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Abstract 5198: 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a

Introductory sentence. Demethylating drugs represent a powerful tool for epigenetic modulation of chromatin structure in cancer cells. 5-azacytidine (5-AZA) and 5-aza-2’-deoxycytidine (decitabine) are promising therapeutic solutions also in mature B-cell neoplasms, although the molecular mechanisms of action have yet to be elucidated. Here we describe their effects on non-Hodgkin lymphoma cells and demonstrate how hypomethylation is not homogeneous across the genome. Experimental procedures. Toledo (GC-derived) and NU-DUL-1 (ABC-like) diffuse large-B cell lymphoma (DLBCL; non-Hodgkin) cell lines. Peripheral blood mononuclear cells (PBMCs) isolation. Epitect methyl II methylation qPCR and global chromatin methylation quantitative assays. Immunoblottings. Proliferation assays. CpG islands bioinformatics analysis. Comet assay and phosphorylation of gamma-H2AX histone assay. Gene silencing through siRNAs. New data. A panel of 9 CpG islands within a set of genes that we previously identified has been investigated as a potential target for hypomethylating activity of 5AZA and decitabine. KLF4, DAPK1 and SPG20 promoters (located on chr. n°9 the first two and n°3 the latter) are not affected by a single dose of 5AZA after 48h or 7 days of incubation (the CpG islands within these promoters are fully unmethylated in healthy PBMCs). However, global chromatin demethylation is clearly visible either after 48h or 7 days in both cell lines. By comparison, decitabine demethylating effects return to the initial values after 7 days. MZB1 promoter results demethylated by both treatments, while MGMT is demethylated only in NU-DUL-1 by decitabine but not 5AZA. These data suggest that hypomethylating agents act selectively on discrete regions of the genome. 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion and activate PARP. Despite DNMT1 downregulation, KLF4, DAPK1 and SPG20 promoters result unaffected. DNMT1 silencing in our cells do not cause any change in the promoter methylation of the selected targets. The de novo methyltransferase DNMT3a is expressed only by NU-DUL-1 and its silencing leads to a partial MZB1 demethylation and to the significant decrease in global chromatin methylation whereas DNMT1 silencing does not. In order to assess an involvement of the genotoxic damage, Comet assays were performed up to 24 hours of incubation with the drugs. We demonstrate that 5AZA does not induce any significant genotoxic damage while decitabine causes a tail formation starting at 6h after drug administration. Conclusions. Our data show that 5AZA and decitabine exhibit different mechanisms of action on lymphoma cells. Different DLBCL-derived cell lines display different changes when exposed to single doses of hypomethylating drugs, underlying the fact that cell of origin may play a role during the response. DNMT3a contributes to chromatin methylation in lymphoma cells. Citation Format: Raffaele Frazzi, Tonia De Simone, Olga Serra, Annamaria Buschini, Laura Canovi, Tiziana De Luca, Francesco Merli. 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5198.