bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association.
Authors: Mohseni Morvarid , Khan Anwar , Chishti Athar H. 
Published: October 2019

Abstract
Abstract 1990
Poster Board I-1012
Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity.

Disclosures:
No relevant conflicts of interest to declare.
Deficiency of MnSOD in Hematopoietic Stem Cells Causes a Sideroblastic Anemia-Like Phenotype.
Authors: Case Adam John , Wagner Brett A. , Buettner Garry R. , Domann Frederick E. 
Published: October 2019

Abstract
Abstract 1991
Poster Board I-1013

Introduction:
Sideroblastic anemia is caused by an inappropriate incorporation of iron into hemoglobin, and as such a phenotype demonstrating ringed sideroblasts, improper iron deposition, and malignancy is observed. The only known hereditary cause of this disease is a mutation in delta-aminolevulinate synthase 2 (ALAS2), which is a mitochondrially located enzyme that catalyzes the first step in heme synthesis, while the causes of the acquired or idiopathic forms remain unknown. With the knowledge that the free radical superoxide (O2•-) is able to displace iron from heme-containing proteins, it is hypothesized that the loss of the mitochondrial anti-oxidant enzyme manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, may cause disruption of proper heme synthesis and as such may contribute to a sideroblastic anemia-like phenotype. Methods: Due to the early lethality of the constitutive MnSOD knock-out mouse, a floxed (flanked by loxP) MnSOD mouse was created. Homozygous hematopoietic stem cell specific SOD2 knock-outs were generated by breeding the floxed MnSOD mice with mice expressing Cre-recombinase under the control of the hematopoietic stem cell specific promoter Vav. Results: Mice harboring the hematopoietic knock-out of MnSOD are born viable, and are able to live to maturity. Complete blood count (CBC) analysis of knock-out mice demonstrated a decrease in red blood cells (RBC), thymic white blood cells, and splenic white blood cells. The knock-outs additionally showed a decrease in hemoglobin to 5 g/dL compared to 11 g/dL in controls, a reduction in hematocrit to 32% versus 43.5% in controls, and a reticulocyte count of 31.6% compared to only 7.9% in controls. CBC analysis also revealed many RBC morphology flags including hypochromasia, ansiocytosis, and hematocrit variance. These findings were visualized in blood smears (see below). Finally, electron paramagnetic resonance spectrometry of plasma from the knock-out mice showed a ∼2-fold decrease in transferrin levels compared to controls. Conclusions: The loss of MnSOD in the hematopoietic stem cell population of a mouse creates a viable organism, but with an apparent sideroblastic anemia phenotype. Additionally, the white blood cells within these organisms also may be affected due to the decrease in total cellularity, and thus could contribute to immunodeficiency and/or malignancy later in the animal's life. Overall, this phenotype is very comparable to those observed in human patients, and as such MnSOD may play an undiscovered role in acquired or idiopathic sideroblastic anemia. Future work will focus on further characterization of the animals, examination of human blood samples for MnSOD mutations, and the use of SOD mimetics to potentially reverse the observed phenotype. This work was supported by NIH grants CA73612 and CA115438

Disclosures:
No relevant conflicts of interest to declare.
Pediatric-Based Therapy for Young Adults with Newly Diagnosed Lymphoblastic Leukemia.
Authors: Rytting Michael , Thomas Deborah A. , Franklin Anna R , Jabbour Elias , Wierda William , Faderl Stefan , Wells Robert , Kadia Tapan , Ravandi Farhad , Ferrajoli Alessandra , Cortes Jorge , O'Brien Susan , Kantarjian Hagop 
Published: October 2019

Abstract
Abstract 2037
Poster Board II-14
Adolescents 14 to 21 years of age with de novo acute lymphoblastic leukemia (ALL) have improved outcomes if treated on pediatric chemotherapy regimens as opposed to adult regimens. Young adults with ALL may also benefit from chemotherapy modeled after pediatric regimens, though toxicities may be limiting. We report on 48 young adult patients between the ages of 12 to 40 years with de novo Philadelphia chromosome negative ALL treated with the augmented Berlin-Frankfurt-Munster (BFM) chemotherapy regimen. All patients have completed at least the initial 28 days of therapy (induction) consisting of high dose prednisone, pegylated asparaginase (PEG-asp), vincristine, daunorubicin and intrathecal treatments. The median age of the group was 20 yrs (14-36); 40 patients had pre-B ALL and 8 T-ALL, No infectious deaths were observed during induction. 45 (95%) patients achieved a remission by 29 days and 2 achieved remission after a 2 week extension of induction. One patient was refractory to therapy. 39 (81%) patients achieved a morphological marrow remission (<5% blasts) by day 15 of treatment (rapid early responders). Minimal residual disease (MRD) assessed by flow cytometry was negative at the end of induction for 30 (62%), positive in 10 (21%), suspicious in 6 (12%) and not available for 2 patients. In the 41 patients who completed 12 weeks of therapy, MRD was negative in 35 (85%) and positive in 6 (15%). 7 (15%) patients have relapsed or have refractory disease; 1 patient died in CR. Admission for fever of unknown origin with neutropenia occurred in 10 (21%) patients, an additional 10 (21%) patients had documented infections. Grade III-IV hepatic toxicity has been prominent: 22 (45%) increased transaminase, 14 (29%) hyperbilirubinemia. 9 (19%) patients had allergic reactions to PEG-asp, and 10 (21%) had thrombotic events. The majority of grade III-IV adverse events have been reversible. The median complete remission duration is 57 weeks (range 5-143). The overall survival at two years is 82%. In summary, this pediatric-based regimen for young adults with ALL effectively induces remission, both by morphology and by flow cytometry. Toxicity has been significant, but mostly transient and tolerable. Longer follow-up is needed to determine the long-term efficacy of this regimen in young adults with ALL.

Disclosures:
Rytting: Enzon: Speakers Bureau. Jabbour:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Factor Xa Dimerization and Prothrombinase Complex Formation Are Competitive Process On a Membrane Surface.
Authors: Chattopadhyay Rima , Koklic Tilen , Lentz Barry 
Published: October 2019

Abstract
Abstract 2123
Poster Board II-100
Exposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factors Xa and on Va promotes prothrombinase assembly and enhances proteolytic activity of factor Xa. PS binding produces significant structural changes in both proteins. Recent studies demonstrate formation of inactive Xa dimer by both soluble, short-chain PS and PS-containing membranes at high Ca+2 concentration (Chattopadhyay et al.,2009, Biophys. J, 974). Association of factor Xa with factor Va in the presence of soluble PS relieves this inactivation. From kinetic measurements, we found that apparent Kd's for the Xa-Va interaction in the presence of 50 mM PS/PC (25/75) membranes increased in with increasing factor Xa concentrations, suggesting that factor Va must compete with factor Xa dimer in order to form the Xa-Va complex on the membrane surface. The fluorescence anisotropy of active site labeled factor Xa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is lowered in the presence of PS membrane on which it forms dimer (Koklic et al., October 2009, Biophys. J., V-97, Issue 8) We report now that addition of factor Va to FEGR-Xa/membrane samples produced a saturating increase in anisotropy, clearly indicating that factor Va competed with factor Xa dimer formation. This experiment was repeated for several Ca2+ concentrations and yielded increasing apparent Kd's for Xa-Va interaction with increasing Ca2+concentration. FEGR-Xa fluorescence anisotropy as well as DEGR-Xa (factor Xa labeled at the active site with Dansyl-GLU-GLY-ARG-chloromethylketone) fluorescence intensity at varying membrane and factor Va concentrations for both 23 nm and 120 nm vesicles were analyzed globally to obtain surface Kd's of dimerization and factor Va-factor Xa interaction. This showed that membrane discreteness (slow distribution of factor Xa between discrete membrane vesicles) had almost no effect on the competition between factor Xa dimer formation and Xa-Va complex formation. We conclude that prothrombinase complex formation can overcome PS-induced factor Xa inhibition through factor Xa dimer formation at Ca2+ concentrations just above plasma concentration. In this way, one can imagine that factor Va is required to overcome factor Xa dimerization and amplify thrombin production in the early platelet plug.
Supported by USPHS grant HL072827 to BRL.

Disclosures:
No relevant conflicts of interest to declare.
Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.
Authors: Zhang Jingyu , Ma Zhenni , Dong Ningzheng , Su Jian , Wang Anyou , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 2133
Poster Board II-110
Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation and Clinical Application of a New Method for Detecting ADAMTS13 Activity.
Authors: Wang Anyou , Dong Ningzheng , Ma Zhenni , Zhang Jingyu , Su Jian , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 2134
Poster Board II-111

Introduction:
A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, and not appropriate for routine laboratories. ADAMTS13 cleaves the von Willebrand factor (VWF) within the domain A2, which locates between the domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structure of VWF before and after the cleavage.

Materials and Methods:
To determine the activity of ADAMTS13, a new simple method has been developed in this study. Firstly, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies (SZ-129 and SZ-125), which are specifically recognize the VWF A1 and A3 domain, respectively, by using a two-site sandwich ELISA. Comparing with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibodies in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.

Results
Our results showed that plasma ADAMTS13 activities determined by the new assay were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays, and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activities of ADAMTS13 autoantibodies were ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.

Conclusions
This new and simple assay for ADAMTS13 activity could be used routinely in clinic to determine the activity of ADAMTS13.

Disclosures:
No relevant conflicts of interest to declare.
Title:Dose-Adjusted EPOCH Plus Rituximab (DA-EPOCH-R) in Untreated Patients with Poor Risk Large B-Cell Lymphoma. A Phase 2 Study Conducted by the Spanish PETHEMA Group.
Authors: Purroy Noelia , López Andrés , Vallespi Teresa , Gironella Mercedes , Bergua Juan , Sancho Juan Manuel 
Published: October 2019

Abstract
Abstract 2701
Poster Board II-677

Background.
Since the development of CHOP chemotherapy, many efforts have been made to improve first line treatment in aggressive lymphomas, mainly in poor prognosis cases. To assess the efficacy and safety of an infusional DA-EPOCH plus Rituximab (DA-EPOCH-R) regimen in poor prognosis large B-cell lymphomas, the Spanish PETHEMA group conducted a phase 2 study in patients with a higher than 1 aaIPI in this setting.

Methods:
Eligibility criteria included histologically documented DLBCL, grade 3b follicular lymphoma or primary mediastinal B-cell lymphoma, IPI higher than 2 or aaIPI higher than 1 (or equal to 1 plus bulky disease). No restriction was made based on ECOG performance status when they could understand and sign the informed consent. Radiation therapy as consolidation was permitted on bulky masses detected at diagnosis.

Results:
Herein, 71 patients were evaluated for safety and outcome: 32 were male and 39 female. Median age was 60 years (range: 21-77). DLBCL was diagnosed in 57 (80%), PMBCL in 6 (9%), and grade 3b follicular lymphoma 8 (11%). Eighty nine percent had advanced (III-IV) CS, and 58% presented B symptoms. Bulky disease (3 10 cm maximum diameter) was present in 18%. ECOG was higher than 2 in 13 patients (18%). b-2-microglobulin was elevated in 62%, and LDH in 90% of patients. AaIPI was: 1 in 7 patients (10%), 2 in 36 (51%), and 3 in 28 (39%). Four hundred and fifteen cycles were given. A grade 3-4 mucositis occurred in 9 occasions, and neutropenic fever or infections in 66 cycles (16%). Fifty nine patients (83%) achieved a CR or CRu, 6 (8%) a PR and 4 (6%) an inadequate response, there were 2 early deaths: a toxic one, due to septic shock, and one due to non-related cause (traumatism). Twenty six percent of patients who achieved a CR relapsed. Twenty one patients (30%) died so far: 76% due to progressive disease, one patient who had undergone an ASCT after relapse died from secondary MDS. With a median follow-up of 40 months the event-free survival and overall survival is 60% and 61% respectively at 5 years. Disease-free survival is 72% at 5 years for patients who achieved a CR or CRu. No statistically significant difference in overall survival were seen according to aaIPI risk factors.

Conclusion:
DA-EPOCH-R is an immunochemotherapy regimen with a clinically significant activity and a very acceptable toxicity profile in patients with poor prognosis aggressive B-cell lymphomas and could somehow overcome IPI risk factors.

Disclosures:
No relevant conflicts of interest to declare.
Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.
Authors: Heinrich Daniel A. , Klein Christian , Decheva Kristina , Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Weiglein Tobias , Hiddemann Wolfgang , Dreyling Martin H. 
Published: October 2019

Abstract
Abstract 2704
Poster Board II-680

Background:
Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively.

Methods:
Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms.

Results:
After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations.

Conclusions:
Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101.

Disclosures:
Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.