EphB4/EphrinB2 Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Involved in Cytoskeletal Proteins

Background and Objective: Through the introduction ofthe tyrosine kinase inhibitor(TKI) significantly improves the prognosis, drug resistance is still a major obstacle to cure chronic myelogenous leukaemia (CML).Many documents have suggested aberrant activation of EphB4/ephrin B2 participate in imatinib-resistant CML patients but the role and mechanism have largely remained elusive. Our previous study found that Homoharringtonine overcome imatinib resistance by blocking the EphB4/RhoA pathway in K562. Then, we established IM resistance CML cell line (K562-R) and EphB4 knock down IM resistance CML cell line (K562-R-EphB4-sh). Furthermore, we performed the experiment to address the hypothesis that aberrant over expressed of EphB4 might play an important role in of Imatinib-resistant chronic myeloid leukemia cells.

Methods and Results: EphB4 receptor was over expressed in CML -Blast Crisis (BC) patients and resistant cell lines by western blot (P <0.05) and mRNA(P <0.01).We evaluated the imatinib response in 22 CML-CP patients (400mg/d or 600mg/d).EphB4 mRNA levels in cancer cells correlated with the corresponding BCR-ABL transcript level of at 3 months in each case (y=24.461x-2.3491 R² =0.8756, P<0.0001).These patients were then equally divided into two groups according to relative EphB4 mRNA median level (0.979217).The level of EphB4 receptor expression was significantly associated with 12-month CCyR(The 12-month CCyR rate in low EphB4 group was 72.7% VS36.3% in high EphB4 groups (P<0.01)).Cell invasive ability decreased in K562-R-EphB4-sh cells (K562-R13.56±2.70;K562-R-EphB4-sh 5.78±2.54 n=3) meanwhile cells restored sensitivity(IC50μM: K562-R 2.7621±0.0154; K562-R-EphB4-sh0.6908±0.0623; K562 0.1179±0.0379) to IM in vitro and in vivo (tumor volume after IM treatment (mm³):K562-R 2301.25±555.76; K562-R-EphB4-sh1630.16±412.01; K562 806.15±202.31);The apoptosis rate of K562-R-EphB4-sh (25.27±1.27)had been showed much higher than that ofK562-R (7.23±1.97). In addition, the proportion of cells in G0/G1 phase was significantly decreased in K562-R-EphB4-sh cells (13.58%)than K562-R( 29.30%), but higher than K562( 6.37%).When co-cultured cells with ephrinB2 ligand, restored sensitivity to IM was observed in K562-R cells( IC50μM : ephrinB2-Fc1.071±0.039; IgG-Fc2.697±0.145; Blank control 2.663±0.102), apoptosis rate (ephrin B2-Fc(42.72 ± 0.95) %;was higher than IgG Fc group (28.77 ± 1.64) % or the control group (28.93 ± 1.49) % (P <0.001) with group treated with IM (2.7 µg/ml, 24 hours), along with significantly increased level of phospho-EphB4 (450nm absorbance:ephrinB2-Fc0.920±0.031;IgG-Fc0.379±0.008; blank control0.381±0.005) and decreased phosphorylation level of cytoskeletal proteins RhoA, Rac1, Cdc42(P <0.05).

Conclusion: Our study illustrated that aberrant activation EphB4/ EphrinB2 may mediate chronic myeloid leukemia resistance involved in cytoskeletal proteins.