bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Src-Abl Kinase Inhibitor Dasatinib (BMS-354825) Shows Anti-Proliferative and Anti-Apoptotic Effects in Chronic Lymphocytic Leukemia (CLL) Cells In Vitro.
Authors: Veldurthy Aditya , Patz Michaela , Pallasch Christian P. , Wendtner Clemens M. , Hallek Michael , Krause Gunter F. 
Published: October 2019

Abstract
Introduction: Dasatinib is an ATP competitive, dual-specific Src-Abl kinase inhibitor used for the treatment of imatinib-resistant Bcr-Abl-positive leukemias. Originally it was developed from the aminothiazole scaffold as an inhibitor of Src family kinases (SFKs). Since the aberrant expression and activity of the SFK Lyn seems to enhance the survival of CLL cells, dasatinib is currently tested in clinical studies for CLL. In order to explore the anti-leukemic potential of dasatinib we tested its effects on freshly isolated CLL cells and on CLL cell lines.
Methods and Results: In freshly isolated CLL cells, dasatinib showed a dose and time dependent reduction of global tyrosine phosphorylation which was paralleled by a decreased phosphorylation of the activating tyrosine residue of SFKs. The comparison of the inhibitory effect of several protein tyrosine kinase inhibitors on overall tyrosine phosphorylation in cellular lysates of freshly isolated CLL cells showed that 0.1 μM dasatinib had a more pronounced effect than 5 μM of nilotinib, or 10 μM of PP1 or imatinib. In the CLL cell lines, Mec1 and JVM-3, 0.1 μM of dasatinib appeared to interfere with survival signaling, since decreased levels of the activated phosphorylated forms of Akt, Erk1/2 and p38 were observed after exposure to dasatinib for 2 hours. In these cell lines, dasatinib treatment increased p53 protein levels and induced PARP cleavage and caspase activity. Pro-apoptotic effects of dasatinib were observed by an increase of annexin V-positive cells and a decrease of metabolic activity measured as XTT reduction, alone and in combination with fludarabine. Among six patient samples, two clones were particularly sensitive to dasatinib and fludarabine-resistant. While in these two samples the apoptosis induction by 5 μM dasatinib surpassed that by 5 μM fludarabine, the same dose of the two drugs led to similar apoptosis induction in the fludarabine-sensitive samples. Dasatinib treatment sensitized CLL cells for fludarabine-induced apoptosis. This enhancement of apoptosis induction was more pronounced in fludarabine-resistant patient samples and JVM-3 cells than in Mec1 and fludarabine-sensitive CLL cells.
Conclusion: The Src-Abl kinase inhibitor dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro.
Marked Sensitivity of Chronic Lymphocytic Leukemia (CLL) to Apoptosis Induced by BCL2 Antagonist ABT-737.
Authors: Vogler Meike , Majid Aneela , Walewska Renata , Dyer Martin J.S. , Cohen Gerald M. 
Published: October 2019

Abstract
Most cases of CLL express BCL2 constitutively at levels comparable to those seen in follicular lymphoma with t(14;18)(q32;q21) and BCL2 is considered to play a pivotal role in the suppression of apoptosis in this disease. The BCL2 antagonist ABT-263 has recently entered phase I/IIa clinical trials for potential treatment of CLL. ABT-263 and ABT-737 are rationally designed BH3 mimetics that are thought to act by displacing pro-apoptotic BIM from BCL2 and thus activating the apoptotic cascade. While being resistant towards some commonly used apoptotic stimuli like etoposide or TRAIL, we found that all primary CLL cells are highly and rapidly sensitive to ABT-737, with an average EC50 in 60 patients of only 6.7 nM and after only 4 h of treatment. ABT-737 induced BAX/BAK oligomerization followed by rapid and extensive cytochrome c release, caspase activation and chromatin condensation. Incubating unfractionated samples in a whole blood assay did not significantly alter sensitivity. Notably, sensitivity of CLL cells to ABT-737 was independent of the patient’s stage of disease, p53 mutation or function, cytogenetics, or IGVH mutational status. Furthermore, ABT-737-induced apoptosis was observed irrespective of previous treatment or in vivo resistance to fludarabine, suggesting that ABT-737 might induce apoptosis even in patients with highly progressive and otherwise resistant disease. We also analyzed expression levels of BCL2 and MCL1 in CLL patients by western blot, since MCL1 is considered to be the most important factor for resistance towards ABT-737. Interestingly, sensitivity to ABT-737 was independent of BCL2 and MCL1 levels in CLL, indicating that expression of MCL1 was not sufficient to render CLL resistant to ABT-737. In addition, we further induced expression of MCL1 by either IL4 or interferon γ treatment, which resulted only in a minor delay of apoptosis that was easily overcome by treatment with slightly higher concentrations of ABT-737. Hence, our data suggest that in contrast to previous reports obtained in a variety of cell lines, MCL1 upregulation is not sufficient to induce resistance to ABT-737 in primary CLL cells. Next, we investigated whether exposure to ABT-737 resulted in a displacement of BH3-only proteins from BCL2. Surprisingly, we could not detect a significant displacement of BIM from BCL2. In contrast, we found that the multidomain proapoptotic protein BAK is directly bound to BCL2 in CLL and this interaction is abrogated by ABT-737, indicating that ABT-737 can directly displace BAK but not BIM from BCL2. Taken together, our results indicate that targeting BCL2 with ABT-263 or ABT-737 may be a highly promising strategy for treatment of CLL.
