bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Comparison Of Hemostatic and Inflammatory Markers In Overt and Non-Overt DIC
Authors: Hoppensteadt Debra , Tsuruta Kazuhisa , Hirman Joe , Kaul Inder , Osawa Yutaka , Pharm M , Fareed Jawed 
Published: October 2019

Abstract

Disseminated intravascular coagulation (DIC) is a complex pathophysiologic syndrome with marked alterations in the hemostatic system, which may lead to microvascular thrombosis and multi-organ failure. The International Society of Thrombosis and Hemostasis (ISTH) classifies DIC into two distinct categories; overt and non-overt DIC (NO). Overt DIC is characterized by a decompensated hemostatic system whereas non-overt is characterized as a stressed, compensated system. DIC is a frequently observed in sepsis patients. An association between the hemostatic aberrations and inflammation has been previously reported. Therefore we studied the level of hemostatic activation and inflammation in patients with sepsis and suspected DIC enrolled in the ART-123, Phase 2b study. The level of inflammatory mediators such as anaphylatoxin C5a (C5a), procalcitonin (PCT), interleukin 6 (IL-6), interleukin 10 (IL-10), myeloperoxidase (MPO), and circulating levels of hemostatic markers including protein C inhibitor (PCI), plasminogen activator inhibitor 1 (PAI-1), and protein C (Pr C) were evaluated in 617 subjects enrolled in a randomized, double-blind, placebo-controlled, Phase-2b study evaluating the safety and efficacy of recombinant thrombomodulin (ART-123) in subjects with sepsis and suspected DIC. When the subjects with overt DIC and with NO DIC were compared, the PrC level was lower and PCT, PAI-1, IL-6 and IL-10 levels were higher, in the overt DIC subjects (Table). The upregulation of the inflammatory cytokines observed in this study may contribute to the decreased levels of Pr C. Inflammatory cytokines may dysregulate the endothelial thrombomodulin resulting impaired hemostatic regulation. Pr C levels are also low due to consumption and impaired synthesis, especially in the overt DIC patients. These results suggest that profiling these mediators may be useful in the diagnostic and prognostic management of sepsis and differentiation between overt and non-overt DIC.BiomarkerGroupNMeanSEMMedianP ValuePr C (%)Normal4094.52.491.80.0043Overt9824.71.828.30.0043NO15746.52.637.00.0043PCTNormal240.260.020.270.0001(ng/ml)Overt9836.87.011.220.0001NO51918.031.45.330.0001PAI-1Normal3120.62.118.7<0.0001(ng/ml)Overt98232.925.0137.5<0.0001NO519147.18.676.0<0.0001Il-6Normal306.80.536.10.0003(pg/ml)Overt981017.6121.7374.40.0003NO519576.739.6174.70.0003IL-10Normal3210.42.43.50.0049(pg/ml)Overt98187.339.146.00.0049NO518108.114.831.00.0049

Disclosures:
Hoppensteadt: Asahi Kasei Pharma: Research Funding. Tsuruta:Asahi Kasei Pharma: Employment. Hirman:Asahi Kasei Pharma: Consultancy. Kaul:Asahi Kasei Pharma: Employment. Osawa:Asahi Kasei Pharma: Employment. Fareed:Asahi Kasei Pharma: Research Funding.
Characterization Of a New Recombinant Human Factor VIIa (LR769)
Authors: Jerry Grandoni , Perret Gerald , Forier Cynthia 
Published: October 2019

Abstract
Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in the replacement therapies. In such cases factor replacement becomes ineffective, and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB/rEVO Biologics was tested. The goal is to provide patients with Hemophilia A and B who develop inhibitors a cost-effective alternative treatment option. Recombinant Human Factor VII was activated during the purification process to yield a highly homogenous rhFVIIa product (LR769). Kinetic enzyme assays and binding studies were used to characterize LR769. Active site titration demonstrated approximately 1 mole of active site per mole of protein. The Km and kcat for activation of FX and FIX were determined using an assay containing recombinant human tissue factor and phospholipid. The Kd for binding to soluble tissue factor was 22.3 ± 1.7 nM as measured using a FX activation assay. The apparent second order rate constant for inactivation by human plasma antithrombin was 5.9 ± 0.4 x103 M-1 sec-1. In all kinetic assays, LR769 behaved as expected for rhFVIIa. Binding studies with isolated human platelets were performed by FACS and indicated that binding was dependent on Ca+2. Activation of the platelets with thrombin and convulxin increased the binding of LR769. Binding of LR769 to Endothelial Protein C Receptor (EPCR) on the surface of cultured HEK 293 cells was determined by FACS analysis and confirmed that LR769 binds to EPCR. This was further demonstrated with a surface plasmon resonance assay using purified soluble EPCR. Binding to EPCR was dependent on Ca+2, and could be stimulated by Mg+2. Specificity of the binding was confirmed by the fact that it was inhibited by excess Protein C.

Disclosures:
No relevant conflicts of interest to declare.
Acquired Von Willebrand Factor Deficiency Caused By Left Ventricular Assist Devices Is Adamts-13 Dependent
Authors: Jilma-Stohlawetz Petra , Heinrich Schima , Stoiber Martin , Quehenberger Peter , Schranz Sabine , Jilma Bernd 
Published: October 2019

Abstract

Background
The high shear rates are induced by artificial heart valves or left ventricular assist devices cause acquired von Willebrand factor syndrome (aVWS). We hypothesised that an ex vivo model could be established to study whether mechanical shear alone causes aVWS or whether this process depends also on the VWF cleavage protein ADAMTS-13.

Methods
Healthy volunteers and two patients with congenital ADAMTS-13 deficiency donated blood. In vitro extracorporeal circuits were established using medically approved left ventricular assist devices (Heartware®) and silicone tubings with blood inlets and outlets. The pump speed and circulating blood volume were adjusted to correspond to in vivo use in humans. Anticoagulated blood circulated for 1-4 hours in the extracorporeal circuits. VWF multimers were quantified by sodium dodecyl sulphate-agarose discontinuous gel (1.2%) electrophoresis followed by Western-Blotting and consequent quantification with a luminescence image analyzer and a commercially available software, VWF antigen was measured by an STA assay and ristocetin co-factor activity (VWF:RCo) was quantified by turbidometry using a commercial kit (BC von Willebrand reagent; Dade Behring/Siemens, Marburg, Germany). Platelet function was measured by multiple electrode aggregometry in whole blood.

Results
The high shear stress in the extracorporeal circulation rapidly decreased VWF:RCo and thereby the VWF:RCo/VWF:Ag ratio by 47% (p<0.01) to pathologically low values. Concomitantly, high molecular weight multimers (HMWM) decreased, resembling the pathophysiological events that occur when an LVAD is implanted into humans. Up to 14-15 mers were visible on the gels at baseline, which were reduced by a maximum of 6-7 mers, corresponding to an average 68% lower densitometry signal of HMWM (p<0.01). This was accompanied by a 3-fold reduction in ristocetin induced aggregation (p<0.01). In contrast, the two patients with congenital thrombocytopenic purpura and virtually complete deficiency of ADAMTS-13 activity had only a minimal or no decrease in multimers (p<0.05 vs. healthy controls).

Conclusion
A model for LVAD associated aVWS was established, which demonstrated that ADAMTS-13 activity is essential for the depletion of HMWM of VWF that occurs in VAD-associated aVWS.

Disclosures:
No relevant conflicts of interest to declare.
RAP-011 Efficiently Rescues Erythropoiesis In Zebrafish Models Of Diamond Blackfan Anemia
Authors: Ear Jason , Huang Haigen , Tehrani Zahra , Sung Victoria , Daniel Thomas , Chopra Rajesh , Lin Shuo 
Published: October 2019

Abstract
Diamond Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count but normal levels of platelets and white blood cells. Ribosomal mutations in RPS19, RPS26, RPL5, and RPL11 have been identified in approximately 50% of all DBA cases. Corticosteriod therapy and bone marrow transplantion are the most common treatment options for DBA patients. However, corticosteroids have severe side effects and bone marrow transplantation is risky; thus, novel therapeutics for DBA are needed. Sotatercept (ACE-011), an activin receptor IIA ligand trap which rapidly increased hemoglobin and hematocrit in both pharmacologic models and in healthy volunteers, is currently being evaluated in diseases of ineffective erythropoiesis such as ß-thalassemia and MDS. Non-clinical studies in mice have demonstrated that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through stimulating expansion of late-stage erythroblasts through a mechanism distinct from EPO. Here, we evaluated the effect of RAP-011 in zebrafish models of ribosome insufficiency in RPS19 and RPL11 that recapitulate the anemic phenotype seen in DBA patients. Treatment with RAP-011 treatment dramatically restored hemoglobin levels compromised by ribosome stress. Furthermore, the beneficial effect of RAP-011 is synergistic with corticosteriod treatment. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by altering the microenvironment of erythroid cells, reducing p21 levels through a p53-independent manner. These findings uncover a novel signaling pathway in the pathogenesis of DBA and support the potential use of Sotatercept for the treatment of DBA patients with ribosomal disorders. Our studies also demonstrate, for the first time, that protein drugs can be effectively evaluated in zebrafish human disease models, which offer a unique opportunity to identify the targets and study their mechanisms of action.

Disclosures:
Sung: Celgene Corp.: Employment. Daniel:Celgene: Employment. Chopra:Celgene: Employment, Equity Ownership. Lin:Celgene: Research Funding.
The Epigenetic Regulators CBP and p300 Facilitate Leukemogenesis and Represent Therapeutic Targets In Acute Myeloid Leukemia (AML)
Authors: Giotopoulos George , Chan Wai-In , Ruau David , Gallipoli Paolo , Fowler Alexis , Göttgens Berthold , Van Deursen Jan , Cole Philip , Huntly Brian 
Published: October 2019

Abstract
Although molecularly and clinically heterogeneous, AML is characterised by aberrant transcription and abnormal epigenetic regulation. The transcriptional co-activators CBP and p300 have well characterised roles in hematopoiesis and hematopoietic stem cell (HSC) function. These are mutated in AML and also bind a number of AML-associated oncogenes. However, despite this, their roles in the induction and maintenance of AML are poorly understood. To address this question, we have combined genetic and pharmacological inhibition of CBP and p300 in AML and normal hematopoiesis.
Using a murine model where Cbp was conditionally deleted from murine hematopoietic stem and progenitor cells (HSPC), either prior to, or following expression of a number of AML-associated oncogenes, we assessed the role of Cbp in the induction and maintenance of AML. We demonstrated that although not an absolute requirement, Cbp confers a selective advantage for robust immortalisation in vitro, and that Cbp is also an important requirement for the generation and maintenance of AML in vivo. Furthermore, redundancy between Cbp and p300 in myeloid transformation was demonstrated, as p300 knockdown further decreased proliferation in Cbp-/- AML cells.
We next validated CBP/p300 as potential therapeutic targets, using a pharmacological strategy. Using a selective small molecule inhibitor (C646) of the lysine acteyltranferase (KAT) activity of CBP/p300, we demonstrated a significant decrease in growth and clonogenic potential across multiple AML subtypes in vitro. This was mediated through induction of apoptosis and cell cycle arrest. Importantly, no alteration in the growth of normal murine and human hematopoietic progenitors was detected at similar doses. We further demonstrated that inhibition of CBP/p300 KAT activity in human leukemia cells alters a transcriptional programme associated with genomic integrity, linking transcriptional changes to the cellular phenotype. Finally, we demonstrated the efficacy of the HAT inhibitors to decrease clonogenic growth across a panel of primary AML patient samples, representing multiple genetic subtypes. Taken together, these data suggest that CBP/p300 are involved in leukemogenesis across multiple subtypes in AML and that targeting these proteins may be possible with an acceptable side-effect profile.

Disclosures:
No relevant conflicts of interest to declare.
NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies
Authors: Nagel Stefan , Ehrentraut Stefan , Meyer Corinna , Kaufmann Maren , Drexler Hans G. , MacLeod Roderick AF 
Published: October 2019

Abstract
Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation.

Disclosures:
No relevant conflicts of interest to declare.
An Epigenetic Mechanism Of Imatinib Via Demethylation Of MiR-203
Authors: Umemura Tsukuru , Shibuta Tatsuki , Honda Emi , Shiotsu Hiromichi , Tanaka Yuka , Vellasamy Shalini , Shiratsuchi Motoaki 
Published: October 2019

Abstract

Background
MicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells.

