As Neoplasias Mieloproliferativas (NMPs) se caracterizam por apresentarem acúmulo de eritrócitos, leucócitos e plaquetas morfologicamente normais e seus precursores. Nos últimos anos vários estudos buscaram conhecer os mecanismos celulares e moleculares envolvidos na fisiopatologia e evolução dessas desordens, com o intuito de encontrar marcadores de diagnóstico, prognóstico e terapias eficazes. A mutação pontual no gene que codifica a enzima Janus Kinase 2 (JAK2 V617F), presente em aproximadamente 90% dos pacientes com PV e em 50% dos pacientes com TE e MF, foi o principal achado genético anormal associado a essas doenças. Essa mutação resulta na ativação constitutiva da enzima JAK2 e na desregulação da proliferação celular e resistência à apoptose. Nosso grupo de pesquisa descreveu em PV, TE e MF a expressão alterada de genes reguladores da apoptose e dados da literatura indicam que a desregulação do ciclo celular contribui para a fisiopatologia das NMPs. Nesse projeto o intuito foi investigar a associação da via de sinalização m-TOR com as alterações do ciclo celular e via JAK/STAT nas NMPs. A via de sinalização m-TOR participa dos processos celulares de sobrevivência e proliferação. A estratégia experimental foi avaliar a expressão de genes e proteínas, reguladores da via m-TOR, em leucócitos de pacientes com NPMC e linhagens celulares JAK2+ tratadas com inibidores de JAK2 e AKT. Para determinar a relação da via m-TOR nas NMPs foi escolhido o gene eIF4E, alterado nessas doenças, para observar sua modulação diante da inibição farmacológica nas linhagens celulares JAK2 positivas. Os resultados desse estudo contribuem para a descrição de novos alvos terapêuticos dependentes e indepentendes da atividade quinase JAK2 e para o melhor conhecimento da participação da via de sinalização m-TOR na fisiopatologia das NMPs. / The myeloproliferative neoplasms (MNPs) are characterized by accumulation of erythrocytes, leukocytes and platelets morphologically normal and their precursors. In recent years several studies have sought to understand the cellular and molecular mechanisms involved in the pathophysiology and progression of these disorders in order to find diagnostic and prognostic markers and effective therapies. The point mutation in the gene encoding the enzyme Janus kinase 2 (JAK2 V617F), present in approximately 90% of PV patients and in 50% of patients with ET and MF was the main abnormal genetic finding associated with these diseases. This mutation results in constitutive activation of JAK2 enzyme and the deregulation of cell proliferation and resistance to apoptosis. Our research group described in PV, ET and MF altered expression of apoptosis regulatory genes and literature data suggest that deregulation of the cell cycle contributes to the pathophysiology of MNPs. In this project the aim was to investigate the signaling pathway of the association m-TOR with the changes of the cell cycle and JAK / STAT in NMPs. The signaling pathway participates in the m-TOR cell survival and proliferation processes. The experimental strategy was to evaluate the expression of genes and proteins, regulators of m-TOR pathway in leukocytes from patients with and NPMC cell lines treated with the JAK2 JAK2 + and AKT inhibitors. To determine the relationship of m-TOR pathway with MNPs has been selected the eIF4E gene deregulated in these disorders to observe their modulation in pharmacologically inhibited cells lines JAK 2 positive. Results of this study contribute to the description of new therapeutic targets of dependent and independent JAK2 kinase activity and to a better understanding of the signaling pathway of participation m-TOR in the pathophysiology of NMPs.

Les néoplasies myéloprolifératives (NMP) sont des maladies hématologiques acquises de la cellule souche hématopoiétique (CSH) caractérisées par une prolifération accrue de cellules sanguines. Une mutation activatrice de la protéine JAK2 (JAK2V617F) a été identifiée chez plus de 50% des patients. La survenue de thromboses au niveau artériel et veineux constitue une des principales complications au cours de ces maladies. Au cours d’un premier axe de travail, nous avons souhaité étudier les conséquences sur la physiopathologie de la thrombose de la présence de la mutation JAK2V617F dans les cellules endothéliales (CE). Utilisant deux approches complémentaires, in vitro avec l’utilisation de cellules endothéliales veineuses, et in vivo avec l’utilisation d’un modèle murin permettant la présence de la mutation JAK2V617F dans le compartiment endothélial, nous avons démontré que la présence de CE JAKV617F favorisait la survenue de thrombose. Cette augmentation de la survenue de thrombose était médiée par une augmentation de l’adhésion des leucocytes au niveau des CE JAK2V617F, secondaire à une augmentation d’expression de P-Sélectine à leur surface. Ce travail a permis de mettre en évidence un nouveau mécanisme de la physiopathologie de la thrombose au cours des NMP JAK2V617F. Au cours d’un second axe de travail, nous avons étudié les polynucléaires neutrophiles (PNN) et le phénomène de NETose (formation de neutrophil extracellular traps ou NET) au cours des NMP. Nous avons démontré que la formation de NET était augmentée dans une cohorte de patients avec NMP, tout comme des marqueurs indirects de NETose (ADN plasmatique, MPO-ADN plasmatique). Nous avons mis en évidence que les taux de MPO-ADN plasmatique étaient plus élevés chez les patients avec antécédents de thrombose par rapport aux patients sans. Nous avons confirmé ces résultats à l’aide de deux modèles murins différents dans lesquels nous avons également retrouvé une formation de NET augmentée. Nous avons mis en évidence que cette formation de NET participait à la physiopathologie de la thrombose, l’administration de DNAse entraînant une réduction de la survenue de thrombose. Enfin, nous avons démontré que les