bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mef2C Is a Lineage-Restricted Target Gene of Scl/Tal1 and Regulates Megakaryopoiesis and B-Cell Homeostasis
Authors: Rhodes Katrin E , Gekas Christos , Gereige Laurraine , Helgadottir Hildur , Ferrari Roberto , Kurdistani Siavash , Montecino-Rodriguez Encarnacion , Basel-Duby Rhonda , Olson Eric , Orkin Stuart H. , Pellegrini Matteo , Mikkola Hanna 
Published: April 2020

Abstract
The bHLH transcription factor stem cell leukemia/T-cell acute leukemia gene (Scl/Tal1) is a master regulator for hematopoiesis, essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. To identify Scl target genes in hematopoietic cells, we performed gene expression analysis on HOX11-immortalized Sclfl/fl fetal liver cell lines. Analysis of the top 50 downregulated genes revealed several genes related to hematopoiesis including erythroid and megakaryocyte development, vasculogenesis, as well as genes/unknown ESTs that have not been previously linked to blood development. One of the top downregulated genes was transcription factor myocyte enhancer factor 2C (Mef2C). Mef2C−/− embryos die at E9.5, the same time as Scl−/− embryos, and exhibit severe defects in cardiac and muscle development. Analysis of Mef2C−/− embryos showed that, Mef2C, in contrast to Scl, is not required for specification into primitive or definitive hematopoietic lineages. To bypass the embryonic lethality, we utilized a conditionally targeted Mef2Cfl/fl strain and crossed it with a hematopoietic cell-specific VavCre strain that deactivates Mef2C shortly after the emergence of HSCs. Interestingly, adult VavCre+Mef2Cfl/fl mice exhibited severe platelet defects highly reminiscent to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reductions of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
Differentiation-Dependent Interactions Between Runx-1 and Fli-1 during Megakaryocyte Development
Authors: Huang Hui , Yu Ming , Moran Tyler B , Tu Nathan , Akie Thomas E , Cantor Alan B 
Published: April 2020

Abstract
The transcription factor Runx-1 is required for the ontogeny of all definitive hematopoiesis, and plays a specific role in megakaryopoiesis during later stages of development. Germline mutations in Runx-1 cause Familial Platelet Disorder with Propensity to Develop AML (FPD/AML), and acquired mutations occur in a subset of patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia. Although many of the reported Runx-1 mutations affect DNA binding and/or its interaction with the cofactor CBF-beta, other mutations occur outside of these binding regions and have unknown mechanistic effects. In this study, we purified Runx-1 containing multiprotein complexes from murine megakaryocytic cells in order to identify potential novel Runx-1 associated factors whose interaction may be altered by Runx-1 mutations. Here we report the identification of the key megakaryocyte ets transcription factor Fli- 1 as a direct Runx-1 binding partner. This interaction involves the negative regulatory DNA binding and activation domains of Runx-1 (amino acids 179–370), and a region around the Ets DNA binding region of Fli-1 (amino acids 281–361). The interaction is lost in the MDS- associated Y254X Runx-1 mutation. We also show that Runx-1 and Fli-1 co-occupy the c-mpl promoter in primary megakaryocytes and act synergistically in transcriptional reporter assays. Interestingly, the interaction between Runx-1 and Fli- 1 occurs in murine L8057 megakaryoblastic cells only after they have been induced to differentiate, even though both proteins are expressed abundantly in uninduced cells. The interaction correlates with assembly of a large multiprotein complex that also includes the key megakaryocyte transcription factor GATA-1 and its cofactor Friend of GATA-1 (FOG- 1) based on gel filtration chromatography experiments. Furthermore, we show that Fli-1 from this large complex lacks phosphorylation of a specific residue that is phosphorylated on non-complexed Fli-1. Mutation of this site to aspartic acid, which mimics constitutive phosphorylation, disrupts the interaction between Fli-1 and Runx-1 and abrogates their transcriptional synergy. We propose that dephosphorylation of Fli-1 is a key event in the transcriptional activation of megakaryocyte terminal maturation by facilitating the assembly of a RUNX-1/FLI-1/GATA-1/FOG-1 enhancesome complex. These findings have implications for the differentiation of other cell types where interactions between Runx and ets family proteins occur.
Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia
Authors: Trappenburg Marijke , van Schilfgaarde Muriel , Marchetti Marina , Spronk Henri , ten Cate Hugo , Leyte Anja , Terpstra Wim , Falanga Anna 
Published: April 2020

Abstract
Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET).
Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay.
Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p&lt;0.001).
Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.
Allogeneic Hematopoietic Cell Transplants in Patients with Myelofibrosis Age 60 and Older
Authors: Samuelson Scott James , Sandmaier Brenda M. , Heslop Helen E. , Popat U. , Boyer Michael , Hickman Kimberly , Prchal Josef T. , Deeg H. Joachim 
Published: April 2020

