Megakaryocytic Lineage Research Articles

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Aurora Kinase A Inhibition Provides Clinical Benefit, Normalizes Megakaryocytes, and Reduces Bone Marrow Fibrosis in Patients with Myelofibrosis: A Phase I Trial.

Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868.

Peri-arteriolar megakaryopoietic microenvironment via reciprocal CLEC-2/PDPN axis in bone marrow

Bone marrow (BM), the tissue specializing in the production of hematopoietic cells, consists of multiple components (e.g., extracellular matrixes, vasculatures, and stromal cells) that generate a complex three-dimensional network and several localized microenvironment. These microenvironments regulate hematopoietic stem and progenitor cells, including megakaryocyte lineage cells. In this review, we first provide an overview of the microenvironment for hematopoietic stem cells as an introduction to bone marrow microenvironment and subsequently summarize the microenvironment for megakaryocyte differentiation and maturation (megakaryopoiesis). In the last portion, we describe megakaryocyte regulation by podoplanin-positive peri-arteriolar stromal cells in the mouse bone marrow.

THE ROLE OF SHEAR STRESS IN THE GENERATION OF DEFINITIVE HAEMATOPOIETIC LINEAGES AND ARTERIAL VASCULATURE FROM HUMAN PLURIPOTENT STEM CELLS AT THE SINGLE-CELL LEVEL

This study aims to characterise the effect of fluid shear stress on human embryonic stem cell (hESC)-derived haematopoiesis and vasculogenesis by applying microfluidics, live cell imaging and single cell RNA sequencing. AGM-like haematopoietic cultures developed by Ng et al. were generated by manipulating WNT and TGFs signalling during mesoderm specification. The effect of pulsatile circulatory flow was studied in a microfluidic dynamic culture system, using SOX17 (Cherry)/RUNX1c(GFP) hESC line to read out arterial endothelial and haematopoietic differentiation, respectively. Microdevices generated cardiac-like, pulsatile flow in a circulatory culture system with a volume of 2–3µL. AGM-like haematopoietic development was observed by time-lapse imaging on chip for more than two weeks. We observed cells entering the circulation from the adherent layer, and the release of lightly tethered SOX17+ cells into the circulation. In parallel bulk differentiation culture using an orbital mixer to mimic the effect of wall shear stress, single cell RNA seq was performed on 8800 single cells at day 18 of culture. Control treatments were static culture and drug vehicle. Hierarchical cluster analysis identified 18 clusters belonging to erythroid-megakaryocytic, cardiovascular and myeloid differentiation pathways. We identified HOXA expressing mesenchymal cells in AGM cultures. Shear treatment promoted proliferative MYB, RUNX1, CD31 expressing blood progenitors, a reduced proportion of unipotent erythroid and megakaryocytic lineages, and increase numbers of myeloid and bipotent megakaryocyte-erythroid progenitors. The production of smooth muscle and cardiomyocytes was promoted by shear in non-AGM culture. This study demonstrates the feasibility of modelling human embryonic blood formation using microfluidic technology.

3167 – DEVELOPMENTAL CHANGES IN THE CHROMATIN ACCESSIBILITY OF HEMATOPOIETIC STEM CELLS ARE ASSOCIATED WITH DISTINCT GENETIC PROGRAMS

Hematopoietic stem cells (HSCs) undergo a developmental switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Molecular determinants like dynamic changes in the chromatin landscape across the switch hold key insights into developmental changes in cell fate and function but remain poorly understood. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) has emerged as a powerful tool to study chromatin accessibility in rare cells and in combination with genome-wide expression profiling has the potential to resolve cell specific transcriptional networks. To explore the regulation of the switch between fetal liver and adult bone marrow HSC states as well as during the first stages of hematopoietic differentiation, we here asses the changes in chromatin accessibility and genome-wide gene expression patterns in immunophenotypically defined HSCs and lymphoid primed multipotent progenitors (LMPPs). Chromatin accessibility unique to adult bone marrow HSCs were found to be enriched for putative binding sites of the megakaryocyte and erythroid lineage transcription factors Nuclear factor erythroid 2 (NFE2). In parallel, adult HSC gene expression and epigenetic profiles were highly enriched for the megakaryocyte gene signature which is strikingly absent in the E14.5 fetal liver. Taken together, our results identify a previously unrecognized hallmark of the developmental switch of the HSC compartment centered around acquisition of a megakaryocyte bias. These age specific features may have important implications in host response to environmental insults.

