bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Prostate Apoptosis Response-4 Tumor Suppressor in the Survival and Growth of Chronic Lymphocytic Leukemia
Authors: Nooti Sunil K , McKenna Mary K , Frissora Frank W , Gachuki Beth W , Alhakeem Sara S , Greene Joseph T , Muthusamy Natarajan , Rangnekar Vivek M , Bondada Subbarao 
Published: October 2019

Abstract
Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5+ CD19+ B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+ CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants)

Disclosures
No relevant conflicts of interest to declare.
The Effect of Proteasome Subunit Beta Type 1 P11A Polymorphism on the Survival of Multiple Myeloma Patients Treated with First Line Bortezomib Based Chemotherapy
Authors: Varga Gergely , Mikala Gabor , Szombath Gergely , Balassa Katalin , Kiss Katalin Piroska , Tordai Attila , Andrikovics Hajnalka , Masszi Tamas 
Published: October 2019

Abstract
Background: Proteasome inhibitors have a fundamental role in the treatment of multiple myeloma however there are still patients who fail to achieve a good response and have a dismal prognosis. Markers predicting the outcome of individual patients are needed to identify those who would benefit more from a different approach. Proteasome subunit beta type 1 (PSMB1) rs12717 single nucleotide polymorphism (C/G substitution resulting in a P11A change) was reported recently to be associated with greater clinical benefit in relapsed follicular lymphoma patients treated with bortezomib containing combination (Coiffier et al Clin Cancer Res 2013). To the best of our knowledge there is only one report with limited number of myeloma patients showing no association between PSMB1 P11A genotype and survival (Lichter et al Blood 2012).
Aims and Methods: We analyzed the association of PSMB1 P11A polymorphism and treatment outcome of 220 consecutive myeloma patients having had first line chemotherapy in our unit. PSMB1 P11A polymorphism was tested using LightCycler melting analysis. Statistical analyses were performed using the SPSS (version 20.0) software package.
Results: The distribution of presentation prognostic markers such as age, ISS and FISH were even among the three genotype groups. 149 patients had bortezomib-based chemotherapy, mainly VTD (n=76) and MPV (n=47); 63% of these patients had ASCT consolidation. 71 patients had non-bortezomib-containing induction with either thalidomide, lenalidomide or other protocols including MP and VAD; only 28% of these patients had ASCT. In a recessive model the median PFS was 727 (635-818) days in carriers of the wild type C allele either in homozygous of heterozygous form (C/C&C/G, n=188), and only 491 (366-615) days in the homozygous variant G allele carriers (G/G, n=32, p=0.01). In multivariate analysis, C allele carriers had favorable PFS compared to G/G genotype patients with a hazard ratio of 1.402 (1.093-1.799), p=0.008 besides age, ISS, FISH, bortezomib treatment and ASCT. When we analyzed separately the bortezomib-treated and non-bortezomib-treated patients the benefit in PFS was only present in the bortezomib group [Fig 1, PFS 747 (656-837) days in C/C&C/G and 448 (279-616) days in G/G patients, p=0.002], and not in patients treated without bortezomib (p=0.559). We performed statistical interaction testing which showed significant interaction between bortezomib exposure and PSMB1 P11A (p=0.035).
Conclusion: PSMB1 P11A is a novel predictive marker in multiple myeloma which could help to identify patients having a suboptimal response to bortezomib who might benefit from alternative management.

Disclosures
No relevant conflicts of interest to declare.
Upregulation of Microparticles, Tissue Factor, Adhesion Molecules, Nitric Oxide and Adiponectin in End Stage Renal Disease
Authors: Fareed Jawed , Bansal Vinod , Syed Daneyal , Hoppensteadt Debra , Guler Nil 
Published: October 2019