Abstract
Allogeneic Hematopoietic Cell Transplants in Patients with Myelofibrosis ≥ 60 years of age. Primary myelofibrosis (PMF) and myelofibrosis arising from polycythemia vera (PV) and essential thrombocythemia (ET) are challenging clinical entities and many patients have a very poor prognosis. For the most part, treatment is palliative and often of limited efficacy. Allogeneic hematopoietic cell transplants (HCT) can effectively control the disease, and reduced intensity conditioning regimens have reduced regimen related complications. However, many studies have excluded patients older than age 65 (or even 60) years. This is problematic as the median age at diagnosis is approximately 67. We retrospectively evaluated a cohort of 26 patients age ≥ 60 years with PMF or post PV or ET myelofibrosis who underwent allogeneic HCT between 1999 and 2007. The median age was 63 (range 60–75) years; 9 of these patients were older than 65 (range 66–75) years. Donors were HLA-matched siblings (n=14) or unrelated (n=12). Conditioning regimens included Fludarabine (Flu)/2 Gy TBI (n=16), Flu/Campath(Cam)/TBI (n=2), Flu/Melphalan/ATG (n=2), Cam/Flu/CD45Mab/4.5 Gy TBI (n=2), Flu/Busulfan (Bu) (n=2), Flu/Bu/ATG (n=1), Flu/Mel/Cam (n=1). The median follow up for the cohort was 200 (range 17 – 1322) days. Eleven of 26 patients were alive at the date of last followup, and 9 were relapse free. The median follow up for living patients was 251 (range 100 – 1322) days. Of the 15 patients who had died, 5 died of disease progression and the remaining 10 of non-relapse causes: infection (n=4), GVHD (n=2), respiratory failure (n=2), multi-organ failure (n=1), dissecting aneurysm (n=1). At last follow up, 4 of 9 patients age &gt;65 were alive a median of 374 (range 132 – 896) days without disease relapse. Five of the nine patients died, 4 from non-relapse causes and one with disease progression. In summary, reduced intensity conditioning and allogeneic transplantation in patients with myelofibrosis more than 60 years of age resulted in relapse-free survival of 40–45%. While the mortality rate was high, the success rate was remarkable in view of the patient age and the frequent presence of co-morbid conditions. We suggest, therefore, that ongoing and future clinical trials of allogeneic HCT for myelofibrosis should allow enrollment of older patients.
Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines
Authors: Bonzheim Irina , Irmler Martin , Anastasov Natasa , Klier Margit , Beckers Johannes , Pittaluga Stefania , Fend Falko , Raffeld Mark , Quintanilla-Martinez Leticia 
Published: April 2020

Abstract
Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling.
Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.
Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a &gt;1,1 and &lt;0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites.
Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.
Prognostic Impact of Microvessel Density in Follicular Lymphoma Patients Treated with Immunochemotherapy
Authors: Taskinen Minna , Bono Petri , Leppä Sirpa 
Published: April 2020

Abstract
Tumor infiltrating mast cells have at least two potential roles in remodeling tumor microenvironment and tumor growth: suppression of immune responses and potentiation of angiogenesis. We have recently shown that accumulation of mast cells in follicular lymphoma (FL) contributes to unfavorable survival after immunochemotherapy. Interestingly, high mast cell content eliminates the positive prognostic value of tumor-associated macrophages (TAMs). Here we investigated whether tumor vascularity, as determined by CD31 immunostaining, is associated with mast cell content and outcome of primary FL patients treated with combination of rituximab (R) and CHOP chemotherapy. We found that increased microvessel density (MVD) correlates positively with the number of tumor infiltrating mast cells (r=0.266, p=0.011). In a cohort of 95 patients, the patients with high MVD (&gt;the lowest tertile) had a worse 4-year progression free survival (PFS) (44% vs 82%, p=0.001). In multivariate analysis with FLIPI, MVD retained its negative predictive value. Additional prognostic impact was especially provided in patients with low FLIPI scores. When MVD related outcome was estimated separately for patients with high and low mast cell contents, the adverse effect of high MVD on the PFS was seen in both subgroups. Taken together, the data demonstrate that MVD is associated with mast cell content and adverse outcome in R-CHOP treated FL patients. In contrast to TAMs, the predictive value of MVD is independent of tumor infiltrating mast cells.
NPM-ALK Modulates the p53 Tumour Suppressor Pathway in a JNK and PI 3-Kinase Dependent Manner: MDM-2 Is a Potential Therapeutic Target for the Treatment of ALK-Expressing Malignancies
Authors: Cui Yuxin , McDuff Fiona , Kerby Alan , Ye Hongtau , Turner Suzanne Dawn 
Published: April 2020

Abstract
Anaplastic large cell lymphoma (ALCL) is in the majority of cases a paediatric disease of a T- or null-cell phenotype and is characterised by the presence of the t(2;5)(p23;q35) or variant translocations involving the ALK gene on chromosome 2. This chromosomal translocation generates the Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) fusion protein, a hyperactive kinase with transforming properties. The p53 tumour suppressor gene is rarely mutated in ALK-expressing ALCL, perhaps one reason why this disease has a good prognosis. However, the mechanism controlling p53 activity in ALCL has not been fully elucidated. We show in patient-derived ALCL cell lines and NPM-ALK transformed BaF3 cells that NPM-ALK induces post-translational modification of the p53 antagonist MDM2, leading to inactivation of the p53 tumour suppressor pathway. Furthermore, we demonstrate that the PI 3-Kinase-Akt pathway downstream of NPM-ALK is responsible for this activity. It therefore follows that inactivation of MDM2 with the specific inhibitor nutlin-3 results in a decrease in proliferation and subsequently apoptosis of NPM-ALK-expressing ALCL cells, a response that is enhanced when cells are exposed to nutlin-3 in conjunction with the PI 3-Kinase inhibitor LY294003. We also demonstrate that NPM-ALK activates JNK by phosphorylation in turn leading to JNK-mediated sequestration and degradation of p53. This activity can be attenuated following administration of a specific JNK inhibitor. We conclude that NPM-ALK regulates the activity of the p53 tumour suppressor pathway via sequestration by JNK and MDM2 leading to its degradation. MDM2 antagonists in combination with JNK/PI 3-Kinase inhibitors may therefore be potential targets for the treatment of ALK-expressing malignancies such as ALCL.
ALL-Associated JAK1 Mutants Activate the JAK/STAT Pathway Via IL-9Rα Homodimers
Authors: Hornakova Tekla , Staerk Judith , Royer Yohan , Flex Elisabetta , Tartaglia Marco , Constantinescu Stefan N. , Renauld Jean-Christophe , Knoops Laurent 
Published: April 2020

Abstract
Point mutations in the JAK2 tyrosine kinase play a major role in the development of myeloproliferative syndromes by activating preformed homodimers of cytokine receptors such as EpoR, TpoR and GM-CSFR. More recently, a series of activating mutations in JAK1 have also been shown to be associated with B and T cell acute lymphoblastic leukemias, but little is known about the mechanisms involved in the constitutive activation of these mutants and about their mode of interaction with cytokine receptors. Here, we studied the ability of several ALL-associated JAK1 mutants (V658F, A634D, R724H, R879C) to activate the JAK/STAT pathway upon ectopic expression in HEK293 cells, alone or together with the other components of the IL-9 receptor complex (IL-9Rα, γc and JAK3). Expression of the JAK1 mutants alone failed to trigger STAT activation, but coexpression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STATs activation, even without γc and JAK3 which are required for IL-9 responsiveness. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with IL-9R and phosphorylate this tyrosine residue to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with deletion or mutation of the JAK-interacting BOX1 region not only failed to promote JAK1 activation, but acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, this process was partially inhibited by the expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Coexpression of wild-type JAK3 partially reverted the inhibitory effect of γc, indicating that JAK3 can cooperate with JAK1 mutants within the IL-9 receptor complex, even in the absence of the cytokine. Similar results were also observed with IL-2Rβ, which binds JAK1, but not for JAK2-associated homodimeric receptors such as TpoR or EpoR. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers can efficiently mediate constitutive activation of ALL-associated JAK1 mutants, even in cells lacking the expression of γc, which is required for the activation of wild-type JAK1 to their respective physiological ligands, IL-9 and IL-2.
Role of CalDAG-GEFI in Calcium-Dependent Thromboxane A2 Generation
Authors: Stefanini Lucia , Roden R. Claire , Bergmeier Wolfgang 
Published: April 2020

Abstract
The second messenger Ca2+ is essential for various platelet functions, including integrin activation, thromboxane A2 (TxA2) generation and granule release. In previous studies, we have identified Ca2+- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a key molecule regulating Ca2+-dependent activation of Rap1 and integrins in platelets. Here we demonstrate that CalDAG-GEFI/Rap1 signaling is required for Ca2+-dependent ERK 1/2 activation and TxA2 generation in platelets. Impaired TxA2 formation was observed in collagen- and convulxin-activated CalDAG-GEFI deficient platelets, both at high and low doses of agonists. In contrast, no defect in TxA2 generation was observed in mutant platelets stimulated with high doses of PAR4-activating peptide, suggesting that CalDAG-GEFI may be particularly important for TxA2 generation in platelets activated through the major collagen receptor, GPVI. Our data further demonstrate a strong correlation between reduced TxA2 generation and impaired activation of ERK1/2 and Rap1 in mutant platelets. Pretreatment of CalDAG-GEFI−'/− platelets with 2-MesAMP, an inhibitor of P2Y12/Gi signaling, blocked the residual TxA2 production. These data suggest that CalDAG-GEFI synergizes with the Gi signaling pathway to control TxA2 generation. Finally, addition of thromboxane A2 analog, U46619, restored dense granule release in CalDAG-GEFI-deficient platelets stimulated with low doses of convulxin, suggesting that impaired TxA2 generation contributes to the defect in granule release previously described for CalDAG-GEFI−/− platelets. In conclusion, our studies strongly support that CalDAG-GEFI is the elusive Ca2+ sensor that links increases in intracellular Ca2+ to the signaling pathways regulating integrin activation and TxA2 formation in stimulated platelets.