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

To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism.

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.

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.

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.

No relevant conflicts of interest to declare.