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ABSTRACT: Although other mutations may predate the acquisition of the JAK2(V617F) mutation, the later 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 (HSC) 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. 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 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 bone marrow and spleen. Altogether, overproduction of ROS is a mediator of JAK2(V617F)-induced DNA damages that promotes disease progression. Targeting ROS accumulation might prevent the development of JAK2(V617F) MPNs.Leukemia accepted article preview online, 5 April 2013; doi:10.1038/leu.2013.102.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 04/2013; · 8.30 Impact Factor
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Blood Cancer Journal 05/2012; 2(5):e70.
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H Moniz,
M Gastou,
T Leblanc,
C Hurtaud,
A Crétien,
Y Lécluse,
H Raslova,
J Larghero,
L Croisille,
M Faubladier,
O Bluteau,
L Lordier,
G Tchernia, W Vainchenker,
N Mohandas,
L Da Costa
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ABSTRACT: Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.
Cell Death & Disease 01/2012; 3:e356. · 5.33 Impact Factor
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ABSTRACT: JAK2(V617F) is a gain of function mutation that promotes cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN). Mutations in p53 are rarely found in these diseases before acute leukemia transformation, but this does not rule out a role for p53 deregulation in disease progression. Using Ba/F3-EPOR cells and ex vivo cultured CD34(+) cells from MPN patients, we demonstrate that expression of JAK2(V617F) affected the p53 response to DNA damage. We show that E3 ubiquitin ligase MDM2 accumulated in these cells, due to an increased translation of MDM2 mRNA. Accumulation of the La autoantigen, which interacts with MDM2 mRNA and promotes its translation, was responsible for the increase in MDM2 protein level and the subsequent degradation of p53 after DNA damage. Downregulation of La protein or cell treatment with nutlin-3, a MDM2 antagonist, restored the p53 response to DNA damage and the cytokine-dependence of Ba/F3-EPOR-JAK2(V617F) cells. Altogether, these data indicate that the JAK2(V617F) mutation affects p53 response to DNA damage through the upregulation of La antigen and accumulation of MDM2. They also suggest that p53 functional inactivation accounts for the cytokine hypersensitivity of JAK2(V617F) MPN and might have a role in disease progression.
Oncogene 07/2011; 31(10):1323-33. · 6.37 Impact Factor
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A-S Gabet,
S Coulon,
A Fricot,
J Vandekerckhove,
Y Chang,
J-A Ribeil,
L Lordier,
Y Zermati,
V Asnafi,
Z Belaid,
N Debili, W Vainchenker,
B Varet,
O Hermine,
G Courtois
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ABSTRACT: Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the
differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal
maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal
differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin
condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are
followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in
early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of
Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light
chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through
caspase-induced ROCK-1 kinase activation
Cell death and differentiation 01/2011; · 8.24 Impact Factor
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A-S Gabet,
S Coulon,
A Fricot,
J Vandekerckhove,
Y Chang,
J-A Ribeil,
L Lordier,
Y Zermati,
V Asnafi,
Z Belaid,
N Debili, W Vainchenker,
B Varet,
O Hermine,
G Courtois
[show abstract]
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ABSTRACT: Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation.
Cell death and differentiation 11/2010; 18(4):678-89. · 8.24 Impact Factor
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J-J Kiladjian,
A Massé,
B Cassinat,
H Mokrani,
I Teyssandier,
J-P le Couédic,
N Cambier,
C Almire,
E Pronier,
N Casadevall, W Vainchenker,
C Chomienne,
F Delhommeau
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 08/2010; 24(8):1519-23. · 8.30 Impact Factor
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C Ragu,
S Boukour,
G Elain,
O Wagner-Ballon,
H Raslova,
N Debili,
E N Olson,
D Daegelen, W Vainchenker,
O A Bernard,
V Penard-Lacronique
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 06/2010; 24(6):1227-30. · 8.30 Impact Factor
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N Carbuccia,
A Murati,
V Trouplin,
M Brecqueville,
J Adélaïde,
J Rey, W Vainchenker,
O A Bernard,
M Chaffanet,
N Vey,
D Birnbaum,
M J Mozziconacci
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 08/2009; 23(11):2183-6. · 8.30 Impact Factor
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[show abstract]
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ABSTRACT: Each day in every human, approximately 1 x 10(11) platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.
Journal of Thrombosis and Haemostasis 08/2009; 7 Suppl 1:227-34. · 5.73 Impact Factor
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ABSTRACT: Background: We have recently reported a novel mutation in the β3 subunit of the platelet fibrinogen receptor (αIIbβ3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated αIIbβ3D723H or αIIbβ3D723A salt bridge mutants, but not fully activated αIIbβ3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of αIIbβ3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant αIIbβ3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type αIIbβ3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO αIIbβ3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO αIIbβ3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.
Journal of Thrombosis and Haemostasis 06/2009; 7(7):1207 - 1217. · 5.73 Impact Factor
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[show abstract]
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ABSTRACT: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions.
Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation.
CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.
Journal of Thrombosis and Haemostasis 06/2009; 7(7):1207-17. · 5.73 Impact Factor
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A Tefferi,
A Pardanani,
K-H Lim,
O Abdel-Wahab,
T L Lasho,
J Patel,
N Gangat,
C M Finke,
S Schwager,
A Mullally,
C-Y Li,
C A Hanson,
R Mesa,
O Bernard,
F Delhommeau, W Vainchenker,
D G Gilliland,
R L Levine
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ABSTRACT: High-throughput DNA sequence analysis was used to screen for TET2 mutations in bone marrow-derived DNA from 239 patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). Thirty-two mutations (19 frameshift, 10 nonsense, 3 missense; mostly involving exons 4 and 12) were identified for an overall mutational frequency of approximately 13%. Specific diagnoses included polycythemia vera (PV; n=89), essential thrombocythemia (ET; n=57), primary myelofibrosis (PMF; n=60), post-PV MF (n=14), post-ET MF (n=7) and blast phase PV/ET/MF (n=12); the corresponding mutational frequencies were approximately 16, 5, 17, 14, 14 and 17% (P=0.50). Mutant TET2 was detected in approximately 17 and approximately 7% of JAK2V617F-positive and -negative cases, respectively (P=0.04). However, this apparent clustering of the two mutations was accounted for by an independent association between mutant TET2 and advanced age; mutational frequency was approximately 23% in patients > or =60 years old versus approximately 4% in younger patients (P<0.0001). The presence of mutant TET2 did not affect survival, leukemic transformation or thrombosis in either PV or PMF; a correlation with hemoglobin <10 g per 100 ml in PMF was noted (P=0.05). We conclude that TET2 mutations occur in both JAK2V617F-positive and -negative MPN, are more prevalent in older patients, display similar frequencies across MPN subcategories and disease stages, and hold limited prognostic relevance.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 03/2009; 23(5):905-11. · 8.30 Impact Factor
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Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 03/2009; 23(6):1186-90. · 8.30 Impact Factor
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B da Costa Reis Monte-Mór,
I Plo,
A F da Cunha,
G G L Costa,
D M de Albuquerque,
A Jedidi,
J-L Villeval,
S Badaoui,
I Lorand-Metze,
K B B Pagnano,
S T O Saad, W Vainchenker,
F F Costa
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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.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 11/2008; 23(1):144-52. · 8.30 Impact Factor
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F Bernard,
V Gelsi-Boyer,
A Murati,
S Giraudier,
V Trouplin,
J Adélaïde,
J Rey,
S Olschwang, W Vainchenker,
M Chaffanet,
N Vey,
M J Mozziconacci,
D Birnbaum
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2008; 23(3):583-5. · 8.30 Impact Factor
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R Chaligné,
C Tonetti,
R Besancenot,
L Roy,
C Marty,
P Mossuz,
J-J Kiladjian,
G Socié,
D Bordessoule,
M-C Le Bousse-Kerdilès, W Vainchenker,
S Giraudier
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ABSTRACT: MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G(1)/S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 07/2008; 22(8):1557-66. · 8.30 Impact Factor
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ABSTRACT: This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.
Bone Marrow Transplantation 04/2008; 41(6):579-84. · 3.75 Impact Factor
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[show abstract]
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ABSTRACT: Megakaryocytopoiesis is the process that leads to the production of platelets. This process involves the commitment of multipotent hematopoietic stem cells toward megakaryocyte (MK) progenitors, the proliferation and differentiation of MK progenitors, the polyploidization of MK precursors and the maturation of MK. Mature MK produce platelets by cytoplasmic fragmentation occurring through a dynamic and regulated process, called proplatelet formation, and consisting of long pseudopodial elongations that break in the blood flow. Recent insights have demonstrated that the MK and erythroid lineages are tightly associated at both the cellular and molecular levels, especially in the transcription factors that regulate their differentiation programs. Megakaryocytopoiesis is regulated by two types of transcription factors, those regulating the differentiation process, such as GATA-1, and those regulating proplatelet formation, such as NF-E2. The humoral factor thrombopoietin (TPO) is the primary regulator of MK differentiation and platelet production through the stimulation of its receptor MPL. Numerous acquired or congenital pathologies of the MK lineage are now explained by molecular abnormalities in the activity of the transcription factors involved in megakaryocytopoiesis, in the Tpo or c-mpl genes, as well as in signaling molecules associated with MPL. The recent development of MPL agonists may provide efficient agents for the treatment of some thrombocytopenias.
Journal of Thrombosis and Haemostasis 08/2007; 5 Suppl 1:318-27. · 5.73 Impact Factor
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ABSTRACT: The BCR-ABL oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias. Here, we report that ectopic expression of p210(BCR-ABL) in the megakaryoblastic Mo7e cell line and in primary human CD34(+) progenitors trigger erythroid differentiation at the expense of megakaryocyte (MK) differentiation. Clonal culture of purified CD41(+)CD42(-) cells, a population highly enriched in MK progenitors, combined with the conditional expression of p210(BCR-ABL) tyrosine kinase activity by imatinib identified a true lineage reprogramming. In both Mo7e or CD41(+)CD42(-) cells transduced with p210(BCR-ABL), lineage switching was associated with a downregulation of the friend leukemia Integration 1 (FLI-1) transcription factor. Re-expression of FLI-1 in p210(BCR-ABL)-transduced Mo7e cells rescued the megakaryoblastic phenotype. Altogether, these results demonstrate that alteration of signal transduction via p210(BCR-ABL) reprograms MK cells into erythroid cells by a downregulation of FLI-1. In addition, our findings underscore the role of kinases in lineage choice and infidelity in pathology and suggest that downregulation of FLI-1 may have important implications in CML pathogenesis.
Leukemia 06/2007; 21(5):917-25. · 9.56 Impact Factor
