bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Genomics in Zebrafish Permits Rapid Characterization of Novel Platelet Membrane Proteins
Authors: Salles Isabelle I , O’Connor Marie N , Watkins Nicholas A. , Walker Adam , Garner Stephen F , Jones Chris I , Macauley Iain , Steward Michael , Zwaginga Jaap Jan , Bray Sarah L , Cvejic Ana , Dudbridge Frank , de Bono Bernard , Deckmyn Hans , Stemple Derek L. , Ouwehand Willem 
Published: April 2020

Abstract
In this study we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell the megakaryocyte, together with nucleated blood cell elements, endothelial cells and erythroblasts, identified novel platelet membrane proteins with hitherto unknown role in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of five of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet Glycoprotein (GP) IIb and the coagulation protein Factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of four of the five novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.
Caspase Activated Rock-1 Allows Erythroblasts Enucleation Independently of Cytokine Signaling
Authors: Courtois Genevieve , Vandekerckhove Julie , Chang Yunhua , Ribeil jean-Antoine , Kersual Joelle , Debili Najet , Vainchenker William , Varet Bruno R. , Hermine Olivier 
Published: April 2020

Abstract
Stem Cell Factor (SCF) and Erythopoietin (Epo) are strictly required for preventing apoptosis and stimulating proliferation, allowing differentiation of erythroid precursors from CFU-E until the polychromatophilic stage. In contrast, terminal maturation generating erythrocytes occurs independently of cytokine signaling by a mechanism poorly understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size and nucleus with chromatin condensation and disappearance of organelles that require transient caspase activation. These events are followed by nuclei extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Two serine/threonine protein kinases, Rock-1 and Rock-2, the main Rho effectors, are key regulators of the actomyosin-based contractility. To determine the role of the Rho/Rock pathway in the terminal stage of erythropoiesis, we used an in vitro model of erythroid differentiation based on human hematopoietic cells derived from cord blood. We showed that in early step, SCF stimulates Rho/Rock pathway that is involved in erythroblasts proliferation and cytokinesis until the basophilic stage. Thereafter, Rock-I is activated independently of Rho signaling by caspase-3-mediated cleavage. To investigate the role of Rock during erythroid terminal differentiation, we used a Rock inhibitor (Y-27632). At day 8 of the CD36+ control culture, 55% of cells exhibited a mature phenotype (30% polychromatic, and 25% orthochromatic erythroblasts). By contrast, in the Y-27632 treated culture, there were no more than 23% mature cells (22% polychromatic and 1% orthochromatic erythroblasts) and 47% exhibited apoptotic features. Most of these apoptotic cells exhibited mature morphology suggesting that apoptosis occurred during the late step of maturation at the time of caspase activation. The percentage of enucleated cells was low in these culture conditions, thus, to determine whether or not Rock was also involved in the enucleation process, we used a coculture on MS5 stromal layer system (Giarratana, Nat Biotechnol 2005). At day 18, the rate of enucleated cells was 30% in the control culture whereas it was two-fold decreased in the Y-27632 treated culture and many of mature cells were instead dying by apoptosis. This finding suggested that Rock inhibition could be also involved in the enucleation process. By using shRNA technique, we demonstrated that Rock-1 is involved in terminal maturation and enucleation through phosphorylation of the light chain of Myosin II (MLC2). Therefore, in this differentiation system, caspase activation allows final maturation independently of cytokine signaling through kinase cleavage and activation.
The Unfolded Protein Response (UPR) Is Activated in Human Acute Myeloid Leukemia (AML) and Suppresses Translation of the CCAAT/Enhancer Binding Protein-Alpha (CEBPA) by Induction of the RNA-Binding Protein Calreticulin
Authors: Schardt Julian , Eyholzer Marianne , Mueller Beatrice U , Pabst Thomas 
Published: April 2020

Abstract
Deregulation of the myeloid key transcription factor CCAAT/enhancer binding protein alpha (CEBPA) is a common event in AML patients. We previously reported that the RNA-binding protein calreticulin efficiently blocks CEBPA translation and is specifically induced in core binding factor (CBF) leukemias. In addition, calreticulin is a crucial component of the unfolded protein response (UPR) which is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In vitro studies suggested that the UPR is activated in some solid cancers and thereby involved in tumor development. The role of the UPR during leukemogenesis has not been addressed so far. Here, we investigated the induction of the spliced variant of the X-box binding protein 1 (XBP1s) as a marker for activated ER stress and determined the expression of key mediators of the UPR such as calreticulin and the 78-kDa glucose-regulated protein (GRP78) in leukemic cells from 92 consecutive AML patients. Increased expression of the XBP1 spliced variant was detected in 16 of 92 AML patients. Consistently, this group also had increased mRNA and protein levels of calreticulin and GRP78. In patients expressing the XBP1 spliced variant, CEBPA protein was hardly detectable in contrast to AML patients not expressing the XBP1 spliced variant. Moreover, treatment of myeloid leukemic cells with compounds activating the UPR – such as thapsigargin - confirmed rapid induction of XBP1s, GRP78 and calreticulin in myeloid cells whereas CEBPA protein levels decreased. In addition, conditional expression of calreticulin in U937 cells suppressed CEBPA protein. At the molecular level, we identified two functional ER stress response elements (ERSE) in the calreticulin promoter, and both elements were found to be necessary for full induction of calreticulin following ER stress. The presence of the tripartite nuclear transcription factor Y (NFY) and activating transcription factor 6 (ATF6), as well as an intact binding site for the YY1 transcription factor (YY1) within these ERSE motifs appeared to be crucial for mediating sensitivity to ER stress. Finally, chromatin-immunoprecipitation assays indicated that binding of NFY and YY1 to the ERSE motifs is induced after induction of ER stress in vivo. Therefore, we conclude that the UPR is activated in a subgroup of AML patients. Activation of the UPR involves induction of calreticulin expression by the ATF6 pathway and ultimately leads to suppressed CEBPA translation, thus contributing to the block in myeloid differentiation in these leukemias.
MMP-14 Mediates Migration of Acute Myelogenous Leukemia Cells
Authors: Shirvaikar Neeta , Jalili Ali , Mirza Imran , Ilnitsky Sara , Korol Chris , Larratt Loree M. , Turner A. Robert , Janowska-Wieczorek Anna 
Published: April 2020

Abstract
Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multiple tumor cell types and is a major mediator of cell migration and invasion. MMP-14 possesses a trans-membrane domain that tethers the enzyme to the plasma membrane and not only activates proMMP-2 but also degrades extracellular matrix (ECM) by pericellular proteolysis and cleaves several non-ECM molecules, including adhesion molecules and chemokines. To assess the role of MMP-14 in leukemic dissemination we evaluated its expression in myeloid cell lines and primary acute myelogenous leukemic (AML) samples. Using RT-PCR, flow cytometry and Western blotting we found that MMP-14 is highly expressed in
leukemic myeloid cell lines THP-1, U937, HEL and K562; and primary samples from 37 out of 40 patients (pts) diagnosed with AML (WHO classification, AML with recurrent cytogenetic translocations: 9 pts; AML with multilineage dysplasia: 4 pts; AML not otherwise categorized: 27 pts).
Moreover, primary AML blasts secreted the 72 kDa proenzyme form of MMP-2 into media (zymography) which became activated after co-culture of AML blasts with bone marrow (BM) stromal cells. This activation was inhibited by the potent MMP-14 inhibitor epigallocatechin-3-gallate (EGCG). We also found that
migration of primary AML cells (trans-Matrigel invasion assay) was inhibited by EGCG; and silencing of MMP-14 by transfecting THP-1 cells and AML blasts with MMP-14 siRNA oligonucleotides resulted in reduced trans-Matrigel migration of these cells.