Materials & Methods
Two CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA.

Results
The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed.

Discussion & Conclusion
We report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Role Of Tescalcin-Sodium/Hydrogen Exchange Axis Underlying Sorafenib Resistance In FLT3-ITD+ AML
Authors: Man Cheuk Him , Leung Anskar Y.H. , Yuen Lam Stephen Sze , Hei Sun Murphy Ka , Hung Chow Howard Chun , Gill Harinder , Kwong Yok Lam 
Published: October 2019

Abstract
Internal tandem duplication (ITD) of FLT3 (fms-like tyrosine kinase 3) in acute myeloid leukemia (AML) is associated with inferior clinical outcome. Sorafenib is effective in targeting chemo-refractory FLT3-ITD+ AML. However, leukemia progresses invariably. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), which activate a sodium/hydrogen exchanger 1 (NHE1) and known to be up-regulated at leukemia progression during continuous sorafenib treatment, may underlie TKI resistance. TESC was over-expressed in FLT3-ITD+ AML lines MOLM-13 and MV4-11. Knocking down TESC by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene) amiloride (HMA). Sorafenib resistant MOLM-13 cell line (M13-RE) was generated by long term culture of sorafenib. M13-RE significantly increased its sensitivity to HMA. HMA also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. Treating MOLM-13, MV4-11 and primary FLT3-ITD+ AML cells with HMA significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of the TESC-NHE1-pHi axis in mediating sorafenib resistance in AML.

Disclosures:
No relevant conflicts of interest to declare.
IDH1 Mutant Inhibitor Induces Cellular Differentiation and Offers a Combination Benefit With Ara-C In a Primary Human Idh1 Mutant AML Xenograft Model
Authors: Yen Katharine , Lemieux Rene , Popovici-Muller Janeta , Chen Yue , Yang Hua , Straley Kim , Choe Sung , Dorsch Marion , Agresta Samuel , Schenkein David P. , Biller Scott , Su Michael , Wang Fang 
Published: October 2019

Abstract
Somatic point mutations in isocitrate dehydrogenase 1/2 have a gain-of-function neomorphic activity that converts alpha-ketoglutarate to the oncometabolite, R (-)-2-hydroxyglutarate (2HG). Prospective studies of AML patients carrying IDH mutations have shown that intracellular concentrations of 2HG can range from 3-10 mM. This abnormal level of 2HG results in dysregulation of alpha-ketoglutarate dependent enzymes leading to alterations in the epigenetic state of hematopoietic progenitor/stem cells and functionally blocks their ability to fully differentiate.
We have developed a potent and selective, orally available IDH1 mutant inhibitor AGI-14100, that is able to reduce intracellular 2HG concentrations to baseline levels found in wildtype cells. Ex vivo treatment of IDH1 mutant-containing primary human AML patient samples with AGI-14100 induced a proliferative burst followed by cellular differentiation as shown by flow cytometry and cytology.
We next treated a primary human IDH1 (R132H)/FLT3-ITD mutant xenograft model with AGI-14100 either alone or in combination with Ara-c. In these studies, AGI-14100 alone significantly decreased tumor burden in the peripheral blood after 1 month of continuous BID treatment. In combination with a short-term, low-dose course of Ara-C, we also observed a decrease in the bone marrow tumor burden that was better than either treatment alone. Furthermore, this response was sustainable for >3 weeks even after dosing of both drugs had been terminated. Taken together, these data suggest that inhibition of mutant IDH1with AGI-14100 and low dose Ara-c could provide a combination benefit for patients with AML.

Disclosures:
Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Lemieux:agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:agios Pharmaceuticals: Employment, Equity Ownership. Chen:agios Pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:agios Pharmaceuticals: Employment, Equity Ownership. Dorsch:agios Pharmaceuticals: Employment, Equity Ownership. Agresta:agios Pharmaceuticals: Employment, Equity Ownership. Schenkein:agios Pharmaceuticals: Employment, Equity Ownership. Biller:agios Pharmaceuticals: Employment, Equity Ownership. Su:agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership.