Synergistic Effect Of Chidamide And Venetoclax On Apoptosis Of Acute Myeloid Leukemia Cells And Its Mechanism

Acute myeloid leukemia(AML)is a hematological malignancy with low remission rate and high recurrence rate.The 5-year overall survival rate is only about 25%.Overexpression of anti-apoptotic protein Bcl-2 is associated with lower overall survival rate in AML patients.Venetoclax is a selective inhibitor of Bcl-2,which has a significant effect in AML,but single drug resistance often occurs due to the high expression of Mcl-1 protein.How to overcome drug resistance and improve the curative effect has become an urgent problem to be solved.Chidamide is a deacetylase inhibitor independently developed in China.Studies have confirmed that chidamide can down regulate the expression levels of Bcl-2 and Mcl-1,and induce apoptosis.We wonder whether the combination of Venetoclax and chidamide can synergistically inhibit AML leukemia.The purpose of this study is to explore the anti leukemia effect and mechanism of Venetoclax combined with chidamide.The following three parts were studied and found:First,Venetoclax combined with chidamide can synergistically promote apoptosis of AML cell lines and primary cells;Second,transcriptome sequencing,RT-qPCR and Western blot showed that chidamide synergistically promotes apoptosis of AML cells by inhibiting the activation of PI3K/AKT pathway and Jak2/STAT3 pathway.Thirdly,Compared with the single drug group,the combination of chidamide and Venetoclax could significantly inhibit the tumor progression and prolong the survival time of mice.The research results of this project provide a new strategy and theoretical basis for solving the problem of drug resistance of Venetoclax in clinic.Part Ⅰ:Synergistic effect of chidamide and Venetoclax on apoptosis of acute myeloid leukemia cellsObjectivesTo observe the effects of Venetoclax combined with chidamide on proliferation,apoptosis and cell cycle of AML cell lines(OCI-AML3,THP-1,MV4;11,MOL13)and primary cells.Methods1.Different concentrations of Venetoclax and chidamide were used to treat AML cell lines(OCI-AML3,THP-1,MV4;11,MOLM13)for 24h,48h and 72h respectively.The proliferation inhibition levels of the four cell lines were detected by CCK method,and the corresponding IC50 values were calculated.2.Different concentrations of Venetoclax and chidamide were used to treat AML cell lines(OCI-AML3,THP-1,MV4;11,MOLM13)for 8h and 24h respectively.The apoptosis rate was detected by flow cytometry,and CI value was calculated in combination group.3.Different concentrations of Venetoclax,chidamide and their combination were used to treat primary AML cells for 24 hours,respectively.The apoptosis was detected by flow cytometry.4.Different concentrations of Venetoclax and chidamide were used to treat AML cell lines for 24h respectively.The changes of cell cycle and mitochondrial membrane potential were detected by flow cytometry.5.The above experiments were repeated three times.SPSS22.0 software was used for statistical analysis.Results1.Both Venetoclax and chidamide could inhibit the proliferation of AML cell lines in a time and concentration dependent manner.2.Both Venetoclax and chidamide could promote the apoptosis of AML cell lines and primary cells in a time and concentration dependent manner.The combined effect of the two drugs was further enhanced,and had a synergistic effect(CI<1).3.The cell cycle of AML cell line was arrested in G1 phase by single drug of chidamide,and in S phase by single drug of chidamide and Venetoclax.The blocking effect was further strengthened by the combination of the two drugs.Both Venetoclax and chidamide could decrease the mitochondrial membrane potential of AML cell lines,and the combination of the two drugs could further enhance the effect.Part Ⅱ:Mechanism of synergistic effect of chidamide and venetoclax on apoptosis of acute myeloid leukemia cellsObjectivesStudy the mechanism of synergistic effect of chidamide and venetoclax on apoptosis of AML cells.Methods1.RT-qPCR and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Mcl-1 in AML cell lines.2.The expressions of apoptosis related molecules at mRNA and protein levels were detected by RT-qPCR and Western blot respectively.3.The most suitable concentration of venetoclax and chidamide were used to treat venetoclax drug-resistant cell line OCI-AML3 for 24 hours.The transcriptome sequencing was performed to screen the differentially expressed genes