EphB4/ephrinB2 Interaction Mediate Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Spreading and Osteogenic Differentiation

Background and Objective: Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment.Recent studies have indicated interaction between acute leukemia cells and MSCs has a major role in cancer progression and resistance to treatment.Our previous study found that EphB4 receptor was over expressed in CML-Blast Crisis (BC) patients and resistant cell lines. Furthermore, we performed the experiment to prove that aberrant over expressed of EphB4 play an important role to change characterize of Imatinib-resistant in chronic myeloid leukemia cells. However,the contribution of over expressed of EphB4 molecules in leukemia cells to change MSCs function remains to be determined. Therefore,we hypothesis that the change of EphB4/ephrinB2 molecules on leukemia cells might play an important role to transform characterize of MSCs through direct contact, which finally support to leukemia progression and disruption of normal hematopoiesis in microenvironment of the bone marrow.

Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or patients' BM and cultures in CyagenBone marrow culture medium at 37 °C, 5% CO2 incubator. EphrinB2(2.628±0.2303 n=3; P<0.05), ALP\RUNX2 (early osteogenesis differentiation genes)(6.430±0.1343, n=3P<0.001; 4.948±0.1418,n=3P<0.001)were over expressed in MSCs (CML patients)in contrast to normal human MSC by QRT-PCR. After osteogenic induced for 2 weeks,MSCs from CML-initial patient showed significantly higher osteogenic differentiation (Osteogenesis Score 4.5P<0.01) and protein (later period osteogenesis differentiation)(2.1669%±0.1443, n=3P<0.001)was overexpressed in MSCs (CML patients) in contrast to normal human MSC(0.2993%±0.1612n=3) by western blot. In functional spreading assay, cultured MSC (CML patient) exposed to EphB4-Fc (5 μg/mL) were significantly rounder (p<0.001) and smaller (p<0.001) as demonstrated with F-actin staining, compared to control and human-Fc.No difference inmorphology was observed when MSC were cultured in the presence of ephrin-B2-Fc.Incubation of MSC with signaling pathway-specific inhibitors before the spreading assay, the PI3Kinase pathway (LY294002) (p<0.001),not the Src kinase pathway (PP2), inhibited MSCs (CML) attachment and spreading. The results revealed that the PI3Kinase pathway was activated upon ephrinB2 reverse signaling in response to EphB4-Fc to promote the contraction and rounding up of MSC. Activation of ephrin-B2 molecules expressed by MSCs (CML)by EphB4-Fc(5 μg/mL), human-Fc(5 μg/mL) or blank control, which cultured in osteogenic media for 2 weeks, more mineral in the presence of EphB4-Fc was visualized by Alizarin Red staining compared to the control human-Fc.Furthermore, after co-cultured respectively with K562-R or K562-R-EPHB4-SH for 72h,ALP\RUNX2 in MSCs (CML) were increased significantly in K562-R group(15.544±2.647; 6.378±2.0775 n=3)compared to K562-R-EPHB4-SH group (6.014±1.19273; 4.045±2.0273 n=3) and control group by QRT-PCR. However, ALP\RUNX2 in normal MSC was unaffected when respectively co-cultured with K562-R(0.741±2.2121; 0.129±0.9194 n=3) or K562-R-EPHB4-SH(0.171±2.062; 0.232±2.0474 n=3) for 72h. The co-cultured assays were also performed using transwells. ALP\RUNX2 in MSCs (CML) were no difference in K562-R group (6.28±1.7875; 4.232±2.4533 n=3) compared to K562-R-EPHB4-SH group (6.107±2.158; 4.139±1.9727 n=3).

Conclusion: Our study illustrated that the change of EphB4/ephrinB2 molecules on leukemia cells may transform MSCs functional spreading and osteogenic differentiation through direct contact involved in PI3Kinase pathway.