Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

FrançOis-Xavier Mahon,ValéRie Lagarde,Sandrine Hayette,Serge Roche,Francis Belloc,CéDric Leroy,Franck Nicolini,Coralie Belanger,Paul W Manley,BéAtrice Turcq,Gabriel Etienne,Jean-Max Pasquet

doi:10.1158/0008-5472.can-08-1008

Abstract

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 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.

Highlights

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome (22qÀ) that results from a t(9;22)(q34;q11) reciprocal translocation [1, 2]
We tested the effect of imatinib on nilotinib-resistant cell lines, showing that the sensitivity to imatinib was modified in the nilotinib-resistant cell lines
The level of Bcr-Abl protein in the different nilotinib-sensitive and -resistant cell lines was studied by immunoblotting. Albeit both AR230 and LAMA84 cell lines did not express endogenous Abl, the nilotinibresistant cell lines showed a significant increase of Bcr-Abl compared with the sensitive parental line as we have previously described for imatinib, it was less pronounced

Summary

Introduction

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome (22qÀ) that results from a t(9;22)(q34;q11) reciprocal translocation [1, 2]. The unifying feature of all these Bcr-Abl fusion proteins is their deregulated protein tyrosine kinase activity that is responsible for leukemogenesis in vitro and in vivo [4, 5]. Imatinib mesylate (previously known as STI571) is a Bcr-Abl tyrosine kinase inhibitor that competes with ATP for binding to the Abl kinase domain and stabilizes the protein in its closed, inactive conformation, thereby inhibiting its activity [6, 7]. One of the main mechanisms of resistance in patients with CML is mutation in the Bcr-Abl tyrosine kinase domain. The second-generation tyrosine kinase inhibitors, have been developed to override the phenomenon [9, 10]

Methods

Results

Conclusion