140 results on '"Villeval JL"'
Search Results
2. Myélofibrose par excès de thrombopoïétine
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Abina, A, primary, Villeval, JL, additional, Cohen-Solal, K, additional, Vainchenker, W, additional, Wendling, F, additional, Haddada, H, additional, and Tulliez, M, additional
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- 1997
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3. Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis
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Cohen-Solal, K, primary, Villeval, JL, additional, Titeux, M, additional, Lok, S, additional, Vainchenker, W, additional, and Wendling, F, additional
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- 1996
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4. Missense mutation of the erythropoietin receptor is a rare event in human erythroid malignancies
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Le Couedic, JP, primary, Mitjavila, MT, additional, Villeval, JL, additional, Feger, F, additional, Gobert, S, additional, Mayeux, P, additional, Casadevall, N, additional, and Vainchenker, W, additional
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- 1996
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5. Autocrine stimulation by erythropoietin (Epo) requires Epo secretion
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Villeval, JL, primary, Mitjavila, MT, additional, Dusanter-Fourt, I, additional, Wendling, F, additional, Mayeux, P, additional, and Vainchenker, W, additional
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- 1994
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6. Retrovirus-mediated transfer of the erythropoietin gene in hematopoietic cells improves the erythrocyte phenotype in murine beta- thalassemia
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Villeval, JL, primary, Rouyer-Fessard, P, additional, Blumenfeld, N, additional, Henri, A, additional, Vainchenker, W, additional, and Beuzard, Y, additional
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- 1994
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7. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients
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Duhrsen, U, Villeval, JL, Boyd, J, Kannourakis, G, Morstyn, G, and Metcalf, D
- Abstract
Hematopoietic progenitor cell levels were monitored in the peripheral blood and bone marrow of 30 cancer patients receiving recombinant human granulocyte-colony stimulating-factor (rG-CSF) in a phase I/II clinical trial. The absolute number of circulating progenitor cells of granulocyte-macrophage, erythroid, and megakaryocyte lineages showed a dose-related increase up to 100-fold after four days of treatment with rG-CSF and often remained elevated two days after the cessation of therapy. The relative frequency of different types of progenitor cells in peripheral blood remained unchanged. The frequency of progenitor cells in the marrow was variable after rG-CSF treatment but in most patients was slightly decreased. The responsiveness of bone marrow progenitor cells to stimulation in vitro by rG-CSF and granulocyte- macrophage colony-stimulating factor did not change significantly during rG-CSF treatment. In patients nine days after treatment with melphalan and then rG-CSF, progenitor cell levels were very low with doses of rG-CSF at or below 10 micrograms/kg/d, but equaled or exceeded pretreatment values when 30 or 60 micrograms/kg/d of rG-CSF was given.
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- 1988
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8. Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold
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Cramer, E, Pryzwansky, KB, Villeval, JL, Testa, U, and Breton-Gorius, J
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Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.
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- 1985
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9. Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias
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Mitjavila, MT, Villeval, JL, Cramer, P, Henri, A, Gasson, J, Krystal, G, Tulliez, M, Berger, R, Breton-Gorius, J, and Vainchenker, W
- Abstract
Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in “serum-free” cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM- CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two “de novo” leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.
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- 1987
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10. Phenotype of early erythroblastic leukemias
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Villeval, JL, Cramer, P, Lemoine, F, Henri, A, Bettaieb, A, Bernaudin, F, Beuzard, Y, Berger, R, Flandrin, G, and Breton-Gorius, J
- Abstract
Nine cases of early erythroblastic leukemia, unidentified by usual criteria, have been diagnosed using a panel of antibodies. Three cases arose in patients with Down's syndrome, one in a patient with therapy- related leukemia, and four patients were in blast crisis of chronic myeloid leukemia; only one case arose de novo. Blast cells could be assigned to two main stages of erythroid differentiation: presence of all erythroid-specific proteins in two patients, a phenotype corresponding to an immature erythroblast; absence of the erythroid markers such as glycophorin A and spectrin in the presence of carbonic anhydrase isoenzyme I, ABH group antigens, and the antigen defined by FA6 152 monoclonal antibody in six patients, a phenotype related to a late erythroid progenitor (CFU-E). One patient had an intermediate phenotype. All patients except one demonstrated a megakaryocytic component. In three patients, chromosomal abnormalities were present, detected both in blasts and in erythroid colonies. In conclusion, these findings indicate that most “cryptic erythroleukemias” are blocked at a “CFU-E-like” stage of differentiation, it may be a frequent event in Down's syndrome and chronic myeloid leukemia, and these erythroleukemias are phenotypically heterogeneous.
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- 1986
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11. A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors
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Edelman, P, Vinci, G, Villeval, JL, Vainchenker, W, Henri, A, Miglierina, R, Rouger, P, Reviron, J, Breton-Gorius, J, and Sureau, C
- Abstract
A murine monoclonal antibody (MoAb) designated FA6–152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6–152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6–152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.
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- 1986
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12. Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation
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Villeval, JL, Testa, U, Vinci, G, Tonthat, H, Bettaieb, A, Titeux, M, Cramer, P, Edelman, L, Rochant, H, and Breton-Gorius, J
- Abstract
The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU- E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti- glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.
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- 1985
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13. Impaired fibrinolysis in JAK2V617F-related myeloproliferative neoplasms.
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Bourrienne MC, Loyau S, Faille D, Gay J, Akhenak S, Farkh C, Ollivier V, Solonomenjanahary M, Dupont S, Choqueux C, Villeval JL, Plo I, Edmond V, Ho-Tin-Noé B, Ajzenberg N, and Mazighi M
- Abstract
Background: Myeloproliferative neoplasms (MPN) are characterized by a high rate of thrombotic complications that contribute to morbi-mortality. MPN-related thrombogenesis is assumed to be multifactorial involving both pro-coagulant and pro-inflammatory processes. Whether impaired fibrinolysis also participates in the pro-thrombotic phenotype of MPN has been poorly investigated., Objectives: We determined whether MPN, particularly JAK2V617F-positive MPN, are associated with fibrinolytic changes., Patients: Tissue plasminogen activator (tPA)-mediated fibrinolysis was evaluated both in whole blood (WB) and plasma from mice with a hematopoietic-restricted Jak2
V617F expression compared to wild type mice (Jak2WT ) using: (1) halo clot lysis, (2) front lysis and (3) plasmin generation assays. tPA-clot lysis assay was performed in the plasma from 65 MPN patients (JAK2V617F mutation, n=50; CALR mutations, n=9) compared to 28 healthy controls., Results: In WB from Jak2V617F mice, we observed a decreased fibrinolysis characterized by a significant lower halo clot lysis rate compared to Jak2WT (95±22 vs 147±39 UA/min, p<0.05). Similar results were observed in plasma (halo clot lysis rate: 130±27 vs 186±29 UA/min; front lysis rate: 2.8±1.6 vs 6.1±1.2 μm.min-1 , p<0.05). Plasmin generation was significantly decreased both in plasma clots and standardized fibrin clots from Jak2V617F mice compared to Jak2WT mice. Among MPN patients, impaired tPA-related fibrinolysis with prolonged clot lysis time was observed in JAK2V617F and CALR patients. Plasminogen activator inhibitor-1 and alpha 2-antiplasmin were significantly increased in plasma from JAK2V617F patients compared to controls., Conclusions: Our results suggest that impaired tPA-mediated fibrinolysis represents an important pro-thrombotic mechanism in MPN patients that requires confirmation on larger studies., (Copyright © 2024. Published by Elsevier Inc.)- Published
- 2024
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14. Exacerbation of thromboinflammation by JAK2V617F mutation worsens the prognosis of cerebral venous sinus thrombosis.
