Structural Changes in the Internal Domain of CD20 Antigen Associated with the Emergence of Rituximab Resistance: Effects of Proteasome Inhibition in CD20 Structure and Rituximab Anti-Tumor Activity in Rituximab-Resistant Cell Lines (RRCL).
Authors: Hernandez-Ilizaliturri Francisco J. , Olejniczak Scott H. , Knight Joy , Czuczman Myron S. 
Published: April 2020

Abstract
Background: The acquirement of resistance to rituximab has been observed 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 in RRCL. In addition we previously reported upregulation in gene/protein expression of members of the ubiquitin-proteasome system in RRCL. Recently, we evaluated the effects of proteasome inhibition in the structure of CD20 antigen and rituximab sensitivity in RRCL.
Methods: RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or with human complement (4RH). Functional assays were performed to demonstrate a decrease in rituximab-associated activity. 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 treatment, lipid raft domains were extracted from Raji and RRCL. Redistribution of CD20 antigen following rituximab exposure was evaluated by Western blotting. RRCL were exposed to 0, 20 and 100nM of Bortezomib for 24 and 48 hrs. Changes in the internal domain of CD20 were evaluated by Western-blotting. Rituximab responses following Bortezomib exposure were determined by flow cytometric analysis in RRCL exposed to rituximab or isotype (10μg/ml) +/− human serum (dilution 1:4).
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, a significant decrease in expression of the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. Redistribution of CD20 into the lipid raft domains was decreased in RRCL following rituximab exposure. In vitro exposure to Bortezomib for 24 and 48 hrs resulted in an increased expression of the C-terminal region of the internal domain of CD20 in RRCL. In addition, Bortezomib-exposed RRCL regained partial sensitivity to rituximab-associated complement mediated cytotoxicity.
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, decreases the redistribution of CD20 antigen into lipid raft domains and negatively impacts rituximab anti-tumor activity. The C-terminal region of CD20 expression is modulated by exposure to Bortezomib. Proteasome inhibition partially improves rituximab responsiveness suggesting the existence of mediators of rituximab-resistance.