| BackgroundAcute lymphoblastic leukemia is a malignant proliferation of lymphoid cells blocked at an early stage of differentiation that can invade bone marrow,blood,and extramedullary sites.It can occur in both children and adults,but mainly occurs in children,with the peak incidence between 1 and 4 years old,accounting for about 80%of all cancers in this age group.Although in the past 30 years,the continuous improvement of diagnosis and treatment technology has made the 5-year overall survival rate of contemporary ALL children generally reach more than 90%,the prognosis of patients with refractory or relapsed ALL is still poor.BCL-2 inhibitor is a novel type of anti-tumor drug,which mainly kills tumor cells by directly targeting the BCL-2 protein that promotes cell survival.Its high efficiency,specificity and good bioavailability as an oral drug make it widely used in various hematological malignancies.Currently,Venetoclax has been approved by Food and Drug Administration(FDA)for the treatment of chronic lymphocytic leukemia(CLL)and acute myeloid leukemia(AML).In ALL,the antitumor activity of Venetoclax has also been validated in some ALL subtypes.However,with the clinical application of BCL-2 inhibitors,acquired drug resistance also appears.Therefore,finding new therapeutic targets to overcome drug resistance is an important strategy to promote clinical treatment effects and improve patient prognosis.Identifying the mechanism of drug resistance and exploring new therapeutic targets largely depend on the construction of tumor drug-resistant cell line models.Therefore,our study aims to use the B-cell acute lymphoblastic leukemia cell line RS4;11 to construct two cell line models that are resistant to BCL-2 inhibitors-Navitoclax and Venetoclax,respectively,and then combine RNA sequencing(RNA-seq)technology and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9(CRISPR/Cas9)technology to study the potential mechanisms of drug resistance to BCL-2 inhibitors.MethodsThe drug-resistant cell line was established by intermittent induction with small doses and low concentrations.For Navitoclax,the initial culture concentration of induction was 10 nmol/L and for Venetoclax,1 nmol/L.After 6 months of intermittent cultivation,MTT assay was used to detecte the viability of RS4;11 parental cell line and drug-resistant cell lines with varying drug doses to confirme the successful establishment of BCL-2 inhibitor-resistant cell lines,RS4;11 Navitoclax-Resistant(RS4;11 N-R)cell line and RS4;11 Venetoclax-Resistant cell line(RS4;11 V-R).Then the effect of Navitoclax and Venetoclax on the distribution of cell apoptosis among RS4;11 parental cell line and drug-resistant cell lines was detected by flow cytometry and the effect of Navitoclax and Venetoclax on the cell cycle of RS4;11 parental cell line and drug-resistant cell lines was detected by EDU.In order to verify whether the mechanism of drug-resistant cell line is related to the overexpression of MCL-1,MTT was used to detect the sensitivity of the parental cell line and drug-resistant cell lines to the MCL-1 inhibitor S63845,and cell apoptosis and cell cycle experiments were performed to verify the results of the MTT assay.The expression of anti-apoptotic proteins of BCL-2 family was detected by western blot experiment.RNA-seq technology was performed on RS4;11 drug-resistant cell lines and parental cell line,and the significantly differential expressed genes were obtained by analyzing the sequencing results,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis to initially explore the possible mechanism of BCL-2 drug resistance.Significantly differential expressed genes were verified from mRNA and protein expression level by RT-qPCR and Western blot.CRISPR/Cas9 technology was used to knock out significantly differential expressed genes,and western blot was used to detect whether the target gene was successfully knocked out,and MTT assay was used to detect the impact of gene knockout on drug-resistant cell lines.ResultsThe results of MTT experiments of Navitoclax and Venetoclax proved the successful construction of RS4;11N-R and RS4;11V-R cell lines.The results of cell apoptosis and cell cycle experiments showed that the parental cell line was significantly inhibited by Navitoclax and Venetoclax(p<0.001),while the apoptosis and proliferation of drug-resistant cell lines were basically not affected by the drugs.The results of MTT assay of MCL-1 inhibitor S63845 showed that the two drug-resistant cell lines were more resistant to S63845 than the parental cell line.Besides,the western blot detecting of the BCL-2 family anti-apoptosis proteins,BCL-2,BCL-XL and MCL-1,found that there was no significant difference in the expression levels of these proteins between the parental and drug-resistant cell lines.The result of RNA-seq showed that the expression of E1Abindingproteinp300(p300,EP300)in the drug-resistant cell lines were higher than that in the parental cell line,and the results of subsequent western blot and RT-qPCR proved that the upregulation of EP300 expression in drug-resistant cell lines from protein level and mRNA level,respectively.Western blot experiments proved that the CRISPR/Cas9 technology successfully knocked out the EP300 gene in two cell lines,RS4;11 N-R and RS4;11 