bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and Epigenetic Contributions to Longevity
Authors: Barzilai Nir
Published: October 2019

Abstract SCI-1

Aging is a natural process that involves a general decline in many physiological functions resulting in loss of function and eventually death, and is also the main risk of death from almost all diseases. Therefore we reason that if we could understand the biology of aging, we could design a therapy that will not affect a single disease at a time but rather will prevent many aging related diseases. Indeed, in the field of aging we have been successful in identifying several mechanism that may be involved in aging, but even more importantly, mechanisms that are protective against aging.

Here we will summarize the efforts to implicated protective pathways in relatively healthy subjects with exceptional longevity. These subjects are part of the LonGenity (or Longevity Genes Study) at Albert Einstein College of Medicine, which gathered the clinical phenotype and performed extensive genotyping on centenarians and their offspring. We will demonstrate that genetic factors that are associated with human longevity are also heritable and may contribute not only to increase life span, but also provide a protection from age dependent disease and exceptional good health.

We also will demonstrate that aging may be modified by epigenetics mechanism and will show results from high throughput methylation studies in aging rodents and humans.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA in Platelet Production and Activation
Authors: Bray Paul F.
Published: October 2019

Abstract SCI-39

Alterations in gene expression are at the heart of both megakaryocytopoiesis and interindividual variation in platelet reactivity. Over the past decade there has been an increasing awareness of the important role played by microRNAs (miRNAs) in these diverse cell biologic and physiologic processes. MiRNAs are noncoding RNAs that target complementary sequences in mRNAs, leading to mRNA degradation or inhibition of translation. More than 1000 miRNAs have been identified, which are estimated to regulate 30%-90% of all coding genes. Expression of miRNAs is cell and developmental stage specific. MiRNAs regulate hematopoietic lineage commitment, as well as B lymphopoiesis, granulopoiesis, erythropoiesis and monocytopoiesis. Recent data also demonstrate a clear role for miRNAs in megakaryocytopoiesis (Meg-poiesis). The molecular genetic basis for the transition from one stage of Meg-poiesis to another requires fine-tuning of the various control elements, and miRNAs act as “rheostats” in this process. MiRNA-150 has a strong effect on Meg-erythroid progenitor cells, inducing commitment to the Meg lineage at the expense of erythropoiesis. MiRNA-150 directly down-regulates MYB, a transcription factor important in thrombopoiesis. Over-expressed miRNA-146a knocks down CXCR4 (receptor for SDF-1), reducing Meg proliferation and maturation. MiR-146a gene expression is negatively regulated by the promyelocytic zinc finger protein, PLZF. Thus, miRNAs act as intermediaries of transcription factors that control Meg-poiesis. An increasing number of genetic diseases are being described due to mutations in miRNA genes or their mRNA targets. Deletion of miR-145 and miR-146a on 5q is sufficient to cause the 5q- phenotype; miR-125b-2 up-regulates Meg-poiesis and may be involved in megakaryoblastic leukemia. Platelets appear to be a particularly rich source of miRNAs, many of which are expressed at very high levels. Notably, platelets contain Dicer, TRBP2 and Ago2, and are capable of synthesizing miRNAs from pre-miRNAs. MiRNAs regulate Bcl-xL and Bak, raising the possibility that platelet miRNAs affect platelet lifespan. Because miRNAs are very stable, they also represent a potential biomarker, and we have found sets of miRNAs that appear to classify platelet reactivity to epinephrine. Using bioinformatic approaches, we have identified miRNA-mRNA pairs differentially expressed in platelets with differing phenotypes. This approach permits functional characterization of novel platelet mRNAs, and elucidation of a potential genetic mechanism for adjusting megakaryocyte/platelet mRNA expression. Thus, information extracted from these RNA networks continue to provide insights into systems biology of higher organisms.

Disclosures:

No relevant conflicts of interest to declare.
The Cellular and Molecular Mechanisms of Neutropenia in Barth Syndrome
Authors: Aprikyan Andrew A , Makaryan Vahagn , Kulik Willem , Vaz Frederic , Allen Christopher , Dror Yigal , Dale David C.
Published: October 2019

Abstract 1105

Barth syndrome (BTHS) is a rare, X-linked recessive disease characterized by progressive cardiomyopathy and neutropenia with life-threatening bacterial infections. BTHS is attributable to loss-of-function mutations in the tafazzin gene, TAZ, a phospholipid acyltransferase localized to the mitochondrial membrane and involved in remodeling cardiolipin. BTHS children exhibit reduced blood levels of total cardiolipin; however, the molecular and cellular mechanisms of BTHS remain largely unknown.

We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrated a significant down-regulation in the TAZ gene expression, mimicking naturally-occurring truncation mutations. FACS analyses of cells with TAZ-specific but not scrambled shRNAs demonstrated nearly two-fold increase in proportion of annexin-V positive cells and significantly increased dissipation of mitochondrial membrane potential (MMP) as determined by DIOC6-staining. Similar pro-apoptotic effects were observed in U937 myeloid progenitors, but not in Jurkat or Ramos human lymphoid cell lines. Further studies revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knock-down. Treatment with caspase-specific inhibitor zVAD-fmk reduced apoptosis to near-normal levels. FACS analysis utilizing cryopreserved bone marrow cells from a BTHS patient showed evidence of accelerated apoptosis compared to corresponding cells from a healthy volunteer. Importantly, human myeloid progenitor cells and cardiomyoblasts/cardiomyocytes depend heavily on mitochondria, which may explain why the loss of mitochondrial protein tafazzin has more severe effects on these specific tissues in BTHS resulting in neutropenia and cardiomyopathy.

These data suggest that neutropenia in BTHS patients is attributable to increased dissipation of MMP, aberrant release of cytochrome c, activation of caspase-3 and accelerated apoptosis, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors. Further studies should evaluate the TAZ–regulated mitochondrial apoptosis pathway using primary cells from BTHS patients.

Disclosures:

Aprikyan: Barth Syndrome Foundation: Research Funding. Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies
Authors: Nalivaiko Kristina , Hofmann Martin , Grosse-Hovest Ludger , Krammer Peter H , Rammensee Hans-Georg , Salih Helmut R. , Jung Gundram
Published: October 2019

Abstract 1114

Antibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells.

Disclosures:

No relevant conflicts of interest to declare.
A Humanin Analog Attenuates Platelet Responses to Vascular Injury
Authors: Ren Lijie , Li Qiang , Zeng Zhao , Mou Peipei , Liu Xiaohui , Tang Xiaorong , Lu Qiongyu , Xu Xingshun , Zhu Li
Published: October 2019

Abstract 1131

Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P<0.05) and 71.9% (P<0.001), respectively. Similarly, HNG significantly reduced ATP release stimulated by collagen or convulxin. Convulxin-induced P-selectin expression and fibrinogen binding on single platelet was inhibited by HNG, as measured by flow cytometry. Moreover, HNG reduced platelet spreading on the fibrinogen coated surface by 62.9 % (P <0.05). Western blot revealed a reduction of platelet AKT phosphorylation by HNG upon collagen stimulation, implying the involvement of PI3K pathway. In addition, MAPK P38 phosphorylation by collagen and convulxin was also reduced by HNG. HNG effects on thrombus formation were tested in vivo in a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of HNG (25μg/kg) to male C57BL6/J mice significantly extended the first occlusion time (7.3±0.4 min, N=10), when compared to the saline injected littermates (5.4±0.7 min, N=12) (P <0.05). Furthermore, the number of mice that formed stable thrombus was less in the HNG–treated group (3/13) than the control group (6/13), while the non-occlusion mouse number was more in the HNG-treated group (3/13) than the control group (1/13). Together, these data show that HNG inhibits platelet activation and arterial thrombus formation. This might suggest that the protective effects of HNG against ischemia reperfusion injury could be, in part, via attenuating platelet activation. Therefore, HNG could be a potential therapeutic agent in thrombotic and cardiovascular disorders.

Disclosures:

No relevant conflicts of interest to declare.
Hypoxia-Induced Pulmonary Arterial Hypertension: The Role of TLR4
Authors: Ma Liping , Chang Jianxing , Wu Hui , Chen Yabing
Published: October 2019

Abstract 1146

Pulmonary arterial hypertension (PAH) is often as a result of COPD and contributes to morbidity and mortality of chronic cor pulmonale. Hypoxemia is its main clinical features. The toll-like receptors are a group of type I transmembrane proteins that play a central role in specific recognition of pathagen-associated molecular patterns and are critical for the induction of innate immunity and inflammation. Expression of these innate immune receptors, especially TLR4 in healthy and pathological arteries has implicated its role in the homeostasis of vasculature. Chronic hypoxia-induced PAH is mediated by increased production of reactive oxygen species (ROS). ROS have been linked to activation of toll-like receptor 4 (TLR4) signaling. The present studies investigated the role of TLR4 in the pathogenesis PAH and the underlying mechanisms.

In vitro, pulmonary arterial smooth muscle cells (PASMC) derived from TLR4−/− mice exhibited increased production of intracellular reactive oxygen species (ROS). Furthermore, under hypoxia, the expression of TLR4 in PASMC from WT mice was decreased, ROS production in PASMC from WT mice were increased, suggesting down-regulation of TLR4 may contribute to hypoxia-induced PAH.

Using a hypoxia-induced PAH model, we found that hypoxia induced PAH and increased pulmonary arterial wall thickness in wild type (WT) mice. In contrast, mice deficient in TLR4 spontaneously developed PAH and increased pulmonary arterial wall thickness, which were not further enhanced by hypoxia. Consistent with the development of PAH in TLR4−/− mice, echocardiography confirmed right ventricular hypertrophy and decreased pulmonary arterial acceleration time compared to WT mice. Above results suggest that TLR4 plays an important role in maintaining normal pulmonary vasculature, and that hypoxia induces PAH via TLR4.

In short, these studies demonstrate an important role of TLR4 in maintaining normal pulmonary vasculature and in the development of hypoxia-induced PAH. Genetic ablation of TLR4 and inhibition of TLR4 expression by hypoxia induces PASMC proliferation and vascular remodeling, in association with redox signaling in the pathogenesis of PAH.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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.
Vacuolin-1 Promotes Enucleation of Cultured Human and Murine Erythroblasts
Authors: Keerthivasan Ganesan , Ghinassi Barbara , Wilmes John S. , Wickrema Amittha , Migliaccio Anna Rita F , Duban Mark-Eugene , Crispino John
Published: October 2019

Abstract 2321

Enucleation of erythroblasts is the final step of differentiation before reticulocytes enter circulation in mammals. In culture, enucleation efficiencies vary widely, between 0 and 90%: improving this process is a necessary step for large-scale production of reticulocytes for transfusion. Recently, we demonstrated that the formation, movement and coalescence of vesicles and vacuoles play a key role in enucleation. Moreover, we showed that vacuolin-1, a small molecule that induces the formation and fusion of vacuoles, increases the enucleation of primary erythroblasts. Separately, we recently discovered that the anti-apoptotic and chromosomal passenger protein survivin plays a novel role in enucleating erythroblasts. Survivin interacts with clathrin and EPS-15, proteins that play important roles in vesicle trafficking, and together these proteins contribute to enucleation. In order to further understand the specific role played by survivin in vesicle trafficking and enucleation, we treated fetal liver Ter119- cells derived from survivin fl/fl mice with TAT-Cre to excise the survivin gene and assess the consequences of survivin loss on enucleation. We observed a significant reduction in the extent of enucleation upon deletion of survivin. Of note, survivin depleted cells harbored fewer numbers of vacuoles compared to control cells, and the vacuoles that were present were smaller than normal. Survivin deleted cells also lacked autophagolysosomes, which are formed by fusion of late endosomes/lysosomes to autophagosomes. Since vacuolin-1 is known to induce fusion of endosomes and lysosomes, we hypothesized that vacuolin-1 would rescue this enucleation defect observed in survivin KO erythroblasts. Indeed, vacuolin-1 reversed the inhibitory effect of survivin KO on enucleation. Surprisingly, we also observed that vacuolin-1 promoted the proliferation of human erythroblasts at the CFU-E stage, leading to a 4-fold increase in the percentage of glycophorin-positive cells on day 12. Thus, we conclude that vacuolin-1 acts at multiple stages of differentiation to improve the production of erythrocytes in vitro. Finally, to gain insights into the mechanism of action of the “hit” vacuolin-1, and to identify more potent small molecules that enhance enucleation in vitro, we designed and studied a further small set of vacuolin-1 structural analogs (N=12). Four vacuolin-1 analogs increase enucleation of erythroblasts with greater potency than that of vacuolin-1, providing us with initial insights into the small molecule structure-activity relationships that might govern this process. We are currently assaying the activity of these next generation compounds in erythroblast cultures as a means to improve production of red blood cells for human use.

Disclosures:

No relevant conflicts of interest to declare.
Runx1 Induced Chromatin Folding of the PU.1 Gene Locus Is Necessary for Adult Long-Term Hematopoietic Stem Cell Maintenance
Authors: Staber Philipp B. , Zhang Pu , Ye Min , Huang Gang , Ruscio Annalisa Di , Ebralidze Alexander K , Tenen Daniel G.
Published: October 2019

Abstract 2364

Background:

PU.1 is a hematopoietic transcription factor that is essential for normal hematopoiesis. PU.1 gene expression is mainly regulated through the proximal promoter (PrPr), and an upstream regulatory element (URE) located –14kb or –17kb upstream of the transcription start site in mice and humans, respectively. We previously demonstrated that PU.1 is a downstream target of core binding factor Runx1 (also called AML1) for megakaryocytic, lymphoid, and myeloid differentiation. Recent reports identified Runx1 to restrict proliferation of hematopoietic stem cells (HSCs) and to prevent HSC exhaustion.

Approach:

By employing conditional (Mx1-Cre) Runx1 knockout mice (Runx1Δ/Δ) and knock-in mice with targeted disruption of all three Runx1 binding sites at the −14kb URE of PU.1 (mRunxki/ki) we aimed to genetically dissect a potential functional role and mechanisms by which Runx1 might regulate PU.1 expression in HSCs.

Results:

Both, Runx1Δ/Δ and mRunxki/ki mice exhibited decreased PU.1 levels of approximately 65% in purified phenotypic HSCs (CD150+CD48-LSKs). In addition, activating histone marks H3K4me3 and H3Ac were significantly decreased at the −14kb enhancer and the PrPr. Mechanistically, we found by quantitative chromosome conformation capturing (3C) that Runx1 binding established a physical interaction of the −14kb enhancer with the PrPr thereby forming an active chromosomal conformation for proper PU.1 transcription. In concordance to Runx1Δ/Δ, phenotypic HSCs of mRunxki/ki mice were significantly increased in number. However, limiting dilution transplantations using the congenic Ly5.1/Ly5.2 system revealed a 25-fold decrease of competitive repopulating units (CRU) in phenotypic HSCs of mRunx-ki/ki mice compared to controls. Long-term reconstitution in serial transplantation assays was also severely impaired indicating HSCs exhaustion in mRunxki/ki mice. HSC homing and engraftment was not affected. Importantly, restoration of PU.1 levels by crossing to a human PU.1 transgenic fully rescued long-term HSC functions, thus demonstrating that HSC maintenance was strictly related to PU.1 levels.

Conclusion:

Runx1 binding to the −14kb enhancer of PU.1 is necessary to maintain an active chromosomal conformation for proper PU.1 transcription in order to prevent HSC exhaustion.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Survivin with YM155 As a Potential Therapy in Pediatric Acute Lymphoblastic Leukemia
Authors: Jemal Abdusebur , Tyner Jeffrey W , Thayer Mathew , Muschen Markus , Druker Brian J. , Chang Bill H
Published: October 2019

Abstract 2490

Background:

Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed ALL. Therefore, survivin may be a potential target for therapy in pediatric ALL. The selective survivin suppressant, YM155 (Astellas) has been shown to inhibit survivin expression and activate cell death in multiple cell lines. Early phase I studies show promise in both tolerability and possible efficacy in B-cell malignancies. Therefore, this drug may have the potential of improving treatment for pediatric B-cell precursor ALL.

Design/methods:

Pediatric lymphoblastic cell lines, fresh primary lymphoblast cells from newly diagnosed patients with ALL and xenografted patient samples were used in this study. Cells were incubated in the presence of YM155 at doses ranging from 1nM to 10μM. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay for cell lines.

Results:

Treatment of ALL cell lines, primary patient samples and xenograft samples show a dose-dependent sensitivity to YM155 by both activation of apoptosis and by cell viability. IC50 doses for the majority of the samples are in the low nanomolar range (Table). Interestingly, there is some variability amongst patient samples suggesting possible variable responses in vivo. Ectopic expression of survivin in cell lines treated with YM155 rescues the effect of the drug. Further, t(9;22) positive ALL samples, including primary patient, xenograft, and dasatinib resistant samples remain significantly sensitive to YM155. For dasatinib sensitive Ph+ALL samples, combination therapy suggest an additive effect by isobologram analysis.

Conclusion:

Pediatric ALL samples remain sensitive to treatment with YM155 in cell lines, primary patient and xenografted samples. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop YM155 as an additional therapy for pediatric acute lymphoblastic leukemia.

Disclosures:

Druker: Cylene:; MolecularMD:; Novartis:; Bristol-Myers-Squibb:.
Fluoroquinolone Prophylaxis in Acute Myeloid Leukemia (AML) Patients Undergoing Post-Remission Chemotherapy Reduces Hospital Admission Rates
Authors: Kusick Karissa , Earl Marc , Gallagher Erika M. , Yeh Jun-Yen , Advani Anjali S. , Kalaycio Matt , Copelan Edward A. , Sekeres Mikkael A.
Published: October 2019

Abstract 2570

Background:

High-dose cytarabine (HIDAC) is a standard treatment for younger AML patients (pts) receiving post-remission chemotherapy. The risk for febrile neutropenia (FN) is high among these pts and considered a medical emergency, requiring prompt hospital admission and treatment. In 2004 our institution adopted the use of fluoroquinolones (FQ) for bacterial prophylaxis in AML pts receiving HIDAC. We compared rates of hospital admission due to FN before and after the initiation of bacterial prophylaxis with FQ. We also evaluated the incidence of blood stream infections (BSI) and the incidence of resistant infections among the two groups.

Methods:

We identified AML (excluding acute promyelocytic leukemia) pts receiving cytarabine-based remission induction chemotherapy at Cleveland Clinic who achieved a complete remission and then were treated with HIDAC post-remission chemotherapy (cytarabine dose of 3000mg/m2 × 6) from 1997–2008. Pts receiving FQ prophylaxis with ciprofloxacin 500mg twice daily or levofloxacin 500mg once daily comprised the treatment group, while AML patients with no prophylaxis served as controls. Pts were excluded if they developed an episode of FN during hospital admission for HIDAC or received non-FQ bacterial prophylaxis. Categorical data and continuous variables were analyzed per chemotherapy cycle using Fishers exact test and independent-sample t-tests, respectively.

Results:

80 pts received HIDAC post-remission chemotherapy during the study period; 52 received FQ prophylaxis; 28 did not. A total of 198 chemotherapy cycles were administered, 148 in the FQ group and 50 in the control group. The mean age (+/−SD) of the entire cohort was 44.8 (11.4) years; 53% were female. Despite the decreased use of granulocyte-colony stimulating factor in the FQ group (12% vs. 50%, p<0.001), the duration of neutropenia was the same (24.6 days vs. 23.8 days, p=0.5) between groups. All other baseline characteristics were similar. FQ treated pts had a 33% absolute reduction in hospital admissions due to FN (50.7% vs. 84%, p<0.001) and when admitted, hospital LOS for FN was shorter for the FQ group (7.9 days vs. 10.5 days, p=0.007). The incidence of BSI was also reduced in the FQ group (23% vs. 42%, p=0.009); however there was an increase in resistant gram-negative (5/17 vs. 0/14, p=0.04) and resistant gram-positive (15/19 vs. 3/9, p=0.03) infections among pts treated with FQ.

Conclusion:

AML pts treated with HIDAC chemotherapy followed by FQ prophylaxis had fewer and shorter hospital admissions due to FN compared to pts with no prophylaxis. FQ treated patients were at increased risk of developing antibiotic-resistant bacterial BSI.

Disclosures:

No relevant conflicts of interest to declare.