The Acquirement of Rituximab Resistance Is Associated with a Global Downregulation of CD54 in B-Cell Lymphoma Cells: The Potential Role of Adhesion Molecules in Rituximab Anti-Tumor Activity

Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration, aggregation, or other cell functions. Preclinical studies have been conducted to investigate the expression profiles of different adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of spread between non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Of interest, CLL cells have lower levels of both adhesion molecules and CD20 when compared to follicular lymphomas (FL). Recently, knockout studies had demonstrated that CD26, an adhesion molecule, modified responses to chemotherapy in B-cell lymphomas. It is unclear if the expression of adhesion molecules affects rituximab activity. To this end, we studied the patterns of cell aggregation and expression of adhesion molecules in a panel of rituximab-sensitive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that had been extensively characterized by our group (Czuczman S. et al. Clin Cancer Res.2008; 14:1561–70). Homotypic adhesion of B-cells is known to, due to the interaction of ICAM-1(CD54) and LFA-1(CD11a). Expression of CD54 and its ligand CD11a was studied by flow cytometry analysis and polymerase chain reaction (PCR, CD54 only). Patterns of cell aggregation in RSCL and RRCL in resting conditions were studied by inverted light microscopy. To define further the role of CD54 in B-cell aggregation and rituximab activity, RSCL (Raji and RL cells) were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy, and photographs were captured at different time intervals. Experiments were conducted with or without the potent pan-caspase inhibitor Q-VD-OPh and performed in triplicates. Cell death was detected by propidium iodine staining and quantified by flow cytometry. Differences in the expression levels of CD54 were observed in the NHL cells tested. RRCL were found to have lower levels of CD54 at the surface protein and gene level. No differences in the CD11a were observed. RSCL aggregate and form clusters under culture conditions whereas RRCL do not aggregate in vitro. In vitro exposures to rituximab lead to a rapid cell clustering in RSCL. Blocking CD54 using mAbs prevented spontaneous and rituximab induced cell clustering, resulting in a phenotype similar to the RRCL. Of interest, in vitro exposure to anti-CD54 mAb and to a lesser degree rituximab resulted in apoptosis of RSCL, suggesting that cell adhesion is important for survival in B-cell lymphomas. The decrease in cell aggregation following CD54 blocking was not reduced by inhibition of caspase activation suggesting that cell death was not the dominant factor in preventing cell clustering in RSCL. In summary, our data suggests that CD54 is important for B-cell lymphoma cell aggregation and survival. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cell lines and may disrupt signaling events that control cell proliferation (i.e. pro- or anti-apoptotic proteins) rendering these cells resistant to rituximab and chemotherapy drugs. Ongoing studies in lymphoma severe combined immunodeficiency mice (SCID) are underway to further define the role of CD54 in the progression of B-cell lymphomas and responses to rituximab activity in vivo.