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, secondgeneration inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance. [Cancer Res 2008;68(23):9809-16]

The nonreceptor tyrosine kinase Src is frequently overexpressed and/or activated in human colorectal carcinoma (CRC), and its increased activity has been associated with a poor clinical outcome. Src has been implicated in growth and invasion of these cancer cells by still not well-known mechanisms. Here, we addressed Src oncogenic signaling using quantitative phosphoproteomics. Src overexpression increased growth and invasiveness of metastatic SW620 CRC cells. Stable isotope labeling with amino acids in cell culture in combination with liquid chromatography tandem mass spectrometry allowed the identification of 136 proteins which exhibited a significant increase in and/or association with tyrosine phosphorylation upon Src expression. These mainly include signaling, cytoskeleton, and vesicular-associated proteins. Interestingly, Src also phosphorylated a cluster of tyrosine kinases, i.e., the receptors Met and EphA2, the cytoplasmic tyrosine kinase Fak, and pseudo-tyrosine kinase SgK223, which were required for its invasive activity. Similar results were obtained with metastatic Colo205 CRC cells that exhibit high endogenous Src activity. We concluded that Src uses a tyrosine kinases network to promote its invasive activity in CRC and this implicates a reverse signaling via tyrosine kinase receptors. Targeting these tyrosine kinases may be of significant therapeutic value in this cancer. [Cancer Res 2009;69(6):2279-86]