bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells
Authors: Jäger Roland , Olcaydu Damla , Berg Tiina , Gisslinger Bettina , Gisslinger Heinz , Kralovics Robert 
Published: April 2020

Abstract
A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.
Specific Inhibition of Syk Is Sufficient to Disrupt Proliferation and Survival of Non-Hodgkin’s Lymphoma Cell Lines without Concomitant Inhibition of JAK.
Authors: Coffey Greg , Luan Peng , Delaney Suzanne , Pandey Anjali , Jia Zhaozhong , Xu Qing , Bauer Shawn , Song Yonghong , Conley Pamela B. , Phillips David , Sinha Uma 
Published: April 2020

Abstract
Recent studies indicate that dual inhibition of the spleen tyrosine kinase (Syk) and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) are clinically efficacious in patients with non-Hodgkin’s lymphoma. Since Syk has been implicated in B cell activation, proliferation, and survival, the present study was designed to determine whether the specific inhibition of Syk alone would suffice to disrupt proliferation and survival of lymphoma cells. To test this, a series of compounds were synthesized and screened to identify those that specifically inhibited Syk in a panel of in vitro multi-kinase assays at 300nM. Of these, three were selected for this study; compounds P459-72, P505-15, and P420-89. All three compounds inhibited purified Syk with IC50’s in the 6-43nM range. P459-72 was highly Syk specific. P505-15 also inhibited purified Lyn with an IC50 of 199nM. P420-89 inhibited multiple kinases in the BCR signaling pathway, in addition to JAK1, 2, and 3; IC50’s of 0.63-6.2nM. Assays using the non-Hodgkin’s lymphoma B cell lines Ramos, SUDHL-4 and -6 showed that each compound also inhibited BCR-induced Syk auto-phosphorylation and BLNK phosphorylation (IC50’s in the 100–400nM range) as well as subsequent Ca2+ flux and ERK phosphorylation (IC50’s in the 50–156nM range). In contrast, the activity of the Src family member Lyn, upstream of Syk in the BCR signaling pathway and responsible for Syk tyrosine phosphorylation at amino acid position 352, was not affected. Cellular proliferation was also attenuated in these lymphomas (IC50’s in the 1–5μM range), and all three compounds induced apoptosis to various extents between 1 and 3μM. Interestingly, while P505-15 did inhibit purified Lyn with an IC50 of 199nM, this did not translate into inhibition of Lyn activity upon BCR cross-linking in cells at concentrations where Syk activity was inhibited. A structurally similar compound with a greater than one hundred fold lower Syk inhibitory activity (P528-85) had no effect on the proliferation and survival of these B cell lines. Finally, the cellular effects of the Syk inhibitors required an active BCR signaling pathway, as the B cell line Toledo which lacks BCR expression was insensitive to these compounds; proliferation was inhibited with IC50’s in the 9-38μM range with no induction of apoptosis below 10μM, the highest concentration tested. Taken together, these data suggest that the specific inhibition of Syk, without concomitant inhibition of JAK, may be sufficient for the treatment of non-Hodgkin’s lymphoma.
A Molecular Network Comprising MicroRNA-223, E2F1 and C/Ebpα in Granulopoiesis and in Acute Myeloid Leukemia.
Authors: Pulikkan John Anto , Dengler Viola , Peerzada Abdul , Bohlander Stefan , Tenen Daniel G. , Behre Gerhard 
Published: April 2020

Abstract
MicroRNAs play crucial roles in gene expression programmes and have been demonstrated to have major influence in various biological processes. Recent findings suggest aberrant regulation of microRNAs is a hall mark of many cancers including leukemia. MicroRNA-223 (miR-223) is regulated by the transcription factor CCAAT enhancer binding protein α (C/EBPα) and is upregulated during granulopoiesis. miR-223 mutant mice display defects in granulopoiesis pointing out the importance of miR-223 during granulopoiesis. Recent studies suggest that loss of function or expression of C/ EBPα is a major step in the development of acute myeloid leukemia (AML). Using an inducible cell line model, we show that C/EBPα upregulates microRNA-223 expression during granulopoiesis. Based on these findings, we hypothesized that miR-223 could be downregulated in human AML. Here we report that miR-223 is downregulated in different subtypes of AML as analysed by quantitative Real-Time RT-PCR. We investigated what are the critical targets of miR-223 during granulopoiesis. Computational analysis suggests that E2F1, the transcription factor that promotes cell cycle progression which is inhibited by C/EBPα during granulopoiesis, could be a putative target of miR-223. By luciferase assay using 3’UTR of E2F1, we show that E2F1 is a potential target of miR-223. miR-223 downregulates E2F1 by translational repression as revealed by reduction in E2F1 protein level. Silencing of miR-223 leads to upregulation of E2F1 protein level as analyzed by Western blot analysis. Proliferation assays as well as cell cycle analysis demonstrate that miR-223 blocks cell cycle progression in myeloid cells. Interestingly, sequence analysis of miR-223 promoter revealed putative E2F1 binding sites. We demonstrate that E2F1 inhibits the microRNA-223 promoter activity through its transactivation domain as shown by promoter assay. Furthermore, overexpression of E2F1 down regulates the expression of miR-223, suggesting E2F1 acting as a transcriptional repressor of the miR-223 gene. Meanwhile, C/EBPα transactivates miR-223 promoter activity. We also report that E2F1 is able to block granulocytic differentiation. Recent studies demonstrate that disruption of E2F1 inhibition by C/EBPα leads to leukemia, pointing out the significance of E2F1 inhibition in the development of AML. Our data support a circuitry comprising miR-223, C/EBPα and E2F1 as major components of the granulocyte differentiation programme, which is deregulated in AML. Manipulation of miR-223 could be therapeutically relevant in AML subtypes in which E2F1 inhibition is deregulated.
Cytoskeletons of X-Linked GATA-1, G208S Macrothrombocytes Are Deficient in Talin.
Authors: White James G. , Burris Steven M. , Thomas Angela 
Published: April 2020

Abstract
Germline mutations in the X-linked transcription factor, GATA-1, cause several hematopoietic disorders resulting in anemia and/or macrothrombocytopenia. Features of the platelet ultrastructural pathology, including hypo- and agranular megathrombocytes (Mtc) in all mutations, tubular membrane sheets often in parallel association in megakaryocytes and Mtc of patients with the V205M and G208S variations, platelets sequestered in Mtc and platelets in platelets in Mtc (as many as five in one Mtc) in the V205M, G208S and R216Q disorders, together with platelets attached to platelets attached to platelets forming large Mtc in the V205M and G208S mutations have helped to explain how Mtc are formed and why they fail to separate from proplatelets of megakaryocytes and enter the circulation as single cells. However, the findings do not explain why patients with GATA-1 mutations often have serious bleeding problems. Major receptors for hemostasis and thrombosis are present on GATA-1 Mtc, although, in some cases, slightly reduced in frequency. Yet, while the cells bind to and spread fully on glass and plastic, they adhere poorly to vWF or collagen coated glass slides under flow conditions, aggregate less well than normal platelets when stirred with collagen or ristocetin in an aggregometer and fail to retract clots. The findings suggest that internal activation mechanisms for the surface receptors are defective. The present study has solubilized GATA-1 Mtc from two males with the G208S mutation and normal platelets in sodium dodecyl sulfate/ EDTA in the presence of protease inhibitors and analyzed their cytoskeletal proteins on reduced, one dimensional polyacrylamide gels using a Mini-Slab Gel Apparatus. Gels were electrophoresed by the method of Laemmli. Proteins were stained with colloidal Coomassie G250 blue. GATA-1 Mtc cytoskeletal proteins were the same as those from the normal subjects, except for the absence of Talin. Western blot analysis to prove the missing protein is Talin was carried out. Normal platelet and GATA-1 Mtc cytoskeletal proteins were transferred to nitrocellulose sheets. Identification and localization of the missing protein was accomplished using a mouse monoclonal antibody against human Talin and a secondary anti-mouse IgG peroxidase conjugate. The Western blot analysis confirmed that the missing Mtc protein is Talin. Recent studies have shown that binding of the 235–245 kD actin-binding protein Talin to the β subunit cytoplasmic tails of integrins is the final step of integrin activation. The absence of Talin in GATA-1 Mtc may result in failure of full integrin activation accounting for functional failure in hemostasis.
A Novel Histidine Tyrosine Phosphatase, TULA-2, Associates with Syk and Negatively Regulates GPVI Signaling in Platelets.
Authors: Thomas Dafydd H , Dangelmaier Carol , Jin Jianguo , Tsygankov Alexander , Kunapuli Satya P. , Daniel James 
Published: April 2020

Abstract
Glycoprotein VI (GPVI) is the primary platelet receptor for collagen signaling. Following damage to the vascular endothelium, the GPVI receptor interacts with the exposed sub-endothelial collagen. This interaction initiates a signaling cascade involving phosphorylation of the dual ITAM motif of the FcRγ chain by Fyn and Lyn, followed by the recruitment, phosphorylation and activation of Syk. This leads to the eventual activation of PLCγ2 and the release of calcium from intracellular stores to cause platelet activation. While a lot is known about the activation processes involved in GPVI signaling less is known about its negative regulation. The T-cell ubiquitin ligand (TULA) family of proteins has been implicated in the negative regulation protein tyrosine kinase (PTK)-dependent signaling pathways. More recently, it has been shown the TULA family member, TULA-2, exhibits phosphatase activity towards PTKs, including Syk, and this activity is responsible for the negative regulation of T-cell receptor signaling (Mikhailik et. al. 2007, Agrawal et. al. 2008). Thus, we investigated the role of TULA-2 in the negative regulation of the GPVI signaling cascade. We show that TULA-2 is expressed in both human and murine platelets. Deletion of TULA-2 in murine platelets manifests itself functionally as enhanced aggregation in response to the GPVI agonist convulxin as well as enhanced dense granule secretion when compared to wild type platelets. No difference was witnessed in response to the PAR4 agonist AYPGKF. TULA-2-deficient platelets also exhibit sustained hyperphosphorylation of Syk at tyrosines 525 and 526 as well as hyperphosphorylation of PLCγ2 at tyrosines 753 and 759, indicative of enhanced kinase and phospholipase activity respectively. GST-pulldown experiments suggest that Syk and TULA- 2 are able to associate in resting and convulxin stimulated platelets and in-vitro phosphatase assays demonstrate that TULA-2 can dephosphorylate Syk at tyrosines 525 and 526. Taken together, these data suggest that TULA-2 is a negative regulator of GPVI signaling and this regulation is mediated by an association of TULA-2 with Syk, allowing the dephosphorylation of Syk at catalytically important tyrosine residues.
Functional Genomics of the Platelet ADP and Collagen Signalling Pathways.
Authors: Garner Stephen F , Jones Chris I , Stephens Jonathan , Bray Sarah L , Angenent Will , Burns Philippa , Farndale Richard W , Rice Catherine , Dudbridge Frank , Watkins Nick , de Bono Bernard , Deloukas Panos , Goodall Alison H , Ouwehand Willem 
Published: April 2020

Abstract
The extent of platelet response to agonists varies between individuals and is to a large extent genetically controlled. Here we present the results of a systems biology study designed to define the association between sequence variation and platelet functional phenotype. The exons and flanking intronic regions of 98 genes, with known function in platelets, were sequenced in 48 Caucasoid CEPH DNA reference samples, identifying 1949 SNPs, approximately half of which were novel. From this enriched data set 1340 SNPs, comprising tagging and non-synonymous validated SNPs, were mounted on an Illumina Golden Gate array and typed in 500 DNA samples from a cohort of healthy subjects whose platelet functional phenotype was measured by flow cytometry, using fibrinogen binding and P-selectin expression in response to pathway-specific ADP (10−7M), or CRP-XL (0.1μg/ mL) stimulation. Thirty-six SNPs in 16 genes showed an association (p&lt;0.005) with one or more platelet functional trait. To explore the mechanism by which sequence variation alters the platelet response, further tests were performed on platelets from individuals recalled on the basis of genotype for the SNPs with the lowest p-value for association in two of the genes, ITPR1 and VAV3. A SNP within the 3’UTR located in a putative transcription factor binding site of the VAV3 gene, for which the minor allele was associated with increased P-selectin expression following ADP stimulation (p=0.004), was associated with a higher VAV3 protein expression in subjects with the minor allele (p=0.03). Conversely, the minor allele of the lead SNP in ITPR1, associated with increased fibrinogen binding and P-selectin expression in response to ADP but not CRP-XL (p=0.003, &gt;0.2, respectively), did not show significant effect on the level of ITPR1 protein, but there was increased mobilisation of [Ca2+]i in response to ADP (but not CRP-XL) with the minor allele (p=0.004). By applying systems biology to platelets we have identified SNPs in 16 genes which together account for up to 60% of the genetic variation underlying platelet response to ADP and CRP-XL. These data provide novel insights into key “hubs” in well-studied platelet signalling networks where sequence variation modifies the response phenotype.
Increased Nucleosomal DNA Fragmentation in Leukocytes of Thalassemia Patients.
Authors: Walter Patrick B , Porter John B , Evans Patricia , Kwiatkowski Janet , Neufeld Ellis J. , Coates Thomas D. , Giardina Patricia , Grady Robert jW , Vichinsky Elliott , Olivieri Nancy F. , Trachtenberg Felicia , Alberti Daniele , Fung Ellen , Ames Bruce , Lui Annie , Harmatz Paul 
Published: April 2020

Abstract
Iron overload has been shown to induce DNA damage and may be implicated in leukocyte apoptosis. Previously we have shown that indirect markers of apoptosis such as caspase activity and Bax levels were higher in leukocytes of thalassemia patients. We have now assessed whether nucleosomal DNA fragmentation, a hallmark of apoptosis, is higher in leukocytes of iron-overloaded thalassemia patients compared with control subjects.
Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, mean age: 21.8 ± 11.1 yrs) were enrolled in the study. Fasting blood samples were obtained
after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator at 1, 6, and 12 months on study.
Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, mean age 24.5 ± 9 yrs) also supplied a blood sample. Plasma, serum and cells were separated by centrifugation. The classic marker for apoptosis, nucleosomal DNA fragmentation was determined by a high throughput ELISA assay that used monoclonal antibodies directed against single- and double-stranded DNA bound to histones (H1, H2A, H2B, H3, and H4) and thus specifically detected mono- and oligonucleosomes (measured in ng/μg leukocyte protein). Detection of free mono- or oligonucleosomes is a direct measurement of apoptotic DNA fragmentation. DNA fragmentation results were log-transformed prior to analysis and means are reported.
Results: At baseline, thalassemia patients had elevated nucelosomes compared to the controls (351.5 vs. 74.9 ng/μg leukocyte protein, p = 0.001). This high level of leukocyte DNA fragmentation was unchanged throughout the study. Furthermore, nucleosomal DNA fragmentation was positively correlated with CRP (r = 0.3, p = 0.01), WBC (r = 0.2, p = 0.05) and ANC (r = 0.2, p = 0.04), suggesting apoptosis is present in increased inflammatory states.
Conclusions: This finding demonstrates that nucleosomal DNA fragmentation is higher in thalassemia compared to control patients and thus provides further evidence that there is increased apoptosis in circulating leukocytes of thalassemia patients.
Is It Possible to Stop Imatinib in Patients with Chronic Myeloid Leukemia? An Update from a French Pilot Study and First Results from the Multicentre « Stop Imatinib » (STIM) Study
Authors: Mahon Francois-Xavier , Huguet Francoise , Guilhot Francois , Legros Laurence , Nicolini Franck E , Charbonnier Aude , Guerci Agnes , Rea Delphine , Varet Bruno R. , Gardembas Martine , Guilhot Joelle , Etienne Gabriel , Milpied Noel-Jean , Aton Emilie , Reiffers Josy , Rousselot Philippe 
Published: April 2020

Abstract
Imatinib (IM) has greatly improved survival rates in chronic myeloid leukemia (CML). However, all patients (pts) must continue treatment for an unknown period of time. A pilot study of the first pts who discontinued IM therapy was previously reported (Rousselot et al. Blood. Blood2007;109:58–60). The new, multicentre « Stop Imatinib » (STIM) study was started in July 2007. The aim of this study is to evaluate in a larger cohort the persistence of complete molecular remission (CMR) after stopping IM, and to determine the factors that could influence the persistence of CMR. The criteria for inclusion were IM treatment for at least 3 years and sustained CMR. Sustained CMR was defined as BCR-ABL/ABL levels below a detection threshold corresponding to a 5-log reduction (undetectable signal using RQ-PCR) for at least 2 years. Molecular relapse, defined as RQ-PCR positivity, was taken into account if confirmed in two successive assessments. In cases of molecular relapse, pts were re-treated with IM at 400 mg dailyIn the pilot study, 7 out of 15 pts relapsed within 6 months, but CMR was re-attained in all cases after IM was re-started. The other 8 pts (4 male, 4 female) are still in CMR, with a median follow up of 37 months (range 26–49 months) after IM discontinuation. All pts were pre-treated with interferon-alpha (IFN) and most responded to IFN before IM treatment. The STIM study included 50 pts from 18 centres (20 male, 30 female), with a median age of 62 years (range 32–81 years). Of these, 25 pts had received no pre-treatment with IFN. By July 2008, 34 pts had a follow up ≥ 6 months. Eighteen pts relapsed within the first 6 months: 3 pts in month 2 (M2), 8 pts in M3, 4 pts in M4, and 3 pts in M5. One patient relapsed after more than 6 months (M8). Among the 19 pts who relapsed, 11 were not IFN pre-treated and 8 were IFN pre-treated (relapse rate 44% vs 32%). Ten IFN pre-treated pts with follow up ≥ 6 months have not relapsed (M12 in 2 pts, M10 in 5 pts, M8 in 1 pt, M7 in 2 pts), and 5 pts with follow up ≥ 6 months who were not IFN pre-treated have not relapsed (M12 in 1 pt, M10 in 1 pt), M8 in 1 pt, M6 in 2 pts).
These studies confirm that CMR can be sustained after discontinuation of IM, particularly in pts pre-treated with IFN with a long follow-up (pilot study). Among pts in the STIM study who were not pre-treated with IFN, more than half have not relapsed, and 20% have reached a follow-up ≥ 6 months and not relapsed. Updated data will be presented but we conclude that it is possible to stop treatment in pts with sustained CMR, even in those treated with IM as a single agent.