Given that AML blasts constitutively secrete TNF-α, we confirmed that
AML blasts express mRNA for TNF-α as well as its receptors TNFR1 and TNFR2; and TNF-α levels are elevated in the plasma of AML patients.
However, we demonstrated that recombinant human (rh) TNF-α further upregulated MMP-14 expression in AML blasts (RT-PCR, Western blot) and increased its incorporation into membrane lipid rafts (confocal microscopy). Moreover, rh TNF-α- stimulated AML blasts
were found to be more potent activators of pro-MMP-2 than unstimulated cells (zymography), indicating that activation may occur via upregulation of MMP-14; had significantly higher migration which was inhibited by EGCG and also by the TNF-α receptor inhibitor Enbrel; and rh TNF-α had no effect on the migration of MMP-14 siRNA-silenced AML cells.
Other factors tested, for example, SDF-1, IL-1 and TGF-β, had no effect on MMP-14 expression in AML cells (flow cytometry). In conclusion, we report here for the first time that MMP-14 is expressed in AML blasts and is modulated by TNF-α. We suggest that, by pericellular degradation of ECM and activation of latent MMP-2 secreted by AML blasts, MMP-14 may contribute to the highly proteolytic BM microenvironment in AML and the invasive phenotype of this malignancy.
Single Cell Molecular Genotyping Reveals Significant Molecular Heterogeneity at Diagnosis and Emergence of Clonal Dominance at Relapse
Authors: Zeng Rong , Meshinchi Soheil 
Published: April 2020

Abstract
Acute myeloid leukemia is a heterogeneous disease with significant variation in morphologic, cytogenetic, and molecular characteristics. We used single cell molecular genotyping to investigate the extent of molecular heterogeneity in individual patients with FLT3/ITD, WT1 and c-KIT mutations. Diagnostic leukemic samples from patients with FLT3/ITD (N=10) with varying FLT3/ITD allelic ratios (proportion of mutant to wild type product, ITD-AR) were flow sorted to immunophenotypic homogeneity (CD45dim/CD34+/CD33+) and individual leukemic blasts were isolated and directly genotyped. Single cell signal recovery as evident by the detection of FLT3 signal ranged from 39% to 88% (median 58%). Single cells from FLT3/ITD-positive samples demonstrated significant molecular heterogeneity with the presence of 3 distinct FLT3 genotypes: heterozygous FLT3/ITD, homozygous FLT3/ITD and wild type FLT3 (FLT3/WT). Proportion of cells with homozygous FLT3/ITD varied from 3% to 80% and directly correlated with the diagnostic ITD-AR, where those with high allelic ratios had a higher proportion of homozygous ITD cells and in those with the highest ITD-AR, with demonstrated 13q uniparental disomy (13q UPD), cells with homozygous ITD were the dominant clone. We further evaluated relapse specimens from 3 patients with FLT3/ITD, where cells with homozygous ITD was a minor clone at diagnosis (15%–30%). Single cell FLT3 genotyping of these specimens revealed significant expansion and clonal dominance of the homozygous ITD at relapse, where &gt;85% of leukemic cells harbored homozygous ITD (and presumed 13q UPD). We demonstrated similar molecular heterogeneity in patients with WT1 and c-KIT mutations, with detection of homozygous mutations in 20–60% of the cells tested. Single cell genotyoing demonstrates significant molecular heterogeneity in AML that may not be evident by conventional methods. This data provides direct evidence that cells with homozygous FLT3/ITD and associated 13q UPD may be present as a minor population at the time of diagnosis and emerge as the dominant clone at relapse. Such a molecular heterogeneity and emergence of clonal dominance by an individual clone may provide insight into mechanism of disease resistance. Extent of molecular heterogeneity may also be important in our interpretation of data obtained from biologic specimens.
Poly ADP Ribose Polymerase (PARP) Inhibitors Induce Apoptosis Alone or Synergistically with Histone Deacetylase Inhibitors in Primary Acute Myeloid Leukemic Patient Cells
Authors: Gaymes Terry J , Shall Sydney , Farzaneh Farzin , Mufti Ghulam J 
Published: April 2020

Abstract
It has been previously reported that cells defective for double strand break (dsb) DNA repair can be selectively targeted for apoptosis through abrogation of their PARP activity. It has also been reported that leukemic cells have inherent defects in dsb DNA repair. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a specific and less toxic form of therapy for a number of hematological malignancies. In order to test the efficacy of PI therapy we analysed primary cells from acute myeloid leukemia (AML) patients and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that from a panel of 12 AML patients, 2 AML patient cells demonstrated abnormal cell cycle profiles and apoptosis in response to the PI, KU-0058948 (1μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI. Clonogenic cytotoxicity assays also showed that these PI sensitive AML patient cells exhibited between 45–55% cell survival compared with 100% cell survival in control cells (p&lt;0.05) in response to PI. The homologous recombination (HR) DNA repair component, rad51 forms foci in response to DNA damage. In HR compromised cells, rad51 foci fail to form. In response to PI, immunofluorescent studies show that the 2 PI sensitive AML patients cells demonstrated severely reduced rad51 foci formation (&lt;1%) compared to PI insensitive and normal control cells (15%). This confirmed that PI targets the HR deficiencies in PI sensitive cells. Histone H2AX, phosphorylated in response to DSB had greatly increased foci formation in PI sensitive cells compared to PI insensitive control cells as a result of unrepaired DNA damage (32.3 vs 19.3%)(p&lt;0.05). We next explored the use of PI in combination with DNMTi or HDACi. Whilst KU-0058948 offered only additive effects on DNMTi cytotoxicity, a non-cytotoxic concentration of KU-0058948 (10nM) behaved synergistically with HDACi potentiating the cytotoxic effect of MS275 by 45% compared to MS275 alone (p&lt;0.05). Furthermore, non-cytotoxic doses of MS275 (50nM) potentiated the cytotoxic effects of KU-0058948 in PI sensitive cells (25–30%) compared to KU-0058948 alone (P&lt;0.05). In conclusion, we have shown that primary AML cells are sensitive to the cytotoxic actions of PI. We have also showed that PI acts synergistically in combination with HDACi. PARP inhibitors can potentially exploit dsb repair defects in leukemic cells paving the way for a targeted therapy for leukemia.
Phase I/II Study of Tipifarnib and Bortezomib in the Treatment of Poor Risk Adult Acute Myeloid Leukemia
Authors: Paolini Stefania , Ottaviani Emanuela , Lama Barbara , Salmi Federica , Clissa Cristina , Curti Antonio , Finelli Carlo , Giannoulia Panagiota , Iacobucci Ilaria , Laterza Claudio , Papayannidis Cristina , Parisi Sarah , Piccaluga Pier Paolo , Vianelli Nicola , Saglio Giuseppe , Fanin Renato , Morabito Fortunato , Zaccaria Alfonso , Lauria Francesco , Amadori Dino , Baccarani Michele , Martinelli Giovanni 
Published: April 2020

Abstract
Background. Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Tipifarnib (Zarnestra, Z) and Bortezomib (Velcade, V) are new, targeted-treatments that might improve current results. In particular, Z is a farnesyltransferase inhibitor effective in AML, allowing complete remission (CR) rates ranging from 8 to 22%. Notably, the response rate appeared superior among patients with higher RASGRP1/APTX gene expression ratio. V is a proteasome inhibitor, possibly effective in AML as for phase I studies. Interestingly, Z and V appeared synergistic in AML cell lines.
Aim. We designed a phase I/II study aiming to assess the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of V in association with standard doses of Z and the efficacy and toxicity of the combination, in AML patients &gt;18 years, unfit for conventional therapy, or &gt;60 years, in relapse. Furthermore, we aimed to assess the correlation between RASGRP1/APTX ratio and treatment response.
Methods. V was administered as weekly infusion for three consecutive weeks (days 1, 8, 15), starting from 0.7 mg/m2 and increasing by 0.3 mg/m2 until the DLT was reached. Z was administered at the daily dose of 600 mg BID for 21 consecutive days. Response was assessed at the end of each cycle (28 days). Patients’ withdrawn was planned in case of progression or stable disease after six cycles. Real-time quantitative PCR (q-PCR) was used for RASGRP1/APTX genes evaluation.
Results. 45 patients were enrolled: 12 in the phase I and 33 in the phase II. 11/12 patients were evaluable in the phase I. Five patients received V at the dose of 0.7 mg/m2 without reporting DLT. Conversely, DLT was reached at the dose of 1.0 mg/m2 due to grade III SNC toxicity, recorded in 1/6 patients.
22/45 patients were evaluable for treatment response; transient reduction of peripheral blasts (&gt; 50% to baseline) was observed in 20/22 cases. 2 patients achieved CR and 1 obtained a partial response (PR). 1 patient had a haematological improvement (HI), 12/22 had a stable disease (SD) and 5/22 showed progressive disease (PD). Interestingly, the 4 responders (CR+PR+HI) had a significant higher RASGRP1/APTX ratio respect to non responder (p=0.017).
Conclusion. We conclude that the MTD of V in association with Z is 1.0 mg/m2. The association seemed to be safe, with response rate (18%) similar to what reported for Z alone. Finally, though in few cases, RASGPR1/APTX ratio was confirmed to be associated to treatment response.
Effects of Recipients’ Body Weight and Total Nucleated Cell Dose on Outcomes of Unrelated Bone Marrow Transplantation: Analysis of Clinical Data of 3,114 Leukemia Patients from the Database of the Japan Marrow Donor Program
Authors: Yamashita Takuya , Akiyama Hideki , Ohashi Kazuteru , Onizuka Makoto , Mori Shin-ichiro , Sao Hiroshi , Miyamura Koichi , Sakamaki Hisashi 
Published: April 2020

Abstract
In allogeneic bone marrow transplantation (BMT) from unrelated donor of Japan Marrow Donor Program (JMDP), the standard bone marrow nucleated cell dose for transplantation is regulated as 3.0×10E8 total nucleated cells (TNC)/recipient’s body weight (RBW) (kg). RBW are also used to estimate the target harvest volume of bone marrow from the donor. So RBW determine the dose of harvested TNC, and these factors will influence the outcomes of BMT. We retrospectively analyzed the clinical data of 3,114 adult transplant patients (median age 33, range 16–70) from extracted the database of JMDP. The diagnosis of these patients were AML (n=1257), ALL (n=927) and CML (n=930). Five years overall survival (OAS) of all cases was estimated as 47.6%. Possible factors that could influence on survival and hematological recovery were analyzed by using proportional hazards analysis. In univariate analysis, TNC&gt;=3.0×10E8/RBW was one of the significant factors to improve OAS (p=0.022). TNC/recipients’ ideal body weight (IBW) whose body mass index (BMI) was over 22 could be also a significant favorable factor for OAS (p=0.014). In multivariate analysis, variables correlated with improving OAS were TNC&gt;=2.0×10E8/IBW (p=0.011), using tacrolimus for GVHD prophylaxis (p=0.011), younger recipient’s age (p=0.013). TNC&gt;=2.0×10E8/IBW were also powerful variables correlated with the recovery of neutrophils, reticulocytes and platelets. These findings suggest that TNC/IBW can substitute for TNC/RBW to evaluate infused cell dose. And in our data set, use of IBW to estimate the harvest volume from the donor brought 10.7% reduction of target bone marrow volume compared with use of RBW. JMDP sets up the upper limit of harvested bone marrow volume based on the donor’s BW, but heavyweight recipients often require larger volume of bone marrow from the donors. Appropriate evaluation of TNC for transplantation and recipients’ BW by using BMI may be one of the useful methods to improve outcomes of unrelated BMT and avoid excess risk and stress of unrelated donors.