EZH2 Mediates Resistance To Apoptosis In Nktl By Activating Nfkb Signaling Through Repression Of TNFAIP3/A20 By H3K27 Trimethylation

Extranodal nasal-type natural killer/T-cell lymphoma (NKTL) is a rare and aggressive form of lymphoma with poor outcome. A better understanding of the molecular pathophysiology of the disease is needed to development better treatment and improve patient’s outcome. We previously showed that EZH2, a histone methyltransferase, is abnormally over-expressed in NK cell lines as compared to normal NK cells, and may therefore be important for the pathogenesis and treatment of NKTL. 3- Deazaneplanocin A (DZNep) is a drug that can deplete EZH2 levels. Downregulation of EZH2 by DZNep induces apoptosis in a panel of NKTL cell lines but not normal NK cells. We perform gene expression studies to identify possible mechanism by which DZNep induce apoptosis in NKTL. There is significant enrichment of NFKB target genes amongst the genes downregulated after DZNep treatment. In addition, 2 inhibitors of the NFKB pathways, TNFAIP3/A20 and NFKBIA were upregulated. The downregulation of these genes were validated by PCR. We further found that NFKB2 was downregulated after DZNep treatment on western blot suggesting that NFKB signaling was downregulated. Similar results were obtained when we silence EZH2 using siRNA suggesting that much of the effect of DZNep is mediated through downregulation of EZH2. When TNFAIP3/A20 was expressed in untreated NKTL cells, apoptosis was induced. Conversed with TNFAIP3/A20 was knocked down in cells treated by DZNep, DZNep induced apoptosis was reduced. These data suggest in NKTL, EZH2 downregulate TNFAIP3/A20 and activate NFKB leading to resistance to apoptosis. We next investigated if EZH2 directly regulate TNFAIP3/A20 expression at its promoter. Using chromatin immunoprecipitation, we found that there is high occupancy of EZH2 and H3K27me3 at the TNFAIP3/A20 promoter in NKTL. Upon EZH2 knockdown, EZH2 and H3K27me3 is markedly reduced at the promoter and there is concurrent increase in TNFAIP3 expression. Consistent with cell lines data, in clinical samples, there is a strong association between H3K27me3 and nuclear p50 expression on a tissue microarray of NKTL (Chi-square p-value 0.01). We have therefore uncovered a novel mechanism by which EZH2 activates the NFKB pathway through its histone methyltransferase activity on the promoter of TNFAIP3/A20 resulting in resistance of NKTL to apoptosis.