and enrich KEGG pathway.4.RT-qPCR and Western blot were used to verify the expression of differential molecules in the enrichment pathway at mRNA and protein levels;5.The above experiments were repeated three times.Spss22.0 software was used for statistical analysis.Results1.Bcl-2 and Mcl-1 were both expressed in AML cell lines(OCI-AML3,THP-1,MV4;11,MOLM13)at mRNA and protein levels.The IC50 value of venetoclax in AML cell lines had no correlation with the expression levels of Bcl-2 and Mcl-1(p>0.05).2.At mRNA level,the expressions of Bcl-2 and Bcl-xl were significantly decreased in the single drug group and the combined group(p<0.05).The expression of Mcl-1 was up-regulated by venetoclax and down regulated by chidamide.The expression of Mcl-1 was further decreased after combination(p<0.05).3.At the protein level,the expressions of cf-caspase3,cf-PARP,γ-H2AX and Bax/Bak in the combination group were significantly higher than those in the control group and single drug group.The expression of Bcl-2 was down-regulated by chidamide.The expression of Bcl-2 was decreased after the combination of chidamide and venetoclax.The expression of Mcl-1 was up-regulated by venetoclax and down regulated by chidamide.The expression of Bim was decreased in the single drug group of venetoclax and chidmide,and the effect was further enhanced after the combination of the two drugs.4.Both venetoclax and chidamide could significantly up-regulate the expression of p21 gene and protein,while chidamide could down-regulate the expression of CDK2 and myc gene and protein,which was further enhanced after combined with venetoclax.5.Transcriptome sequencing showed that the differentially expressed genes were mainly enriched in PI3K-AKT pathway and JAK2/STAT3 pathway in combination group compared with venetoclax monotherapy group.6.RT-qPCR and Western blot confirmed the results of transcriptome sequencing:1)Chidamide down-regulated the expression of AKT gene and p-AKT protein,up-regulated the expression of p21,down regulated the expression of CDK2 and myc.2)The expression of SOCS3 gene and protein was up-regulated by chidamide,which down regulated the expression of JAK2 gene and p-JAK2 protein,then up-regulated the expression of p21,down regulated the expression of CDK2 and c-myc,and down regulated the expression of Bcl-2 and Mcl-1.3)The expression of HDAC1 gene and protein was up-regulated and the expression of p21 was up-regulated in the single chidmide and combined groups.Part Ⅲ:Synergistic effect of chidamide and Venetoclax on apoptosis of acute myeloid leukemia cells in vivoObjectivesEstablish the mouse model of AML xenotransplantation and observe the anti-AML leukemia effect of chidmide combined with venetoclax in vivo.MethodsThe mouse model of AML xenotransplantation was established by NSG.The mice were randomly divided into four groups:control group,monotherapy group of venetoclax and chidamide and combination group.The weight of mice in each group was monitored;the changes of tumor load in vivo were observed by vivo imaging;the extramedullary infiltration of AML cells was detected by immunohistochemistry;the survival time of mice was observed,and the survival curve was drawn.ResultsCompared with single drug group and control group,chidamide combined with venetoclax could significantly inhibit the disease progression and prolong the survival time of AML mice.Conclusion1.Both venetoclax and chidamide inhibited the proliferation of AML cells,decreased the mitochondrial membrane potential and promoted the apoptosis of AML cells in a time and concentration dependent manner.2.Chidamide may synergistically promote apoptosis of AML by reducing the expression of Mcl-1 and Bcl-2.3.Chidamide combined with venetoclax may inhibit the proliferation of AML cells by blocking the cell cycle in G1 phase and the primary cells in S phase.4.Chidamide may inhibit the expression of AKT,a key molecule of PI3K-AKT signaling pathway;up-regulate the expression of SOCS3,a key molecule of JAK2/STAT3 pathway,then up regulation of p21 expression,down-regulation of CDK2 and myc expression to cause cell cycle arrest and inhibit cell proliferation.5.Chidamide may up regulate HDAC1 and deacetylated H3 protein expression,increase the degree of histone acetylation,and inhibit cell proliferation by up regulating p21 gene level.6.Chidamide combined with venetoclax can significantly inhibit the tumor progression and prolong the survival time of AML xenograft mice.