| ObjectivesAcute leukemia develops rapidly and prognosis is poor.According to the origin of the affected cells,it can be classified into two groups:acute lymphoblastic leukemia(ALL)and acute myeloid leukemia(AML).It is of great significance to analyze the novel mechanism and targets of diagnosis and treatment.Abnormal epigenetic regulation based on histone demethylation is an important reason for the occurrence and development of leukemia.PHF8 and KDM7A are members of the KDM7 family of histone demethylase carrying JmjC domain.The role they play in the pathogenesis of adult acute leukemia and its related mechanism remains unclear.The purpose of this study is to:1.To clarify the function and regulatory mechanism of histone demethylase PHF8 involved in the development and progression of adult ALL.2.To explore the role of histone demethylase KDM7A as a tumor suppressor in adult AML and its function as a diagnostic and prognostic marker for AML.Methods and Results1.The histone demethylase PHF8 promotes adult acute lymphoblastic leukemia through interaction with the MEK/ERK signaling pathway.(1)PHF8 is up-regulated in adult ALL and associated with its progression.By comparing 710 ALL bone marrow samples and 73 normal bone marrow samples from GEO database,we found that PHF8 was significantly up-regulated in ALL patients,which confirmed that PHF8 might be involved in the occurrence of ALL disease.Comparison the PHF8 expression of bone marrow samples from newly diagnosed,complete remission,relapsed adult ALL patients and controls showed a positive correlation between the expression of PHF8 and the progression of ALL.The PHF8 protein level detected in patients with ALL at different stages and the PHF8 expression level in same patients at different ALL stages further confirmed this conclusion,(2)PHF8 knockdown inhibits proliferation and promotes apoptosis of ALL cells in vitro and attenuates cell growth in vivo.EdU assays and Annexin V/7-AAD assays showed that cell proliferation was distinctly suppressed and apoptosis was significantly increased upon PHF8 knockdown.To further explore whether PHF8 knockdown suppresses ALL tumorigenesis in vivo,we performed tumor xenograft experiments and found tumor size was significantly reduced upon PHF8 knockdown.(3)PHF8 positively regulates MEK1 in ALL cells in vitro and in vivo.qRT-PCR and Western blot showed that PHF8 knockdown inhibited the expression of MEK1.ChIP assay showed that PHF8 transcriptionally activated MEK1 expression at least partly by binding directly to the stream of TSS in MEK1 promoter and subsequently activated the MEK/ERK pathway.qRT-PCR and IHC further confirmed this conclusion in vivo.(4)The MEK/ERK pathway inhibitor PD 184352 inhibited PHF8 expression subsequently,and we elucidated a novel positive regulatory loop between PHF8 and the MEK/ERK pathway.The immunofluorescence assay showed that PHF8 expression decreased with increasing concentrations of PD 184352 in ALL cells.(5)Synergistic cooperation between PHF8 knockdown and the MEK inhibitor PD184352 enhanced lethality in ALL cells.EdU assays and Annexin V/7-AAD assays showed that cell proliferation was distinctly suppressed and apoptosis was significantly increased by PD184352 and PHF8 knockdown enhanced the lethal effects in ALL cells.2.The low expression of histone demethylase KDM7A is related to multiple clinical and biological characteristics of AML patients and may act as a new diagnostic and prognostic marker in AML.(1)Epigenetic spectrum abnormalities was found in AML patients.To explore the role of epigenetic molecules in the pathogenesis of AML,the expression of 429 epigenetic genes in 501 AML patients and 73 normal bone marrow samples in GEO database was analyzed.It was found that there were extensive abnormalities in the expression of epigenetic genes in AML patients.A total of 111 epigenetic genes were expressed differently in AML patients and controls(Fold Change>1.2,adjusted P<0.05).Among them,71 genes were up-regulated,including SMARCC1,KAT2An1,ELMSAN1,DNMT3B,SMYD3,etc.40 genes were down-regulated.The down-regulated genes were KDM7A,KAT2B,CBX7,MYBL2,TADA2B,etc.,among which KDM7A down-regulation was the most obvious.(2)KDM7A is involved in the pathogenesis and progression of AML as a tumor suppressor gene.The expression of KDM7A of bone marrow or peripheral blood samples in three independent sets including AML group and control group was detected and it was shown that KDM7A was decreased in AML patients.The expression of KDM7A in leukemia stem cell positive population was significantly lower than that in negative group.Compared bone marrow samples from patients with newly diagnosed,complete remission and recurrence showed a negative correlation between the expression of KDM7A and the disease progression of AML.Compared KDM7A expression in newly diagnosed with relapse stage AML samples from same patients,confirming that KDM7A may be involved in the progression of AML.(3)The abnormal expression of KDM7A is related to various clinical and biological characteristics of AML patients.By analyzing the different clinical and biological characteristics of KDM7A low expression group and high expression group in bioinformatics databases,it was found that the low expression of KDM7A was associated with AML FAB typing,the proportion of immature cells in peripheral blood and bone marrow,and was correlated with the expression of many known predictors of AML,such as ERG,WT1,FLT3,etc.(4)The low expression of KDM7A can be used as a poor prognostic marker of AML.By analyzing GEO and other database,it is proved that the patients with lower expression of KDM7A had shorter survival time and worse prognosis.This result was also confirmed in AML patients with normal karyotype.Conclusions1.PHF8 plays an oncogenic role and participates in disease progression in ALL.PHF8 regulates proliferation and apoptosis of ALL cells in vitro as well as tumor growth in vivo,through the interaction between PHF8 and the MEK/ERK pathway.PHF8 may serve as a potential epigenetic target for adult ALL,and the synergism of PHF8 intervention and MEK/ERK pathway inhibitors may serve as a new therapeutic strategy for adult ALL.2.This study confirmed that there were extensive abnormalities in epigenetic spectrum in patients with AML,among which the down-regulation of KDM7A expression was the most obvious.The low expression of histone demethylase KDM7A was associated with the clinical and biological characteristics of many AML patients and can be used as a new prognostic maker for AML patients.Therefore,this study suggests that KDM7A can be used as a potential target for AML diagnosis,treatment,disease progression monitoring and prognosis evaluation. |
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