Phase I Clinical Trial of a Novel, Long-Acting Antithrombotic Drug: A Human Monoclonal Antibody Partially Inhibiting FVIII Activity.
Authors: Verhamme Peter , Pakola Steve , Jacquemin Marc , Saint-Remy Jean Marie , Stassen Jean Marie , Collen Desire , Riisbro Rikke , Jensen Thomas , Glazer Steven 
Published: October 2019

Abstract
Current treatments for preventing thrombotic disease are still inconvenient and associated with a high risk of bleeding. Improved anticoagulant agents are therefore required. TB-402 is a monoclonal antibody (fully human IgG4-antibody) that targets factor VIII (FVIII) and represents a novel type of anticoagulant agent. In vitro, TB-402 only partially inhibits human FVIII even when TB-402 is in excess over FVIII. Preclinical studies confirmed this plateau inhibition and also established the antithrombotic efficacy of TB-402 (Jacquemin M, et al. J Thromb Haemost.2006; 4:1047). Plateau inhibition even in setting of excess TB-402 may allow for improved safety with decreased risk of overdose and decreased need for monitoring. The long half-life of the antibody creates the possibility for a once a month administration. TB-402 may therefore represent a safer and more convenient agent than other available anticoagulants. A Phase I study has completed enrolment of 56 healthy male volunteers. This randomised, double-blind, placebo-controlled, single dose, dose escalation study evaluated the safety and the pharmacokinetic/pharmacodynamic profile of TB-402. Volunteers were treated with a single intravenous administration of placebo or TB-402 at doses of 0.015, 0.1, 0.5, 2.5, 12.5, 37.5, 188, 620 or 1,860 μg/kg. All doses were evaluated in 18–45 year old volunteers, with the highest dose also evaluated in an older age, 55–75 years, cohort. Study drug has been well tolerated in both the young and the older age cohorts, with no safety issues observed. A plateau effect in terms of FVIII level inhibition has been observed. At plateau, FVIII levels were generally decreased by 1/3 to 2/3 from baseline. aPTT prolongation (generally 1.1–1.2 times baseline) was also observed whereas the Prothrombin time (PT) was not modified. aPTT prolongations at doses of ≥ 620 μg/kg were generally maintained for at least 4 weeks. Long half-life of drug and associated anticoagulant effect are supported by the finding of consistent and prolonged effect on aPTT. In conclusion, this study demonstrates that in healthy young and older volunteers a single administration of TB-402 results in a prolonged anticoagulation effect without the risk of overdosing or spontaneous hemorrhage. TB-402 will next be evaluated in patients at risk for venous thromboembolism.
High Dose Methylprednisolone and Rituximab Is an Effective Therapy in Advanced Refractory Chronic Lymphocytic Leukaemia Resistant to Fludarabine Therapy.
Authors: Dungarwalla Moez , Kanagasabapathy Pamala , Kulkarni Samar , Evans Steve O. , Riley Unell , Morgan Gareth J. , Catovsky Daniel , Dearden Claire E. , Matutes Estella 
Published: October 2019

Abstract
The management of refractory advanced chronic lymphocytic leukaemia (CLL) continues to pose a considerable challenge to the clinician. CLL patients who are refractory or become resistant to fludarabine containing regimens currently have a poor prognosis. High dose methylprednisolone (HDMP) has been shown to be an effective therapeutic option in patients who are resistant to fludarabine particularly those with bulky lymphadenopathy or refractory cytopenias who are unsuitable for alemtuzumab therapy. We have used HDMP in combination with the anti-CD20 monoclonal antibody, Rituximab, to treat 14 patients with advanced refractory CLL. Eight of fourteen patients were male. The median age was 62.5 years (range 30–71). Twelve patients had bulky lymphadenopathy with nodal masses greater than 5 centimetres in diameter. Nine patients had Binet stage C and the remainder were stage B. Autoimmune manifestations were present in five of the patients, and in one of them fludarabine therapy was contraindicated due to active autoimmune haemolysis. FISH analysis was performed to detect 17p, 11q and 13q deletions and showed 11q23 deletions in five patients. The overall response rate was 93% with a median progression free survival (PFS) of seven months. Two patients achieved a CR, another a nodular PR and 10 patients had a PR. Responses were seen in all 4 patients who had not responded to alemtuzumab as a single agent. Only one patient failed to respond. The median survival was 20 months. All five patients with 11q23 deletions responded, and the mean progression free survival in this subgroup was 10.5 months. These results compare extremely favourably with our own previous study (Thornton et al, 1998) that used HDMP alone in the management of advanced/refractory CLL where only 43% of patients responded and no patients achieved a CR. We have demonstrated that the combination of HDMP with rituximab is more effective than HDMP alone (p<0.01) in advanced refractory CLL. Although HDMP in combination with rituximab causes little or no myelosuppression, 6/14 patients (43%) developed opportunistic fungal or viral infections and this included fungal mould infections, fungal yeast infections and opportunistic viral infections. Three patients died of pulmonary fungal infections, although 2 of these patients had been retreated with alternative regimens. While the immunosuppressive effects of high dose steroids are well recognised, it appears that the addition of rituximab has made these patients more prone to opportunistic infections. In summary, HDMP in combination with rituximab is an effective rescue regime in purine analogue refractory CLL particularly in patients with bulky lymphadenopathy who are unsuitable for alemtuzumab, and in patients with 11q23 deletions.
The Influence of CYP2C9 and VKORC1 1173C/T Genotype on Reversal of Anticoagulation (INR) Following Phytonadione Administration.
Authors: Limdi Nita A. , Acton Ronald T. , Beasely Timothy M. , Adler Brian 
Published: October 2019

Abstract
Context: Excessive anticoagulation increases the risk of hemorrhagic complications associated with warfarin therapy. Oral, subcutaneous or parenteral phytonadione is used to reverse both symptomatic and asymptomatic excessive anticoagulation. Multiple reports have addressed the influence of phytonadione dose and route of administration on time course of anticoagulation reversal.
Objective: To determine the influence of CYP2C9 and VKORC1 1173C/T on anticoagulation reversal following phytonadione administration.
Design and Setting: Prospective cohort study of 578 patients newly initiated on warfarin (target INR: 2–3).
Main Outcome measures: INR difference (pre-treatment INR- post-treatment INR) produced by phytonadione after accounting for age, gender, race, CYP2C9 and VKORC1-1173C/T genotype, warfarin dose, comorbidity, concomitant therapy with CYP2C9 inhibitors, phytonadione dose and time (hours) between pre-treatment and post-treatment INR measurement.
Results: During follow-up (August 2003-April 2007) various treatment modalities were used to reverse anticoagulation. Phytonadione monotherapy was administered 28 times to reverse anticoagulation in 24 patients (15 women, 14 African American), mean age 66.4±16.9 yrs. The mean phytonadione dose was 4.1mg (±2.7) and the mean duration between pre-treatment and post-treatment INR measurements was 53.4 (±26.7) hours. Administration of phytonadione produced a median INR decline of 5.5 (range 0.2–10.9). Because 3 of the 24 patients were measured more than one time, a linear mixed model using SAS PROC MIXED was performed in order to account for the dependency in the observations. The mixed model statistically adjusted for age, gender, race, CYP2C9 genotype, warfarin dose, comorbidity, concomitant therapy with CYP2C9 inhibitors, phytonadione dose and time (hours) between pre-treatment and post-treatment INR measurement. The extent of anticoagulation reversal (INR decline in response to phytonadione administration) was significantly smaller among patients with variant VKORC1-1173C/T genotype compared to those with wild-type genotype (p=0.0027). The INR decline was also smaller among patients with variant CYP2C9 genotype, although this finding was of marginal statistical significance (p=0.069).
Conclusion: The efficacy of phytonadione in reversing anticoagulant effect of warfarin is significantly influenced by the individuals VKORC1-1173C/T genotype.
Immunogenicity of Human Factor VIII in Rhesus and Cynomolgus Monkeys.
Authors: Skupsky Jonathan , Lozier Jay N. , Donahue Robert E. , Metzger Mark N. , Azimzadeh Agnes M. , Pierson Richard N. , Scott David W. 
Published: October 2019

Abstract
Up to 30% of hemophilia A patients form inhibitory antibodies to therapeutic doses of human factor VIII (fVIII), thus rendering therapy ineffective. Advances in identifying the nature of the fVIII mutations (inversion, stop codons, etc), HLA linkage and other factors have suggested these as important predictors of inhibitor responsiveness in patients. The ability to predict which patients may respond with inhibitors would be important in order to identify suitable patients for tolerance therapies prior to exposure to fVIII and the development of high inhibitor titers. Animal models to evaluate the immunogenicity of fVIII would also be useful in this regard. We and others have previously shown that mice with normal hemostasis produce antibodies to fVIII in response to therapeutic doses of human fVIII given intravenously (i.v.), a normally tolerogenic route (Lin, Soukhareva, and Scott, ASH 2004) or in an adenovirus vector (Brown et al. JTH 2004; Lozier and Zhang, ASH 2005). To establish whether hemostatically normal non-human primates would respond to human fVIII and provide a model for future tolerogenic therapy, we immunized rhesus and cynomolgus monkeys with fVIII using a variety of routes. In the case of rhesus monkeys, fVIII was administered via three routes: i.v. in PBS, intramuscularly (i.m.) in alum, or i.m. in a fVIII-expressing adenoviral vector. All three routes led to significant titers as measured by ELISA, and all six animals responded to i.v. boosting with fVIII in PBS. Cynomolgus macaques (3) that received multiple doses of fVIII i.v. also produced antibody to fVIII, albeit of lower titer. Importantly, T cell proliferation to human fVIII in vitro could be elicited from cynomolgus peripheral blood cells. These results set the stage for future tolerogenic therapies in both naïve and fVIII primed non-human primates.
Dendritic Cells as a Novel Target for Erythropoietin: Studies in a Human System.
Authors: Sagiv Sari Prutchi , Lifshitz Lilach , Orkin Ruth , Newmann Drorit , Mittelman Moshe 
Published: October 2019

Abstract
The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system have been described for some time; however, the mechanism of action by which EPO affects lymphocyte number and function has yet to be elucidated. Our search for possible mechanisms by which EPO affects these parameters led us to the novel discovery that EPO receptors (EPO-R) are expressed in dendritic cells (DCs), the most potent antigen presenting cells and most efficient T cell primers. Furthermore, we show that EPO has direct effects on the phenotype and function of human DCs. When added in vitro, EPO increased the percentage of peripheral blood originated DCs that express the co-stimulatory molecules CD80, CD86 and CD40. We also show that EPO up-regulates the level of expression of these molecules, as well as that of HLA-DR in monocyte-derived DCs. When added to immature DCs, EPO alone can induce their maturation. Furthermore, we demonstrate that EPO enhances DC function, as revealed by increased antigen uptake, secretion of interleukin (IL)-12 and stimulatory function in allogeneic mononuclear cell proliferation. We propose that DCs may represent a missing link which might explain previously observed immunomodulatory actions of EPO. Hence, our findings are of crucial importance and may open new clinical avenues for EPO in the optimization of ex-vivo DC-based vaccines, or when administered to patients in order to enhance immune system responses.
PHF23: A Novel Erythropoietin-Induced Gene Associated with AML and MDS.
Authors: Hodges Vivien M. , Rainey Susan , Smyth Victoria , Mills Ken , Lappin Terence R.J. , Maxwell A. Peter 
Published: October 2019

Abstract
Chromosomal translocations in a number of hematopoietic malignancies, including AML and MDS, result in the formation of NUP98 gene chimeras. Recently, fusion of NUP98 with a novel gene, PHF23 or plant homeodomain (PHD) finger 23, was reported in a cryptic translocation in a patient with AML (Reader et al., 2007). PHF23 is one of three known NUP98 fusion partners, associated with AML, which contain PHD finger motifs. In this study, we report a more detailed characterization of PHF23, which we originally identified by differential display PCR as an erythropoietin-induced gene in a murine model of terminal erythroid differentiation. In silico sequence analysis revealed a five exon gene containing a nuclear localization signal (NLS) as well as the C-terminal PHD motif. PHF23 mapped to chromosome 17p13.1, <450 kb from the TP53 gene, in a region frequently disrupted in cancer. Quantitative PCR analysis indicated Phf23 expression is not restricted to hematopoietic cells but demonstrates widespread tissue distribution, with increased expression levels in more proliferative tissues (∼6 fold increase in gene expression in the testis over mRNA levels in the liver). A ubiquitously expressed Phf23 splice variant (with mRNA concentrations ∼30–45% lower than full-length Phf23 gene expression levels) was generated using an alternative 3′ splice site within exon four, which exactly corresponded to the chr17 translocation breakpoint in the AML patient described. PHF23 is evolutionarily conserved, particularly within the PHD finger domain (>76% sequence identity between human and zebrafish PHD motifs), and is significantly related to another potentially oncogenic protein, PHF13/SPOC-1, associated with ovarian cancer. PHD motifs act as zinc fingers and the expanding family of PHD-containing proteins are implicated in chromatin regulation. Cellular localization studies using fluorescently tagged PHF23 indicated that both the full-length and spliced isoforms are located exclusively within the nucleus. We predicted that PHF23 has a similar structure to PHD motifs of known histone effector proteins, which influence transcription through recognition of specific epigenetic marks. This was demonstrated by immunoprecipitation and histone peptide binding assays showing that PHF23 associated with histone H3, primarily di- and tri-methylated H3K4, which are characteristic marks of active transcription. Microarray analysis of PHF23 mRNA expression levels in AML and MDS patient samples indicated no defined association with AML, however a positive correlation was demonstrated with a subset of MDS patients exhibiting erythroid dysplasia. This study suggests that PHF23, a novel histone effector protein, may play a role in aberrant transcription during defective hematopoiesis.
siRNA Mediated Knockdown of the Chimeric Transcription Factor E2A-PBX1 Identifies Potential Target Genes.
Authors: Chen Chun-Liang , Connell Harkness , Racoma Ira , Regula Brandi , Foley Katherine , Townsend Jessica , Ross Mary Elizabeth 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Several chromosomal translocations have prognostic significance and are used to classify patients for risk-directed therapy. The translocation t(1;19) which results in the fusion of TCF3 (E2A) and PBX1 genes occurs in 5% of pediatric ALL. E2A-PBX1 has been shown previously to have transcriptional activity. However, very little is known about what genes have altered expression in the presence of E2A-PBX1. To explore this question, we assessed genome wide gene expression after siRNA mediated knock down of E2A-PBX1. An E2A-PBX1 containing cell line (697) was transfected with E2A-PBX1 specific siRNA utilizing an Amaxa nucleofector2. Biologic replicates were performed by transfection of independent cultures. 70–80% reduction of E2A-PBX1 at mRNA and protein levels in 697 cells was reproducibly achieved using nucleofection and a combination of E2A-PBX1 specific siRNAs. Genome wide gene expression was assessed by Affymetrix U133 2.0 Plus arrays. Hybridizations were prepared and performed according to current Affymetrix protocols in the Functional Genomics Core at the Research Institute. Microarray data was normalized with RMA. Differentially expressed genes were selected using Significance Analysis of Microarrays (SAM). 78 probe sets demonstrated change in expression in E2A-PBX1 siRNA mediated knockdown relative to both mock transfection and nontargeting siRNA controls at a FDR of ≤ 5%. The 78 probe sets represent 49 known genes and 8 ESTs. Genes of specific interest upregulated by E2A-PBX1 include WNT-16, ANKS1B(EB-1), FAT, and RORB. Other investigators have previously cloned these genes by representational differential display from E2A-PBX1 expressing cell lines. We confirmed knockdown of these messages by E2A-PBX1 siRNA using qRT-PCR. Gene expression was calculated using the ΔΔCt method and normalization to ABL. Additionally, these genes are part of the E2A-PBX1 gene expression profile derived from primary pediatric leukemia samples previously published. [Ross, Blood 2003] An additional 34 candidate genes were further verified using qRT-PCR. The direction of change in gene expression correlated with microarray results in 30/34 genes evaluated. Other classes of genes upregulated by E2A-PBX1: kinases (FGFR2, MAP3K1), phosphtases (PPPIR14C), transcription factors (FOSL2, IRX2, EBF3, BMI1, BCL6), cell cycle-related genes (FBXW7, ETV5), Ras and Rho family genes (RAB8B). While B cell surface antigen expression (HLA-DRA, CD22) was decreased by E2A-PBX1. We have utilized siRNA to E2A-PBX1 to identify potential target genes of E2A-PBX1. This data suggests E2A-PBX1 can alter expression of a variety of classes of genes. Some of these genes may be potential targets for molecularly targeted therapy.