EZH2 Overexpression And Its Biological Function In Systemic Anaplastic Large Cell Lymphoma

Background and ObjectiveAnaplastic large cell lymphoma(ALCL)refers to a group of CD30-positive T-cell non-Hodgkin lymphomas that display great heterogeneity in their clinical manifestation,molecular features,and outcomes.Enhancer of Zeste Homolog2(EZH2),an epigenetic regulator,specifically methylates Histone H3 at lysine27(H3K27me3)via its histone methyltransferase activity.EZH2 was overexpressed in various neoplasms and its overexpression was frequently associated with aggressive behavior and poor clinical outcomes in different types of solid tumours,as well as some hematologic neoplasms.The aim of this study was to investigate the mechanisms of EZH2 overexpression and its biological function in systemic ALCL(S-ALCL).Firstly,we performed immunohistochemistry staining for EZH2,MYC,p-STAT3,p-ERK1/2 and H3K27me3 on all 62 cases of S-ALCL to explore the potential mechanisms of EZH2 overexpression.Subsequently,we downregulated EZH2 expression in S-ALCL cell lines via lentivirus mediated RNA interference to further investigate the detail function of EZH2 in S-ALCL.Finally,we inhibited EZH2 expression by DZNep,an inhibitor of EZH2,to illustrate that EZH2 may be a potential therapy target of S-ALCL.Materials and Methods1.Patients and cell lines selection: We retrospectively reviewed the morphology and immunophenotype features of 62 S-ALCL caces from multiple centers between2010 and 2018.Clinical and pathological data were retrived from electronic medical records.In vitro,two ALCL cell lines,Karpas-299 cell line and MAC-1cell line were selected to verify the results.2.Immunohistochemistry staining for EZH2,H3K27me3,MYC,p-STAT3 and p-ERK1/2 was performed on all 62 cases of S-ALCL including 19 cases of ALKpositive ALCL,9 cases of DUSP22-rearranged ALCL,34 cases of double negative ALCL,to investigate the possible mechanisms of EZH2 overexpression in systemic ALCL.3.EZH2 expression was down-regulated by lentivirus mediated RNA interference in Karpas-299 cells,which confirmed by RT-qPCR and Western blot analysis.Cell growth was measured with a CCK-8 based cell viability assay.4.JAK/STAT3 signaling pathway inhibitors(Tofacitinib and Stattic)were cultured with S-ALCL cell lines to observe their effect on the expression and the biological function of EZH2 by using western blot analysis.5.After treating with EZH2 inhibitor(DZNep),the expression of EZH2 in S-ALCL cell lines was tested by western blot analysis.Besides,cell growth were measured with a CCK-8 based cell viability assay.6.All analyses were performed using SPSS 19.0 software.Differences were considered statistically significant when the 2-sided P value was less than 0.05.The Spearman rank-correlation coefficient test was used to correlate the expression of p-STAT3 and H3K27me3.Results1.Immunohistochemical analysis: EZH2 was homogenous strong positive in all S-ALCL cases(62/62,100%).The expression of MYC,p-STAT3,p-ERK1/2 and H3K27me3 were positive in 56/62(90%),39/62(63%),1/62(2%)and 21/62(34%),respectively.In cases of ALK-positive ALCL,18/19(95%)and 19/19(100%)were positive for MYC and p-STAT3,whereas only 1/19(5%)and 1/19(5%)were positive for p-ERK1/2 and H3K27me3.In cases of DUSP22-rearranged ALCL,7/9(78%)and 8/9(89%)were positive for MYC and H3K27me3,whereas1/9(11%)and none were positive for p-STAT3 and p-ERK1/2.In cases of double negative ALCL,31/34(91%)and 19/34(56%)were positive for MYC and p-STAT3,whereas 12/34(35%)and none were positive for H3K27me3 and p-ERK1/2.H3K27me3 showed a negative association with p-STAT3(P<0.001,Spearman r=-0.652).2.The pathophysiological changes of Karpas-299 cell line after EZH2 was knocked out: Lentivirus shRNA vector against EZH2 was successfully constructed,with a transfection efficiency over 80% in Karpas-299 cell line.The transcription and expression of EZH2 was significantly decreased in both EZH2 shRNA-1 group and EZH2 shRNA-2 group by comparing with control group.The proliferation of Karpas-299 was significantly inhibited.3.The effect of the JAK/STAT3 pathway on EZH2: Reduction of p-STAT3 in MAC-1 cell line and Karpas-299 cell line by Stattic treatment was accompanied with increased H3K27me3(P<0.05),and EZH2 expression was not affected(P>0.05).MAC-1 cell line by Tofacitinib treatment has the same result.EZH2,pSTAT3 and H3K27me3 expression was not affected by Tofacitinib in Karpas-299 cell line.4.The changes of biological function in S-ALCL cell lines by DZNep treatment:EZH2 protein expression,cell expansion were decreased in S-ALCL cell lines by DZNep 5μM and 10μM treatment,and there was a statistical difference(P<0.05).Conclusions1.EZH2 was overexpressed in S-ALCL,which may mediate by MYC-associate signaling pathway.2.EZH2 may promote the oncogenesis of S-ALCL.3.EZH2 plays complicated functions in S-ALCL.Its canonical EZH2 typical methyltransferase function mainly impacted DUSP22-rearranged ALCL,but for most cases of double negative ALCL and ALK-positive ALCL.4.JAK/STAT3 signaling pathway activation may switch EZH2 the biological function in S-ALCL.5.EZH2 may be a potential therapy target in S-ALCL.