Megakaryocyte Precursors Research Articles

Rker-050 Rescued Ruxolitinib (Rux)-Associated Reductions in Red Blood Cell Volume

Myelofibrosis (MF) is characterized by the dysfunctional Janus kinase/signal transducers and activators of transcription signaling (JAK/STAT) pathways leading to progressive proliferation of granulocytic and megakaryocytic cells in the bone marrow at the expense of other hematopoietic lineages. Clinical signs of MF include cytopenias, splenomegaly and transformation to acute leukemia.Jakafi® (ruxolitinib, or rux), a JAK2 inhibitor, is a therapeutic for MF and functions to impair the activated mutations that cause the expansion of megakaryocytic precursors. However, JAK2 also transduces signals of the erythropoietin receptor, thrombopoietin receptor, and the granulocyte colony-stimulating factor receptor. Therefore, individuals being treated with rux are susceptible to treatment-associated effects on normal hematopoiesis resulting in thrombocytopenia, neutropenia and anemia.The TGF-β superfamily plays a vital role in the regulation of hematopoiesis; specifically, SMAD 2/3 activation results in cell quiescence and inhibits precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA extracellular domain fused to the Fc of human IgG1, is designed to inhibit ligands including activin A, activin B, GDF8 and GDF11, that activate SMAD 2/3. In a preclinical study, administration of KER-050 in mice led to upregulation of erythropoiesis by mobilizing early- and late-stage erythroid precursors and facilitating their terminal maturation into red blood cells (RBCs). In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to sustained increases in RBCs and hemoglobin (HGB) along with increases in platelets. Given the observed effect of KER-050 on increasing RBCs, we evaluated whether treatment with a research form of KER-050 (RKER-050) could reverse rux-associated reductions in RBCs. Additionally, preclinical studies have shown that KER-050 potentially functioned as a muscle anabolic by increasing lean mass in rodents.We first established anemia in C57Bl/6 mice by dosing with rux before administering RKER-050. Anemia was confirmed on study day 37; mice receiving 120 mg/kg rux via oral gavage (PO) BID had significantly lower RBC (-7.4%, p=0.0001), HGB (-4.0%, p=0.002) and hematocrit (HCT; -5.7%, p=0.0006) levels compared to the control group. Treatment with RKER-050 was initiated on study day 41 and mice received 7.5 mg/kg RKER-050 or vehicle intraperitoneally (IP) twice weekly for approximately 14 days. Mice receiving rux alone continued their decline in RBCs and, on day 55, continued to have significant reductions in RBC (-6.7%, p<0.0001), HGB (-6.0, p<0.00001) and HCT (-5.6%, p=0.0002) levels compared to the control group. These findings are consistent with the progressive effect of JAK2 inhibition on suppressing erythrocyte development and production. In contrast, treatment with RKER-050 abrogated the observed rux-associated reductions in RBCs, HGB, and HCT in the rux-RKER-050 cohort with significant observed increases (+15.8%, +12.2%, +11.2%, respectively, all p<0.0001) when compared to the rux-vehicle group. The rux-RKER-050 cohort also had significantly increased body mass, measured between study day 41 and study day 55 versus the rux-vehicle group (+9.9%, p= 0.006, and +0.69%, respectively).These data demonstrate that rux treatment reduced RBCs, HGB, and HCT in mice, and that coadministration of RKER-050 reversed rux-associated reductions in RBC parameters. Therefore, treatment with KER-050 has the potential to mitigate the dose limiting effects of rux and enhance duration of therapy in MF patients. RKER-050 also increased body weight in mice receiving rux through its anabolic effect on muscle, a potential benefit in elderly MF patients.These data support the potential benefit of KER-050 as a monotherapy and in combination with rux in patients with MF and anemia. KER-050 will be assessed in a Phase 2 clinical trial (KER050-MF-301), which we expect to commence in 2021. DisclosuresNathan: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Feigenson: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lamora: Keros Therapeutics: Current Employment. Tseng: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Pathophysiology, classification, and complications of common asymptomatic thrombocytosis in newborn infants.

We frequently encounter newborn infants with thrombocytosis in the neonatal intensive care unit. However, neonatal thrombocytosis is not yet fully understood. Thrombocytosis is more frequently identified in newborns and young infants, notably more often in those younger than 2 years than in older children or adults. The production of megakaryocytes (megakaryopoiesis) and platelets (thrombopoiesis) is mainly regulated by thrombopoietin (TPO). Increased TPO levels during infection or inflammation can stimulate megakaryopoiesis, resulting in thrombopoiesis. TPO concentrations are higher in newborn infants than in adults. Levels increase after birth, peak on the second day after birth, and start decreasing at 1 month of age. Initial platelet counts at birth increase with gestational age. Thus, preterm infants have lower initial platelet counts at birth than late-preterm or term infants. Postnatal thrombocytosis is more frequently observed in preterm infants than in term infants. A high TPO concentration and low TPO receptor expression on platelets leading to elevated plasma-free TPO, increased sensitivity of megakaryocyte precursor cells to TPO, a decreased red blood cell count, and immaturity of platelet regulation are speculated to induce thrombocytosis in preterm infants. Thrombocytosis in newborn infants is considered a reactive process (secondary thrombocytosis) following infection, acute/chronic inflammation, or anemia. Thrombocytosis in newborn infants is benign, resolves spontaneously, and, unlike in adults, is rarely associated with hemorrhagic and thromboembolic complications.

In and out: Traffic and dynamics of thrombopoietin receptor.

Thrombopoiesis had long been a challenging area of study due to the rarity of megakaryocyte precursors in the bone marrow and the incomplete understanding of its regulatory cytokines. A breakthrough was achieved in the early 1990s with the discovery of the thrombopoietin receptor (TpoR) and its ligand thrombopoietin (TPO). This accelerated research in thrombopoiesis, including the uncovering of the molecular basis of myeloproliferative neoplasms (MPN) and the advent of drugs to treat thrombocytopenic purpura. TpoR mutations affecting its membrane dynamics or transport were increasingly associated with pathologies such as MPN and thrombocytosis. It also became apparent that TpoR affected hematopoietic stem cell (HSC) quiescence while priming hematopoietic stem cells (HSCs) towards the megakaryocyte lineage. Thorough knowledge of TpoR surface localization, dimerization, dynamics and stability is therefore crucial to understanding thrombopoiesis and related pathologies. In this review, we will discuss the mechanisms of TpoR traffic. We will focus on the recent progress in TpoR membrane dynamics and highlight the areas that remain unexplored.

Evaluating the Role of Autologous Platelet-Rich-Plasma in The Treatment of Dry Eye Disease

Background: Platelets are irregularly shaped, non-nucleated cytoplasmic bodies derived from fragmentation of megakaryocyte precursors. Objective: This study aimed to evaluate the role of autologous platelet-rich-plasma (PRP) as a monotherapy versus artificial tears (Hyaluronic acid) use for better management of dry eye disease. Patients and Methods: A prospective clinical randomized trial study was conducted on 62 patients with moderate to severe dry eye disease (Schirmer`s test outcomes of 5.5 mm or lower). Patients had been recruited from the Outpatient Clinics of Ophthalmology Department in Zagazig University Hospital during the period from February 2020 to January 2021. They were divided into two equal groups; group A was treated with PRP and group B was treated with artificial tears. Results: There was a significant decrease regarding ocular surface disease index (OSDI) in both groups after treatment, but the improvement was more significantly in PRP group compared to artificial tears group. There was a significant increase in tear break-up time (TBUT) in PRP group only after treatment; although, there was an increase in TBUT in artificial tears group but without statistical significance. There was a significant increase in BCVA in both groups after treatment, but the improvement was more significantly in PRP group compared to artificial tears group. Conclusion: PRP is an interesting alternative therapy in symptomatic dry eye.

A Review of Romiplostim Mechanism of Action and Clinical Applicability.

Thrombocytopenia results from a variety of conditions, including radiation, chemotherapy, autoimmune disease, bone marrow disorders, pathologic conditions associated with surgical procedures, hematopoietic stem cell transplant (HSCT), and hematologic disorders associated with severe aplastic anemia. Immune thrombocytopenia (ITP) is caused by immune reactions that accelerate destruction and reduce production of platelets. Thrombopoietin (TPO) is a critical component of platelet production pathways, and TPO receptor agonists (TPO-RAs) are important for the management of ITP by increasing platelet production and reducing the need for other treatments. Romiplostim is a TPO-RA approved for use in patients with ITP in the United States, European Union, Australia, and several countries in Africa and Asia, as well as for use in patients with refractory aplastic anemia in Japan and Korea. Romiplostim binds to and activates the TPO receptor on megakaryocyte precursors, thus promoting cell proliferation and viability, resulting in increased platelet production. Through this mechanism, romiplostim reduces the need for other treatments and decreases bleeding events in patients with thrombocytopenia. In addition to its efficacy in ITP, studies have shown that romiplostim is effective in improving platelet counts in various settings, thereby highlighting the versatility of romiplostim. The efficacy of romiplostim in such disorders is currently under investigation. Here, we review the structure, mechanism, pharmacokinetics, and pharmacodynamics of romiplostim. We also summarize the clinical evidence supporting its use in ITP and other disorders that involve thrombocytopenia, including chemotherapy-induced thrombocytopenia, aplastic anemia, acute radiation syndrome, perisurgical thrombocytopenia, post-HSCT thrombocytopenia, and liver disease.

Apoptotic profiling of chronic myeloid leukaemia patients' platelets ex vivo before and after treatment with Imatinib.

Chronic myeloid leukaemia (CML) is a malignancy of the haematopoietic stem cells. The first line of treatment for CML, especially in developing countries, remains the first-generation tyrosine kinase inhibitor, Imatinib. Patients with CML are frequently diagnosed with platelet abnormalities. However, the specific mechanism of platelet abnormalities in CML remains unclear and poorly understood. The aim of this study was therefore to determine the apoptotic profiles of CML patients ex vivo on platelets before and after treatment with Imatinib. Blood samples of healthy volunteers and CML patients at diagnosis and after 6months treatment with Imatinib were collected. Platelet counts, viability and activation were determined. Results showed that CML patients' platelet counts were elevated upon diagnosis and these levels statistically significantly decreased after 6months of treatment. Platelet activation was significantly increased after 6months of treatment compared to levels at diagnosis (P-value < .05). Similarly, platelet apoptosis was also increased after 6months of treatment. Abnormalities in platelet functioning found in this study may partly be due to clonal proliferation of haematopoietic cells in CML patients, specifically of megakaryocyte precursors as well as the inhibition of platelet tyrosine kinase's and the inhibition of platelet-derived growth factor.

The Effects of Thrombopoietin on Megakaryocyte-CFC, Megakaryocytes, and Thrombopoiesis: With Studies of Ploidy and Platelet Size

The effects of partially purified thrombopoietin (TPO), prepared from the plasma of thrombocytopenic rabbits, upon megakaryocyte colony-forming cells (Meg-CFC), megakaryocytes, and platelets were evaluated. The thrombopoiesis-stimulating activity of both types of preparations of TPO used was established by their ability to increase the levels of selenomethionine- 75Se ( 75SeM) in the circulating platelets of mice, as described previously. TPO did not increase the frequency or total numbers of Meg-CFC or granulocyte-macrophage-CFC (GM-CFC) in the bone marrow or spleen of recipient mice. Similarly, the addition of TPO directly to 0.3% agar cultures of bone marrow or splenic cells did not alter the frequency of either Meg-CFC or GM-CFC, although in some experiments, concentrations greater than required to stimulate thrombopoiesis in vivo were used. TPO did not enhance the ability of suboptimal concentrations of spleen conditioned medium to support colony growth. Human erythropoietin (EPO) at concentrations from 0.6 U/ml to 8 U/ml, in vitro, did not increase the frequency of bone marrow or splenic Meg-CFC or GM-CFC. As previously reported, two types of megakaryocyte colonies were present in soft agar cultures. The proportion of 16N cells was significantly increased in the more mature appearing, big cell colonies derived from mice that had previously received thrombopoietin. There was no detectable change in the ploidy of heterogeneous colonies. Addition of TPO directly to cultures produced a slight, but not statistically significant increase in the proportion of 16N, 32N, and 64N megakaryocytes in big cell colonies. Although 16N megakaryocytes remained the most common ploidy group among recognizable megakaryocytes in the bone marrow of mice that had received TPO, there was an increase in the proportion of 32N and 64N megakaryocytes. Both preparations of TPO significantly increased the mean platelet volume of recipient mice, although the platelet counts remained unchanged. Similar protein fractions, prepared from the plasma of normal donors, did not increase levels of 75SeM in circulating platelets or increase mean platelet volume. Our data indicate that thrombopoietin produces a variety of effects, some of them perhaps indirect, on megakaryocyte precursors, megakaryocytes, and platelets.

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)

The RNA Splicing Factor U2AF1 Controls Hematopoietic Stem Cell Survival and Function

Somatic mutations in U2AF1 have been identified in ~11% cases of MDS. U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. Most U2AF1 mutations are found in two hotspots (S34 and Q157) within the first and second zinc finger domains. Transgenic and knock-in mice expressing U2AF1 S34F mutant exhibit impaired hematopoiesis. However, the role of wild-type U2AF1 in regulating hematopoietic stem cell (HSC) function and normal hematopoiesis has remained unknown. To determine the role of U2AF1 in normal hematopoiesis, we generated a new conditional U2af1 knockout (floxed) mouse. We crossed U2af1 floxed mouse with Mx1-Cre mouse and the expression of Cre recombinase was induced with polyinosine-polycytosine (pI-pC) injection at 5 to 6 weeks after birth. We observed that deletion of U2af1 significantly reduced white blood cell, neutrophil, red blood cell and platelet counts in their peripheral blood compared with control animals within 10-14 days after pI-pC injection. Histopathologic analysis of the BM sections from U2af1-deficient mice showed severe BM aplasia. Flow cytometric analyses revealed a marked decrease in myeloid, erythroid and megakaryocytic precursors in the BM of U2af1-deficient mice compared with control animals. We also observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2af1-deleted mice. Hematopoietic progenitor colony assays showed a significant decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2af1-deficient mice.Together, these data suggest that loss of U2af1 causes severe defects in hematopoiesis. We performed both non-competitive and competitive BM transplantation assays using U2af1-deficient BM to determine the role of U2af1 in HSC function. There was marked reduction of HSC, progenitors and all types of blood and BM cell precursors upon U2af1 deletion (by pI-pC administration) in the transplanted animals. Also, U2af1-deficient HSCs were unable to compete with WT HSCs and there was rapid loss of hematopoietic progenitors/precursors derived from the U2af1-deficient HSCs. Since U2af1 deletion resulted in rapid decrease of hematopoietic progenitors in the BM, we asked whether deletion of U2af1 insulted the genome and induced apoptosis to hematopoietic cells in the BM. We observed significantly increased apoptosis in the total BM as wells as in c-kit+, Gr1+, Ter119+and CD41+cells suggesting that hematopoietic progenitors and precursors of multiple cell lineages underwent apoptosis upon U2af1 deletion. We also performed gamma-H2AX assay using imaging flow cytometry to evaluate DNA damage in total BM, Gr1+(myeloid) and CD71+(erythroid) cells in control and U2af1-deleted mice. We observed markedly elevated gamma-H2AX in total BM, Gr1+and CD71+cells from U2af1-deficient mice compared with control mice.In addition, we observed increased Chk1 phosphorylation (ser345), a hallmark for activation of the ATR pathway, and increased histone H2A K119 ubiquitination (H2AK119Ub), a marker for DNA damage response, in the BM of U2af1-deficient mice. Thus, depletion of U2af1 causes insult to the genome and induces DNA damage and increased cell death. To gain insights into severe hematopoietic defects observed in U2af1-deficient mice, we performed transcriptome profiling of sorted LSK cells from U2af1 wild type (control) and U2af1-deleted mice. GSEA analysis of RNA sequencing data revealed significant downregulation of genes related to HSC maintenance in U2af1-deficient LSK. GSEA also revealed enrichment for cell cycle and DNA damage response-related genes, consistent with decreased proliferation and increased DNA damage and apoptosis observed in U2af1-deficient hematopoietic progenitors. We also determined the effects of U2af1 deletion on RNA splicing. Interestingly, we observed significant changes in gene expression as well as splicing alterations in several genes important for HSC survival and function. In conclusion, our results suggest a crucial role for U2af1 in the survival and function of HSC. Disclosures Mohi: Tolero Pharmaceuticals Inc.: Research Funding.

Multifaceted Actions of GFI1 and GFI1B in Hematopoietic Stem Cell Self-Renewal and Lineage Commitment.

Growth factor independence 1 (GFI1) and the closely related protein GFI1B are small nuclear proteins that act as DNA binding transcriptional repressors. Both recognize the same consensus DNA binding motif via their C-terminal zinc finger domains and regulate the expression of their target genes by recruiting chromatin modifiers such as histone deacetylases (HDACs) and demethylases (LSD1) by using an N-terminal SNAG domain that comprises only 20 amino acids. The only region that is different between both proteins is the region that separates the zinc finger domains and the SNAG domain. Both proteins are co-expressed in hematopoietic stem cells (HSCs) and, to some extent, in multipotent progenitors (MPPs), but expression is specified as soon as early progenitors and show signs of lineage bias. While expression of GFI1 is maintained in lymphoid primed multipotent progenitors (LMPPs) that have the potential to differentiate into both myeloid and lymphoid cells, GFI1B expression is no longer detectable in these cells. By contrast, GFI1 expression is lost in megakaryocyte precursors (MKPs) and in megakaryocyte-erythrocyte progenitors (MEPs), which maintain a high level of GFI1B expression. Consequently, GFI1 drives myeloid and lymphoid differentiation and GFI1B drives the development of megakaryocytes, platelets, and erythrocytes. How such complementary cell type- and lineage-specific functions of GFI1 and GFI1B are maintained is still an unresolved question in particular since they share an almost identical structure and very similar biochemical modes of actions. The cell type-specific accessibility of GFI1/1B binding sites may explain the fact that very similar transcription factors can be responsible for very different transcriptional programming. An additional explanation comes from recent data showing that both proteins may have additional non-transcriptional functions. GFI1 interacts with a number of proteins involved in DNA repair and lack of GFI1 renders HSCs highly susceptible to DNA damage-induced death and restricts their proliferation. In contrast, GFI1B binds to proteins of the beta-catenin/Wnt signaling pathway and lack of GFI1B leads to an expansion of HSCs and MKPs, illustrating the different impact that GFI1 or GFI1B has on HSCs. In addition, GFI1 and GFI1B are required for endothelial cells to become the first blood cells during early murine development and are among those transcription factors needed to convert adult endothelial cells or fibroblasts into HSCs. This role of GFI1 and GFI1B bears high significance for the ongoing effort to generate hematopoietic stem and progenitor cells de novo for the autologous treatment of blood disorders such as leukemia and lymphoma.

The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis

AbstractStudies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes gray platelet syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Coimmunoprecipitation (co-IP) experiments using tagged proteins expressed in human HEK293 and megakaryocytic immortalized megakaryocyte progenitor (imMKCL) cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-IP, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798 to 1903, where 2 GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B coexpressed in HEK293 cells. CRISPR/Cas9-mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.

The Landscape of Coding and Noncoding RNAs in Platelets.

Significance: Levels of platelet noncoding RNAs (ncRNAs) are altered by disease, and ncRNAs may exert functions inside and outside of platelets. Their role in physiologic hemostasis and pathologic thrombosis remains to be explored. Recent Advances: The number of RNA classes identified in platelets has been growing since the past decade. Apart from coding messenger RNAs, the RNA landscape in platelets comprises ncRNAs such as microRNAs, circular RNAs, long ncRNAs, YRNAs, and potentially environmentally derived exogenous ncRNAs. Recent research has focused on the function of platelet RNAs beyond platelets, mediated through protective RNA shuttles or even cellular uptake of entire platelets. Multiple studies have also explored the potential of platelet RNAs as novel biomarkers. Critical Issues: Platelet preparations can contain contaminating leukocytes. Even few leukocytes may contribute a substantial amount of RNA. As biomarkers, platelet RNAs have shown associations with platelet activation, but it remains to be seen whether their measurements could improve diagnostics. It also needs to be clarified whether platelet RNAs influence processes beyond platelets. Future Directions: Technological advances such as single-cell RNA-sequencing might help to identify hyperreactive platelet subpopulations on a single-platelet level, avoid the common problem of leukocyte contamination in platelet preparations, and allow simultaneous profiling of native megakaryocytes and their platelet progeny to clarify to what extent the platelet RNA content reflects their megakaryocyte precursors or changes in the circulation. Antioxid. Redox Signal. 34, 1200-1216.

Downregulated Platelet miR-1233-5p in Patients with Alzheimer's Pathologic Change with Mild Cognitive Impairment is Associated with Aβ-Induced Platelet Activation via P-Selectin.

MicroRNAs (miRNAs) have been proposed as a promising biomarker for various diseases including Alzheimer’s disease (AD). More attention has recently been focused on the diagnosis and treatment at earlier stage of mild cognitive impairment (MCI) for preventing its progression to AD. To identify potential pathologic markers for Aβ(+)MCI (Alzheimer’s pathologic change with MCI), we investigated miRNA expression profiles in the platelets from patients with Aβ(+)MCI, in comparison with those from Aβ(−)MCI (Non-Alzheimer’s pathologic change with MCI) and CNI (cognitively normal individuals). We found that let-7i-5p, miR-125a, miR-1233-5p, and miR-6787-5p were significantly downregulated, while miR-6880-5p expression was upregulated. Of these, only miR-1233-5p was significantly downregulated by Aβ treatment in both human platelets and their precursor megakaryocytes (MEG-01 cells). We explored the role of miRNAs by using miRNA mimics or inhibitors, and found that the diminished level of miR-1233-5p was associated with Aβ-induced increase in the expression of P-selectin and cell adhesion to fibronectin. Our results further indicated that Aβ-induced increase in platelet/MEG adhesion to fibronectin is likely mediated via P-selectin. In conclusion, this study suggests the downregulation of platelet-derived miR-1233-5p as a pathologic marker for Aβ(+)MCI.

Role of Brain-Derived Neurotrophic Factor in Cognitive Function and Exercise

Brain-Derived Neurotrophic Factor (BDNF) is a member of neurotrophins and it plays important role in both peripheral nervous system (PNS) and short-term, as well as long-term plasticity in central nervous system. Physical exercise fosters neurocognitive functions and brain plasticity at all ages. However, there was an interesting finding stated that BDNF concentration was found to be lower in the trained athletes compared to that of control. Platelet BDNF is found to be acquired from the blood circulation rather than the megakaryocyte precursor cells. This review directs attention to the role of BDNF on cognitive function intensified by exercise for future perspectives.&#x0D; &#x0D; &#x0D; &#x0D; Keywords: BDNF, cognitive function, exercise

Sighting acute myocardial infarction through platelet gene expression

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

Mouse Erythroid Cells Originate from a Megakaryocyte Precursor in Common Myeloid Progenitors

The hierarchical model of hematopoiesis posits that hematopoietic stem and progenitor cells produce common myeloid progenitors (CMP). CMP can become granulocyte/monocyte progenitors (GMP) or bipotential megakaryocyte/erythroid progenitors (MEP). MEP can produce megakaryocytic (Mk) or erythroid (Ery) cells. However, we and others have shown that early mouse and human progenitor populations express many Mk genes (Heuston, Epig. Chrom., 2016), while single cell studies have identified lineage-specific colony forming cells in progenitor populations thought to be multipotent (Psaila, Genome Biol., 2016). To identify the earliest mouse Ery and Mk cells, we performed single cell RNASeq on 10000 stem and progenitor cells (Lin-Sca1+Kit+), 12000 CMP (Lin-Sca1-Kit+CD16/32-CD34+), 6000 MEP (Lin-Sca1-Kit+CD16/32-CD34-) and 8000 GMP (Lin-Sca1-Kit+CD16/32+CD34+). TSNE analysis of expression in the 4 populations identified 33 clusters, which were correlated to biological functions using gene set enrichment analysis. In LSK, no cells with an Ery RNA profile were found, while 56% of cells co-expressed Mk-associated (e.g., Meis1, Fli1) and lymphoid genes. In CMP, 12% of the cells co-expressed Ery (e.g., Gata1, Fog1) and Mk (e.g., Pf4, Cd41) genes, while 23% had an Mk-specific profile (e.g., Fli1, Cd41) enriched for platelet biology processes (p&amp;lt; 3E-18). Unlike traditional models, over 94% of MEP had Ery RNA profiles enriched for ribosome synthesis and heme-biology processes (p&amp;lt; 4E-10). To establish developmental relationships, we performed pseudotime analysis using the Monocle and Scanpy software packages. These programs model differentiation by mapping similar transcriptomes together. Map nodes indicate lineage commitment points and cells further from a node are more differentiated. Combined analysis of LSK, CMP, and MEP generated a model with a single node and 2 trajectories. LSK with Mk and lymphoid RNA profiles diverged at the node, as did 14% of CMP. 31% of CMP with an Mk RNA profile were downstream of the node. Further downstream were cells with mixed Ery/Mk profiles, and furthest from the node were MEP with Ery profiles. A separate pseudotime analysis of CMP only 2 trajectories: one with decreasing Mk- and increasing Ery RNA profiles, and a second with an early Mk endomitotic RNA profile. Pseudotime analysis of MEP only identified a linear trajectory: cells at one end expressed early Ery RNA profiles, and cells at the other end had RNA profiles similar to those of burst-forming unit-erythroid (BFU-E). We generated a predictive set of RNAs for each TSNE cluster. We used index-sorting with 11 markers (Kit, Sca1, CD34, CD16/32, CD36, CD41, CD48, CD123, CD150, CD9, Flk2) to isolate single cells for custom high-throughput multiplex qPCR. This allowed confirmation of cell frequency within TSNE clusters while identifying surface markers for prospective isolation of cell subsets. We focused on 2 populations: CMP-E, which had an Ery RNA profile (10% of clustered CMP and 12% of CMP in the qPCR assay), and CMP-MkE, which had Mk and Ery RNA profiles (12% of clustered CMP and 13% of CMP in the qPCR assay). We prospectively isolated CMP-E and CMP-MkE to compare RNASeq profiles, ATACSeq profiles, and colony forming ability against those of bulk CMP, Ery, and Mk. In CMP-E, 54% of RNAs were expressed in both CMP and ERY, while 41% were expressed only in CMP (p &amp;lt; 6E-72). In contrast, 41% of CMP-E ATACSeq peaks were present in CMP and ERY, while 57% of CMP-E peaks were present only in CMP (p &amp;lt; 1E-3). We conclude that in CMP-E, the RNASeq profile is more erythroid than the ATACSeq profile. In CMP-MkE, 89% of RNAs were expressed in both CMP and Mk, while 7% were expressed only in CMP (p &amp;lt; 8E-190). Likewise, 88% of CMP-MkE ATACSeq peaks were present in both CMP and Mk, while 3% were present only in CMP (p &amp;lt; 1E-3). We conclude that in CMP-MkE, the RNASeq and ATACSeq profiles are equivalent. In soft agar assays, 21% of CMP-E and 3% of CMP-MkE colonies contained BFU-E, compared to 9% of control colonies. We conclude that the CMP-E and CMP-MkE populations are skewed towards the ERY and MK lineages, but are not erythro-megakaryocyte restricted. Our data support a model in which there are two megakaryocyte precursor populations and no erythroid populations in LSK. A third megakaryocyte population in CMP gives rise to erythroid cells. Finally, our data show that transcriptional changes precede chromatin accessibility changes in the earliest erythroid cells. Disclosures No relevant conflicts of interest to declare.

Critical Requirement of U2AF1 in the Maintenance and Function of Hematopoietic Stem Cells

Mutations in the RNA spliceosome genes have been frequently found in myelodysplastic syndromes (MDS). U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. U2AF1 mutations have been identified in ~11% cases of MDS and are associated with poor prognosis. However, the role of wild type U2AF1 in normal hematopoiesis has remained unknown. To determine the role of U2AF1 in hematopoietic stem/progenitor cell (HSPC) function and normal hematopoiesis, we have generated a conditional U2AF1 knockout (floxed) mouse. We crossed the U2AF1 floxed mouse with Mx1Cre mouse and the expression of Cre recombinase was induced with pI-pC injection at 4 weeks after birth. All induced Mx1Cre;U2AF1fl/fl (U2AF1-deleted) mice became moribund or died between 11-12 days after pI-pC induction. U2AF1-deleted mice exhibited marked decrease in bone marrow (BM) cellularity and significantly reduced numbers of WBC, neutrophil, RBC and platelet counts in their peripheral blood compared with control animals. Flow cytometric analyses revealed a dramatic decrease in myeloid, erythroid and megakaryocytic precursor cells in U2AF1-deficient mice compared with control animals. Hematopoietic progenitor colony assays showed a marked decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2AF1-deficient mice. Histopathologic analysis revealed severe BM aplasia in U2AF1-deficient mice. Together, these data suggest that deletion of U2AF1 results in profound defects in hematopoietic development. The fatal BM failure in U2AF1-deficient mice prompted us to examine the HSPC compartments in the BM of these animals. We observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as early progenitors including common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2AF1-deficient mice, indicating a defect at the earliest stage of adult hematopoietic development. To determine whether the loss of HSCs in U2AF1-deficient animals is cell autonomous, BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice were transplanted into lethally irradiated WT C57BL/6 mice. Six weeks after transplantation, recipients were injected with pI-pC to induce the deletion of U2AF1. All the recipients of U2AF1-deficient BM became moribund within 14 days after pI-pC induction. Deletion of U2AF1 in the recipient animals resulted in pancytopenia and marked decrease in HSC/progenitors, myeloid, erythroid and megakaryocytic cells similar to that observed in the primary U2AF1-deficient mice, suggesting that the hematopoietic defects in U2AF1-deficient HSCs is cell intrinsic. We performed competitive repopulation assays to further evaluate the function of U2AF1-deficient HSCs. BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice (CD45.2+) were mixed with CD45.1+competitor BM cells at a ratio of 1:1 and then transplanted into lethally irradiated congenic recipient animals (CD45.1+). Chimerism analysis in the transplanted animals revealed that U2AF1-deficient mice BM cells were completely unable to compete with WT BM cells. The percentages of U2AF1-deficient CD45.2+(donor-derived) LSK, myeloid, B and T cells were markedly reduced in the recipient animals compared with wild type U2AF1 BM donor at 16 weeks after transplantation, indicating that U2AF1-deficiency impairs the repopulation capacity of the HSCs. To gain insights into the mechanism by which U2AF1controls HSC maintenance and function,we performed RNA-sequencing on purified LSK cells from control and U2AF1-deleted mice. Analysis of RNA-sequencing data revealed significant down regulation of genes related to HSC maintenance, cell cycle and JAK-STAT pathway in U2AF1-deficient LSK cells compared with control LSK. RNA sequencing also identified significantly altered splicing events in several important genes in U2AF1-deficient LSK cells. The most commonly altered splicing events were exon skipping/inclusion. We also observed increased phospho-H2AX and DNA damage in U2AF1-deficient BM cells. Overall, our results suggest an essential role for U2AF1 in the maintenance and function of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

