Megakaryocytes Research Articles

Post-translational polymodification of β1-tubulin regulates motor protein localization in platelet production and function

In specialized cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviors. The mechanisms by which b1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell derived MK, and healthy human donor platelets. We find distinct patterns of polymodification in MK and platelets, mediated by the antagonistic activities of the cell specific expression of tubulin tyrosine ligase like enzymes and cytosolic carboxypeptidase enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganization required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Lung megakaryocytes display distinct transcriptional and phenotypic properties.

Megakaryocytes (MKs) are responsible for platelet biogenesis, which is believed to occur canonically in adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may contribute significantly to circulating platelet mass. Although a diversity of cells specific to the BM is known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single-cell RNA sequencing, coupled with histological, ploidy, and flow cytometric analyses, to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts, with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors that are known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed toward roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. The immune phenotype displayed by lung MKs also introduces their potential role in microbial surveillance and antigen presentation.

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)

Lysyl oxidase inhibition in primary myelofibrosis: A renewed strategy.

Primary myelofibrosis (PMF) is a type of myeloproliferative neoplasm (MPN) that portends a poor prognosis and has limited options for treatment. PMF is often driven by clonal mutations in one of three genes that regulate the JAK-STAT signaling pathway, leading to hyperactivation of this signaling pathway and over-proliferation of megakaryocytes (MKs) and their precursors. PMF presents with debilitating symptoms such as splenomegaly and weight loss. The few available treatments for PMF include a JAK2 inhibitor, ruxolitinib, which causes side effects and is not always effective. The extracellular matrix (ECM) and bone marrow (BM) microenvironment may play an important role in the pathogenesis of PMF. Lysyl oxidase (LOX), an enzyme that plays a key role in the ECM by facilitating the cross-linking of collagen and elastin fibers, has been shown to be upregulated in MKs of PMF mice and in PMF patients, suggesting its role in the progression of BM fibrosis. Recently, LOX has been identified as a potential novel therapeutic target for PMF and the development of new small molecule LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, has shown some promise in slowing the progression of PMF in pre-clinical studies. Given that these inhibitors displayed an ability to target the dysregulation of the ECM via LOX inhibition, they show promise as therapeutic agents for an underappreciated aspect of PMF.

The (Patho)Biology of SRC Kinase in Platelets and Megakaryocytes.

Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.

Bone marrow megakaryocytic activation predicts fibrotic evolution of Philadelphia-negative myeloproliferative neoplasms.

Philadelphia-negative chronic myeloproliferative neoplasms (MPN) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPN prognosis needs to be personalized. We identified a new morphological parameter, defined as megakaryocytic activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) cluster formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPN cohorts of patients with polycythemia (PV) (n=64) and non-PV patients (including essential thrombocythemia, and early/prefibrotic primary myelofibrosis [PMF]) (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell count, and lactate dehydrogenase serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<0.0001). Interestingly, M-ACT was not found in the subgroup of essential thrombocythemia patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPN and can represent a new morphologic parameter with a predictive value for progression of MPN.

Abstract 15499: The Increase of Proplatelets and Tpo in Circulation May be a New Pathological Mechanism of Thrombosis

Introduction: Platelets are produced from megakaryocytes, which mainly regulated by thrombopoietin (TPO) and 5-HT. We hypothesized that the secretion of inflammatory cytokines increased in patients with thrombosis under stress or inflammation, leading to increased TPO levels, which in turn increased production capacity of platelet, and increased the number of platelet intermediates proplatelets into the circulation. Therefore, the increase in the number of proplatelets in the circulation, and the increase in plasma TPO levels may be a new pathological mechanism of thrombosis. Methods: TPO was detected by ELISA. Proplatelets from patients were stained with CD41-FITC and further confirmed by a fluorescence microscope and flow cytometry. The in vitro proplatelets were tested by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients' plasma TPO levels were significantly higher with acute inflammation state compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p&lt;0.001, n=65). The plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml, n=20) were significantly higher than that of the control group (122.3±10.2 pg/ml, n=20). The number of platelets in patients with cerebral infarction (222.11±8.55 х10 9 /L, n=20) was slightly higher than that in the control group (206.55±8.83 х10 9 /L, n=20). More large platelets or proplatelets were found in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8, p&lt;0.05). In vitro study confirmed that in 200 megakaryocytes (MKs), more proplatelets were produced in the TPO group (100 ng/ml, 18±2.5%) and in the 5-HT group (100 nM, 15±3.2%), while the control group was only (7±3.2) % (n=5). The effects of TPO/ 5-HT were interfered with by its receptor blockers, confirming that their effects were mediated by its receptors. Both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The studies showed that the increase of proplatelets in circulation and TPO levels in plasma may be a new pathological mechanism of cerebral infarction or thrombosis.

