Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.

Abstract 2175

Poster Board II-152

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl.