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.
IntroductionThe BCR-ABL-negative chronic myeloproliferative disorders (MPDs) include polycythemia vera (PV), essential thrombocytosis (ET), and primitive myelofibrosis (PMF). 1 These disorders are hematologic malignancies characterized by a clonal proliferation of one or several myeloid lineages. 2 PV, ET, PMF, and chronic myeloid leukemia (CML), defined as the classical MPDs, are considered to arise from the transformation of a multipotent hematopoietic stem cell. [3][4][5][6] However, the stem cell origin of the malignant clone had been demonstrated only in CML by the detection of either the characteristic t(9;22) translocation or the BCR/ABL transcript in all hematopoietic lineages. 7 More recently, detection of the JAK2 V617F mutation in a lympho/myeloid progenitor has been shown in some PV and PMF. 8 The JAK2 V617F mutation is found in almost all patients with PV, in 35% to 70% with ET, and in 50% with PMF. 9-13 Recently, we have detected the JAK2 V617F mutation in B and natural killer (NK) cells in approximately half of the PMF patients and in a minority of PV patients. Using the fetal thymus organ culture (FTOC) and B/NK/myeloid assays, we also have demonstrated that immature lympho/myeloid progenitors from PV and PMF carry the JAK2 V617F. 8 In 2006, 2 novel MPL somatic mutations (MPL W515L and MPL W515K) have been discovered in 5% and 1%, respectively, of JAK2 V617F-negative PMFs. The MPL W515L mutation confers to hematopoietic cells a cytokine-independent proliferation capacity, and results in a constitutive activation of JAK-STAT signaling. 14 The MPL W515K was later identified by sequencing granulocytes from 1182 MPD patients, but its precise effects on signaling are presently unknown. 15 W515 is the key amino acid located in a unique amphipathic domain that prevents spontaneous activation of MPL. 16 The substitution of W515 to an alanine or a serine has been shown to induce a factor-independent growth of the Ba/F3 cell line. 14,16 Therefore, it is expected that other amino acid changes in 515, such as the W515K, may induce a spontaneous MPL signaling.The growth factor-independent cellular proliferation of the megakaryocytic lineage was suggested to be based on the spontaneous homodimerization of MPL. [17][18][19][20][21][22] The viral oncogenic form of MPL, v-MPL, is characterized by the deletion of its extracellular domain. Its homodimerization results from the fusion of the truncated MPL sequence with the virus envelope. 18,23 Furthermore, introduction of a cysteine residue at codon 368 leads to a covalent disulfide-bonded homodimerization 19 and ligand-independent receptor activation. Similarly, the forced dimerization of MPL using monoclonal antibodies or the FK506-binding protein FKPB12 induces factor-independent proliferation. 24 W515 seems to play an important role in the prevention of basal MPL homodimerization. Indeed, the amphipathic domain of MPL, more precisely W515, prevents 2 receptors from coming close to each other in the absence of thrombopoietin (TPO). 16 In in vivo models, transpla...
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