Structural changes in the internal domain of the CD20 antigen is associated with the development of rituximab resistance: Effects on signaling and redistribution of CD20 into lipid raft domains in rituximab-resistant cell lines (RRCL)

Abstract

2502 Backgound: Following rituximab binding, CD20 antigen redistributes into lipid raft domains (LRD) and initiates signaling events leading to apoptosis in malignant B-cells. The development of resistance to rituximab has been observed clinically in lymphoma patients. To define the molecular basis for rituximab resistance we developed various RRCL and studied changes in CD20 structure, membrane reorganization and signaling events following rituximab therapy. Methods: RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or with human complement (4RH). Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Following rituximab exposure, LRD were extracted from Raji and RRCL. Redistribution of CD20 antigen and signaling events following rituximab exposure were evaluated by Western blotting. Results: No significant changes were observed in the expression of the external domain of CD20 antigen between rituximab-sensitive and RRCL, as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. While redistribution of CD20 into the LRD was similar between Raji cells and RRCL, a decrease in p38 expression was observed following rituximab therapy on RRCL. Conclusions: Our data strongly suggests that the acquirement of rituximab resistance is associated with significant changes in the structure of the internal domain of the CD20 antigen. The abnormal production of truncated forms of the CD20 antigen, while not affecting rituximab binding or redistribution of the CD20 antigen into LRD, results in a decrease in signaling events. Impairment of anti-CD20 signaling activation theoretically renders the cells resistant to rituximab. No significant financial relationships to disclose.