Regulation of CD20 in rituximab-resistant cell lines (RRCL) and B-cell non-Hodgkin's lymphoma (B-NHL)
MetadataShow full item record
Reduced CD20 expression has been associated with decrease in rituximab activity in DeNovo and/or relapsed/refractory B-cell lymphomas. Using pre-clinical models, we found that the chronic exposure of lymphoma cells lead to a global down regulation of surface CD20 and rituximab resistance. Besides changes in CD20 expression, we demonstrated that rituximab resistant cell lines (RRCL) exhibit changes in the expression of Bcl-2 family members leading to cross-resistance to chemotherapy agents. In this thesis, we investigated the mechanisms responsible for CD20 down regulation in RRCL and to what degree changes in CD20 expression contribute to sensitivity to rituximab in B-cell lymphomas. Using RRCL and their precursors we found a gradual loss of CD20 surface expression that directly correlated with a decrease in rituximab associated complement mediated cytotoxicity (CMC) and determined a CD20 surface expression threshold necessary for an efficient rituximab CMC in vitro. Subsequently, we investigated mechanisms leading to CD20 mRNA and protein expression observed in RRCL. No changes in CD20 mRNA half-life were observed in RRCL. Of interest, CD20 promoter activity was lower in RRCL. Detailed analysis of different CD20 promoter fragments indicated that low CD20 promoter activity occurred within the first 300 base pairs, suggesting a lack of positive regulatory factor. No differences in PU.1 or Oct-2 levels were found between RSCL and RRCL. Exposure to interleukin-4 (IL-4) induced higher CD20 promoter activity, CD20 expression improved rituximab activity in RRCL and in primary tumor cells isolated from patients with B-cell lymphomas. CD20 transfection studies restored cytoplasmic but not surface CD20 levels suggesting the existence of a defect in the CD20 protein transport partly responsible for the down regulation of CD20 observed in RRCL. Transient CD20 knockdown in rituximab-sensitive cell lines showed only a 20% loss of rituximab-CMC. Together our data suggest that, while CD20 expression is important for rituximab activity, additional factors may affect rituximab responses in B-cell lymphomas. In addition, we identified two additional novel mechanisms responsible for CD20 downregulation in RRCL. The characterization of the mechanisms leading to rituximab resistance is most likely to lead to the development of novel therapeutic strategies to improve the clinical outcomes of patients with relapsed/refractory B-cell lymphoma in the post-rituximab era.