bluuebunny/crossref_metadata_2025_split huggingface dataset:
RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia
Authors: Pippa Raffaella , Dominguez Ana , Marcotegui Nerea , Malumbres Raquel , Guruceaga Elizabeth , Odero Maria D. 
Published: October 2019

Abstract
INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation.
AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation.
RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene.
CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias.

Disclosures
No relevant conflicts of interest to declare.
Dual Inhibition of Bcr-Abl and Hsp90 By C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells
Authors: Wu Lixian , Yu Jing , Liu Yang , Liguang Lou , Wu Yong , Chen Yuanzhong , Xu Jianhua 
Published: October 2019

Abstract
Purpose: Although such tyrosine kinase inhibitors (TKIs) as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of CML patients, TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition.
Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation and immunoblotting were applied to examine Hsp90 function; Colony-forming unit (CFU), long-term culture-initiating cells (LTC-ICs), and flow cytometry were used to test CML progenitor and stem cells.
Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, inhibited the growth of both imatinib-sensitive and resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38- cells in vitro. Moreover, C086 could decreased the number of CD45+, CD45+CD34+CD38+ and CD45+CD34+CD38- cells in CML NOD-SCID mice.
Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs.

Disclosures
No relevant conflicts of interest to declare.
The New Targeted Nanoparticles: Tf-Peg-PLL-PLGA Combined with Daunorubicin Overcome Hypoxia Induced Drug Resistance of K562 Cells
Authors: Liu Ping , Guo Liting , Wang Fei , Xia Guohua , Wang Yonglu , Li Xueming , Song Huihui , Chen Wei , Yin Haixiang , Chen Baoan 
Published: October 2019

Abstract
Drug resistance is a major obstacle in cancer chemotherapy, especially in the treatment of leukemia. Hypoxia is an inseparable component of the solid tumor microenvironment as well as the bone marrow (BM) microenvironment. In this study, we investigated the effect of the new nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) (PEG-PLL-PLGA) modified by transferrin (Tf) combined with daunorubicin (DNR) on K562 cells under hypoxia. We found the IC50 of DNR and DNR-PEG-PLL-PLGA in K562 cells increased under hypoxia, while the IC50 of DNR-Tf-PEG-PLL-PLGA in K562 cells decreased under hypoxia according to the results of MTT assay. The apoptosis of cells and the intracellular concentration of DNR were detected by flow cytometry (FCM). From the datas, we found that the new targeted nanoparticles can overcome hypoxia induced drug resistance of K562 cells. By targeting the transferrin receptor (TfR) on the surface of K562 cells, the new targeted drug led to an increase in intracellular accumulation of DNR. Transcription and Protein of P-glycoprotein (P-gp) and TfR increased in K562 cells under hypoxia, which probably had a relationship with the decreased degradation of the Hypoxia-Inducible Factor-1α (HIF-1α). We further detected the levels of HIF-1α, Bcl-2, Bax and Caspase-3 of K562 cells in transcription and protein under hypoxia regulated by DNR, DNR-PEG-PLL-PLGA and DNR-Tf-PEG-PLL-PLGA, respectively. DNR-Tf-PEG-PLL-PLGA had the best effect when compared with other groups. Furthermore, intravenous injection of DNR-Tf-PEG-PLL-PLGA expressed the best effect of suppressing proliferation of K562 cells injected to nude mice among the three drugs. Therefore, the new drug DNR-Tf-PEG-PLL-PLGA may be a potential cancer therapy for human leukemia.

Disclosures
No relevant conflicts of interest to declare.
Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo
Authors: Stalker Timothy J. , Wu Jie , Brass Lawrence F. 
Published: October 2019

Abstract
Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo.
Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy.
Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core.
Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute.

Disclosures
Stalker: Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.
Genome and Epigenome Editing: A Revolution in Science and Medicine
Authors: Joung J. Keith 
Published: October 2019

Abstract
Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. This talk will focus on our recent work on both improving and defining the genome wide specificities of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, a robust platform for introducing targeted genome sequence alterations. The talk will also briefly describe the creation and validation of new technologies for modifying specific epigenomic marks on histones and DNA that can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing new classes of therapeutics based on targeted alterations of gene sequence and/or gene expression.

Disclosures
Joung: Editas Medicine: Consultancy, Equity Ownership, Other, Patents & Royalties; Transposagen Biopharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Horizon Discovery: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Dual Face of Immune Microenvironment in Myeloma
Authors: Dhodapkar Madhav V. 
Published: October 2019

An important property of human multiple myeloma (MM) is the growth of tumor cells and their precursors in the bone marrow microenvironment. This specialized microenvironment includes a distinct component of immune cells that includes cells of innate as well as adaptive immune system. Growing body of evidence points to a central role for cross-talk between this immune microenvironment and tumor cells in regulating the growth of tumor cells in myeloma. These interactions play a dual role-with some interactions promoting tumor growth while others suppress growth of tumor cells. Cross talk between T and B cells has been implicated in normal B/plasma cell differentiation and may also play a role in pathogenesis of myeloma, explaining increased risk of plasma cell malignancies in certain settings such as Gaucher disease. The bone marrow of MM patients is often infiltrated with innate cells such as dendritic cells (DCs) and cross-talk between myeloid/plasmacytoid DCs or macrophages may promote growth and survival of MM cells. This cross-talk may also have fundamental implications for inducing genomic instability in tumor cells, such as via the induction of cytidine deaminases in tumor cells. Genomic instability is an early feature of human MM and many of the mutations found in MM tumor cells are also present in the precursor states. The immune system has the capacity to recognize these precursor lesions and even in the setting of clinical MM, the tumor bed contains T cells that can be recruited to kill tumor cells. Specific targets of tumor immunity may differ between preclinical and clinical malignancy. Immunity to certain stem cell-associated antigens are in particular associated with reduced risk of progression to clinical MM. With advanced disease, tumor infiltrating T cells encounter several immune inhibitory checkpoints which leads to suppression of function of tumor-infiltrating T cells. In addition to T cells, cells of the innate immune system such as natural killer and natural killer T cells may also be attractive targets of protective tumor immunity in MM. Immunologic approaches may also be combined with other strategies such as other immunomodulatory drugs (IMiDs) already in clinical use in MM. Recent advances in mouse modeling provide new opportunities to better understand how to harness the immune system against human MM. Such approaches have the advantage of the potential for durable control of tumor cells via immunologic memory. Such approaches are now entering the clinic as attractive targets to restore anti-tumor immunity, both in the context of therapy or prevention of MM.

Disclosures
Dhodapkar: Celgene: Research Funding. Off Label Use: checkpoint blockade drugs, imids.
A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)
Authors: Perdomo Jose , New Jaa Yien , Ahmadi Zohra , Jiang Xing-Mai , Chong Beng H 
Published: October 2019

Abstract
Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions.
Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec.
Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1).
Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition.

Disclosures
No relevant conflicts of interest to declare.
Structural Basis for PECAM-1 Homophilic Binding
Authors: Paddock Cathy , Zhou Dongwen , Lertkiatmongkol Panida , Newman Peter J. , Zhu Jieqing 
Published: October 2019

Abstract
PECAM-1 is a 130 kDa member of the immunoglobulin gene (Ig) superfamily that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its two amino-terminal Ig homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to sense flow, regulate leukocyte transendothelial migration, maintain vascular integrity, and transmit survival signals into the cell. Mutagenesis studies have implicated amino acids on either side of the face of IgD1 in homophilic binding, however the spatial orientation of this domain, the role that IgD2 plays in positioning IgD1 to participate in PECAM-1 homophilic interactions, and the contribution of its sialylated glycans to homophilic PECAM-1/PECAM-1 interactions remain to be critically explored. Complicating things further, PECAM-1 forms dimers and oligomers within the plane of the plasma membrane. The structural features of PECAM-1 responsible for maintaining the PECAM-1/PECAM-1 homophilic contacts necessary for carrying out these activities, however, have heretofore remained unresolved. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic binding domain. The structure of IgD1-D2 was determined by multi-crystal anomalous diffraction and refined at 2.8 Å resolution. Interestingly, PECAM-1 crystallized as a strand-swapped dimer, a feature that has been seen in the crystal structures of many other members of the Ig superfamily, including cadherins, NCAM2, CTLA-4, and CD47. The homophilic interface between adjacent IgD1 domains was large, with a total buried interface of 1608 Å2. In addition to the large IgD1/IgD1 interface, interdomain contacts were found between IgD1/IgD2, and IgD2/IgD2 that likely reinforce the PECAM-1/PECAM-1 homophilic interactions that form as a consequence of the large number of PECAM-1 molecules that become concentrated at endothelial cell-cell borders as a result of diffusion trapping. Taken together, these results provide atomic-level detail to the unique architecture of the interface that forms the most abundant component of the endothelial cell intercellular junction.

Disclosures
No relevant conflicts of interest to declare.
Thrombocytopenia in Hospitalized Non-ICU Patients
Authors: Fountain Eric , Arepally Gowthami M 
Published: October 2019

Abstract
Thrombocytopenia is common in patients admitted to the intensive care unit, with a reported prevalence of 8-68% and incidence of 13-44% in published series. Those who develop thrombocytopenia in an ICU have a statistically increased risk of ICU mortality, duration of mechanical ventilation, and platelet, RBC, and FFP transfusions. Few studies have examined the occurrence and risk factors for thrombocytopenia in non-ICU hospitalized patients. The only systematic investigation of thrombocytopenia in the non-ICU hospitalized medical population dates to 1989, predating modern diagnostic assays for a variety of infectious and drug-induced causes of thrombocytopenia. We, therefore, undertook this study to characterize the incidence and causes of thrombocytopenia in the general medical, non-ICU patient population at a tertiary care hospital. For this study, we performed a single-institutional retrospective analysis of patients admitted to a general medical ward at a tertiary care medical center (Duke University Hospital). Inclusion criteria included all adult patients (>18 years) admitted from the emergency department to the general medicine floors during the calendar year defined as 01/2014-01/2015. Exclusion criteria included pre-existing thrombocytopenia, ICU admission, or patients undergoing chemotherapy. Primary endpoints included patients with incident thrombocytopenia, defined as a platelet count on admission greater than 150 x 10^9/L with subsequent platelet counts decreasing to < 150 x 10^9/L. Patients meeting these criteria were evaluated for admission diagnosis, pertinent past medical history, cause of thrombocytopenia (infection, splenic sequestration, drugs, surgery, liver disease), cost of admission, and prognosis. Preliminary evaluation of patients admitted to the general medical service in 2014 reveals ~711 patients who meet inclusion criteria. Of patients developing absolute thrombocytopenia, 56% experienced a platelet count fall of greater than 30%. Those with incident thrombocytopenia greater than 30% had a statistically significant increase in mortality (n=36/399) compared to those with milder thrombocytopenia (n=13/312), (9.0% versus 4.2%, p<0.05), and had an increased length of stay (12.6 days versus 6.8 days, p<0.01). In patients who developed absolute thrombocytopenia, review of diagnosis codes associated with thrombocytopenia reveal a predominance of infectious etiologies (43%; sepsis, severe sepsis, septic shock, bacteremia, urinary tract infection/pyelonephritis, pneumonia, Clostridium difficile colitis). These results are similar to prior published data which suggests infection as a leading cause of incident thrombocytopenia in non-ICU hospitalized patients. Additional studies are underway to delineate non-infectious causes of thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.