Abstract 161: xCT expression alters global DNA methylation levels in lung adenocarcinoma cells

Metabolic reprogramming and genomic instability are two key components in driving carcinogenesis. While it has been theorized that these two processes influence each other, the mechanisms by which metabolic reprogramming can induce genomic instability remain unknown. Our lab previously demonstrated xCT, a cystine/glutamate antiporter, is highly expressed in NSCLC and induces metabolic reprogramming. However, its influence on genomic instability is unknown. In this study, we aim to examine how xCT could potentially contribute to genomic instability by first investigating its influence on the epigenetic landscape, markedly DNA methylation. We hypothesize that changes in intracellular metabolite concentrations due to high xCT expression have a downstream effect on the levels of metabolites involved in DNA methylation, and such changes are significant enough to induce alterations in global DNA methylation. Global methylation levels were quantified through a colorimetric assay in A549 wildtype (WT) and xCT knockout (KO) cells. Next, xCT KO cells were treated with propargylglycine (PAG), a cystathionase inhibitor, and measured global methylation. By inhibiting cystathionase, we blocked cells9 ability to synthesize cysteine through the utilization metabolites in the methionine cycle. Quantified methylation levels reported 51.2 ± 11.4% and 25.6 ± 11.0% in WT and xCT KO cells respectively- an average of a 50.0% decrease in methylation upon knocking out xCT. Furthermore, initial data showed DNA methylation more than tripling (234.8% increase) in xCT KO cells when treated with PAG, notably demonstrating 43.4% higher methylation levels than that of treated and untreated WT. Additionally, a western blot revealed that compared to WT, xCT KO cells express much higher levels of cystathionine beta synthase, another enzyme linking cysteine biosynthesis and the methionine cycle. These results support the notion that metabolic activity in the context of xCT can change DNA methylation, potentially through altered metabolic demand between the transsulfuration pathway and methionine cycle. Further studies investigating key metabolite concentrations in xCT KO versus WT cells, as well as analyzing deregulated genes resulting from the altered methylation landscape, will help elucidate the extent of xCT-induced metabolic reprogramming. Understanding the full scope xCT overexpression and the underlying mechanisms leading to epigenetic changes may provide crucial insight to the interplay of metabolic reprogramming and other cancer drivers like genomic instability. By understanding the true origin and influences of such hallmarks of carcinogenesis, more informed approaches can be applied to early preventative therapies. Citation Format: Dalton Hill, S.M. Jamshedur Rahman, Christien Kluwe, Pierre P. Massion. xCT expression alters global DNA methylation levels in lung adenocarcinoma cells [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 161.