Prolonged inhibition and incomplete recovery of mitochondrial function in oxazolidinone-treated megakaryoblastic cell lines

Thrombocytopenia is commonly seen in patients receiving linezolid for >14 days. Linezolid is a reversible inhibitor of mitochondrial function in various cell types. This study investigated the inhibitory effects of linezolid and tedizolid, and their potential recovery on (i) CYTox I expression (subunit I of cytochrome c-oxidase; encoded by the mitochondrial genome), (ii) cytochrome c-oxidase activity and (iii) mitochondrial respiration (Seahorse bioanalysis) in two megakaryocytic cell lines [UT-7 WT (human acute megakaryoblastic leukaemia cells) and UT-7 MPL (transduced to express the thrombopoietin receptor)]. Cells were exposed to linezolid (0.5-25 mg/L) or tedizolid (0.1-5 mg/L) for up to 5 days and recovery followed after drug removal. Both oxazolidinones caused concentration- and time-dependent inhibition of CYTox I expression, cytochrome c-oxidase activity and mitochondrial spare capacity. On electron microscopy, mitochondria appeared dilated with a loss of cristae. Globally, tedizolid exerted stronger effects than linezolid. While CYTox I expression recovered completely after 6 days of drug washout, only partial (linezolid) or no (tedizolid) recovery of cytochrome c-oxidase activity, and no rescue of mitochondrial spare capacity (after 3 days) was observed. Thus, and in contrast to previous studies using a variety of cell lines unrelated to megakaryocytic lineages, the inhibitory effects exerted by oxazolidinones on the mitochondrial function of megakaryoblastic cells appear to be particularly protracted. Given the dynamics of platelet production and destruction, these results may explain why oxazolidinone-induced thrombocytopenia is one of the most common side effects in patients exposed to these antibiotics.

Biological Characteristics and Regulation of Early Megakaryocytopoiesis.

For decades, megakaryocytopoiesis is believed to occur following a classical binary hierarchical developmental model. This model is based on an analysis of predefined flow-sorted cell populations by using cell surface markers. However, this classical model has been challenged by increasing evidences obtained with new techniques which integrating flow cytometric, transcriptomic and functional data at single-cell level and with lineage tracing technique. These recent advances in megakaryocytopoiesis proposed that commitment of haematopoietic stem cells (HSCs) towards megakaryocytic lineage occurs in much earlier stage than that postulated in the classical model. There may exist multipotent but megakaryocyte (MK)/platelet-biased HSCs within HSC compartment and even HSCs can directly differentiate into MKs in steady state or in response to stress. In this review, we focus on recent findings about differentiation from commitment of HSCs to MK and its regulation, and discuss future directions in this research field.

SYSTEMIC MASTOCYTOSIS: CLINICAL AND MORPHOLOGICAL ANALYSIS FIVE PATIENTS WITH A RARE DISEASE

The central component of the diagnosis of systemic mastocytosis is a morphological study of the affected organ, which is aimed at assessing the volume of tumor infiltration and the pattern of damage, which may reflect the biological properties of the tumor and the prognosis of the disease. The material for the study was trephination biopsies and bone marrow smears of 5 patients with systemic mastocytosis, aged 17 to 68 years. Paraffin sections were stained with hematoxylin and eosin, azure by Romanovsky, and immunohistochemistry was performed with antibodies to CD25 (Interleukin-2 receptor alpha chain), CD2 (T-cell surface antigen T11/Leu-5), CD117 (Mast/ stem cell growth factor receptor) and tryptase. Bone marrow smears stained by Romanovsky-Giemsa. At the time of the diagnosis, the 2016 WHO revision classification was used. In all patients, a tumor was detected in SM. In the indolent form, the bone marrow diseases were located singly and discretely, and they also formed perisinusoidal and perivascular clusters up to 10-15 cells, the total number of which did not exceed 15% of all nucleated cells. On the contrary, in case of smouldering form (n=2), a nodular lesion pattern was revealed, in which mas-tocytes formed para- and intertrabecular foci of various shapes with sizes up to 200-300 cells, with a total volume of tumor infiltration of 36 and 43%. In two patients with an aggressive form of the disease, the infiltration volume of CM was 65 and 75%, while in both cases diffuse growth was observed, with a subtotal substitution of most of the bone marrow lacunae, with narrowing of hemopoiesis, as well as the appearance of secondary dysplasia features in erythroid and megakaryocytic lineages. Thus, the clinical manifestations of the disease correlate with the volume and pattern of CM damage in SM.