Prognostic significance of mutated genes in megakaryocytic disorders

Megakaryopoiesis is a process during which platelets that play a major role in hemostasis are produced due to differentiation and maturation of megakaryocytic precursors. Several genes, including oncogenes and tumor suppressor genes, play a role in the regulation of this process. This study was conducted to investigate the oncogenes and tumor suppressor genes as well as their mutations during the megakaryopoiesis process, which can lead to megakaryocytic disorders. Relevant literature was identified by a PubMed search (1998-2019) of English language papers using the terms ‘Megakaryopoiesis’, ‘Mutation’, ‘oncogenes’, and ‘Tumor Suppressor’. According to investigations, several mutations occur in the genes implicated in megakaryopoiesis, which abnormally induce or inhibit megakaryocyte production, differentiation, and maturation, leading to platelet disorders. GATA-1 is one of the important genes in megakaryopoiesis and its mutations can be considered among the factors involved in the incidence of these disorders. Considering the essential role of these genes (such as GATA- 1) in megakaryopoiesis and the involvement of their mutations in platelet disorders, study and examination of these changes can be a positive step in the diagnosis and prognosis of these diseases.

Combined Pharmacological Inhibition of Bruton's Tyrosine Kinase (BTK) and Phosphoinositide 3-Kinase (PI3K) p110δ Rescues Monocytosis, Thrombocytopenia, and Splenomegaly in a Genetic Mouse Model for JMML

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN). It has no effective treatments and the only treatment that prolongs survival is allogeneic hematopoietic stem cell transplant (HSCT). However, even with this aggressive intervention, approximately 50% of patients relapse with JMML within five years. The most commonly mutated gene in JMML patients is PTPN11, which encodes for the non-receptor protein tyrosine phosphatase SHP2. Gain-of-function (GOF) mutations in SHP2 lead to hyperactive RAS signaling and PTPN11 is recognized as a well-established oncogene in various leukemia's. Our lab has shown that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase (PI3K), plays an important role downstream of SHP2-signaling. Recently, we have shown that in vivo treatment with a specific pharmacologic p110δ inhibitor significantly prolongs the overall survival and reduces the splenomegaly seen in GOF Shp2-expressing mice. Having observed the effectiveness of PI3K p110δ inhibition in correcting the mutant Shp2-induced leukemia phenotype in vitro and in prolonging survival of mice in vivo, we next explored signaling molecules with which p110δ may be interacting to promote the aberrant Shp2 signaling in myeloid cells. In recent years, a key player in B cell receptor (BCR) signaling, Bruton's tyrosine kinase (BTK), has come under intense study in the field of lymphocytic leukemia and lymphoma research. Ibrutinib, a small molecule inhibitor targeting BTK, has proven to be very effective and has received FDA-approval for the treatment of a variety of B cell malignancies. Although research on BTK up to this point has focused on B cell malignancies, BTK is also highly expressed in myeloid cells and mice lacking BTK are defective in many myeloid cell functions. Thus, it is possible that BTK signaling may also play an important role in myeloid malignancies, such as JMML. Given the collaboration of BTK and p110δ in BCR signaling, the key role of p110δ in GOF Shp2-induced leukemia, and the high expression of BTK in myeloid cells, we hypothesized that BTK and p110δ function cooperatively in GOF Shp2-expressing myeloid cells to promote MPN. To test this hypothesis, we examined the potential collaboration of GM-CSF-stimulated BTK and p110δ in GOF Shp2-expressing myeloid cells and tested the inhibitor combination in vivo. We show that BTK cooperates with p110δ to promote GOF Shp2-induced leukemia. We show a role for B cell adaptor for PI3K (BCAP) in BTK upregulation of PI3K activity. In mutant Shp2 macrophages, BCAP phosphorylation is increased specifically in the larger isoforms regulating PI3K activation, and BTK inhibition results in a dose-dependent reduction in this phosphorylation. Our findings show that the MPN caused by GOF Shp2 is due to cooperative signaling between p110δ and BTK, which forms a positive feedback loop with BCAP, thus leading to more Akt/Erk hyperphosphorylation and hyperproliferation in response to GM-CSF.Given these in vitro observations, we treated GOF Shp2 bearing mice with a combination of a PI3K p110δ-specific inhibitor and, a BTK-specific inhibitor, or single agents alone and performed hematopoietic analysis. The combination treatment scheme completely rescued monocytosis and uniquely ameliorated thrombocytopenia compared to single agent treatment of diseased mice. In addition, splenomegaly was also completely rescued in all three treatment groups compared to vehicle. Detailed flow cytometric analysis of bone marrow hematopoietic stem and progenitor cell populations revealed significant repression of LSK, HPC1 and granulocyte macrophage progenitors (GMPs) with a concomitant increase in the megakaryocyte precursors (MKPs) in drug combination treated mice vs. other groups. Consistent with these observations, a significant reduction in mature myeloid cells was noted in the spleen and a complete rescue in the production of peripheral blood platelets was observed in the mutant mice treated with a combination of the two drugs. Thus, combination therapy with PI3K p110δ- and BTK-specific inhibitors profoundly rescues disease state hallmarks of JMML, and may warrant further clinical investigation. DisclosuresNo relevant conflicts of interest to declare.

