MS4A1 (CD20) Gene Expression Is Down-Regulated by Recruiting the Histone Deacetylase Protein Complex to the Promoter in the CD20-Negative B-Lymphoma Cells After Treatment with Rituximab.

Rituximab, a mouse/human chimeric-monoclonal antibody, is now one of the critical molecular targeting drugs for treatment of CD20-positive B-cell lymphomas. Although the survival benefit of rituximab has been proved for several types of CD20-positive B-cell malignancies, resistance to rituximab has also become a considerable problem. Very recently, we reported that down-modulation of CD20 protein expression in CD20-positive B-cell lymphoma patients after treatment with rituximab-containing combination chemotherapies had been observed in 26.3% of re-biopsied patients under relapsed/progress disease (RD/PD) condition (Hiraga et al., 2009, Blood). Interestingly, CD20 expression and the rituximab sensitivity were partially restored by some epigenetic drugs in vitro, suggesting that aberrant down-regulation of MS4A1 gene expression by epigenetic mechanisms may be related to the loss of CD20 protein expression.

Analyses of the molecular mechanisms of MS4A1 gene down-regulation after treatment with rituximab-containing chemotherapies.

Primary B-lymphoma cells and RRBL1 cells (Hiraga et al., 2009, Blood; Tomita et al., 2007, Int J Hematol.), that showed CD20-negative phenotype after using rituximab, were analyzed in these assays. CD20 mRNA and protein expression was partially stimulated by decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, and the expression was enhanced by trichostation A (TSA), a histone deacetylase (HDAC) inhibitor. Immunoblot analysis indicated that DNMT1 expression was once down-regulated one day after treatment with DAC, and reversed within 3 days. On the other hand, IRF4/Pu.1, the transcription regulators of MS4A1 gene expression, were consistently present with or without DAC. Bisulfite sequencing was performed to check the CpG methylation status of MS4A1 gene promoter, with the result that no significant methylation was confirmed in CD20-negative transformed cells without DAC. Chromatin immunoprecipitation (ChIP) assay indicated that Sin3-HDAC1 co-repressor complex was recruited to MS4A1 gene promoter without DAC/TSA. In the presence of those drugs, Sin3-HDAC1 recruitment was dissociated from the promoter and the histone acetylation of the promoter was confirmed. Under these conditions with/without DAC/TSA, IRF4 and Pu.1 were constantly recruited to the promoter. Immunoprecipitation using whole cell lysate of RRBL1 cells indicated that endogenous Sin3-HDAC1 forms a protein complex, but IRF4 and/or Pu.1 interaction with the complex was not confirmed under this condition. To explore the critical factors for CD20 transcription regulation, expression-profiling assay using cDNA micro array was performed. mRNA from RRBL1 cell with/without DAC/TSA was harvested, and expression profiles were compared. In the presence of DAC or DAC+TSA, 0.7% and 7.0%, respectively, of genes were significantly activated. We are now analyzing some candidates that are critical for the transcription regulation of MS4A1 gene expression.

Our data indicate that the transcription repression of MS4A1 gene in the CD20-negative phenotypic change after treatment with rituximab is, in part, introduced by the recruitment of Sin3-HDAC1 co-repressor protein complex, not by CpG methylation of the promoter. However, the direct interaction of the complex with IRF4/Pu.1 transcription factors was not confirmed in our assay, and the existence of other transcription factors that interact with Sin3-HDAC1 complex was suggested. To confirm the key regulators of CD20 expression is quite useful for exploring some strategies to overcome the rituximab resistance through CD20-negative transformation after using rituximab.

Kiyoi:Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Wyeth K.K.: Research Funding.