The CXCR4/SDF-1 Axis Regulates Migration and Adhesion in Waldenstrom Macroglobulinemia.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), and lymphadenopathy in 20% of the patients, implying continuous trafficking of WM cells into and out of the BM and lymph nodes. The normal process of B-cell homing is regulated by cytokines, chemokines, and adhesion molecules. One of the most extensively studied chemokines in migration is stromal derived factor SDF-1 and its receptor CXCR4. Here we study the role of chemokine receptors, and the SDF-1/CXCR4 axis on migration and adhesion in WM.

Methods: Flow cytometry for CXC and CC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6 and CCR7), and adhesion molecules (VLA-4 and LFA-1) on WM cell lines (BCWM.1 and WM-WSU) and patient samples was performed. Migration was determined using the transwell migration assay (Costar, NY). Cells were placed in the upper chambers of the migration assay with 1% FCS medium in the presence of serial concentrations of SDF-1 in the lower chambers. After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96-well plated coated with fibronectin. Immunoblotting for proteins downstream of CXCR4 was performed. The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO) and Gi protein inhibitor pertussis toxin PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling.

Results: The following chemokine receptors were expressed on patient CD19+WM cells with over 30% expression: CXCR1 (mean 60%), CXCR2 (mean 47%), CXCR4 (mean 47%), CXCR5 (mean 69%), CCR4 (mean 54%) and CCR6 (mean 61%). Similar expression was observed on WM cell lines. We next determined the effect of SDF-1 on migration and signaling pathways in WM. SDF-1 (10–100nM) induced migration in a bell-shaped curve with 30nM inducing maximum migration (110% compared to control). SDF-1 30nM induced a rapid activation of signaling pathways downstream of CXCR4 including pERK1/2, pAKT, and pPKC at 1 min, with maximum activation at 5min. The CXCR4 inhibitor AMD3100 inhibited migration of BCWM.1 in the presence of 30nM SDF-1, with AMD3100 10uM inhibiting migration at 59% of control, and 20 to 50uM leading to a plateau in inhibition of migration at 54% of control. AMD3100 inhibited pERK and pPKC activation, downstream of CXCR4 in a dose-dependent fashion. Similar results were observed using PTX, with inhibition of migration of WM cells at 50% compared to control. To determine the role of SDF-1 on adhesion, we first demonstrated that WM cells from patients and cell lines expressed high levels of surface VLA-4 expression (mean 95% surface expression). WM cells had an increase in adhesion to fibronectin (VLA-4 ligand) compared to BSA control. AMD3100 10uM inhibited adhesion to fibronectin (63 % of control), indicating that the SDF-1/CXCR4 axis regulates adhesion.

Conclusion: CXCR4 is highly expressed on WM cells and regulates migration and adhesion, indicating a potential role in regulating WM trafficking into the BM and lymph nodes. These studies provide the preclinical framework to study CXCR4 inhibitors in the regulation of homing and adhesion in WM.