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Bourrienne MC, Le Cam Duchez V, Faille D, Farkh C, Solo Nomenjanahary M, Gay J, Loyau S, Journé C, Dupont S, Ollivier V, Villeval JL, Plo I, Edmond V, Jandrot-Perrus M, Labrouche-Colomer S, Cassinat B, Verger E, Desilles JP, Ho-Tin-Noé B, Triquenot Bagan A, Mazighi M, and Ajzenberg N
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- Animals, Mice, Humans, Prognosis, Disease Models, Animal, Male, Female, Neutrophils metabolism, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Sinus Thrombosis, Intracranial genetics, Inflammation genetics, Mutation
- Abstract
Abstract: Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms, has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological, and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared with wild-type Jak2 mice. In parallel, we studied a human cohort of JAK2V617F-positive or -negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICHs) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase plasma level. Concurrently, immunohistological and brain homogenate analysis showed higher neutrophil infiltration and increased blood-brain barrier disruption. Similarly, patients with JAK2V617F-positive CVST tended to present higher thrombotic burden and had significantly higher systemic immune-inflammation index, a systemic thromboinflammatory marker, than patients who were JAK2V617F-negative. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH, and mortality. The exacerbated thromboinflammatory response, observed both in mice and patients positive for JAK2V617F, could contribute to hemorrhagic complications., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
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- 2024
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15. SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.
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Willekens C, Laplane L, Dagher T, Benlabiod C, Papadopoulos N, Lacout C, Rameau P, Catelain C, Alfaro A, Edmond V, Signolle N, Marchand V, Droin N, Hoogenboezem R, Schneider RK, Penson A, Abdel-Wahab O, Giraudier S, Pasquier F, Marty C, Plo I, Villeval JL, Constantinescu SN, Porteu F, Vainchenker W, and Solary E
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- Animals, Mice, Janus Kinase 2 genetics, Janus Kinases genetics, Mutation, RNA-Binding Proteins genetics, Signal Transduction, STAT Transcription Factors genetics, Hematopoietic Stem Cell Transplantation, Myeloproliferative Disorders genetics, Primary Myelofibrosis genetics
- Abstract
Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2
P95H with Jak2V617F , we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFβ1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H , an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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16. In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target.
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Murakami S, Barroca V, Perié L, Bravard A, Bernardino-Sgherri J, Tisserand A, Devanand C, Edmond V, Magniez A, Tenreira Bento S, Torres C, Pasquier F, Plo I, Vainchenker W, Villeval JL, Roméo PH, and Lewandowski D
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- Animals, Hematopoietic Stem Cells, Janus Kinase 2 genetics, Mice, Carbonic Anhydrases, Hematologic Neoplasms pathology, Polycythemia Vera drug therapy
- Abstract
Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV., Significance: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265., (©2022 American Association for Cancer Research.)
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- 2022
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17. Lessons from mouse models of MPN.
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Benlabiod C, Dagher T, Marty C, and Villeval JL
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- Animals, Disease Models, Animal, Disease Progression, Genetic Predisposition to Disease, Humans, Mice, Mutation, Receptors, Thrombopoietin genetics, Thrombocythemia, Essential genetics
- Abstract
Over the past decades, a variety of MPN mouse models have been developed to express in HSC the main mutations identified in patients: JAK2
V617F , CALRdel52 or ins5 and MPLW515L . These models mimic quite faithfully human PV or ET with their natural evolutions into MF and their hemostasis complications, demonstrating the driver function of these mutations in MPN. Here, we review these models and show how they have improved our general understanding of MPN regarding (1) the mechanisms of fibrosis, thrombosis/hemorrhages and disease initiation, (2) the roles of additional mutations and signaling pathways in disease progression and (3) the preclinical development of novel therapies. We also address controversial results between these models and remind how these models may differ from human MPN onset and also how basically mice are not humans, encouraging caution when one draw lessons from mice to humans. Furthermore, the contribution of germline genetic predisposition, HSC and niche aging, metabolic, oxidative, replicative or genotoxic stress, inflammation, immune escape and additional mutations need to be considered in further investigations to encompass the full complexity of human MPN in mice., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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18. Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.
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Mosca M, Hermange G, Tisserand A, Noble R, Marzac C, Marty C, Le Sueur C, Campario H, Vertenoeil G, El-Khoury M, Catelain C, Rameau P, Gella C, Lenglet J, Casadevall N, Favier R, Solary E, Cassinat B, Kiladjian JJ, Constantinescu SN, Pasquier F, Hochberg ME, Raslova H, Villeval JL, Girodon F, Vainchenker W, Cournède PH, and Plo I
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- Calreticulin genetics, Hematopoietic Stem Cells metabolism, Hematopoietic Stem Cells pathology, Humans, Immunologic Factors pharmacology, Interferon-alpha pharmacology, Janus Kinase 2 genetics, Longitudinal Studies, Myeloproliferative Disorders genetics, Myeloproliferative Disorders pathology, Prospective Studies, Receptors, Thrombopoietin genetics, Tumor Cells, Cultured, Hematopoietic Stem Cells drug effects, Immunologic Factors therapeutic use, Interferon-alpha therapeutic use, Mutation drug effects, Myeloproliferative Disorders drug therapy
- Abstract
Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose., (© 2021 by The American Society of Hematology.)
- Published
- 2021
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19. PPARγ agonists promote the resolution of myelofibrosis in preclinical models.
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Lambert J, Saliba J, Calderon C, Sii-Felice K, Salma M, Edmond V, Alvarez JC, Delord M, Marty C, Plo I, Kiladjian JJ, Soler E, Vainchenker W, Villeval JL, Rousselot P, and Prost S
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- Animals, Bone Marrow Cells metabolism, Bone Marrow Cells pathology, Disease Models, Animal, Hematologic Neoplasms genetics, Hematologic Neoplasms metabolism, Hematologic Neoplasms pathology, Mice, Mice, Transgenic, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Neoplasms, Experimental genetics, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, PPAR gamma genetics, PPAR gamma metabolism, Primary Myelofibrosis genetics, Primary Myelofibrosis metabolism, Primary Myelofibrosis pathology, Tumor Microenvironment drug effects, Tumor Microenvironment genetics, Antineoplastic Agents pharmacology, Hematologic Neoplasms drug therapy, Neoplasm Proteins agonists, Neoplasms, Experimental drug therapy, PPAR gamma agonists, Primary Myelofibrosis drug therapy
- Abstract
Myelofibrosis (MF) is a non-BCR-ABL myeloproliferative neoplasm associated with poor outcomes. Current treatment has little effect on the natural history of the disease. MF results from complex interactions between (a) the malignant clone, (b) an inflammatory context, and (c) remodeling of the bone marrow (BM) microenvironment. Each of these points is a potential target of PPARγ activation. Here, we demonstrated the therapeutic potential of PPARγ agonists in resolving MF in 3 mouse models. We showed that PPARγ agonists reduce myeloproliferation, modulate inflammation, and protect the BM stroma in vitro and ex vivo. Activation of PPARγ constitutes a relevant therapeutic target in MF, and our data support the possibility of using PPARγ agonists in clinical practice.
- Published
- 2021
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20. JAK2V617F myeloproliferative neoplasm eradication by a novel interferon/arsenic therapy involves PML.
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Dagher T, Maslah N, Edmond V, Cassinat B, Vainchenker W, Giraudier S, Pasquier F, Verger E, Niwa-Kawakita M, Lallemand-Breitenbach V, Plo I, Kiladjian JJ, Villeval JL, and de Thé H
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- Animals, Cell Line, Cell Line, Tumor, Disease Models, Animal, Humans, Leukemia, Promyelocytic, Acute drug therapy, Leukemia, Promyelocytic, Acute metabolism, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myeloproliferative Disorders metabolism, Arsenic Trioxide pharmacology, Interferon-alpha pharmacology, Janus Kinase 2 metabolism, Myeloproliferative Disorders drug therapy, Promyelocytic Leukemia Protein metabolism
- Abstract
Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene., Competing Interests: Disclosures: B. Cassinat reported a pending patent to WO2018134260. W. Vainchenker reported a patent to JAK2V617F licensed "Quiagen." V. Lallemand-Breitenbach reported a pending patent to WO2018134260. J. Kiladjian reported personal fees from AOP Orphan during the conduct of the study and personal fees from Novartis outside the submitted work. In addition, J. Kiladjian had a pending patent to WO2018134260 naming Hugues de Thé, Bruno Cassinat, Valérie Lallemand-Breitenbach, Isabelle Plo, Jean-Luc Villeval, and Jean-Jacques Kiladjian as inventors. H. de Thé reported personal fees from Vectorlab outside the submitted work; in addition, H. de Thé had a pending patent to WO2018134260. No other disclosures were reported., (© 2020 Dagher et al.)
- Published
- 2021
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21. Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN.
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Benlabiod C, Cacemiro MDC, Nédélec A, Edmond V, Muller D, Rameau P, Touchard L, Gonin P, Constantinescu SN, Raslova H, Villeval JL, Vainchenker W, Plo I, and Marty C
- Subjects
- Animals, Calreticulin metabolism, Disease Models, Animal, Female, Hematopoietic Stem Cells metabolism, Homozygote, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Male, Mice, Mice, Inbred C57BL, Mutagenesis, Insertional, Phenotype, Primary Myelofibrosis metabolism, Sequence Deletion, Thrombocythemia, Essential metabolism, Calreticulin genetics, Primary Myelofibrosis genetics, Thrombocythemia, Essential genetics
- Abstract
Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.
- Published
- 2020
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22. Megakaryocytes tame erythropoiesis with TGFβ1.
- Author
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Villeval JL and Vainchenker W
- Subjects
- Cell Count, Stem Cells, Erythropoiesis, Megakaryocytes
- Published
- 2020
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23. Erythrocyte-derived microvesicles induce arterial spasms in JAK2V617F myeloproliferative neoplasm.
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Poisson J, Tanguy M, Davy H, Camara F, El Mdawar MB, Kheloufi M, Dagher T, Devue C, Lasselin J, Plessier A, Merchant S, Blanc-Brude O, Souyri M, Mougenot N, Dingli F, Loew D, Hatem SN, James C, Villeval JL, Boulanger CM, and Rautou PE
- Subjects
- Animals, Antioxidants pharmacology, Aorta, Thoracic drug effects, Aorta, Thoracic physiopathology, Cardiovascular Diseases etiology, Cardiovascular Diseases genetics, Cardiovascular Diseases physiopathology, Cell-Derived Microparticles physiology, Femoral Artery drug effects, Femoral Artery physiopathology, Humans, In Vitro Techniques, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myeloproliferative Disorders complications, Oxidative Stress, Simvastatin pharmacology, Vasoconstriction drug effects, Vasoconstriction physiology, Erythrocytes physiology, Gain of Function Mutation, Janus Kinase 2 genetics, Janus Kinase 2 physiology, Myeloproliferative Disorders genetics, Myeloproliferative Disorders physiopathology
- Abstract
Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPNs). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPNs suggests that vascular function is altered. The consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice resulting from a disturbed endothelial NO pathway and increased endothelial oxidative stress. This response was reproduced in WT mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for their effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes suppressed their effect on oxidative stress. Antioxidants such as simvastatin and N-acetyl cysteine improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPNs are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears to be a promising therapeutic strategy in this setting.
- Published
- 2020
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24. Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.
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Lopez CK, Noguera E, Stavropoulou V, Robert E, Aid Z, Ballerini P, Bilhou-Nabera C, Lapillonne H, Boudia F, Thirant C, Fagnan A, Arcangeli ML, Kinston SJ, Diop M, Job B, Lecluse Y, Brunet E, Babin L, Villeval JL, Delabesse E, Peters AHFM, Vainchenker W, Gaudry M, Masetti R, Locatelli F, Malinge S, Nerlov C, Droin N, Lobry C, Godin I, Bernard OA, Göttgens B, Petit A, Pflumio F, Schwaller J, and Mercher T
- Subjects
- Adolescent, Age Factors, Animals, Child, Child, Preschool, Female, Humans, Infant, Leukemia, Myeloid, Acute genetics, Mice, Neoplasm Transplantation, Transcription Factors, Tumor Cells, Cultured, Leukemia, Myeloid, Acute pathology, Oncogene Proteins, Fusion genetics
- Abstract
Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2 , are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2 -induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state. See related commentary by Cruz Hernandez and Vyas, p. 1653 . This article is highlighted in the In This Issue feature, p. 1631 ., (©2019 American Association for Cancer Research.)
