Abstract
Abstract Background. DNA methyltransferases (DNMTs) are key enzymes necessary to the cells in order to establish and maintain methylation marks across the genome during both embrional development and adult cell divisions. DNMT1 is the main target of hypomethylating drugs 5-azacytidine (5-AZA) and 5-aza-2'-deoxycytidine (decitabine). These drugs have been used for a long time even though some mechanisms of action are yet unknown. We previously demonstrated that 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion, activate PARP and casp-3/7. However, 5AZA does not affect the methylation of selected targets, namely KLF4, DAPK1 and SPG20, who had been shown to play a role in lymphoma malignancy. We also observed that human PBMCs display an unmethylated profile that is opposite to the lymphoma cells. Experimental procedures. Toledo (Germinal center-derived) and NU-DUL-1 (Activated B-cell like) diffuse large-B cell lymphoma (DLBCL) cell lines. Epitect methyl II methylation qPCR. Immunoblottings. Gene silencing through siRNAs. Illumina Infinium 450K methylation arrays. Bioinformatics analysis for CpG doublets identification. Results. In order to dissect the molecular contribution of the main DNMTs during methylation maintenance, we individually silenced DNMT1 and DNMT3a in DLBCL cell lines. We show that neither DNMT1-, DNMT3a- nor double-silencing significantly affects the promoter methylation of KLF4, DAPK1 and SPG20. Genome-wide methylation analysis on a panel of 45 B-cell lymphoma cell lines reveals that the SPG20 locus presents hypermethylated CpG doublets upstream of the transcription starting site (open sea regions) while is mainly hypomethylated within the first exon. An intronic CpG island maps within SPG20 locus and presents heterogeneous beta-values among CpG sites. After 7 days from transfection, surprisingly, DNMT1, 3a and 3b gene expressions increase above the controls in Toledo cells. Furthermore, DNMT3a-silencing leads to DNMT1 protein upregulation in Toledo and NU-DUL-1 cells, suggesting a feedback regulatory mechanism. These data support a mechanism through which the methylation maintaining capacity of the cells is preserved even under pharmacological pressure. Conclusions. DNMT1- and DNMT3a-silencing exert a different effect on DNMTs regulation in lymphoma cells and evidences a functional redundancy among family members. Our data overall suggest that DNMT3a exerts suppressive effect on DNMT1, possibly by participating to the formation of a multi-factorial repressor complex. Citation Format: Raffaele Frazzi, Vincenza Ylenia Cusenza, Luciano Cascione, Andrea Rinaldi, Francesco Bertoni, Mariaelena Pistoni, Francesco Merli. DNA methyltransferase modulation in lymphoma cells involves functional redundancy and maintains hypermethylation of selected regions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 163.
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