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p&amp;lt;0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

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

Plasmacytoid Dendritic Cells Surveil Megakaryocyte Sialic Acid to Regulate Thrombopoiesis

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases. Recent data suggest that altered Sia may cause platelet destruction because treatment with the sialidase inhibitor Tamiflu increases platelet count in healthy and thrombocytopenic patients. In humans, genetic mutations involving Sia synthesis and transport, and atypical cell surface sialylation, unrelated to any genetic mutation, are associated with reduced platelet count, supporting the role of Sia in regulating platelet count. Immune cells, including classical dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and subsets of T cells (CD8+, CD4+, and Treg cells) can also affect immune thrombocytopenia pathogenesis. Like many other immune cells, cDCs, and pDCs express Siglecs (sialic-acid-binding immunoglobulin-like lectins), which often contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that act as immunosuppressors. Whether BM immune cells monitor MKs via glycan-lectin receptors, including Siglecs and Sia interactions, to control platelet production is unclear. To investigate the role of the TF-antigen in thrombopoiesis, we generated St3gal1MK-KO mice (Pf4-Cre) that display increased TF-antigen specifically in megakaryocytes (MK) and platelets. St3gal1MK-KO mice developed significant thrombocytopenia, but had normal platelet half-life, suggesting that the TF-antigen affected BM thrombopoiesis. In vitro MK maturation and proplatelet production from primary ST3Gal1MK-KO mouse BM cells were also normal, pointing to extrinsic factors in the BM environment affecting thrombopoiesis. Platelet counts of St3gal1MK-KO mice were restored to wild-type levels by 1) crossing St3gal1MK-KO mice with Jak3KO mice that have impaired of lymphoid cell development, 2) by treatment with anti-inflammatory dexamethasone, and 3) treatment with a depleting anti-CD4 antibody. Immunofluorescence staining of the St3gal1MK-KO BM revealed proplatelet structures positive for GPIba+ and the TF-antigen, being infiltrated by mononuclear cells resembling lymphocytes. We speculated that immune cells surveil megakaryocytes to control thrombopoiesis. Bulk RNAseq of CD4+ cells in St3gal1MK-KO BM confirmed a population bias for Type I interferon (IFN-I)-releasing pDCs, a cell type regulated by unique sialic acid binding lectins (Siglecs). Inhibition of IFN-I activity, by a blocking receptor antibody improved platelet counts in St3gal1MK-KO mice. Co-cultures of pDCs with MKs show inhibited pro-platelet formation when TF-antigen is present on MKs with elevated IFN-I levels. Gene set enrichment analysis of BM pDCs single cell RNASeq (scRNAseq) data further confirmed that TF-antigen exposure by MKs up-regulates IFN-I transcripts. scRNAseq also reveals a new population of immune cells with pDC transcript signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm. In conclusion, the data shows that recognition of aberrant MK sialylation by pDCs regulates thrombopoiesis through IFN-I secretion. Disclosures No relevant conflicts of interest to declare.

M2 Macrophages, but Not M1 Macrophages, Support Megakaryopoiesis Via up-Regulating PI3K-AKT Pathway

Background Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×109/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated. Aims To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients. Methods This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway. Results Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily. Summary/Conclusion The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients. Disclosures No relevant conflicts of interest to declare.