- Published
- 2019
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25. Comparison of endothelial promoter efficiency and specificity in mice reveals a subset of Pdgfb-positive hematopoietic cells.
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Kilani B, Gourdou-Latyszenok V, Guy A, Bats ML, Peghaire C, Parrens M, Renault MA, Duplàa C, Villeval JL, Rautou PE, Couffinhal T, and James C
- Subjects
- Alleles, Animals, Brain metabolism, Hemostasis, Integrases metabolism, Kidney metabolism, Liver metabolism, Lung metabolism, Mice, Mice, Transgenic, Myocardium metabolism, Polymerase Chain Reaction, Retina metabolism, Tamoxifen pharmacology, Thrombosis metabolism, Endothelial Cells cytology, Hematopoietic Stem Cells cytology, Lymphokines genetics, Lymphokines metabolism, Platelet-Derived Growth Factor genetics, Platelet-Derived Growth Factor metabolism
- Abstract
Essentials To reliably study the respective roles of blood and endothelial cells in hemostasis, mouse models with a strong and specific endothelial expression of the Cre recombinase are needed. Using mT/mG reporter mice and conditional JAK2
V617F/WT mice, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Comparison of recombination efficiency and specificity towards blood lineage reveals major differences between endothelial transgenic mice. Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2;JAK2V617F/WT transgenic mice. SUMMARY: Background The vessel wall, and particularly blood endothelial cells (BECs), are intensively studied to better understand hemostasis and target thrombosis. To understand the specific role of BECs, it is important to have mouse models that allow specific and homogeneous expression of genes of interest in all BEC beds without concomitant expression in blood cells. Inducible Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 transgenic mice are widely used for BEC targeting. However, issues remain in terms of recombination efficiency and specificity regarding hematopoietic cells. Objectives To determine which mouse model to choose when strong expression of a transgene is required in adult BECs from various organs, without concomitant expression in hematopoietic cells. Methods Using mT/mG reporter mice to measure recombination efficiency and conditional JAK2V617F/ WT mice to assess specificity regarding hematopoietic cells, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Results Adult Cdh5(PAC)-CreERT2 mice are endothelial specific but require a dose of 10 mg of tamoxifen to allow constant Cre expression. Pdgfb-iCreERT2 mice injected with 5 mg of tamoxifen are appropriate for most endothelial research fields except liver studies, as hepatic sinusoid ECs are not recombined. Surprisingly, 2 months after induction of Cre-mediated recombination, all Pdgfb-iCreERT2;JAK2V617F/ WT mice developed a myeloproliferative neoplasm that is related to the presence of JAK2V617F in hematopoietic cells, showing for the first time that Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2 transgenic mice. Conclusion This study provides useful guidelines for choosing the best mouse line to study the role of BECs in hemostasis and thrombosis., (© 2019 International Society on Thrombosis and Haemostasis.)- Published
- 2019
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26. Rare type 1-like and type 2-like calreticulin mutants induce similar myeloproliferative neoplasms as prevalent type 1 and 2 mutants in mice.
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Toppaldoddi KR, da Costa Cacemiro M, Bluteau O, Panneau-Schmaltz B, Pioch A, Muller D, Villeval JL, Raslova H, Constantinescu SN, Plo I, Vainchenker W, and Marty C
- Subjects
- Animals, Calreticulin chemistry, Cell Line, Disease Models, Animal, Humans, Janus Kinase 2 genetics, Mice, Myeloproliferative Disorders metabolism, Receptors, Thrombopoietin metabolism, STAT Transcription Factors genetics, Transcriptional Activation, Calreticulin genetics, Calreticulin metabolism, Mutation, Myeloproliferative Disorders genetics
- Abstract
Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.
- Published
- 2019
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27. Vascular endothelial cell expression of JAK2 V617F is sufficient to promote a pro-thrombotic state due to increased P-selectin expression.
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Guy A, Gourdou-Latyszenok V, Le Lay N, Peghaire C, Kilani B, Dias JV, Duplaa C, Renault MA, Denis C, Villeval JL, Boulaftali Y, Jandrot-Perrus M, Couffinhal T, and James C
- Subjects
- Animals, Disease Models, Animal, Endothelial Cells pathology, Gene Expression Regulation, Enzymologic drug effects, Humans, Hydroxyurea pharmacology, Janus Kinase 2 genetics, Mice, Mice, Transgenic, P-Selectin genetics, Thrombosis drug therapy, Thrombosis genetics, Thrombosis pathology, Endothelial Cells metabolism, Janus Kinase 2 biosynthesis, P-Selectin biosynthesis, Thrombosis metabolism
- Abstract
Thrombosis is the main cause of morbidity and mortality in patients with JAK2
V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F -expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F -expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F -expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F -positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses., (Copyright© 2019 Ferrata Storti Foundation.)- Published
- 2019
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28. Endothelial JAK2 V617F does not enhance liver lesions in mice with Budd-Chiari syndrome.
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Poisson J, Hilscher MB, Tanguy M, Hammoutene A, Boulanger CM, Villeval JL, Simonetto DA, Valla D, Shah VH, and Rautou PE
- Subjects
- Amino Acid Substitution, Animals, Budd-Chiari Syndrome pathology, Disease Models, Animal, Endothelial Cells metabolism, Female, Humans, Liver metabolism, Liver pathology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Missense, Recombinant Proteins genetics, Recombinant Proteins metabolism, Budd-Chiari Syndrome genetics, Budd-Chiari Syndrome metabolism, Janus Kinase 2 genetics, Janus Kinase 2 metabolism
- Published
- 2018
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29. HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.
- Author
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Sevin M, Kubovcakova L, Pernet N, Causse S, Vitte F, Villeval JL, Lacout C, Cordonnier M, Rodrigues-Lima F, Chanteloup G, Mosca M, Chrétien ML, Bastie JN, Audia S, Sagot P, Ramla S, Martin L, Gleave M, Mezger V, Skoda R, Plo I, Garrido C, Girodon F, and de Thonel A
- Subjects
- Animals, Bone Marrow Cells immunology, Bone Marrow Cells pathology, Bone Marrow Transplantation, Cell Line, Tumor, Disease Models, Animal, Female, HEK293 Cells, HSP27 Heat-Shock Proteins immunology, Humans, Janus Kinase 2 immunology, K562 Cells, Leukocytes drug effects, Leukocytes immunology, Leukocytes pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Molecular Targeted Therapy, Mutation, Primary Myelofibrosis immunology, Primary Myelofibrosis pathology, STAT5 Transcription Factor immunology, Thrombopoietin genetics, Thrombopoietin immunology, Transduction, Genetic, Whole-Body Irradiation, HSP27 Heat-Shock Proteins genetics, Janus Kinase 2 genetics, Oligonucleotides pharmacology, Primary Myelofibrosis drug therapy, Primary Myelofibrosis genetics, STAT5 Transcription Factor genetics
- Abstract
Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34
+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.- Published
- 2018
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30. Endothelial-to-Mesenchymal Transition in Bone Marrow and Spleen of Primary Myelofibrosis.
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Erba BG, Gruppi C, Corada M, Pisati F, Rosti V, Bartalucci N, Villeval JL, Vannucchi AM, Barosi G, Balduini A, and Dejana E
- Subjects
- Animals, Disease Models, Animal, Humans, Megakaryocytes pathology, Mice, Bone Marrow pathology, Primary Myelofibrosis pathology, Spleen pathology
- Abstract
Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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31. Inhibitors of the PI3K/mTOR pathway prevent STAT5 phosphorylation in JAK2V617F mutated cells through PP2A/CIP2A axis.
- Author
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Bartalucci N, Calabresi L, Balliu M, Martinelli S, Rossi MC, Villeval JL, Annunziato F, Guglielmelli P, and Vannucchi AM
- Abstract
Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in JAK2V617F mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in JAK2V617F mutated cell lines, primary patients' cells, and JAK2V617F knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials., Competing Interests: CONFLICTS OF INTEREST The authors disclose no potential conflicts of interest
- Published
- 2017
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32. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.
- Author
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Marty C, Pecquet C, Nivarthi H, El-Khoury M, Chachoua I, Tulliez M, Villeval JL, Raslova H, Kralovics R, Constantinescu SN, Plo I, and Vainchenker W
- Subjects
- Animals, Calreticulin genetics, Frameshift Mutation, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Megakaryocytes pathology, Mice, Mice, Mutant Strains, Primary Myelofibrosis etiology, Primary Myelofibrosis genetics, Primary Myelofibrosis pathology, Receptors, Thrombopoietin genetics, Thrombocytosis complications, Thrombocytosis genetics, Thrombocytosis pathology, Calreticulin metabolism, INDEL Mutation, Megakaryocytes metabolism, Primary Myelofibrosis metabolism, Receptors, Thrombopoietin metabolism, Thrombocytosis metabolism
- Abstract
Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation., (© 2016 by The American Society of Hematology.)
- Published
- 2016
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33. JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice.
- Author
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Debeurme F, Lacout C, Moratal C, Bagley RG, Vainchenker W, Adrian F, and Villeval JL
- Subjects
- Animals, Disease Progression, Mice, Platelet Count, Polycythemia Vera blood, Polycythemia Vera physiopathology, Primary Myelofibrosis blood, Primary Myelofibrosis physiopathology, Splenomegaly drug therapy, Thrombocythemia, Essential blood, Thrombocythemia, Essential physiopathology, Janus Kinase 2 antagonists & inhibitors, Polycythemia Vera drug therapy, Primary Myelofibrosis drug therapy, Protein Kinase Inhibitors administration & dosage, Pyrrolidines administration & dosage, Sulfonamides administration & dosage, Thrombocythemia, Essential drug therapy
- Abstract
JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2(V617F) mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2(V617F) allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)
- Published
- 2015
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34. p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.
- Author
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Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, Villeval JL, Louache F, Gonin P, Debili N, Plo I, Vainchenker W, Gilles L, and Raslova H
- Subjects
- Animals, Bone Marrow metabolism, Bone Marrow pathology, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Cell Count, Cell Differentiation genetics, Cell Lineage genetics, Cyclin-Dependent Kinase Inhibitor p19 deficiency, Cyclin-Dependent Kinase Inhibitor p19 metabolism, Hematopoiesis, Mice, Mice, Knockout, Osteosclerosis genetics, Osteosclerosis pathology, Primary Myelofibrosis genetics, Primary Myelofibrosis pathology, Resting Phase, Cell Cycle genetics, Stromal Cells metabolism, Cellular Senescence genetics, Cyclin-Dependent Kinase Inhibitor p19 genetics, DNA Damage, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Stem Cell Niche genetics
- Abstract
Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
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35. Hemostatic disorders in a JAK2V617F-driven mouse model of myeloproliferative neoplasm.
- Author
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Lamrani L, Lacout C, Ollivier V, Denis CV, Gardiner E, Ho Tin Noe B, Vainchenker W, Villeval JL, and Jandrot-Perrus M
- Subjects
- Animals, Aorta metabolism, Aorta pathology, Aorta physiopathology, Bleeding Time, Blood Platelets metabolism, Flow Cytometry, Gene Knock-In Techniques, Humans, Immunoblotting, Mice, Transgenic, Myeloproliferative Disorders blood, Platelet Activation genetics, Platelet Membrane Glycoproteins genetics, Platelet Membrane Glycoproteins metabolism, Polycythemia Vera blood, Polycythemia Vera genetics, Primary Myelofibrosis blood, Primary Myelofibrosis genetics, Thrombosis blood, Thrombosis genetics, Vasodilation genetics, von Willebrand Factor metabolism, Disease Models, Animal, Hemostatic Disorders genetics, Janus Kinase 2 genetics, Mutation, Missense, Myeloproliferative Disorders genetics
- Abstract
Thrombosis is common in patients suffering from myeloproliferative neoplasm (MPN), whereas bleeding is less frequent. JAK2(V617F), the main mutation involved in MPN, is considered as a risk factor for thrombosis, although the direct link between the mutation and hemostatic disorders is not strictly established. We investigated this question using conditional JAK2(V617F) knock-in mice with constitutive and inducible expression of JAK2(V617F) in hematopoietic cells, which develop a polycythemia vera (PV)-like disorder evolving into myelofibrosis. In vitro, thrombosis was markedly impaired with an 80% decrease in platelet-covered surface, when JAK2(V617F) blood was perfused at arterial shear over collagen. JAK2(V617F) platelets presented only a moderate glycoprotein (GP) VI deficiency not responsible for the defective platelet accumulation. In contrast, a decreased proportion of high-molecular-weight von Willebrand factor multimers could reduce platelet adhesion. Accordingly, the tail bleeding time was prolonged. In the FeCl3-induced thrombosis model, platelet aggregates formed rapidly but were highly unstable. Interestingly, vessels were considerably dilated. Thus, mice developing PV secondary to constitutive JAK2(V617F) expression exhibit a bleeding tendency combined with the accelerated formation of unstable clots, reminiscent of observations made in patients. Hemostatic defects were not concomitant with the induction of JAK2(V617F) expression, suggesting they were not directly caused by the mutation but were rather the consequence of perturbations in blood and vessel homeostasis., (© 2014 by The American Society of Hematology.)
- Published
- 2014
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36. Use of the 46/1 haplotype to model JAK2(V617F) clonal architecture in PV patients: clonal evolution and impact of IFNα treatment.
