The Hepatic Asialoglycoprotein Receptor Regulates Platelet Homeostasis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2025-2025
Author(s):  
Renata Grozovsky ◽  
Gerard Jansen ◽  
Karin M. Hoffmeister
Keyword(s):  
Sialic Acid ◽  
Blood Loss ◽  
Human Body ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Massive Blood Loss ◽  
Platelet Survival ◽  
Sialidase Inhibitor

Abstract Abstract 2025 The human body produces and removes more than a 100 billion of platelets every day. The mechanisms responsible for platelet homeostasis are subject to speculation since the 1950's. The most popular hypothesis to date has been antibody-mediated clearance, platelet consumption due to massive blood loss and an internal “senescence timer”. We and others have recently demonstrated that sialic acid deficient platelets due to external triggers such as sepsis or chilling are cleared by hepatic asialoglycoprotein receptors (ASGPR) independently of macrophages. Here, we investigated whether loss of sialic acid mediates platelet clearance in vivo. We show that 1) Injection of the specific sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lengthened the survival of biotinylated platelets by ∼50% (T1/2 of 72h), compared to mock treated (PBS injected) control mice (T1/2 of 49h); 2) Similarly, biotinylated platelet survival in ASGPR-null mice was prolonged by ∼ 50% (T1/2 of 74h) compared to platelet survival in wild type (WT) mice (T1/2 of 48h); 3) ASGPR-null mice have significantly increased platelet counts, compared to WT (p=0.0004) and platelets isolated from ASGPR-null mice are ∼15% smaller than WT (p=0.03); 4) Platelets isolated from ASGPR-null mice showed significant increased in b-galactose exposure (∼50% increase, i.e. decrease of sialic acid), compared to WT, as evidenced by binding of the b-galactose specific lectin (RCA-I). These data show that the ASGPR not only removes desialylated platelets due to sepsis or chilling, but also regulates platelet homeostasis. Disclosures: No relevant conflicts of interest to declare.

Sialic Acid Loss Regulates Platelet Survival and Integrity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3504-3504
Author(s):  
Renata Grozovsky ◽  
Gerard Jansen ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Platelet Survival ◽  
Sialidase Inhibitor ◽  
Glycan Degradation

Abstract It becomes increasingly apparent that, besides the intrinsic apoptotic machinery, surface glycan modifications regulate platelet survival. Platelets with reduced α2,3-linked sialic acid during sepsis due to S. pneumoniae infection, after cold storage, or in mice lacking the sialyltransferase ST3GalIV are cleared by the hepatic Ashwell-Morell receptor (AMR, ASGPR1/2). Platelet survival in Asgr2-/- mice was increased by ∼35% when compared to that of WT mice, which results in a ∼50% increase in circulating platelet counts, despite a loss of surface sialic acid. We reasoned that sialidase activity increases on the surface of circulating platelets as they age, a process that would facilitate sialic acid hydrolysis and removal from the circulation. To test this hypothesis, we directly injected the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) into WT mice and determined endogenous platelet circulatory times. Platelet survival was prolonged by ∼30% (T1/2 of 62.0 ± 2.7 h) in DANA-treated mice, compared to that of mock-treated mice (T1/2 of 47.5 ± 4.3 h). DANA injections decreased terminal sialic acid loss on circulating platelets by ∼40% by day 2, compared to control platelets, as evidenced by binding of RCA-I lectin that specifically recognizes terminal β1-4 galactose moieties exposed by sialic acid removal. Freshly isolated, resting platelets from Asgr2-/- mice (AMR-platelets) were significantly smaller in size (22%) and had increased sialidase Neu1 (∼5 fold), but not Neu3 surface expression, when compared to WT platelets or St3gal4-/- platelets, as measured by flow cytometry. We next investigated if AMR-platelets age/deteriorate faster upon in vitro storage. Platelets were isolated from WT, Asgr2-/- and St3gal4-/- mice and stored for 24hrs at room temperature, and sialidase expression (Neu1 and Neu3) as well as microvesiculation were measured by flow cytometry. Although significant Neu1 and Neu3 surface expression increase was measured on platelets from all phenotype after storage, Neu1 and Neu3 surface expression was significantly higher in AMR-platelets (∼2 and 4 fold, respectively) when compared to WT and St3gal4-/- platelets. AMR-platelets, but not St3gal4-/- platelets microvesiculated upon storage, consistent with a faster deterioration of aged AMR-platelets. We next injected into WT and Asgr2-/- mice the BH3 mimetic, ABT-737, which binds and inhibits the pro-apoptotic Bcl-2, Bcl-xL and Bcl-w. After injection of ABT-737, platelets in the Asgr2-/- mouse were cleared more efficiently (∼20%) from the circulation when compared to those in WT mice. Collectively, our data show that blood borne sialidases contribute to loss of sialic acid during circulation to regulate platelet survival. Our data also suggest that platelet glycan degradation, i.e. sialic acid loss, may trigger the intrinsic apoptotic machinery in platelets, linking glycan degradation and intrinsic apoptotic machinery in the clearance mechanisms regulating platelet survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2346-2346
Author(s):  
Irene Marini ◽  
Jan Zlamal ◽  
Lisann Pelzel ◽  
Wolfgang Bethge ◽  
Christoph Faul ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Life Span ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Cell Interaction ◽  
Bleeding Tendency ◽  
Sialidase Inhibitor ◽  
Von Willebrand ◽  
The Impact

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hsiangyu Hu ◽  
Nirmalya Saha ◽  
Yuting Yang ◽  
Sierrah Marie Grigsby ◽  
Rolf Marschalek ◽  
...  
Keyword(s):  
Cell Growth ◽  
Acute Leukemia ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Fusion Partner ◽  
Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

ABCG2 C.421C>A Is Associated with Higher Trough Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 110-110
Author(s):  
Naoto Takahashi ◽  
Masatomo Miura ◽  
Stuart A Scott ◽  
Kenichi Sawada
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Plasma Levels ◽  
Myeloid Leukemia ◽  
Drug Transport ◽  
Conflicts Of Interest ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Large Patient

Abstract Abstract 110 [Background] Despite the excellent efficacy of imatinib for the treatment of chronic myeloid leukemia (CML), trough imatinib plasma levels can vary widely among patients. This may be due, in part, to inter-individual variation in imatinib metabolism and drug transport efficacy. To investigate the role of genetic variation in the pharmacokinetics of imatinib, we analyzed common single nucleotide polymorphisms within important imatinib pathway genes including ABCG2 (BCRP), ABCB1 (MDR1), ABCC2 (MRP2), CYP3A5, and SLC22A1 (OCT1) in 67 CML patients treated with imatinib. In addition, trough imatinib plasma levels were determined using high-performance liquid chromatography-tandem mass spectrometry. [Results] Distinct imatinib pharmacokinetics were identified in association with ABCG2 c.421C>A (p.Q141K; rs2231142) genotype. Specifically, the presence of the variant c.421A allele was significantly (p=0.024) associated with higher imatinib concentrations [median Cmin/Dose 2.70 (range: 1.50-8.30) ng/ml/mg; n=25] compared to patients with the wild-type ABCG2 (c.421C/C) genotype [median Cmin/Dose 2.27 (range: 0.37-5.30) ng/ml/mg; n=42]. ABCG2 is an efflux transporter for many xenobiotics, including imatinib, and is expressed at high levels in the human liver. Previous studies indicate that c.421A causes a 40% reduction in imatinib transport in vitro when compared to the wild-type genotype. Our data suggest that CML patients with ABCG2 c.421A allele may have deficient ABCG2 activity in vivo, resulting in reduced hepatic excretion of imatinib. Of note, although less common among Africans and individuals of European decent, the ABCG2 c.421C>A allele occurs at a high frequency in the Japanese (0.311) and Han Chinese (0.289) populations. [Conclusion] The association of ABCG2 c.421C>A with imatinib pharmacokinetics may explain why some Japanese CML patients administered less than 400 mg/day of imatinib have clinically sufficient trough imatinib plasma levels. Prospective studies are warranted to confirm the association between ABCG2 genotype and imatinib pharmacokinetics in large patient populations. Disclosures: No relevant conflicts of interest to declare.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 111-111
Author(s):  
Meghedi N Aghourian ◽  
Catherine A Lemarie ◽  
Francois-Rene Bertin ◽  
Mark D Blostein
Keyword(s):  
Endothelial Cells ◽  
Venous Thromboembolism ◽  
Microarray Analysis ◽  
Conflicts Of Interest ◽  
Prostaglandin E ◽  
Wild Type ◽  
Prostaglandin E Synthase ◽  
Murine Lung

Abstract Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE. Disclosures No relevant conflicts of interest to declare.

hnRNP K: A Tumor Suppressor or Oncogene?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 259-259
Author(s):  
Miguel Gallardo ◽  
Hun Ju Lee ◽  
Carlos E. Bueso-Ramos ◽  
Xiaorui Zhang ◽  
Laura R. Pageon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Chromosome 9 ◽  
Wild Type ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Hnrnp K ◽  
Differentiation And Proliferation

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene. The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML. To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K. Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression). Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Palmitoyl Acyltransferase DHHC9 Is Required for Efficient Induction of Leukemia By Oncogenic NRAS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 893-893
Author(s):  
Ping Liu ◽  
Bo Jiao ◽  
Ruihong Zhang ◽  
Huanbin Zhao ◽  
Chun Zhang ◽  
...  
Keyword(s):  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Myeloproliferative Disease ◽  
Ras Proteins ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Palmitoyl Acyltransferase

