The Mechanism Research Of Transcription Factor Mediated Differentiation Of M2b Leukemia Cells

All-trans retinoic acid(ATRA)induced differentiation therapy,a breakthrough for acute promyelocytic leukemia(APL)treatment,has attracted worldwide attention.However,differentiation therapy for other leukemias is still waiting to be conquered.In this project,we used Kasumi-1 cell line,which derived from an acute myeloid leukemia(AML]patient with t(8;21)chromosomal translocation(M2b type of AML),as a model to look for new treatment approaches by studying induced differentiation mechanism in M2b leukemia cells.After screening a series of hematopoiesis related transcription factors,we found that CEBPA overexpression could induce granulocytic differentiation of Kasumi-1 cells.In order to further clarify the mechanism,transcriptome sequencing(RNA-seq)of wide-type and CEBPA-overexpressed Kasumi-1 cells were performed.Total 733 differentially expressed genes were identified between the two groups.Among these,a set of hematopoietic stem cell(HSC)self-renewal associated genes were down-regulated,and many granulocytic differentiation associated genes were up-regulated remarkably in the CEBPA-overexpressed Kasumi-1 cells as compared with the control group.KEGG and Gene Set Enrichment Analysis(GSEA]also revealed that the CEBPA-overexpressed Kasumi-1 cells exhbited granulocytic maturation but not HSC characteristic.More interestingly,there emerged a significant correlation between the CEBPA regulated genes and the target genes of AML1-ETO-containing transcription factor complex(AETFC),which is critical for M2b leukemogenesis.Consistent with this,Western Blot showed that CEBPA down-regulated the protein levels of most members of the components of AETFC in Kasumi-1 cells.Further analysis revealed that there was only one AETFC component,LYL1,appearing to be down-regulated at mRNA level in the CEBPA-overexpressed group.And,furthermore GSEA results also indicated a significant correlation between the CEBPA-overexpressed group and our previous LYL1 knockdown data of Kasumi-1 cells.At cellular level,LYL1 could facilitate AML1-ETO to recruit LDB1 and LMO2,and LYL1 knockdown would affect the stability of AETFC,leading to decrease of the AETFC components at peotein level.Indeed,lueiferase reporter gene assays indicated that CEBPA suppressed the transcription activity of LYL1 promoter.Overexpression of LYL1 partially rescued cell differentiation induced by CEBPA,Knockdown of LYL1 increased the sensitivity of Kasumi-1 cells to VD3 derivative which induces Kasumi-1 cells to granulocytic differentiation.Taken together,our research revealed a new mechanism of differentiation of M2b leukemia cells,CEBPA partially affects AETFC stability through regulating LYL1,leading to granulocytic differentiation of the leukemia cells.