EZH2 Inhibition Mediates Proptosis Via DHX9 and S100A9 in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) is considered as an archetypal epigenetic disorder due to the aberrant epigenome, frequent epigenetic regulator mutations and the most responsive disease to DNA methylation inhibitors. Among numerous epigenetic regulators, Enhancer of zeste 2 homologue 2 (EZH2) occupies an important position owing to its involvement in multiple epigenetic modifications. In MDS, EZH2 mutation frequency is 5%-10%. Given that the EZH2 gene is located at 7q36.1, abnormalities of chromosome 7q disturb EZH2 as well. To investigate the expression and effects of EZH2 in MDS patients without EZH2 mutation and chromosome 7 abnormalities, qPCR was performed. We found that EZH2 transcript level was significantly higher in MDS patients and its expression was highest in very high risk group, followed by the high risk group. The survival time of EZH2 high expression was shorter than the low expression group. Multivariable Cox regression showed that EZH2 expression was an independent prognostic factor. Next, we treated MDS cell lines MDS-L and SKM-1 with EZH2 inhibitor DZNep and found that DZNep inhibited cell proliferation in a dose- and time-dependent manner. To determine which pattern of cell death played the most important role, we pretreated different cell death inhibitors for 1 hour before adding DZNep. Compared with the group without cell death inhibitors, pyroptosis inhibitor (VX765) was the most effective in restoring cell viability. Besides, both DZNep and knockdown of EZH2 could trigger pyroptosis. To elucidate the possible mechanism of pyroptosis induced by EZH2 inhibition, we searched for RNA-seq data from tumor cells treated with EZH2 inhibitors by using the GEO database. We overlapped 53 genes by analyzing GSE151576, GSE80239 and GSE119088, of which DHX9 was the highest expressed in BM and attracted attention.Meanwhile, down-regulation of DHX9 was observed after pharmacological inhibition or EZH2 knockdown. There was a positive correlation between EZH2 and DHX9 in MDS patients. Furthermore, DHX9 expression was increased in MDS patients in contrast to control, and its overexpression was connected with shorter OS. Knockdown of DHX9 with two shRNAs induced pyroptosis. CUT&RUN and qPCR assay results validated that EZH2 and H3K4me3 occupancy at DHX9 promoter. Moreover, with RNA sequencing we found DHX9 can regulate S100A9 and confirmed by western blot. Overexpression of S100A9 could cause pyroptosis of MDS cell lines, which could be reversed by a S100A9 inhibitor. DHX9 and S100A9 are negatively correlated in MDS patients. Finally, we established xenograft mouse models by intravenously injecting luciferase-labeled SKM-1 cells. Intraperitoneal injection of 2mg/kg DZNep daily could significantly reduce the tumor burden and prolong the survival time of mice. We further confirmed that inhibition of EZH2 regulated DHX9 and S100A9 and induced pyroptosis in mouse bone marrow cells. In conclusion, we found that EZH2 expression in de novo MDS patients without EZH2 mutation and chromosome 7 abnormality was higher than that of the control group, and its high expression was correlated with inferior prognosis. EZH2 suppression inhibited tumor growth and induced pyroptosis both in vitro and in vivo. Inhibition of EZH2 triggered pyroptosis by reducing its binding in the DHX9 promoter and upregulating S100A9. In this present study, we reveal a novel molecular mechanism by which EZH2 inhibition induced pyroptosis and provide a basis for the treatment of MDS with EZH2 inhibitor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal