bluuebunny/crossref_metadata_2025 huggingface dataset:
The Notch Signaling Pathway as a Suppressor of Myeloid Transformation
Authors: Aifantis Iannis
Published: October 2019

Abstract SCI-13

Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase complex (γSE) complex. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations. We identified somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis reveals that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. These studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue. These observations also suggest that Notch activity is not simply a promoter of the T cell lineage in the thymus but that Notch signaling thresholds could regulate commitment and/or survival of distinct hematopoietic lineages in the bone marrow. To address these issues in vivo, we have generated Notch receptor lineage tracing and activity reporter genetic tools. Analysis of these animal models identified unique novel functions for the Notch pathway during early bone marrow hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Role of Notch Signaling in Hematopoietic Stem Cell Function
Authors: Espinosa Lluis , Bigas Anna
Published: October 2019

Abstract SCI-15

The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Insights Derived From B-Cell Immunodeficiency Disorders
Authors: Rawlings David
Published: October 2019

Abstract SCI-19

Over the last four decades, the genetic defects leading to more than 140 distinct inherited immune disorders have been discovered. A subset of these diseases primarily impact B cell development, homeostasis, and/or activation. This talk will provide an overview of these B cell disorders. It will highlight new information regarding human immune function that has been gained from studying these disorders including: the role of key receptors or signaling effectors in immune development or activation, how such changes may impact pathogen specific versus autoimmune responses, and how targeting these pathways may provide a means to modulate other more common human immune diseases. Finally, we will provide a brief overview of efforts to correct one of these disorders in patients using genetic therapies.

Disclosures:

No relevant conflicts of interest to declare.
p53-Mediated Stress and Tissue-Dependent Cell Fate Decisions; Implications for p53 Targeting
Authors: Lozano Guillermina (Gigi)
Published: October 2019

Abstract SCI-3

The activity of the p53 tumor suppressor is tightly regulated by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation. Loss of Mdm2 in embryos or in individual tissues in vivo results in cell lethal events that are p53-dependent. A hypomorphic allele of p53, p53515C (encoding the p53R172P protein), rescues the embryonic lethality of Mdm2–/– mice. Mdm2–/– p53515C/515C mice, however, die by postnatal day 13 due to hematopoietic failure. In these mice, hematopoiesis is normal during embryogenesis. After birth, these mice had elevated reactive oxygen species (ROS), which activated p53R172P. p53R172P, in turn, induced ROS, creating a loop that caused cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment. The cell cycle inhibitor, p16, was also stabilized due to ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Cytokines and Vascular Diseases
Authors: Weber Christian
Published: October 2019

Abstract SCI-36

During the past decade it has become increasingly clear that platelets exert important functions in the context of inflammation, beyond their role in hemostasis. Platelets may adhere to intact endothelial cells and promote local vascular inflammation by recruiting leukocytes via direct interactions or by secreting inflammatory mediators such as cytokines and chemokines. Accordingly, platelet-derived chemokines play a crucial role in directing leukocytes to sites of vascular injury or dysfunction, thereby contributing to neointimal hyperplasia or atherosclerosis. In addition, platelet-derived cytokines can shape the local inflammatory environment. In this overview, I will discuss the function of platelets as immune cells that potentiate vascular inflammation with a special focus on platelet-derived chemokines: their effects and interactions and their potential quality as targets for the treatment and/or prevention of cardiovascular disease.

Disclosures:

Weber: Carolus Therapeutics: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.
Physiology of HIF Regulation of Erythropoiesis
Authors: Johnson Randall S.
Published: October 2019

Abstract SCI-38

Current research related to hypoxic response and the role of the HIF transcription factors with regards to their function in erythropoiesis will be presented and discussed.

Disclosures:

No relevant conflicts of interest to declare.
Changes in Hemoglobin and Hematocrit May Predict Mortality in Patients with Acute Coronary Syndromes.
Authors: Alirhayim Zaid , Qureshi Waqas , Donthireddy Vijaya , Hassan Syed , Khalid Fatima
Published: October 2019

Abstract 3171

Introduction:

Changes in plasma volume, the intravascular portion of the extracellular fluid volume, can be estimated by measuring changes in the levels of hemoglobin and hematocrit in the blood. In addition to hemoglobin & hematocrit levels, we are also able to use standard dilution techniques with radiolabeled albumin to accurately measure plasma volume changes. It is not known if plasma volume changes influence outcomes in patients with acute coronary syndromes. The aim of this study was to evaluate the effects of plasma volume changes in patients presenting with the acute coronary syndromes.

Methods:

Consecutive patients presenting to a single tertiary care center from January 2001 to December 2010 with non ST elevation myocardial infarction (NSTEMI) or ST elevation myocardial infarction (STEMI) were enrolled. Admission hemoglobin (Hbpre) & hematocrit (Hctpre) and discharge hemoglobin (Hbpost) and hematocrit levels (Hctpost) were obtained. Plasma volume changes were measured and a validated formula (ΔPV = ((Hbpre/Hbpost) × (100-Hctpost/100-Hctpre)-1) × 100%) was used to calculate the changes in plasma volumes. A detailed chart review was performed to collect information about baseline variables such as age, gender, hypertension, diabetes, hyperlipidemia, smoking status, and congestive heart failure. The Framingham Risk score was also calculated for each individual. Survival analysis was carried out for plasma volume changes of -20% - 0%, 0 – 20%, and ≥20%. Mortality data was collected from the social security death index for the first 60 days post-discharge.

Results:

A total of 9770 patients with confirmed NSTEMI or STEMI (mean age 61.8 ± 4.8 years, 48.8% women) were included in the final analysis. Mean pre admission hemoglobin (Hbpre) was 10.2 ± 1.4 g/dl and post admission hemoglobin (Hbpost) was 10.4 ± 1.3 g/dl. Change in plasma volume, ΔPV, was categorized into one of four categories, with 131 (1.3%) ≤20%, 6126 (62.7%) -20% - 0%, 3393 (34.7%) 0 – 20%, and 120 (1.2%) ≥20%. There were 509 deaths within 60 days of discharge. Change in plasma volume was found to be an independent predictor of mortality (HR 5.71; 95% CI 4.75 – 6.86, p = 0.0001) in a Cox proportional hazard model. Most of the deaths occurred during the first thirty days as demonstrated by the Kaplan – Meier's survival curve (Figure 1). Receiver operating curve showed an area under the curve of 0.876 for changes in plasma volume.

Conclusion:

This study shows that hemoglobin and hematocrit, although simple tests, can provide important prognostic information strongly predictive of short term mortality in patients with acute coronary syndromes. Further studies are required to see if monitoring of plasma volume and correction with pharmacological agents such as diuretics may lead to better outcomes.

Disclosures:

No relevant conflicts of interest to declare.
Temporal Differences in Terminal Erythroid Differentiation of Human CD34+ Cells Derived From Cord Blood and Adult Peripheral Blood
Authors: Jaffray Julie , Hu Jingping , Li Jie , An Xiuli , Narla Mohandas
Published: October 2019

Abstract 3200

Purified CD34+ cells derived from either cord blood (CB) or peripheral blood (PB) are currently being used to further our molecular and mechanistic understanding of human terminal erythroid differentiation. What is unclear is whether there are differences in the kinetics of terminal erythroid differentiation of CD34+ cells from these two sources. In the present study, we document that terminal differentiation in cultured CD34+ cells purified from peripheral blood is faster than that of CD34+ cells from cord blood. For these studies, we optimized an 18 day, three phase, in vitro culture system using CD34+ cells to obtain enucleated reticulocytes. In this system, proerythroblasts are generated starting at day 6 which further differentiate during the duration of culture to eventually generate reticulocytes. Based on the expression of various membrane surface markers, we used flow cytometry to quantitatively monitor terminal erythroid differentiation from proerythroblasts to enucleated reticulocytes during culture. The three surface markers, alpha-4 integrin, band 3 and CD36 enabled us to clearly distinguish between all distinct stages of terminal erythroid differentiation – proerythroblasts, early- and late- basophilic erythroblasts, polychromatic and orthochromatic erythroblasts. These analyses enabled us to show that CD34+ cells purified and cultured from peripheral blood underwent terminal erythroid differentiation at a faster rate than CD34+ cells from cord blood. Terminal erythroid differentiation in cord blood cultures was delayed on an average of 2 to 3 days compared to peripheral blood. For example, the surface protein expression pattern seen on days 11–12 of cell culture of peripheral CD34+ cells was not achieved in cord blood cultures until day 14. This delay in terminal differentiation was also reflected by increased extents of enucleation in peripheral blood cultures compared to cord blood (culture day 12: 33% enucleation in PB and 7% in CB and on day 14: 45% enucleation in PB and 19% in CB). These findings have enabled us to document significant differences between the kinetics of terminal erythroid differentiation of CD34+ cells derived from fetal cord and adult peripheral blood. While at the present time we do not have a mechanistic understanding for this difference, we are currently exploring if the observed differences may be related to differences in cell cycle dynamics between fetal and adult erythropopiesis.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Method Using Extracted Human Neutrophil Antigens From HNA Gene-Transfected Cell Lines for Detection of Antibodies Against Human Neutrophil Antigens
Authors: Onodera Rie , Nakamura Kazuhiro , Taniguchi Kikuyo , Kurita Emi , Hiraoka Asako , Yasui Kaduta , Matsuyama Nobuki , Hirayama Fumiya , Kobayashi Masao
Published: October 2019

Abstract 3272

Antibodies against Human Neutrophil Antigen (anti-HNA antibodies) have been implicated in immune neutropenia and transfusion-related acute lung injury. Those have been usually analyzed by Granulocyte indirect immunofluorescence test by flow-cytometry (GIFT-FCM), which requires fresh blood for panel neutrophils that cannot be preserved more than a few hours. Furthermore, specificity for anti-HNA antibodies cannot be easily identified by GIFT-FCM because of several antigens on membrane of panel neutrophil. In this study, we have developed a novel method designated as KY-Luminex Method, which was modified from Monoclonal antibody-specific immobilization of granulocyte antigens.

For KY-Luminex Method, extracted human neutrophil antigens (eHNAs) were established from gene-transfected cell lines (KY-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b) that express HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b, respectively. These eHNAs were immobilized by monoclonal antibodies on microspheres (eHNA-microspheres). Sera were tested to the eHNA-microspheres in wells of a 96-well microplate, respectively. The reactivities of the sera were analyzed by Luminex100.

From May 2010 to March 2012, sera were obtained from 101 patients with autoimmune neutropenia in children. The age distribution was from 2 months to 72 months. Sixty-three patients were females and 38 were males.

As shown in Table, the sera from 74 patients showed positive reaction both in standard GIFT-FCM and KY-Luminex method. The sera from 11 patients showed positive in GIFT-FCM, and negative in KY-Luminex. These sera finally proved to have antibodies against antigens other than neutrophil such as anti-HLA antibody. The sera from 13 patients showed negative in GIFT-FCM, and positive in KY-Luminex. The sera from 3 patients showed negative both in GIFT-FCM and KY-Luminex. Sera with positive reaction against each panel neutrophil in GIFT-FCM have tendency to react against multiple antigens in KY-luminex method. Especially, majoryty of analyzed sera had antibodies against HNA-4a, 4b, 5a and 5b. No antibodies against each antigens were detected in sera from normal controls by KY-luminex method.

These results indicate that KY-luminex method has superior to GIFT-FCM in both sensitivity and specificity of anti-HNA antibodies. Furthermore, eHNA-microspheres are stable at 4 centigrade for more than a half year, and many samples such as 96 samples could be analyzes at once in simple and easy method.

Table. Relationship of reactivities of patient's sera between GIFT-FCM and KY-luminex method. Data represent the number in 101 patients GIFT-FCM KY-Luminex method positive negative positive 74 11 negative 13 3

Disclosures:

No relevant conflicts of interest to declare.
Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway
Authors: Betapudi Venkaiah , McCrae Keith R.
Published: October 2019

Abstract 3305

Background and objective:

High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis.

Results:

We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid.

Conclusion:

These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Complement Activation in Patients with Acquired Thrombotic Thrombocytopenic Pupura (TTP)
Authors: Yang Shangbin , Cataland Spero R , Holers V. Michael , Lundberg Ante S , Wu Haifeng
Published: October 2019

Abstract 3323

Background:

Although severe deficiency(<10%) of the ADAMTS13 protease is central to the pathophysiology of acquired TTP, recently published data have suggested a role for complement activation in the pathogenesis of acquired TTP (Reti et al, JTH 2012; 10:791–798).

Patients:

We performed a retrospective study in longitudinally banked plasma samples from 9 patients with acquired TTP and severely deficient ADAMTS13 activity at presentation. Patients were randomly selected from our cohort of acquired TTP patients who achieved remission and were followed longitudinally with plasma samples banked at regular intervals. The presenting platelet count, LDH, serum creatinine, and ADAMTS13 activity of these 9 subjects were comparable to the entire cohort of acquired TTP patients in our research program at the time of their initial presentation.

Methods:

Samples were obtained at the time of their initial presentation prior to the initiation of plasma exchange therapy (PEX), at remission (median 2 weeks after the last PEX), and at sustained remission (median 2 months after remission sample) were utilized in this study. The complement proteins Bb (alternative pathway), C3a and C3 (all pathways of complement activation), and C5b-9 (terminal pathway) were measured.

Results:

At presentation, 4 subjects had evidence of complement activation as defined by increased levels of C3a and decreased C3, and all 9 subjects exhibited activation of the alternative pathway (Bb). In serially measured samples, the complement protein Bb was significantly higher, and C3 significantly lower, at presentation, returning to normal at the time of sustained remission (Table 1). C5b-9 and C3a demonstrated similar trends as Bb. ADAMTS13 activity, severely deficient in all subjects at presentation, was also significantly higher at remission and sustained remission.

Conclusions:

These data from a cohort of ADAMTS13 deficient, acquired TTP patients provide additional evidence for the role of complement activation in the pathogenesis of acquired TTP. Further study of our complete cohort of patients with acquired TTP is presently underway to better define the role complement activation in patients with acquired TTP.

Disclosures:

Cataland: Alexion: Consultancy, Research Funding. Holers:Alexion: Consultancy. Lundberg:Alexion: Employment. Wu:Alexion: Consultancy, Research Funding.
The Molecular Mechanisms of Serotonin in the Regulation of Megakaryocytopoiesis and TPO Production
Authors: Yang Mo , Meng Fanyi , Ye Jie Yu , Xu Yue , Xiao Bin , Chong Beng
Published: October 2019

Abstract 3347

We have reported that serotonin (5-HT) show a promoting effect on cord blood CD34+ stem/progenitor cells (Yang et al, Stem Cells 2007). We also demonstrated that serotonin enhances murine megakaryopoiesis via 5-HT2 receptors (Yang et al. Blood Coagul Fibrinol 1996). In this present study, we explored how serotonin regulated human megakaryocytopoiesis, proplatelet formation, and thrombopoietin (TPO) production. Our results indicated that serotonin significantly promoted human CFU-MK formation and reduced apoptosis in megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryocytopoiesis through stimulating release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO by q-PCR, ELISA and cytokine-array assays. Our findings demonstrated an important role of serotonin played on megakaryocytopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs, which indirectly promoted megakaryopoiesis. In present studies, we have demonstrated a positive “feed-back” control loop between MK-derived granule- serotonin and megakaryocytopoiesis. These findings improved our knowledge on megakaryocytopoiesis regulation and provided new clues on identifying novel thrombopoietic agents. It also deepened our understandings on how TPO production is regulated.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Thrombomodulin to Endothelial ICAM-1 Rather Than PECAM-1 Allows Partnering with the Endothelial Protein C Receptor and Enhances Protection of Endothelial Barrier Function
Authors: Greineder Colin F , Zaytsev Sergei , Zern Blaine , Chacko Ann Marie , Muzykantov Vladimir R
Published: October 2019

Abstract 3351

The thrombomodulin-protein C pathway has important antithrombotic and anti-inflammatory roles in a variety of disease models and human illnesses, including severe sepsis, acute lung injury, and focal ischemia & reperfusion. We recently reported that anchoring recombinant thrombomodulin (TM) to PECAM-1 on the luminal surface of the vascular endothelium is protective in a mouse model of inflammatory lung injury. To this point, it has been unclear whether targeted thrombomodulin is able to partner with the endothelial protein C receptor (EPCR) in the same way as its endogenous counterpart. Here we demonstrate that anchoring TM to endothelial ICAM-1, rather than PECAM-1, results in approximately 10-fold greater activation of protein C. Furthermore, blocking protein C binding to EPCR results in marked reduction of protein C activation when TM is targeted to ICAM-1, whereas protein C activation is largely independent of EPCR when PECAM-1 is used as the target ligand. Consistent with this in vitro observation, anti-ICAM/TM fusion protein provides greater in vivo protection in a mouse model of inflammatory lung injury than its PECAM-targeted analogue, more potently blocking expression of pro-inflammatory cytokines and more effectively stabilizing endothelial barrier function. Since ICAM, endogenous TM, and EPCR are all thought to localize to microdomains on the apical surface of endothelial cells, we hypothesize that ICAM-targeting more effectively mimics the natural configuration and brings TM within sufficient proximity to allow access to its membrane co-factor. These observations could have profound implications for the therapeutic efficacy of a whole series of endothelial-targeted proto-drugs and their potential for translational success.

Disclosures:

No relevant conflicts of interest to declare.
Structure-Activity Relationship of Pro- and Anticoagulant Effects of Fucus Vesiculosus Fucoidan
Authors: Dockal Michael , Till Susanne , Zhang Zhenqing , Knappe Sabine , Reutterer Sabrina , Quinn Catherine , Redl Christoph , Ehrlich Hartmut J. , Scheiflinger Friedrich , Szabo Christina
Published: October 2019

Abstract 3353

Fucoidans are sulfated polysaccharides extracted from brown algae. Fucoidans have a wide variety of biological activities including pro- and anticoagulant activities, which occur at different concentration ranges. Therefore, fucoidans have also been described as non-anticoagulant sulfated polysaccharides (NASPs, Liu et al. Thromb Haemost 2006; 95:68). Fucoidans have complex structures due to their large molecular weight (Mw), wide Mw distribution, variable degree and pattern of sulfation, diverse monosaccharide composition, branching of the sugar chain and different monomer linkages. This structural complexity challenges identification of the components responsible for fucoidan activities.

