bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Red Cells in Thrombosis and Hemostasis
Authors: Weisel John W. 
Published: October 2019

Until recently it has been assumed that red blood cells (RBCs) play mainly a passive role in hemostasis and thrombosis, but now some studies have uncovered several novel roles for them in both of these processes. The best-known effects of RBCs in clotting in vivo are rheological, involving laminar shearing plus aggregation and deformability of RBCs. In addition, RBCs have specific receptors for platelets and fibrin(ogen) that may be involved in thrombosis. Furthermore, in the contraction of blood clots, the trapping of RBCs in the platelet-fibrin network and subsequent physical changes may be important for hemostasis and thrombosis. Contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral RBCs within. The structure and properties of contracted clots vary depending on the relative amounts of platelets, fibrinogen and RBCs and the conditions of clotting. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, have also been observed in human arterial and especially venous thrombi taken from patients. The mechanical nature of this RBC shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Many experiments suggest that polyhedrocytes are more prevalent under conditions associated with thrombosis and could be a marker of prothrombotic conditions. Consistent with this hypothesis, RBC membranes have been found to be a major source of exposure of phosphatidylserine, which forms a procoagulant surface to generate thrombin. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from RBCs and to compress these cells into a tightly packed array. Clots formed after puncture of the mouse saphenous vein are composed primarily of polyhedrocytes, suggesting that they may be important in venous hemostasis. Moreover, the permeability of contracted clots is nearly the same as that of the endothelial cell lining, consistent with their functional significance. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and also to restore flow past obstructive thrombi. On the other hand, they also help explain how fibrinolysis is greatly retarded after clots contract, as a result of these striking structural changes. In summary, RBCs may perform a dual role, both helping to stem bleeding but at the same time contributing to thrombosis in a variety of ways, including when the tightly packed array of RBCs in a contracted clot blocks blood flow and resists dissolution.

Disclosures
No relevant conflicts of interest to declare.
Therapeutic Strategies for the Treatment of Telomere-Associated Diseases
Authors: Blasco Maria A. 
Published: October 2019

Abstract
Aplastic anemia is a rare but life-threatening disorder often accompanied by the presence of short telomeres in the hematopoietic compartment in both the acquired and the inherited form. We recently generated a mouse model that faithfully recapitulates the bone marrow phenotype observed in aplastic anemia patients. In this model, incomplete conditional Trf1 gene deletion specifically in the hematopoietic system causes acute telomere uncapping and persistent activation of a DNA damage response at telomeres, leading to a fast elimination of those HSCs lacking Trf1 and a compensatory hyperproliferation along with rapid telomere shortening in those which retain intact Trf1. Thus, this model recapitulates the high turnover and hyperproliferation observed in aplastic anemia patients of autoimmune origin, as well as presence of very short telomeres owing to mutations in telomere maintenance genes. We are currently employing this model to test two different therapeutic strategies for the reactivation of telomerase in the hematopoietic system, therefore counteracting telomere attrition and alleviating the aplastic anemia phenotype owed to the presence of short telomeres. First, we explored the possibility that the recently described regulation of telomerase activity by sex hormones may be the responsible mechanism for the frequently observed induction of remission in aplastic anemia patients following hormone therapy. We show that androgen therapy results in telomerase up-regulation, improved blood counts, and a significant extension of life-span in our mouse model. Importantly, longitudinal follow-up studies revealed longer telomeres in peripheral blood in mice subjected to hormone treatment. In addition to this chemical approach for telomerase activation we are also testing the efficacy of increasing telomerase gene dosage by virtue of AAV9 gene therapy. Our preliminary data indicate that AAV9-Tert treatment leads to a significantly reduces aplastic anemia related morbidity which coincides with telomere elongation in blood and bone marrow cell compared to empty vector treated mice. In summary, we used a mouse model of aplastic anemia produced by short telomeres and demonstrate two different strategies of telomerase activation which are effective in treating the disease. Thus this work may pave the way for the development of novel treatment options for aplastic anemia in humans.

