FAS-AS1 Intronic Lncrna and Ibrutinib Negatively Regulate Soluble Fas Production and Sensitize B-Cell Lymphomas To Fas-Mediated Apoptosis

The death receptor Fas is responsible for cell homeostasis, elimination of defective cells, and more recently promotion of cancer. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas- mediated apoptosis is a recognized basis for primary and acquired resistance to chemotherapy. Impaired Fas-mediated apoptosis is associated with poor clinical outcome and chemoresistance in patients with lymphoma. Evasion of immune surveillance and resistance in Fas apoptosis occur in part through interference with FasL binding either by Fas binding proteins (nucleolin and human herpesvirus 8 K1) or by release of soluble Fas (sFas), which is produced by skipping of exon 6. The first intron of Fas pre-mRNA contains a long-noncoding RNA (lncRNA) termed FAS-AS1 implicated in gene expression. The presence of sFas in serum is associated with poor clinical outcome of patients with non-Hodgkin's lymphoma (NHL). However, the mechanisms that control Fas alternative splicing are incompletely understood.

The levels of FAS-AS1 lncRNA are suppressed in primary NHL and B-cell lymphoma cell lines and inversely correlate with production of sFas. ChiP assay revealed hyper- methylation of FAS-AS1 promoter that is responsible for the reduced expression of FAS-AS1 lncRNA.

Histone methyltransferase EZH2 is responsible for transcriptional repression via trimethylation of lysine 27 on histone H3. EZH2-mediated hyper-methylation of the FAS-AS1 promoter correlated with production of sFas, which was reversed by the ectopic expression of FAS-AS1 or pharmacological inhibition of EZH2 by DZNep. Inhibition of EZH2 methyltransferase activity elevated FAS-AS1 lncRNA levels and promoted its binding to RBM5 in RNA-Immunoprecipitation assay. Sequestering RBM5 by FAS-AS1 lncRNA inhibited RBM5-mediated skipping of exon 6 and thus sFas production. Decreased production of sFas was accompanied by increased levels of surface Fas and FasL binding in lymphoma cell lines that translated into enhanced Fas-mediated apoptosis.

Bruton tyrosine kinase (BTK) is important for B cell receptor signaling and was proposed and demonstrated to be a feasible target for lymphoma treatment. BTK inhibitor ibrutinib decreased levels of EZH2 and RBM5 and correspondingly decreased levels of sFas and enhanced Fas-mediated apoptosis.

Taken together, we show here for the first time that the FAS-AS1 intronic lncRNA negatively regulates production of sFas. The oncogenic proteins EZH2 and RBM5, overexpressed in lymphoma cells, cooperate to produce sFas through down regulation of FAS-AS1 expression and stimulation of skipping of exon 6 with production of sFas. Targeting of EZH2 by DZNep and ibrutinib reduces sFas expression and sensitizes cells to Fas-mediated apoptosis. We anticipate that these drugs will play a key role in effective therapy regimens by enhancing Fas-signaling and account in part for the enhanced chemotherapy responses in hematologic malignancies.

No relevant conflicts of interest to declare.