Megakaryocyte-Derived TGF-β1 Negatively Regulates Megakaryopoiesis in Mice

Megakaryocytes (MK) and platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Mice with conditional deletion of Tgfb1 in megakaryocytes (PF4Cre/Tgfb1flox/flox) resulted in &amp;gt;90% reduction of TGFβ1 in platelets and 50% reduction of TGFβ1 in plasma. TGFβ1 has been shown to play an inhibitory role in megakaryopoiesis in vitro, and inhibiting TGFβ1 increased megakaryopoiesis in vivo. However, the source of TGFβ1 in megakaryopoiesis is unknown. In this study, we tested whether megakaryocyte-derived TGFβ1 contributes to megakaryopoiesis in bone marrow (BM) by comparing three groups of mice: PF4Cre/Tgfb1flox/flox, littermate control Tgfb1flox/flox, and WTC57Bl/6 mice. Bones (femurs) from these mice (n=12) (age 15-30 weeks, males 60% and females 40%) were harvested, fixed, decalcified, sectioned, and H&amp;E stained. Whole stained BM areas of the sectioned femurs were scanned with an Aperio slide scanner to quantify the number of megakaryocytes and the demarcation membrane system (DMS) and ploidy (nucleus size) of the megakaryocytes were quantified by manually counting megakaryocytes and tracing their DMS and nucleus. The percentage of MK among total BM cells was calculated by dividing total numbers of BM cells in the total area of a BM section with the number of MK in the section. Freshly isolated BM cells were cultured in vitro in culture medium (DMEM+10%FBS) in the presence of thrombopoietin (TPO, 100 ng/ml) with and without TGFβ1 (20 ng/ml) or with a neutralized antibody against the active form of TGFβ1 (AF-101; 2 ug/ml). TGFβ1 and TPO levels in plasma, BM exudates, and cells were measured by ELISA. PF4Cre/Tgfb1flox/floxmice had &amp;gt;50% reduction in TGFβ1 levels in BM cells and exudates (TGFβ1 levels in BM exudates were 1.4 ± 0.033 ng in WT and 0.68 ± 0.065 ng in PF4CreTgfb1flox/floxmice, p&amp;lt;0.01; and in BM cells 50 ± 9 ng/ml in WT and 22 ± 4.2 ng/ml in PF4CreTgfb1flox/flox; p&amp;lt;0.001). MK numbers were ~25% higher in PF4Cre/Tgfb1flox/floxmice (n=6) compared to combined littermate controls (n=3) and WT (n=3) (MK was 0.30 ± 0.02% in PF4Cre/Tgfb1flox/flox and 0.23 ± 0.16% in combined controls; p&amp;lt;0.001 (n=6), whereas blood platelet counts were only marginally higher in PF4Cre/Tgfb1flox/flox (1114 ± 300) vs. controls (806 ± 116; p&amp;lt;0.05). There was a ~2-fold higher plasma TPO levels in PF4CreTgfb1flox/floxmice vs. WT (p=0.04, n=4). Increased DMS and nucleus areas in MK have been shown to correlate with proplatelets formation and platelet production. However, DMS and nuclear areas remained unchanged between genotypes [(DMS area was 197 ± 46 in PF4CreTgfb1flox/flox and 228 ± 50 um2 in combined WT and littermate controls (p=0.3), and nucleus size was 154 ± 23 in PF4CreTgfb1flox/flox and 160 ± 33 um2 in controls (p=0.7)], indicating that the role of TGFβ1 is limited to megakaryopoiesis. To test whether the in vivo phenotype was recapitulated, we cultured whole BM isolated from WT and PF4Cre/Tgfb1flox/flox mice, which showed a ~2.5-fold increase in MK numbers vs. WT when cultured for 5 days in TPO-supplemented medium. The addition of recombinant TGFβ1 in culture medium inhibited MK numbers, and a neutralizing antibody against TGFβ1 resulted in increased MK numbers. We conclude that MK-derived TGFβ1 negatively regulates megakaryopoiesis in mice. Further investigation is needed to determine the mechanism by which TGFβ1 regulates TPO-induced megakaryopoiesis. Our study may be important in megakaryocyte generation in vitro and may have important implications in vivo under normal and stress-inducing conditions where variable megakaryopoiesis is observed, such as essential thrombocythemia and primary myelofibrosis. Disclosures No relevant conflicts of interest to declare.

