Study On The Clinical Significance And Mechanism Of BRD4 In Pediatric T-lineage Acute Lymphoblastic Leukemia

Part one.Clinical significance and expression pattern of BRD4 gene in Tlineage acute lymphoid leukemiaObjective:In this study,the relative expression of BRD4 mRNA in bone marrow of pediatric Tlineage acute lymphoblastic leukemia(T-ALL)patients was detected by transcriptome sequencing and real-time quantitative PCR,and the correlation between its expression and the clinical characteristics of pediatric T-ALL was analyzed.In brief,the aim of this study was to clarify the clinical significance of BRD4 in pediatric T-ALL.Methods:1.Through tumor cells database(https://portals.broadinstitute.org/ccle),BRD4 mRNA expression in different types of tumor cell lines were obtained;The expression of BRD4 mRNA in acute leukemia cell lines was detected by real-time quantitative PCR.2.Sixty-two newly diagnosed T-ALL patients who received the protocol of CCLG-ALL 2008 and 9 healthy donors from the Children’s Hospital of Soochow University between June 2012 and December 2017 were selected.The expression level of BRD4 gene in 16 TALL samples,9 normal bone marrow and various leukemia cell lines was detected by realtime quantitative PCR.The transcriptome level of BRD4 gene in 46 children with T-ALL was detected by transcriptome sequencing.Combined with clinical data,the expression profile of BRD4 gene in T-ALL and its correlation with prognosis were analyzed.Results:1.The expression profile of BRD4 mRNA in different types of tumor cell lines showed high,but no specificity for different types of tumor cells.2.In pediatric T-ALL,increased BRD4 mRNA expression was associated with poor prognosis.Patients with high BRD4 mRNA expression had significantly shorter overall survival than those with low BRD4 expression(p=0.045).Patients with high BRD4 expression didn’t show significant difference in relapse-free survival compared with those with low BRD4 expression,but the trend was obvious.3.The risk of death in patients with high BRD4 expression was 4 times higher than that in patients with low BRD4 expression analyzed by multivariate logistic regression analysis.Conclusion:The BRD4 gene is commonly expressed in tumor cells.In pediatric T-lineage acute lymphoid leukemia,high expression of BRD4 gene is a poor prognostic factor.Part two.Study on the biological function of BRD4 gene in T-ALLObjective:Preliminary clinical data showed that BRD4 was a poor prognostic factor in T-ALL patients,but the mechanism remains unclear.In this study,we investigated the biological function of BRD4 gene in T-ALL cells by silencing the expression of BRD4 in vitro and in vivo.Methods:1.BRD4 knockdown T-ALL cells were received by lentivirus and treatment with ARV825.The expression of BRD4 in the cell lines was further identified by real-time quantitative PCR and Western blot.2.CCK-8 assay was used to detect the effect of BRD4 on the proliferation of T-ALL leukemia cells and primary cells.Flow cytometry was used to detect the effects of BRD4 on cell cycle and apoptosis,and Western blot was employed to detect the expression of apoptotic-related proteins.3.Virus particles were obtained by knocking down and overexpressing CRBN plasmid in HEK293T cells.Then 6T-CEM,Jurkat and Molt4 cells were infected with CRBNknockdown and CRBN-overexpression virus particles,respectively.The cell viability of ARV-825 in CRBN-overexpression and CRBN-knockdown cells were detected by CCK-8 assay.After treatment with protease inhibitor MG132,the BRD4 protein of these cells were detected by Western blot.4.The xenograft model was established by subcutaneously inoculating leukemia cells in nude mice.The drug toxicity and the effect of ARV-825 on the xenograft were analyzed in vivo.Western blot was used to detect the level of BRD4 protein in tumor tissues.Meanwhile,the levels of Ki67 and activated caspase 3 in tumor tissues were detected by immuohistochemical stainin.Results:1.The BRD4 downregulated T-ALL cells were confirmed by real-time quantitative PCR and Western blot.BRD4 reducing cells with ARV-825 treatment were proved through Western blotting validation.2.CCK-8 assay showed that reduced BRD4 expression significantly inhibited proliferation of T-ALL cells.3.After BRD4 reduced,cells were mainly blocked in the G0/G1 phase.Meanwhile,flow cytometry showed that the proportion of cell apoptosis was also increased when BRD4 expression was reduced,and the apoptotic marker proteins PARP and caspase 3 were also activated,which was consistent with the phenotype.4.By degrading BRD4 protein,ARV-825 could inhibit the proliferation and induce the apoptosis of T-ALL cells.The anti-tumor activity of ARV-825 is related to the expression level of CRBN,and the protease inhibitor MG 132 can reverse the degradation of BRD4 protein by ARV-825.5.The BRD4 degrader ARV-825 didn’t show the effect on the body weight and important organs in the xenograft model.BRD4 degrader ARV-825 can significantly degrade the expression of BRD4 protein in tumor tissues,induce the inhibition of Ki67 and promote the activation of caspase 3,thereby inhibiting the growth of T-ALL xenograft tumor.Conclusion:In vitro and in vivo studies showed that BRD4 is an important gene to maintain the survival of T-ALL cells.With the help of the protease CRBN,the BRD4 degrader ARV-825 can effectively degrade the expression of BRD4 and produce anti-tumor effect.Part three.Study on the molecular mechanism of BRD4 in T-ALLObjectives:Previous studies have found that BRD4 is a prognostic factor for T-ALL,which inhibits cell proliferation and promotes cell apoptosis,but its detailed molecular mechanism is still not fully understood.In this study,transcriptome sequencing combined with ChIP sequencing was used to explore the mechanism.Methods:1.The differential genes after BRD4 interfered were explored by transcriptome sequencing technology,and the differential gene signaling pathway was enriched by GSEA.The expression of BRD4 and c-Myc protein at the cellular level was further verified by Western blot.2.The super enhancer related genes in BRD4 interfering cell lines and pediatric T-ALL specimens were analyzed by ChIP-seq technique.Further enhancer analysis and gene expression analysis were combined to obtain BRD4 downstream target genes,which were verified by real-time quantitative PCR.3.The IGLL1-shRNA vector was constructed by lentiviral vector.The 6T-CEM cells stably expressing IGLL1 were identified by real-time quantitative PCR,and the proliferation of cells with reduced IGLL1 expression was detected by CCK-8 assay.The effect of IGLL1 on cell apoptosis was detected by flow cytometry.Results:1.Analysis of transcriptome sequencing data showed that after degrading BRD4,ARV825 mediated c-Myc transcriptional inhibition through H3K27Ac,and c-Myc signaling molecules were further verified at the cellular level.2.IGLL1 was found to be a super enhancer related gene in primary T-ALL cells by ChIP-seq analysis.Combined with the ChIP-seq and RNA-seq data of BRD4-reduced cell lines,the results showed that IGLL1 was the most significantly affected gene by BRD4.At the cellular level,the expression level of IGLL1 gene was found to be related to cell proliferation.Conclusion:The effect of interfering BRD4 on T-ALL is related to the inhibition of transcription of target gene c-Myc.In addition,the reduced cell viability caused by BRD4 interference may also be caused by the decrease of the transcription level of the target gene IGLL1,which requires more studies to verify in the future.