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.

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.

Protein kinase D-dependent CXCR4 down-regulation upon BCR triggering is linked to lymphadenopathy in chronic lymphocytic leukaemia.

In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser324/325. Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro. In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

Down-regulation of CXCR4 and CD62L in chronic lymphocytic leukemia cells is triggered by B-cell receptor ligation and associated with progressive disease.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases.