bluuebunny/crossref_metadata_2025_split huggingface dataset:
RNAi-Mediated Silencing of p190Bcr-Abl Inactivates Stat5 and Cooperates with Imatinib Mesylate and 17-Allylamino-17-Demetoxygeldanamycin in Selective Killing of p190Bcr-Abl-Expressing Leukemia Cells.
Authors: Futami Muneyoshi , Hatano Toshiyuki , Soda Yasushi , Kobayashi Seiichiro , Miyagishi Makoto , Tojo Arinobu 
Published: October 2019

Abstract
The 190kD (p190) and 210kD (p210) Bcr-Abl proteins are responsible for the development and progression of Philadelphia chromosome (Ph)-positive leukemia. We applied RNA interference (RNAi) technology to specific killing of p190+ cells. After screening a series of small hairpin RNA (shRNA) targeting p190, we determined the optimal sequences for gene silencing in the BCR (shBCR), junctional (shE1A2) and ABL (shABL) region of p190, respectively. Then, p190+ (KOPN30), p210+ (K562) and both negative (HL60) cell lines were infected with lentiviral vectors encoding these shRNAs, resulting in an efficient killing of KOPN30 cells. K562 cells were also sensitive to the two other than junction-specific shRNA and none of the anti-p190 shRNAs could affect survival of HL-60 cells, although shABL appeared to have some inhibitory effects on their growth, suggesting that Abl may be positively involved in proliferation of HL60 cells. Furthermore, colony formation derived from CFU-GEMM, BFU-E and CFU-GM was not influenced by shBCR. On the contrary, CFU-GEMM and BFU-E-derived colony formation was significantly suppressed by shABL, suggesting that Abl may play a certain role especially in normal erythropoiesis. Nonetheless, anti-p190 shRNAs may become therapeutic options in Ph-ALL by improvement of their delivery system such as CD19-targeted liposome formulation which we previously developed. In p190-transformed Ba/F3 cells, RNAi-mediated silencing of p190 specifically inhibited tyrosine phospohorylation of stat5 prior to their death, but did not affect phosphorylation of either Jak2, Akt, MEK1/2 or ERK1/2. These results offer another evidence for the critical role of Stat5 in BCR-ABL-induced transformation of hematopoietic cells. It is likely that p190 directly phosphorylates Stat5 and that Jak2 is remote from activation of Stat5 in this cell context, although the possible involvement of Src family kinases such as Hck and Lyn has not been excluded. In contrast, down-regulation of p190 by their treatment with 17-allyl-amino-geldanamycin (17-AAG) was associated with reduced phosphorylation of not only Stat5 but also Jak2 and Akt. shRNA targeting p190 also collaborated additively with imatinib and 17-AAG in growth inhibition of Ba/F3-p190 and BaF/3-p190Y253H cells. Collectively, RNAi-mediated silencing of p190 is a promising option both for therapeutic application and for delineating signal transduction in p190-expressing leukemia.
Figure Figure Figure Figure
The Novel Histone Deacetylase Inhibitor, LBH589, Induces Expression of DNA Damage Response Genes and Apoptosis in Ph-Acute Lymphoblastic Leukemia Cells.
Authors: Anna Scuto , Kirschbaum Mark H. , Atadja Peter , Juhasz Agnes , Pullarkat Vinod , Bhatia Ravi , Forman Stephen J. , Yen Yun , Jove Richard 
Published: October 2019

Abstract
We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph−) acute lymphoblastic leukemia (ALL). Two model human Ph− ALL cell lines (T-cell MOLT-4, and non-T non-B cell Reh) were treated with LBH589 and evaluated for biological and gene expression responses. Low nM concentrations (IC50 5–20 nM) of LBH589 induced cell cycle arrest, apoptosis and histone (H3 and H4) hyperacetylation. PCR array analysis revealed that LBH589 treatment increased mRNA levels of pro-apoptosis, growth arrest and DNA damage repair genes. Quantitative real-time PCR confirmed that LBH589 induces expression of FANCG, FOXO3A, GADD45A, GADD45B and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. Chromatin immunoprecipitation (ChIP) assays demonstrated increased histone acetylation at the GADD45G promoter following LBH589 treatment. Finally, treatment with LBH589 was active against cultured primary Ph− ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%). Thus, LBH589 possesses potent growth inhibitory activity against Ph− ALL cells associated with upregulation of genes critical for DNA repair. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph− ALL.
Leukemia-Associated Antigen Specific T-Cell Responses Following Combined PR1 and WT1 Peptide Vaccination in Patients with Myeloid Malignancies.
Authors: Rezvani Katayoun , Yong Agnes S.M. , Mielke Stephan , Savani Bipin N. , Musse Laura , Superata Jeanine , Jafarpour Behnam , Boss Carol , Barrett A. John 
Published: October 2019

Abstract
The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation (SCT) is evidence that T lymphocytes can eradicate leukemia. The successful identification of a range of leukemia-associated antigens such as proteinase 3 (PR3) and Wilms tumour-1 (WT1) has stimulated efforts to induce leukemia-specific T-cell responses to these antigens using peptide vaccines. Here we describe the safety and immunogenicity of a combined vaccine of two leukemia-associated antigenic peptides, PR1 and WT1. Eight HLA-A*0201 positive patients with myeloid malignancies (2 myelodysplasia, 5 acute myeloid leukemia and 1 chronic myeloid leukemia) received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients completed 4 weeks follow-up to monitor toxicity and immunological responses. Toxicity was limited to grade 1–2. All remain alive at a median of 252 days (range 105–523). We analyzed the immunological response to vaccination using PR1/HLA-A*0201 and WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular interferon-gamma (IFN-γ) in samples obtained pre- and weekly post-vaccination. A significant CD8+ T-cell response to the vaccine was defined as the emergence of PR1 or WT1-specific CD8+ T-cells when the pre-study analysis was negative or a twofold increase in frequencies when responses were present pre-vaccination. Following vaccination, a significant CD8+ T-cell response to PR1 was seen in 7/8 patients (median 0.34%, range 0.04–0.48%), to WT1 in 5/8 patients (median 0.29%, range 0–0.42%) and to one or both antigens in 8/8 patients. Vaccine-induced CD8+ T-cells were seen as early as 1 week post-vaccination, produced IFN-γ and were preferentially expanded in the effector compartment (CD45RO+/-CD27−). Post-vaccination, there was a strong correlation between the emergence of PR1 or WT1+CD8+ T-cells and a reduction in WT1 mRNA expression, a marker of minimal residual disease, suggesting a vaccine-driven anti-leukemia effect. Loss of response was associated with reappearance of WT1 transcripts (P<0.01). Two patients with detectable CD8+ T-cell responses to PR1 who failed to have a reduction in MRD relapsed 3–6 months following completion of vaccination. This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. Based on these results we have initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies.
Role of High-Dose Chemotherapy with Autologous Stem Cell Transplantation in Primary Systemic Amyloidosis: A Systematic Review and Meta-Analysis.
Authors: Behera Madhusmita , Kumar Ambuj , Kharfan-Dabaja Mohamed A. , Djulbegovic Benjamin 
Published: October 2019

Abstract
Background: Primary systemic amyloidosis (AL) is a rare plasma cell clonal disorder(8/million) characterized by extracellular deposits of material composed mainly of fragments of light chain immunoglobulin throughout a body. Standard chemotherapy (e.g. melphalan and prednisone) is associated with poor outcomes (typical median survival is between 12–18 months with less than 5% survive 10 years). Autologous stem cell transplant (ASCT) has been increasingly advocated for treatment of AL. However, it is uncertain whether ASCT is better than standard chemotherapy. To address this uncertainty, we undertook a systematic review/meta-analysis to evaluate the efficacy of high-dose chemotherapy and autologous stem-cell transplant (HSCT) versus conventional chemotherapy in patients with AL.
Methods: Data search of published studies included Medline [all randomized controlled trials (RCTs)], Cochrane library and hand search of references. Studies were included if they were comparison trials of HSCT versus conventional chemotherapy, regardless if they were RCTs, prospective studies with historical control, or single arm studies. The studies were eligible if patients had biopsy proven AL with at least one major organ involved. Data were extracted on benefits as well as harms (overall survival, event-free survival, response, treatment related mortality, treatment-related morbidity).
Results: Out of 34 identified studies only 13 met the inclusion criteria for the current systematic review (2 RCTs, 2 prospective non-randomized trials involving historical control, and 9 single arm trials). Altogether these trials enrolled 1056 patients. Pooled data from 4 trials with controls (RCT and non-RCT) found similar overall survival for ASCT and conventional therapy arms [hazard ratio (HR) of 1.10 (95% CI 0.88, 1.36, p=0.4); p= 0.6]. Analysis of data according to trial design also did not find any difference in survival [HR for RCTs was 1.10 (95% CI 0.88, 1.37) and for non RCTs HR was 0.98 (95% CI 0.29, 3.35)]. The complete hematological response was also similar in both arms in RCTs (Odds ratio [OR]=1.38, 95%CI 0.67, 2.85; p=0.4) and non RCTs (OR=1.78, 95%CI 0.22, 14.65; p=0.32). The pooled proportion of treatment-related deaths in the single arm studies for AHCT was 0.119 (95% CI = 0.09 to 0.14)].
Conclusion: The results from the meta-analysis indicate that there is no statistically significant difference between the treatment effects from high-dose chemotherapy with ASCT and conventional chemotherapy. Hence, the efficacy of ASCT in improving overall survival and complete hematological response remains to be proven.
ADAMTS-13 Levels Do Not Predict Outcome in Thrombotic Thrombocytopenic Purpura: Protein S Levels Are Maintained during Plasma Exchange with Solvent Detergent Treated Plasma.
Authors: Rock Gail A. , Anderson David , Benny Barrett , Barth David , Clark William , Foley Ronan , Sutton David , Leblond Pierre 
Published: October 2019

Abstract
Introduction The ultimate therapy for TTP is not yet defined. While approximately 85% of patients respond to plasma exchange with FFP, the use of alternative solutions such as cryosupernatant plasma, which is deficient in the high molecular weight forms of vWF. has not been proven by appropriate randomized prospective sample trials. However, it is intellectually appealing. Additionally, the use of the ADAMTS 13 level either to predict disease or outcome has not been resolved.
Methods Cryosupernatant plasma (CSP) was compared to solvent detergent treated plasma (SDP) over one cycle (nine days) of plasma exchange (PE) and subsequent procedures as required. Throughout treatment, ADAMTS 13 and protein S levels were followed.
Results At the primary end point of one month, 3 of 35 of the CSP and 1 of 27 of the SDP patients died. The average platelet count was 184 × 109 per litre for CSP and 209 × 109 per litre for SDP. While vWF and FVIII were elevated in all patients at entry, no unusually large vWF multimers were seen at any time. At entry 9 patients had ≤ 0–10% ADAMTS 13 and 14 of 62 had normal levels with the rest in between. At presentation only 9 patients had 100% inhibitor activity with no inhibitor in 18 patients. Of the four patients who died, two who received CSP, had 100% enzyme activity at presentation whereas one had no ADAMTS-13 and another had 10%. All 4 patients died within 10 days. Fifteen patients with less than 10% enzyme activity had >1.5 vWF. However, elevated (>3u/mL) vWF was seen in the presence of normal ADAMTS-13 levels. Six months after remission, and in the presence of a normal platelet count, ADAMTS13 remained low in 5 patients and normal in 15.Protein S levels in 41/62 patients studied showed little variation during therapy with values on day 3 and 5, similar to those seen on entry.
Conclusion In idiopathic TTP, ADAMTS 13 levels do not correlate with disease, and are not predictive of outcome. While protein S levels in the SDP were lower than those found in FFP, this did not appear to have clinical significance.
Inactivation of Human White Blood Cells in Red Blood Cell Products Using the MIRASOL® System for Whole Blood.
Authors: Fast Loren D. , Marschner Susanne , DiLeone Gilbert , Doane Suzann , Fitzpatrick Christy , Ray Goodrich 
Published: October 2019

