Rituximab Resistance Is Associated with Global Downregulation of CD20 and CD54 In B-Cell Lymphoma Cells: Implication of CD20 Signaling on CD54

Abstract Abstract 5077 Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration and aggregation. Pre-clinical studies have been conducted to investigate differences in the expression of adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of disease dissemination 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). It is unclear if the expression of adhesion molecules affects rituximab activity or if rituximab disrupts cell-to-cell interactions. To this end, we studied cell aggregation and expression of adhesion molecules in a panel of rituximab-senstive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that have been extensively characterized by our group (Czuczman MS. 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 Amnis ImageStream Technology analysis not only in RSCL and RRCL, but also in several intermediate passages obtained during the generation of RRCL process. To further define the role of CD54 in B-cell aggregation and rituximab activity, RSCL were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (mAb) (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy and photographs were captured at different time intervals. Cell death and down-stream signaling was evaluated at various times after rituximab treatment. Differences in the expression levels of CD54 correlated with CD20 levels in NHL samples. RRCL were found to have lower mean CD54 density. Gradual loss of CD54 was observed during repeated exposure to escalating doses of rituximab, indicating potential CD54 regulation through rituximab-CD20 signaling. No difference in the CD11a was observed between RSCL and RRCL. RSCL aggregate and form clusters under typical culture conditions whereas RRCL do not aggregate in vitro. Exposure of RSCL to rituximab induced 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 resulted in apoptosis of RSCL, suggesting 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 CD54-neutralized RSCL. In summary, we observed a loss of CD54, CD20 and cell aggregation during the process of acquiring resistance to rituximab. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cross-resistant cell lines and may disrupt signaling events thereby contributing to resistance to rituximab and chemotherapy drugs. Disclosures: No relevant conflicts of interest to declare.