Publication: Nuevas dianas terapéuticas en neoplasias mieloproliferativas philadelphia negativas
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2013-02-06
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Universidad Complutense de Madrid
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Abstract
JAK2 is a non-receptor tyrosine kinase that acts as an important signal transducer in cytokine signalling, promoting growth, survival and differentiation of various cell types. A somatic point mutation in JAK2 leading to the expression of the JAK2 V617F mutant occurs with high frequency in myeloproliferative neoplasm (MPN) patients (>95 % in polycythemia vera (PV), >50 % in essential thrombocythemia (ET) and primary myelofibrosis (PMF). While clinical trials treating MPN patients with JAK inhibitors have shown positive results, success has primarily been due to improved patient symptomatology with one clinical trial also showing an increased survival rate but a limited reduction of the tumour clone. Therefore additional advances in understanding MPN cell biology could be utilized to further improve clinical outcomes and treatment optimization. The aim of this work was to investigate genes and proteins involved in the physiopathogenic events and phenotype divergence between PV and ET, in order to obtain a better knowledge of MPN and for a future application as diagnostic tools or therapeutic targets. To this end, we performed a gene and protein screening looking for differences between ET and PV at different levels: gene expression microarray epigenetic profile (methylation microarray) and protein expression (by GEL 2D-DIGE/MS). This approach has shown several genes with a diferential expression rate that allow us to select several inhibitors targeted against their product proteins. Then, we determine in cell models and primary cells of patients diagnosed with Philadelphia negative myeloproliferative neoplasms (MPN), the antitumor effects, mechanisms of action at the molecular level and potential synergies with Ruxolitinib of the tyrosine kinase inhibitors selected: Dasatinib, Sorafenib, perifodine and KNK437, HSP70 inhibitor The cytotoxic and cytostatic effects of these different compounds were identified in JAK2V617F positive cell lines, in methylcellulose cultures of mononuclear cells and in stem cell (CD34 positive) cells cultures from bone marrow. Ruxolitinib, Sorafenib, and KNK437 were able to inhibit proliferation in cell line models and in cells from patients NMP. Furthermore, the cytotoxic effect on CD34 positive cells from patients was significantly higher than the presented by CD34 control culture. Dasatinib, Sorafenib and KNK437 showed a strong synergistic effect in combination with Ruxolitinib. By western blot we confirmed that Sorafenib inhibits activation of ERK and p38, and consequently, STAT5, Ruxolitinib blocks the activation of ERK and STAT5 and Dasatinib inhibits the activation of SRC and STAT5.
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Medicina Interna, leída el 19-11-2012