| Objective To evaluate the prognostic factors,chemotherapy efficacy and the clinical outcome of acute myeloid leukemia(AML).Meanwhile,to explore the drug-resistance mechanism of AML,especially the role and the significance of tumor necrosis factor receptor-associated factor 2(TRAF2)in AML cells(including AML patients leukemia cells and AML cell lines),and expect to solve the problem of clinical drug-resistance of AML and to improve the clinical efficacy of relapsed and refractory AML(R/R AML).Methods 1.Clinical study of AML patients: 1.1 R/R AML: Clinical data(including chromosome karyotype,risk stratification,disease status,etc.)of 49 R/R AML patients diagnosed in our hospital from June 2014 to December 2019 were collected,and the prognostic factors were analyzed.Kaplan-meier survival curve was used to analyze overall survival(OS),and the chemotherapy efficacy difference among the different salvage treatment groups were evaluated.1.2 FLT3-ITD mutated AML patients’ data: Clinical information of 103 adult AML patients(including 23FLT3-ITD mutated patients)were collected.The difference of treatment response rate,relapse-free survival(RFS)and OS between the following paired groups were compared respectively,FLT3-ITD mutated and FLT3-ITD unmutated patients’ group,FLT3-ITD mono-mutated and FLT3-ITD mutation combined with other molecular abnormalities group,normal karyotype and abnormal karyotype group.The sorafenib treatment efficacy in FLT3-ITD mutated AML patients was also evaluated.2.AML patients’ samples and AML cell line experiments: 2.1 Differentially expressed proteins screening: BMMCs(bone marrow mononuclear cells)from 3 chemotherapy susceptible and 3 R/R AML patients were used for the proteomics experiments.The differentially expressed proteins were screened by the data analysis process of liquid-mass spectrometry,protein database construction,protein identification and protein function annotation.KEGG(Kyoko Encyclopedia of Genes and Genomes,a database for the systematic analysis of the metabolic pathways and functions of gene products and compounds in cells)signaling pathway enrichment analysis was carried out on the differentially expressed proteins(TRAF2 was found to be up-regulated in R/R AML).2.2 Clinical specimen validation: The expression level of TRAF2 protein in BMMC samples of AML patients was detected by Western Blot,and the correlation between the TRAF2 expression level and the prognosis of AML patients was analyzed.2.3 Design and synthesis of the TRAF2 si RNA: Based on the gene sequence information from gene bank,3 pairs of TRAF2 si RNA and negative control were designed.2.4 TRAF2 functional experiments: TRAF2 expression was knockdown in HL-60 cells.The experiments were divided into three groups: blank cells,negative control,and TRAF2 si RNA.Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry methods.2.5 The role of TRAF2 in drug resistance of AML: RNA expression levels of TRAF2 and MDR1 were detected by fluorescence quantitative PCR.Western Blot was used to detect the protein expression of P-glycoprotein(P-gp),TRAF2,PI3 K,AKT,NFκB,Bcl-x L,C-IAP1 and Caspase-3.Results 1.Clinical research results: 1.1 49 R/R AML patients were treated with salvage chemotherapy,15 patients achieved CR and 34 patients were remained in no remission(NR),the CR rate was 30.61%(15/49),and 16 patients were still alive by the follow-up date.Multivariate analysis showed that the risk stratification,CD7 expression,chromosome karyotype and FLT3 gene mutation were the important prognostic factors that affecting induction remission rate and OS of AML patients.Survival analysis showed that the median OS of the new drug chemotherapy group was 20 months,which was longer than that of the traditional drug chemotherapy group,but there was no statistical significance in two groups(20 months vs 11.5months,p = 0.091).1.2 Compared with FLT3-ITD mono-mutated AML patient,the FLT3-ITD mutation combined with the good prognosis of molecular biological abnormalities AML patients had significantly longer median OS(12 months vs 3months,p = 0.036)and RFS(12 months vs 4 months,p = 0.043).Sorafenib combined with chemotherapy could improve the CR rate and the overall response rate(ORR)of FLT3-ITD mutated AML patients,but could not improve the OS and RFS of these patients.2.AML proteomics and clinical sample results: Proteomics experiments showed that TRAF2 expression was up-regulated(12.33 times)in R/R AML patients,and clinical samples verification analysis showed that TRAF2 expression was lower in chemotherapeutic sensitive patients than in R/R AML patients,and the survival analysis suggested that patients with high expression level of TRAF2 had shorter OS.3.AML cell line experimental results: 3.1 The apoptosis rates of following experiment groups,blank cell,negative control,and TRAF2 si RNA transfected group were(7.1333±0.1785)%,(13.0333±0.4522)% and(30.8000±0.6978)%,respectively,there was a significant difference between the negative control and TRAF2 si RNA transfected group(p = 0.021),this result suggested that knockdown TRAF2 could promote the apoptosis of HL-60 cells.3.2 At different transfection time(12,24,36,48,60,72 hours),the cell viability of the transfected TRAF2 si RNA group and the negative control were(0.7400±0.0396)% and(0.8717±0.0224)%,respectively(p= 0.010).3.3 The expression level of TRAF2 and MDR1 m RNA in TRAF2 si RNA transfected HL-60 cells were lower than controls.The expression of TRAF2,PI3 K,AKT,NFκB,Bcl-x L,C-IAP1 and P-gp proteins were down-regulated and the expression of Caspase-3 protein was up-regulated in HL-60 cells by TRAF2 knockdown.Conclusion 1.High expression of CD7,chromosome karyotype and FLT3-ITD mutation were the key factors that affecting the chemotherapy efficacy and the prognosis of AML.2.Molecular abnormalities with a good prognosis can partly overcome the risk of FLT3-ITD mutation.AML patients with FLT3-ITD mutation combined with other poorly prognostic molecular or cytogenetic abnormalities had very poor clinical outcome.3.Novel targeted drugs such as sorafenib in combination with chemotherapy can improve the remission rate of R/R AML,but could not improve the OS and RFS of these patients.4.TRAF2 expression level was increased in R/R AML patients,and patients with high expression of TRAF2 had poor prognosis.5.TRAF2 knockdown by TRAF2 si RNA could promote the apoptosis and inhibit the proliferation of HL-60 cells.6.Through affecting PI3K/AKT and NFκB signaling pathways,TRAF2 could mediate AML drug-resistance by upregulating PI3K、AKT、NFκB、P-gp、Bcl-x L and C-IAP1 expression and downregulating Caspase-3 expression in AML cells. |
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