Selectin Inhibition Disrupts Multiple Myeloma Cells Interaction with the Bone Marrow Microenvironment and Sensitizes Them to Therapy

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications.

Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 < MGUS, n=20 < smoldering MM, n=23 < MM, n=68 p<0.01). Moreover, it was shown that both BMSCs (isolated from MM patients and HS5 cell line) and ECs (isolated from MM patients and HUVECs) had high expression of P-selectin. SDF1 treatment increased the expression of P-selectin on ECs but it had no effect on the expression of PSGL on MM cells. The interaction of PSGL and P-selectin played a major role in the adhesion of MM cell to both BMSCs and ECs, and the inhibition of this interaction either by the pan-selectin inhibitor GMI-1070 (500uM, 3hrs) or by knock-down of P-selectin expression significantly decreased (50-60%) the adhesion of MM cells to BMSCs and ECs. The CXCR4 inhibitor AMD3100 (25uM, 3hrs) similarly induced similar inhibition of adhesion, and the combination of AMD3100 and GM1070 had more profound inhibition of MM adhesion to BMSCs and ECs (p = 0.006). Both AMD3100 and GMI1070 induced MM cell de-adhesion from BMSCs and ECs, but the combination of both drugs was not additive. AMD3100, GMI1070 or their combination prevented BMSCs or ECs mediated induction of proliferation of MM cells. Moreover, it was shown that the co-culture of MM cells with BMSCs and ECs reduced their sensitivity to bortezomib (5nM, 24hrs) and dexamethasone (25nm, 24hrs) compared to MM cells cultured alone. Importantly, GMI1070 restored the sensitivity of MM cells to bortezomib and dexamethasone to the level observed without co-culture with BMSCs or ECs. These effects were next tested in vivo using an orthotopic xenograft model of MM. SCID-beige mice were injected with luciferase-expressing MM1S cells, and tumor burden was assessed bioluminescence imaging. Mice with established disease were divided into treatment groups (n=10 per group) treated with vehicle, GMI1070 by osmotic pump, velcade at 1.5 mg/kg IP weekly, or a combination of GMI1070 and bortezomib. Tumor burden was determined by bioluminescence imaging. Treatment with GMI1070 alone was not different from vehicle treated control mice. While treatment with bortezomib alone had a minimal delay in tumor progression, the combined treatment of bortezomib and GMI1070 resulted in synergistic anti-tumor efficacy (p=0.0017).

We have shown that PSGL-1 is highly expressed in MM cells as compared to normal plasma cells, and that it plays a major role in the interaction of MM cells with the BM microenvironment in relation with the SDF1/CXCR4 axis in vitro, an effect which was inhibited by the pan-selectin inhibitor GMI1070. We also demonstrated that selectin inhibition by GMI1070 reduced MM cell proliferation induced by BMSCs and ECs sensitized MM cells to bortezomib and dexamethasone in vitro, and significantly increased the sensitivity of MM tumors to bortezomib in vivo. This information provides the rationale for future clinical trials for increasing efficacy of existing therapies through a combination with selectin inhibitors for the treatment of myeloma.

Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.