V-R,which were successfully induced and constructed to be resistant to BCL-2 inhibitors and two cell lines,Reh and Nalm6,which were naturally resistant to BCL-2 inhibitors.In addition,the results of MTT experiment showed that the above four cell lines resistant to BCL-2 inhibitors became more sensitive to BCL-2 inhibitors(Navitoclax,Venetoclax),BCL-XL inhibitors(A1331852)and MCL-1 inhibitors(S63845)after EP300 gene was knocked out,suggesting that the mechanism of drug resistance of B-ALL cell lines to BCL-2 inhibitors may be related to the up-regulation of EP300 expression.ConclusionsBy establishing drug-resistant cell lines model in vitro,combining with RNA sequencing technology and CRISPR/Cas9 technology,it was determined that the overexpression of EP300 is the drug resistance mechanism leading to the B-ALL cell lines that were resistant to BCL-2 inhibitors.BackgroundAcute lymphoblastic leukemia(ALL)is the most common hematological malignancy in children.Although the cure rate of contemporary children with ALL has exceeded 90%due to the implementation of risk stratified diagnosis and the optimization of treatment schemes,there are still a considerable number of children with chemotherapy resistance and tumor recurrence.Since chemotherapy drugs exert anti-leukemic effects mainly by inducing apoptosis,the increase of anti-apoptotic activity of leukemia cells is one of the most common relapse mechanisms.Ferroptosis is an iron-dependent cell death caused by abnormal accumulation of lipid peroxidation products.Because it is different from other types of programmed death,such as apoptosis,necroptosis,and autophagic cell death,inducing ferroptosis in tumor cells has become a new tumor treatment strategy,especially for aggressive,recurrent malignancies that do not respond well to conventional therapies.Studies have shown that induction of ferroptosis may be an effective method for the treatment of relapsed ALL,therefore,our study aimed to identify the prognostic ferroptosis-related genes(FRGs)in children with relapsed ALL and construct a risk assessment model.MethodWe obtained gene expression data of 256 children with ALL from the Therapeutically Applicable Research to Generate Effective Treatments(TARGET)database,and the children were divided into relapse group and non-relapse group.268 FRGs were obtained from FerrDb database,and the differentially expressed FRGs(DEFRGs)were obtained by combining the differentially expressed genes(DEGs)between the relapse group and the non-relapse group of children with ALL patients.The 191 patient samples with complete clinical information were randomly divided into a training cohort(n=96)and a testing cohort(n=95).Univariate Cox regression analysis and Lasso regression analysis of DEFRGs were performed on the training cohort to determine prognostic-related DEFRGs,followed by multivariate Cox regression analysis and risk model construction.According to the constructed risk model,the training cohort was divided into high-risk and low-risk groups.and verify the accuracy of the risk model in the testing cohort and the whole sample cohort.DEGs in high and low risk groups were compared and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed on them to analyze the function of differential genes between high and low risk groups.ResultWe screened 43 DEFRGs from 256 pediatric ALL samples,and these genes were significantly upregulated in the relapsed group.28 DEFRGs associated with overall survival(OS)were screened using univariate Cox analysis on the training cohort.Then,Lasso regression analysis screened 8 prognostic-related genes for constructing the prognostic risk model,and further multivariate Cox regression analysis identified 5 optimal prognostic genes(MT1G,GPT2,TFAP2C,GCLM,and HSBP1).Risk scores and survival analysis were calculated for the high and low risk groups in the training cohort,testing cohort,and the whole sample cohort,and the results showed that high risk scores were associated with poor prognosis in each group.The receiver operating characteristic curve(ROC)showed good predictive accuracy(the AUC values of the prognostic model in the training cohort,the testing cohort and the whole sample cohort were 0.817,0.870 and 0.918 at 1 year,0.652,0.770 and 0.782 at 3 years,0.743,0.825,and 0.851 at 5 years,respectively,).Therefore,the prognostic risk model that we constructed was successfully validated in training cohort,testing cohort,and whole sample cohort.Difference analysis and enrichment analysis between high-risk and low-risk groups suggested that the prognosis of ALL patients was related to immune activity.Single-sample gene enrichment analysis(ssGSEA)between high-risk and low-risk groups showed that there were immune cell infiltration and activation of immune-related pathways in the high-risk group.ConclusionThe prognostic risk model of FRGs constructed in pediatric relapsed ALL can effectively divide patients into high-risk and low-risk groups and predict patient prognosis.Besides,the difference in prognosis between high-risk and low-risk groups may be related to tumor immune infiltration,which has great significance for predicting the prognosis of clinical ALL patients and the optimization treatment of individualized... |
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