Vandetanib Drives Growth Arrest and Promotes Sensitivity to Imatinib in Hematologic Malignancies by Targeting Ephrin Type-B Receptor 4

Abstract Background: The oncogenic role of ephrin type-B receptor 4 (EphB4) has been reported in many types of tumors. However, little is known about its role in chronic myeloid leukemia (CML). Methods: A pool of clinically approved tyrosine kinase inhibitors (TKI) were screened for the antitumor activity using various hematologic cancer cell lines with different levels of EphB4 expression. To investigate whether EphB4 serves as a major therapeutic target in vandetanib-mediated inhibition on CML, the binding affinity of vandetanib to the active sites of extracellular and intracellular domains of EphB4 was evaluated using affinity chromatography and surface plasmon resonance. The binding regions were defined by molecular dynamics simulations and CRISPR/Cas9. Furthermore, the anticancer activity of vandetanib was evaluated by in vitro MTT assay and in vivo therapeutic study.Results: Here, Gene Expression Omnibus (GEO) data and blood analyses of CML patients revealed higher levels of EphB4 expression in CML patients than in healthy subjects. EphB4 knockdown inhibited K562 cell growth in vitro. In addition, transient transfection of EphB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EphB4 transfection. To develop an anti-EphB4 therapy with rapid clinical translation for CML, vandetanib from a pool of clinically approved TKIs were screened. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EphB4 protein level via ubiquitin-proteasome pathway and inhibited the signaling pathways of PI3K/AKT and MAPK/ERK in K562 cells. Vandetanib alone led to significant inhibition of tumor growth in a K562 xenograft model. More importantly, combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib-resistant K562 cells in vitro and in vivo. Conclusions: These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EphB4.