Restoration of Rituximab Sensitivity in CD20 Negative B-Cell Lymphoma Cells; Epigenetic Change of CD20 Expression after Treatment of Rituximab.

Abstract

CD20 surface antigen is widely expressed on normal B-lineage cells and B-lymphoid malignant cells. Rituximab, which is a chimeric monoclonal antibody specifically recognizing CD20 surface antigen, works as a molecular targeting drug against CD20 positive lymphoid malignancies. Although combination chemotherapy with Rituximab has significantly improved the survival of CD20 positive lymphoma patients, CD20 negative tumor regression and transformation becomes a considerable problem. Recently, we have identified a patient with CD20(−) diffuse large B-cell lymphoma (DLBCL) transformed from CD20(+) follicular lymphoma after chemotherapies including Rituximab. Here we established RRBL1 (Rituximab resistant B-cell lymphoma 1) cells from this patient's CD20(−) B-cell lymphoma cells, and analyzed the mechanisms of negative CD20 surface antigen expression. CD20 was not expressed as determined by immunoblotting and FACS analysis, and resistance to Rituximab was observed after cell culture analysis with/without Rituximab. Expression of wild type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that CD20 mRNA expression level was almost 100 times lower than that of CD20 positive B-cell lymphoma cells obtained from DLBCL patients. These data suggest that lower expression level of CD20 mRNA is closely related to the negative CD20 surface antigen expression, and the aberrant transcription regulation by epigenetic mechanisms can be anticipated. With these data, we thought that CD20 expression could be restored by modulating the transcription regulation. As we expected, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression after the treatment with specific molecular targeting drugs, and the sensitivity for the Rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of co-repressor complexes to CD20 promoter might be related to the regulation of CD20 expression.Our data may provide us with a new therapeutic strategy, epigenetic therapy or differentiation therapy, combined with molecular targeting therapy using Rituximab, for Rituximab resistant CD20(−) B-cell malignancies.