Introductory sentence. The layer of regulation represented by gene promoter methylation is being investigated in Non-Hodgkin lymphoma (NHL) cell lines, primary B lymphocytes and peripheral blood mononuclear cells (PBMCs). The aim is to assess whether the panel of differentially methylated genes we previously identified is able to distinguish normal B lymphocytes from lymphoma cells.
Experimental procedures. Quantitative, gene-specific methylation assays. Immunosorting of human B lymphocytes. Proliferation assays. 5-azacytidine treatment. NHL-derived cell lines.
New data. Here we show that NHL-derived cell lines display a characteristic profile of promoter methylation on specific tumor suppressors and target genes. A panel of 9 target promoters was selected previously and assessed on a series of healthy and tumor primary B lymphocytes. 4 out of 5 NHL-cell lines show a highly methylated profile on KLF4, DAPK1 and SPG20, at variance with normal B lymphocytes (lymph-node derived) and PBMCs where these targets are completely unmethylated. Interestingly, PBMCs from healthy donors display the same methylation pattern as the one shown by B lymphocytes immunologically sorted from follicular hyperplasias. Spartin (SPG20) emerges here as a differentially methylated gene associated to lymphocyte malignancy, at variance with other tumor suppressors in our panel that show variability among different cell lines. SPG20 is completely or highly methylated in all the tested NHL-cell lines. According to this observation, SPG20 results not methylated in PBMCs as well as sorted non-tumor B lymphocytes. On the contrary, the methylation status of three different BCL6 CpG islands (assessed with the same technique) may vary among different cell lines. In order to investigate what's the functional role of these specific promoters methylation, we treated Toledo and NU-DUL-1 cell lines with the demethylating agent 5-azacytidine (5AZA). We observe that the dose-dependent inhibition of proliferation upon 5AZA is accompanied by a small but reproducible decrease in SPG20 and MZB1 promoter methylation. Experiments aimed at assessing any involvement of DNA methyltransferases DNMT1 and DNMT3a are currently ongoing.
Conclusions. Our data show a consistent methylation profile of 9 gene promoters on 5 different NHL-derived cell lines. Spartin emerges as a completely methylated promoter in the tested tumor cells. PBMCs show a consistent unmethylated profile of Spartin and of all the target genes. The epigenetic regulation of these genes in B-derived lymphomas warrants further investigation.
Citation Format: Raffaele Frazzi, Tonia De Simone, Mariaelena Pistoni, Francesco Merli. Spg20 (Spartin) promoter methylation identifies tumor B lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5334.