Inhibition of P-Selectin Rescues the Phenotype of a Novel Genetic Animal Model for Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory disorder which leads to progressive fibrosis and lung failure within 2-5 years1,2. Approximately 30% of IPF patients develop Chronic Kidney Disease (CKD) with renal dysfunction and fibrosis3. The treatment options for IPF are still limited and the disease is an unmet clinical need. The only animal model for IPF is represented by mice treated with bleomycin, a compound used for chemotherapy which has lung toxicity as its major adverse effect, that develop progressive fibrosis. Recent data indicate that bleomycin triggers lung fibrosis by increasing the release of the pro-inflammatory cytokine TGF-β by the resident megakaryocytes (MK)4. Using these observations as foundation, we investigated whetherGata1lowmice, a model for myelofibrosis induced by a TGF-β/P-selectin (P-sel) circuitry established by the abnormal MK5, includes fibrosis in lungs and kidney and whether these traits are rescued by genetic ablation (P-selnullmutation) and antibody neutralization (by RB40.34) of P-sel. MK were observed in lungs from wild-type (WT, n=9),Gata1low(n=22),P-selnull(n=4) andP-selnullGata1low(n=10) littermates. In frequency, MK were significantly lower inGata1low andP-selnullGata1lowthan in WT andP-selnulllittermates (median=1.3-1.5 vs 3-3.7/0.144mm2, p&amp;lt;0.003) and mostly immature. In addition, lungs and kidney fromGata1low mice, but not those from the other experimental groups, contained great levels of TGF- β (Fig.1). Interstitial pneumonia was observed in lungs from WT (n=12, age=3-13 months, males=8, females=4),P-selnull(n=9, age=3-16 months, males=4, females=5),Gata1low(n=29, age=2-24 months, males=16, females=13) andGata1lowP-selnull(n=18, age=3-16 months, males=7, females=11) mice while nephritis was observed only in kidneys fromGata1low mice. Controlling for the effects of sex, there are statistically significant differences in severity of inflammation in kidney (p=0.001) but not in lungs (p=0.86) betweenGata1low and WT mice. These results indicate that lung inflammation is a baseline condition of the CD1 background used for the study while kidney inflammation is a trait exclusive toGata1low mutants independently from their gender. Great levels of fibrosis were observed in lungs and kidney fromGata1low mice but not in those from WT littermates. There are statistically significant differences in lung (p=0.01) and kidney (p&amp;lt;0.001) fibrosis between the two groups; these differences remained after adjusting for age or sex. By contrast, there is insufficient evidence to conclude there exists a relationship between age and fibrosis grade in the other experimental groups. Age was found to be a statistically significant effect modifier on the relationship between genotype and fibrosis in lung (p=0.024) and kidney (0.004). In addition, although there is insufficient evidence for a relationship between sex and fibrosis grade in lung (p=0.62), there is a statistically significant effect of gender in kidney fibrosis inGata1low mice (p&amp;lt;0.001). These results indicate that fibrosis in lung and nephritis and fibrosis in kidney are novel traits ofGata1low mice developed independently from the underlying state of inflammation and which are rescued by ablation ofP-sel. To further clarify the role of P-sel in lung and kidney fibrosis, 14Gata1lowmice (7 males, 7 females) were treated with either vehicle (8 mice) or RB40.34 (30 μg/mouse/day per 3 days/week by tail vein injection as described6). Mice were sacrificed on day 5 (males) and 12 (females) and histopathology, inflammation, fibrosis and TGF-β content in lungs and kidney analyzed. Treatment with RB40.34 restored the morphology and reduced inflammation, fibrosis and TGF-β content in the lungs and in kidney with respect to vehicle (Fig.1). In conclusion, Gata1low mice are a novel animal model for IPF with CKD. The phenotype is associated with highTGF-βcontent in lung and kidney and is rescued by targeting P-sel, suggesting that treatment with antibodies against P-sel may cure IPF in man. REF: 1Richeldi et al.Lancet389: 1941, 2017; 2Hutchinson et al.EurRespirJ46, 795, 2015; 3Ikezoeet al.Respiration94: 346, 2017; 4Zhouet al.CellDeathDis10:648, 2019: 5Spangrudeet al.StemCells34: 67, 2016; 6Emburyet al.Blood104: 3378, 2004. Disclosures Migliaccio: Novartis:Research Funding.

