bluuebunny/crossref_metadata_2025_split huggingface dataset:
Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice
Authors: Bergmeier Wolfgang , Paul David S , Stefanini Lucia , Robledo Raymond F. , Chan E. Ricky , Getz Todd M , Piatt Raymond , Boulaftali Yacine , Adams Mark D , Peters Luanne L. 
Published: October 2019

Abstract

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Disclosures
No relevant conflicts of interest to declare.
Epithelial (E)-Cadherin Is a Novel Regulator of Platelet Function
Authors: Krause Diane , Halene Stephanie , Booth Carmen Jane , Jin Huiyan , Zou Siying , Teixeira Alexandra M , Zhang Ping-Xia 
Published: October 2019

Abstract
Epithelial (E-) cadherin is an adhesion molecule that mediates cell-cell interactions, and is important in pluripotent stem cell reprogramming. We are investigating the role of E-cadherin in megakaryocyte differentiation and platelet function, and propose that E-cadherin mediates interactions that facilitate the essential roles of platelets. We have evidence that mature megakaryocytes and platelets express E-cadherin, and therefore generated a megakaryocyte-specific E-cadherin knockout mouse using the PF4-Cre system. E-cadherin deleted mice are viable and fertile. Despite having normal platelet counts and mean platelet volume, platelet function is abnormal. E-cadherin deletion in platelets significantly increases bleeding time in adult mice, with wild type (Ecadw/w) bleeding times of 225±52 secs, and homozygous deletion (Ecadf/f) times of 880±134 secs (p=0.005). In vivo platelet depletion using systemic administration of anti-CD42b antibody in the Ecadf/f mice causes death, likely due to hemorrhage and failure of hemostasis, which is not observed in Ecadw/w with similar levels of platelet depletion, suggesting a platelet function defect that becomes more evident under stress. We performed immunofluorescence to probe platelet structure as a potential explanation for the phenotypes, and observed disrupted b-tubulin architecture in the E-cadherin null platelets. Also, static platelet adhesion assays revealed that E-cadherin deficient platelets have impaired adhesion on fibrinogen, relative to a BSA substrate (p=0.0005). We have extended our studies to the human system, and initial studies using an E-cadherin blocking antibody in human platelets show impairment of aggregation in response to ADP, epinephrine, and thrombin. Taken together, the results demonstrate that E-cadherin contributes to the delicate balance between bleeding, hemostasis, and thrombosis. Future studies will focus on identifying how E-cadherin regulates hemostasis, with an emphasis on the interactions mediated by E-cadherin, whether between platelets, or with other cells in the blood.

Disclosures
No relevant conflicts of interest to declare.
Basic Mechanisms of Arterial and Venous Thrombosis
Authors: Renné Thomas 
Published: October 2019

Abstract
Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. This system starts a cascade of procoagulant and proinflammatory reactions via the intrinsic pathway of coagulation, and the bradykinin producing kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood, however its in vivo functions are just beginning to emerge. This presentation will summarize roles of the FXII-driven contact system in vivo. Genetically altered mice and large animal models have shown that FXII is essential for thrombus formation while being dispensable for hemostatic processes that terminate blood loss. Challenging the dogma of a coagulation balance, targeting FXII protected from cerebral ischemia without interfering with hemostasis. In contrast, excess FXII activity is associated with a life threatening inflammatory disorder, hereditary angioedema. Platelet polyphosphate (an inorganic polymer), neutrophil extracellular traps (NETs) and mast cell heparin activate FXII with implications on the initiation of thrombosis and edema. A key aspect of the talk will be the analysis of common principles, interactions and cross-talk between coagulation and inflammation, and the use of the novel FXII blocking antibody 3F7 in cardiopulmonary bypass system. Elucidating the FXII-driven contact system offers the exciting opportunity to develop strategies for safe interference with both thrombotic and inflammatory diseases.

Disclosures
No relevant conflicts of interest to declare.
How Do We Define the Genomic Landscape?
Authors: Dave Sandeep S. 
Published: October 2019

High throughput sequencing is a revolutionary technology for the definition of the genomic features of tumors. This talk will provide a review of the relevant methodologies for non-experts in the field. The presentation will include a discussion of how high throughput sequencing is performed, its relative strengths and weaknesses, and how it is applicable to formalin-fixed and fresh/frozen tissue samples. The talk will also describe future directions in the genomic analysis of tumors.

Disclosures
No relevant conflicts of interest to declare.
Clinical Implications of the New Molecular Knowledge for Treatment of Diffuse Large B-Cell Lymphoma
Authors: Friedberg Jonathan W. 
Published: October 2019

For more than 35 years, the standard induction therapy for patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Until recently, efforts to improve upon this regimen were empiric and without success, and consisted of adding additional cytotoxic drugs, intensifying doses and schedules of drugs, and consolidating initial responses with high dose therapy and autologous stem cell support. The addition of rituximab to standard CHOP therapy (RCHOP) more than a decade ago improved progression-free and overall survival, and now the majority of patients presenting with DLBCL are cured with induction therapy. Biological insights from gene expression profile (GEP) studies indicate substantial heterogeneity of DLBCL, which is largely responsible for treatment failure. We are now at the cusp of being able to utilize rational targeted therapies in biologically-defined subgroups of patients of DLBCL. In addition to cell-of-origin defined subgroups, GEP has defined stromal signatures and pathway-specific signatures in DLBCL which also predict outcome and potentially define a group of patients for a biologically-targeted therapy approach. At present, clinical development strategies include combining novel agents with standard RCHOP treatment and/or evaluating novel agents as maintenance or consolidation after RCHOP treatment. Two recent failures illustrate the challenges inherent in these approaches: RCHOP + bevacizumab (targeting angiogenesis) and R-CHOP followed by enzastaurin (targeting protein kinase C-b). Ongoing randomized trials are evaluating R-CHOP + bortezomib (proteasome inhibitor targeting NFkB), R-CHOP + ibrutinib (Bruton’s tyrosine kinase inhibitor targeting B-cell receptor signaling pathway), and R-CHOP + lenalidomide (immunomodulator with pleotropic effects) in specific GEP (or immunohistochemistry surrogates for GEP)-defined groups of patients. Finally, the majority of treatment failures in DLBCL appear to occur in patients with overexpression of c-MYC oncogene (often in combination with BCL-2 protein), which represents up to one third of patients when defined using immunohistochemistry. This unique subgroup occurs across cell-of-origin designations, and represents a substantial opportunity to improve outcome in DLBCL with the addition of novel therapeutics. This talk will review the considerable challenges in translating the exciting biology defined by GEP into the clinic for patients with DLBCL, provide an update on current and future trial designs, and conclude with a forward looking time to when GEP, or an accurate surrogate, is a component of the standard diagnostic algorithm to define optimal therapeutic approach to DLBCL.

Disclosures
Off Label Use: Novel agents for DLBCL.
Platelets Induce Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury
Authors: Looney Mark R. 
Published: October 2019

Abstract
Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality in the U.S. and a major cause of transfusion-associated morbidity including increased time on mechanical ventilation and length of stay in the intensive care unit and the hospital. Neutrophils have been identified as critical cellular mediators in the pathogenesis of TRALI in both clinical studies and in experimental settings using a variety of injury models. Platelets have been implicated as a blood product that can trigger TRALI, and endogenous platelet activation contributes to lung injury. Platelets bind to the surface of neutrophils to form heterotypic aggregates, and activated platelets can trigger the formation of neutrophil extracellular traps (NETs), which is a new mode of neutrophil death that is distinct from apoptosis and necrosis. NETs are produced in experimental TRALI and are increased in post-transfusion plasma from patients who develop TRALI. Blocking platelet activation reduces the production of NETs and lung injury, and inhibiting NETs by blocking extracellular histones or dismantling the NET structure with DNase1 are strongly protective in TRALI. In conclusion, TRALI is an immune-mediated event in which activated platelets, neutrophils, and NETs, contribute to injury and are therefore targets for therapeutic intervention.

Disclosures
No relevant conflicts of interest to declare.
Inherited Predisposition to Myelodysplastic Syndrome and Acute Leukemia
Authors: Churpek Jane E. 
Published: October 2019

