AMD3100 disrupts the cross-talk between chronic lymphocytic leukemia cells and a mesenchymal stromal or nurse-like cell-based microenvironment: pre-clinical evidence for its association with chronic lymphocytic leukemia treatments

Abstract

Background: Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface.

Design and methods: Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model.

Results: AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies.

Conclusions: Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.

Figure 1.

AMD3100 decreases CXCR4 expression signal. MNC isolated from CLL patients were incubated with AMD3100 for 30 min and then incubated with anti-CXCR4 antibody and analyzed by flow cytometry. Percentage of CXCR4⁺ cells (A) and mean fluorescence intensity ratio (MFIR representing the CXCR4 signal/isotypic signal) (B) for normal B cells (n=8) or CLL cells (n=15) treated with increasing concentrations of AMD3100. (C) Representative histogram showing the decrease in CXCR4 expression signal after AMD3100 treatment. Each bar represents the mean ± SEM of 8–15 experiments.

Figure 2.

AMD3100 interferes with CLL cell trafficking. (A) Intracellular F-Actin was measured using FITC-labeled phalloidin in CD19-pre-labeled CLL cells after the addition of SDF-1α (100 ng/mL) at different time points without any drugs, in the presence of AMD3100 (5 μg/mL) or in the presence of pertussis toxin (200 ng/mL) as control. All time points are plotted relative to the mean fluorescence of the sample before addition of the chemokine. (B) CLL cells were pre-treated or not with AMD3100 for 30 min before being plated onto 5-μm Transwell microporous membranes for the migration assay. Results are expressed as the mean ± SEM migration index of 8 experiments. Migration index was calculated as the number of cells transmigrating in the presence of the chemoattractant divided by the number of transmigrating cells in the absence of the chemoattractant. (C) 5×10⁶ untreated or AMD3100-treated cells were added to stromal layers, and after a 3-h incubation, cells that had migrated to the stromal layer were counted as described in the Online Supplementary Appendix.

Figure 3.

AMD3100 restores apoptosis and decreases the viability of CLL cells in the presence of an MSC-based or NLC-based microenvironment or SDF-1α. MNC isolated from CLL patients were treated or not with AMD3100 and then cultured alone or with SDF-1α (A, D), MSC (B, E) or NLC (C, F), and apoptosis and cell viability were measured after 48 h. The rates in the figure include early and late apoptosis. Viability rates were obtained after normalizing the results to those for untreated cells. Each bar represents the mean ± SEM of 8–10 experiments. (G) MCL1 and FLIP expression were evaluated by flow cytometry in 4 patients in different conditions (with/without AMD3100; with/without NLC) and MFIR representing MCL1 or FLIP expression over isotypic signal were plotted for each case. A representative case of intra-cellular staining for MCL-1 and FLIP protein on CD19⁺ cells is provided for the different situations.

Figure 4.

AMD3100 sensitizes CLL cells to drug-induced apoptosis in a CLL/MSC co-culture model. MNC isolated from CLL patients were treated or not with AMD3100 and then cultured with or without various drugs. Apoptosis and cell viability were measured after 48 h, as described in the Design and Methods section. The rates in the figure include early and late apoptosis. The drug-induced apoptosis rates were obtained after subtracting the spontaneous apoptosis, while the viability rates were obtained after normalization to untreated cells. Each bar represents the mean ± SEM of 8 experiments.