Eph-B2 Receptor Tyrosine Kinase Is Overexpressed in Waldenstrom's Macroglubulinemia and Plays a Major Role in Its Interaction with the Bone Marrow Microenvironment.

Waldenstrom's macroglobulinemia (WM) is a low-grade lymphoma characterized by the presence of lymphoplasmacytic lymphoma cells in the bone marrow (BM). The BM microenvironment was shown to promote growth and proliferation of WM cells. The Eph receptor family are receptor tyrosine kinases RTKs activated by ephrin which a cell-surface protein, and the interaction between the receptor on one cell and the ligand on other cells promotes the activity of the receptor. Eph receptors are known to control processes such as cell growth, proliferation, migration, and invasion, and their expression level was shown to be elevated in a wide range of solid tumors. However, their role in WM was never explored.

Using phosphor-RTK array kit, we have screened the activity of 42 RTKs in CD19+ cells from 4 WM patients, WM cell line BCWM1, IgM secreting cell lines MEC1 and RL, and CD19+ cells from the BM of 4 healthy donors. We found that one of the most significant RTKs which showed high activation in all WM cells from patient sample and cells, compared to all normal cells was Eph-B2 receptor. We have used ephrin-B2, the ligand of the receptor Eph-B2, to test the signaling pathways involved in the activity of this receptor. We found, by immunoblotting, that ephrin-B2 induce phosphorylation of the receptor in a bell-curve manner, with the activity peaking in 100 ng/ml, while the expression of the total form of the receptor was unchanged over arrange of 0-1000 ng/ml of ephrin. A comparable activation was found for several cell-adhesion related proteins including FAK, SRC, paxillin, P130 and cofillin. These finding indicated a major role of the Eph-B2 receptor cell adhesion of WM cell. We have coated plates with increasing concentration of ephrin-B2 and tested the adhesion of BCWM1, MEC1 and RL to ephrin. Again, we found the adhesion peaked at the concentration of 100nM of ephrin. In contrast, ephrin-B2 had no chemotactic effect on WM cells. BM microenvironment components including BM stromal cells (BMSCs) and endothelial cells were shown to enhance the proliferation of WM cells detected by thymidine uptake assay. Ephrin-B2 was shown to be expressed on both endothelial cells and BMSCs. The inhibition of either the ephrin-B2 on endothelial cells and BMSCs, or inhibition of the Eph-B2 receptor on WM cell reduced the adhesion of WM cell to both endothelial cells and BMSCs and decreased the increase of WM cell proliferation induced by endothelial cells and BMSCs. The combination of both inhibition ephrin-B2 and Eph-B2 did not have an additive effect compared to each of them alone. Moreover, the inhibition of either ephrin-B2 or Eph-B2 reduced the activation of cell adhesion-related proteins.

In conclusion, we showed that the Eph-B2/ephrin-B2 axis was activated in WM cells and that it is important for the adhesion and proliferation of WM cells induced by the BM microenvironment. These findings provide a novel therapeutic target for WM.

Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.