Bone Marrow Interaction in Multiple Myeloma Pathogenesis: Phenotypical Analysis, Kinetics and Novel Therapy Approaches Based On CXCR4 Inhibition.

The interaction between malignant plasma cells and their microenvironment is central in multiple myeloma (MM) pathogenesis. Binding of MM cells to bone marrow (BM) stroma cells triggers the expression of adhesion molecules and secretion of chemo- and cytokines, promoting MM cell growth, drug resistance and migration. Stromal-derived factor-1 (SDF-1) and its receptor CXCR4 are essential for normal hematopoietic progenitor cell movement and adherence within the BM microenvironment. In leukemia and lymphoma, oncoproteins may inhibit SDF-1-dependent cell trafficking within the BM through a mechanism that is not fully understood. For that reason, understanding SDF-1-dependent cell trafficking within the BM and targeting MM-cell - host-BM interactions display a promising approach for the development of novel therapeutic strategies.

BM samples of MM patients (n=59) were analysed using flow cytometry and compared to MGUS patients (n=3) and healthy volunteers (n=7). We compared patient samples with low BM infiltration (≤5%; n=13) intermediate (5–30%; n=29) and high infiltration rates (≥30%; n=17). We also assessed expression of adhesion molecules in MM patients with long-term disease control (n=20) vs. both newly diagnosed (n=16) and symptomatic MM patients (n=23) as previously grouped by San Miguel et al. (Haematologica July 6,2012). We also sought to elucidate in vitro, whether specific anti-MM agents (bortezomib, vorinostat, pomalidomide, EGCG), with and without M210B4 stroma support, and with and without the CXCR4 inhibitor AMD3100, target the interaction of MM cells. Experiments were performed using MM cell lines (U266, RPMI8226, L363, NCI-H929), the control T-cell line MOLT-4 and MM-patient BM samples. Cell viability was assessed via Trypan Blue- and AnnexinV/PI-staining. CD138, CXCR4 (SDF1-receptor), CD49d (VLA-4), CD11a (LFA-1) and CD44 (HERMES antigen) expression was evaluated by flow cytometry and ScanR microscopy.

In BM samples of MM patients as compared to MGUS and healthy volunteers, the CXCR4/CD138- (p=.036), CD49d/CD138- (p=.0013) and CD44/CD138-expression (p=.0072) was significantly amplified and correlated with increasing BM infiltration rates (p=.001). Both newly diagnosed and symptomatic MM patients confirmed significantly increased CXCR4/CD138-, CD49d/CD138- (p=.0013) and CD44/CD138-expression as compared to patients with long-term disease control. Of note, in newly diagnosed patients, the expression of adhesion molecules was even more enhanced than in symptomatic myeloma patients, underlining their critical and future potential role as targets for novel therapeutics. Comparison of MM cell lines' adhesion and migration markers with that of MM-patient BM specimens revealed U266 as the cell line most closely resembling human specimens. Cytotoxic effects with use of MM cell lines and bortezomib, vorinostat and pomalidomide confirmed prior cytotoxic concentrations. Cocultivation with stroma substantially reduced apoptosis and induced tumor protective effects. Additional AMD3100 treatment restored sensitivity to bortezomib, vorinostat and pomalidomide. CXCR4 expression was substantially reduced after AMD3100 treatment, while that of CD49d, CD44 and CD11a remained widely unchanged. Toxic or therapeutic effects of AMD3100 monotherapy were excluded for used doses of 50μM. Additional use of ScanR microscopy visualized co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. ScanR microscopy results correlated with flow cytometry-determined CXCR4 expression. Ongoing analyses of both ScanR microscopy and flow cytometry will allow the detailed assessment of treatment studies with and without anti-MM agents and AMD3100.

Our findings underline the critical role of adhesion and migration molecules in MM and may pave the way for novel therapeutic approaches targeting these microenvironmental mediators.

No relevant conflicts of interest to declare.