- Author
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Hasan S, Cassinat B, Droin N, Le Couedic JP, Favale F, Monte-Mor B, Lacout C, Fontenay M, Dosquet C, Chomienne C, Solary E, Villeval JL, Casadevall N, Kiladjian JJ, Vainchenker W, and Plo I
- Subjects
- Alleles, Clonal Evolution, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells metabolism, Humans, Interferon-alpha therapeutic use, Models, Biological, Polycythemia Vera drug therapy, Haplotypes, Janus Kinase 2 genetics, Mutation, Polycythemia Vera genetics, Polycythemia Vera metabolism
- Published
- 2014
- Full Text
- View/download PDF
37. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.
- Author
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Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, and Vannucchi AM
- Subjects
- Animals, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Apoptosis drug effects, Cell Proliferation drug effects, Drug Screening Assays, Antitumor, Drug Synergism, Female, Hematologic Neoplasms drug therapy, Hematologic Neoplasms enzymology, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells physiology, Humans, Imidazoles administration & dosage, Inhibitory Concentration 50, Janus Kinase 2 antagonists & inhibitors, Janus Kinase 2 metabolism, K562 Cells, Mice, Mice, SCID, Mice, Transgenic, Molecular Targeted Therapy, Mutation, Missense, Myeloproliferative Disorders enzymology, Neoplasm Transplantation, Nitriles, Phosphatidylinositol 3-Kinases metabolism, Pyrazoles administration & dosage, Pyrimidines, Quinolines administration & dosage, Splenomegaly prevention & control, TOR Serine-Threonine Kinases metabolism, Antineoplastic Combined Chemotherapy Protocols pharmacology, Janus Kinase 2 genetics, Myeloproliferative Disorders drug therapy, Phosphoinositide-3 Kinase Inhibitors, TOR Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)
- Published
- 2013
- Full Text
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38. A role for reactive oxygen species in JAK2 V617F myeloproliferative neoplasm progression.
- Author
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Marty C, Lacout C, Droin N, Le Couédic JP, Ribrag V, Solary E, Vainchenker W, Villeval JL, and Plo I
- Subjects
- Acetylcysteine pharmacology, Animals, Antioxidants pharmacology, Blotting, Western, Bone Marrow Transplantation, Case-Control Studies, DNA Damage drug effects, Disease Progression, Female, Flow Cytometry, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells metabolism, Humans, Immunoenzyme Techniques, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Myeloproliferative Disorders genetics, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Janus Kinase 2 physiology, Myeloproliferative Disorders metabolism, Myeloproliferative Disorders pathology, Point Mutation genetics, Reactive Oxygen Species metabolism
- Abstract
Although other mutations may predate the acquisition of the JAK2(V617F) mutation, the latter is sufficient to drive the disease phenotype observed in BCR-ABL-negative myeloproliferative neoplasms (MPNs). One of the consequences of JAK2(V617F) is genetic instability that could explain JAK2(V617F)-mediated MPN progression and heterogeneity. Here, we show that JAK2(V617F) induces the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment of a knock-in (KI) mouse model and in patients with JAK2(V617F) MPNs. JAK2(V617F)-dependent ROS elevation was partly mediated by an AKT-induced decrease in catalase expression and was accompanied by an increased number of 8-oxo-guanines and DNA double-strand breaks (DSBs). Moreover, there was evidence for a mitotic recombination event in mice resulting in loss of heterozygosity of Jak2(V617F). Mice engrafted with 30% of Jak2(V617F) KI bone marrow (BM) cells developed a polycythemia vera-like disorder. Treatment with the anti-oxidant N-acetylcysteine (NAC) substantially restored blood parameters and reduced damages to DNA. Furthermore, NAC induced a marked decrease in splenomegaly with reduction in the frequency of the Jak2(V617F)-positive hematopoietic progenitors in BM and spleen. Altogether, overproduction of ROS is a mediator of JAK2(V617F)-induced DNA damages that promote disease progression. Targeting ROS accumulation might prevent the development of JAK2(V617F) MPNs.
- Published
- 2013
- Full Text
- View/download PDF
39. Combination treatment for myeloproliferative neoplasms using JAK and pan-class I PI3K inhibitors.
- Author
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Choong ML, Pecquet C, Pendharkar V, Diaconu CC, Yong JW, Tai SJ, Wang SF, Defour JP, Sangthongpitag K, Villeval JL, Vainchenker W, Constantinescu SN, and Lee MA
- Subjects
- Animals, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Drug Screening Assays, Antitumor, Drug Synergism, Female, Gene Knock-In Techniques, Hematologic Neoplasms drug therapy, Hematologic Neoplasms enzymology, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Mice, Mice, Nude, Mice, Transgenic, Mutation, Missense, Myeloproliferative Disorders enzymology, Neoplasm Transplantation, Nitriles, Phosphatidylinositol 3-Kinases metabolism, Pyrazoles administration & dosage, Pyrimidines, Pyrrolidines administration & dosage, Signal Transduction drug effects, Sulfonamides administration & dosage, Triazines administration & dosage, Tumor Cells, Cultured, Antineoplastic Combined Chemotherapy Protocols pharmacology, Janus Kinase 2 antagonists & inhibitors, Myeloproliferative Disorders drug therapy, Phosphoinositide-3 Kinase Inhibitors
- Abstract
Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)
- Published
- 2013
- Full Text
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40. JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.
- Author
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Hasan S, Lacout C, Marty C, Cuingnet M, Solary E, Vainchenker W, and Villeval JL
- Subjects
- Animals, Bone Marrow Cells cytology, Bone Marrow Transplantation, Cell Cycle, Crosses, Genetic, Disease Models, Animal, Humans, Lymphocytes cytology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myeloproliferative Disorders genetics, Phenotype, Polycythemia Vera genetics, Primary Myelofibrosis metabolism, Stem Cells cytology, Apoptosis, Hematopoietic Stem Cells cytology, Interferon-alpha metabolism, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Mutation
- Abstract
The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.
- Published
- 2013
- Full Text
- View/download PDF
41. Thrombopoietin receptor down-modulation by JAK2 V617F: restoration of receptor levels by inhibitors of pathologic JAK2 signaling and of proteasomes.
- Author
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Pecquet C, Diaconu CC, Staerk J, Girardot M, Marty C, Royer Y, Defour JP, Dusa A, Besancenot R, Giraudier S, Villeval JL, Knoops L, Courtoy PJ, Vainchenker W, and Constantinescu SN
- Subjects
- Amino Acid Substitution genetics, Amino Acid Substitution physiology, Animals, Cell Membrane drug effects, Cell Membrane metabolism, Cells, Cultured, Down-Regulation drug effects, Drug Evaluation, Preclinical, HEK293 Cells, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Missense physiology, Phenylalanine genetics, Receptor Cross-Talk drug effects, Receptor Cross-Talk physiology, Receptors, Thrombopoietin metabolism, Signal Transduction drug effects, Valine genetics, Janus Kinase 2 antagonists & inhibitors, Janus Kinase 2 physiology, Proteasome Inhibitors, Protein Kinase Inhibitors pharmacology, Receptors, Thrombopoietin genetics
- Abstract
The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.
- Published
- 2012
- Full Text
- View/download PDF
42. The cell cycle regulator CDC25A is a target for JAK2V617F oncogene.
- Author
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Gautier EF, Picard M, Laurent C, Marty C, Villeval JL, Demur C, Delhommeau F, Hexner E, Giraudier S, Bonnevialle N, Ducommun B, Récher C, Laurent G, Manenti S, and Mansat-De Mas V
- Subjects
- Amino Acid Substitution physiology, Animals, Cell Cycle genetics, Cells, Cultured, Enzyme Activation genetics, Gene Expression Regulation, Leukemic genetics, Hematologic Neoplasms genetics, Hematologic Neoplasms pathology, Hematopoietic Stem Cells metabolism, Humans, Janus Kinase 2 metabolism, Mice, Mutant Proteins genetics, Oncogenes genetics, Phenylalanine genetics, Up-Regulation, Valine genetics, cdc25 Phosphatases metabolism, Janus Kinase 2 genetics, cdc25 Phosphatases genetics
- Abstract
The JAK2(V617F) mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2(V617F) is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2(V617F) has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2(V617F)-positive cell lines, JAK2-mutated patient CD36(+) progenitors, and in vitro-differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2(V617F) knock-in mice compared with wild-type littermates. By using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2(V617F)-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2(V617F)-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2(V617F) patients, making this protein an attracting potential therapeutic target.
- Published
- 2012
- Full Text
- View/download PDF
43. JAK2V⁶¹⁷F/TET2 mutations: does the order matter?
- Author
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Pronier E, Quivoron C, Bernard OA, and Villeval JL
- Subjects
- Amino Acid Substitution, Clone Cells metabolism, Dioxygenases, Erythroid Precursor Cells metabolism, Hematopoietic System metabolism, Humans, Time Factors, DNA-Binding Proteins genetics, Janus Kinase 2 genetics, Mutation, Polycythemia Vera genetics, Proto-Oncogene Proteins genetics
- Published
- 2011
- Full Text
- View/download PDF
44. Thrombospondin-1 is not the major activator of TGF-β1 in thrombopoietin-induced myelofibrosis.
- Author
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Evrard S, Bluteau O, Tulliez M, Rameau P, Gonin P, Zetterberg E, Palmblad J, Bonnefoy A, Villeval JL, Vainchenker W, Giraudier S, and Wagner-Ballon O
- Subjects
- Animals, Blood Platelets metabolism, Blood Platelets pathology, Bone Marrow metabolism, Bone Marrow pathology, Female, Male, Megakaryocytes metabolism, Megakaryocytes pathology, Mice, Mice, Inbred C57BL, Mice, Knockout, Primary Myelofibrosis metabolism, Spleen metabolism, Spleen pathology, Primary Myelofibrosis chemically induced, Primary Myelofibrosis pathology, Thrombopoietin adverse effects, Thrombospondin 1 physiology, Transforming Growth Factor beta1 metabolism
- Abstract
Transforming growth factor-β1 (TGF-β1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-β1 activation, we investigated the role of TSP-1 in the TPO(high) murine model of myelofibrosis. Two groups of engrafted mice, WT TPO(high) and Tsp-1-null TPO(high), were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-β1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-β1 in Tsp-1-null TPO(high) mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPO(high) mice. Our results show that TSP-1 is not the major activator of TGF-β1 in TPO-induced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.
- Published
- 2011
- Full Text
- View/download PDF
45. Notch/Delta4 signaling inhibits human megakaryocytic terminal differentiation.
- Author
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Poirault-Chassac S, Six E, Catelain C, Lavergne M, Villeval JL, Vainchenker W, and Lauret E
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Calcium-Binding Proteins, Cells, Cultured, Coculture Techniques, Erythroid Precursor Cells metabolism, Fetal Blood cytology, Fetal Blood metabolism, Flow Cytometry, Humans, Intercellular Signaling Peptides and Proteins genetics, Megakaryocytes metabolism, Mice, RNA, Messenger genetics, Receptors, Notch genetics, Reverse Transcriptase Polymerase Chain Reaction, Stromal Cells metabolism, Cell Differentiation, Intercellular Signaling Peptides and Proteins metabolism, Megakaryocytes cytology, Receptors, Notch metabolism, Signal Transduction
- Abstract
The effects of Notch signaling on human megakaryocytic and erythroid differentiation were investigated by exposing human CD34(+) progenitor cells to an immobilized chimeric form of the Notch ligand, Delta-like4 (Dll4Fc). Exposure of human cord blood CD34(+) cells to Dll4Fc induced a modest enhancement of erythroid cell production. Conversely, under megakaryocytic culture conditions, Dll4Fc strongly impaired platelet production by reducing the generation of mature CD41a(+)CD42b(+) megakaryocytes (MKs) and platelet-forming cells. The inhibitory activity of Dll4 on terminal MK differentiation was confirmed by culturing CD34(+) cells onto Dll-4-expressing stroma cells (engineered to express the membrane-anchored form of Dll4). The reduced production of mature CD41a(+)CD42(+) cells was rescued by inhibiting Notch signaling either with the N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester γ-secretase inhibitor or the dominant-negative version of Mastermind. Dll4 impaired the generation of mature CD41a(+)CD42b(+) cells and proplatelet formation without affecting earlier steps of MK differentiation, such as production of megakaryocytic/erythroid progenitors and colony-forming units-MKs. This blockade was accompanied by a modulation of the transcriptional program of megakaryocytic differentiation. All these results indicate that Dll4/Notch signaling inhibits human terminal MK differentiation.
- Published
- 2010
- Full Text
- View/download PDF
46. Myeloproliferative neoplasm induced by constitutive expression of JAK2V617F in knock-in mice.
- Author
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Marty C, Lacout C, Martin A, Hasan S, Jacquot S, Birling MC, Vainchenker W, and Villeval JL
- Subjects
- Amino Acid Substitution, Animals, Bone Marrow pathology, Cell Lineage, Crosses, Genetic, Gene Knock-In Techniques, Heterozygote, Humans, Hyperplasia, Janus Kinase 2 physiology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Missense, Myeloproliferative Disorders enzymology, Myeloproliferative Disorders genetics, Point Mutation, Polycythemia Vera enzymology, Primary Myelofibrosis enzymology, Primary Myelofibrosis etiology, Primary Myelofibrosis genetics, Spleen pathology, Thrombocythemia, Essential enzymology, Thrombocythemia, Essential genetics, Janus Kinase 2 genetics, Myeloproliferative Disorders etiology, Polycythemia Vera genetics
- Abstract
The Jak2(V617F) mutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2(V617F) in knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2(V617F) in mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.
- Published
- 2010
- Full Text
- View/download PDF
47. Selective reduction of JAK2V617F-dependent cell growth by siRNA/shRNA and its reversal by cytokines.
- Author
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Jedidi A, Marty C, Oligo C, Jeanson-Leh L, Ribeil JA, Casadevall N, Galy A, Vainchenker W, and Villeval JL
- Subjects
- Antigens, CD34 biosynthesis, Apoptosis, Cell Line, Cell Line, Tumor, Cell Proliferation, Cell Survival, Humans, K562 Cells, Mutation, Phosphorylation, RNA Interference, Cytokines metabolism, Gene Expression Regulation, Janus Kinase 2 metabolism, RNA, Small Interfering metabolism
- Abstract
The JAK(V617F) mutation is responsible for the majority of breakpoint cluster region (BCR)/Abelson (ABL)-negative myeloproliferative disorders. Ongoing clinical trials of Janus kinase 2 (JAK2) inhibitors in myeloproliferative disorder patients use small molecules targeting both wild-type and mutated JAK2. To selectively target malignant cells, we developed JAK2(V617F)-specific small interfering RNAs or short hairpin RNAs. Expression of these RNAs in cell lines or CD34(+) cells from patients reduced JAK2(V617F)-driven autonomous cell proliferation. Mechanisms of inhibition involved selective JAK2(V617F) protein down-regulation, and consequently, decrease in signal transducer and activator of transcription 5 phosphorylation, cell-cycle progression, and cell survival. However, the addition of high concentrations of cytokines to cell lines or erythropoietin to patient cells greatly reduced growth inhibition. Similarly, the efficacy of a JAK2 small molecule inhibitor on cell line and patient cell proliferation dose dependently decreased with the addition of cytokines. Our results demonstrate that it is possible to specifically target JAK2(V617F) by RNA interference (RNAi) strategies. In addition, cytokines partially reverse the inhibition induced by both RNAi and small molecule approaches. This strongly suggests that patient cytokine levels in current JAK2 inhibitor clinical trials modulate the outcome of these therapies.
- Published
- 2009
- Full Text
- View/download PDF
48. Ligand-independent thrombopoietin mutant receptor requires cell surface localization for endogenous activity.
- Author
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Marty C, Chaligné R, Lacout C, Constantinescu SN, Vainchenker W, and Villeval JL
- Subjects
- Animals, Cell Line, Tumor, Cell Membrane genetics, Dimerization, Endoplasmic Reticulum genetics, Endoplasmic Reticulum metabolism, Golgi Apparatus genetics, Golgi Apparatus metabolism, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Ligands, Mice, Mice, Nude, Neoplasms, Experimental, Phosphorylation genetics, Primary Myelofibrosis genetics, Primary Myelofibrosis metabolism, Protein Structure, Tertiary physiology, Receptors, Thrombopoietin genetics, Thrombocythemia, Essential genetics, Thrombocythemia, Essential metabolism, Amino Acid Substitution, Cell Membrane metabolism, Mutation, Missense, Receptors, Thrombopoietin metabolism, Signal Transduction
- Abstract
The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.
- Published
- 2009
- Full Text
- View/download PDF
49. Constitutive JunB expression, associated with the JAK2 V617F mutation, stimulates proliferation of the erythroid lineage.
- Author
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da Costa Reis Monte-Mór B, Plo I, da Cunha AF, Costa GG, de Albuquerque DM, Jedidi A, Villeval JL, Badaoui S, Lorand-Metze I, Pagnano KB, Saad ST, Vainchenker W, and Costa FF
- Subjects
- Bone Marrow pathology, Cell Lineage, Erythropoiesis, Humans, Polycythemia Vera genetics, Proto-Oncogene Proteins c-jun physiology, Tumor Cells, Cultured, Cell Proliferation, Erythrocytes pathology, Janus Kinase 2 genetics, Mutation, Missense, Myeloproliferative Disorders etiology, Proto-Oncogene Proteins c-jun genetics
- Abstract
The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes constitutive activation of JAK2 and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. In this study, we performed a large-scale gene expression study using serial analysis of gene expression in bone marrow cells of a newly diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells of healthy donors. JUNB was one of the genes upregulated in PV, and we confirmed, by quantitative real-time PCR, an overexpression of JUNB in hematopoietic cells of other JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, we found that JUNB was transcriptionally induced after erythropoietin addition and that JAK2 V617F constitutively induced JunB protein expression. Furthermore, JUNB knockdown reduced not only the growth of Ba/F3 cells by inducing apoptosis, but also the clonogenic and proliferative potential of human erythroid progenitors. These results establish a role for JunB in normal erythropoiesis and indicate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders.
- Published
- 2009
- Full Text
- View/download PDF
50. Activating mutations in human acute megakaryoblastic leukemia.
- Author
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Malinge S, Ragu C, Della-Valle V, Pisani D, Constantinescu SN, Perez C, Villeval JL, Reinhardt D, Landman-Parker J, Michaux L, Dastugue N, Baruchel A, Vainchenker W, Bourquin JP, Penard-Lacronique V, and Bernard OA
- Subjects
- Adult, Aged, Animals, Cell Line, Tumor, Child, Child, Preschool, Down Syndrome metabolism, Female, Humans, Infant, Infant, Newborn, Leukemia, Megakaryoblastic, Acute metabolism, Male, Mice, Mice, Inbred BALB C, Middle Aged, Neoplasm Proteins biosynthesis, Neoplasm Transplantation, Down Syndrome genetics, Leukemia, Megakaryoblastic, Acute genetics, Mutation, Neoplasm Proteins genetics
- Abstract
Oncogenic activation of tyrosine kinase signaling pathway is recurrent in human leukemia. To gain insight into the oncogenic process leading to acute megakaryoblastic leukemia (AMKL), we performed sequence analyses of a subset of oncogenes known to be activated in human myeloid and myeloproliferative disorders. In a series of human AMKL samples from both Down syndrome and non-Down syndrome patients, mutations were identified within KIT, FLT3, JAK2, JAK3, and MPL genes, with a higher frequency in DS than in non-DS patients. The novel mutations were analyzed using BaF3 cells, showing that JAK3 mutations were activating mutations. Finally, we report a novel constitutively active MPL mutant, MPLT487A, observed in a non-Down syndrome childhood AMKL that induces a myeloproliferative disease in mouse bone marrow transplantation assay.
- Published
- 2008
- Full Text
- View/download PDF
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