Drug Transporters in Imatinib Resistance: A New Therapeutic Targeted Approach In CML

Abstract

5010 Chronic myeloid leukaemia (CML) is a mieloproliferative disorder characterized by the presence of the BCR-ABL gene fusion, which encodes an oncoprotein with a deregulated tyrosine kinase activity. This oncoprotein became the main therapeutic target of the disease. In fact, the first-line treatment is Imatinib, a specific tyrosine inhibitor that blocks BCR-ABL tyrosine kinase activity. Although, the good results with Imatinib, the development of drug resistance is a reality and can be mediated by different pathways, besides the BCR-ABL mutations, like the unbalance between influx and efflux drug transporters expression. Therefore, the knowledge of these changes would enable the use of new therapeutics approaches in CML. The aims of this study are to evaluate the role of drug influx and efflux transporters in Imatinib resistance and the potential therapeutic of Reversin 205 in overcoming the resistance to Imatinib. For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and generated two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. To obtain these resistant cells, K562 cells were exposed to the drug following two strategies: continuous exposure of increasing concentrations of Imatinib (K562-RC cells) and discontinuous exposure, with interchanged mediums with and without Imatinib (K562-RD cells). Drug influx and efflux transporters expression levels, namely OCT1, OCTN2, PgP and BCRP, were evaluated by flow cytometry (FC) using monoclonal antibodies labeled with fluorescent probes. Functional activity of PgP was assessed by a kinetic study using the radiosensitizer 99mTc Sestamibi. To evaluate the effect of Imatinib and Reversin 205, a P-gP inhibitor, on cell viability, all cell lines were treated in the absence and presence of different drug's concentrations and analyzed by the resazurin assay. Cell death was analyzed by optical microscopy (May-Grünwald staining) and by FC using the Annexin V and Propidium Iodide double staining and caspases expression levels. Our results show that the half maximal inhibitory concentration (IC50) of Imatinib in K562 sensitive cells is 75nM, whereas in K562-RC cells this value is increased eight times (to 605nM) and in K562-RD cells is around eighteen times higher (1389nM). On the other hand, we observed a decrease in OCT1 and OCT2 expression levels and an increase in PgP and BCRP expression in resistant cells compared with the sensitive cell line, which may contribute to the resistance to Imatinib. Besides the alterations in proteins expression levels we found, in resistant cell lines, a lower uptake profile of the radiosensitizer, demonstrating the functional activity of the MDR protein efflux transporter, Pgp. Concordantly with these results, we observed that when resistant cell lines were exposed to the combination of Reversin 205 (5μM), plus increasing concentrations of Imatinib, the IC50 decreased to 170nM in K562 RC and to 367nM in K562 RD. These results suggest that Reversin 205 may circumvent resistance to imatinib in CML cells. On the other hand, at higher doses, this compound revealed a cytotoxic effect in sensitive and resistant cells, and induces cell death by apoptosis in a time- and dose- dependent manner. The IC50 at 48 hours of exposure was of 12μM to K562, 18,9 μM to K562-RD and 18,7 μM to K562-RC cells. In conclusion, our results suggest that discontinuous drug exposure induces higher resistance levels and drug's transporters are involved in the acquisition of resistance to Imatinib. The use of Reversin 205 or other drug efflux transporters inhibitors might circumvent this resistance and could be used as a new potential therapeutic targeted approach in CML. Disclosures: No relevant conflicts of interest to declare.