bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Cdc42 Interacting Protein 4, a Member of the Membrane-Associated BAR Family, Decreases Platelet Microparticle Formation – a Possible Role for Dynamin
Authors: Awomolo Arinola , Chen Yolande , Aardema Jorie , Corey Seth J. 
Published: October 2019

Abstract
Abstract 1153
Our lab isolated Cdc42 interacting protein 4 (CIP4) in a yeast two-hybrid screen using the Src kinase Lyn as bait. CIP4 is a Bin/Amphiphysin/Rvs (BAR) protein with a C-terminal SH3 domain that interacts with Wiskott Aldrich Syndrome protein (WASp) or dynamin. BAR proteins contain a domain that interacts with membrane lipids generating membrane curvature or tubulation. WASp and dynamin, on the other hand, function in actin polymerization and membrane vesicle scission respectively. WASp is defective in boys with the eponymous syndrome and is characterized by thrombocytopenia, which are small in size. Dynamin-3 has been shown to function during megakaryocyte development (Reems et al Exp Hematol. 36:12, 2008). We have observed that CIP4−/− mice displayed thrombocytopenia comparable to that observed in WAS-/ mice. Also, cultured megakaryocytes from CIP4 and WASp knockout mice showed decreased proplatelet extensions.
As a result of those observations, we hypothesized that defects in membrane dynamics due to loss of CIP4 might decrease platelet production. To better understand the role of CIP4 in platelet biogenesis, CHRF-288-11 cell line, a megakaryoblastic cell line was used for studying CIP4's interaction with WASp and dynamin. CIP4's interaction with WASp and dynamin-3 was investigated by co-immunoprecipitation and microscopy in CHRF cells. The cells were induced to form proplatelets by 100 ng/ml phorbol myristate acetate (PMA) or fibronectin-coated plates. Activated cells showed CIP4/dynamin-3 co-precipitation in a time-dependent manner. Also, immunofluorescent images showed diffuse distribution of CIP4 and dynamin-3 prior to activation with PMA or fibronectin. However after activation, co-localization of both proteins were observed at the membrane. These findings suggest possible interaction of CIP4-dynamin in platelet biogenesis. Platelet microparticles may serve as a surrogate marker for membrane deformation, rigidity, and cleavability. PMP are formed during cellular activation or apoptosis from various cell types such as platelets. The process of PMP formation is incompletely understood. We hypothesized that circulating levels of platelet microparticles (PMP) would be affected by the loss of CIP4 and its affects on membrane remodeling. Using flow cytometric analysis we observed decreased circulating levels of CD41+/ AnnexinV+ PMP in 3–6 month old C57BL/6 male CIP4 −/− and CIP4−/−,WAS-/ mice when compared to controls. However, WAS-/ mice showed similar levels to control mice.
We conclude that CIP4, a membrane deforming protein, promotes PMP formation. However, this is independent of its interaction with WASp. Because of dynamin's role in promoting membrane scission and its association with CIP4, we are currently investigating the role of dynamin in PMP formation.

Disclosures:
No relevant conflicts of interest to declare.
Gain-of-Function ADAMTS13 Variants Resistant to Anti-ADAMTS13 Autoantibody Inhibition: A Potential Novel Therapy for Acquired Autoimmune Thrombotic Thrombocytopenic Purpura
Authors: Jian Cui , Xiao Juan (Jenny) , Gong Lingjie , Skipwith Christopher G. , Jin Sheng-Yu , Kwaan Hau C. , Zheng X. Long 
Published: October 2019

Abstract
Abstract 1188
Spacer domain of ADAMTS13 is essential for recognition and proteolytic cleavage of von Willebrand factor. It also contains major antigenic epitope targeted by anti-ADAMTS13 autoantibodies in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). Through site-directed mutagenesis and recombinant protein strategy, we identified two novel ADAMTS13 variants, which exhibited enhanced specific activity, but reduced inhibition by anti-ADAMTS13 autoantibodies. Among twenty-three ADAMTS13 variants tested, ADAMTS13-M4 (R660K/F592Y/R568K /Y661F) and ADAMTS13-M5 (R660K/F592Y/R568K /Y661F/Y665F) exhibited increased specific activity by 4∼5 fold and 12∼16 fold in cleavage of FRETS-VWF73 and plasma-derived multimeric VWF under denaturing conditions, respectively. Moreover, these two novel ADAMTS13 variants (i.e. M4 and M5) were resistant to inhibition by anti-ADAMTS13 IgG autoantibodies from 10 out of 12 acquired TTP patients tested. In contrast, proteolytic activity of wild-type ADAMTS13 and several other mutants (i.e. M1: R660K, M2: R660K/F592Y, and M3: R660K/F592Y/R568K) were significantly inhibited by same amount of anti-ADAMTS13 IgGs in all 12 patients' plasmas. The resistance to inhibition of these variants was the result of reduced binding interaction between the ADAMTS13 variants (M4 and M5) and the anti-ADAMTS13 IgGs. These findings demonstrate for the first time that an exosite modification in the spacer domain may be a viable strategy for identification of novel ADAMTS13 variants with more desirable enzymatic properties. The results provide a potential tool for treatment of acquired (autoimmune) TTP and perhaps other arterial thrombotic disorders.

Disclosures:
No relevant conflicts of interest to declare.
RNAi-Mediated Inhibition of Activated Protein C- A New Approach for Hemophilia Treatment
Authors: Toudjarska Ivanka , Cai Zuhua , Racie Tim , Hettinger Julia , Milstein Stuart , Bettencourt Brian R , Sah Dinah WY , Bumcrot David , Maraganore John , Vaishnaw Akshay 
Published: October 2019

Abstract
Abstract 1204
Protein C is a major anticoagulation factor that serves as a key regulator of the clotting cascade. Activated protein C (APC), which is formed by limited proteolysis of the zymogen protein C by the thrombin–thrombomodulin complex, modulates thrombin generation. The anticoagulant effect of APC is due to subsequent inactivation of both activated factors V (FVa) and VIII (FVIIIa). The important role of APC in coagulation is highlighted in FV Leiden (FVL), the most common inherited form of thrombophilia. FVL is caused by a mutation in the FV gene at the initial APC cleavage site, which results in FVL being inactivated at approximately one tenth the rate of normal FVa, leading to high thrombin levels that create a procoagulant state. Several clinical studies have suggested that the severity or onset of bleeding phenotype in hemophilia patients is substantially reduced in association with impaired inactivation of FVa by APC in the presence of the FVL mutation. Transgenic animal studies also show that hemophilic mice, either heterozygous or homozygous for FVL, have improved clotting times with the ability to form clots at sites of laser-induced injury in a microvascular bed injury model, supporting the role of the FVL mutation in enhancing hemostasis. Therapeutic strategies aimed at reducing the level of protein C and activated protein C, thereby increasing levels of FVa and thrombin could thus prove efficacious in hemophilia A and B. Here we investigate the systemic administration of lipid nanoparticle (LNP) formulated siRNA directed against Protein C (encoded by Proc). We have designed potent siRNA sequences against human, cynomologous monkey, dog, mouse and rat Proc mRNA. The lead human/cynomologous candidate has an IC50 of 5pM. The lead mouse/rat candidate has an IC50 of 30 pM and an ED50 of 20 ug/kg. We demonstrate robust and durable inhibition of Proc mRNA in normal mouse liver- a single injection of 0.3 mg/kg resulted in 90% silencing of Proc mRNA and reduction of more than 75% which was sustained for more than two weeks. We also demonstrate significant reduction of circulating protein as measured by Western blot. Further testing using genetic models of hemophilia is being conducted to provide proof-of-concept for developing a Proc siRNA as a novel therapeutic agent in hemophilia management.

Disclosures:
Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment. Maraganore:Alnylam pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment.
In Vivo Imaging of Hematopoietic Stem Cell Engraftment in the Mouse Tibia Bone
Authors: Kim Seungbum , Lin Li , Hosaka Koji , Scott Edward W 
Published: October 2019

Abstract
Abstract 1286
A fundamental question in stem cell biology is where stem cells reside and how stem cell niches control stem cell activity. Although the hematopoietic stem/progenitor cell (HSPC) is well characterized, our current knowledge of where and how transplanted HSPCs become engrafted is very limited. HSPC transplantation is now routinely done in clinics to correct a variety of bone marrow deficiencies. Considering its extensive use, understanding the HSPC engraftment process has now become critical if we are to improve the treatment strategies. A key to understanding HSPC engraftment is to be able to observe the process in vivo. We aimed to visualize fluorescent HSCs in the mouse tibia bone directly by grinding one side of the tibia until the bone was sufficiently thin for direct observation. By making a “window” into the tibia bone, we were able to observe the early engraftment process of a single HSC forming a colony in real time with high resolution. The Sca-1+, c-Kit+, Lin- (SKL) cells preferred to lodge and proliferate mainly on the osteoblastic niche. In contrast, further purified SKL, CD48-, CD150+ population (SLAM-SKL) was mostly observed in the perivascular niche. When mice were co-transplanted with DsRed+ SKL and GFP+ SLAM-SKL populations, SKL cells were 5–7 times more than SLAM-SKL cells in the bone marrow at Day 7. However, contribution of each population to the blood circulation at the same time was similar, which suggests that the SLAM-SKL cells engrafted in the perivascular niche can produce blood much quicker. We were not only able to observe the engrafted cells but also visualize donor derived cells circulating in the bone marrow in real time. Our study shows a novel technique to understand and highlight the dynamic process of the stem cell engraftment in complex microenvironment of the bone marrow.

Disclosures:
No relevant conflicts of interest to declare.
Interferon-Gamma Induces Monopoiesis and Inhibits Neutrophil Development During Inflammation
Authors: de Bruin Alexander M. , Valkhof Marijke G. , Boon Louis , Touw Ivo P. , Nolte Martijn A. 
Published: October 2019

Abstract
Abstract 1320
Steady-state hematopoiesis is altered upon infection, but the cellular and molecular mechanisms driving these changes are largely unknown. Modulation of hematopoiesis is essential to increase the output of the appropriate type of effector cell required to combat the invading pathogen. Here we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFNγ) is involved in orchestrating inflammation-induced myelopoiesis. Using both mouse models and in vitro assays we show that IFNg induces differentiation of monocytes over neutrophils at the level of myeloid progenitors. We show that acute viral infection induces monopoiesis in WT mice, but a strongly increased neutrophil production in IFNγ−/− mice. When exploring the underlying molecular mechanism, we found that IFNγ increases expression of the monocyte-inducing transcription factors PU.1 and IRF8 in granulocyte-macrophage progenitors (GMPs) and enhances the response of these cells to M-CSF. On the contrary, IFNγ inhibits proliferation and differentiation of GMPs in response to G-CSF. We demonstrate that IFNγ reduces G-CSF-induced phosphorylation of STAT3, an important transcription factor in neutrophil development. Moreover, IFNγ also induces expression of SOCS3, which is a negative feedback regulator of G-CSFR signaling; by using G-CSFR mutants we demonstrate that the IFNγ-mediated inhibition of G-CSF-driven STAT3 phosphorylation is dependent on the recruitment of SOCS3 to the G-CSFR.
In conclusion, our findings illustrate that IFNγ is an important factor in shaping the hematopoietic response during inflammation. IFNγ is able to regulate myelopoiesis in the bone marrow upon viral infection by promoting the production of the appropriate myeloid cell type, but also by actively suppressing formation of cells less important for anti-viral defense. In addition, our data provide a molecular explanation for the observed aberrant hematopoietic remodeling observed in pathogen-challenged IFNγ-deficient mice. As both monocytes and neutrophils play important, but distinct roles in the defense against numerous pathogens, this study provides important new insight in the mechanism that regulates the formation of these vital myeloid cell types during infections.

Disclosures:
No relevant conflicts of interest to declare.
CALM Insufficiency Impairs Leukemia Cell Proliferation by Limiting Iron Uptake
Authors: Lavau Catherine P. , Heath Jessica L , Scotland Paula B. , Wechsler Daniel S. 
Published: October 2019

Abstract
Abstract 1478

Background:
The CALM-AF10 fusion protein that arises from the t(10;11) chromosomal translocation is found in both acute myeloid leukemia and 8–10% of T-cell acute lymphoblastic leukemia and is associated with a poor clinical outcome. The interaction of AF10 with the DOT1L histone methyltransferase has been shown to be necessary for CALM-AF10 mediated transformation. However, the role of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein in leukemogenesis remains uncertain. CALM plays a role in clathrin-mediated endocytosis. This process mediates the entry of various growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin.

Objective:
We hypothesize that loss of CALM function, as a result of CALM haploinsufficiency and/or a dominant negative CALM-AF10 effect, may contribute to transformation.

Design/Methods:
To study the effects of CALM deficiency, we used genetically modified fit1 mice that harbor a Calm gene mutation that results in a severely truncated and nonfunctional Calm protein. Fetal liver (FL) cells from Calm−/−, Calm+/− or CalmWT/WT E14 embryos were retrovirally transduced with MLL-ENL or CALM-AF10 and studied in vitro or transplanted into syngeneic mice. Interleukin-3 dependent cell lines were established from Calm−/− or CalmWT control FL cells immortalized by CALM-AF10 or MLL-ENL.

Results:
CALM-AF10 leukemias induced in Calm−/− or Calm+/− precursors were considerably delayed compared with those induced in CalmWT controls. Clathrin-mediated endocytosis of fluorescently labeled transferrin receptor (TfR) was impaired in Calm−/− cells. This was accompanied by a 2–4 fold increase in surface expression of TfR by flow cytometry and an increase in total TfR protein as measured by Western blot in Calm−/− cells. Calm -deficient cells also displayed lower levels of the iron storage protein ferritin, suggestive of an iron deficient state. Each of these features was reversed by re-expression of CALM via retroviral transduction of Calm−/− cells with a CALM expression vector. Paralleling the prolonged latency of Calm−/− leukemias, we found that Calm -deficient cells proliferated at a considerably slower rate than CALM -rescued cells. This cell proliferation defect could be rescued by supplementing the cells with iron (ferric ammonium citrate, 50 μM), indicating that reduced iron availability limits the expansion of Calm -deficient cells. Intriguingly, Calm−/− cells were significantly more sensitive to the growth inhibitory effect of iron chelation (deferoxamine, 5 μM) than CALM -rescued cells.

Conclusions:
These observations suggest that loss of CALM function impairs iron import and consequently limits the rate of cell proliferation. This raises the possibility that CALM haploinsufficiency present in CALM-AF10 leukemias might render cells particularly responsive to iron chelation therapy.

Disclosures:
No relevant conflicts of interest to declare.
Concurrent Radiotherapy and Dexamethasone, Etoposide, Ifosfamide, and Carboplatin (DeVIC) for Patients with Localized Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type: A Single Institution Analysis in Japan
Authors: Nara Eriko , Yokoyama Masahiro , Takeuchi Kengo , Nakano Kenji , Ueda Kyoko , Nishimura Noriko , Sakajiri Sakura , Mishima Yuko , Tsuyama Naoko , Oguchi Masahiko , Takahashi Shunji , Terui Yasuhito , Hatake Kiyohiko 
Published: October 2019

Abstract
Abstract 1611

Background:
Extranodal natural killer/T-cell lymphoma (ENKL), nasal type, is a recognized a rare distinct entity strongly associated EBV infection, accounting for 3% to 10% of malignant lymphomas in East Asia. Recent studies suggest that concurrent chemoradiotherapy was effective as first-line therapy for patients with localized ENKL, nasal type. We assess treatment results with concurrent chemoradiotherapy for ENKLs.

Patients and methods:
From December 2007 to July 2010, newly diagnosed localized ENKL, nasal type, and treated with concurrent radiotherapy (median 50 Gy; range 46–56 Gy) and 3 cycles of dexamethasone, etoposide, ifosfamide, and carboplatin (DeVIC) at the Cancer Institute Hospital, Tokyo, Japan were analyzed retrospectively. All the histopathological samples were reviewed according to the WHO classification by expert hematopathologists.

Results:
Total of sixteen patients was identified. Baseline patients. characteristics included a median age of 56.5 years (range; 30–76 years), eleven men and five women, thirteen patients (81%) with stage IE and three (19%) with stage IIE. For IPI, patients with low and low-intermediate risks were thirteen patients (81%) and three (19%), respectively. For NKIPI, patients with group 1, 2, and 3 were six patients (37%), seven (44%), and three (19%), respectively. All patients received concurrent chemoradiotherapy, and eleven patients (69%) achieved complete remission, with 2 partial remissions. The overall response rate was 81%. At a median follow-up of 17 months (range; 2–44), the 2-year progression-free survival and overall survival rates were 54.7% and 61.1%, respectively. Seven patients were confirmed progression disease, and six died from progression of lymphoma. Grade 3/4 adverse events were leukopenia (100%), neutropenia (100%), anemia (19%), mucositis (63%), anorexia (81%), and febrile neutropenia (25%), respectively. Seven patients were confirmed progression disease, and six died from progression of lymphoma. No treatment-related deaths were observed.

Conclusions:
Concurrent radiotherapy and DeVIC for patients with localized ENKL, nasal type, demonstrated favorable outcomes. We are now investigating what factors indicate good or poor prognosis in this regimen.
Progression free survival of concurrent radiotherapy and DeVIC regimen

Disclosures:
Yokoyama: CHUGAI PHARMACEUTICAL CO.,LTD: Consultancy. Mishima:CHUGAI PHARMACEUTICAL CO.,LTD: Consultancy.
Exportin 7 (RanBP16) Plays An Essential Role in Terminal Erythroid Chromatin Condensation and Enucleation
Authors: Hattangadi Shilpa , Burke Karly , Eng Jennifer , Cooney Jeffrey D. , Wang Junxia , Murata-Hori Maki , Paw Barry H. , Lodish Harvey F. 
Published: October 2019

Abstract
Abstract 178
In contrast to compacting chromatin into highly condensed mitotic chromosomes, the quite distinct process of global chromatin condensation culminating in enucleation that occurs during terminal erythroid development is still poorly understood. By examining the protein composition of the erythroid nucleus from early erythroblast to ultimate extrusion, I observed that extruded nuclei are largely depleted of all nuclear proteins. Given my previous observations that the highly-regulated but reportedly nonspecific nuclear export protein, Xpo7 or RanBP16, is highly induced during erythropoiesis and highly erythroid-specific, I hypothesized that its role may be to remove almost all nuclear proteins in order to allow the terminal erythroid chromatin to condense. Knockdown of Xpo7 using shRNA in primary fetal liver erythroid progenitors resulted in severe inhibition of chromatin condensation and enucleation but had little effect on hemoglobin accumulation or erythroid cell surface marker expression. As expected based on my hypothesis, proteomic analysis of nuclei from Xpo7-knockdown cells revealed largely all nuclear proteins, some of which may be responsible for the process of histone redistribution during chromatin condensation. Xpo7 is also highly regulated: besides its promoter being bound directly by the erythroid master regulators GATA1 and Klf1 (unpublished data), it is also the target of a miRNA whose level decreases during erythropoiesis, miR-181, and whose overexpression has been shown to result in the inhibition of terminal enucleation. Because chromatin condensation occurs in lower vertebrates without subsequent enucleation, I have also explored the localization and function of Xpo7 in zebrafish using in situ hybridization and morpholinos, respectively, and found that this export function is specific to mammalian chromatin condensation, providing evidence that condensation and enucleation are inextricably linked processes in mammals.

Disclosures:
No relevant conflicts of interest to declare.