Role of Selectins in the Pathogenesis of Multiple Myeloma.

Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role.

We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib.

In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma.

Patton:GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.