Cbl Controls the Expression of Axl and Lyn Tyrosine Kinases Mediating Resistance to Nilotinib in Chronic Myeloid Leukemia Cells

Abstract 3734

The second generation of Abl tyrosine kinase inhibitor (TKI) nilotinib has been developed to overcome resistance to imatinib, which was up to now the gold standard treatment of chronic myeloid leukemia (CML). Although nilotinib has been approved recently as the front line treatment of CML, there are still some unresponsiveness nilotinib CML cells. The generation of nilotinib-resistant K562 CML cells led us to characterize the underlying mechanism regulating one of such cell resistance. We have previously shown that overexpression of the Lyn tyrosine kinase (TK) could explain this resistance both in vitro and in vivo (Mahon et al, 2008). Analysis by “Stable isotope labeling with amino acids in cell culture” (SILAC) further showed that this activity of Lyn is associated to overexpression of the TK Axl and the hyper activation of the Spleen TK Syk (Gioia et al, 2011). The inhibition step by step of these TK confirmed the relevance of each TK in the resistance to nilotinib. Similar mechanisms were detected in primary cells from nilotinib-resistant CML patients. Although the mechanism of deregulation of Lyn is currently unknown, is correlated to a transcriptional upregulation. The knockdown of Axl expression in nilotinib-resistant K562 cells (K562-rn shRNA Axl) overcomes resistance that correlates with a decrease of Lyn overexpression and an increase of c-Cbl expression. Interestingly, SILAC analysis also showed a down-regulation of several signaling proteins including the ubiquitin E3-ligase c-Cbl. Since c-Cbl targets several TK for lysosomal recycling or proteosomal degradation, we addressed whether c-Cbl down-regulation plays a role in Lyn and Axl protein accumulation. c-Cbl depletion in K562 cells (shRNA) induced a large increase of Axl and Lyn protein levels. In addition, the depletion of c-Cbl also induced an emerging resistance to nilotinib as assessed in viability and apoptosis assays. Conversely, the overexpression of c-Cbl in nilotinib-resistant K562 cells (K562-rn) dramatically reduced Axl and Lyn levels expression and re-sensitized K562-rn cells to nilotinib. These effects were dependent upon an intact E3-ligase activity of c-Cbl. Looking at c-Cbl mutations in K562 cells and in CML patients who overexpressed Lyn and Axl did not revealed any mutations. Altogether, these results suggest that c-Cbl down-regulation plays a critical role in the increase of Axl and Lyn levels that control cell resistance to nilotinib in CML cells.