Résumé
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 of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress.
In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.