A major determinant in platelet production is the megakaryocyte (MK) size that is regulated both by ploidization and the increase in cytoplasmic volume at the end of maturation. Here we investigated the involvement of the mammalian target of rapamycin (mTOR) pathway in the regulation of megakaryopoiesis. We show that phosphorylation of mTOR, p70S6K1, and 4E-BP1 was diminished in thrombopoi IntroductionDuring megakaryocyte (MK) differentiation, the MK progenitor switches from a classic mitosis to an endomitosis corresponding to DNA replication without karyokinesis and cytokinesis. This process leads to a giant cell containing a single polylobulated nucleus with a 2N ploidy. The principal role of ploidization is to increase the MK size, particularly the volume of the cytoplasm, which may increase more strikingly than the ploidy level. As platelets arise from MK cytoplasm fragmentation, ploidization is an efficient manner to amplify the MK mass and thus platelet production.Thrombopoietin (TPO), the ligand for the Mpl receptor, stimulates the proliferation and differentiation of MK progenitor cells in vitro and in vivo and promotes their ploidization and their cytoplasmic maturation. Although TPO is not directly involved in proplatelet formation, this growth factor increases platelet production by augmenting MK number, polyploidization, and by inducing cytoplasm maturation. The binding of TPO to Mpl activates various types of intracellular signaling pathways that play important roles in the regulation of megakaryocytopoiesis, such as the Janus kinase (JAK)/ signal transducer and activator (STAT), Ras/Raf/mitogenactivated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI-3K), and protein kinase C (PKC) pathways. Indeed, activated STAT3 promotes the expansion of megakaryocytic progenitor cells, 1 the STAT5 and Ras pathways are involved in TPO-induced proliferation, and prolonged activation of the MAPK/extracellular signal-related kinase (ERK) pathway is required for TPO-induced megakaryocytic differentiation. 2 Activation of PKC-␣ is required for the process of proplatelet formation, 3 and TPO-induced PI-3K activity is essential for optimal cell survival and cell cycling (G 1 /S transition) of MK progenitors. 4 Protein kinase B (AKT) has been shown to be phosphorylated in normal human megakaryoblasts after stimulation by TPO in a PI-3K-dependent manner and to protect the cells from apoptosis. 5 One downstream target of PI3-K/AKT/Rheb is mTOR (mammalian target of rapamycin; also known as RAFT , FRAP [FKBP12-rapamycin-associated protein], or RAPT). 6-8 mTOR is a Ser/Thr kinase that regulates cell growth (cell mass and cell size) and cell-cycle progression through the G 1 /S transition, 9-11 2 processes shown to be coordinated in yeast, 12 Drosophila, 13,14 and mammals. 15 The binding of rapamycin (Rapa), an immunosuppressive drug, to its cellular receptor FKBP12 directly inhibits mTOR-dependent downstream signaling. mTOR regulates cell growth via 2 effector proteins critical for ribosomal biogenesis and translat...
Human T-cell Lymphotrophic Virus 1 (HTLV-1) is the etiologic agent of Adult-T cell Leukemia/Lymphoma (ATL). Therapeutic options for ATL patients are very limited and in aggressive forms of the disease survival rate is only 10% to 30% with conventional chemotherapies and bone marrow transplantation. Although some clinical trials gave encouraging results regarding the efficacy of new treatments, most of them are lifelong, aggressive and failed to achieve a significant impact on long-term survival. Consequently, new treatments for ATL patients are needed to limit relapses and side effects. Specific HTLV-1 cellular immune response is dramatically impaired in ATL patients, which could favor the initiation and the progression of the disease. Hence, stimulating immune responses against HTLV-1 can be an appropriate therapeutic option to treat ATL. THERAVECTYS has developed an anti-HTLV-1 vaccine, based on its lentiviral vector technology inducing a broad, intense and long-lasting cellular immune response after intra-muscular injection. THERAVECTYS was the first company to have launched a clinical trial based on lentiviral vectors technology with the THV01 vaccine for the treatment of HIV (NCT02054286). Results obtained demonstrated both safety and immunogenicity of THV01 in human, with polyfunctional and multi-specific CD4 and CD8 T-cells responses. The anti-HTLV-1 lentiviral vector, THV02 vaccine, encodes for a unique polypeptide derived from Tax, HBZ, p12I and p30II proteins, involved in HTLV-1 pathogenicity and known to be recognized by the immune system of HTLV-1 infected patients. Our preclinical results have demonstrated that THV02 can induce a cellular immune response in C57Bl/6j and BalbC mice and in Sprague Dawley rats, as demonstrated by IFN-γ Elispot. Safety of the THV02 vaccine has been demonstrated during carcinogenicity and regulatory GLP preclinical toxicity studies. Biodistribution and shedding studies demonstrated the very limited diffusion of THV02 after injection, its fast clearance and a non-dissemination in body fluids. As no relevant ATL immunocompetent animal model is available to assess the anti-tumor effect of THV02, THERAVECYTS is developing an ex-vivo efficacy model using blood samples of ATL patients. Briefly, monocyte-derived dendritic cells (MDDC) from blood of ATL patients are purified by isolation of CD14 positive cells from PBMC and differentiation in the presence of IL4 and GM-CSF. MDDC are then transduced with lentiviral vectors encoding for the anti-HTLV-1 antigen and maturation is induced upon TNFa and PGE2 exposure before the co-culture with autologous CD8+ T-cells for stimulation of the cellular immune response. Then, stimulated CD8+ are co-cultured with autologous CD4+ CD25+ ATL cells and the cytotoxic activity is monitored by flow cytometry. Preliminary results demonstrated that MDDC from a chronic ATL patient can be efficiently transduced and matured as attested by the CD40, CD86, HLA-DR, -A, -B and C markers on their surface. In addition, we have observed a specific stimulation of the CD8+, ie an increase of IFNg, TNFa, IL2 and perforin in the media of the co-culture of CD8+ with MDDC expressing anti-HTLV-1 antigen. These data are very encouraging and demonstrate for the first time the feasibility to develop an ex vivo model to assess vaccine efficacy using ATL blood sample. The development of this model is ongoing using several ATL donors representing the different subtypes of the disease and will be presented at the meeting. Regarding the indication and the safety profile of THV02, THERAVECTYS plans to begin a clinical trial in Q4 2015. This assay will be an open-label, dose escalation phase I/II study to assess the safety and the immunogenicity (cellular immune response) of the THV02 vaccination as a treatment of ATL patients. All ATL subtypes will be considered since THV02 vaccine can be combined with conventional ATL treatments. In addition, as the THV02 antigen contains peptides derived from Tax but also HBZ, p12I and p30II viral proteins, all ATL patients can be treated whatever the status of Tax expression. As secondary objectives, both humoral immune response and clinical effect will be assessed. HTLV-1 RNA expression and clonality of HTLV-1 infected cells will be studied as exploratory objectives. Finally, up to 16 patients will be enrolled in France, UK, French Guiana, Martinique and Guadeloupe before doing a phase of extension cohort in US. Disclosures No relevant conflicts of interest to declare.
Platelets are formed from mature megakaryocytes (MKs) and arise from the development of cytoplasmic extensions called proplatelets (PPT). Proliferation and full maturation of MKs require TPO, but it is dispensable for platelet shedding. To precisely define the role of different signaling pathway activated by TPO on proplatelet formation (PPF), chemical inhibitors of ERK (PD98059), p38 (SB 203580) and a pan PKC inhibitor (GF109203X) were added to MK derived from human CD34+ cells. As previously reported, the PKC inhibitor leads to a marked decrease in PPF and the p38 inhibitor has no effect on PPF. Addition at day 8 of an inhibitor of the ERK pathway (PD98059) to the purified CD34+CD41+ cultured cells leads to an early PPF associated to a 3-fold increase of PPT. As observed with PD98059, an overexpression of a dominant-negative (DN) form of MEK1 in primary MKs led to a 2-fold increase of PPT. Inhibition of the ERK pathway in the immature MKs surprisingly increased MK mean ploidy, (5.7N in the control versus 10.9N in presence of PD98059). Especially, the highest classes of ploidy (8N, 16N and 32N) were significantly increased. Altogether, those results suggest that the inhibition of MAP/ERK1/2 is an essential step for PPF in mature MKs but also that MAPK/ERK1/2 is a negative regulator of endomitosis. As PPF occurs in the presence of TPO, we investigated whether MAPK activation was down-regulated during MK differentiation. Western blot and flow cytometry analysis were performed and show that ERK activation induced by TPO was dramatically decreased at the late stages of megakaryocytopoiesis. In addition, inhibition of MEK prior to PPF was associated with an activation of caspase 3 in the absence of apoptosis suggesting a direct role of the ERK pathway in the regulation of proplatelet. Altogether, those results show a differential response of MAP kinase pathway to TPO depending on the maturation stage. ERK downregulation occurs when PPF begins and MEK inhibition is associated with increased PPF.
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