bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of hTid1 as a Molecular Partner and a Negative Regulator of the Transcription Factor Stat5B
Authors: Nyga Remy , Dhennin-Duthille Isabelle , Yahiaoui Saliha , Munger Karl , Regnier Aline , Gouilleux-Gruart Valerie , Lassoued Kaiss , Gouilleux Fabrice 
Published: October 2019

Abstract
Stat5A and 5B transcription factors play a major role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia and solid tumors. However, their mechanisms of action remain poorly understood and their molecular partners still need to be identified. Since Stat5A and 5B mainly differ in their carboxy-terminal transactivation domains, we sought to identify molecules that specifically bind to this domain, using a bacteria-match two-hybrid assay to screen a human Jurkat T cells library. This approach enabled us to isolate hTid1, a DNA-J protein homologous to a drosophila tumor suppressor, that interacts with Stat5B but not Stat5A. Specific interaction between Stat5B and hTid1 was confirmed by immunoprecipitation using various mouse and human lymphoïd cell lines and Cos-7 cells transfected with plasmids encoding both molecules. Using various deleted mutants, we showed that the cysteine-repeats containing region (CRR) of hTid1 was required for this interaction. Moreover, we found that the complex formed by hTid1 and STAT5B was dissociated upon cytokines- (IL-3, IL-7) or Tel-Jak2-induced tyrosine phosphorylation of STAT5B. We also brought evidence for a down-regulation of STAT5B transcriptional expression and activity by hTid1 in human hematopoietic cells. Indeed, overexpression of hTid1 but not of its CRR deleted form in Ba/F3 cells, resulted in a sharp reduction of Stat5B expression, a process that probably involves a proteasome-dependent degradation of Stat5B. By contrast, treatment of Ba/F3 cells with siRNA of hTid1 induced an increase of Stat5B expression. Finally, Cos-7 cells cotransfected with the prolactin receptor and Stat5B expression vectors and with a Stat5 specific luciferase constructs enabled us to demonstrate that overexpression of hTid1 inhibits of Stat5B transcription activity in a dose dependent manner. In conclusion, our findings define hTid1 as a novel negative regulator of Stat5B.
A Rhesus Macaque Model to Optimize Adoptive NK Cell Therapy
Authors: Lopez Rebecca , Lundqvist Andreas , Sellers Stephanie , Berg Maria , Ramanathan Muthalagu , Vasu Sumithira , Smith Aleah , Dunbar Cynthia E. , Childs Richard W. 
Published: October 2019

Abstract
NK cell based immunotherapy represents a promising treatment approach for patients with cancer. Although preliminary clinical trials in humans suggest NK cell infusions can mediate anti-tumor effects, animal models are needed to provide insight into methods to enhance both the function and in vivo longevity of adoptively infused NK cells. Research conducted in our laboratory has shown that ex vivo expanded human NK cells are highly activated, up-regulating NKG2D, Granzyme B, TRAIL and Fas-ligand expression making them much more cytotoxic to tumor cells compared to freshly isolated NK cells. However, important questions remain regarding whether in vitro expansion alters the capacity of these cells to replicate, and traffic to tissues in vivo following their adoptive infusion into recipients. Differences in the genotype and phenotype of mouse NK cells compared to human NK cells limit the value of murine animal models to address these questions. In contrast to mice, Rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model system for evaluating questions related to adoptive NK cell therapy. We developed an in vitro method to expand macaque NK cells to characterize their in vivo longevity and tissue trafficking following adoptive infusion. Macaque NK cells were enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads and were then expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used to expand NK cells from humans. NK cell cultures expanded 50- to 100-fold over 7 to 20 days, were greater than 99% CD3 negative, and had a similar phenotype to human NK cells including a large proportion of CD16/CD56 double positive cells, and ubiquitous expression of NKG2D, KIR2D, LFA-1, granzyme B, and CXCR3. In contrast to mice but analogous to human NK cells, macaque expanded NK cells upregulated surface expression of TRAIL and were highly cytotoxic to K562 cells and other human tumor lines (Figure). CFSE labelling of expanded NK cells did not alter their phenotype or tumor cytotoxic function. Data characterizing the longevity, proliferative capacity, and tissue trafficking patterns in the blood, bone marrow and lymph node of in vitro expanded and adoptively infused CFSE labeled NK cells (up to 1 × 108 NK Cells/kg i.v.) in macaque recipients will be presented from this analysis.
Figure Figure
Oxidative Modification of Von Willebrand Factor Inhibits Its Cleavage by ADAMTS13
Authors: Chen Junmei , Fu Xiaoyun , Ling Minhua , McMullen Brad , Kulman John , Chung Dominic , Lopez Jose A 
Published: October 2019

Abstract
During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways:
by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, and by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product.
We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.
Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis
Authors: Booten Sheri , Knox Daniel , Alvarado Luis , Guo Shuling , Monia Brett P. 
Published: October 2019

Abstract
Abstract 1047
Hereditary hemochromatosis (HH) is a genetic disorder in which hyperabsorption of dietary iron leads to accumulation of iron in multiple tissues including liver and heart. A common clinical manifestation in HH patients is cirrhosis and hepatocellular carcinoma as a result of iron-mediated injury in liver. The most prevalent genetic defect for HH is the failure to up-regulate hepcidin, a peptide hormone that inhibits the absorption of iron in duodenum and the release of iron from intracellular iron storage such as macrophages. Mutations in a number of genes have been identified as the cause for HH, including hepcidin itself. However, the most common mutation is C282Y mutation in HFE, which is a positive regulator for hepcidin expression. C282Y mutation represents about 85% of the HH population. HFE C282Y HH is an autosomal recessive disease with a ∼50% penetrance. Currently, the only treatment available for iron overload is phlebotomy which will continue throughout the patient's life.
Hepcidin is mainly expressed and secreted by the liver and its expression is regulated predominantly at the transcription level. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor for hepcidin expression. It's been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of TMPRSS6 in Hfe−/− mice could ameliorate the iron overload phenotype (Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg KE et al., Blood, 2011).
Using second generation antisense technology, we identified antisense oligonucleotides (ASOs) targeting mouse TMPRSS6 for the treatment of HH. These compounds were first identified through in vitro screens in mouse primary hepatocytes. After 4 weeks of treatment in C57BL/6 mice on normal chow, we observed an 80% to 90% reduction of liver TMPRSS6 mRNA with a subsequent 2–3 fold induction of liver hepcidin mRNA. Serum iron and transferrin saturation levels were reduced by ∼50%. These ASOs are currently being evaluated in a diet-induced iron overload model and an Hfe−/− iron overload model. Our preliminary results demonstrate that targeting TMPRSS6 is a viable approach for the treatment of hereditary hemochromatosis and possibly other iron-loading diseases associated with suppressed hepcidin levels.

Disclosures:
Booten: Isis Pharmaceuticals: Employment. Knox:Isis Pharmaceuticals: Summer Intern. Alvarado:Isis Pharmaceuticals: Employment. Guo:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment.
BCL11A Polymorphism and HbF Response to Hydroxyurea in Adult Patients with Sickle Cell Disease
Authors: Sobash Philip , Kutlar Ferdane , Brown Harrison C. , Clair Betsy , Kutlar Abdullah 
Published: October 2019

Abstract
Abstract 1051
Several genetic polymorphisms are known to influence baseline hemoglobin F (HbF) levels in patients with sickle cell disease (SCD). These include XmnI polymorphisms in the Gγ globin promoter, polymorphisms of HBS1L-MYB and more recently BCL11A. Over the past several years, many studies have shown that polymorphisms in the BCL11A gene are strongly associated with baseline HbF levels in various populations with SCD. The strongest association has been found with a SNP (G→A) in the intron2 of the BCL11A gene (rs4671393). SCD patients on hydroxyurea (HU) therapy display a wide range of HbF responses. The factors underlying this heterogeneity are not clearly delineated. We undertook a study of the BCL11A rs4671393 polymorphism in a population of adult SCD patients who have been on HU therapy for an extended period. Fifty-nine African-American patients with SCD (25 male, mean age 33.9 and 34 female, mean age 34.5) were studied for BCL11A rs4671393 polymorphism and its correlation to HbF response (percent change in HbF from baseline to maximal response). The minor allele frequency (MAF) of A was 0.288. The distribution of HbF response (δHbF) was plotted against the genotype and is shown in the figure. As can be seen, patients with an A allele had a higher δHbF (12.1%) as opposed to those with the wild type (G/G), which was at 10.6%. Those homozygous for the minor allele (A/A) showed a 10.8% increase in HbF in response to HU therapy. These results are suggestive of a higher HbF response to HU when an A allele is present at this position. The number of homozygous for the minor allele are too small to reach a definite conclusion.

Disclosures:
No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Negatively Modulates Fatty Acid-Induced Inflammation in Macrophages Via PI3K/AKT Pathway
Authors: Aerbajinai Wulin , Chin Kyung , Zhu Jianqiong , Rodgers Griffin P 
Published: October 2019

Abstract
Abstract 1096
The macrophage-mediated inflammatory response plays a critical role in the development of obesity-related tissue inflammation and insulin resistance. Free-fatty acids (FFAs) are well-characterized factors causing production of inflammatory factors and insulin resistance. Glia maturation factor-gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in the macrophages immune response remains unclear. In this study, we investigated whether GMFG may affect FFAs-induced inflammatory reaction in macrophages. We show here that silencing of GMFG (80% inhibition of the endogenous expression levels) by transfected with GMFG siRNA significantly enhanced FFAs-induced production of proinflammatory cytokines and chemokines, including TNF-α, IL-6 and IL-8 compared to transfected non-targeting silencing siRNA in human peripheral blood monocytes-derived macrophage (1.2 × 104 ± 224.2 vs. 4.6 × 103 ± 98.4 molecules/ng, P < 0.001; 1.2 × 103 ± 56.1 vs. 2.3 × 102 ± 8.4 molecules/ng, P =0.001; 1.3 × 107 ± 5.8 × 105 vs. 4.9 × 106 ± 2.8 × 105 molecules/ng, P < 0.001) as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay (TNF-α: 9.4 × 102 ± 38.6 vs. 5.6 × 102 ± 31.1 ng/mL, P < 0.01; IL-6: 4.5 × 102 ± 31.3 vs. 2.2 × 102 ± 22.7 ng/mL, P < 0.01; IL-8: 2.2 × 103 ± 205.5 vs. 1.3 × 103 ± 113.9 ng/mL, P < 0.01). These increased inflammatory cytokines resulted from an increased activation of NF-κB and the ERK1/2 MAPK signaling pathway (based on increased NF-κB p65 phosphorylation, IκBα loss and enhanced phosphorylation of ERK1/2 in Western blot analysis) and a reduced phosphorylation of the PI3K/Akt/GSK3-β pathway following FFAs stimulation. Overexpression of GFP-GMFG in GMFG-silenced cells abrogated enhanced phosphorylation of NF-κB p65, ERK1/2 MAPK and reduced phosphorylation of Akt and GSK3-β. Furthermore, in cells in which GMFG was knockdown, FFAs induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway, suggesting increased SHIP1 expression may be responsible for the augmentation of inflammatory cytokines following FFAs stimulation. We also show that silencing of GMFG enhances the oxidized low density lipoprotein (oxLDL) induced expression of TNF-α and IL-6 (2.9 × 104 ± 204.8 vs. 1.3 × 104 ± 153.9 molecules/ng, P < 0.01; 2.5 × 103 ± 31.3 vs. 9.2 × 102 ± 22.7 ng/mL, P < 0.01) in MDM. Taking together with the data that GMFG is constitutively expressed in macrophages and its expression is down-regulated by FFAs stimulation, GMFG might function as a novel negative regulator through participating in the PI3K/Akt signaling pathway, suggesting that macrophage-specific modulation of GMFG may be beneficial in the treatment of FFAs induced inflammation.

Disclosures:
No relevant conflicts of interest to declare.
LDL and HDL Counteract Hematopoietic Stem/Progenitor Cells in Regulation of Inflammation and Atherosclerosis
Authors: Feng Yingmei 
Published: October 2019

Abstract
Abstract 1288
Low-density lipoprotein (LDL) is an independent risk factor in cardiovascular disease. Hypercholesterolemia-associated monocytosis and transformation of monocytes into macrophages are the main features underlying the proatherogenic property of LDL. Different from LDL, high-density lipoprotein (HDL) and its major apolipoprotein, apoA-I, counteract atherosclerosis by reversing cholesterol transport, inhibiting inflammatory cell adhesion to plaques, maintaining endothelial integrity, inhibiting oxidation, and as has recently been shown, suppressing hematopoietic stem cell (HSC) proliferation. As inflammatory cells in atherosclerotic plaques are exclusively derived from hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM), we hypothesized that differential effects of LDL and HDL on HSPC proliferation and differentiation may play a role in atherosclerotic plaque development. We explored the effect of HDL and LDL on HSPC number and function using LDL receptor knockout (LDLr ko) mice, fed with high fat diet, and C57BL/6 mice infused with purified apoA-I and or reconstituted (r)HDL. Compared to mice on normal diet, the number of lineage−/cKit+Sca-1+ (KLS) cells was 5-fold higher in LDLr ko mice on high-fat diet. By contrast, infusion of either 8 mg/kg apoA-I or 8, 12 and 16 mg/kg rHDL decreased the KLS cell frequency in BM. Using BrdU incorporation, we demonstrated that LDL induced but HDL inhibited KLS cell proliferation. When lineage negative cells, exposed to LDL or rHDL were used in competitive repopulation studies, HDL-treated cells supported greater chimerism than untreated controls, whereas LDL-treated cells competed less well. In addition, for HDL-treated Lin− cells, skewing of reconstitution to the B-lymphoid lineage at the expense of the granulocyte/monocyte lineage was seen. In vitro, LDL promoted KLS cell differentiation towards the monocytic lineage, which was abrogated by addition of HDL. In conclusion, LDL and HDL have opposing effects on HSPC number and function. These differential effects may contribute to the opposing effects of HDL and LDL on atherosclerosis development.

Disclosures:
No relevant conflicts of interest to declare.
Pivotal Role for Gsk3 in Hematopoietic Stem Cell Homeostasis
Authors: Huang Jian , Klein Peter S. 
Published: October 2019

Abstract
Abstract 1292
Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our recent study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a μ-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by μ-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. These findings identify unexpected functions for GSK3 in HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects of lithium, a widely prescribed GSK3 inhibitor. In the following study, we found that the combination of Gsk3 inhibitor and mTOR inhibitor can expand phenotypic HSCs in vivo and maintain functional HSC in ex vivo culture. This study will provide the basis for a new clinical approach to improve the efficiency of bone marrow transplantation.

Disclosures:
Klein: Follica: Consultancy.
The Cell Cycle Regulator CDC25A Is a Target for JAK2V617F Oncogene
Authors: Gautier Emilie-Fleur , Recher Christian , Laurent Camille , Marty Caroline , Villeval Jean-Luc , Demur Cécile , Delhommeau Francois , Hexner Elizabeth , Giraudier Stephane , Bonnevialle Nicolas , Ducommun Bernard , Laurent Guy , Manenti Stéphane , Mansat- De Mas Véronique 
Published: October 2019

Abstract
Abstract 1385
The JAK2V617F mutation is present in the majority of polycythemia vera (PV) patients and half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signalling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell cycle transition, is constitutively over-expressed in JAK2V617F -positive cell lines, JAK2-mutated patient CD36+ progenitors and in vitro differentiated proerythroblasts. Accordingly, CDC25A is also overexpressed in bone marrow and spleen of Jak2V617F knock-in mice compared to wild-type (WT) littermates. Using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was not caused by increased transcription or CDC25A stability or the involvement of its known upstream regulators AKT and MAPK. Instead, our results suggest that CDC25A is originaly regulated at the translational level through the implication of the transcription factor STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing erythroid progenitors, while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making of this protein an attracting potential therapeutic target.

Disclosures:
No relevant conflicts of interest to declare.
MicroRNA-146a Deficiency Leads to Increased Myeloid Cell Proliferation and Activation
Authors: Zhao Jimmy L. , Rao Dinesh S. , Boldin Mark , Taganov Konstantin , Baltimore David 
Published: October 2019

Abstract
Abstract 2815
NF-κB is a key regulator of inflammation and the immune activation against pathogens. On the other hand, constitutive and dysregulated NF-κB activation has been shown to lead to hematologic malignancies. Thus, NF-κB activity has to be properly regulated to achieve this delicate balance. Recent studies on microRNA-146a (miR-146a) suggest it to be an important negative regulator of NF-κB activation by targeting TRAF6 and IRAK1. To characterize the function of miR-146a in the context of NF-κB activation in the hematopoietic system, we have created a knockout (KO) mouse with germline deletion of miR-146a gene. We have shown that over time miR-146a KO mice develop a myeloproliferative condition in spleen and bone marrow that progresses to splenic myeloid sarcoma and bone marrow fibrosis. The myeloproliferative disease is driven by dysregulated NF-κB activation as the KO spleen and bone marrow cells show increased NF-κB activity and reduction in NF-κB level effectively rescues the myeloproliferative disease. In addition, the KO mice exhibit a chronic inflammatory state with increased cytokine production and macrophages from the KO are hyper-activated upon primary LPS stimulation and secondary re-stimulation. In this study, we have provided genetic evidence that miR-146a functions as a tumor suppressor in the hematopoietic system and highlighted the important role of miR-146a as a negative regulator of NF-κB activation and inflammation.

Disclosures:
Taganov: Regulus Therapeutics Inc: Employment.