bluuebunny/crossref_metadata_2025_split huggingface dataset:
JQ 1, a Selective Bromodomain Inhibitor, Decreased the Expression of the Tolerogenic Molecule PDL1 in Antigen-Presenting Cells (APCs) and Restores the Responsiveness of Anergic CD4+ T Cells
Authors: Wang Hongwei , Cheng Fengdong , Xing Limin , Zhao Xiaohong , Villagra Alejandro , Pinilla-Ibarz Javier , Tao Jianguo 
Published: October 2019

Abstract
Bromodomain and extraterminal (BET) is a protein domain that recognizes acetylated lysine residues such as those on the N-terminal tails of histones. This recognition is often a prerequisite for protein-histone association, chromatin remodeling and gene transcription. The role of BET proteins in regulating the response of inflammatory cytokine genes through translation of histone marks is poorly understood.
Given that the inflammatory status of the APC is critical in determining T-cell activation versus T-cell tolerance and that epigenetic modifications of specific genes in the APC play a key role in this process, we recently determined the functional consequences of inhibiting BET in APCs.
First, we evaluated the effects of JQ 1, a selective small-molecule BET bromodomain inhibitor on APC’s function and its regulation of antigen-specific CD4+ T-cells response. In vitro treatment of peritoneal elicited macrophages (PEM) or bone marrow derived dendritic cells (DCs) with increasing concentrations of JQ 1 resulted in decreased expression and protein production of the anti-inflammatory cytokine IL-10 and IL-6 in response to LPS stimulation. At the concentration used, JQ 1 did not affect the viability of treated APCs. Second, analysis of the expression of MHC class molecules and co-stimulatory molecules revealed a decreased expression of the tolerogenic PDL1 molecule in JQ 1- treated APCs as compared to untreated APCs. Third, we evaluated the ability of JQ 1 treated APCs to present cognate antigen to naïve or tolerant antigen-specific CD4+ T-cells. We found that treatment of either PEM or DC with JQ 1 enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, JQ 1- treated APCs were able to restore the responsiveness of tolerant CD4+ T-cells isolated from lymphoma bearing hosts.
Taken together, we have found that APCs treated with the Bromodomain specific inhibitor JQ 1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and more importantly, are capable of restoring the responsiveness of tolerant T-cells. Our studies therefore have unveiled a previously unknown immunological effect of BET inhibitors and have broadened their clinical scope as promising adjuvants in cancer immunotherapy.

Disclosures
No relevant conflicts of interest to declare.
Modeling Warfarin Associated Intracerebral Hemorrhage (ICH) in Cynomolgus Monkeys
Authors: Liu June , Li Zhiqun , Ni Jia , Deng Jilin , Cui Guoqiang , Wang Song , Feng Jianming , Li Hanjian , Tu Rong , Zhang Kevin 
Published: October 2019

Abstract
Intracerebral hemorrhage (ICH) accounts for about 15% of strokes and leads to higher mortality. However, its underlying pathophysiologic mechanism remains gloomy, partially due to the lack of suitable animal models in research. Approximately 20% ICH cases are associated with the use of oral anticoagulants, warfarin for the most instances in clinic, especially following the long-term treatment. Even though the coumarin drugs has been used in rodents for comparison of standard-of-care effects in ICH studies, the elucidation of results must bear the significant differences of gyrencephalic brain structure, brain vasculature and various biological interactions affecting the hemorrhage and hemostasis between rodents and human being. Quite a few works have been reported in study of strokes, particularly, the cerebral ischemic stroke in cynomolgus monkeys (Macaca fascicularis) for its high homology to human. In order to establish a more clinical relevant model for ICH and saver anticoagulant research, we studied ICH in cynomolgus monkeys by injection of collagenase in brain and characterized the effect of warfarin treatment by MRI measurement. Following injection of type IV collagenase into internal capsule, distinct acute, sub-acute, and chronic phases of ICH were observed, with the highest level of cerebral hematoma volume at 3 days after ICH induction measured by a GE signa excite 1.5T MRI scanner. Pretreatment with 0.3 mg/kg of warfarin daily for 6 days significantly increased the hematoma volumes to proximal 10% of cerebral hemisphere compared to the 4% with saline treatment. In addition, the AUC of hematoma volume changes was also remarkably enlarged with the warfarin treatment as measured at different time points following ICH. Transfusion of 30 ml of autologous fresh frozen plasma (FFP) and injection of 1 mg Vitamin K reduced the effects of warfarin treatment in hematoma and AUC enlargement, and ameliorated ICH stroke associated symptoms. The results of coagulation tests were all in agreement with the warfarin associated effects on cerebral hematoma in cynomolgus monkeys. Our data demonstrated the successful establishment of ICH model in cynomolgus monkeys and the clinical relevant treatments, such as warfarin and FFP, can be incorporated as the controls for proper researches in ICH and its therapeutics. With the high similarity in brain and high cross reactivity in target to human, the cynomolugs monkey ICH model adds a valuable tool to evaluate new therapeutic approaches and the safety of novel anticoagulants, especially for the human target-specific interventions, such as plasma products, biologics, oligonucleotides, and peptides.

Disclosures
No relevant conflicts of interest to declare.
Efficacy of a Transdermal Granisetron Patch in Controlling Chemotherapy-Induced Nausea and Vomiting (CINV) in Hematologic Cancer Patients
Authors: Niesvizky Ruben , Fernández Velasco Ana , Wong Doris S , Braccia Deborah 
Published: October 2019

Abstract
Introduction: Multiday, highly emetogenic chemotherapy regimens are commonly used in the management of hematologic malignancies. Despite progress in prophylaxis, CINV, especially during the delayed phase, can remain a significant barrier to attaining planned chemotherapy dose on time for some patients. A granisetron transdermal system (GTS) has been shown to be as effective as oral granisetron in controlling CINV across multiple tumor types. This post-hoc analysis specifically examined the efficacy and safety of GTS in patients with hematologic malignancies.
Methods: A randomized, phase 3 study has been published comparing GTS (7 day application) to oral granisetron (2 mg/day) in patients receiving either moderately or highly emetogenic chemotherapy for 3-5 days. Data for this analysis were limited to patients with a primary tumor diagnosis of lymphoma (n=51), leukemia (n=27), or multiple myeloma (n=5). The rates of complete control (CC; no vomiting, mild nausea, no rescue medication) and complete response (CR; no vomiting, no rescue medication) using either GTS or oral granisetron were compared during both the acute (first 24 hours) and delayed phase (days 2 to 5) following chemotherapy. Need for rescue medication and patient assessment of response to therapy with GTS were also compared.
Results: 83 hematologic cancer patients (31 GTS, 52 oral granisetron) were included. The majority of patients received a non-cisplatin regimen with corticosteroids (59 patients; 71%). Patients received chemotherapy for 3 days (37; 45%) or 4 to 5 days (46 patients; 55%). During the acute phase of CINV, the CC rate of 94% and CR rate of 94% in the GTS group were similar to rates in the overall population (94% and 95%, respectively). There was no difference in CC and CR between GTS and oral granisetron in the acute phase (P=0.90 and 0.59, respectively). In the delayed phase, GTS resulted in CC in 87% and CR in 90%; compared with CC in 77% and CR in 81% of patients given oral granisetron (P=0.26 and 0.25, respectively). The use of rescue medication over the entire study period and patient assessment of overall response to therapy were not different between arms (P=0.60 and 0.92, respectively). GTS was well tolerated; the only treatment related adverse event was one case of mild constipation.
Conclusion: This retrospective analysis suggests GTS may be an appropriate option for prevention of CINV in hematologic cancer patients. A trend toward improved control in the delayed phase was observed and further investigation of a benefit in delayed CINV for hematologic malignancies may be warranted.

Disclosures
Fernández Velasco: ProStrakan: Employment. Wong:ProStrakan: Employment. Braccia:ProStrakan: Employment.
Pomalidomide Induces Mir-125b with Subsequent Downregulation of P53 in CD34+cells Conferring Decreased Apoptosis: A Potential Mechanism of Secondary Primary Malignancies
Authors: Zheng Yijie , Li Shirong , Fu Jing , Mapara Markus , Lentzsch Suzanne 
Published: October 2019

Abstract
Introduction:IMiD® compounds such as lenalidomide (LEN) and pomalidomide (POM) represent a novel class of agents in the treatment of multiple myeloma. Increasing concerns about its potential toxicity on stem cells resulting in an increase of secondary malignancies primarily affecting the myeloid cells have been reported. However, the exact mechanism is unclear. MicroRNAs (miRNAs) are emerging as important regulators in stem cell development. Therefore, the present study investigated the effect of IMiD® compounds on stem cell renewal/proliferation and the role of miRNAs involved in this process.
Methods and Results: When human CD34+ cells were cultured with POM or DMSO under conditions favoring myeloid development, POM significantly increased the percentage of CD34+cells in S phase in cell cycle analysis with concomitant dramatic decrease of apoptotic cells. This resulted in a strong and significant proliferation of CD34+cells as well as induction of colony formation. Systematic screening of miRNAs expression profile by microarray showed that POM significantly enhanced the expression of miR-125b. Upregulation of miR-125b was further confirmed by realtime PCR. Interestingly, recently miR-125b was shown to function as an oncomir by inducing tumorigenesis in myeloid cells by So et al., Blood 2014. In accordance with that we found that overexpression of miR-125b in CD34+ cells exhibited similar effects as POM on CD34+ cells, such as increased cell number in S-phase, decreased cell apoptosis resulting in significant induction of colony formation and the absolute number of hematopoietic progenitors. Bioinformatics prediction models revealed a potential binding site of miR-125b on 3'UTR of P53. Indeed subsequent experiments showed that P53 was downregulated when miR-125b was overexpressed in CD34+cells or when treated with POM. Further analysis of the P53 down stream targets revealed that POM decreased the expression of PUMA and BAK1 all critical for control of cell proliferation and apoptosis. The POM-induced upregulation of mir-125b was independent of the CRBN/IKZF1 induced protein degradation.
Conclusion: Based on our results we postulate that POM upregulates the oncomir miR-125b in CD34+cells. Mir-125b controls proliferation and apoptosis by downregulating P53 and genes involved in the P53 pathway including BAK1 and PUMA. Thus, our findings on the effects of POM on the miR-125b/P53 axle provide for the first time a potential mechanism for the development of secondary hematologic malignancies in patients treated with IMiD® compounds.

Disclosures
Lentzsch: Celgene: Consultancy, Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy.
The Fifth Region of Epidermal Growth Factor like-Domain of Thrombomodulin Is Essential to Produce Cytoprotection
Authors: Ikezoe Takayuki , Yang Jing , Nishioka Chie , Nakamura Keigo , Yurimoto Hiroya , Sakai Yasuyoshi , Honda Goichi , Yokoyama Akihito 
Published: October 2019

Abstract
We previously found that the fourth and fifth regions of epidermal growth factor-like domain of thrombomodulin (TME45) protects vascular endothelial cells from calcineurin inhibitor-induced cell damage in association with activation of extracellular signal-regulated kinase (ERK)/myeloid cell leukemia-1 (Mcl-1) signal pathways via activated protein C (APC) independent manner (Arterioscler Thromb Vasc Biol. 2012;32:2259-70). The present study attempted to identify the minimum region of TME45 that is essential to mediate the cytoprotective function of thrombomodulin. The blocking antibody for TME5 but not for TME4 hampered the ability of TME45 to increase levels of Mcl-1 in human umbilical vein endothelial cells (HUVECs), indicating the important roles of TME5 in cytoprotection. We next divided TME5 into three parts and synthesized oligonucleotides for each region. None of the fragment was as potent as TME5 to induce Mcl-1 in HUVECs, suggesting that TME5 was essential for induction of Mcl-1. Importantly, TME5 was not able to produce APC. Furthermore, TME5 counteracted calcineurin inhibitor-induced vascular permeability and stimulated angiogenesis in vitro as well as in vivo. Taken together, TME5 may be useful to prevent or treat endothelial cell damage associated lethal complications developing after hematopoietic stem cell transplantation such as sinusoidal obstruction syndrome and thrombotic microangiopathy.

Disclosures
Honda: Asahi Kasei Pharma: Employment.
Gas6 Is Required for Thrombin-Induced Expression of VCAM-1 through Foxo-1 in Endothelial Cells
Authors: Bertin Francois-Rene , Robins Richard , Lemarie Catherine A , Blostein Mark D 
Published: October 2019

Abstract
Venous thromboembolism (VTE) is a leading cause of morbidity and mortality. Gas6-/- mice are protected against VTE suggesting an important role for Gas6 in the pathophysiology of this disorder. VCAM-1 is a key adhesion molecule, expressed on the endothelium, implicated in the pathophysiology of venous thrombosis. We previously demonstrated that Gas6 induced the phosphorylation of Forkhead box O1 (FoxO-1) through the activation of the PI3K/Akt signaling pathway. Thus, we hypothesize that Gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO-1 in endothelial cells. Thrombin induces VCAM-1 mRNA and protein expression in WT but not in Gas6-/- endothelial cells. Silencing FoxO-1, using siRNA, enhances VCAM-1 expression both in WT and in Gas6-/- endothelial cells. Thrombin induces FoxO-1 phosphorylation and nuclear exclusion in WT but not in Gas6-/- endothelial cells. Inhibition of FoxO-1 phosphorylation by overexpression of a phosphorylation resistant FoxO-1 mutant (Tm-FoxO-1) decreases VCAM-1 expression, both in WT and Gas6-/- endothelial cells. Inhibition of Akt by LY294002 prevents FoxO-1 phosphorylation and VCAM-1 expression. Finally, thrombin-induced VCAM-1 expression in WT endothelial cells was associated with an increase in bone marrow mononuclear cell (BM-MC) adhesion. BM-MC adhesion was further increased when endothelial cells were transfected with a FoxO-1 siRNA and decreased when transfected with the Tm-FoxO-1 plasmid. Thus, we demonstrate that Gas6 regulates VCAM-1 expression through phosphorylation and nuclear exclusion of FoxO-1, thereby promoting BM-MC adhesion. These data suggest that the Gas6/FoxO-1 signaling axis plays an important role in VCAM-1 expression during thrombosis.

Disclosures
No relevant conflicts of interest to declare.
Characterization of Antithrombin-Specific RNA Aptamers for Use in Anticoagulant Therapy
Authors: Fortenberry Yolanda 
Published: October 2019

Abstract
Historically, unfractionated heparin (a complex polysaccharide classified as a glycosaminoglycan) has been used in the treatment and prevention of thromboembolic disorders. However, due to complications associated with heparin (such as bleeding and heparin-induced thrombocytopenia), low molecular weight heparins (LMWHs) have been developed and are being used in place of unfractionated heparin. LMWHs have proven effective as antithrombotic agents; however, the effects of LMWHs cannot be readily reversed, while unfractionated heparin can be reversed by protamine sulfate. The overall goal of this project was to characterize an antithrombin (AT)-specific RNA aptamer that will combine the safety and efficacy of LMWH with the reversibility of unfractionated heparin. I have developed an RNA molecule (aptamer) that binds to AT. Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) we created a combinatorial library consisting of single stranded RNA molecules with 20-40 randomized positions resulting in approximately 1014 different sequences. The RNA libraries after round 12 consisted of several different aptamers, and there was a progressive decrease in library complexity. We isolated four individual RNA molecules that bind in the nanomolar range to AT. Each displayed the same general properties, they accelerated factor Xa inhibition by AT in a dose-dependent manner. It should be noted that as anticipated, the aptamers did not promote the enhanced inhibition of thrombin by AT. Overall, our results show that these molecules are able to enhance the inhibition of factor Xa by AT. We suspect that these aptamers mimic the action of LMWH by binding to the D-helix of AT. Our previous AT specific RNA aptamer, Aptamer 7-4.16 was shown to prolonged bleeding in vivo in a vascular injury model. These data showed that Aptamer 7-4.16 is as effective as heparin in preventing clotting after vascular injury. We suspect that these new aptamers will have a similar effect in vivo. Proof of the concept was established by the work of Rusconi et al. (Nature, 2002), who developed an RNA aptamer and RNA aptamer antidote directed against coagulation factor IXa. The factor IXa aptamer completely inhibits the activation of factor X. Its anticoagulant action is controlled by the antidote, which is able to reverse the effects of the factor IXa aptamer within seconds. This aptamer/antidote pair is currently being tested clinically. Our data suggesting that antidotes to our AT aptamers have the ability to reverse its activity. If successful, this aptamer/antidote pair will have advantages over LMWHs because not only will it be able to control thrombosis in a fashion similar to LMWHs, but the availability of an antidote will allow for better therapeutic regulation and intervention.

Disclosures
No relevant conflicts of interest to declare.
PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis
Authors: LIU Fan , Cheng Guoyan , Perna Fabiana , Haiming Xu , Hamard Pierre-Jacques , Luciani Luisa , Nimer Stephen 
Published: October 2019

Abstract
Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology.

Disclosures
No relevant conflicts of interest to declare.
An Erythroid-Specific Intron Retention Program Regulates Expression of Selected Genes during Terminal Erythropoiesis
Authors: Pimentel Harold , Parra Marilyn , Gee Sherry , Mohandas Narla , Pachter Lior , Conboy John G. 
Published: October 2019

Abstract
Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proerythroblasts differed substantially from that in orthochromatic erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation.

Disclosures
No relevant conflicts of interest to declare.