Abstract
Transfusion of blood products containing white blood cells (WBC) can result in the induction of immune responses that can negatively impact the recipient. An approach that would mitigate these consequences would be beneficial. Previous studies had shown that exposure of platelet concentrates to light in the presence of riboflavin was able to inhibit immune responses mediated by WBC. To make this protocol more widely applicable the effect of treating whole blood units with riboflavin and varying amounts of light was tested. Human peripheral blood mononuclear cells were purified by Ficoll-Hypaque discontinuous centrifugation from aliquots of nonleukoreduced whole blood units that were untreated or exposed to Mirasol treatment using varying light dosages. Viability and phenotype of treated cells was unchanged compared to untreated controls. The results showed that exposure of whole blood units to 33J/mL red blood cells (RBC) UV light in the presence of riboflavin completely inhibited proliferation of WBC in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Additional assays showed that treated WBC were unable to induce proliferation of normal responder cells in an MLC. Treated cells did not produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. In addition, treatment was found to inhibit T cell activation as evidenced by the lack of CD69 expression in treated compared to untreated control cells when incubated with phorbol 12-myristate 13-acetate. These treatment conditions did not induce crossmatch incompatibility. Methemoglobin levels and hemolysis in RBC units stayed below 1% during storage for 42 days in AS-3. Platelet and plasma units separated from whole blood after treatment showed acceptable cell and protein quality over 5 days in storage or as fresh frozen plasma, respectively. In summary, Mirasol treatment was able to functionally inactivate WBC in whole blood products without adversely affecting the quality of the RBC, platelets and plasma. This technique offers potential means to achieve inactivation of WBC in whole blood units that can subsequently be separated into RBC, platelet and plasma components.
The Intracellular ABC Transporter A3 Contributes to Imatinib Resistance of CML Cells by Subcellular Sequestration.
Authors: Chapuy Bjoern , Albrecht Melanie , Radunski Ulf , Koch Raphel , Haase Detlef , Truemper Lorenz , Wulf Gerald G. 
Published: October 2019

Abstract
Inhibition of bcr-abl tyrosine kinase activity has evolved as a mainstay of therapy for patients with chronic myeloid leukaemia (CML). However, a fraction of leukaemia cells persists under targeted therapy and can lead to disease progression on cessation of treatment. Analysing leukemic progenitor cells with the side population phenotype we found that the bcr-abl positive leukemic clones contributed to this compartment in CML patients, and that the intracellular ABC Transporter A3 is strongly expressed in such leukemic SP cells. Subsequently we found ABCA3 expressed in all of 39 whole bone marrow and peripheral blood samples from 39 CML patients, with transcripts numbers substantially exceeding those of patients with AML or normal volunteers. Similarly, the CML cell lines K562, Lama-84 and BV17.3 showed high endogenous ABCA3 expression levels, allowing specific downregulation by siRNA. As measured in viability and colony forming assays, the functional knock down of ABCA3 induced a significantly increased susceptibility to imatinib. Furthermore, we applied subcellular fractionation techniques to investigate the subcelluar distribution of imatinib, and found the majority of [14C]-labelled imatinib to accumulate in the lyosomal fraction, in K562 and Lama84 leukemic cells as well as the HEK293 model system. In conclusion, the intracellular ABC transporter A3 is expressed consistently and at high levels expressed in CML cells, and may contribute to imatinib resistance via lysosomal sequestration.
PD-1 Signaling on Chronic Myeloid Leukemia-Specific T Cells Results in T Cell Exhaustion and Disease Progression.
Authors: Mumprecht Sabine , Schwaller Juerg , Solenthaler Max , Ochsenbein Adrian F. 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells with a characteristic chronic phase of several years before progression to acute myeloid leukemia. The immune system may contribute to disease control at this stage. Here we analyzed leukemia-specific immune responses in a murine retroviral bone marrow transduction and transplantation model using the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen. We found that CML-specific cytotoxic T cells (CTLs) became exhausted after initial activation and expansion. Only a small fraction of CML-specific CTLs persisted longterm. They maintained some limited cytotoxic activity but did not produce IFNγ or TNFα or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking PD-1 signaling in CML mice by transferring BCR/ABL-NUP98/HOXA9 transduced bone marrow cells to PD-1 deficient recipient mice resulted in improved CML disease control and prolonged survival. In addition, we extended our findings from the preclinical CML model to human patients. PD-1 was expressed at significantly higher levels on CD8+ T cells of CML patients when compared to healthy donors. These data identify PD-1 as a potential target to restore the function of exhausted CML-specific CTLs and therefore to treat CML.
Altered Expression of PP2A Regulatory Subunits in Chronic Myelogenous Leukemia: Identifying Targets for Improved Therapies.
Authors: Roberts Kathryn G. , Ashman Leonie K. , Sim Alistair T.R. , Verrills Nicole M. ,   (Intr. by Danilo Perrotti)
Published: October 2019