Palbociclib Prevents CDK4/6 Dependent-Myeloproliferation and Myelofibrosis

Introduction Primary and secondary myelofibrosis (MF) present the worst prognosis in the group of BCR-ABL-negative myeloproliferative neoplasms. Presently there is no really efficient therapy of myelofibrosis, except allogenic bone marrow transplantation. In PMF patients, the JAK inhibitors ruxolitinib and fedratinib do not significantly reduce the MF itself or the natural progression of the disease. This could be due to the absence of direct link between JAK2 signaling level and the development of MF. However, ruxolitinib only partially inhibits JAK2 signaling and a reactivation of JAK/STAT signaling through heterodimerization of JAK2 with other JAK may occur (Koppikar et al, Nature, 2012). Complete inhibition of JAK2 signaling would likely induce a profound anemia and thrombocytopenia and at long-term an aplastic anemia. It therefore remains of great interest to identify and inhibit specific downstream targets dysregulated by JAK2 activation that may be responsible of the MF to circumvent the absence of a specific JAK2V617F inhibitor. JAK2V617F mutation leads to the autonomous activation of homodimeric type 1 cytokine receptors and their downstream signaling that induces G1/S cell cycle transition by activating the cyclin-dependent kinases CDK4/6. Cdk6 loss in Jak2V617F KI mice delays the development of the MPN and increases mouse survival (Uras et al, Blood, 2019). We hypothesized that intensifying the JAK2V617F-mediated CDK4/6 increased activity could potentially accelerate the MF development, which is a late event in the Jak2V617F KI model. Our study aims to explore the role of CDK4/6 overactivation or inhibition in the disease and MF development. Methods Since p19INK4D is a cyclin dependent kinase inhibitor, which inhibits CDK4 and to a lesser extent CDK6 and which specifically regulate megakaryocyte (MK) cell cycle and the arrest of endomitosis, we established a pre-clinical MF model by crossing p19Ink4d knock-out (KO) mice with heterozygous inducible Jak2V617F KI mice to obtain p19Ink4d-/-/Jak2FLEXV617F/+SCL-Cre+/-mice called here KOKI mice. With this model, we studied the development of the myeloproliferative disorder. To prevent a MF development in this model, a CDK4/6 inhibitor Palbociclib was administered from 2 to 4 weeks post-Jak2V617F induction. Results In contrast to Jak2V617F (KI) mice that developed a severe MF, in both bone marrow and spleen, at 24 weeks post-Jak2V617F induction, all KOKI mice developed a grade I MF as early as 3 weeks after Jak2V617F induction that progressed to severe fibrosis at 8 weeks (grade III). KOKI mice were characterized by the rapid onset of an extramedullary hematopoiesis (EMH) and megakaryocytic hyperplasia in bone marrow and spleen. As MK are known to be a major source of TGF-β1 in the development of MF, we further focused on the MK lineage. At 1 week post-Jak2V617F induction, prior to MF development, the numbers of MK progenitors (MK-P) were markedly increased in the spleen of KOKI mice (11-fold) while only 3.9-fold increase was observed for KI mice as compared to WT littermates. A significant 2.6-fold increase in the number of MK in the spleen was only visible in KOKI mice, in accordance with splenomegaly and EMH. The analysis of mean ploidy level (&amp;gt;4N) revealed that it increased to 34.8N in KI, 43.7N in KO p19 and 59.4N in KOKI mice as compared to 19N in WT mice. Total TGF-β1 bone marrow levels were moderately elevated in KOKI mice, with a higher proportion of activated TGF-β1. Treatment of KOKI mice with a CDK4/6 inhibitor, Palbociclib, prevented the development of MF and markedly decreased the EMH, the abnormalities of the megakaryocyte differentiation including the ploidy level and the total and activated TGF-β1 levels. Conclusions Altogether, using a KOKI mouse model we show that the deregulation of CDK4/6 mainly in megakaryocytes is sufficient to induce a severe MF strongly suggesting that CDK4/6 inhibitors could be a very promising therapeutic issue for MF patients (Figure). Disclosure of Prior Presentation/Publication : The abstract will not have been presented and published between January 1, 2020 and December 5, 2020. Figure 1 Disclosures No relevant conflicts of interest to declare.

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Preclinical Rationale for the Use of Crizanlizumab (SEG101) in Myelofibrosis

Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative Neoplasms, is still an unmet clinical need. Treatment with the JAK1/2 inhibitor ruxolitinib ameliorates symptoms and improves overall survival but it is unclear whether it is effective in improving the clinical progression of these patients. The bone marrow (BM) and spleen from MF patients contain great numbers of impaired megakaryocytes (MK) which have been hypothesized to be responsible for their clinical manifestation by expressing on their surface high levels of the adhesion receptor P-selectin which triggers a process of pathological neutrophil emperipolesis leading to increased bioavailability of the pro-fibrotic transforming growth factor-β (TGF-β) in the microenvironment1,2. We have peviously reported that ablation of P-selectin cures myelofibrosis in Gata1low mice, a bona fide model for MF3. Using these data as a foundation, we tested whether Crizanlizumab, an antibody targeting human P-selectin that effectively prevents pain crisis in patients with Sickle Cell Disease4, represents an effective option to treat MF. To this aim, we treated Gata1low mice with RB40.34, an antibody against murine P-selectin used in the pre-clinical study that supported the clinical trial with crizanlizumab5, alone or in combination with ruxolitinib. Twenty-four 11-months old Gata1low mice were microchipped, randomly separated in four groups (3 males and 3 females/group) and treated as follow: Group 1: vehicle; Group 2: biotinylated RB40.34 (30 μg/mouse/day per 3 days/week by tail vein injection); Group 3: ruxolitinib (45mg/Kg twice per day by gavage); Group 4: biotinylated RB40.34 and ruxolitinib in combination. Mice were treated for 2 cycles of 5 consecutive days with two days of rest in between and sacrificed on day 5 (I end-point) and 12 (II end-point). End-points: Day 5: all mice were weighed and bled for blood counts determinations and presence of labeled-RB34.40 on platelets. Males were sacrificed for signaling studies and determination of the presence of RB40.34 on MK from BM and spleen. Day 12: females were weighed, bled for blood counts determinations and sacrificed for histopathology analyses. None of the treatments induced changes in body weight that remained similar to those observed before treatment, suggesting that overall all the treatments were well tolerated. None of the treatments rescued the platelet deficiency of Gata1low mice, which remained thrombocytopenic, nor induced anemia by day 12, inspite they had been bleed 1 week earlier. This lack of anemia indicates that the treatments do not impair the ability of the mice to recover from erythroid stress. Atday 5, high levels of RB40.34 were observed on the platelets and on the MK of mice treated with the antibody, alone or with ruxolitinib. However, only the two drugs in combination significantly normalized the TGF-β signaling in BM by reducing the abnormally high content of TFGβRII and SMAD2/3 and the phosphorylation levels of ERK in this organ. These results indicate that when used in combination, the two drugs are effective in rescuing the abnormal TGF-β signaling of BM from these mice. Byday 12, high levels of RB40.34 were observed on BM and spleen of mice treated with the antibody alone or with ruxolitinib but again only treatment with the two drugs in combination greatly improved the cellularity of the BM medulla (Hematoxilin-eosin staining), which is typically reduced in Gata1low mice, and reduced the massive fibrosis (Gomory staining) and high TGF-β content (immunostaining) observed in the diaphysis area of the long bones (Fig. 1). Treatment with the two drugs in combination also reduced fibrosis and TGF-β content in spleen restoring the architecture of this organ which regained a normal appearance with well recognizable red and white pulp zones. When used alone, neither RB40.34 nor ruxolitinib had any effect of the MF phenotype of this mouse model. In conclusion, RB40.34 in combination with ruxolitinib rescues the MF phenotype of Gata1low mice with limited toxicity providing pre-clinical evidence that treatment with crizanlizumab in combination with ruxolitinib may be more effective than that with ruxolitinib alone to cure MF patients.

