The Role Of FTO And ALKBH5 Mediated M~6A RNA Demethylation In The Development Of Human Acute Myeloid Leukemia

Objective:Acute myeloid leukemia(AML)is a hematologic malignancy with an unfavorable prognosis.Abetter understanding of AML pathogenesis and chemotherapy resistance at the molecular level is essential for the development of new therapeutic strategies.Apart from DNA methylation and histone modification,RNA epigenetic modification,another layer of epigenetic modification,also plays a critical role in gene expression regulation.Among more than 150 kinds of RNA epigenetic modifications,N6-methyladenosine(m~6A)is the most prevalent internal mRNA modification in eukaryotes and is involved in various biological processes,such as circadian rhythms,adipogenesis,Tcell homeostasis,spermatogenesis,and the heat shock response.As a reversible and dynamic modification,m~6A is deposited on specific target RNAs by methyltransferases and is removed by demethylases.Moreover,m~6A binding proteins recognize m~6A modifications,influencing RNA splicing,stability,translation,nuclear export,and localization at the posttranscriptional level.Emerging evidence suggests that the dysregulationof m~6A modification is implicated in tumorigenesis,including that of AML.Demethylases,also known as erasers,are proteins that remove m~6A methylation modifications for reversible and dynamic regulation;these erasers include fat mass and obesity-associated protein(FTO)and Alk B homolog 5(ALKBH5).However,the roles of FTO and ALKBH5 in AML are not entirely clear.Therefore,ourresearch focused onm~6A RNA demethylation mediated by FTO and ALKBH5 to explore their roles in the development of AML.Part 1.The expression of FTO and ALKBH5 in adult acute myeloid leukemia patients and normal and malignant myeloid differentiationMaterials and Methods:We evaluated the expression levels of FTO and ALKBH5 in AML patients and healthy volunteers using GSE13204.We also used GSE24759 to evaluate FTO gene expression during normal hematopoiesis.The acute promyelocytic leukemia(APL)cell line NB4 was selected as the model cell for studying the myeloid differentiation of AML,using all trans retinoic acid(ATRA)to induce cell differentiation.Western Blot and quantitative real time polymerase chain reaction(qPCR)were used to detect the protein and mRNA expression levels of FTO and ALKBH5.Peripheral blood or bone marrow samples were collected from newly diagnosed AML patients in our institute.The expression level of FTO and ALKBH5 were detectedby qPCR.Results:1.The demethylases(FTO,ALKBH5)were all highly expressed in various subtypes of AML patients.2.FTO was significantly overexpressed in AML patients carrying common chromosomal translocations such as t(15;17)or11q23.ALKBH5,another m~6A demethylase,was significantly upregulated in AML patients with t(8;21),t(15;17)and 11q23.3.FTO mRNA was highly expressed in human hematopoietic stem/progenitor cells but downregulated in mature differentiated myeloid cells.4.FTO and ALKBH5 were downregulated during myeloid differentiation in AML cells.Conclusion:1.FTO and ALKBH5 were all highly expressed in AML patients.2.FTO and ALKBH5 were all highly expressed in various subtypes of AML patients.3.FTO was downregulated during normal hematopoiesis.4.FTO and ALKBH5 were downregulated during myeloid differentiation in AML cells.Part 2.The biological effect of FTO and ALKBH5 on acute myeloidleukemia cell linesMaterials and Methods:Stably FTO or ALKBH5 knockdown in AML cell lines,such as NB4,HL-60/S4 and K562,were constructed with a lenti-viral system.MTT assayto determine cell proliferation;colony-forming assay for cell tumorigenic ability;flow cytometry for cell differentiation,apoptosis and chemoresistance.Global m~6A methylation level was measured in cells.Results:1.Western Blot and qPCR detection revealed that FTO and ALKBH5 cell knockdown models were successfully constructed in AML cell lines(NB4,HL-60/S4 and K562).2.The Epi Quik m~6A RNA methylation quantification kit and LC-MS mass spectrometry were used to detect changes in the overall m~6A level of cells after FTO and ALKBH5 knockdown.It was found that the overall m~6Alevels of NB4,HL-60/S4 and K562 cells after FTO and ALKBH5 knockdown increased.3.The MTT assay showed that the proliferation of NB4,HL-60/S4 