The aim of the presented study was to fractionate Fucus vesiculosus (F.v.) fucoidan by size and to de- and oversulfate it to obtain compounds of varying Mw or degree of sulfation (DS), respectively. The fucoidans were then to be analyzed for their pro-and anticoagulant activities and structure, and the effect of modified fucoidan on the target protein Tissue Factor Pathway Inhibitor (TFPI) investigated.

Two approaches were applied to generate F.v. fucoidan with different structural properties to the original. Firstly, fucoidan was fractionated by size using ultrafiltration. The Mw of the resulting fractions ranged from 8–180 kD. NMR, elemental analysis and HPAEC showed that other structural features, such as sulfate content and monosaccharide composition, were similar to those of the original fucoidan. Thrombin generation (CAT) assays showed an EC50 for procoagulant activity of 0.3–3 μg/mL; aPTT increased by 50% at 4–25 μg/mL. Generally, higher Mw increased procoagulant activity. Below 15 kD, this activity was markedly reduced. The minimum active length of F.v. fucoidan was 70 carbohydrate units.

De- and oversulfated F.v. fucoidans were used to investigate the impact of charge on pro- and anticoagulant activities. In the CAT assay, oversulfated fucoidans showed improved procoagulant activity with an EC50 of 0.09–0.12 μg/mL, while desulfated fucoidans demonstrated reduced procoagulant activity compared to the original. A DS of 0.5 was estimated to be the limit for procoagulant activity. Inhibition of TFPI by fucoidan was assessed with a dilute prothrombin time assay (dPT) with added full-length TFPI. TFPI-blocking activity was mainly dependent on the DS and less on the fucoidans' Mw. Interestingly, oversulfation also stimulated an undesired activation of the contact pathway.

The presented study determined the minimal structural requirements for procoagulant activity of fucoidan molecules and identified features causing undesired biological effects.

Disclosures:

No relevant conflicts of interest to declare.
Role of Von Willebrand Factor Triplet Bands in Glycoprotein Ib-Dependent Platelet Adhesion Under Flow Conditions
Authors: Schwartz Bruce A. , Kannicht Christoph , Fuchs Birte , Kröning Mario , Solecka Barbera
Published: October 2019

Abstract 3356

Objective:

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer respectively have one shorter or longer N-terminal peptide sequence. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Distribution of VWF triplet bands is significantly altered in some types of VWD, however, the impact of triplet structure on VWF function has not been investigated so far.

Methods:

Platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition obtained by size exclusion in addition to heparin affinity chromatography were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, while preparation B predominantly contained larger triplet bands. Collagen- and GPIb-binding was determined by surface plasmon resonance (SPR). Platelet adhesion under flow was determined using flow-chamber models.

Results:

Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb, but an unchanged affinity for collagen type III, when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored.

Conclusion:

Thus, VWF forms lacking larger size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.

Disclosures:

Schwartz: Octapharma: Employment. Kannicht:Octapharma: Employment. Fuchs:octapharma: Employment. Kröning:octapharma: Employment. Solecka:Octapharma: Employment.
Expression of Let-7 Micro RNAs in Transgenic Mice Overexpressing Truncated Hmga2
Authors: Ikeda Kazuhiko , Harada Kayo , Ogawa Kazuei , Mason Philip J , Bessler Monica , Takeishi Yasuchika
Published: October 2019

Abstract 3470

Expression of HMGA2 is negatively regulated by binding of let-7-family micro RNAs (miRNAs) to specific sites in 3' untranslated region (UTR) of HMGA2. Either downregulation of let-7 or truncation of HMGA2 is thought to cause overexpression of HMGA2. Overexpression of HMGA2 mRNA due to truncation of its 3'UTR by chromosomal translocation was reported in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH; Inoue et al, Blood, 2006). Accordingly, we recently established transgenic ΔHmga2 mice with 3'UTR-truncated Hmga2 cDNA, which revealed a clonal growth advantage of hematopoietic cells overexpressing Hmga2 mRNA that may lead to expansion of a PNH cell (Ikeda et al, Blood, 2011). It has also been reported that HMGA2 mRNA is overexpressed whereas let-7b and −7c are downregulated in peripheral blood cells from the majority of patients with PNH (Murakami et al, BJH, 2012). Although downregulation of let-7-family miRNAs may in part explain overexpression of HMGA2 mRNA, not only full-length HMGA2 mRNA but also short transcript variants of HMGA2 mRNA without 3'UTR are overexpressed in most of these patients. Thus, we aimed to investigate the influence of overexpression of 3'UTR-truncated Hmga2 mRNA, which mimics its short transcript variant, on expression of let-7-family miRNAs. We evaluated expression of let-7a, -7b, and -7c miRNAs in bone marrow (BM) and spleen cells of 12 week-old ΔHmga2 mice (n= 3–4) and wild-type (WT) mice (n= 3–4) using quantitative real-time RT-PCR. Relative expression levels of let-7a (mean ± SD, 1.14 ± 0.57 and 1.62 ± 1.05, respectively), -7b (0.76 ± 0.38 and 1.14 ± 0.77), and -7c (0.62 ± 0.47 and 0.96 ± 0.51) of BM cells were not significantly different between WT mice and ΔHmga2 mice. However, relative expression level of let-7c was significantly higher in spleen cells of ΔHmga2 mice (11.75 ± 2.10) compared with WT mice (1.47 ± 1.19; P < .01). Expression of let-7a (2.46 ± 2.72 and 20.20 ± 25.22) and −7b (1.43 ± 1.62 and 5.27 ± 4.05) was not significantly different in spleen between WT mice and ΔHmga2 mice. Relative expression levels of let-7c were higher in spleen cells compared with those in BM cells in ΔHmga2 mice (P < .01). Therefore, differences in cell lineage or differentiation status might alter expression of let-7-family miRNAs and 3'UTR-truncated Hmga2 may further influence let-7 expression in a tissue specific manner. However, the cause of downregulation of let-7b and -7c, and overexpression of 3'UTR truncated variants of HMGA2 in patients with PNH remains to be elucidated.

Disclosures:

No relevant conflicts of interest to declare.
C/EBPα-Induced Microrna-30c Directly Targets Notch1 During Granulopoiesis and Is Repressed in Acute Myeloid Leukemia
Authors: Katzerke Christiane , Madan Vikas , Gerloff Dennis , Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Wurm Alexander Arthur , Mueller-Tidow Carsten , Schnittger Susanne , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Abstract 3514

The transcription factor CCAAT Enhancer Binding Protein alpha (C/EBPα) is crucial for normal granulopoiesis and frequently disrupted in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported for about 10% of all AML. Loss of function of C/EBPα leads to a block of myeloid differentiation. MicroRNAs (miR) inhibiting translation of mRNA into protein were identified as critical players in granulocytic differentiation. We and others have already shown that C/EBPα exerts its effects by regulating miRNAs such as miR-223 and miR-34a. In a global microRNA-array we found miR-30c as a novel most important target of C/EBPα during granulopoiesis. Wild-type C/EBPα-p42 up regulates miR-30c expression, whereas the C/EBPα-p30 mutant, found in AML, does not. Furthermore, miR-30c expression is up regulated by G-CSF during granulocytic differentiation of primary human CD34-positive progenitor cells and down regulated in various subtypes of AML. Among these, miR-30c is significantly down regulated in samples from AML with normal karyotype and CEBPA mutations compared to AML patients with CEBPA wildtype. In mice, miR-30c shows a high expression in GMP and granulocytes. An induced tissue specific knock-out of C/EBPα in mice leads to a significant suppression of miR-30c expression in bone marrow cells. A luciferase reporter assay identifies NOTCH1 as a direct target of miR-30c as evident by its binding to the 3'UTR. Recent studies show that Notch1 blocks protein expression of C/EBPα-p42 by activation of Tribbles homolog 2 (Trib2). On the other hand, Proteins of Notch1 and Trib2 are down regulated by C/EBPα-p42. A block of miR-30c by locked nucleic acid (LNA) oligonucleotides prevents C/EBPα–induced downregulation of Notch1 protein expression, thus, miR-30c is necessary for C/EBPα to block NOTCH1. In this direction, the network leads to a block of granulopoiesis and further to leukemogenesis. Our study indicates that C/EBPα-induced miR-30c inactivates Notch1 during granulopoiesis and is down regulated in AML. This data stress the important role of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Disclosures:

Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership.
Microrna-143 Blocks ERK5 Signaling During Granulocytic Differentiation of Hematopoietic Stem Cells and Is Downregulated in AML
Authors: Hartmann Jens-Uwe , Braeuer-Hartmann Daniela , Schödel Cindy , Gerloff Dennis , Katzerke Christiane , Müller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: October 2019

Abstract 3516

Mitogen-activated protein kinase (MAPK) pathways are a family of related and sometimes interconnected pathways and one of the most studied. Over the last years, extensive work has established that these proteins play a critical role in G-CSF mediated maturation of neutrophil granulocytes. Understanding the mechanisms by which the MAPK pathways are regulated represents an important area of investigation. MicroRNAs, a class of small non-coding RNAs, have been found to play an important role in the regulation of diverse cellular processes by binding to target mRNAs leading to their translational repression. Deregulation of certain microRNAs, thereby, may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and development of leukemia remains largely unknown. In this study we performed a global screen to identify microRNAs involved in G-CSF-regulated MAPK-pathways in primary human CD34+ hematopoietic progenitor cells. Here we found microRNA-143 (miR-143) to be frequently upregulated in G-CSF stimulated CD34+ cells with a strong correlation to CD15 expression. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models and acute myeloid leukemia (AML) patient samples. Especially, AML patient samples FAB M4 and M5, which show monocytic phenotypes, had a significant lower expression level of miR-143 compared to the AML FAB types M0, M1, M2, and M3. In general, miR-143 expression was shown to be downregulated in AML patient samples in comparison to normal CD34+ hematopoietic progenitor cells. Most interestingly, we show that miR-143 expression is upregulated in acute promyelocytic leukemia (APL) patients after ATRA treatment. By in silico prediction we found MAPK protein family members (eg. MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. Finally, by transient overexpression of miR-143 we could show a strong downregulation of ERK protein expression in NB4 cells. Our study suggest that miR-143 upregulation by G-CSF may be an important regulatory step for permitting neutrophil differentiation. MicroRNA-143 blocks ERK5 signaling in G-CSF-induced granulopoiesis of CD34+ hematopoietic stem cells, is downregulated in myelo-monocytic acute myeloid leukemia subtypes, and upregulated after ATRA treatment in APL patients. This information may prove useful for the understanding of conditions in which neutrophil proliferation/differentiation balancing is dysregulated, such as in myeloid leukemia and myelodysplastic disorders.

Disclosures:

No relevant conflicts of interest to declare.
Possible Association Between Acute Myelogenous Leukemia and Thrombopoietin Receptor Agonist in Immune Thrombocytopenia Patients: A Preliminary Signal Report
Authors: Yuji Koichiro , Tanimoto Tetsuya , Tojo Arinobu , Hinomura Yasushi , Yasuo Oshima
Published: October 2019

Abstract 3541

Background

One of the concerns of potential adverse reactions of thrombopoietin receptor (MPL) agonists (romiplostim and eltrombopag) is development of myeloid malignancies. Progression from myelodysplastic syndrome to acute myelogenous leukemia (AML) has been reported in romiplostim. However, development of AML in immune thrombocytopenia (ITP) patients has not been reported. The objective of this study is to clarify whether the concern of AML in MPL agonists in patients with ITP is worth further investigation.

Method

We performed a disproportionality analysis on patients with ITP registered in an adverse event database, AERS between 2002 and 2011. We compared characteristics between AML case and others.

Results

Total of 4,821 subjects were identified as ITP and 62 AML cases among them in the AERS. The proportion of AML reporting in romiplostim and eltrombopag were 1.73% (95%CI; 1.30–2.26%) and 1.35% (0.62–2.54%), respectively. It should be noted that all AML was reported with one or more MPL agonist(s). Other factors associated with AML were male gender (OR 2.01; 95%CI 1.03–3.91) and age between 60 and 69 (OR 2.22; 95%CI 1.16–4.24).

Conclusion

We reported possible association between AML and MPL agonist use in ITP subjects. Due to limitations of spontaneous reporting, further studies are warranted to determine whether the detected signal is real risk or not. Prescribing physicians should not alter their prescribing behaviors based on only this preliminary analysis.

Disclosures:

Tojo: Novartis Pharma K.K.: Research Funding; Bristol-Myers Squibb Company: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Dainippon Sumitomo Pharma Co., Ltd.: Research Funding; Celgene K.K.: Consultancy. Yasuo:sanofi-aventis K.K.: Employment.
Azacitidine Plus Lenalidomide for Untreated AML Patients Ineligible for Conventional Chemotherapy
Authors: Pollyea Daniel A , Zehnder James L. , Coutre Steven , Gotlib Jason , Gallegos Leonel , Greenberg Peter , Zhang Bing , Liedtke Michaela , Levine Ross L. , Medeiros Bruno C.
Published: October 2019

