| Acute myeloid leukemia(AML)is a heterogeneous,haematologically aggressive bone marrow tumor characterized by irreversible expansion of differentiated and functionally defective precursor myeloid cells,which exhibit maturation arrest at specific stages of hematopoietic differentiation and are characterized by different subtypes of leukemia,classified according to the FAB classification as M0-M7.According to studies,there are two main types of mutated genes in AML,those associated with the failure of blood cells to differentiate normally and those associated with abnormal proliferation of immature blood cells,including FLT3,a receptor tyrosine kinase that plays a regulatory role in the physiological processes of normal haematopoietic stem cells and is expressed on AML cells,and mutations in the FLT3 gene are among the most Mutations in the FLT3gene are one of the most common mutations in patients with intermediate-risk AML,most often associated with a normal karyotype,and are located on chromosome 13q12,which contains 24 exons.Two-thirds of the total number of mutations are internal tandem duplications of the proximal structural domain(ITD),a proto-oncogene whose mutations cause abnormal expansion of malignant cells and,through activation of the tyrosine kinase region,phosphorylate FLT3 itself leading to the activation of multiple signaling pathways that promote proliferation and survival of AML cells.The E2F family encodes nuclear transcription factors that are critically involved in the regulation of the cell cycle and eight genes have been identified,E2F1-8.All members of the family contain a DNA-binding structural domain(DBD)that binds to the gene promoter to initiate transcription.E2F1 remains a controversial prognostic factor,but its high expression in the disease is strongly associated with cell proliferation and influences the clinical outcome.Database and clinical analyses have found that E2F1 is overexpressed in most solid tumors,except for brain and central nervous system cancers,such as liver,lung,breast,colorectal,kidney,and ovarian cancers.In addition to its important role in regulating key cellular processes including cell cycle arrest and apoptosis,the transcription factor E2F1 has also been reported to be closely associated with cellular energy metabolism.The results of previous pre-experiments suggest that E2F1 expression is abnormally up-regulated in AML patients,especially in AML patients carrying the FLT3-ITD mutation.The study aimed to elucidate the role and possible molecular mechanism of the E2F1 gene in AML with FLT3-ITD mutation and to provide possible targets for the potential treatment of AML.Objective:To investigate the role of E2F1 in the differentiation and proliferation of FLT3-ITD mutated AML cells and the related molecular mechanisms.Methods:1.E2F1 expression in FLT3-ITD~+AML cells and patient samples and during in vitro differentiation of CD34~+HSPCsWestern blot was used to detect the difference in E2F1 gene expression in peripheral blood samples from healthy volunteers and AML patients.The FLT3-ITD-positive cell lines Molm-13 and Mv4-11,and FLT3-ITD-negative cell line THP-1 were cultured in vitro,and the changes in m RNA and protein levels of the E2F1 gene in the three cell lines with different degrees of mutation were examined by Western blot and Q-PCR.CD34-positive cells were isolated from cord blood using a high-gradient magnetic cell sorting system(MACS),and differentiation was induced with hematopoietic growth factor G-CSF and IL-3 in a liquid culture system.q-PCR was used to detect the expression of E2F1in CD34+HSPCs cells during myeloid differentiation at different time points.2.Effects of E2F1 knockdown and overexpression on the proliferation and differentiation of AML cellsWe separately transfected two independent lentiviral short hairpin RNAs(sh E2F1#1and sh E2F1#2)to construct a stably silenced cell model and used the Ubi-E2F1-MCS-SV40-PURO lentivirus to construct an exogenous stable overexpression of E2F1 in the cell line.After the cell models were successfully constructed,the protein expression levels of proliferation-related genes(PCNA,CDK2)were measured by Western blot,the proliferation activity of the cells was measured by CCK-8,the cell proliferation was assessed by Ki-67 fluorescence staining,the cell cycle progression,and the cell differentiation ratio were determined by Flow Cytometry(FCM),and the contraction of the nuclei was observed by Richter-Gimza Flow Cytometry(FCM)to determine cell cycle progression and cell differentiation ratios,Richter-Gimza staining to observe nuclear contraction,and Colony Formation Unit(CFU)to determine cell clonogenic capacity.3.FLT3-ITD-driven hyperproliferation of AML cells was inhibited by E2F1 deletionChanges in protein and m RNA levels of the E2F1 gene were detected by Western blot,q-PCR after treatment with AC220 at different time points using specific phosphatase inhibition of FLT3-ITD,and a lentiviral system was constructed to induce ectopic expression of FLT3-ITD in human CD34~+HSPCs and THP-1 cells(FLT3-WT).Western blot was performed to detect the protein expression levels of cell proliferation-related genes(PCNA,CDK2),CCK-8 to detect the proliferative activity of the cells,immunofluorescence staining to detect the expression of E2F1,and colony formation assay(CFU)to determine the clonogenic ability of the cells.4.FLT3-ITD reprograms E2F1-dependent transcription of genes involved in purine metabolismImmunofluorescence staining was used to detect the effect of FLT3-ITD overexpression on E2F1 expression.nucleoplasmic proteins were extracted and verified using a nucleoplasmic isolation kit,and the fluorescence changes of E2F1 were observed by laser confocal.PCR was performed to detect changes in the expression of the enriched genes.5.FLT3-ITD/E2F1 axis initiates purine metabolism transcriptional programsLiquid chromatography-mass spectrometry(LC/MS)analysis was performed to compare changes in energy metabolites between experimental groups.6.The absence of E2F1 inhibits the progression of AML cells in NSG miceNSG mice were injected with the E2F1 knockout Molm-13 cell line in the tail vein.The leukemic burden of the mice was assessed by spleen size and live imaging,the infiltration of organs was observed by hematoxylin-eosin(HE)staining and the proportion of Molm-13 cells in the peripheral blood of the mice was detected by flow cytometry to compare the length of survival between the two groups.Results:1.E2F1 gene was highly expressed in AML patients compared with healthy controls.Compared with highly differentiated CD34 negative HSCS,E2F1 was highly expressed in CD34 positive HSCS and was significantly down-regulated during differentiation.2.E2F1 silencing inhibited cell proliferation and induced cell differentiation,and E2F1deletion enhanced the proportion of AML cells with differentiation induced by differentiation agents.3.The expression of E2F1 gene was regulated by FLT3-ITD,and the FLT3-ITD-driven AML cell proliferation was reduced after E2F1 knockdown.4.The overexpression of FLT3-ITD promoted the expression of the E2F1 gene,which was significantly increased in the nucleus.The sequencing results showed that the genes binding to E2F1 were closely related to purine metabolism.5.Targeted metabolomics analysis showed that the expression of purine metabolism-related metabolites decreased after further knockdown of E2F1.6.The results of animal experiments suggested that silencing the E2F1 gene could delay the progression of AML,the survival rate of leukemia cells was reduced,and the survival time of mice was significantly prolonged.Conclusion:E2F1 is highly expressed in FLT3-ITD mutated cell lines and patient samples,and the expression of E2F1 is dependent on FLT3-ITD activity and regulates cell proliferation and differentiation.Silencing E2F1 can inhibit the malignant transformation of AML cells driven by FLT3-ITD+by blocking purine metabolism... |
PDF Full Text Request