Mechanism Study On EZH2-Mediated Cell Proliferation And Anti-Leukemia Effect Of EZH2 Inhibitor In T-cell Acute Lymphoblastic Leukemia

Objective:Zest gene enhancer homolog 2?EZH2?,a histone methyltransferase,which can regulate chromatin conformation and silence target gene transcription,has important,extensive and diverse effects in physiological and pathological processes.In solid tumors and hematological malignances including acute lymphoblastic leukemia?ALL?,EZH2 overexpression is highly related to disease development,high invasiveness,and poor prognosis.EZH2 inhibitors have been initially confirmed to have anti-tumor effects in cancer cells such as lymphoma.Also,EZH2 mutation may lead to dysfunction,affecting ALL development.The aim of this study was to investigate the effect of EZH2 point mutation on EZH2 expression and cell proliferation and also the efficacy of EZH2 inhibitor GSK126 on cell proliferation,cell apoptosis and cell cycle of T-cell acute lymphoblastic leukemia?T-ALL?.Methods:The EZH2^K466T lentiviral plasmid was constructed using the point mutation kit.The human T-ALL?CEM?cells were transfected with mutant EZH2(EZH2^K466T)wild-type EZH2(EZH2^WT)and blank vector(EZH2^control)to establish the stable cell lines.The Cell Counting Kit-8?CCK-8?was used for detection of the optical density?OD?value to evaluate the cell proliferation in the resulted stable cell lines and EZH2inhibitor treatment.Effect of the EZH2 inhibitor GSK126 on cell apoptosis and cell cycle arrest was measured by Annexin PE/7-AAD staining and propidium iodide?PI?staining,respectively,following flow cytometry analysis in the cells.The difference of mRNA and protein level of EZH2 in EZH2^K466T,EZH2^WT,and EZH2^control CEM cells were detected by quantitative polymerase chain reaction?qPCR?and Western blot?WB?.The relative mRNA and protein expression levels of tumor-associated genes[EZH2,PTEN?phosphatase and tensin homolog?],apoptosis-related genes[Bcl-2?B-cell lymphoma-2?,Bcl-xl?Bcl2-like 1?]and cell cycle-related genes[p15?cyclin dependent kinase inhibitor 2B?,p16?cyclin dependent kinase inhibitor 2B?]by GSK126in CEM were measured by qPCRand WB,respectively.Results:The stable CEM cell line transfected with EZH2^K466T were successful generated.The OD values of EZH2^K466T,EZH2^WT and EZH2^control CEM cells were increased with the prolongation of culture time.The OD values of EZH2^K466T were higher than those of EZH2^WT at each time point.After 3 days of culture,the difference of OD values was statistically significant?P<0.01?.In addition,the OD value of EZH2^WT at each time point was also higher than that of EZH2^control,and the difference of OD values between the two was statistically significant after 3 days of culture?P<0.01?.The relative expression of EZH2 mRNA and EZH2 protein in EZH2^K466T was significantly higher than that in EZH2^WT?P<0.001?.EZH2 inhibitor GSK126 reduced the cell proliferation of CEM.The 50%inhibitory concentration?IC50?for CEM treated with GSK126 for 24 hours was 14.10?M.We also observed the possible mechanism underlying GSK126 anti-tumor effect in T-ALL cells.GSK126 can reduce EZH2 mRNA and protein level in CEM cells.At the same time,GSK126 can up-regulate PTEN mRNA and protein level and down-regulate expression of anti-apoptotic proteins Bcl-2 and Bcl-xl to induce apoptosis of CEM cells.In addition,GSK126 can enhance the mRNA and protein levels of Cyclin-dependent kinase inhibitors p15 and p16to arrest CEM cells in G1 phases.Conclusion:EZH2^K466T CEM showed an enhanced cell proliferation capacity and an increase in EZH2 mRNA and protein expression levels.This indicates that EZH2^K466T466T is a gain-of-function mutation.EZH2 inhibitor GSK126 can inhibit the cell proliferation,induce apoptosis by down-regulating EZH2 expression?affecting PTEN signaling and suppressing the expression of Bcl-2 and Bcl-xl,and also cause cell cycle arrest by enhancing expression of p15 and p16 in T-ALL CEM cells.Our results indicate that EZH2 inhibitor has the in vitro anti-tumor effect in T-ALL,which suggest it may play an important role in the control of the development and progression of T-ALL and might be a potential new drug in the treatment of this disease.