Abstract 3575

There are limited treatment options for older patients with acute myeloid leukemia (AML) and prognosis has remained poor, thereby warranting development of novel therapeutic strategies. The optimal treatment for these patients requires the consideration of multiple factors, such as patient and disease-related factors that can help to determine the appropriate therapy. Previous studies have shown that both azacitidine and lenalidomide have single-agent activity in patients older than 60 years with untreated AML and have non-overlapping mechanisms of action that may complement each other. We evaluated the efficacy and safety of the combination of azacitidine plus lenalidomide as front-line therapy for untreated, older AML patients. In this phase 2 study, patients 60 years of age or older with untreated AML received azacitidine 75mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 day on 42-day cycles. Patients received continued therapy until disease progression, an unacceptable adverse event, or completion of 12 cycles. Forty-one AML patients (median age, 74 years) were enrolled with equal distribution according to ELN risk. Overall response rate (ORR) was 41% (CR/CRi rate was 29%). The median time to CR/CRi was 12 weeks, and duration of CR/CRi was 28 weeks (range, 4- >104 weeks). The median OS was 20 weeks (range, 1–121+) for the entire cohort and 69 weeks (range, 10–121+) for responders (Figure 1A; responders had better OS compared to non-responders (69 vs. 15 weeks, p-value <0.01). Baseline characteristics, such as age at diagnosis, baseline WBC count, bone marrow blast percentage or ELN risk classification, or molecular markers were not predictive of responses. Overall survival was similar according to ELN classification. Therapy-related AML and high hematopoietic cell transplantation comorbidity index were the only negative predictors of response. Early death was noted in 14% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosupression. Sequential combination of azacitidine plus lenalidomide has evidence of clinical activity as initial therapy for older AML patients, and further study of this combination in AML and MDS is warranted. This study is registered at www.clinicaltrials.gov as # NCT00890929.

Disclosures:

Gotlib: Celgene: Research Funding. Liedtke:Celgene: Research Funding. Medeiros:Celgene: Consultancy, Research Funding. Off Label Use: Treatment of AML.
Health Related Quality of Life of Bosutinib in Imatinib-Resistant or Imatinib-Intolerant Patients with Advanced Chronic Myeloid Leukemia
Authors: Whiteley Jennifer , Reisman Arlene , Kelly Virginia , Cortes Jorge E , Cella David
Published: October 2019

Abstract 4448

Purpose:

Bosutinib is a dual Src/Abl tyrosine kinase inhibitor (TKI), which has demonstrated efficacy in a phase I/II study of patients with Advanced Phase Chronic Myeloid Leukemia (CML). The objective was to evaluate the effect of bosutinib on health -related quality of life (HRQoL) in patients with advanced CML after failure with imatinib.

Methods:

Patient reported HRQoL was an exploratory objective in the clinical trial and measured using the 44-item Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). The FACT-Leu is a modular approach to assess patient HRQoL using a core set of general cancer questions as well as a cancer site specific leukemia subscale with 5 domains: Social Well-being (SWB), Emotional Well-being (EWB), Physical Well-being (PWB), Functional Well-being (FWB) and Leukemia Subscale (LeuS); and 3 summary scales: FACT-General, FACT Trial Outcome Index (TOI) and FACT-Leu Total. The item responses for each scale are summed to provide scores; higher scores indicate better HRQoL. The FACT-Leu was completed at weeks 4, 8, 12 and every 12 weeks thereafter, as well as treatment completion. Within cohort comparisons were assessed using paired t-tests.

Results:

Of the 164 patients with advanced leukemia included in the trial, 76 had accelerated phase (AP) CML and 64 blast phase (BP) CML. At 24 weeks, AP patients reported statistically significant improvements in PWB (p=0.02), EWB (p<0.001), LeuS (p<0.001), FACT-G (p<0.001), FACT-Leu (p<0.001) and FACT-TOI (p<0.001) with the PWB, FACT-G, LeuS, TOI and FACT-Leu exceeding minimally important differences (MID) at 24 weeks. Blast phase patients reported significant improvements in PWB (p=0.02), EWB (p=0.02), FWB (p=0.04), LeuS (p=0.01), FACT-G (p<0.001), FACT-Leu (p<0.001) and FACT-TOI (p=0.01) at 24-weeks with all scales exceeding MID except the SWB. At 48-weeks the AP patients continued to have statistically significant improvements in PWB (p=0.05), EWB (p=0.02), and FACT-G (p=0.03) and PWB, FACT-G, TOI and FACT-Leu exceeded the MID at 48 weeks. There were no statistically significant deteriorations in HRQoL through week 48 (Figure 1)

Conclusions:

These data suggest that CML patients treated with bosutinib demonstrate improved HRQoL. Confirmation in a controlled study is needed.

Disclosures:

Whiteley: Pfizer Inc: Employment, Equity Ownership. Reisman:Pfizer Inc: Employment, Equity Ownership. Kelly:Pfizer Inc: Employment, Equity Ownership. Cortes:Novartis, Bristol Myers Squibb, Pfizer, Ariad, Chemgenex: Consultancy, Research Funding. Cella:Pfizer Inc: Honoraria, Research Funding.