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Mécanismes de résistance aux inhibiteurs de tyrosine kinase sur le modèle de leucémie myéloïde chronique

Abstract : Chronic myeloid leukemia (CML) is a malignant transformation of hematopoietic stem cell, characterized by an acquired genetic abnormality: the Philadelphia chromosome (Ph), resulting from the reciprocal translocation t(9; 22)(q34,q11), and its equivalent molecular the oncogene BCR-ABL. The BCR-ABL protein has a constitutive tyrosine kinase activity that acts on downstream survival and proliferation pathways. CML progression to accelerated and blastic phases is accompanied by additional genetic abnormalities markers of an increasing genomic instability. The responsibility of BCR-ABL in the genesis of this genomic instability is strongly suspected. Derived from 2-phenylamino-pyrimidine, Imatinib inhibits the BCR-ABL tyrosine kinase (TK) activity and the proliferation of BCR-ABL+ cell lines and induces apoptosis. Imatinib resistance is due to various mechanisms that are not fully characterized. New inhibitors of tyrosine kinase (TKIs) or associations with Imatinib have been developed to overcome Imatinib resistance. However, cross and multiple resistances remain difficult to treat and require a better understanding of resistance mechanisms to eradicate the disease. Our work showed that the presence of genetic abnormalities (indicating instability) in leukemic progenitor CD34+ cells of patients treated with Imatinib correlated with the loss of response to Imatinib in patients diagnosed in chronic phase, suggesting that Imatinib resistance may be correlated with genetic alterations that occur early during the disease development. 113 affected genes were detected, some of which, are located in areas known to be hypervariable (CNV), suggesting a role for these regions in the development of CML or Imatinib resistance. We have demonstrated the presence of a recurrent acquired abnormality in such region located in 8p23.1, which groups the genes of Defensin B family. This cryptic anomaly is not associated with Imatinib resistance since it is found in both cell population CD34+ Ph+ and Ph-. It can therefore be considered as a marker of instability of a cellular clonal sub-population of CD34+ presenting a high predisposition to acquire additional genetic alterations, as the t(9; 22)(q34,q11) translocation. Our studies show that secondary abnormalities may be the support for Imatinib resistance. Imatinib resistance may then be linked to deregulation of signaling pathways upstream or downstream of BCR-ABL, thus affecting proliferation, survival, differentiation and genome integrity. It seems that treatment with Imatinib alone is not sufficient to eradicate the disease. For this reason, new molecules associated with Imatinib are currently being evaluated. Among these approaches, inhibitors of Ras pathway have shown some effectiveness in restoring the proapoptotic activity of Imatinib. It was therefore interesting to search for therapeutic targets that regulate negatively this pathway to overcome Imatinib resistance. GILZ (Glucocorticoid-Induced Leucine Zipper) inhibits Ras/Raf pathway through direct interaction. It also inhibits NFκB pathway of which some antagonists are capable to overcome Imatinib resistance. Our results show for the first time that GILZ expression is decreased in human and mouse cell lines expressing BCR-ABL sensitive or resistant for TKIs and in dormant cells isolated from a mouse model developed in our laboratory. Enhanced GILZ expression in these resistant cell lines restores Imatinib sensitivity. Our results show that the mechanism involves inhibition of the mTORC2/Akt Ser473 pathway. It is due to direct interaction of GILZ with mTORC2 allowing activation of FoxO3a and induction of expression of the proapoptotic protein Bim. In this model, GILZ enhances Bim expression, while Imatinib decreases Mcl-1 expression by its Off-target effects, thus reversing the ratio antiapoptotic/proapoptotic of Bcl-2 family members and resulting in apoptosis. The sequential treatment with glucocorticoids and then Imatinib induced apoptosis of stem cells from resistant patients by the same mechanism, suggesting that the modulation of GILZ expression may represent a new therapeutic strategy to target the resistant or residual leukemic cells. Regulating the mTORC2/Akt Ser473 pathway seems to be crucial in BCR-ABL+ cell survival, as well as ITKs resistance. Among the methylated genes in our study, we find a gene involved in the regulation of this pathway and which codes for the phosphatase PHLPP (PH domain and Leucine rich repeat Protein Phosphatase). This phosphatase dephosphorylates serine 473 of Akt and promotes its inactivation. Our results show that the re-expression of variant 1 of this phosphatase (PHLPP1) restores Imatinib sensitivity and that the repression of PHLPP1 expression is not exclusively secondary to BCR-ABL activity but also to epigenetic modifications. Our studies have identified genes involved in CML resistance to TKIs and offer new treatments to overcome this resistance by blocking the AktSer 473 pathway
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Sami Joha. Mécanismes de résistance aux inhibiteurs de tyrosine kinase sur le modèle de leucémie myéloïde chronique. Sciences du Vivant [q-bio]. Université du Droit et de la Santé - Lille II, 2009. Français. ⟨tel-00451045⟩

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