Bleeding Diathesis in Mice Lacking JAK2 in Platelets

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells such as hematopoietic stem and progenitor cells (HSPCs), megakaryocytes (MKs), and platelets. Individuals expressing somatic activating JAK2 mutations such as JAK2V617F commonly develop myeloproliferative neoplasms (MPNs) associated with serious complications, including venous and arterial thrombosis, a leading cause of mortality. Here, we investigated the role of JAK2 in hemostasis and thrombosis using Jak2fl/flPf4-Cre (Jak2Plt-/-) mice specifically lacking JAK2 within the platelet lineage. Jak2Plt-/- mice developed severe thrombocytosis with a 5-fold increase in circulating platelet number, MK hyperplasia, and splenomegaly. Jak2Plt-/-platelets were of normal size and the expression of major membrane surface glycoproteins was indistinguishable from controls, except for the integrin β3, which was reduced by 20%. Despite the thrombocytosis, Jak2Plt-/- mice had a severe bleeding diathesis, as evidenced by: 1) prolonged tail bleeding time; 2) failure to occlude in a ferric chloride-induced carotid artery injury model; and 3) failure to form stable thrombi in a laser-induced cremaster muscle injury model. Jak2Plt-/- platelets spread poorly on immobilized collagen or on immobilized fibrinogen following GPVI stimulation with the collagen-related peptide (CRP). Jak2Plt-/- platelets had defective α-granule secretion and integrin αIIbβ3 activation following stimulation with CRP, but not thrombin, and showed aggregation defects with low-doses of CRP. Together, the data support a GPVI-specific impairment in platelets lacking JAK2, a notion that was supported by impaired intracellular signaling following GPVI stimulation, as assessed by protein tyrosine phosphorylation. Jak2Plt-/- platelets adhered poorly to type I collagen under arterial shear rates in whole blood. However, JAK2 deletion in platelets did not alter plasma von Willebrand factor (VWF) levels or botrocetin-mediated binding of plasma VWF to GPIbα. Together, the results underline a critical role for JAK2 in platelet GPVI signaling and hemostatic function, which likely contributes to venous and arterial thrombosis observed in patients with MPNs with the activating JAK2V617F mutation. Disclosures No relevant conflicts of interest to declare.

Increased Circulating Proplatelets and Elevated Plasma TPO Levels: A Novel Pathological Mechanism of Cerebral Infarction