GATA‐1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

PB1751 HOW WE TREAT PATIENT WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND ACUTE MYELOID LEUKEMIA DEVELOPED FROM MYELODYSPLASTIC SYNDROME

Background:According to various authors, the frequency of concurrent myelodysplastic syndrome (MDS) in patients with chronic lymphocytic leukemia (CLL) is 5%. The observation of patients with combination of lympho‐ and myeloproliferative diseases is clinical interest, due to the rare occurrence, poorer outcomes and absence of optimal treatment strategy for this category of patients.Aims:We present a case of a 71‐year‐old woman with CLL and acute myeloid leukemia (AML) developed from MDS.Methods:The baseline diagnosis CLL III stage (Rai) was established in 2016 at 68 years. FISH: trisomy12 – 9%; del13 – 20.5%; del17p – 17.5%. CT scan demonstrated lymphadenopathy (axillary, epitrochlear, mediastinal, paraaortic). The first line therapy: Ibrutinib 420 mg daily, and Rituximab 375 mg/m2 D1 C1 and 500 mg/m2 D1 C2‐6 of each 28‐day cycle. Despite the treatment, patient still had moderate/severe anemia without reduction of transfusion dependency. The disease evaluation (DE) after cycle 6 of therapy showed the presence of 13.4% blasts and dysplasia in erythroid, granulocyte and megakaryocyte lines (≥10%) in BM. Flow cytometry (FCM) of BM demonstrated the population of myeloid blasts. The BM's biopsy showed combined lesions: MDS with excess of blasts‐2 and CLL (5‐7% of infiltrate). The lymph nodes, spleen and liver were normal sizes by CT scan. The response of CLL was evaluated as partial remission (PR) and the second diagnosis was established: MDS with excess of blasts‐2 (IPSS: high risk, IPSS‐R: very high risk). Azacytidine 75 mg/m2 D1‐7 of each 28‐day cycle, SC was started. Simultaneous, therapy with ibrutinib 420 mg daily was ongoing. After 4 cycles of treatment the patient became transfusion independent; HB level stabilized at 110–115 g/L; BM blasts decreased to 9.8%, and dysplasia changes persisted only in granulocyte line. We observed the decrease of minimal residual disease (MRD) of CLL: 8.6%→5.4%→3.8%. The toxicity grade 3‐4 during therapy was not observed. However, after 8 cycle of therapy the results of laboratory tests showed transformation to AML: HB–73 g/L, WBC–1.6x109/L (NEUT–27%, BANDS–1%, LYM–66%, MON–6%), PLT–44x109/L; BM blasts–24%; FCM of BM ‐ population of myeloid blasts; normal karyotype 46, XX; mutations in genes FLT3, NMP1, BCR/ABL, CBFb/MYH1, RUNX1, TP53, IDH1/2, c‐KIT, ASXL1 were not detected. Treatment with ibrutinib was stopped. Cytozar 38 mg D1‐10 of each 28‐day cycle, SC, and venetoclax 600 mg daily were started.Results:After 1stcycle of therapy: complete remission (CR) with incomplete BM recovery. After 2ndcycle – complete BM recovery. By FCM MRD negative status (MRDneg) AML and MRD positive status (MRDpos) CLL were detected. Patient is ongoing to intake cytozar and venetoclax. Presently, the MRDneg CR of AML and MRDneg PR of CLL are lasting.Summary/Conclusion:Our findings suggest that use of targeted drug in the treatment of patients with AML and CLL is possible and very effective. Also, treatment combination of low dose cytozar with venetoclax is powerful and shows clinical significance. Due to the small number of patients with this combined pathology, further studies are needed to confirm the results obtained.

PB2348 IMMUNE RECONSTITUION OF ΓΔT CELLS AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA – IMPACT OF REFRACTORINESS/RELAPSE AND PREVIOUS THERAPEUTICS ON RECOVERY

