Epigenetic Down-Regulation of NFkB-Mediated FAS Gene Transcription During Progression of Myelodysplastic Syndromes
Authors: Ettou Sandrine , Humbrech Catherine , Benet Blandine , Billot Katy , Mariot Virginie , Kosmider Olivier , Lacombe Catherine , Mayeux Patrick , Solary Eric , Fontenay Michaela 
Published: October 2019

Abstract
Abstract 3503

Background
CD95/Fas, a member of the death receptor family, can induce cell death upon binding of its ligand, FasL Suppression of Fas signaling leads to autoimmune diseases while overexpression of Fas and FasL accounts for inappropriate apoptosis of erythroid cells in low grade myelodysplastic syndromes (MDS). The progression of MDS into secondary acute myeloid leukaemia (sAML) is associated with inhibition of hematopoietic progenitor cell death which involves the methylation of FAS gene promoter. We have previously demonstrated that FAS gene expression was increased in MDS and down-regulated when disease progresses to sAML. We further investigated the molecular mechanisms of FAS gene extinction during MDS progression. The transcription factor NFκB is functionnal as homo or heterodimer and is composed of proteins p65 or p50. The constitutive activation of NFkB pathway identified in solid tumors and in diverse hematological malignancies was suggested to promote tumor cell survival.
The aim of the present work was to further investigate the interplay between FAS gene and NFkB during the progression of MDS to sAML.

Methods
85 consecutive patients with a diagnosis of MDS and 15 age-matched controls with normal bone marrow (BM) were included between 2005 and 2011. Using cell lines and primary normal and leukemic bone marrow cells, we explored the p65/RelA NFkB subunit localisation by immunofluorescence and immunoblotting, inhibited NFkB pathway with the IKKa inhibitor BAY11–7082, quantified FAS transcript and protein by RT-qPCR and flow cytometry respectively, and explored p65/RelA binding to DNA by pull-down and chromatin immunoprecipitation (ChIP).

Results
In order to explore the role of NFkB in FAS gene expression, we used the HL-60 human leukemic cells which spontaneously express Fas at their surface and the SW-480 colon carcinoma cells which do not. We show that TNFα increased FAS gene and Fas receptor expression only in HL-60 cells indicating a cell type-dependent regulation of FAS gene expression. We conclude that NFkB is responsible for FAS gene expression in HL60 cell line.
The FAS gene promoter contains putative NFκB sites in the 5' UTR region spreading between −899 and the transcription start site. By using in vitro DNA affinity precipitation experiments, we show that P65/RelA bound to each of the 3 putative NFκB binding sites on FAS promoter upon TNFa treatment. We conclude that p65/RelA binds to the chromatin at FAS promoter in a cell type-dependent manner to regulate FAS gene expression in cells.
Then, we hypothesized that DNA methylation in FAS gene 5'UTR regulatory regions may prevent the docking of NFkB. ChIP Experiments identified one active mark H3K9/14Ac and none of the two repressive marks H3K27me3 and H3K9me2 in HL60 cell line while the repressive mark H3K9me2 was expressed in SW480 cell line These results suggested that the chromatin conformation at FAS promoter was open in Fas-positive HL-60 cells, and repressed in Fas-negative SW-480 cells. By treatment with azacitidine, which stimulated the expression of Fas in SW-480 but not in HL-60 cells and by experiments using TNFa, we show that NFkB-dependent FAS gene expression is epigenetically regulated.
We used ChIP to explore the binding of p65/RelA to the chromatin at FAS promoter in 4 Fas-expressing and 6 Fas-negative MDS samples. The expression of Fas was proportional to the recruitment of p65/RelA at the gene promoter. Among 16 MDS samples where FAS protein expression was positive, BAY11–7082 was able to decrease Fas expression in 12 of them. In addition, we show that azacitidine rescues NFκB-induced Fas expression in primary MDS/sAML cells. Altogether, NFκB controls the transcription of FAS gene in primary BM cells from MDS or sAML and azacitidine can induce FAS promoter demethylation, thus facilitates p65/RelA binding and FAS gene transcription in myeloid cells.

Conclusion
The present study demonstrates that NF-kB directly promotes FAS gene expression in normal and myelodysplastic hematopoietic progenitor cells and that, when MDS progress into sAML. The inhibition of FAS gene expression observed in sAML is due to the methylation of NFkB binding site in the gene promoter, which can be reversed by the demethylating agent 5-azacitidine. The increased expression of FAS gene induced by 5-azacitidine now deserves to be tested as a biomarker of the drug activity.

Disclosures:
No relevant conflicts of interest to declare.