Colavita, Irene (2010) Unravelling the mechanisms of resistance to imatinib mesylate in chronic myeloid leukemia: a proteomic approach. [Tesi di dottorato] (Inedito)

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Abstract

Imatinib mesylate is a potent inhibitor of the Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. The aim of this study was to obtain further insights into the Bcr-Abl activity-independent mechanisms underlying imatinib resistance, in chronic myeloid leukemia. The imatinib-resistant KCL22R and sensitive KCL22S cells were used as experimental model. None of the already described resistance mechanisms has been detected so far in KCL22R cells; therefore additional mechanisms independent of Bcr-Abl kinase activity could be envisaged. Moreover, KCL22S cells exhibited typical features of the quiescent hematopoietic Ph+ stem cells, thereby representing a good experimental model to investigate imatinib resistance. To this aim differentially expressed proteins between KCL22S and KCL22R cells were characterized using a proteomic approach: two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Tandem Mass Spectrometry. 51 proteins were identified: 27 over-expressed and 24 under-expressed in KCL22R cells versus KCL22S cells. Bioinformatic analysis with GeneSpring and Ingenuity Pathway Analysis (IPA) softwares showed that several of these proteins were involved in the modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kB and Ras-MAPK signaling. Since the Erk pathway has been shown to influence chemotherapeutic drug resistance of hematopoietic cells, the level of activation of Erk in KCL22R and KCL22S cells was investigated. This analysis demonstrated that continuous activation of Erk occurred in KCL22R cells as compared to sensitive cells. Interestingly, examination of the most statistically significant protein network showed that several differentially expressed proteins, between KCL22R and KCL22S cells, were directly or indirectly connected with Erk. In particular, among them, this study focused on two SH2-containing, non-receptor protein tyrosine phosphatases: Shp1 (PTPN6) and Shp2 (PTPN11). It has been shown that Shp2 positively regulates the Ras-Erk pathway and is activated by phosphorylation. This study demonstrated that the level of phosphorylation and hence of activation of Shp2 in KCL22R cells was higher than in KCL22S cells. In addition the knock-down of Shp2 expression, in combination with imatinib treatment, significantly reduced the activation of Erk 1/2 in KCL22R cells and produced a reversion of the KCL22R phenotype, suggesting that Shp2 plays a role in the Bcr-Abl activity-independent mechanisms of imatinib resistance. Interestingly this study also demonstrated that Shp1, that was found down-regulated in KCL22R cells, interacted with Shp2 and that Shp1 played a negative role in the Shp2 activation in KCL22S cells. Moreover Annexin A1 and Hsp70, belonging to the same protein network, were found down-regulated in KCL22R cells. They could also play a role in imatinib resistance by the direct or indirect interaction with Shp2. Taken together these results suggest that a reduced Shp1 expression in KCL22R cells could contribute to a continuous Shp2 activation, sustaining a Bcr-Abl activity-independent pathway of proliferation and survival to imatinib treatment. These two proteins could be used as putative biomarkers to evaluate the efficacy of imatinib treatment and to develop new combinatorial therapeutic approaches.

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