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

Acute myeloid leukemia(AML)is a hematopoietic tumor characterized by the rapid growth of myeloid clonal cells.It accounts for about 70% of acute leukemia.The incidence of AML is about 3.4/100,000,and it has been increasing in recent years.With the advancement of treatment,about 60% to 80% of adult AML patients can obtain complete remission(CR)through the induction chemotherapy,while most AML patients will show resistance to chemotherapy at some stage in the course of the disease.About 20 % of AML patients appear to be primary refractory,more than 50%of young AML patients and up to 90% of elderly AML patients will eventually relapse.The prognosis of relapsed and refractory AML is extremely poor.In addition to clinical trials,the main treatments for relapsed and refractory AML include salvage chemotherapy,target and immunotherapy,and allogeneic hematopoietic stem cell transplantation(Allo-HSCT).The anti-apoptotic Bcl-2 family members,such as Bcl-2,Bcl-x L,and Mcl-1,BH3-only proteins such as Bim that promote apoptosis,prevent the activation of proapoptotic proteins Bax and Bak,and ultimately prevent the outer mitochondrial membrane from permeating,cytochrome c release and apoptosis.The BCL-2 gene was discovered as an anonymous partner of the immunoglobulin heavy chain locus in the common translocation t(14;18)of follicular lymphoma.The survival of AML cells depends on the expression of BCL-2.Overexpression of BCL-2 protein protects cells from apoptosis,and is related to chemotherapy resistance.Therefore,inhibiting antiapoptotic Bcl-2 family members is promising in the treatment of AML.Venetoclax is an oral selective small molecule inhibitor targeting BCL-2.It is an only-BH3 protein mimic.The BH3 mimic inhibits anti-apoptotic proteins,such as BCL-2,BCL-w and BCL-XL,by binding Bcl-2 protein to replace Bim and other proapoptotic proteins,thereby changing the permeability of the outer mitochondrial membrane and activating the cysteine protease,which promotes the activation of the intrinsic apoptotic pathway and causes cell death.Venetoclax has high efficacy and safety in different blood diseases,especially in chronic lymphocytic leukemia(CLL)and AML.In clinical trials,the CR rate of venetoclax monotherapy for R/R AML was only 19%.Studies have shown that the resistance mechanism of venetoclax may be related to the up-regulation of Mcl-1.Can it be combined with other drugs to achieve better clinical treatment effects? Homoharringtonine(HHT)has been used for the treatment of hematological malignancies for the past 30 years as a domestic drug in China.It can down-regulate the expression of Mcl-1 to promote cell apoptosis,and its combined application with venetoclax may have a synergistic effect.The purpose of this project is to explore the effect and mechanism of venetoclax and homoharringtonine in combination with anti-leukemia.The research is mainly divided into the following three parts: First,Venetoclax combined with HHT has a synergistic pro-apoptotic effect on AML cell lines and primary cells;Secondly,transcriptome sequencing found that HHT inhibits PI3K/AKT,MAPK/ERK pathways and activates p53 pathways cooperating with venetoclax to promote AML cell apoptosis,it was verified by RT-q PCR,Western blot and cycle experiments.Thirdly,venetoclax combined with HHT can significantly inhibit the tumor growth of AML xenograft mice and prolong its survival period compared with the single-agent group.The results of this study provide a theoretical basis for exploring the combined treatment strategy of Venetoclax in AML.Part Ⅰ Synergistic effect of Homoharringtonine and Venetoclax on apoptosis of acute myeloid leukemia cells ObjectivesTo observe the effects of Venetoclax combined with HHT for AML cell lines(THP-1,OCI-AML3,MV4;11,MOLM13)and AML primary cells on the proliferation,apoptosis and mitochondrial membrane potential.Methods1.The CCK-8 test was used to detect the proliferation inhibition levels of AML cell lines treated with different concentrations of Venetoclax and HHT for different time(24h,48 h,72h),and the IC50 values were calculated respectively.2.The flow cytometry was used to detect the apoptosis rate of AML cell lines treated with different concentrations of Venetoclax and HHT singlely and together for different time(8h,24h),and the CI values were calculated.3.The flow cytometry was used to detect the apoptosis rate of primary AML cell s treated with different concentrations of Venetoclax and HHT singlely and together for 24 h,and the CI values were calculated.4.Flow cytometry was used to detect the mitochondrial membrane potential of AML cell lines treated with different concentrations of Venetoclax and HHT singlely and combined for 24 h.5.The above experiment was repeated three times independently.The data were described by mean±standard deviation,independent or paired-sample t-test was used for comparison between groups,P<0.05 was considered statistically significant,and SPSS 18.0 software was used for statistical analysis.Results1.Venetoclax and HHT could inhibit the proliferation of AML cell lines(THP-1,OCI-AML3,MV4;11,MOLM13)in a time and concentration dependent manner.2.Venetoclax and HHT could promote the apoptosis of AML cell lines(THP-1,OCI-AML3,MV4;11,MOLM13)in a time and concentration dependent manner.The apoptosis-promoting effect of the combination of the two drugs was further enhanced,and showed synergistic pro-apoptotic efficacy(CI <1).3.Venetoclax and HHT could promote the apoptosis of primary AML cells in a time and concentration dependent manner.The pro-apoptotic effect of the combination of the two drugs was further enhanced,and showed synergistic proapoptotic efficacy(CI<1).4.Venetoclax and HHT alone could reduce the mitochondrial membrane potential of AML cell lines(THP-1,OCI-AML3,MV4;11,MOLM13)in a concentration dependent manner,and the combination of the two drugs further enhanced the effect(P<0.05).Part Ⅱ Mechanism of synergistic effect of HHT and venetoclax on apoptosis of acute myeloid leukemia cells ObjectivesTo study the mechanism of synergistic effect of HHT and Venetoclax on promoting AML cell apoptosis.Methods1.The expression of Bcl-2,Mcl-1,and Bcl-xl at the m RNA and protein levels in AML cell lines(THP-1,OCI-AML3,MV4;11,MOLM13)and AML primary cells(AML#04,AML#05,AML#06,AML#07)were detected by RT-q PCR and Western blot respectively.2.The expression of apoptosis-related molecules at m RNA and protein level of AML cell lines and primary AML cells treated with Venetoclax and HHT monotherapy and combination were detected by RT-q PCR and Western blot.3.The optimal concentration of Venetoclax and HHT were used to treat OCIAML3 cells for 24 h,transcriptome sequencing analysis was performed to screen the relevant differentially expressed genes and enrich KEGG pathways and Gene Ontology analysis.4.Flow cytometry was used to detect cell cycle changes of AML cell line after treating with Venetoclax and HHT for 24 h.5.The expression changes of cycle-related genes and proteins at the m RNA and protein levels were detected by RT-q PCR and Western blot.6.The expression of differential molecules in the enrichment pathway at m RNA and protein levels were detected by RT-q PCR and Western blot.7.The above experiments were repeated three times.SPSS 18.0 software was used for Statistical analysis.Results1.Bcl-2,Mcl-1 and Bcl-xl were all expressed in AML cell lines(THP-1,OCIAML3,MV4;11,MOLM13)and AML primary cells(AML#04,AML#05,AML#06,AML#07)at m RNA and protein levels,there was no correlation between the IC50 value of Venetoclax and the expression levels of Bcl-2,Mcl-1,and Bcl-xl proteins(p>0.05).2.Venetoclax decreased the Bcl-xl gene expression of MV4;11 cells(p<0.05),and increased the Mcl-1 gene expression of AML#07 primary cells(p<0.05);HHT decreased the Bcl-xl gene expression of OCI-AML3 and AML#06 cells(p<0.05).3.Venetoclax increased the expression of cf-Caspase3,cf-PARP1,γH2AX protein,and the expression was further increased after combining with HHT.4.At the protein level,Venetoclax increased the expression of Mcl-1,HHT decreased the expression of Mcl-1,and the expression of Mcl-1 in the combination group decreased compared with the Venetoclax group.Venetoclax and HHT motherapy increased the expression of Bim,and the combination of the two drugs further increased the expression of Bim.5.By transcriptome sequencing,the differentially expressed genes were mainly enriched in PI3K-AKT,p53 pathway and MAPK/ERK pathway compared the combination group with the Venetoclax group.The differentially expressed genes mainly participated in cell cycle regulation by GO enrichment.6.HHT could block the cell cycle of AML cell lines in G1 phase,and the effect was further enhanced by the combination of HHT and Venetoclax.7.HHT single agent and combined with Venetoclax could significantly upregulate the expression of P21 at m RNA and protein level,and could down-regulate the expression of CDK2 and Myc at protein level.8.At the m RNA level,Venetoclax increased the expression of AKT and decreased the expression of p53,HHT increased the expression of p53 gene and decreased the expression of ERK gene.Compared with the Venetoclax group,the combination group decreased the AKT and ERK expression,increased the p53 expression,and the difference was statistically significant(P<0.05).9.At the protein level,Venetoclax decreased the expression of p-AKT,HHT decreased the expression of p-AKT and p-ERK,increased the expression of p53.Compared with the Venetoclax group,the combination group increased the p53 expression and decreased the p-AKT,p-MEK,p-ERK expression.Part III Synergistic effect of HHT and Venetoclax on apoptosis of acute myeloid leukemia cells in vivo ObjectivesEstablish an AML xenograft mouse model an and observe the anti-AML effect of HHT in cooperation with Venetoclax in vivo.MethodsThe mouse model of AML xenotransplantation was established with NSG mouse.The mice were randomly divided into 4 groups: the control group,the monotherapy of venetoclax group,the monotherapy of HHT group and the combination group.The weight of mice in each group was monitored,and the tumor load changes in vivo were observed by vivo imaging.The extramedullary infiltration of AML cells were detected by immunohistochemistry,the h CD45+ cell ratio of bone marrow was detected by flow cytometry,the survival time of mice were observed and recorded and the survival curve was drawn.ResultsHHT combined with Venetoclax could significantly inhibit the disease progression of AML mice,and significantly prolong the survival time of AML mice.Conclusions1.Both Venetoclax and HHT can inhibit the proliferation of AML cells,reduce the mitochondrial membrane potential of AML cells,and have a synergistic effect on promoting AML cell apoptosis,which is time-and concentration-dependent.2.HHT plays a synergistic effect of Venetoclax to promote AML cell apoptosis by reducing the expression of Mcl-1 protein.3.HHT combined with Venetoclax may inhibit cell proliferation by blocking the AML cell cycle in the G1 phase.4.HHT may enhance the pro-apoptotic effect of Venetoclax by inhibiting the PI3K-AKT,MAPK/ERK signaling pathway,and activating the p53 pathway.5.HHT combined with Venetoclax can significantly inhibit the progression of leukemia in AML xenograft transplanted mice and prolong the survival time of AML mice.