and K562 cells was significantly slowed down after FTO and ALKBH5 gene knockdown.4.Flow cytometry detected cell differentiation,and found that FTO and ALKBH5 gene knockdown promoted the differentiation of NB4,HL-60/S4 and K562 cells or increased the sensitivity of leukemia cells to phorbol myristate acetate(PMA)induced differentiation.5.Flow cytometry detected cell apoptosis,and found that FTO and ALKBH5 gene knockdown promoted the apoptosis of NB4,HL-60/S4 and K562 cells or increased the sensitivity of leukemia cells to homoharringtonine.6.The results of the soft agar clone formation experiment showed that after FTO and ALKBH5 gene knockdown,the number of clones in NB4 and K562 cells was decreased,and the clone volume became smaller,suggesting that the clone formation ability was significantly reduced.Conclusion:1.FTO or ALKBH5 knockdown significantly inhibited AML cell proliferation,decreased colony numbers,suppressed chemoresistance,as well as promoted apoptosis and differentiation in vitro.2.As m~6A demethylases,knockdownof FTO or ALKBH5 can also increase the global m~6A modification level.Part 3.FTO regulates m~6A methylation of S100A9 mRNA in acute myeloid leukemiaMaterials and Methods:Transcriptome sequencing and methylated RNA immunoprecipitation sequencing(MeRIP-Seq)were performed to find potential target genes of FTO knockdown.We evaluated the expression levels of FTOand S100A9 in AML patients using datasets such as The Cancer Genome Atlas(TCGA)and GSE37642.Gene-specific m~6A qPCR assay was performed to quantify m~6A methylation level of target mRNA.The half-lifeof mRNA was detected by RNA stability assay.Stably overexpressing S100A9 in AML cell lines,such as NB4 and HL-60/S4,were constructed with a lenti-viral system.Flow cytometry detected cell differentiation,apoptosis and chemotherapy resistance.Results:1.Gene set enrichment analysis(GSEA)showed the enrichment of differentially expressed genes between NB4-sh FTO-2 and NB4-sh CTR.Upon FTO knockdown,there was an increase in the expression levels of the genes down-regulated by leukemia oncogenes HOXA9 and MEIS1 and the gene set down-regulated in APL pathogenesis.2.MeRIP-Seq revealed thatthe m~6A sites were mainly enriched at the motif GGACU in the exon and near stop codon of mRNA.3.The 549 mRNAs with significantly higher methylation peaks and differential expression in the NB4-sh FTO group were potential mRNA targets after FTO knockdown.4.The downstream target gene S100A9 was negatively correlated with the expression level of FTO in the AML patients datasets such as TCGA,GSE37642-GPL96 and GSE37642-GPL570.5.Using GSE24759,it was found that the expression level of S100A9 was up-regulated during normal hematopoietic differentiation.6.S100A9 wasup-regulated during myeloid differentiation in AML cells.7.The qPCR results of specimens in our institution found that the expression level of S100A9 mRNA in healthy volunteers was significantly higher than that in newly diagnosed AML patients.In AML patients,the expression levels of S100A9 and FTO were also negatively correlated.8.After knockdown FTO in NB4 and HL-60/S4 cells,the mRNA and protein levels of S100A9 were up-regulated.9.MeRIP-Seq data and gene-specific m~6A qPCR assayfound that knockdown FTO in NB4 cell can promote m~6A methylation on the downstream target S100A9.10.The half-life of S100A9 mRNA in NB4 and HL-60/S4 cells was significantly prolonged after FTOknockdown.11.Western Blot and qPCR results found that the S100A9 overexpression cell models were successfully constructed in NB4 and HL-60/S4 cell lines.12.Flow cytometry detected cell differentiation,and found that S100A9 overexpression promoted the differentiation of NB4 and HL-60/S4 cells.13.Flow cytometry detected cell apoptosis,and found that S100A9 overexpression promoted the apoptosis of HL-60/S4 cell and increased the sensitivity of NB4 cell to homoharringtonine.Conclusion:Mechanistically,FTO knockdown promoted m~6A methylation on its downstream target S100A9,which therefore increased S100A9 mRNA stability through an m~6A dependent mechanism.Upregulation of S100A9 caused by FTO knockdowninhibited AML progression.FTO maintains the undifferentiated state of AML cells by suppressing S100A9 mRNA stability through decreasing m~6A modification of its transcript.