Abstract
Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NFκB levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3Y640F and STAT3G618R mutants compared to KAI3 NK cells overexpressing STAT3WT. Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments.
Highlights
Monocyte chemoattractant protein 1 (MCP-1) levels were upregulated in patients with STAT3 mutations, whereas for example IL-2 and IL-4 levels were similar between patients and healthy controls (Fig. 1A)
IL-15 expression levels were significantly higher in STAT3 mutated T-Large granular lymphocyte leukemia (LGLL), whereas only borderline differences in expression levels were observed for IL-6 and MCP-1 (Fig. 1B), suggesting that other cell types contribute to circulating cytokine levels [13]
Activating STAT3 somatic mutations can be found both in patients with T-cell LGLL (40-50% of cases with CD8 + CD3 + phenotype) and in CLPD-natural killer (NK) (20-30% of patients) [3, 6]
Summary
LGLL originates from the expansion of natural killer (NK) cells (chronic lymphoproliferative disorder of NK cells, CLPDNK). Both T-cell LGLL and CLPD-NK are chronic and indolent diseases, but instances of aggressive leukemias have been reported [2]. As the role of STAT3 in regulating DNA methylation in LGLL is currently unknown, we investigated whether inflammatory cytokine stimulation and STAT3 activation modify the epigenetic machinery in CD8+ T cells and could thereby affect disease pathogenesis in LGLL. Using LGLL patients samples, healthy human CD8+ T cells, and KAI3 NK cell line, we show that cytokine stimulation or overexpression of GOF STAT3 variants lead to increased protein levels of epigenetic regulators, increased global DNA methylation levels, and increased reactive oxygen species (ROS) production.
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