Although generally thought of as pediatric conditions, inherited forms of myelodysplastic syndrome (MDS) and acute leukemia (AL) are increasingly recognized among adult patients. At present, at least nine genes, including ANKRD26, CEBPA, GATA2, PAX5, RUNX1, SRP72, TERC, TERT, and TP53, are known to cause familial MDS and/or AL syndromes. Several other promising candidate genes are emerging from ongoing research on the many pedigrees identified without a mutation in one of these already recognized genes. Clinical recognition of individuals with these syndromes is essential for optimal care of the patient and his/her at-risk family members and requires familiarity with the subtle clinical features of each syndrome and a high index of suspicion by the treating physician. Once recognized, genetic testing should be performed to identify the specific syndrome present as each can have unique aspects to their clinical care. For example, individuals with familial MDS/AL due to TERT or TERC abnormalities require monitoring of lung function and screening for head and neck and anogenital cancers, whereas individuals with platelet dysfunction due to familial platelet disorder/RUNX1 or ANKRD26 mutations require careful planning prior to surgical procedures to prevent bleeding complications. Due to significant overlap in the clinical presentation, often a multigene-based approach to genetic testing is necessary. Unique aspects of genetic testing in this population include: 1) tissue type selection as many of the genes that cause the familial MDS/AL syndromes are also somatically mutated in hematologic malignancies so results from DNA derived from peripheral blood or bone marrow in an individual with MDS or AL are difficult to interpret; and 2) urgency as allogeneic hematopoietic stem cell transplantation may be pursued quickly and requires knowledge of the specific mutation in the family to identify the optimal stem cell donor. The management of affected individuals who have not yet developed hematologic malignancies can be challenging as many may show morphologic signs of dysplasia in the bone marrow that may not truly represent overt malignancy. The decision of when to pursue allogeneic hematopoietic stem cell transplantation with curative intent is especially difficult. Ongoing research to define the specific events that trigger malignant transformation and how to optimally detect these events is underway. Practical algorithms for the clinical recognition, genetic testing, and management of individuals with these syndromes based on currently available knowledge as well as research seeking to improve the clinical care of these patients will be explored. A summary of the yield of next generation sequencing-based genetic testing strategies for familial presentations of MDS/AL will also be provided.

Disclosures
No relevant conflicts of interest to declare.
Predisposition to Myeloproliferative Neoplasms
Authors: Skoda Radek C. 
Published: October 2019

Familial forms of myeloproliferative neoplasms (MPN) and genetic contribution to sporadic cases of MPN have long been recognized. In the majority of cases, familial MPN is inherited as an autosomal dominant trait. The penetrance varies from around 20% to up to 100% in some pedigrees. We can distinguish two types of familial MPN. Type 1 has high penetrance, polyclonal hematopoiesis and hyperproliferation of a single hematopoietic lineage, caused by mutations in a single gene and usually manifesting at birth or early childhood. Examples are mutations in the genes for the erythropoietin receptor, thrombopoietin or its receptor, MPL. The type 2 familial MPNs are characterized by clonal hematopoiesis, low penetrance and manifestation beginning in most cases later in adult life. These type 2 familial MPNs are classical examples of inherited predisposition to a clonal malignant disease, in which acquired somatic mutations in hematopoietic cells are required for disease manifestation. Affected family members typically display the same acquired driver mutations in the genes for JAK2 (JAK2-V617F or JAK2-exon12), MPL (MPL-W515), or calreticulin (CALR) as patients with sporadic MPN. The mutated genes and the mechanism of how these inherited germline mutations predispose to MPN have not yet been elucidated. The search for these germline mutations has been hampered by the low penetrance of MPN manifestation and the rare occurrence of pedigrees that are large enough for genetic studies. Furthermore, the few candidate gene mutations that have been identified to date do not map to one gene locus and the function of the candidate genes does not fall into a common category. Two models of how the germline predisposition interacts with acquired driver mutations can be considered. First, the germline mutation may increase the mutation rate for gene mutations in JAK2, MPL, and CALR. Second, the germline mutation functionally synergizes with mutations in JAK2, MPL, and CALR and promotes disease initiation. The current state of our studies and studies in other laboratories will be discussed.

Disclosures
Skoda: Novartis: Consultancy; Sanofi: Consultancy.
Regulated RNA Expression and Normal Erythropoiesis
Authors: Higgs Douglas 
Published: October 2019

Although a small number of the vast array of long non-coding RNAs (lncRNAs) have known effects on cellular processes, in general their contribution to development, differentiation and disease remains unknown. We have shown that some intragenic enhancers, when active, behave as alternative promoters producing lncRNA transcripts that are processed using the canonical signals of their host gene. More recently we have also analyzed intergenic lncRNAs to determine the extent to which they too might originate from intergenic enhancers. We find that intergenic lncRNAs in erythroid cells are almost evenly divided between those arising from enhancer-associated or promoter-associated elements and that these RNAs differ with respect to their conservation and tissue specificity. Of considerable interest we find that expression of lncRNAs arising from enhancers is associated with enhanced expression of neighboring protein coding genes during erythropoiesis.

Disclosures
No relevant conflicts of interest to declare.
Stem Cell Paradigms in Tissue Regeneration and Cancer
Authors: Fuchs Elaine 
Published: October 2019

Abstract
Stem cells (SCs) have the ability to self-renew long term and differentiate into one or more tissues. Typically, SCs are used sparingly to replenish cells during normal homeostasis. However, even SCs that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How SCs balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans. The skin is an excellent model system to understand how SCs function in normal tissue generation and how this process goes awry in cancer. Many of the principles we have unearthed by studying skin stem cells have parallels to the hematopoietic system. We have identified quiescent and “primed”/active SCs within the skin and shown that primed SCs have a lower threshold for activation to enter tissue regeneration. We have learned that during normal homeostasis, SC behavior is controlled not only through cues received from their microenvironment but also through signals emanating from their differentiating lineages. We have been dissecting how extrinsic signaling to SCs trigger a cascade of transcriptional changes that govern SC activation during tissue development, homeostasis and hair regeneration. Our findings provide new insights into our understanding of the process of SC activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. Our goal is to understand how SCs start and stop making tissue, and how this changes in cancer. Our recent discoveries on this topic have led us to the realm of identifying and characterizing cancer SCs (tumor-initiating cells) of squamous cell carcinomas (SCCs) of the skin. We developed a new method to knockdown genes specifically in skin and oral progenitors, enabling us to screen not only the differences between cancer and normal SCs, but also the myriad of gene alterations surfacing from the Human Cancer Sequencing project. Our screens have illuminated new oncogenes and tumor suppressors for SCCs, among the most prevalent and life-threating cancers world-wide that include cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. Our findings are unearthing new targets for cancer therapeutics.

Disclosures
No relevant conflicts of interest to declare.
Epigenetic Mechanisms of Stem Cell Aging and Rejuvenation
Authors: Rando Thomas A. 
Published: October 2019

Abstract
For most tissues, stem cell numbers decline negligibly with age; nevertheless, there is an age-dependent decline in stem cell functionality. Many molecular, biochemical, and functional features of stem cells have been characterized across a broad range of tissues, and these changes have been assumed to be largely irreversible and inevitable accompaniments of aging. However, in studies both in vivo and in vitro we have demonstrated a reversibility of the functional and, in some cases, molecular characteristics of aged stem cells. Supported by compelling data from studies of heterochronic parabiotic pairings of mice, it is clear that the aged phenotype can be modified when aged cells are exposed to a youthful systemic milieu. These findings challenge the fundamental tenet of aging as an irreversible process and raises the question of whether, or to what extent, the aged phenotype is epigenetically determined. We have begun to examine the epigenetic profiles of young, old, and “rejuvenated” old stem cells to attempt to define youthfulness and aging in epigenetic terms. To the extent that aging can be “reprogrammed” back to youthfulness in somatic tissues, it has parallels to the resetting of the aging clock that occurs with somatic cell nuclear transfer. Elucidating the underlying molecular features, both genetic and epigenetic, of aged stem cells will provide a framework for understanding the fundamental molecular mechanisms of aging and the mechanisms by which environmental influences, such as those that occur in the setting of heterochronic parabiosis, can reverse the mechanisms of aging.

Disclosures
No relevant conflicts of interest to declare.
Asymmetry and Cell Fate in Stem Cells and Cancer
Authors: Reya Tannishtha 
Published: October 2019

Abstract
Our research focuses on the signals that control stem cell self-renewal and how these signals are hijacked in cancer. Using genetic models, we have shown that classic developmental signaling pathways such as Wnt and Hedgehog play key roles in stem cell growth and regeneration and are dysregulated during leukemia development. In addition, we have used real-time imaging strategies to show that stem cells have the capacity to undergo both symmetric and asymmetric division, and that shifts in the balance between these modes of division are controlled by the microenvironment and subverted by oncogenes. This work led to the discovery that regulators of asymmetric division, such as the cell fate determinant Musashi, can promote aggressive leukemias and may serve as critical targets for diagnostics and therapy in hematologic malignancies. Most recently, we have developed a high resolution in vivo imaging system that has allowed us to begin to map the behavior and interactions of stem cells with the microenvironment within living animals and to define how these change during cancer formation.

Disclosures
No relevant conflicts of interest to declare.