bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation of Erythropoiesis.
Authors: Weiss Mitchell J. 
Published: October 2019

Abstract
Abstract SCI-7
Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas.

Disclosures
No relevant conflicts of interest to declare.
The Reduced and Altered Activities of PAX5 Are Linked to the Protein-Protein Interaction Motif (coiled-coil domain) of the PAX5-PML Fusion Protein In t(9;15)-Associated Acute Lymphocytic Leukemia.
Authors: Qiu Jihui , Chu Huimin , Dong Shuo 
Published: October 2019

Abstract
Abstract 1022
The paired box domain of PAX5 was reported to fuse with the sequence of promylocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia (B-ALL). In the present studies, we found, by gel-shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction (RQ-PCR) analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil protein interaction domain located within PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared to wild-type PAX5. Furthermore, the dimerization domain (coiled-coil) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the coiled-coil domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins.

Disclosures:
No relevant conflicts of interest to declare.
Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.
Authors: Lehotzky Erica A. , Chiang Mark Y. 
Published: October 2019

Abstract
Abstract 1026
Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL.

Disclosures:
No relevant conflicts of interest to declare.
SOCS1 Cooperates with FLT3-ITD In the Development of Myeloproliferative Disease by Promoting the Escape From External Cytokine Control.
Authors: Brandts Christian H. , Reddy Pavankumar , Sargin Bülent , Choudhary Chunaram , Muller-Tidow Carsten , Berdel Wolfgang E. , Serve Hubert 
Published: October 2019

Abstract
Abstract 1054
Activating mutations of FLT3 such as FLT3-ITD are frequently found in AML patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control, which regulates normal hematopoiesis. We have recently demonstrated that FLT3-ITD, when localized to the endoplasmic reticulum, aberrantly activates STAT5. Here we show that one of the target genes induced by STAT5 is SOCS1, a potent negative regulator of cytokine signaling and known tumor suppressor gene. Importantly, a significantly increased SOCS1 expression was found in FLT3-ITD+ AML. While SOCS1 expression in murine bone marrow severely impaired cytokineinduced colony growth, it failed to inhibit FLT3-ITD supported colony growth, indicating resistance of FLT3-ITD to SOCS1. Furthermore, SOCS1 co-expression inhibited interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-γ mediated growth inhibitory effects. In a murine bone marrow transplantation model, the co-expression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared to FLT3-ITD alone (p<0.01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting malignant transformation. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Disclosures:
No relevant conflicts of interest to declare.
Effectiveness of a Short Course of Complex Lymphedema Therapy or Graduated Compression Stocking Therapy In the Treatment of Post-Thrombotic Syndrome (PTS): A Randomized Control Trial.
Authors: Holmes Chris E. , Bambace Nadia M. , Lewis Patricia , Callas Peter , Cushman Mary 
Published: October 2019

Abstract
Abstract 1087

Background:
Investigations of treatment options for established PTS are limited. Graduated compression stocking therapy (GCS) is often used in clinical practice despite limited research on effectiveness. We postulated that the venous hypertension seen in PTS leads to lymphatic overload and secondary lymphedema. Therefore, in patients with established PTS, we compared the efficacy of GCS alone to complex lymphedema therapy, a treatment that includes compression stocking use, exercise, patient education, skin care and manual lymphatic drainage.

Methods:
In an investigator-blind randomized controlled trial of 31 patients with a clinical diagnosis of PTS and prior history of DVT, we compared GCS alone using 30–40 mm Hg compression to complex lymphedema therapy. Primary outcomes were the 1- and 3-month changes in PTS severity by the Villalta score (higher score indicates more severe PTS) and disease-specific quality of life by the VEINES QOL score (high score indicates better QOL). Analysis was by intent-to-treat.

Results:
Overall, 39% of patients enrolled in the study had moderate or severe PTS: baseline mean PTS severity score was 9.9 (SD 7.1) in the lymphedema group and 10.9 (SD 5.3) in the GCS group (p=0.66). The complex lymphedema therapy group had a reduction in mean PTS severity score of -2.4 (p=0.02) at 1 month and -2.3 (p=0.05) at 3 months follow-up. The GCS group had a reduction in PTS severity score of -2.1 (p=0.03) at 1 month and -3.3 (p=0.03) at 3 months. The improvement in PTS severity score did not differ significantly between treatment groups (p=0.61 at 3 months). Neither group had a significant change in VEINES QOL score. Patients not routinely using GCS prior to trial entry derived a greater benefit from either intervention with a decrease in PTS severity score (-8.8 in nonusers vs -1.5 in those using GCS at baseline, p=0.07) and an increase in VEINES QOL score (+1.9 vs -2.0, p=0.02). At 3 months, average self-reported GCS use was 6.7 days/wk (lymphedema therapy) and 5.9 days/wk (GCS) with 78% of patients in the lymphedema therapy group receiving manual lymphatic drainage (home or therapist). Four patients experienced recurrent thrombosis on the trial (3 in GCS group; 1 in lymphedema therapy group); 1 stocking allergy and ulcer formation was also seen in the GCS group with no cardiopulmonary complications reported in either group.

Conclusion:
In this first clinical trial of complex lymphedema therapy for patients with established PTS, a short course of lymphedema therapy was safe and had a similar effect on PTS severity as compression stocking use.

Disclosures:
No relevant conflicts of interest to declare.
Increased Platelet Binding of NN1731, a Factor VIIa Variant with Enhanced Tissue Factor-Independent Activity.
Authors: Hoffman Maureane R. , Persson Egon , Ezban Mirella , Monroe Dougald M. 
Published: October 2019

Abstract
Abstract 1133

Introduction:
Platelet surface activity plays an important role in the efficacy of FVIIa as a bypassing agent in hemophilia. An analog of FVIIa with increased tissue factor (TF) independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain: V158D, E296V, and M298Q. Modeling suggests that Gln in position 298 and Asp in position 158 form a hydrogen bond network with a water molecule that stabilizes the proteolytically active conformation of the molecule. The Val introduced at position 296 avoids electrostatic repulsion between a native Glu residue and the introduced Asp at 158. These changes result in a FVIIa variant that partially mimics the conformation of TF-bound FVIIa. However, in the presence of TF the activity of the wild type FVIIa and NN1731 are indistinguishable. Previous studies suggested that the increased intrinsic activity of NN1731 was not directly reflected in the level of activity on the platelet surface. The goal of the current work was to compare the platelet binding and platelet surface activity of NN1731 and wild type FVIIa.

Methods:
Mutant FVIIa molecules were prepared as described previously (PNAS 98:13583-8, 2001). We assessed binding to purified human platelets by flow cytometry and activity by measuring FXa generation.

Results:
FVIIa and NN1731 had identical binding to phospholipid vesicles designed to have lipid composition similar to platelets. However, the two exhibited different binding to platelets. At all concentrations, more NN1731 bound to thrombin or thrombin-plus-convulxin activated platelets. This was primarily because NN1731 bound to a greater number of sites per platelet. It appears that NN1731 and FVIIa bind similarly to a higher affinity site, but NN1731 binds to an additional low-affinity site. Removal of the Gla domain abolished binding. Thus, the protease domain of NN1731 was insufficient to mediate binding to the low affinity site. Two additional FVIIa mutants were tested: the single M298Q (“Q”) variant, and the double E296V and M298Q (“VQ”) variant. These variants bound to platelets identically to FVIIa.

Conclusion:
While the Gla domain is essential for FVIIa binding to platelets, the three amino acid changes in the protease domain in NN1731 enhance platelet binding as well as proteolytic activity. Since NN1731 and FVIIa bind similarly to phospholipid vesicles, characteristics in addition to lipid composition likely contribute to platelet binding. The platelet surface components that contribute to NN1731 binding remain to be defined.

Disclosures:
Hoffman: Novo Nordisk: Research Funding. Persson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment. Monroe:Novo Nordisk: Research Funding.
Characterizing Membrane-Bound Forms of Coagulation Factors at Atomic Resolution: Exploring Differences In Membrane Affinity.
Authors: Arcario Mark J , Tajkhorshid Emad 
Published: October 2019

Abstract
Abstract 1139

Membrane binding constitutes a key step in the activation of coagulation factors. This process largely hinges on specialized “floating” domains, most prominently the GLA and the C1/C2 domains found in almost all coagulation proteins. Currently, the details of how these domains interact with the membrane are largely unknown, due primarily to the lack of atomic models describing the proteins' membrane-bound forms. In sharp contrast to integral membrane proteins, studying insertion of membrane floating proteins has proven extremely challenging, mainly due to the unknown depth of membrane penetration. Although several computational studies have attempted to study the process using conventional models of the membrane, these methods are often prohibitively costly and inefficient due, in part, to the slow diffusion of the lipid molecules, which is required to accommodate the insertion of the floating domain. Here, we present a study on the membrane-bound forms of several important hemostasis factors, namely, human factor IX (hfIX), human factor × (hfX), human protein C (hPrC), human protein S (hPrS), and human protein Z (hPrZ), which utilize the GLA domain for membrane binding, as well as human factor V (hfV) and human factor VIII (hfVIII) in which the C1/C2 domain plays this role. To make the binding event accessible to molecular dynamics (MD) simulations, we have developed a novel membrane mimetic system, composed of a highly mobile hydrophobic core and explicit head groups that allow one to study specific interactions between the floating domains and lipid molecules. These interactions determine not only the specificity of binding to certain biological membranes, but also the molecular basis of the variable binding affinity of coagulation factors. These two factors are of utmost importance in physiology of blood coagulation, in designing mutations of altered affinity, and in developing novel pharmacological agents for thrombotic disorders. The floating domains were placed in the membrane-mimetic system and simulated under physiological conditions. Given the efficiency of the method, we have been able to capture spontaneous binding of these coagulation factors to the membrane (Figure 1) multiple times, despite starting from various initial orientations ensuring that each coagulation factor converged to the same final, membrane-bound structure over multiple trials. From these simulations, we have been able to elucidate the residues important for association with the membrane, as well as several unique phosphatidylserine (PS) specific binding sites in hfIX, hfX, hPrC, hPrS, and hPrZ. Using the membrane-bound structures of these coagulation factors together with free energy calculations, we have been able to elucidate important differences between the binding of high-affinity and low-affinity coagulation factors to the membrane.

Disclosures:
No relevant conflicts of interest to declare.
Expansion of Liver-Infiltrating CTL Clone In Concert with the Development of Hepatitis-Associated AA.
Authors: Ikawa Yasuhiro , Nishimura Ryosei , Mase Shintaro , Kuroda Rie , Araki Raita , Maeba Hideaki , Shimizu Masaki , Wada Taizo , Toma Tomoko , Koizumi Shoichi , Yachie Akihiro 
Published: October 2019

Abstract
Abstract 1159

Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which an episode of hepatitis precedes AA, namely hepatitis/aplastic anemia syndrome (HAAS). In AA, cytotoxic T-lymphocytes (CTL) play a central role in bone marrow (BM) destruction, and oligoclonal expansion of CTL correlates with disease activity. Immune mediated mechanisms have also been postulated in the pathogenesis of fulminant hepatitis (FH). Therefore we hypothesized that common CTLs recognizing similar target antigens against both liver and BM cells exist during the early period of FH, and subsequently selective proliferation of clones that are highly tropic to BM cause HAA at later stages. To this end, T cell phenotypes infiltrating liver, BM and peripheral blood (PB) samples obtained from 1-year-old boy with HAAS over time were determined by complementarity-determing region 3 (CDR3) size distribution patterns by polymerase chain reaction. Subsequently, CDR3 nucleotide sequences of predominant products were determined. Extremely skewed pattern was observed in Vb23 in BM and PB specimens at the onset of HAA (3 months after FH). A Vb23 clone with identical CDR3 nucleotide sequence predominated (clone 1) in BM and PB (34% and 60% within Vb23, respectively). This clone constituted a minor, but definitive clone in liver at the onset of FH (16% within Vb23). In contrast, initially predominant clones in liver (clone 3; 47% within Vb23) contracted to a minor subpopulation in BM and PB when the patient developed HAA (17% and 10% within Vb23, respectively).
This patient had a very good response to immunosuppressive therapy (IST) using antithymocyte globulin, cyclosporine, and granulocyte-colony stimulating factor. Two months after starting IST, the pattern of CDR3 size distribution was just recovering to normal and initially predominant clones in both liver and BM almost disappeared in Vb23 in PB. However, extremely skewed pattern of CDR3 region was still observed and abnormal clone 1 had still occupied 71% within Vb23 in BM. As of 8 months after starting IST, CDR3 size distribution recovered to normal patterns (Gaussian distribution) and CTL clones against both liver and BM almost disappeared in both BM and PB. These suggest that it would take a relatively long time to recover immunological parameters completely even in IST good responders.
In conclusion, our data is the first report to suggest that oligoclonal CTL clones simultaneously directed against liver and BM are involved in the pathogenesis of HAAS.

Disclosures:
No relevant conflicts of interest to declare.
Binding of FVIII to Recombinant VWF
Authors: Schrenk Gerald , Varadi Katalin , Rottensteiner Hanspeter , Ehrlich Hartmut J. , Scheiflinger Friedrich , Turecek Peter 
Published: October 2019

Abstract
Abstract 1408
Von Willebrand factor (VWF) forms a non-covalent complex with FVIII in the circulation, which not only stabilizes the heterodimeric structure of FVIII but also protects this coagulation protein from early proteolytic degradation and cellular uptake. By convention, the physiological ratio in plasma between VWF and FVIII is 1 IU to 1 IU, corresponding to a stoichiometry of approximately 1 FVIII molecule bound per 50 VWF monomers. However, since each VWF monomer contains one FVIII binding site, this ratio does not seem to reflect the genuine binding capacity of VWF for FVIII.
We therefore investigated the interaction between FVIII and a recombinant VWF (rVWF) drug candidate under different conditions. The FVIII binding capacity of VWF (VWF:FVIIIB) was measured by an ELISA system, where the VWF-bound FVIII was quantified by a FVIII chromogenic assay. Under this condition, VWF:FVIIIB of rVWF was similar to that of VWF derived from human normal plasma. The binding affinity of VWF for FVIII was determined using a Biacore system in which VWF is immobilized onto the sensor chip surface. The FVIII binding affinities were similar for rVWF KD=0.50) and pdVWF (KD=0.49 nM) and agreed with data from the literature.
Plasma-derived VWF was reported to show increased FVIII binding when in solution (instead of being immobilized to a surface). To find out whether this also holds true for rVWF, we developed a new assay system based on Biacore technology which measures the binding of FVIII to VWF in solution without any surface binding step of the VWF-FVIII complex. A mixture of rVWF and rFVIII is thereby injected into the flow cell of a Biacore sensor chip with immobilized rVWF. In this indirect assay system, the rFVIII that forms a complex with rVWF in solution is not available for binding to the immobilized rVWF. Under these conditions, rVWF was endowed with a 30-times higher FVIII binding capacity than could be anticipated from the physiological ratio and similar results were obtained for pdVWF.
The combined results showed that the ability to serve as the physiological chaperone protein for FVIII is fully preserved in rVWF, which can therefore be expected to stabilize FVIII in the circulation in a similar manner to VWF in plasma. Results from an ongoing phase 1 study show the good FVIII stabilizing properties of rVWF also when administered in humans with type 3 VWD.

Disclosures:
Schrenk: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment.