bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamics of Genomic Organization.
Authors: Groudine Mark T. , Rajapakse Indika , Scalzo David , Perlman Michael , Kooperberg Charles L. , Kosak Steven 
Published: October 2019

Abstract
Abstract SCI-17
We have investigated the relationships between lineage-specific gene expression, and total genomic organization during hematopoiesis. First, we determined the linear chromosomal distribution of genes that are co-regulated (identified via microarray analysis) when murine hematopoietic progenitor cells (FDCP-mixA) are differentiated to the erythroid and neutrophil lineages, as well as the organization of all chromosomes (in the form of rosettes) in the three cell types. Our analysis revealed a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. This led us to hypothesize that the genome—at the level of chromosomes—may self-organize to facilitate coordinate gene regulation during cellular differentiation. We tested this hypothesis by applying the approaches of distance matrices and coupled oscillators to our datasets of gene expression and chromosomal associations from the differentiation of the progenitor to the erythroid and neutrophil lineages. Our analysis revealed that coordinate gene expression undergoes a phase transition—characterized by an increase in entropy—upon commitment of the progenitor. As differentiation continues, there is a gradual loss of entropy, culminating in a highly ordered state in the differentiated cell types. The coregulated gene sets of the semi-ordered progenitor and ordered erythroid and neutrophil lineages are significantly correlated with lineage-specific chromosomal association patterns. Furthermore, by transforming the gene expression networks along the time course to corresponding chromosomal association matrices, we found that chromosomal topologies change dynamically during differentiation but, as with gene expression, result in a more highly ordered state in the differentiated cell types. Our analysis demonstrates that the networks of co-regulated gene expression and chromosomal association are mutually related during differentiation, resulting in the self-organization of lineage-specific chromosomal topologies.

Disclosures
No relevant conflicts of interest to declare.
Enucleation.
Authors: Lodish Harvey F. , Ji Peng 
Published: October 2019

Abstract
Abstract SCI-18
Late erythroblasts undergo terminal cell cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division, but the signaling pathways and genes involved in these last steps of erythropoiesis are not known. Last year we showed that enucleation of mouse fetal erythroblasts requires both Rac GTPases and mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments. Rac and mDia2 are required for formation of the cortical actin ring that apparently powers the separation between the membrane- enveloped nucleus and forming reticulocyte. In collaboration with Drs. Tzutzuy Ramirez Hernandez and Maki Murata-Hori of the Temasek Life Sciences Laboratory in Singapore we showed by time-lapse microscopy that extrusion of the nucleus begins with microtubule- dependent “pushing” of the nucleus to one pole of the cell. Final budding off of the nucleus happens quickly – requiring only about 5 minutes. By analyzing fractionated erythroblasts at all stages of differentiation we confirmed that the nucleus undergoes gradual condensation before being extruded out of the cytoplasm. To investigate how chromatin condensation affects condensation and enucleation, we treated cultured erythroblasts with trichostatin A, a pan-histone deacetylase inhibitor, and discovered that enucleation is completely blocked. We further demonstrated that HDAC1, HDAC2 and HDAC3 are highly expressed in erythroid cells. Using a retroviral system to express shRNAs in developing erythroblasts, we found that downregulation of HDAC2 partially blocked enucleation. We also focused on the role of HDAC6 on enucleation since previous reports showed that HDAC6 forms a complex with mDia2. We confirmed the interaction between HDAC6 and mDia2 and demonstrated that HDAC6 can deacetylate mDia2. This may activate mDia2 and eventually promote enucleation. Our hypothesis is that HDACs play two distinct roles in enucleation: HDAC1, HDAC2 and HDAC3 (class I HDACs) are necessary for the condensation of the chromatin which is required for enucleation; on the other hand, HDAC6 (class II HDAC) interacts with and deacetylates mDia2 to promote enucleation.

Disclosures
Lodish: Amgen: Consultancy, Research Funding.
Factor VIII Biosynthesis and the Unfolded Protein Response.
Authors: Kaufman Randal J. 
Published: October 2019

Abstract
Abstract SCI-19
Factor VIII is the protein deficient in the × chromosome-linked bleeding disorder hemophilia A. Previous studies demonstrated that FVIII expression in mammalian cells is limited due to protein misfolding of the newly synthesized polypeptide in the lumen of the endoplasmic reticulum (ER). Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of FVIII to elucidate the relationship between protein misfolding and oxidative stress. Accumulation of misfolded FVIII in the lumen of the ER activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Disclosures
No relevant conflicts of interest to declare.
The NLR (NBD-LRR containing) Family of Intracellular Sensors in Inflammation and Immune Response.
Authors: Ting Jenny P-Y. 
Published: October 2019

Abstract
Abstract SCI-22
The NLR gene family encodes proteins that are comprised of conserved nucleotide-binding domain (NBD) and leucine rich-repeats (LRR). These genes are conserved from plants, lower animals to mammals. Family members include CIITA, NOD1, NOD2, and NLRP3. Several of these genes are genetically linked to immunodeficiency, inflammatory and immune diseases. This large gene family controls a number of innate responses, including inflammasome function leading to IL-1b/IL-18 production, cell death and type I IFN production. This talk will discuss the broad function of this family of genes, with emphasis on their regulation of inflammation and innate immunity, and their divergent roles in pathogen responses.

Disclosures
No relevant conflicts of interest to declare.
Hepcidin Regulation by the BMP Pathway.
Authors: Babitt Jodie L. 
Published: October 2019

Abstract
Abstract SCI-25
Systemic iron balance is regulated by the key iron regulatory hormone hepcidin. Secreted by the liver, hepcidin inhibits iron absorption from the diet and iron mobilization from body stores by decreasing cell surface expression of the iron export protein ferroportin. Iron administration increases hepcidin expression, thereby providing a feedback mechanism to limit further iron absorption, while anemia and hypoxia inhibit hepcidin expression, thereby increasing iron availability for erythropoiesis. Hepcidin excess is thought to have a role in the anemia of inflammation, while hepcidin deficiency is thought to be the common pathogenic mechanism underlying the iron overload disorder hereditary hemochromatosis, due to mutations in the genes encoding hepcidin itself (HAMP), HFE, transferrin receptor 2 (TFR2), or hemojuvelin (HFE2). Notably the precise molecular mechanisms by which iron levels are “sensed” and how this iron “signal” is transduced to modulate hepcidin expression have remained elusive. We have recently discovered that hemojuvelin is a co-receptor for the bone morphogenetic protein (BMP) signaling pathway, and that hemojuvelin-mediated BMP signals increase hepcidin expression at the transcriptional level. In addition to patients with HFE2 mutations and Hfe2 knockout mice, other genetic mouse models associated with impaired hepatic BMP signaling through a global knockout of the ligand Bmp6, or selective hepatic knockout of an intracellular mediator of BMP signaling, Smad4, also cause inappropriately low hepcidin expression and iron overload. Exogenous BMP6 administration in mice increases hepatic hepcidin expression and reduces serum iron, while BMP6 antagonists inhibit hepatic hepcidin expression, mobilize reticuloendothelial cell iron stores and increase serum iron. Not only does the BMP6-hemojuvelin-SMAD pathway regulate hepcidin expression and thereby systemic iron homeostasis, but also the BMP6-SMAD pathway itself is regulated by iron. Acute iron administration in mice increases phosphorylation of Smad proteins in the liver, and chronic changes in dietary iron modulate hepatic Bmp6 mRNA expression and phosphorylation of Smad proteins concordantly with Hamp mRNA expression. Together, these data support the paramount role of the BMP6-hemojuvelin-SMAD signaling pathway in the iron-mediated regulation of hepcidin expression and systemic iron homeostasis, and suggest that modulators of this pathway may be an alternative therapeutic strategy for treating disorders of iron homeostasis. Recent work elucidating the role of the BMP signaling pathway in hepcidin regulation and systemic iron homeostasis will be presented.

Disclosures
Babitt: Ferrumax Pharmaceuticals, Inc.: Equity Ownership.
Matriptase-2, A Novel Suppressor of Hepcidin.
Authors: Camaschella Clara , Nai Antonella , Pagani Alessia , Silvestri Laura 
Published: October 2019

Abstract
Abstract SCI-26
Hepcidin inhibition is a process essential to increase iron release from duodenal cells and macrophages to plasma in conditions of high iron requests such as iron deficiency, hypoxia and erythropoietic expansion. Among the reported potential inhibitors of hepcidin the serine protease matriptase-2 has a strong effect in vivo both in mice and in humans. Matriptase-2 is a member of the transmembrane serine protease (TTPS) family, encoded by TMPRSS6 gene, whose expression is tissue restricted, mainly to the liver and to a lesser extent to kidney and olfactory epithelium. The role of matriptase-2 in iron metabolism was first defined in the Mask mouse, which after birth develops a type of iron deficiency anemia refractory to oral iron and a peculiar pattern of hair loss, because of inappropriate overexpression of hepcidin and impaired iron absorption, a phenotype due to the deletion of the matriptase-2 serine protease domain. An identical phenotype is reported in Tmprss6 -/- mice and a correspondent phenotype is observed in iron-refractory iron-deficiency anemia (IRIDA) patients. We have shown that matriptase-2 cleaves through a proteolytic processing the bone morphogenetic protein (BMP) co-receptor hemojuvelin from plasma membrane in vitro, indicating that the suppression of the BMP signaling is essential for hepcidin inhibition. The finding also suggests that the BMP pathway requiring hemojuvelin as coreceptor is the main iron-dependent pathway of hepcidin regulation. The hepcidin inhibitory effect is observed also in zebrafish and is abolished in the human homologue of Mask. We have investigated several missense TMPRSS6 mutants affecting different domains of the protein, reported in IRIDA patients, in comparison with wild type matriptase-2 and Mask. Transient overexpression in hepatoma cells shows that all mutants are deficient in the ability to inhibit the hepcidin promoter in a classic luciferase-based assay and that the decreased hepcidin inactivation broadly corresponds to the inability to cleave hemojuvelin from plasma membrane. Studies of the in vitro processing of the mutants indicate that determinants of the pathogenic effect others than the protease activity are the intracellular processing and the ability of self-activation of matriptase-2. These results have implications for the molecular pathogenesis of IRIDA.

Disclosures
No relevant conflicts of interest to declare.
Development of Hepcidin Agonists and Antagonists.
Authors: Nemeth Elizabeta 
Published: October 2019

Abstract
Abstract SCI-27
Hepcidin binding to its receptor, the iron exporter ferroportin, leads to degradation of the ligand-receptor complex and decreased flow of iron into plasma from duodenum, macrophages and hepatocytes. Dysregulated production of hepcidin or its aberrant interaction with ferroportin causes a number of iron disorders. Hepcidin deficiency, either relative or absolute, underlies excessive iron loading in hereditary hemochromatosis, beta-thalassemia and hepatitis C. A rare type of hereditary hemochromatosis is also caused by ferroportin resistance to hepcidin. On the other side of the spectrum, hepcidin excess causes iron restriction and anemia, as is observed in patients with iron-refractory iron deficiency anemia. Hepcidin also contributes to development of anemia in different inflammatory disorders and chronic kidney disease, and may cause erythropoietin resistance. Hepcidin agonist and antagonists thus may be useful for treatment of different iron disorders. Antagonist strategies for lowering hepcidin levels have been reported, and include hepcidin-neutralizing monoclonal antibodies or agents interfering with the BMP-related hepcidin production (soluble hemojuvelin and dorsomorphin). To undertake the rational design of drug leads, we analyzed the critical structural features that determine the interaction of hepcidin and Fpn. We showed that the thiol form of Fpn residue Cys326 is essential for hepcidin binding, as is the presence of a disulfide bond on hepcidin. The mechanism of binding appears to involve disulfide exchange between the two molecules. Treatment of ferroportin-expressing cells with thiol-reactive agents prevented hepcidin binding and allowed continuous export of iron from cells. Systematic mutagenesis of additional residues on hepcidin showed that His3, Phe4 and Phe9 are also important for binding to ferroportin. To provide leads for hepcidin agonists, we then synthesized a mini-hepcidin containing only the 9 N-terminal hepcidin residues (DTHFPICIF), and showed that it displayed significant activity in vitro. Proteolysis-resistant analogs of this peptide were also active in vivo after intraperitoneal injection in mice. The small size of these peptides may allow oral bioavailability. Understanding the mechanisms of hepcidin regulation and its interaction with ferroportin will facilitate the development of new agents for the treatment of iron overload disorders and iron-restricted anemias.

Disclosures
Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Patient-Specific Pluripotent Stem Cells for Hematologic Disease.
Authors: Daley George Q. 
Published: October 2019

Abstract
Abstract SCI-40
Pluripotent stem cells can be isolated from embryos (embryonic stem cells; ES cells) or generated by direct reprogramming of somatic cells (induced pluripotent stem cells; iPS cells). Both types can be differentiated into a multitude of cell lineages to serve disease research and cell replacement therapies. Additionally, genetically matched pluripotent stem cells generated via nuclear transfer (ntES cells), parthenogenesis (pES cells), or direct reprogramming (iPS cells) are a possible source of histocompatible cells and tissues for transplantation. We have used customized ntES cells to repair genetic immunodeficiency in mice (Rideout et al., Cell 2002); however, generation of ES cells by nuclear transfer remains inefficient, and to date has not been achieved with human cells. We have also generated ES cells with defined histocompatibility loci by direct parthenogenetic activation of the unfertilized oocyte (Kim et al., Science 2007). Compared to ES cell lines from fertilized embryos, pES cells display comparable in vitro hematopoietic activity, but appear compromised in repopulating hematopoiesis in irradiated adult mouse recipients. We are currently comparing the performance of ntES, pES, and iPS cells in murine models of thalassemia. We have generated human iPS cells by direct reprogramming of human somatic cells with OCT4, SOX2, MYC, and KLF4 (Park et al., Nature 2008), and have generated disease-specific iPS cells from patients with a number of hematologic conditions (Park et al., Cell 2008; Agarwal et al., submitted). Applications of disease-specific cells for investigating the mechanisms of reprogramming and for probing aspects of human bone marrow disorders will be discussed.

Disclosures
Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.
Genetic Modifiers of Thrombosis in Mice.
Authors: Ginsburg David 
Published: October 2019

Abstract
Abstract SCI-44
The genetic factors responsible for the highly variable clinical course of inherited bleeding disorders including von Willebrand disease and hemophilia are largely unknown. Similar factors are also likely to contribute to the variability of common thrombotic disorders, including factor V Leiden. Studies by our lab over the past 10 years have used the power of mouse genetics to identify genes contributing to this variability (referred to as ‘modifier‘ genes). By performing genetic crosses between inbred strains of mice with elevated plasma levels of von Willebrand Factor (VWF) and other strains with low levels, we have mapped a total of 6 genetic factors contributing to the control of murine plasma VWF levels. Similar studies in ADAMTS13-deficient mice are in progress aimed at characterizing genes modifying susceptibility thrombotic thrombocytopenic purpura. We have also conducted large scale mutagenesis studies in the mouse in an effort to identify larger numbers of genes contributing to thrombosis risk in the setting of Factor V Leiden, and most recently are extending this approach to similar genetic screens in zebrafish. Finally, recent advances in human genetics are expanding the potential opportunities for directly identifying bleeding and thrombosis modifier genes in humans.

Disclosures
No relevant conflicts of interest to declare.
The Lymph Node Niche.
Authors: Mempel Thorsten R. 
Published: October 2019

Abstract
Abstract SCI-51
Lymph nodes provide specialized stromal environments that support the maintenance and homeostasis of T and B lymphocyte populations and are also staging grounds for lymphocyte effector responses against pathogens and transformed cells. They serve as immune information hotspots by collecting lymph fluid from peripheral tissues, especially our external and internal epithelial body surfaces, thus displaying a condensed representation of foreign and self-antigens at these sites in addition to integrating innate alarm signals that report tissue damage or pathogen invasion. Naïve B and T cells constantly traffic through these environments via the blood stream and efferent lymphatic vessels, which allows for efficient matching of their antigen receptor repertoires with the regional antigenic landscape. Depending on the absence or presence of signs of a potential threat to the organism, the result may be either tolerance or immunity towards the origin of these antigens. The architecture of lymph nodes is optimized to facilitate the presentation of lymph-borne antigen in various forms and to guide naïve lymphocytes in their search for 'their' cognate antigen in the form in which they are able to 'see' it. It also facilitates the cellular crosstalk with other immune cell populations that shape and regulate an ensuing adaptive response if cognate antigen is encountered in an immunogenic context. Our conception of how these various tasks are accomplished has recently been enriched through new methodological approaches that include the dynamic in situ or in vivo visualization of cellular and molecular processes using modern microscopy technology. We will review some recent insights into the function of lymph nodes derived from these studies.

Disclosures
No relevant conflicts of interest to declare.
A Key Role for Tumor Suppressor BTG1 In the Regulation of Metabolic Stress Responses: Implications for Asparaginase Resistance In Pediatric Acute Lymphoblastic Leukemia.
Authors: van der Meer Laurens , Levers Marloes , van Emst Liesbeth , van Galen Joost C. , van Kessel Ad Geurts , Kuiper Roland P. , Hoogerbrugge Peter , van Leeuwen Frank N. 
Published: October 2019

Abstract
Abstract 1028

Background:
L-Asparaginase (ASNase) is a key component of multi-agent chemotherapy regimens used in the treatment of pediatric Acute Lymphoblastic Leukemia (ALL). This bacterially-derived enzyme lowers blood asparagine levels by catalyzing the hydrolysis of L-asparagine, leading to specific killing of leukemic blasts. However, relapses and associated therapy resistance occur in about 20% of the patients. Although the molecular mechanisms that contribute to ASNase resistance are not well understood, increased expression of asparagine synthethase (ASNS), the enzyme responsible for asparagine synthesis, has been linked to ASNase resistance both in ALL cell lines and in primary leukemias. We recently reported that micro-deletions affecting the B-cell translocation gene 1 (BTG1) occur in about 10% of pediatric pre-B ALL cases. Here we show that BTG1 loss contributes to ASNase resistance by up-regulation of ASNS.

Results:
Using RNA interference, we show that loss of BTG1 expression promotes cell growth and renders pre-B ALL cells completely refractory to ASNase induced cell death. Resistance to ASNase in the BTG1 knockdown cells is accompanied by increased expression of ASNS, while knockdown of this metabolic enzyme is sufficient to reverse therapy resistance, indicating that upregulation of ASNS is required for the observed resistance phenotype. We further show that BTG1 associates with and regulates the activity of the transcription factor ATF4, a key regulator of metabolic stress responses and a central component in the regulatory network that controls ASNS expression.

Conclusion:
Together, our experiments identify BTG1 as an important regulator of ASNS expression which acts by modulating ATF4 function. We expect that a detailed molecular understanding of how BTG1 loss contributes to ASNase resistance, will lead to the identification of pharmacological targets that can be used to improve treatment responses in therapy-resistant ALL.

Disclosures:
No relevant conflicts of interest to declare.