Disclosures
No relevant conflicts of interest to declare.
Clonal Heterogeneity and Genetic Instability of Multiple Myeloma and Impact on Treatment
Authors: Keats Jonathan J 
Published: October 2019

Multiple myeloma is a pathological definition for a series of distinct genetic entities with similar phenotypic and clinical characteristics. Multiple studies have now identified distinct subtypes of the disease, which are associated with different clinical outcomes. The genetic complexity underlying these different subtypes is very diverse. Some subtypes like those characterized by immunoglobulin translocations targeting cyclin D1 are associated with very minimal changes or no copy number changes. Conversely, subtypes defined by translocations targeting WHSC1/MMSET or the MAF and MAFB transcription factors often have highly complex and diverse copy number changes. With the substantial advances in DNA sequencing technology we now know there is a diverse array of somatic mutations in multiple myeloma tumors. Through the integration of copy number changes and somatic mutations multiple groups have now shown the existence of multiple co-existing subclones within individual tumors. Additional studies following patients through their individual disease courses have shown these subclones can ebb and flow with time through multiple rounds of therapeutic selection. This session will highlight our current understanding of how the interplay between tumor evolution and clonal heterogeneity should influence our treatment decisions, particularly when applying a personalized medicine approach.

Disclosures
No relevant conflicts of interest to declare.
Checkpoint Blockade with Neoantigen Cancer Vaccines for Personalized Immunotherapy
Authors: Wu Catherine J. 
Published: October 2019

Methods for harnessing the exquisite specificity of the immune system to eliminate tumors have been under development since the start of the 20th century, with numerous studies in animal models and trials in humans. Despite accumulating evidence, there has been widespread skepticism in the cancer community about whether tumors could be controlled by spontaneous immunity, and whether effective immunotherapy would be feasible to develop. However, the field has now passed an inflection point as new results have quelled these doubts, including: (i) several large clinical studies that have identified infiltrating CD8+ T cells as one of the best predictors of overall survival in human cancers, with strong validation in a large study of colorectal cancers; (ii) adoptive T cell therapy that effectively eradicates some types of B cell leukemias and (iii) novel checkpoint blockade (CPB) therapies inducing unprecedented long-term cures for patients with solid tumors, and more recently with blood malignancies. With the availability of these novel immunologic agents, the priority has shifted to understanding the mechanisms of and predicting responses to each treatment. Fundamental to this endeavor is the understanding of the crosstalk between tumor cells and immune cell populations within the tumor microenvironment and the selective pressure they mutually exert on each other. At the heart of cancer and host immune cell coevolution is the tumor antigen and host antigen-specific T cell interaction. The cytotoxic T cell-cognate antigen interaction forms the mechanistic basis for immune-mediated recognition and the killing of malignant cells, and provides selective pressure on the cancer cell to evolve. While the search for immunogenic tumor antigens has been the subject of decades-long studies, multiple lines of evidence have convincingly demonstrated tumor neoantigens as an important class of immunogenic tumor antigens. Neoantigens arise from amino acid changes encoded by somatic mutations in the tumor cell and have the potential to bind to and be presented by personal HLA molecules. With the recent availability of next-generation sequencing, systematic approaches to identify mutations leading to amino acid changes have become feasible. This development, together with the improved sensitivity of well-validated neural network/machine learning-based algorithms that predict binding to class I MHC, has enabled the implementation of neoantigen discovery pipelines. Recent studies by us and others in humans and mice have leveraged these tools to demonstrate evidence of protective immunity when targeting tumor neoantigens. We have further incorporated this approach to discover antigen targets to generate a multi-epitope personalized therapeutic cancer vaccine, that we are now testing in phase I clinical trials. Altogether, effective targeting of tumor neoantigens occurs spontaneously and during CPB, and offers prospects of a powerful, new immunotherapy.

Disclosures
No relevant conflicts of interest to declare.
High Resolution Mapping of Human Lymphopoiesis Reveals a Common Lymphoid Progenitor (CLP) Population
Authors: Saxena Nivedita Shankar , Davis Kara L. , Inlay Matthew Arthur , Junger Astraea , Yang Yujun , Krampf Mark L. , Dejene Beruh , Nolan Garry P. , Weissman Irving L , Weinberg Kenneth I. 
Published: October 2019

Abstract
In vivo reconstitution studies have established the phenotype of murine CLP, a progenitor population with restricted developmental potential for T, NK, B lymphoid and plasmacytoid dendritic cell (pDC) lineages. Co-transplantation of murine CLP with purified HSC more rapidly restores lymphopoiesis, immune function and resistance to viral challenge in experimental models of HSC transplantation. Thus, the identification of human CLP would have translational impact in clinical HSCT. Based on the expression of IL-7Rα (CD127) as a key distinguishing marker of murine CLP, we studied the CD127+ subset of immature CD34+ Lin- CD38+ cells for lymphoid committed progenitors, comprising approximately 0.1-0.2% of total marrow. Like murine CLP, the CD34+ Lin- CD38+ CD127+ cells also expressed Kit and Flt3. OP9 stromal cells were transduced with a vector expressing a doxycycline inducible delta-like ligand 1 (dll1) gene to test the differentiation potential in a single-well switch culture. Progenitors co-cultured with uninduced OP9Δdll1 cells can differentiate into B and myeloid cells; after induction of dll1, the culture is permissive for T and NK differentiation in the same well. In the presence of Kit and Flt3 ligands, erythromyeloid growth factors (IL-6, IL-3, GM-CSF, EPO, TPO) and lymphoid factors (IL-7, IL-15), CD34+ Lin- CD38- CD127- HSC generated erythroid (E), myelomonocytic (MM), myeloid DC (mDC), T, NK, and B lymphocytes and pDC. In contrast, CD34+ Lin- CD38+ CD127+ cells generated T/NK/B/pDC progeny, distributed in varying proportions, but did not generate E/MM/mDC. Singly plated CD34+ Lin- CD38+ CD127+ cells generate T/NK/B cells in the same well, consistent with these cells being human CLP. CD34+ Lin- CD38+ CD127- "non-CLP" generated E/MM/mDC, but not T/NK/B/pDC progeny. Limiting dilution analyses under lymphoid conditions demonstrated that the CD34+ Lin- CD38+ CD127+ CLP had approximately 2-3-fold greater efficiency of lymphocyte generation than HSC under the same conditions. Transcriptional profiling of the human CLP showed patterns of gene expression distinct from that of HSC, CD34+ Lin- CD38+ CD127- progenitors and immature CD3-CD4-CD8- thymocytes. Approximately 50% of the CD34+ Lin- CD38+ CD127+ CLP also express CD10, a subpopulation which CyTOF analyses revealed to have other markers of B-cell commitment and/or differentiation, e.g., CD179a. Human CD34+ Lin- CD38+ CD127+ marrow cells are a unique multipotent, lymphoid-restricted progenitor population for T/NK/B/pDC cell development, and within which subsets of cells biased for individual lymphoid lineages may exist. Identification of human CLP will permit further analyses of lymphoid specification, commitment to lymphoid lineages, and potential adoptive transfer as a short-term source of polyclonal lymphocytes.

Disclosures
Nolan: Fluidigm: Consultancy.
Dual-Method Clone Tracking in Nonhuman Primates Confirms Long-Term Hematopoietic Reconstitution Initiated By Early Engrafting Clones
Authors: Adair Jennifer E , Norgaard Zachary K. , Haworth Kevin G , Schefter Lauren E , Tam Kenric , Porteus Matthew , Kiem Hans-Peter 
Published: October 2019

Abstract
Summary:
Several models of hematopoietic reconstitution after transplant have been proposed with multiple methods of tracking individual clones in both animal models and patients. Lentivirus-mediated gene therapy is the most widely applied strategy for marking individual cells, tracked by either provirus integration site analysis (ISA) or by proviral DNA barcode sequencing (DBS). Two previous studies have applied one of each of these methods to track hematopoiesis in a nonhuman primate model of myeloablative autologous transplantation. The report using ISA described long-term, multi-lineage hematopoietic clones emerging at 1 year after transplantation [Kim S. et al., Cell Stem Cell, 2014]. The report using DBS described only early engraftment up to 9 months after transplant, precluding long term analysis [Wu C. et al., Cell Stem Cell, 2014]. We applied a combination of ISA and DBS methods to follow longitudinal hematopoiesis in a total of six nonhuman primates following autologous transplant in the myeloablative setting, tracking >120,000 individual clones for up to 10 years. We measured hematopoietic lineage reconstitution, as well as spatial and temporal contributions of individual clones within the bone marrow niche and peripheral blood. Here we applied a stringent definition of the hematopoietic stem cell (HSC) phenotype in vivo (i.e. shared clonal identity between short-lived granulocytes and long-lived B cells with multiple time points of detection), to assess HSC contributions after engraftment. Strikingly, we demonstrate sustained contribution of HSC clones occurs as early as 1 month after transplant and that minor clonal waves continuously emerge when the measureable clonal pool is small in size, but not when the initial engrafting clonal pool is robust. Nearly one half of all HSC clones identified over the length of follow-up were detectable in the periphery within a few months after transplant by both ISA and DBS methods. However, we also identify technical differences between these clone tracking methods that can obscure biological interpretation. This study demonstrates an early and robust phase of multi-lineage hematopoietic reconstitution after myeloablative transplantation in the nonhuman primate, suggesting a single subpopulation of CD34+ cells which maintains clonal stability over the lifetime of the recipient in the autologous setting.

Disclosures
Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.
GATA Factor Switching during Erythroid Differentiation Is Facilitated By FBW7 Mediated Clearance of GATA2
Authors: Ghamari Alireza , Pregerning Gabriela , Fraenkel Ernest , Cantor Alan B. 
Published: October 2019

Abstract
Erythroid differentiation is controlled by the dynamic exchange of GATA family transcription factors. During early erythroid maturation, high GATA2 levels activate progenitor genes such as c-kit-, c-myc, and GATA2 itself. In contrast, GATA1 levels are low in early progenitor cells, but rise during terminal maturation. During this process GATA1 turns off GATA2 controlled early progenitor genes and activates terminal maturation genes, such as globin genes, heme biosynthesis enzymes, and iron transporters. This involves the exchange of GATA1 for GATA2 at key chromatin sites, the so-called "GATA factor switch". GATA factor switching is facilitated by the much shorter half-life of GATA2 (~30-60 min) compared to GATA1 (>4-6 hrs). We and others recently demonstrated that the E3 ubiquitin ligase adaptor protein FBW7 contributes to GATA2's relative instability. This prompted us to dissect the role of FBW7 during GATA switching and erythroid differentiation. We deleted the Fbw7 gene using CRISPR/Cas9 gene editing in the inducible G1-ER murine erythroid cell line. This resulted in the delayed clearance of GATA2 during differentiation. RNA-seq analysis at an early time points (7 hr) demonstrated impaired repression of GATA2 regulated genes and reduced activation of GATA1 target genes. Globally, altered gene expression was enriched for GATA factor switch genes. This ultimately resulted in delayed erythroid maturation. We also found that Fbw7 mRNA transcript levels increase during erythroid maturation in wild type cells. We identified a site ~40kb upstream of the Fbw7 gene transcriptional start site, which is itself a GATA factor switch site. We propose that FBW7 facilitates GATA factor switching by promoting the clearance of GATA2 from GATA factor switch sites. Moreover, we suggest that GATA factor switching at the Fbw7 locus itself reinforces the commitment of erythroid cells to terminal maturation, by enhancing the clearance of GATA2 and other Fbw7 progenitor target gene proteins such as c-Myc and c-Myb. As Fbw7 recognition of GATA2 requires phosphorylation of GATA2's degron motif, this suggests that signaling pathways, acting through Fbw7, may modulate erythroid maturation kinetics.

Disclosures
No relevant conflicts of interest to declare.
Deletion of Pleiotrophin in LepR+ Perivascular Cells Depletes Hematopoietic Stem Cells
Authors: Himburg Heather A , Chang Vivian Y. , Sasine Joshua , Kan Jenny , Zhao Liman , Chute John P. 
Published: October 2019

Abstract
Pleiotrophin (PTN) is a heparin binding growth factor which is expressed by bone marrow vascular endothelial cells (BM ECs) and perivascular stromal cells. Treatment of murine or human HSCs ex vivo promotes HSC expansion (Nat Med. 2010 Apr;16(4):475-82) and constitutive deletion of PTN depletes LT-HSCs in steady state and markedly impairs HSC regeneration following myeloablation (Cell Rep. 2012 Oct 25;2(4):964-75; JCI. 2014;18(7):1123-1129). Here, we sought to determine which BM microenvironment cell is responsible for PTN-mediated maintenance of the HSC pool. Utilizing the Cre-loxP system, we deleted PTN from VE-cadherin+ ECs, leptin receptor+ (lepR+) perivascular stromal cells, osteocalcin+ osteoblasts, and vav1+ hematopoietic cells and examined the effects on hematopoiesis. We observed no differences in steady state hematopoiesis or HSC content as measured by long-term competitive repopulation assays in mice lacking PTN expression in osteocalcin+ cells, vav1+ hematopoietic cells or VE-cadherin+ BM ECs. However, deletion of PTN from lepR+ BM perivascular cells caused a significant decrease in BM c-kit+sca-1+lin- cells (KSL cells) and BM SLAM+KSL HSCs, and colony forming cell (CFC) content compared to PTN+/+ controls (*p = 0.04, 0.04, and 0.001, respectively). Importantly, deletion of PTN in lepR+ cells, caused a significant reduction in long-term HSC content as measured by primary and secondary competitive repopulation assays (*P<0.01 for all time points through 20 weeks). These data suggest that LepR+ BM perivascular cells, rather than VE-cadherin+ ECs are the primary source of PTN in the BM niche which contributes to the maintenance of the HSC pool.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Chronic Pancytopenia and Increased Bone Density Due to TBXAS1 Deficiency
Authors: Sharma Richa , Schwartz Jennifer E. , Nalepa Grzegorz 
Published: October 2019

Abstract
Non-dysmorphic 32-year old Caucasian female developed malaise, fluctuating body aches and progressive transfusion-dependent pancytopenia with peripheral blood morphology consistent with myelodysplastic syndrome (MDS). Reportedly, she had an episode of "low blood counts" of unclear etiology 23 years prior. Multiple attempts at bone marrow biopsy were unsuccessful due to technical difficulties with penetrating the bone, necessitating surgical biopsy by an orthopedic surgeon. Bone densitometry revealed increased bone density with Z-score of 4.5, consistent with mild thickening of long bone diaphysis noted via x-ray. Strikingly, the bone marrow biopsy revealed haphazard ossification, immature cartilage formation and thickened trabeculae replacing hematopoiesis within the marrow cavity. Next-generation sequencing revealed two novel biallelic mutations within the thromboxane synthase gene TBXAS1 (c.266T>C; c.989T>C). Both mutations affected conserved amino acid residues and were predicted to be disruptive to the protein function by two independent bioinformatics algorithms (Provean and PolyPhen-2).
Thromboxane cascade regulates pathways of inflammation, coagulation and osteoclast activation, suggesting a mechanistic explanation of how thromboxane synthase malfunction causes abnormal bone remodeling and bone marrow failure. TBXAS1 disruption had been described in a rare autosomal recessive syndrome Ghosal hematodiaphyseal dysplasia (GHDD) associated with increased bone density and bone marrow failure. GHDD due to TBXAS1 mutations was genetically confirmed only in four families of Northern African and Middle Eastern descent (Nat Genet 2008; 40: 284-286). To our knowledge, this is the first genetically validated case of TBXAS1-/- Ghosal syndrome in the Caucasian population.
Consistent with the well-established role of thromboxane in platelet function, our patient's platelets displayed impaired aggregation in response to arachidonic acid and structural abnormalities by transmission electron microscopy, revealing a functional platelet defect. Steroid therapy rendered the patient transfusion-independent and alleviated chronic pain and inflammation. A long-term follow-up will be needed to determine whether steroid treatment will result in stable improvement in hematopoiesis and decreased bone density, similar to other GHDD patients. This case illustrates how establishing the diagnosis of an underlying rare inherited bone marrow failure syndrome allows personalized, evidence-based treatment with lower risk of morbidity compared to cytotoxic chemotherapy and hematopoietic stem cell transplantation.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.