Abstract Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high-affinity plasma membrane binding of NRAS, HRAS and KRAS4A. NRAS mutations are common in human hematological malignancies. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for RAS related cancers. In previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate the modification reaction. Protein S-palmitoylation is catalyzed by the DHHC family of palmitoyl acyltransferases (PATs). Thus far 24 mammalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9) is the ortholog of yeast Ras2 PAT and constitutes a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancers. In this study we investigated the role of DHHC9 in normal hematopoiesis and NRAS leukemogenesis in vivo. We found that DHHC9 is not the only PATs for RAS palmitoylation in vivo and the Zdhhc9 knockout mice were born and grew similarly as the wild type mice. The frequency of lineage-specific populations and hematopoietic stem cell phenotypes were also similar in mice with knockout alleles of Zdhhc9 as that of wild type mice. However, loss of DHHC9 prolonged the survival of mice with myeloproliferative disease or T-cell acute lymphoblastic leukemia evoked by oncogenic NRAS expressed either from the endogenous locus or from retroviral promoter. These results demonstrate that DHHC9 is dispensable for normal hematopoiesis, but plays an important role in the pathogenesis of NRAS-induced leukemias. The findings suggest that palmitoyl acyltransferase DHHC9 may serve as a safe and effective target for developing therapies against NRAS-related cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Ashwell-Morell Receptor Regulates Hepatic Thrombopoietin Production Via JAK2-STAT3 Signaling in Vivo and in Vitro

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2-2
Author(s):  
Renata Grozovsky ◽  
Antonija Jurak Begonja ◽  
John H. Hartwig ◽  
Herve Falet ◽  
Karin M Hoffmeister
Keyword(s):  
Bone Marrow ◽  
Sialic Acid ◽  
Mrna Expression ◽  
Hepg2 Cells ◽  
Mrna Levels ◽  
Platelet Production ◽  
Platelet Survival ◽  
Post Infusion

Abstract The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of platelet precursors, bone marrow megakaryocytes. Hepatocytes are a major source of production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. Here, we provide experimental evidence that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor), thereby regulating platelet survival and hepatic TPO levels. These conclusions are based on the following evidence: 1) Mice lacking the AMR Asgr2 subunit had increased platelet survival, compared to wild type (WT) mice. Platelets from Asgr2-null mice showed increased loss of sialic acid, as evidenced by flow cytometry using the galactose specific lectins RCAI and ECL, showing that removal of desialylated platelets by the AMR regulates in vivo platelet survival. 2) Livers isolated from Asgr2-null mice had TPO mRNA levels decreased by 40%, compared to WT mice. In contrast, liver TPO mRNA levels were increased by 30% in St3gal4-null mice lacking the sialyltransferase ST3GalIV, where desialylated platelet clearance is increased and specifically mediated by the AMR. Both plasma TPO levels and platelet TPO contents were similarly altered in both mutant mice. Thus, desialylated platelet uptake by the AMR regulated liver TPO levels. 3) Desialylated platelets isolated from St3gal4-null or Asgr2-null mice infused into WT mice increased hepatic TPO mRNA levels as early as 12h post-infusion. Plasma TPO concentrations and bone marrow megakaryocyte numbers increased in parallel with TPO mRNA levels, peaking by day 2 post-infusion, followed by new platelet release at day 10 post-infusion. In contrast, desialylated platelets infused into Asgr2-null mice had no effect on TPO mRNA synthesis, TPO plasma levels and bone marrow megakaryocyte numbers. 4) Incubation of human hepatoma cell line, HepG2 cells, with human desialylated platelets by sialidase treatment resulted in TPO mRNA expression increase by 2.2 and 2.9-fold after 4 and 6h, respectively, followed by significant increase in TPO secretion. 5) The signaling pathways activated by uptake of desialylated platelets by the AMR to induce TPO mRNA transcription were investigated in vivo and in vitro. Major polypeptides of 60-70 and 125 kDa were highly tyrosine phosphorylated in WT liver cells, as evidenced by SDS-PAGE. Using a specific antibody directed against JAK2, we identified the 125-kDa phosphoprotein as the tyrosine kinase JAK2 in mouse liver cells and human HepG2 cells. Analysis of liver samples revealed a marked reduction in JAK2 phosphorylation in Asgr2-null mice and significant increase in St3gal4-null mice. 6) The JAK1/2 inhibitor AZD1480 significantly decreased phosphorylation of JAK2, phosphorylation and translocation to the nucleus of the acute phase response transcription factor STAT3, TPO mRNA expression and TPO secretion in HepG2 cells incubated with desialylated platelets. In vivo treatment of WT mice with AZD1480 blocked TPO mRNA increase promoted by injection of endogenously desialylated platelets. Therefore we conclude that platelets desialylate as they circulate, thereby becoming the primary AMR ligand and providing a novel physiological feedback mechanism to regulate plasma TPO levels and platelet production in vivo and in vitro. Disclosures No relevant conflicts of interest to declare.

Role of Sialic Acid for Platelet Lifespan and Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2864-2864
Author(s):  
Anne Louise Sørensen ◽  
Viktoria Rumjantseva ◽  
Sara Nayeb-Hashemi ◽  
Sunita Patel-Hett ◽  
Jennifer Richardson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Wild Type ◽  
Platelet Surface ◽  
Lectin Domain ◽  
Liver Macrophages ◽  
Reticulated Platelets

Abstract Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.

Sign in / Sign up

Export Citation Format

Share Document