bluuebunny/crossref_metadata_2025_split huggingface dataset:
Truncated Human EpoR Causes Polycythemia in Fetal Erythropoiesis through Stat5 Hyperactivation.
Authors: Yoon Donghoon , Watowich Stephanie S. , Prchal Josef T. 
Published: October 2019

Abstract
Gain-of-function mutations causing truncations of the cytoplasmic domain of the human erythropoietin receptor (EPOR) result in a dominantly inherited disorder-primary familiar congenital polycythemia. The consequence of EpoR truncation is polycythemia and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR gene with a wild-type human EPOR (whEPOR) gene or a mutant human EPOR gene (mthEPOR) that we initially identified in a family with polycythemia. This mutation produces a truncated EpoR mutant with deleted cytoplasmic C-terminal domain after the first tyrosine residue of the intracellular domain. We reported that the mice containing mthEPOR allele have polycythemia and the adult whEPOR mice have anemia. To study the effects of these genotypes on fetal erythropoiesis, we examined fetuses E14.5 homozygotes wild type mouse EpoR, and human EPOR alleles. Hematocrit results show: MthEPOR fetuses were polycythemic compared to mouse EpoR (mthEPOR vs mouse EpoR; 34.58 ± 1.24 % vs 27.49 ± 0.90 %, P<0.001 by t-test), and whEPOR were anemic (whEPOR vs mouse EpoR; 18.61 ± 0.79 % vs 27.49 ± 0.89 %, P<0.001 by t-test). To study the pathophysiology of these phenotypes, we analyzed the erythroid differentiation using differential expression of CD71 and TER119 surface antigens by FACS in the fetal liver cells (FLs). FLs from mthEPOR homozygote had increased population on early and late basophilic erythroblast stage compared to mouse EpoR, but the populations of differentiated erythroid progenitors from mthEPOR homozygote were significantly lower than mouse EpoR and whEPOR homozygotes. We proposed that the erythroid progenitors from mthEPOR exit fetal liver to fetal blood circulation prematurely. The peripheral blood from mthEPOR and mouse EpoR s was analyzed by morphology and FACS. We show that the majority of population in peripheral blood from mthEPOR homozygote were earlier erythroid progenitors (high CD71/low TER119 population in mthEPOR 47.53 %, vs 13.14 % mouse EpoR; high CD71/high TER119 population from mthEPOR 11.49 % vs 65.00 % mouse EpoR). To evaluate the Epo signal transduction we tested the kinetics and dose responses of Epo at Stat5 phosphorylation level. The mthEPOR expressing FLs showed a markedly increased and sustained Epo sensitivity compared to mouse EpoR expressing FLs. In contrast, in whEPOR expressing FLs, Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. We conclude that mthEPOR causes fetal polycythemia and is associated with premature exit of early erythroid progenitors from fetal liver to fetal circulation. In contrast whEPOR is associated with more severe anemia than we observed in adult erythropoiesis. The degree of Epo induced Stat5 activation plays an important role in generation of anemic and polycythemic phenotypes.
siRNA Versus Antisense Locked Nucleic Acids: Stay Single!.
Authors: Kearney Phil , Westergaard Majken , Hansen Henrik F. , Staarup Ellen M. , Koch Troels , Ørum Henrik , Hansen Jens Bo 
Published: October 2019

Abstract
Much discussion has centred around the utility and benefits of siRNA in both target validation and as a therapeutic option. This has been driven by significant publications including that of Soutcheck et al (Nature432, 173–177 2004), which demonstrated liver targeting as well as in vivo efficacy when siRNA against ApoB was tethered to a cholesterol moiety.
Santaris Pharma has developed a third generation nucleic acid chemistry referred to as locked nucleic acid (LNA) which delivers unmatched affinity and stabiliy benefits, largely overcoming the drawbacks associated with traditional antisense molecules. We therefore sought to compare this chemistry with targets which siRNA has been successfully used in in vivo/in vitro settings. The same motif used in the Soutcheck study was targeted with a LNA molecule, and the free siRNA activity was compared to the cholesterol linked and free LNA molecules in their ability ot down regulate ApoB expression. LNA (SPC3197) inhibited ApoB expression by 90% while at an equimolar concentration siRNA was ineffective in the liver and jejunum. Cholesterol linked siRNA was only effective in the jejunum (c50% reduction in mRNA) Fig1. Only the LNA mediated inhibition of ApoB expression was paralleled by a drop in serum cholesterol in the host animal.
In a second model siRNA molecules targeting Hif-1a mRNA (Yu et al Lab Invest84, 553–561 2004) were compared to our lead LNA molecule targeting Hif-1a, SPC2968. Interestingly in in vitro analyses these 2 molecules were equally effective. However in a murine model the increased half life of the LNA molecules translated to a potent inhibition of Hif-1a as measured by QPCR. This effect was noted in jejunum and liver, and persisted for at least 4 days. Hif-1a inhibition mediated by siRNA was not seen in any tissue analysed (Fig 2).
Finally a 3rd molecule targeting Bcl-2 has entered clinical Phase 1 trials, and data will be presented documenting its improved affinity and stabitily in relation to competitor molecules such as Genasense.
Figure Figure
Survival Impact of Time Repeated bcl2/IgH Rearrangement Measurements in Follicular Lymphoma Patients Treated with Front-Line Autotransplantation in the GELF-94 Trial by the GELA.
Authors: Florence Broussais , Nicolas Mounier , Marie-Hélène Delfau , Jean-Marie Cayuela , Elisabeth MacIntyre , Frederic Davi , Catherine Sebban , Coralie Belanger , Nicole Brousse , Pauline Brice , Corinne Haioun , Andre Tilly , Pierre Feugier , Andre Bosly , Bertrand Coiffier , Gilles Salles 
Published: October 2019

Abstract
In the autotranplantation framework, an undetectable MRD before ASCT seems to protect for disease relapse and to increase survival. Using data from the GELF94 trial, which randomized consolidation treatment after achieving clinical response between front line ASCT and chemotherapy, we aimed to assess the impact of MRD on survival controlling for consolidation treatment and time repeated measurement. Of 401 patients included from 07/94 to 03/01, 209 received 12 cycles of CHVP (cyclophosphamide, doxorubicin, teniposide, and prednisone) plus interferon alfa in 18 months (CHVP-I arm) and 192 received 4 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) then high-dose therapy with total body irradiation and ASCT (CHOP-HDT arm). Bcl2/IgH rearrangement was prospectively assayed by PCR in bone marrow (BM) and in blood at diagnosis and repeated every 6 months during the first year then annually. Time repeated measurement was stopped at clinical relapse or instigation of a new treatment. At diagnosis, 225 patients had material available for bcl2/IgH rearrangement analysis: BM for 182 (45 %) patients and blood for 199 (50%). In the latter, 105 of the 199 patients (53%) were found to have a bcl2/IgH rearrangement in the MBR breakpoint, whereas bcl2/IgH rearrangement in mcr was observed in 5 patients (3%) and no rearrangement at MBR or mcr in the remaining 94 patients (44%). No differences were found according to bcl2/IgH rearrangement in terms of response rate (82% for bc2- vs 80% for bcl2+) and.5 yrs survival (85% for bcl2- and 79% for bcl2+). Time repeated bcl2/IgH rearrangement measurements were available for 142 patients (ASCT n=75, chemo n=67): in BM for 79 patients and in blood for 85 patients. The clinical characteristics were well balanced between patients with/without time repeated measurement. By time dependant Cox’s model, the significant prognostic factors for survival were age below 40 yrs (RR= 21, p=0,005), complete clinical response (RR=5, p=0.02) and bcl2/IgH rearrangement negativity (RR=4, p=0.03). There was no treatment impact. These findings confirm the importance of molecular response in addition to the clinical response as a critical factor for prognosis. The results of this study suggest that in FL patients, the eradication of cells bearing the Bcl2/IgH rearrangement is highly desirable early in the treatment and should be maintained as long as possible.
Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.
Authors: Mojica-Henshaw Mariluz P. , Laverdiere Caroline , Prchal Jaroslav F. , Prchal Josef T. 
Published: October 2019

Abstract
Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband’s parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.
MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.
Authors: Georgantas Robert W. , Hildreth Richard , Alder Jonathan , Croce Carlo M. , Calin George A. , Civin Curt I. 
Published: October 2019

Abstract
MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.
Structural Basis and Cellular Consequences of Potent Inhibition of the Aurora Kinases, Wild Type Abl Kinase and a T315I Multi-Drug Resistant Mutant Form of Abl Kinase by MK-0457 (VX-680).
Authors: Pollard John R. , Charlton Peter A. , Cheetham Graham M.T. , Falcon Susanna C. , Golec Julian M.C. , Griffiths Matthew R. , Weber Peter 
Published: October 2019

Abstract
MK-0457 (VX-680) is a potent small molecule inhibitor of the Aurora-family of protein kinases that inhibits cell proliferation through aberrant mitosis and failed cytokinesis. Affected cells are subsequently deleted by apoptosis. Tumor regression and increased survival have been reported in multiple in-vivo models and in January 2005 MK-0457 entered Phase I clinical trials as the first in class Aurora inhibitor. Although MK-0457 demonstrates significant selectivity over the vast majority of kinases tested, it has been shown to potently inhibit Flt-3 and Abl, both of which are targets for anti-cancer therapies. Most notably MK-0457 potently inhibits a series of mutant Abl kinases including the multi-drug resistant T315I form that is commonly expressed in Gleevec refractory CML and ALL. We report here the first MK-0457 / Aurora co-complex crystal structure and show that it adopts a closed inactive conformation. By comparison with other reported kinase structures we provide a structural basis for the cross-reactivity with wild type and drug resistant mutants of the Abl kinase. We also show that MK-0457 blocks both Aurora and Abl cellular kinase activity and displays potent cytotoxic activity against a range of leukemic cell lines, including Gleevec resistant cells that are reliant on mutant Abl for growth and survival. Similar observations have recently been made in the clinic, where new data show that MK-0457 has activity in patients with BCR-ABL T315I mutant CML and Philadelphia positive ALL.
Tal1 and a DNA Binding Mutant (Tal1R188G;R189G) Cooperate with LMO2 To Induce Leukemia in Mice.
Authors: Draheim Kyle M. , Erdkamp Kimberly , Arous Eward , Calvo Jennifer A. , Kelliher Michelle A. 
Published: October 2019

Abstract
LMO2 is a member of the “LIM only” protein family and required for primitive erythropoiesis and adult vasculogenesis and angiogenesis. In erythroid cells, LMO2 interacts with TAL1 and E47 and LDB1 and GATA-1, thereby forming a transcriptional complex whose target genes include EKLF, CKIT, and p4.2 (protein 4.2). LMO2 was first implicated in leukemogenesis when it was identified in chromosomal translocations t(11;14)(p13;q11) and t(7;11)(q35;p13) found in T cell acute lymphoblastic leukemia (T-ALL) patients. Ectopic expression of LMO2 in mice recapitulates the human disease, albeit at low penetrance and following a long latency. LMO2 has been shown to synergize with TAL1, yet the mechanism of oncogene cooperativity is unknown. Two models have been proposed: the first suggests that LMO2 and TAL1 synergize by forming an active transcriptional complex that induces expression of target genes such as retinaldehyde dehydrogenase 2 (RALDH2) and TALLA1 (a surface marker of T-ALL). The second model proposes that LMO2 and TAL1 sequester the E47/HEB heterodimer, resulting in inhibition of E47/HEB-mediated transcription. To distinguish between these models, we mated our Tal1 transgenic mice and our DNA binding mutant of Tal1(R188G:R189G) with Lmo2 transgenic mice. As expected, transgenic expression of Lmo2 induced disease in 23% of mice after 302 days. Similar to published studies, Tal1 and Lmo2 expression dramatically inhibited thymocyte development and induced T cell leukemia in 100% of the mice with a mean latency of 108 days. To test whether the DNA binding properties of tal1 were required to cooperate with LMO2, we mated mice expressing a DNA binding mutant of Tal1(R188G;R189G) with Lmo2 transgenic mice and found that tumors were induced with similar kinetics; 100% of mice developed disease with an average latency of 107 days. These data suggest LMO2 does not require the DNA-binding properties of Tal1 to induce leukemia in mice and support the model that LMO2 contributes to leukemia through E47/HEB sequestration and inhibition.
Myc Promotes Tumorigenesis by Supressing Nfkb2.
Authors: Keller Ulrich , Huber Juergen , Nilsson Jonas , Hall Mark , Peschel Christian , Cleveland John L. 
Published: October 2019

Abstract
Rel/NF-kappaB transcription factors are mediators of immune responses, cell survival, and transformation, and are frequently deregulated in cancer. The NF-kappaB2 subunit is associated with chromosomal translocations or deletions in lymphoid malignancies, and deletion of the COOH-terminal ankyrin domain of NF-kappaB2 results in increased lymphocyte proliferation. Here, we report that activation of the Myc oncogene leads to suppression of Nfkb2 expression in early passage mouse embryonic fibroblasts and primary bone marrow-derived B cells. Accordingly, transgenic expression of c-Myc in the Eμ-Myc model of human Burkitt lymphoma results in reduced nfkb2 transcript and NF-kappaB2 p100 and p52 protein levels in pre-cancerous B cells. Nfkb2 expression is further reduced in the majority of Eμ-Myc lymphomas and in human Burkitt lymphoma. Nfkb2 suppression by Myc occurs at least in part by transcriptional repression as shown by promoter studies. To evaluate the relevance of Myc-mediated suppression of Nfkb2 for tumorigenesis, consequences of complete Nfkb2 loss were evaluated in vivo. In pre-cancerous B cells of Myc-transgenic mice, loss of Nfkb2 affects Myc-induced apoptosis while B cell proliferation is unaffected. Deletion of Nfkb2 results in an acceleration of lymphoma development in Eμ-Myc transgenic mice. Therefore, Myc-induced Nfkb2 suppression promotes lymphomagenesis.
Application of High Throughput Screening To Identify Novel Small Molecule Inhibitors of αIIbβ3-Mediated Platelet Adhesion to Fibrinogen.
Authors: Blue Robert , Jiroušková Markéta , Karan Charles , Coller Barry S. 
Published: October 2019

Abstract
Two separate conformational changes have been proposed to accompany activation of platelet αIIbβ3: 1) leg separation leading to extension of the head region composed of the αIIb propeller and β3 βA (I-like) domains, and 2) a swing-out motion at the junction of the β3 βA (I-like) and hybrid domains. Small molecule inhibitors of αIIbβ3 competitively block the RGD ligand binding site and variably induce conformational changes in αIIbβ3 as judged by the binding of ligand-induced binding site (LIBS)-specific monoclonal antibodies. In an attempt to identify molecules that may inhibit αIIbβ3 activation without initiating the conformational changes associated with ligand binding, we screened 33,264 compounds from four different chemical libraries (Prestwick, Chembridge, Cerep and ChemDiv) for their ability to inhibit the adhesion of washed platelets in HEPES-modified Tyrode’s buffer with 1 mM Ca2+/0.5 mM Mg2+ to immobilized fibrinogen adsorbed from a 50 μg/ml solution. When tested at a final concentration of 16 μM, a total of 102 compounds (0.31%) demonstrated greater than 50% inhibition of platelet adhesion, and two of these (Figure 1) demonstrated >30% inhibition of the initial wave of ADP-induced aggregation of platelets in citrated platelet-rich plasma. IC50s for inhibition of ADP (5 μM)-induced platelet aggregation for compounds 1 and 2 were 13 ± 4.5 and 17 ± 5 μM (n=3), respectively. Compounds 1 and 2 also inhibited fibrinogen binding to platelets induced by the activating LIBS antibody AP5 with IC50s of 27 and 30 μM, and 20 and 27 μM, respectively, in two experiments. Since AP5 binds to and directly activates αIIbβ3, it is likely that the compounds’ inhibitory effects are due to direct binding to αIIbβ3 rather than inhibition of signal transduction. In two separate experiments, compound 1 at 15 - 20 μM produced variable increases in the binding of LIBS mAbs AP5, PMI-1 or LIBS1 to unactivated and ADP-activated platelets, whereas tirofiban (20 μM) consistently increased the binding of each mAb. Compound 2 did not increase the binding of any of the mAbs. Neither compound contains a negatively charged carboxyl group, which mediates the interaction of the Asp group in RGD ligands with the β3 MIDAS metal ion, but compound 1 has a carbonyl group that may potentially interact with the MIDAS metal ion. Compound 1 resembles 1,2-fused pyrimidine derivatives that have previously been demonstrated to inhibit platelet aggregation (Roma et al., Bioorg. Med. Chem. 2003, 11, 123). We conclude that high throughput screening of molecular libraries can identify novel compounds that inhibit αIIbβ3 and that one of them appears to inhibit αIIbβ3 without inducing conformational changes in the receptor.
Figure Figure