Homoharringtonine Exhibits Potent Anti-tunor Effect And Affects DNA Epigenome By Targeting TET1/5hmC/FLT3 Axis

BackgroundHomoharringtonine(HHT)is a cytotoxic alkaloid originally isolated from the cephalotaxus hainanensis and has been approved for treatment of chronic myeloid leukemia(CML)with resistance and/or intolerance to tyrosine kinase inhibitors by US Food and Drug Administration(FDA),but not for AML treatment.It has been used for leukemia treatment for more than 30 years in China due to the lower price and higher efficiency.Moreover,our institute initially started the HHT-based induction regimen HAA(HHT 4 mg/m2/day,days 1-3;cytarabine 150 mg/m2/day,days 1-7;aclarubicin 12 mg/day,days 1-7)in treating de novo AML and achieved 83%complete remission rate in 2006.Afterwards,a multicenter,open-label,randomized,controlled phase 3 trial were carried out and confirmed that modified HAA regimen(HHT 2 mg/m2/day,days 1-7;cytarabine 100 mg/m2/day,days 1-7;aclarubicin 20 mg/day,days 1-7)as an alternative in treating de novo AML,especially for those with favorable and intermediate prognosis.In CML,HHT mainly inhibits protein synthesis by binding to the A site of the ribosome,however in AML,the related mechanism of HHT is still largely unknown.AML is a common disease and characterized with blockage of myeloid differentiation,malignant proliferation of immature myeloid blasts,and poor outcome.In United States,19,950 cases are diagnosed as AML with an estimated 10,430 deaths per year.Despite the common myeloid background,molecular aberrations as well as cytogenetic alterations contribute to the heterogeneity of the disease.With the development of next generation sequence,a recent study has identified 5,234 driven mutations in 1,540 AML patients,with FLT3,NPM1,DNMT3A,NRAS and complex ranking top 5.Besides,the epigenetic landscape of AML is becoming acknowledged and a new functional category including DNMT3A,TET2,IDH1 and IDH2 mutations is classified due to their important roles in formation and conversion of DNA methylation.In clinical,two DNA methyltransferase inhibitors,Azacitidine and Decitabine,are recommended by the European Leukemia Net(ELN)to patients not tolerable for intensive chemotherapy.ObjectiveTo identify the biological function of TET1 in treating human AML cell lines with different backgrounds and further confirm the result in AML mouse model.To analyze the dynamic change of the 5-methylcytosine(5mC)and 5-hydroxymethylation(5hmC)of DNA cytosine,and to explore the direct target gene and to predict its transcriptional factor.Knockdown of TET1 by shRNAs to observe the functional change and sensitivity to HHT in AML cell lines.By analyzing the 5hmC-seq results and RNA-seq results together,seeking the downstream target of TET1 and further analyzing the gene and its subsequent signaling pathway.Methods(1)We measured the IC50 value in human AML cell lines with different backgrounds in vitro by MTT,and further analyzed the cell apoptosis,cell cycle and cell differentiation after HHT treatment;we checked the sensitivity of primary mouse AML cells to HHT treatment by colony forming assay ex vivo and observed the survival curve of two AML mouse model with or without HHT treatment in vivo.(2)Explore the alteration of DNA methylation and hydroxymethylation in human AML cell lines by dot blot assay,and identify the responsive gene TET1(not TET2/TET3)by qPCR and RNA-seq data;perform nuclear run-on assay to check the initial rate of transcription,predict the functional transcriptional factor after HHT treatment and confirm the results by drug affinity responsive stability assay(DARTs)and chromatin immunoprecipitation assay(ChIP).(3)Apply the shRNAs to establish the stable TET1 knockdown human AML cell lines,and study the biological functions in these cells,including cell apoptosis,cell cycle,cell differentiation and sensitivity to HHT treatment.(4)Combined analysis of the DNA 5hmC-seq results and RNA-seq results to identify the target gene FLT3;integrative analysis of our HHT-treatment RNA-seq data with published Tetl knockout RNA seq data to identify pathways or gene sets that were commonly affected by both HHT treatment and Tetl knockout;verify the results with chromatin immunoprecipitation assay and Western blots;finally confirm the results in primary human AML patients。Results(1)HHT displays potential therapeutic significance in AML by inducing cell apoptosis,cell cycle arrest,as well as cell differentiation in vitro;HHT significantly suppressed colony-forming activity and decreased cell proliferation of primary leukemic cells in a dose-dependent manner ex vivo;HHT can effectively delay onset of leukemia,inhibit leukemic progression,and prolong survival of AML mice in vivo.(2)HHT downregulates global 5hmC level by targeting TET1 in leukemic cell.(3)Knockdown of TET1 displays similar phenotypes with HHT treatment in AML.(4)FLT3 is a direct target of HHT-1 TET1/5hmC axis,and MYC was identified as a crucial downstream target of HHT-1 TETl/5hmC/FLT3 axis;AML cells and patients with FLT3 mutations are quite sensitive to HHT treatment.ConclusionIn this study,we show that HHT is effective in treating AML with different backgrounds both in vitro and in vivo.What’s novel,we found HHT reduces global 5hmC abundance via targeting TET1,a member of ten-eleven translocation(TET)family which promotes DNA demethylation by converting 5mC to 5hmC,5-formylcytosine(5fC)and 5-carboxylcytosine(5caC).Cytosine methylation is a major modification in genomic DNA and is crucial in transcription regulation.Direct binding between HHT and SP1 inhibits transcriptional efficiency of TET1,and by this way,HHT suppresses TET1 expression and leads to global down-regulation of 5hmC levels.Via 5hmC-seq along with RNA-seq,we demonstrate that FLT3 is a direct target of HHT-I TET1/5hmC.Moreover,primary FLT3-ITD positive patients display high sensitivity to HHT treatment.Altogether,our study identifies a specific mechanism involving HHT-induced decreased 5hmC modification in treating AML and proposes the application of HHT be a promising treatment for AML patients,especially those with FLT3 mutations.