Role of Stromal Microenvironment In Non-Pharmacological Resistance of CML to Tyrosine Kinase Inhibitors through Lyn/CXCR4 Interactions In Lipid Rafts.

Abstract 3390

In chronic myeloid leukemia (CML), the mechanisms of resistance to tyrosine kinase inhibitors (TKIs) beyond the Bcr-Abl mutations are not well understood. We have previously reported that TKI imatinib induces cell-surface expression of the chemokine receptor CXCR4, which results in enhanced migration towards CXCL12-producing BM stromal cells, promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). Bcr-Abl tyrosine kinase directly activates Src-related kinase Lyn known to frequently localize in lipid raft plasma membrane microdomains and interact with CXCL12/CXCR4 signaling and is directly activated by p210^Bcr-Abl. In this study, we investigated the effects of TKIs on the localization and interaction of CXCR4 and Lyn in the lipid rafts, and the role of lipid rafts as the signal transduction platform for CML cell migration. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that in CML cells CXCR4 primarily localized in the non-raft cell surface regions, while Lyn was present both in the lipid raft and non-raft fractions. In turn, the active, phosphorylated form (p-)Lyn^Tyr396 is present within the lipid rafts, while inactive p-Lyn^Tyr507 in non-raft fractions. Imatinib treatment under co-culture with mesenchymal stem cells (MSC) induced CXCR4 clustering in lipid raft fractions, which was directly co-immunoprecipitaed with Lyn. Under these culture conditions, imatinib repressed p-Lyn^Tyr507, but failed to deplete p-Lyn^Tyr396. Knock-down of Lyn by siRNA, Src inhibitor treatment, or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12 without affecting CXCR4 surface expression. Consistent with its effects on Src, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 compared with imatinib or nilotinib (p =0.04). In summary, our data indicate that stromal cells interfere with inhibitory effects of TKI on active Lyn (p-Lyn)^Tyr396 in CML cells and promote clustering of CXCR4 in lipid rafts where it co-localizes with p-Lyn^Tyr396 and facilitates migration of CML cells to the MSC monolayer. Lipid raft disruption by cholesterol depletion inhibit CML cells migration, suggesting that lipid rafts represent one of the key signaling modules responsible for interactions of CML cells with cells of BM niche. We propose that pharmacological disruption of lipid rafts may eliminate BM-resident CML cells through interference with microenvironment-mediated resistance.