Abstract
Over 90% of chronic myelogenous leukemia (CML) cases are associated with constitutive activation of the BCR/ABL oncoprotein, which induces transformation of hematopoietic stem cells. Inhibition of BCR/ABL with imatinib has shown remarkable success in treating CML patients. However, the emergence of resistance is becoming a major clinical problem. To direct the development of improved therapies, signaling pathways activated by BCR/ABL must be identified. The aim of this project is to determine whether the activity and expression of the tumor suppressor protein phosphatase 2A (PP2A) is altered in the presence of BCR/ABL. PP2A is a serine/threonine phosphatase that is composed of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B), of which there are three different families. Each family of PP2A-B subunits has a number of isoforms that determine the substrate specificity, subcellular localization and catalytic activity of the enzyme. In this study we show that transfection of BCR/ABL into FDC-P1, a mouse factor-dependent myeloid cell line, increased tyrosine phosphorylation on the PP2A-C subunit, and this correlated with a reduction in enzymatic activity. Imatinib treatment rescued PP2A activity in wild type BCR/ABL+ cells, but not the imatinib-resistant Y253F BCR/ABL+ cells. Importantly, pharmacological enhancement of PP2A by forskolin and FTY720 resulted in reduced proliferation in both cell lines. Furthermore, we have demonstrated for the first time that BCR/ABL increases the expression of three specific PP2A-B subunit isoforms - B55α, B56α, and B56δ. It is predicted that enhanced levels of these subunits cause displacement of alternate PP2A-B isoforms to alter enzyme composition and hence the specificity of PP2A. To determine the functional role of each individual B subunit, shRNA studies are being performed. These findings confirm that functional inactivation of PP2A is an essential component of BCR/ABL-induced leukemogenesis in CML, and provide novel insight into the mechanisms by which this may occur. Elucidating the importance of each regulatory subunit may identify novel targets to develop improved therapies for CML patients.
Deletions Adjacent to BCR and ABL Breakpoints Occur in a Substantial Minority of Chronic Myeloid Leukaemia Patients with Masked Philadelphia Rearrangements.
Authors: De Melo Valeria A. , Kotzampaltiris Paul , Nacheva Elisabeth , Marin David , Apperley Jane F. , Reid Alistair G. 
Published: October 2019

Abstract
Chronic myeloid leukaemia (CML) results from the malignant transformation of haematopoietic stem cells by the BCR-ABL fusion gene, generated by a reciprocal translocation between chromosomes 9 and 22, the Philadelphia (Ph) translocation, or variations thereof. Common permutations of the Ph include translocations involving one or more additional chromosomes to 9 and 22 (“variant” Ph), and rearrangements resulting in cryptic insertion of chromosome 9 into chromosome 22, or vice versa, leaving apparently normal chromosomes 9 and 22 (“masked” Ph). Recently the development of new FISH techniques led to the identification of unexpected deletions at the Ph translocation breakpoints in approximately 15% of CML patients. The deletions, encompassing sequences 3′ of the BCR breakpoint and/or 5′ of the ABL breakpoint, were associated with a shorter duration of chronic phase and shorter survival in patients treated with interferon therapy, although their impact in patients treated with tyrosine kinase inhibitors is currently unclear. Interestingly, the incidence of deletions has been shown to vary for different cytogenetic subgroups of CML, with a significantly higher incidence of deletion in patients with a variant Ph translocation. The frequency of such events in patients with masked Ph rearrangements, however, has not yet been explored because of limitations inherent to the widely adopted dual-colour BCR-ABL FISH approach. We report the evaluation of 14 patients with masked Ph-positive CML for the presence of deletions extending 3′ from BCR and 5′ from ABL using two 3-colour BCR-ABL probes. Deletions were identified in 3 patients in total (21%), encompassing sequences 5′ to ABL in two of these and sequences 3′ to BCR in the remaining patient, thus demonstrating that the phenomenon is a significant feature of the masked Ph CML subgroup. Furthermore, our findings are consistent with the notion that loss of genomic material is a potential side effect of any DNA breakage event at the 9q34.1 and 22q11.2 chromosomal regions, regardless of the subsequent mechanism of chromosomal rearrangement.