Overexpression of the EphB4 Receptor Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Through Regulating Mlcp and VAV1

Abstract Abstract 4421 Objective: To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism. Methods: We derived IM-resistant cells, K562-R cells, from wild K562 cells under gradually increasing IM concentrations. We analysed expression level of EphB4 in CML patients, wild K562 and K562-R cell lines by real-time reverse transcription PCR and Western blot analysis. Then we established stable under-expressing EphB4 cell (K562-R-EphB4-sh) lines. We analysed the sensitive for IM of K562, K562-R, K562-R-EphB4-sh cell lines by CCK8 assay. Microarray analysis was used to screen differential expression genes between K562-R and K562-R-EphB4-sh cell lines. Results: The mRNA and protein of EphB4 were significantly increased in IM resistant CML patients compared to IM sensitive CML patients (p<0.05). The Similar results were observed in K562-R and K562 cells (p<0.01). To analyze the role of EphB4 in IM resistance, EphB4 was knocked down with shRNA expressed by pLL3.7 lentivirus vector. We established stable under-expressing EphB4 cell line K562-R-EphB4-sh. RT-PCR and western blot analysis showed that mRNA and protein expression of EphB4 in K562-R-EphB4-sh cells were reduced (p<0.05). CCK8 assay found K562 cells (IC50 0.1207±0.0234μM), K562-R-EphB4-sh cells (IC50 0.7228±0.04752μM) were sensitive to IM but K562-R (IC50 2.8101±0.04674μM) still showed IM resistance (p<0.05). Those suggested K562-R-EphB4-sh cells resensitize to IM when the expression of EphB4 was down regulated. However, these cells were still less sensitive than K562 cells. Microarray analysis between K562-R and K562-R-EphB4-sh cell lines found 641 differential expression genes, most of them were related to cell adhesion and cell cytoskeleton. We confirmed MLCP and VAV1 were down regulated in K562-R-EphB4-sh cells compared to K562-R cell lines by western blot analysis. Conclusion: Our study suggest EphB4 receptor contributes to IM-resistant in CML through regulating cell adhesion molecular MLCP and VAV1, which may provide new biomarkers and contribute to] developping new drugs for the disease. Disclosures: No relevant conflicts of interest to declare.