Abstract 7015: Crosstalk between histone and DNA methylation in the development and progression of ALK+ ALCL
Authors: Lobello Cosimo , Wang Shengchun , Rao Shilpa , Pawlicki Jan , Mansoor Nashwa , Riley James L. , Nejati Reza , Mariusz Wasik A. 
Published: March 2024

ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) is a T-cell non-Hodgkin lymphoma characterized by the presence of the fusion oncogene nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Tumor cells control the expression of key genes in different manners: mutations, translocations or epigenetically. Our group have shown that in this tumor a plethora of genes are silenced by NPM-ALK through DNA methylation. However, recent evidence showed that some of the genes downregulated by DNA methylation might have already been silenced prior to the appearance of DNA methylation. Herein, we investigate the possible role of histone methylation in the silencing of genes and its relationship to DNA methylation. To better understand the possible players in the silencing of genes in ALK+ ALCL we used T-cell transformed with NPM-ALK and recorded over time the changing in gene expression, DNA methylation and histone marks. Using RNAseq comparing transformed cells at different time points, we noticed that downregulated genes in ALK+ ALCL became gradually silenced also in our system. Next, we investigated the DNA methylation (meDNA) on their promoters, and we confirmed a progressive increase of meDNA but clearly slower compared to the silencing of the genes, or if compared to the meDNA status of established ALK+ ALCL cell lines. Finally, we explored the presence of silencing histone marks on the promoter of the silenced genes using qPCR-ChIP. Among the repressive histone marks evaluated, H3K27me3 stood up as possible candidate for the gene silencing. Therefore, we look at the different gene expression changes after the inhibition of either meDNA, using a DNMT1 inhibitor (GSK3685032), or histone methylation, by EZH2 inhibitor (EPZ6438). RNAseq data showed the re-expression of downregulated genes after EZH2 and/or DNMT1 inhibition providing clear insights on their epigenetic regulation. The co-presence of H3K27me3 and meDNA on the promoters of those genes points towards a crosstalk between DNA and histone methylation machineries. Despite it is clear how NPM-ALK regulates DNA methylation, nothing is known about NPM-ALK and EZH2. To investigate their relationship, we treated ALK+ cells with ALK inhibitor, and we observed a decrease of EZH2 expression at protein and mRNA level. STAT3 inhibition, through napabucasin or by STAT3 silencing using Crispr-cas9 technology, resulted, likewise, in the downregulation of EZH2. Moreover, ChIPseq showed STAT3 presence on EZH2 promoter, further confirming this mechanism. Altogether, those data showed that NPM-ALK regulates EZH2 expression through STAT3. In conclusion, we provide, not only novel information on the mechanism in the downregulation of genes in ALK+ ALCL, but also new insight on the understanding of the epigenetics machinery and the crosstalk between histone and DNA methylation, for long time considered two mutually exclusive mechanisms, or even antagonist.

Citation Format: Cosimo Lobello, Shengchun Wang, Shilpa Rao, Jan Pawlicki, Nashwa Mansoor, James L. Riley, Reza Nejati, Wasik A. Mariusz. Crosstalk between histone and DNA methylation in the development and progression of ALK+ ALCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7015.