Background: Platelets are produced by megakaryocytes and are primarily regulated by thrombopoietin (TPO). In the conditions of inflammation or stress, increased 5-HT and other inflammatory cytokines may affect liver and bone marrow to increase TPO and platelet production (Yang et al., Stem Cells, 2007; 2014). Mature megakaryocytes are fragmented in pulmonary circulation to generate more platelet intermediate proplatelets, then entered the circulation. We hypothesized that in patients with cerebral infarction, the secretion of inflammatory cytokines increases under stress or inflammatory conditions, increasing circulating TPO levels, followed by an increase in platelet production, and an increase in the number of platelet intermediate proplatelets. At the same time, platelets also have varying degrees of activation. Therefore, the increase in the number of proplatelets in the circulation and the increase in TPO levels may be the new pathological mechanism of cerebral infarction. Methods: The plasma levels of TPO were detected by ELISA. The in vivo proplatelets from patients were stained with CD41-FITC, which was further confirmed under a fluorescence microscope and flow cytometry. The in vitro proplatelets were observed in a culture system by CD41-FITC staining under a fluorescence microscope and flow cytometry detection. Results: Our clinical data demonstrated that patients with acute inflammation states, plasma TPO levels were significantly higher compared with healthy subjects (181.1 ± 35.38 vs 96.1 ± 9.7 pg/ml, p&amp;lt;0.001, n=65), which may act as an acute response protein to protect the body. We also detected 20 patients with cerebral infarction and 20 normal controls and found that the plasma TPO levels of cerebral infarction patients (186.2±12.5 pg/ml) was significantly higher than that of the control group (122.3±10.2 pg/ml). The number of platelets in patients with cerebral infarction (222.11±8.55 ×109/L, n=20) was slightly higher than that in the control group (206.55±8.83 ×109/L, n=20). More importantly, we found more large platelets or proplatelets in the circulating blood of patients with cerebral infarction, which was significantly different from the control group (n=8). Furthermore, in vitro study confirmed that in 200 megakaryocytes (MKs), (18±2.5)% of MKs producing proplatelets in the TPO group (100 ng/ml), and in 5-HT group (100 nM), (15±3.2)% of MKs producing proplatelets, while the control group was only (7±3.2)% (n=5). At the same time, the effects of TPO and 5-HT were interfered by the corresponding receptor blockers, confirming that their effects were mediated by its receptors. The study further confirmed that both TPO and 5-HT affect the cytoskeleton by activating p-ERK1/2, reorganizing F-actin to generate proplatelets, and its role was blocked by PD98059. Conclusions: The study demonstrated that increased numbers of proplatelets in circulation and elevated plasma TPO levels may be novel pathological mechanisms of cerebral infarction Disclosures No relevant conflicts of interest to declare.

Sialylated Glycans Regulate MUC13 and the Proto-Oncogenes Pim-1 and Myc to Control Hematopoietic Stem and Progenitor Cell Numbers

The enzyme β-1-4 galactosyltransferase 1 (β4GalT1) plays a critical role in thrombopoiesis by modulating sialo-glycan (sialyl N-acetyl-lactosamine or LacNAc) content and function of the β1 integrin on megakaryocytes (MKs) (Nat. Commun. 2020;11(1):356). Recent data, however, point to a more complex role for β4GalT1 in hematopoiesis, as the promoter region of its conserved gene, B4galt1, is rich in enhancer sequences for transcription factors associated with cell identity and pro-oncogenic regulatory programs. Here, we investigated the homeostasis of hematopoietic stem and progenitor cells (HSPCs) in B4galt1-/- mice. We demonstrate that lack of Sialylated LacNAc synthesis perturbs HSPCs beyond the homing deficiency associated with lack of homing sialo-glycan motifs. Flow cytometry analysis showed that B4galt1-/- mouse bone marrows have increased numbers of Long-Term HSCs (LT-HSCs, defined as LineageNeg/Sca-1Pos/c-KitPos/CD150Pos/CD48Neg). The increase in HSC numbers led us to investigate their phenotypic and functional features further. While quiescence markers in B4galt1-/- LT-HSCs were indistinguishable relative to controls, LT-HSCs expressed more of the platelet marker CD41 on their surface, supporting a highly expanded CD41+ subset of LT-HSCs. Platelet-bias of LT-HSCs has been associated with inflammation and aging. However, our data do not support an increased cytokine inflammatory profile in the bone marrow. Instead, single-cell RNA sequencing (scRNA seq) of sorted β4galt1-/- LineageNeg/Sca-1Pos/c-KitPos (LSK) cells showed a significantly increased expression of the proto-oncogene Pim-1, its target, Myc, and the heavily O-glycosylated transmembrane receptor mucin 13 (MUC13), compared to control cells. Analysis of LT-HSC glycan expression using lectin microarray showed the expected decrease in N-glycosylation associated with B4galt1 deficiency, but also an increase in O-glycans, consistent with overexpression of MUC13, expression of which was enriched compared to other surface mucins. The data show that B4galt1 deletion leads to overexpression of the proto-oncogenes Pim-1, Myc, and MUC13 in HSPCs. The data suggest that MUC13-associated O-glycans and glyco-synthetic genes are potential therapeutic targets for hematologic malignancies since mucins have anti-inflammatory functions and alterations in mucin expression are with inflammation and cancer. Disclosures No relevant conflicts of interest to declare.