bluuebunny/crossref_metadata_2025_split huggingface dataset:
Histone Deacetylase 11 (HDAC11) Regulates Cytotoxic T-Cell Function and Memory Phenotype
Authors: Woods David M , Woan Karrune V. , Sahakian Eva , Powers John , Cheng Fengdong , Wang Hongwei , Rock-Klotz Jennifer , Weber Jeffrey S. , Villagra Alejandro , Pinilla-Ibarz Javier , Sotomayor Eduardo M. 
Published: October 2019

Abstract
Abstract 840
T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Rasgrf1 Overexpression Amplifies B Cell Receptor Signaling in Chronic Lymphocytic Leukemia Specimens
Authors: Liao Wei , Jordaan Gwen , Sharma Sanjai 
Published: October 2019

Abstract
Abstract 926
The B-cell receptor (BCR) signaling is a hallmark of chronic lymphocytic leukemia cells and results in cell proliferation, survival and resistance to apoptosis. B cell receptor (BCR) signaling in CLL specimens activates the Ras/ERK/MAPK pathway and is critical for survival and growth of CLL cells. Rasgrf1 is a guanine exchange factor that accelerates the removal of inactive GDP from Ras so that active GTP can bind Ras that results in Ras activation. In an Affymetrix microarray expression analysis screen of CLL specimens (n=5) and peripheral blood normal B cells (n=2), Rasgrf1 was found to be over-expressed (ranked within top 100 overexpressed genes) in CLL. This finding was further confirmed by quantitative RT-PCR as a 5–300 fold overexpression in CLL specimens (n=20) was noted as compared to normal B cells. Western blot analysis data also showed over-expression of Rasgrf1 protein in CLL specimen lysates but interestingly the expressed protein is of a smaller size in CLL specimens (70-80 kDa) as compared to the Rasgrf1 in normal peripheral blood mononuclear cells (140 kDa) This truncated protein retains the important c-terminal GEF (guanine exchange factor) domain but lacks the regulatory domains and is known to function as a hyperactive GEF in other tumor model systems. Knockdown of Rasgrf1 in CLL specimens (n=4) resulted in inhibition of the BCR-Ras/ERK/MAPK pathway as determined by a decrease in ERK phosphorylation. Similar Rasgrf1 knockdown experiments with BCR crosslinking also showed identical results in additional CLL specimens. Transient SiRNA mediated Rasgrf1 knockdown also resulted in higher percentage of annexin positive or apoptotic CLL cells as compared to the scrambled SiRNA control transfection. Lysates from the Rasgrf1 knockdown cells showed an increase in PARP cleavage by western blot analysis as well indicating that continued expression of this protein was necessary for survival of the leukemic cells. To confirm that the Rasgrf1 knockdown resulted in a decrease in the GEF function, the active Ras (Ras-GTP) in the CLL specimens was evaluated by an ELISA assay. In two CLL specimens the assay confirmed that the active Ras in the CLL cells was decreased with Rasgrf1 knockdown. Overall the findings implicate a novel GEF protein, Rasgrf1 as an amplifier of BCR signaling in CLL cells.

Disclosures:
No relevant conflicts of interest to declare.
Erythropoietin-Directed Erythropoiesis Depends Upon Serpin Inhibition of Erythroblast Lysosomal Cathepsins
Authors: Wojchowski Don M. , Dev Arvind , Byrne Susan M , Verma Rakesh , Ashton-Rickardt Philip G 
Published: October 2019

Abstract
Abstract 981
EPO is used to treat anemia associated with chronic kidney disease, and cancer chemotherapy. However, EPO has hypertensive and thrombolytic side-effects and can impact on innate immunity, diabetes, vasculogenesis, and the progression of certain cancers. These observations, together with the clinical emergence of new EPOR agonists provide compelling reasons to better understand key EPO/EPOR actions. This includes effects on erythroid progenitor cells (EPC's) as a prime target in which EPO/EPOR actions remain incompletely understood. To discover novel EPO action modes we profiled the transcriptome of primary EPC's. We report Serpina3g/Spi2A as a major EPO/EPOR target, and a key EPC survival factor. In knockout mice, loss of Spi2A sharply worsened anemia due to hemolysis, radiation or transplantation (p = 0.008; p = 0.0001; p = 0.010, respectively). EPO-induced red cell formation also was limited to 8.6 + 0.7% of wild-type control responses. Unexpectedly, maturing erythroblasts proved to require Spi2A for cytoprotection, with iron and ROS defined as cytotoxic agents. Notably, Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by the genetic co-deletion of lysosomal cathepsin-B. In vivo, pharmacological inhibition of cathepsin B/L enhanced EPO's effects on hematocrits by 5+ points (d4 and d6 post- EPO). Overall, we define an unexpected EPO action mode via an EPOR- Spi2A serpin- cathepsin axis in maturing erythroblasts, with oxidants (and iron) as cytotoxic potentiators, and lysosomal cathepsins as novel therapeutic targets.

Disclosures:
No relevant conflicts of interest to declare.
Divalent Metal Transporter 1 (DMT1) Regulates EPO Receptor Gene Expression Via GATA-1
Authors: Burda Pavel , Curik Nikola , Horvathova Monika , Divoky Vladimir , Stopka Tomas 
Published: October 2019

Abstract
Abstract 991

Introduction:
Erythroid differentiation and iron metabolism are interconnected processes in order to produce sufficient numbers of appropriately hemoglobinized red cells. Patients carrying mutations of divalent metal transporter DMT1 display severe microcytic anemia and iron overload. In vivo, this defect can be partially rescued by stimulation of erythropoiesis by erythropoetin (EPO). In vitro, addition of EPO together with iron-saturated salicylaldehyde isonicotinoyl hydrazone (Fe-SIH, a non-transferrin iron donor) to the cultures significantly improved the growth of patient's DMT1-mutant erythroid progenitors (Horvathova et al, 2012). Regulation of erythropoiesis by EPO and its receptor (EPOR) involves transcription factor GATA-1. Our earlier data (Burda et al, 2009) showed that in erythroblasts GATA-1 transcriptionally regulates both Epor and Dmt1 and that this activation is blocked by negative regulator of erythroid differentiation, transcription factor PU.1. This suggests that simultaneous expression of EPOR and DMT1 is required for erythroid differentiation and survival of erythroid cells. We hypothesize that deficiency of DMT1 negatively affects expression of EPOR, thus leading to inhibition of EPO/EPOR signaling. We suppose that this inhibition involves GATA-1 and PU.1.

Methods:
We used mouse erythroleukemia (MEL) cells expressing 17-OH-estradiol-inducible transgenes of GATA-1 (GER) or PU.1 (PUER). mRNA was quantitated by RT-PCR and protein occupancy on DNA was determined by chromatin immunoprecipitation (ChIP). Downregulation of Dmt1 was achieved by siRNA.

Results:
Using ChIP we established that GATA-1 and PU.1 regulate Epor gene directly. By activating GER or PUER in MEL cells we observed that promoter region of Epor is enriched and depleted respectively by acetylated histone H3 lysine (K) 9. Furthermore, inhibition of Epor by PU.1 coincided with recruitment of PU.1 to the Epor promoter. Similar ChIP analyses of Dmt1 promoter are undergoing. We next activated GER in MEL cells in the presence or absence of Dmt1 siRNA and observed that Dmt1 expression is needed for GATA-1-dependent upregulation of Epor mRNA expression during erythroid differentiation.

Conclusion:
We found that GATA-1 stimulates both Epor and Dmt1 expression and that PU.1 blocks directly these effects. We also found that erythroid transcriptional regulation of Epor via transcription factor GATA-1 is severely diminished in Dmt1-knockdown MEL cells. We are currently testing whether this effect involves abnormal transport of iron or other divalent metals.
(Grant support: P305/11/1745, P301/12/P380, P305/12/1033. Institutional funding: PRVOUK-P24/LF1/3, UNCE 204021, SVV-2012–264507, GAUK 251135 82210).

Disclosures:
No relevant conflicts of interest to declare.
Next-Generation Sequencing: A Discovery Tool for Blood Disorders
Authors: Mardis Elaine R. , Ding Li , Westervelt Peter , Welch John S. , Klco Jeffery M. , DiPersio John F. , Wilson Richard K , Ley Timothy J. 
Published: October 2019

Abstract
Abstract SCI-10
The advent and evolution of next-generation massively parallel sequencing (MPS) has radically altered our approaches to studying the cancer genome and transcriptome. By using unbiased and comprehensive MPS in the context of clinically annotated samples from leukemia cases, rapid progress has resulted in our understanding of the mutational spectrum of hematopoietic malignancies, the heterogeneity of disease presentation, and the impact of chemotherapy on the cancer genome, among others. The complexity of the transcriptome, while daunting from an analytical standpoint, further reveals the nuances of gene expression changes in leukemia that often cannot be predicted simply by studying the genome. By applying the digital nature of MPS to explore tumor heterogeneity and tumor evolution, we have shown that de novo acute myeloid leukemia (AML) presents either as a mono- or multiclonal disease, and that the relapse presentation in the same patient is an evolved genetic derivation of the de novopresentation, often with novel driver mutations that have been acquired during the course of chemotherapy (1). New data from whole genome sequencing of hematopoietic stem cells in healthy volunteers indicates that somatic mutations largely are acquired during aging and occur randomly, carrying forward in the transformed cell. This baseline is important for the further comparison of AML subtypes, and provides a context for understanding tumor biology. Last, by studying 200 AML cases using whole-genome and exome sequencing, RNA-seq, miRNA-seq and array-based methylation, we have begun an integrated characterization of AML in an effort to inform tumor biology. These studies and the accompanying technologies set the stage for precision treatment of each AML patient according to the additional information provided by the person's integrated “omic” profile.

Disclosures:
No relevant conflicts of interest to declare.
von Willebrand Factor-ADAMTS13 Interactions
Authors: Zheng X. Long 
Published: October 2019

Abstract
Abstract SCI-18
ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) is a plasma metalloenzyme that cleaves von Willebrand factor (vWF), thereby limiting excessive platelet adhesion and aggregation and attenuating the rate of thrombus formation at the site of injury. Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome, while mild to moderate deficiency of plasma ADAMTS13 activity is associated with an increased risk for myocardial infarction and stroke. Adamts13 null mice in the ApoE null background exhibit a dramatically increased systemic inflammation and early atherosclerosis. Thus, ADAMTS13 plays a crucial role in pathogenesis of arterial thrombotic disorders such as TTP and chronic inflammatory diseases such as atherosclerosis. vWF is the only known substrate for ADAMTS13 to date. Structure-function analysis demonstrates that the N-terminal portion of ADAMTS13 (MDTCS) is sufficient for proteolytic cleavage of vWF, while the C-terminal portion of ADAMTS13 (T2–8 and CUB) inhibits shear-dependent platelet aggregation via free thiols (-SH). The proteolytic cleavage of vWF by ADAMTS13 is dramatically accelerated by binding of Factor VIII (FVIII) and/or platelets under shear stress. A B-domainless FVIII (FVIII-SQ) or a light chain of FVIII (FVIII-LC) appears to be sufficient for the enhanced proteolysis of vWF by ADAMTS13 in vitro and in vivo using a murine model. In contrast, a heavy chain of FVIII (FVIII-HC) or a light chain lacking an acidic region (a3) (FVIII-LC#x2610;¢a3) exhibits no rate enhancing effect under the same conditions. These results suggest that the light chain, particularly the acidic (a3) region that contains a high-affinity vWF binding site is required for the cofactor activity in accelerating vWF proteolysis. Consistent with this hypothesis, mutations in the D'D3 domain of vWF naturally occurring in patients with type 2N von Willebrand disease that reduces FVIII binding also impair the FVIII-dependent proteolysis by ADAMTS13 under fluid shear stress. To develop novel therapeutics, we have constructed, expressed, and screened for a panel of ADAMTS13 variants with a modification of the exosites (motif A and motif B) in the spacer domain. We identified several ADAMTS13 variants that have enhanced specific activity and are also resistant to inhibition by autoantibodies against ADAMTS13 in patients with acquired idiopathic TTP. These gain-of-function and autoantibody-resistant ADAMTS13 variants may be further developed as potential therapeutics for acquired TTP with inhibitors.

Disclosures:
No relevant conflicts of interest to declare.
Discovery Science and Integrating Molecular Diagnosis into Clinical Practice
Authors: Ebert Benjamin L. 
Published: October 2019

Abstract
Abstract SCI-31
Recent discoveries have yielded profound insights into the genetic basis of myeloid malignancies. A central challenge is now to translate these discoveries into clinical practice. Genetic lesions are powerfully associated with specific aspects of clinical phenotype, including hematologic parameters, particular morphologic abnormalities, response to therapy, and overall survival. Moreover, gene expression profiling, evaluation of microRNA expression, and epigenetic analyses add further biological, prognostic, and predictive information. Current research studies aim to integrate existing clinical prognostic scoring systems with molecular data to refine the taxonomy of myeloid malignancies, to improve the prediction of prognosis and response to specific therapies, and to develop novel therapeutic strategies based on insights into disease biology.

Disclosures:
Ebert: Celgene: Consultancy; Genoptix: Consultancy.
Induced Pluripotent Stem Cells and Erythrocyte Production
Authors: Slukvin Igor 
Published: October 2019

Abstract
Abstract SCI-38
Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups.

Disclosures:
Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Inflammation, Obesity, and Thrombosis
Authors: Samad Fahumiya , Badeanlou Leylla , Furlan-Freguia Christian , Yang Guang , Ruf Wolfram 
Published: October 2019

Abstract
Abstract SCI-45
Clinical and epidemiological studies have long established a connection between obesity and thrombosis leading to increased cardiovascular complications, in part attributed to elevated expression of the prothrombotic molecules plasminogen activator inhibitor 1 and circulating tissue factor (TF). Obesity also increases the risk for metabolic dysfunction, and emerging evidence indicates that coagulation proteases offer a range of signaling pathways that control multiple aspects of the metabolic syndrome, including weight gain, insulin resistance, type 2 diabetes, and hepatic steatosis. While adipose inflammation due to recruitment and activation of macrophages and secretion of inflammatory cytokines promotes systemic hypercoagulability and insulin resistance, pathways that sustain adipose macrophage inflammation in obesity are unclear. Our studies suggest that TF signaling via the G-protein-coupled receptor, protease activated receptor 2 (PAR2), represents a novel link between obesity, coagulation, and associated adipose inflammation and insulin resistance. Mice lacking the cytoplasmic domain of TF or PAR2 were protected from high fat diet-induced weight gain, adipose inflammation, and insulin resistance. Genetic loss of TF cytoplasmic domain and PAR2 in myeloid cells attenuated adipose macrophage inflammation and increased insulin sensitivity, an effect that was also achieved by acute pharmacologic inhibition of TF-PAR2 signaling in macrophages. In contrast, TF signaling in nonhematopoietic cells specifically promoted obesity via its effects on energy metabolism. In adipocytes, TF-VIIa signaling suppressed basal and insulin-mediated AKT phosphorylation, with concordant transcriptional changes in genes regulating lipid and glucose metabolism. Thus, the procoagulant state induced by obesity can in fact contribute to the metabolic syndrome, and adipose TF signaling may be at the intersection between obesity, inflammation, and thrombosis.

Disclosures:
No relevant conflicts of interest to declare.
Intestinal Microbiota in Bone Marrow Transplantation
Authors: Jenq Robert R. 
Published: October 2019

Abstract
Abstract SCI-51
Regulators of the intestinal flora include diet, antibiotics, and importantly, intestinal inflammation. As a result, the cause-effect relationships between intestinal inflammation and changes in microbiota have been difficult to define. The success of allogeneic bone marrow transplantation (allo BMT), a standard therapy for conditions including hematopoietic malignancies and inherited hematopoietic disorders, is limited by graft-versus-host disease (GVHD) morbidity and mortality. With GVHD, vigorous activation of donor immune cells, most importantly T cells, leads to damage of the skin, liver, hematopoietic system, and gut. The major sources of immune activation are histocompatibility complex differences between donor and recipient. Combinations of chemotherapy and radiation also contribute, as damage to the intestinal epithelium results in systemic exposure to microbial products normally sequestered in the intestinal lumen. We have demonstrated in murine and human recipients of allo BMT that intestinal inflammation secondary to GVHD is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice prior to BMT aggravated GVHD, while reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized the gut flora of patients during onset of intestinal inflammation due to GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos soon after allo BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation, and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allo BMT recipients.

Disclosures:
No relevant conflicts of interest to declare.