Background:γδT cells are a minor circulant effector/cytotoxic population involved in immune surveillance, with evidence supporting their anti‐multiple myeloma (MM) role. Although lymphocyte recover after autologous hematopoietic stem cell transplantation (aHSCT) has been extensively studied, there are limited data about γδT cells, with no information on the impact of previous therapeutic in its recovery.Aims:To evaluate γδT cells recovery in MM patients(pts), its impact in posttransplant recovery and the effect of previous treatment/response in γδT subsets reconstitution.Methods:We prospectively analysed 44 MM pts submitted to aHSCT (2016‐2018), previously to conditioning with melphalan 200 mg/m2 (Dc) and at 30 (D30), 60 (D60), and 100 (D100) days after aHSCT. The proportion (%) of γδT cells, performed by flow cytometry based on the expression of CD3/CD45/Vδ1/Vδ2/Vγ9/CD27, indicated the proportion of these cells among all T cells. Pts were divided into 3 groups: those who receive just 1‐line therapy previous aHSCT (NEW) and those who receive more than 1 line due to primary refractory disease (REFAC) or relapse after response (RECID), according to IMWG criteria. Toxicities were graded according to CTCAEv4.03.Results:Median age was 63 years (40‐69), with 64.0% of males, 27.3% ISS III. Median of previous therapeutic lines was 1(1‐4) – all pts had received bortezomib, 27.3% lenalidomide, 18.8% thalidomide, 9.1% daratumumab, and 4.6% pomalidomide; 36.4% received ≥1 novel anti‐MM agent.Median time diagnosis‐aHSCT was 11.1 months(5.3‐112.1) – 68.2%(n = 30) NEW, 16.6%(n = 6) REFAC and 18.2% (n = 8) RECID. The median number of previous lines was higher in REFAC vs RECID (2vs2; p = 0.015). There were no other characteristics differences at diagnosis between the 3 groups. There was no significant difference between NEW vs REFAC vs RECID in achieving at least very good partial response (VGPR) or higher previous to aHSCT (p = 0.224) or at D100 pos‐aHSCT (p = 0.905).RECID group presented a higher % of Vδ1 + γ9 + T cells (vs REFAC)(p = 0.007) and a lower of Vδ2 + γ9 + T(vs REFAC and NEW)(p = 0.060 and p = 0.027, respectively) at D100. There were no other differences between the 3 groups.Time to neutrophils >0.5 × 109/L (TTN) was higher in RECID group (vs NEW and REFAC) (p = 0.003 and p = 0.015, respectively). A higher TTN was positively correlated with a higher %Vγ9 + T (Coef. 21,4;p = 0.012), Vδ1 + γ9 + T(Coef 17.7;p = 0.007) and Vδ1 + γ9 + CD27 + T(Coef 35.9;p = 0.008)(D60), even after adjusting for the study group. Time to platelets >20 × 109/L (TTP) was higher in RECID vs NEW (p = 0.005). REFAC group needed more platelet transfusion vs NEW (p = 0.011). A higher % Vγ9 + T(D60) was associated with higher platelet transfusion requirement (Coef 7.4;p = 0.016), even after group adjusting. Despite a positive association between TTP and Vγ9 + T (D60)(Coef 20.8; p = 0.019), there was a loss significance after adjusting for group (p = NS).For a median follow up of 17.6 months, progression free survival (PFS) at 1‐year post‐aHSCT was lower in REFAC, although not significant(p = 0.276). None subset of Vγδ + T cells presented impact in PFS.Summary/Conclusion:Our results, although conditioned by a small sample size and a short follow‐up, show a higher % of Vδ1 + Vγ9 + T and lower Vδ2 + Vγ9 + T cells in patients’ group RECID at D100. Furthermore, our data suggests that γδT subsets (particularly Vδ1+, Vδ1 + Vγ9+ and Vγ9+) seem to have a role in delaying haematological recovery, mainly in megakaryocytic line. Further studies are needed to evaluate the relationship between these T cells subpopulations and the response to therapeutics/aHSCT, as well as the benefit of its modulation.

G-CSF causes decrease in peripheral blood platelet counts unrelated to leukapheresis during autologous stem cell mobilization

Background & Aim Autologous transplant utilizing G-CSF mobilized peripheral blood stem cells (PBSC) is an established treatment for various conditions. PBSC collection has been reported to be associated with decrease in platelet counts, attributed to leukapheresis. Whether G-CSF directly affects platelet counts is unclear and studies show conflicting findings. We studied the change in platelet count, prior to start of leukapheresis, in patients receiving G-CSF for PBSC mobilization for autologous stem cell transplant Methods, Results & Conclusion Myeloma patients undergoing PBSC collection in our institution were retrospectively studied. Patients received 10 μg/kg G-CSF SQ daily. Stem cell collection started on day 4 (after 3 doses of G-CSF) if blood CD34 count on day 4 was at least 10 cells/μL (Good mobilizer). If not (Poor mobilizer), patients received plerixafor and another dose of G-CSF on day 4 followed by collection starting day 5. Complete blood count was measured before G-CSF on day 0 and on days 2,3,and 4 prior to start of leukapheresis. 163 patients were studied, median age 61 yr (39-74), 99(61%) were men. Platelet count (Mean ± SE) on day 0 was 230±5.9, which dropped to 192±4.4 (p We show a direct effect of G-CSF on platelet counts unrelated to leukapheresis. The decline is more severe in poor mobilizers. This together with known association of thrombocytopenia with poor mobilization, and expected further drop after leukapheresis is an important consideration when assessing safety and efficacy of G-CSF based mobilization in patient with low platelet count. The mechanism of G-CSF induced thrombocytopenia is likely multifactorial. Possibilities include G-CSF induced differentiation and proliferation of myeloid progenitors at the expense of megakaryocytic lineage, G-CSF induced activation and consumption (Platelets are known to have functional G-CSF receptors), or sequestration from G-CSF induced splenomegaly.

Characterization of Mice with a Platelet-Specific Deletion of the Adapter Molecule ADAP.

The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knockout mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knockout mice (ADAPfl/fl PF4-Cretg) (PF4, platelet factor 4). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even under stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and transforming growth factor β1 (TGF-β1). In vitro, platelets from these mice revealed reduced P-selectin expression, lower levels of TGF-β1 release, diminished integrin αIIbβ3 activation, and decreased fibrinogen binding after stimulation with podoplanin, the ligand of C-type lectin-like receptor 2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of phospholipase Cγ2 (PLCγ2) and extracellular signal-regulated kinase 1/2 (ERK1/2). Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-β1 early after T cell transfer reduced EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.

ACUTE MEGAKARYOBLASTIC LEUKEMIA

Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia triggered by megakaryocytes. Acute megakaryoblastic leukemia is divided into three groups, AMKL in children with Down syndrome (DS-AMKL), AMKL in children who do not have Down Syndrome (non-DS-AMKL), and AMKL in non-DS adults (AMKL adults).The basis of the diagnosis of AMKL or AML-M7, according to FAB, is the presence of megakaryocyte line cells as many as 30% or more of all cells. Meanwhile, the diagnosis of AMKL, according to the 2016 WHO guidelines, is acute leukemia with blasts, about > 20%, > 50% of which is megakaryocyte line cells. Megakaryocyte cells can be more clearly seen with electron microscopes that react positively to platelet peroxidase or use marker antibodies to CD41/gpIIb, CD42b/gpIb, CD61/gpIIIa, von Willebrand factors, and linker for T cell activation.Based on the results of this research, there are differences in cytogenetics between the three types of AMKL according to their different pathophysiology. The World Health Organization (WHO) argued that AMKL was categorized into not otherwise specific (NOS) AML criteria. These criteria exclude AML with myelodysplasia (AMLMRC), AML associated with therapy, and AML with recurrent genetic abnormalities, such as AML with t (1; 22) (p13.3; q13.1), inv (3) (q21.3q26.2), or t (3; 3) (q21.3; q26.2). DS-AMKL is also classified into myeloid leukemia associated with DS. In conclusion, AMKL in adults is not only considered as a rare subtype of AMKL, only 1% of AML cases in population-based clinical experiments and data but also has a poor prognosis.

Aberrant Clonal Hematopoiesis following Lentiviral Vector Transduction of HSPCs in a Rhesus Macaque.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murinestem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

Platelet bioenergetics and its implications in disease

Bioenergetics drives energy production for mechanical processes that are critical to platelet functions, e.g., adhesion, spreading, aggregation, clot formation and contraction. Storage and pathological conditions e.g., stroke, sepsis, diabetes etc., alters platelet energetics and function; however, the specific mechanisms governing these alterations are not clear. It is not well understood which metabolic fuels can be used and which are required. We are investigating the roles of different metabolic pathways in the energy consuming process of clot retraction. Using a spectrum of techniques from in vitro platelet functional assays to biochemical methods, whole animal models, and measurement of bioenergetic functions of mitochondria we are elucidating platelet bioenergetics and its implications in disease. We developed a simple method for measuring clot contraction kinetics using time lapse imaging. Our system is efficient, high throughput and is favorable to study platelet bioenergetics of clot formation and retraction. We hypothesize that, upon activation with thrombin, fuel use can be highly plastic, depending on the fuels available. We found that glycolysis and oxidative phosphorylation can both contribute to provide energy for platelet function. Inhibitors of glycolysis and oxidative phosphorylation using 2‐Deoxyglucose and oligomycin inhibited clot retraction showing that both these processes are important.We have also begun to examine bioenergetics in platelets from diabetics. Platelets from type2 diabetes patients have been shown to have abnormal mitochondrial membrane potential, membrane fluidity, ATP content, and oxidative stress. However, the impact of diabetes on platelet bioenergetics is less clear. We have developed a mouse model to probe the role of mitochondria and oxidative phosphorylation in platelet function. TFAM (Mitochondrial Transcription factor A) regulates mitochondria DNA copy number and transcription of mitochondrial DNA encoded proteins. The regulation of mitochondrial DNA is necessary for mitochondrial biogenesis. We have selectively deleted TFAM in the megakaryocyte lineage using the Platelet factor 4 promoter driven Cre‐Lox system. This model will be very useful, and it supports our in vitro studies with metabolic inhibitors in understanding the role of mitochondria and oxidative phosphorylation in platelet function in platelet bioenergetics contributing to the thrombosisSupport or Funding InformationSupported by HL56652, HL138179 and VA Merit AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD226 is involved in megakaryocyte activation and early-stage differentiation