plaquettes JAK2V617F stimulaient la production de NET par les PNN JAK2V617F, suggérant une collaboration de ces deux types cellulaires au cours de la physiopathologie de la thrombose des NMP. / Myeloproliferative neoplasms (MPN) are acquired hematologic diseases of the hematopoietic stem cell (HSC) characterized by blood cell proliferation. An activating mutation of the protein JAK2 (JAK2V617F) has been identified in more than 50% of patients. The occurrence of arterial and venous thrombosis is one of the main complications during these diseases. We first studied the consequences of the presence of the JAK2V617F mutation in endothelial cells (EC) on the physiopathology of thrombosis. We used two complementary approaches, in vitro with the use of human venous endothelial cells, and in vivo with the use of a mouse model allowing the presence of the JAK2V617F mutation only in the endothelial compartment: the PDGFb-iCreERT2;JAK2V617F/WT model. We first observed that PDGFbiCreERT2; JAK2V617F/WT mice displayed a higher propensity for thrombosis. This increased thrombus formation was mediated by an increased leukocyte adhesion in presence of JAK2V617F EC, secondary to an increase in P-Selectin expression at their surface. This work made it possible to highlight a new mechanism of the physiopathology of thrombosis during JAK2V617F MPN. Then we studied neutrophils and the phenomenon of NETosis (formation of neutrophil extracellular traps or NET) during MPN. We demonstrated that NET formation was increased in a cohort of patients with MPN, such as indirect markers of NETosis (plasmatic DNA and MPO-DNA). We found that plasma MPO-DNA levels were higher in patients with a history of thrombosis compared to patients without. We confirmed these results using two different mouse models in which we also found increased NET formation. We found that NET formation was involved in the physiopathology of thrombosis, as DNAse administration resulted in a reduction of thrombosis occurrence. Finally, we demonstrated that JAK2V617F platelets stimulated NET formation by JAK2V617F neutrophils, suggesting collaboration of these two cell types during the physiopathology of thrombosis during MPN.

As Neoplasias Mieloproliferativas (NMPs) se caracterizam por apresentarem acúmulo de eritrócitos, leucócitos e plaquetas morfologicamente normais e seus precursores. Nos últimos anos vários estudos buscaram conhecer os mecanismos celulares e moleculares envolvidos na fisiopatologia e evolução dessas desordens, com o intuito de encontrar marcadores de diagnóstico, prognóstico e terapias eficazes. A mutação pontual no gene que codifica a enzima Janus Kinase 2 (JAK2 V617F), presente em aproximadamente 90% dos pacientes com PV e em 50% dos pacientes com TE e MF, foi o principal achado genético anormal associado a essas doenças. Essa mutação resulta na ativação constitutiva da enzima JAK2 e na desregulação da proliferação celular e resistência à apoptose. Nosso grupo de pesquisa descreveu em PV, TE e MF a expressão alterada de genes reguladores da apoptose e dados da literatura indicam que a desregulação do ciclo celular contribui para a fisiopatologia das NMPs. Nesse projeto o intuito foi investigar a associação da via de sinalização m-TOR com as alterações do ciclo celular e via JAK/STAT nas NMPs. A via de sinalização m-TOR participa dos processos celulares de sobrevivência e proliferação. A estratégia experimental foi avaliar a expressão de genes e proteínas, reguladores da via m-TOR, em leucócitos de pacientes com NPMC e linhagens celulares JAK2+ tratadas com inibidores de JAK2 e AKT. Para determinar a relação da via m-TOR nas NMPs foi escolhido o gene eIF4E, alterado nessas doenças, para observar sua modulação diante da inibição farmacológica nas linhagens celulares JAK2 positivas. Os resultados desse estudo contribuem para a descrição de novos alvos terapêuticos dependentes e indepentendes da atividade quinase JAK2 e para o melhor conhecimento da participação da via de sinalização m-TOR na fisiopatologia das NMPs. / The myeloproliferative neoplasms (MNPs) are characterized by accumulation of erythrocytes, leukocytes and platelets morphologically normal and their precursors. In recent years several studies have sought to understand the cellular and molecular mechanisms involved in the pathophysiology and progression of these disorders in order to find diagnostic and prognostic markers and effective therapies. The point mutation in the gene encoding the enzyme Janus kinase 2 (JAK2 V617F), present in approximately 90% of PV patients and in 50% of patients with ET and MF was the main abnormal genetic finding associated with these diseases. This mutation results in constitutive activation of JAK2 enzyme and the deregulation of cell proliferation and resistance to apoptosis. Our research group described in PV, ET and MF altered expression of apoptosis regulatory genes and literature data suggest that deregulation of the cell cycle contributes to the pathophysiology of MNPs. In this project the aim was to investigate the signaling pathway of the association m-TOR with the changes of the cell cycle and JAK / STAT in NMPs. The signaling pathway participates in the m-TOR cell survival and proliferation processes. The experimental strategy was to evaluate the expression of genes and proteins, regulators of m-TOR pathway in leukocytes from patients with and NPMC cell lines treated with the JAK2 JAK2 + and AKT inhibitors. To determine the relationship of m-TOR pathway with MNPs has been selected the eIF4E gene deregulated in these disorders to observe their modulation in pharmacologically inhibited cells lines JAK 2 positive. Results of this study contribute to the description of new therapeutic targets of dependent and independent JAK2 kinase activity and to a better understanding of the signaling pathway of participation m-TOR in the pathophysiology of NMPs.