Congenital X-Linked Myelodysplasia with Tetraploidy Is Associated with De Novo Germline C-Terminal Mutation of SEPT6, a Septin Filament Protein

Private germline mutations affecting hematopoiesis can cause progressive myelodysplasia and thus constitute pre-leukemic states. These can remain undetected, progressively transform and reveal themselves as infant leukemias, which can be linked to translocations involving the mixed lineage-leukemia (MLL) gene. Septin proteins play key roles in mammalian cell division and cytokinesis and are found as fusion partners of MLL in infant and early childhood acute myeloid leukemia (AML). We identified and describe a human germline disorder of septins in a newborn with myelodysplasia who required early hematopoietic stem cell transplant (HSCT) to prevent progressive disease.Materials & Methods: A Caucasian newborn male with no birth defects/malformations or suspicious family history presented with severe progressive neutropenia and was found to have bone marrow (BM) dysplasia with tetraploidy of myeloid progenitors. The patient developed unfavorable clonal aberrations (trisomy 7,8,9) and increased tetraploidy. Due to his progressive cytopenias and concern about risk of leukemic transformation, he underwent an allogeneic busulfan-cyclophosphamide/ATG conditioned DQ-mismatched unrelated HSCT at age 1 yo. He is currently 8 years post-HSCT with normal trilineage hematopoiesis (full donor chimerism), no graft versus host disease or any other non-hematological phenotype. To investigate the genetic etiology of this unique phenotype, we performed family trio germline exome/whole genome next-generation sequencing (NGS), and somatic pre-HSCT BM NGS for the index case. An established algorithm filtered for significant candidates following a de novo germline model. Immunohistochemical (IHC) staining of the pre- and post-HSCT BM biopsies for the candidate protein was performed. To validate the germline origin of the candidate mutation, we generated patient and control skin fibroblasts and induced pluripotent stem cells (iPSCs) that underwent fidelity testing by murine injection teratoma assays and 16-marker immunofluorescence (IF) staining. We then studied hematopoietic progenitor cells (HPCs) derived from iPSC-embryoid bodies (EB) in methylcellulose assays. To further determine the pathogenic nature of the mutation, we generated CRISPR/Cas9 knock-out of the human erythroleukemic cell line (TF-1) and studies these cells by cytomorphology, DNA and cell cycle assays. In-silico protein analysis of the candidate mutation and its effects on septin filament formation was performed.Results: Family trio and disease-tissue NGS identified a novel, germline C-terminal mutation in SEPT6, which was acquired de novo in the patient, and was not found in any database of common polymorphisms. IHC of pre-HSCT patient BM showed reduced Septin-6 staining in megakaryocyte and granulocyte precursors compared to post-HSCT and controls. Patient-derived iPSCs carried the mutation, were cytogenetically normal and bona-fide pluripotent by IF and teratoma assays. EB-derived HPCs from these cells recapitulated the patient's phenotype as they differentially failed to produce granulocyte vs erythroid colonies (fold-reductions CFU-M:8, CFU-G:36, CFU-GM:46, BFU-E:6, see Figure). Despite multiple approaches, SEPT6 CRISPR/Cas9 knock-out/in of the patient's mutation was not tolerated in iPSCs and human myeloid (granulo-/myelocytic) cell lines (HL-60, Molm-13, K562), and only tolerated in erythroid TF-1 cells. Analysis of SEPT6 knock-out TF-1 single-clone lines revealed a propensity to poly-nuclearity/lobation, as observed in the patient's BM. SEPT6 knock-in of C-terminal mutations caused cell death, consistent with existing literature. In silico protein analysis (incl. previously published crystallographic data) suggests that the mutation a) most likely modulates the key role of the coiled-coil SEPT6 domain in septin filament stabilization/bundling/bending, and thus deleteriously impacts cytokinesis, and b) perturbs the equilibrium of splice variants, possibly conferring tissue specificity.Conclusions: Mutation of the C-terminus of human SEPT6 causes aberrant cytokinesis in HPCs leading to a severe congenital neutropenia with tetraploidy and progressive myelodysplasia and cytogenetic aberrations. This report implicates a human germline disorder of SEPT6, and further investigations are required to elucidate the role septins in normal and disordered myelopoiesis. DisclosuresWilliams:Bluebird Bio: Research Funding.