Abstract
Introduction: End stage renal disease (ESRD) represents the final stage of chronic kidney disease characterized by kidney failure (GFR <15 mL/min/1.73 m2). To understand the pathophysiology of hemostatic dysregulation in ESRD, we measured the circulating levels of microparticles (MP), tissue factor (TF), adhesion molecules, such as p-selectin (P-Sel), soluble ICAM (s-ICAM), nitric oxide (NO) and adiponectin (AD).
Methods: Citrated blood plasma samples were collected from 119 ESRD patients undergoing maintenance hemodialysis to profile various thrombotic inflammatory biomarkers. 100 normal plasma samples were collected from healthy individuals and were used as controls. MP levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France). NO were measured using a kit from R&D systems (Minneapolis, Minnesota) and ELISA based methods for TF, P-Sel, s-ICAM and adiponectin were obtained from R&D systems. Chromogenic substrate methods were used to measure the heparin levels and thrombin generation.
Results: MP levels were elevated in the ESRD group (28.1nm + 6.1nm vs. the control 8.9nm +1.3nm). Tissue factor levels were found to be increased in the ESRD group (20.4±6.1pg/ml) vs the control (11.9±2.8pg/ml). The nitric oxide level was markedly higher in the ESRD group (32±17uM) vs the controls (7±3uM). The p-selectin levels were also elevated in the ESRD group (46±20ng/ml) vs the control (31±3ng/ml). The soluble ICAM levels were higher in the ESRD group (250±112ng/ml) vs the control (180±19ng/ml). Interestingly, the adiponectin levels were also increased in the ESRD group (19.2±9.3ug/ml) vs the control (11.2±4.1ug/ml). Measured variations in thrombin generation parameters were noted. Detectable levels of heparin (.05 - 0.20 U/ml) were present in the ESRD group.
Summary/Conclusions: These studies suggest that MP, TF, NO, p-selectin and s-ICAM levels are increased in the ESRD patient. It is of interest to note that despite that a significant number of ESRD patients were diabetic; the AD levels were increased in this group. These results also suggest that while ESRD represents a pro-inflammatory/hypercoagulable state, the repeated administration of heparin and other drugs may contribute to the regulation of the hemostatic process and inflammatory balance in these patients.

Disclosures
No relevant conflicts of interest to declare.
Enhanced NF-Kb Non-Canonical Signaling Impairs Hematopoietic Stem Cell Self-Renewal
Authors: Xiu Yan , Zhao Chen 
Published: October 2019

Abstract
We previously demonstrated that the NF-B non-canonical signaling way positively and intrinsically regulates hematopoietic stem/progenitor cell (HSPC) self-renewal and maintains stromal/osteoblastic niches (Stem Cells 2012 30:709-18). These results lead us to think that persistent activation of NF-B non-canonical signaling would have favorable effects on the HSPC pool size and self-renewal capacity. NF-B-inducing kinase (NIK) plays a critical role in non-canonical NF-B signaling by directly phosphorylating IKK. It is constitutively degraded by TRAF3 in unstimulated cells to prevent unwanted NF-B activation. To investigate the enhanced NF-kB non-canonical signaling specifically in hematopoietic cells, we crossed Vav-Cre mice with a mouse strain in which a mutated form of NIK lacking the TRAF3-binding domain is expressed under the control of the ROSA26 promoter after Cre-mediated deletion of the LoxP-flanked STOP cassette (NIKΔT3Cre mice). In contrast to what we expected in these preliminary studies, the NIKΔT3Cre mice rapidly developed anemia, pancytopenia, with a reduced HSPC pool and marrow cellularity and postnatal lethality, mimicking many of the findings in humans with bone marrow failure syndrome, and different from recently published mice with deficiency in A20, which also activates NF-B signaling. Furthermore, the NIK activated HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Further analysis showed that the mutant cells are proliferate faster and predispose to apoptosis than wild type cells. These observations suggest that finely controlled NF-B activity is crucial for HSC maintenance. Currently, we are focusing on the analysis of the underlying molecular mechanisms.

Disclosures
No relevant conflicts of interest to declare.
Distinct Regulatory Networks Govern Human Hematopoietic Stem Cell Across Development
Authors: Zandi Sasan , Dick John E. , Notta Faiyaz , Takayama Naoya 
Published: October 2019

Abstract
Introduction: Much of our fundamental understanding of stem cell biology comes from studies of hematopoiesis where single cells produce differentiated progeny while still retaining the ability to produce daughter stem cells (self-renewal). The cardinal property of a stem cell, whether normal or malignant, is self-renewal; the key biological process that ensures the ability of the stem cell to maintain long-term clonal growth. However, our understanding of the molecular basis of self-renewal in human hematopoiesis is limited.
At the embryonic stage fetal liver is the main source of hematopoiesis; from week 6 of gestation until before birth. At this stage HSCs are in a different microenvironment but capable of self-renewing and differentiation to the full spectrum of blood lineages. While murine studies uncovered several intrinsic differences between fetal and adult HSCs, a comprehensive analysis of human HSC compartment across development is lacking.
In this study we have combined HSC purification methods and xenograft quantitative assay in conjunction with low input RNA sequencing and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to provide a comprehensive functional and molecular outlook of human stem cell compartment across development.
Results: We followed the dynamics of four sub-fractions of CD34+CD38- divided by CD90 and CD49f expression across human blood development: fetal liver (hFL) and adult bone marrow (hBM). Using xenograft model, we identified human long, intermediate and short term HSCs in hFL and hBM. 5 single CD90+CD49f+ hFL cells were capable of sustaining the multilineage graft for over 52 weeks up to tertiary recipient, while BM cells only last for 20 weeks in the primary recipient. The frequency of LT-HSC in the CD90+CD49f+ compartment goes from 1/8 in hFL to 1/50 in hBM. hFL CD90-CD49f+ cells showed an intermediate repopulation capacity up to 44 weeks in secondary recipient.
On average 10% of hFL long term HSC (LT-HSC) were in S/G2/M phase, in contrast only 0.4% of BM LT-HSC were in S/G2/M phase indicating that hFL HSCs are 20 times more in cycle compare to BM.
We found that 320 genes were expressed differentially between LT-HSC and multipotent progenitors (MPP) in hBM as oppose to only 32 genes found to be differentially expressed in hFL (FDR<0.1). Interestingly, we found only 2 genes in common between these two groups.
ERRBS showed an overall increase in methylation of HSC compartment in hBM compare to hFL and gradual demethylation of lineage associated genes in MPP.
Conclusion: Our data indicate that there are distinct regulatory networks that govern hFL and hBM HSC self-renewal. We found very little differences in gene expression between all hFL HCS compartments (average 20 genes) compare to hBM (average 224), indicating that by adulthood self-renewal is becoming more restricted to the LT-HSC compartment.

Disclosures
No relevant conflicts of interest to declare.
A Dynamic Intron Retention Program in the Mammalian Megakaryocyte and Erythrocyte Lineages
Authors: Edwards Christopher R , Middleton Rob , An Xiuli , Mishra Tejaswini , Mohandas Narla , Hardison Ross C. , Rasko John , Blobel Gerd A. 
Published: October 2019

Abstract
Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing.

Disclosures
No relevant conflicts of interest to declare.
CD34 Is a Ligand for Vascular Selectins on Human Hematopoietic Stem/Progenitor Cells
Authors: AbuSamra Dina Bashir 
Published: October 2019

Abstract
The cell surface glycoprotein CD34 is widely recognized as a marker of hematopoietic stem/progenitor cells (HSPCs), but its physiological role on HSPCs remains elusive. Although CD34 is well recognized for mediating homing of L-selectin+ naïve T cells to high endothelial venules of secondary lymphoid organs, the natural ligand for HSPC specific-CD34 is unknown. Studies in both humans and mice indicate that vascular selectins (E-selectin and P-selectin) are constitutively expressed on marrow endothelial cells and intravital studies revealed that HSPC migration to marrow occurs at specialized microvascular beds expressing E-selectin. Given that the identity of these glycoprotein E-selectin ligands (E-selL) on human HSPCs is limited, we used mass spectrometry (MS)-based proteomics to fully characterize all these potential ligands expressed on primary human CD34+ HSPCs and on the CD34+ KG1a cell line (human acute myelogenous leukemia that serves as a model for human HSPCs). We obtained a very rich resource for further investigation and chose to highlight CD34 as an E-selL candidate. The physiological binding of CD34 to E-selectin was addressed at both the molecular level and cellular level using novel binding assays. At the molecular level we developed a novel-binding assay to capture endogenous candidate glycoprotein ligands from whole cell lysates prepared from human HSPCs onto a surface plasmon resonance (SPR) chip and characterized its binding kinetics to recombinant E-sel. At the cellular level we used the blot rolling assay to monitor the ability of cells stably transfected with E-selectin (CHO-E) to roll on CD34 immunoprecipitated from HSPCs to demonstrate that CD34 binds to E-selectin with affinity comparable to well-described E-selectin ligands. Further biochemical analysis demonstrated that E-selectin binding was restricted to a specific glycoform that expresses sialyl Lewis X (sLex). This interaction was dependent on calcium, sialylation and O-glycans as removal of these abolished binding to E-selectin. CD34 binding to the other vascular selectin, P-selectin was dependent on O-glycan and tyrosine sulfation making CD34 the first selectin ligand since P-selectin glycoprotein ligand-1 (PSGL-1) reported to bind all three selectins. Among the stem cell specific processes such as self-renewal and differentiation that CD34 has been implicated to play a significant role in, these data are the first to define a role for human CD34 in mediating the direct migration of HSPCs to the bone marrow and suggests that a natural ligand for CD34 on human HSPCs are vascular selectins.

Disclosures
No relevant conflicts of interest to declare.
A Subcutaneously Administered Investigational RNAi Therapeutic (ALN-CC5) Targeting Complement C5 for Treatment of PNH and Complement-Mediated Diseases: Interim Phase 1 Study Results
Authors: Hill Anita , Taubel Jorg , Bush Jim , Borodovsky Anna , Kawahata Noriyuki , Mclean Helen , Powell Christine , Chaturvedi Prasoon , Warner Garvin , Garg Pushkal , Sorensen Benny 
Published: October 2019

Abstract
Introduction: Uncontrolled complement activation plays a pivotal role in a variety of disorders such as PNH and aHUS. ALN-CC5 is a subcutaneous (SC) investigational RNAi therapeutic targeting complement C5 (C5). In preclinical studies, ALN-CC5 has demonstrated decreased terminal complement activity. Based on the literature, preventing the generation of the terminal complex protects against intravascular hemolysis and complement-mediated tissue damage. The purpose of this study is to evaluate the safety and tolerability of ALN-CC5 in normal healthy volunteers.
Material and methods: A multi-centered, placebo controlled, double blind phase 1 clinical study in healthy volunteers is ongoing. Several cohorts of healthy volunteers in Part A, a single ascending dose study and Part B, a weekly multiple ascending dose study have been completed. Primary endpoints are safety and tolerability. Secondary endpoints are pharmacokinetics, reduction of circulating C5, reduction in hemolytic, CAP and CCP activity.
Results: In Part A, 20 healthy volunteers were randomized (1:3) to placebo or single SC dose of 50, 200, 400, 600 or 900mg of ALN-CC5 and followed for at least 70 days. In Part B, 12 healthy volunteers were randomized (1:3) to placebo or 5 weekly doses of 100, 200 or 400mg of ALN-CC5. No SAEs or study discontinuations occurred and overall ALN-CC5 was considered safe and generally well tolerated. A dose dependent and 94% mean maximum C5 knockdown was achieved following weekly administration. Updated safety and tolerability data as well as C5 knockdown, and changes in CAP, CCP and hemolytic activity from the study will be presented.
Conclusion: Collectively, these initial results suggest that the use of a novel RNAi therapeutic targeting C5 is a promising approach for inhibiting complement in PNH, aHUS and other complement-mediated diseases. The subcutaneous route of administration and infrequent dosing make this a potentially encouraging therapy.

Disclosures
Hill: Alnylam: Consultancy. Off Label Use: ALN-CC5 is an investigational RNAi therapeutic targeting complement C5.. Borodovsky:Alnylam Pharmaceuticals: Employment, Equity Ownership. Kawahata:Alnylam Pharmaceuticals: Employment, Equity Ownership. Mclean:Alnylam Pharmaceuticals: Employment, Equity Ownership. Powell:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chaturvedi:Alnylam Pharmaceuticals: Employment, Equity Ownership. Warner:Alnylam Pharmaceuticals: Employment, Equity Ownership. Garg:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.