressions were higher in CD62L-high CMPs, which further corroborated the role of CD62L as a marker for refining the differentiation fate of CMPs. Second, at GMP level, we found that part of CD62L-neg GMPs possess remaining CMP potential and CD62L-low GMPs were skewed to granulocyte differentiation. CD62L expression on GMPs was mostly positive, but when we defined lowest 10% of GMPs as CD62L-neg GMPs, colony-forming cell assay in mice showed that part of CD62L-neg GMPs produced erythroid and megakaryocytic colonies (3.9 ± 2.6%), which suggested that this subset still possesses CMP potential. In vivo transplantation experiment using GFP-positive mice showed that CD62L-neg GMPs produced GFP-positive platelets slightly (0.02 ± 0.01%), but no platelets from CD62L-positive GMPs. Also, single-cell RNA-seq data revealed that part of CD62L-neg GMPs had CMP-specific gene expression pattern, suggesting that the bona fide GMPs were restricted to CD62L-positive GMPs. Next, we divided GMPs into CD62L-low and high GMPs, and performed colony-forming cell assay. CD62L-low GMPs produced granulocyte colony (CFU-G) 73.2 ± 6.1% and macrophage colony (CFU-M) 17.4 ± 3.3%, whereas CD62L-high GMPs produced CFU-G 46.6 ± 1.7% and CFU-M 42.9 ± 2.7%, which suggested that CD62L-low GMPs were granulocyte-skewed population. Also, we performed in vivo transplantation assay using Ly5.1 and Ly5.2 mice. On day 5 after transplantation, murine CD62L-low GMPs produced more neutrophils (87.6 ± 3.1%) in spleen than bulk GMPs (78.9 ± 1.9 %), which further confirmed this hypothesis. In summary, our in vitro and in vivo experiment and transcriptome analysis revealed that CMPs and GMPs had high heterogeneity. CD62L expression level refines the definition of CMPs and GMPs in both mice and humans, and elucidates the differentiation mechanism of myeloid cells in more detail. Disclosures Nakahara: Bristol-Myers Squibb Company: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria. Kagoya:NIPPON SHINYAKU CO.,LTD.: Research Funding; Bristol-Myers Squibb Company: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding. Kurokawa:Eisai: Research Funding, Speakers Bureau; Teijin: Research Funding; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Shire Plc: Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Sanwa-Kagaku: Consultancy; Pfizer: Research Funding; Otsuka: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

RUNX1 Mutation Leads to Megakaryocyte-Primed Hematopoietic Stem Cell Expansion and Familial Platelet Disorder

Backgrounds: Recently it is shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of VWF and CD41+ at the apex of the hematopoietic stem cell hierarchy. This subset of HSCs, termed mega-HSCs, can give rise to megakaryocytes and platelets directly by bypassing the traditional trajectory of megakaryocyte development from HSC via MPP and MEP. To date, aside from phenotypic marker and transplantation studies, there has been limited understanding of the mechanisms involved in the regulation of mega-HSCs. Runx1 belongs to the RUNT domain transcription factors, and is a key regulator of hematopoiesis especially for the megakaryocyte and platelet differentiation. Loss of RUNX1 in mice causes thrombocytopenia through a blockage of megakaryocyte maturation. One allele RUNX1 loss-of-function mutation is associated with familial platelet disorder (FPD) with a predisposition to developing leukemia. Here we hypothesize that Runx1 plays a role in regulating mega-HSCs to impact on platelet generation, and correcting RUNX1 mutation that causes FPD can therapeutically rescue thrombocytopenia in a mouse model. Methods: We have examined the hematopoiesis and cellularity of various bone marrow (BM) stem/progenitor populations and peripheral blood (PB) in two mouse models. Firstly, conditional knock-out of Runx1flox/flox was mediated by Mx1-Cre upon poly I:C induction. Secondly, the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the collagen a1 locus and the mice was crossed to MLL-PTD knock-in. The mutant RUNX1 is only expressed when the mice are fed with doxycycline and the RUNX1 mutant is "corrected" upon doxycycline withdrawn. In the second model, PB and BM were also tracked after a removal of DOX-induced RUNX1 mutation. The LSK CD150+ HSCs from the mouse BM were isolated by FACS sorting, and 10x Genomics' single-cell RNA-seq (scRNA-seq) analyses were performed to define and track HSPCs. We applied the Louvain algorithm to the scRNA-seq data to identify the cell type clusters and annotated the cell types using the marker genes of each cell cluster. MAST was employed to identify the cell-type specific, differentially expressed genes. Results: In the Runx1 KO mice, two weeks after deletion of Runx1 platelet count showed an ~2-fold decrease to 400~600 k/ul in PB. In the LSK CD150+CD48- or the LSK CD34- FLT3- compartment of BM, the CD41+ mega-HSCs increased ~2-3 fold. In the RUNX1 mutant-on model, mutant mice developed thrombocytopenia 16 weeks after DOX induction with the average platelet count dropping to ~600 k/ul and being maintained at this level. In transplant recipients, the RUNX1 mutant-on mice contained drastically increased mega-HSCs compared with RUNX1 mutant-off mice, synchronous with a decrease of the platelet count in PB. Consistent with these observations, sc-RNAseq data show that in Runx1 KO, among the sequenced LSK CD150+ cells ~70% are HSCs and ~15% are MPP2. Among the HSCs, mega-primed HSCs consist ~33% while non-mega primed HSCs are ~67%. The mega-primed HSCs are distinct by virtual of upregulated platelet-related genes and relative dormant cell cycle status. In the RUNX1 mutant-on model, we found that among the sequenced LSK CD150+ cells ~50% are HSCs and ~12% are MPP2. Interestingly, the mega-primed HSCs are significantly increased in RUNX1 mut-on HSCs compared with mut-off HSCs. Similarly, we saw highly upregulated platelet-driven pathways in mega-HSCs in the mutant-on HSCs. Finally, one month after a withdraw of DOX from the RUNX1 mutant expressing mice when the RUNX1 mutant gene became undetectable, the platelet count returned from ~600 to ~1,000 k/ul in PB, a reversal of the thrombocytopenia phenotype caused by the RUNX1 S291fsX300 expression. Importantly, the correction of Runx1 mutation significantly decreased the proportion of mega-HSCs and restored platelet related pathways in this subset of HSCs. Conclusions: Mega-HSCs contain a high level of platelet-driven gene expression. In addition to its role in regulating the HSC-MPP-MEP mediated megakaryocyte development, RUNX1 is important in regulating mega-HSCs by maintaining proper expression of mega-platelet leaning genes. Correction of RUNX1 mutation that causes FPD can rescue mega-HSC population and revert FPD, providing a rationale for future treatment strategies by gene editing in RUNX1 mutation bearing FPD patients. Disclosures No relevant conflicts of interest to declare.