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N–4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA‐binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.

Mediastinal germ cell tumor with associated myeloid sarcoma: An exceptional co-occurrence

The association of mediastinal germ cell tumor (GCT) and hematological malignancy is a well-documented but extremely rare phenomenon. This syndrome is characterized by the occurrence of nonseminomatous mediastinal germ cell tumor and an associated hematological neoplasm that usually involve the megakaryocytic lineage. The hematopoietic malignancies can involve the mediastinum or present as infiltration of bone marrow or lymphoid organs. In majority of cases, the hematological malignancy was detected in the bone marrow simultaneously or within 6 months after the detection of mediastinal germ cell tumor. There are very few limited case reports on the presence of hematopoietic or leukemic cells within the germ cell tumor itself and mass formation due to myeloid sarcoma is exceptionally rare. Herein we report the case of a 36-year-old male patient with a large mediastinal mass, excision biopsy of which showed areas of yolk sac tumor, immature teratoma and a separate round cell component. On immunohistochemical examination, the round cell component showed positivity for CD45 (dim), CD43, MPO, CD11c, and CD68 and were negative for CD117, CD20, CD5, CD34, Tdt, CD30, CD123, and CD61. A diagnosis of mediastinal germ cell tumor with yolk sac tumor and immature teratoma components and associated myeloid sarcoma was given. Peripheral smear and bone marrow examination showed no evidence of leukemic involvement.

AUER RODS IN MATURE NEUTROPHILS IN A CASE OF ACUTE PROMYELOCYTIC LEUKEMIA

Acute promyelocytic leukemia is characterized by the presence of atypical promyelocytes in bone marrow and peripheral blood. Neoplastic promyelocytes, which often have Auer rods, are most commonly seen in early myeloid precursors in certain subtypes of myeloid leukemia. A 63-year-old male patient,was admitted to our clinic with the complaints of malaise and weight loss for several months, presented with leucopenia and mild anemia. There were no findings of organomegaly, lymphadenopathy and bleeding diathesis and no evidence of infection. Bone marrow aspiration and biopsy which was performed with the preliminary diagnosis of myelodysplastic syndrome revealed a slightly increased Myeloid/Erythroid ratio, distinct maturation arrest in the cells of myeloid lineage 15-20% of which have nucleocytoplasmic asynchrony, course cytoplasmic granules and Auer rods. Auer rods were also present in the cytoplasm of mature neutrophils. Additionally, megakaryocytes were also dysplastic. The immunohistochemical staining was positive for CD117 in 10% of the cells and there was no staining for CD34. The diagnosis was obscure until the fluorescein in situ hybridization results which was positive for PML-RARa fusion t(15;17) (q24.1;q21.2) was received. The patient was eventually diagnosed with acute promyelocytic leukemia. This unusual case of APL was characterized with rarity of atypical promyelocytes in the bone marrow, distinct dysplasia in the myeloid and megakaryocytic lineages and silent clinical course without coagulopathy which caused delay in diagnosis, and well response to treatment.

Post-Translational Polymodification of β1 Tubulin Regulates Motor Protein Localisation in Platelet Production and Function

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which β1 tubulin, the platelet and megakaryocyte lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find distinct patterns of polymodification in megakaryocytes and platelets, mediated by the cell specific expression of effecting (Tubulin Tyrosine Ligase Like - TTLL) and reversing (Cytosolic Carboxypeptidase - CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.