The Mechanism Of Drug-resistance Induced By Nrf2 Overexpression In Acute Myeloid Leukemia Via Inhibiting MSH2

Background:Nuclear factor E2-related factor 2（Nrf2）is a key transcription factor that can reduce the body’s oxidative damage.In recent years,Nrf2 has been shown to promote tumor development and cause tumor cells to be resistant to chemotherapy,which brings huge challenges to clinical treatment.Acute myeloid leukemia（AML）is a malignant hematopoietic stem cell disease with abnormal differentiation of myeloid primordial immature cells,in which the treatment of relapsed/refractory AML is still a challenge.Primary or secondary drug resistance of acute leukemia cells to a variety of chemotherapeutic drugs is an important cause of relapse and refractory,and gene mutation is closely related to poor prognosis and relapse/refractory of AML.However,it is still unclear whether Nrf2 leads to chemotherapy resistance in AML by affecting genetic instability.Objective:The purpose of this study was to verify the effect of Nrf2 expression on gene mutation frequency,and to explore the molecular mechanism that mediates gene instability-dependent chemoresistance in AML.Methods:1.Clinical sample collection.According to the“adult acute myeloid leukemia（Non-APL）guidelines of China（2018 edition）”,“the recurrence of refractory acute myeloid leukemia guidelines of China（2017 edition）”,newly diagnosed and relapsed/refractory patients are included AML patients are the subject of research.Collect bone marrow blood samples that have not been treated for newly diagnosed or relapsed.Ficoll separation fluid separates bone marrow mononuclear cells.RNA was extracted by Trizol and reversed into cDNA for the detection of transcriptome expression level.DNA of bone marrow AML stem progenitor cells was extracted by DNA extraction kit,and common AML gene mutations were detected by next generation sequencing.2.The correlation between Nrf2 expression and AML chemotherapy resistance and gene mutation frequency was analyzed.Real-time PCR was used to detect the expression of Nrf2 in the newly diagnosed samples of patients with complete remission and relapse-resistant AML,and the expression of Nrf2 was calculated according to 2^-ΔΔCT,which was divided into high and low groups.Gene mutations in mononuclear cells of each sample were detected by whole exome sequencing.Western blot and real-time PCR were used to detect the expression of Nrf2 in AML samples from the mutant group and the non-mutant group.Meanwhile,the Oncomine database was used to analyze the transcription level of Nrf2 in AML patients from other centers.3.Clinical samples were detected by transcriptome sequencing to analyze the differentially expressed signal pathways of Nrf2 gene between the two groups:In this study,transcriptome sequencing was performed on mononuclear cells（3 with high Nrf2 expression and 4 with low Nrf2 expression）from 7 AML patient samples.Heat maps and Kyoto Encyclopedia of Genes and Genomes（KEGG）related signaling pathways were analyzed.Western blot,real-time PCR and immunocytochemistry were used to detect the relationship between Nrf2 and DNA mismatch repair genes in AML clinical samples.4.In vitro cell line experiment to verify the RNA-seq test results:two human AML cell lines—Kasumi-1 and THP1 were cultured in RPMI-1640 medium with10%fetal bovine serum.Lentiviral transfection of Kasumi-1 and THP-1 cells was performed by constructing lentivirus with up-regulated/silenced Nrf2 expression according to the operating instructions.The corresponding empty vector（EV）was used as the control group of Nrf2 overexpression and silencing group respectively.The cells were treated with 2μM cytarabine（Ara-C）for 24h.Apoptosis was detected by Hoechst33342 staining and Annexin V-FITC/PI,and intracellular ROS production was detected by DCFH-DA.5.18-25g NOD/SCID/IL2Rγc-deficient female mice were selected as the model object.By subcutaneous injection of THP1 cells stably transfected with Nrf2lentivirus into the left anterior axilla of mice,the mice were divided into four groups:Nrf2-THP1 group,EV-THP1 group,Nrf2-THP1+Ara-C group and EV-THP1+Ara-C group.The tumor load was observed regularly by intraperitoneal injection of luciferase substrate Dmure Luciferin.The tumor size was monitored three times a week and the survival time of mice was recorded.After sacrifice,tumor masses were collected and paraffin-embedded pathological sections were observed.6.Through Gene MANIA database and western blot detection,it was analyzed that the related signal pathway of Nrf2-mediated gene unstable drug resistance was related to JNK signal pathway.The cells were pretreated with SP600125（10μM）,an inhibitor of JNK pathway,for 24h to detect the expression of protein in the pathway,and the gray value of each protein band was analyzed by ImageJ software.7.Statistical analysis of data:SPSS20.0,Graph Pad Prism 7.0 and ImageJ software were used for statistical analysis.All the data were expressed as mean±standard deviation（（?）±s）,and the data were in line with normal distribution.Differences between two groups were analyzed using an unpaired two-tailed Student’s t test.The comparison among three or more groups was analyzed using one-way ANOVA.A P value of less than 0.05 was considered statistically significant.Results:1.Firstly,we found that the gene expression of Nrf2 was significantly increased in relapsed/resistant AML samples,and AML samples with high Nrf2 expression had a higher tumor mutation load.The gene and protein expressions of Nrf2 were increased in the gene mutation group.2.Nrf2 expression was associated with DNA mismatch repair pathways.DNA mismatch repair genes（MSH2,MLH1,POLD2,RFC4,PMS2 and MSH6）were detected in AML clinical samples with high/low expression of Nrf2,and the results showed that the gene expression of MSH2 was significantly reduced in the Nrf2-high expression group.At the protein expression level,the MSH2 protein expression was decreased in the Nrf2-high expressing AML patients.3.Overexpression of Nrf2 protected AML cell lines from Ara-C-induced apoptosis in vitro and inhibited the expression of MSH2 protein,while silencing Nrf2in AML cell lines increased the sensitivity of tumor cells to Ara-C,and the expression of MSH2 protein was increased.Further studies showed that intracellular ROS was decreased in the Nrf2 overexpression group,and the protein expression of MSH2 was not increased in the Nrf2-overexpression group treated with Ara-C in combination with H₂O₂.However,in the Nrf2-silenced group,MSH2 protein expression was decreased after ROS scavenging by N-acetylcysteine（NAC）pretreated cells.4.In AML mouse model,the growth of AML cell tumor in Nrf2 overexpression group was faster than that in control group,and it was insensitive to Ara-C,and the survival time was significantly shortened.At the same time,the expression of MSH2in Nrf2 overexpression group was significantly decreased.5.The JNK/c-Jun signaling pathway was activated in Nrf2-overexpressed cells and the protein expression of MSH2 was inhibited.When the signaling pathway was blocked with the JNK inhibitor SP600125,the protein expression of MSH2 in the Nrf2 overexpression group was higher than before.Conclusions:1.High expression of Nrf2 increases the risk of gene mutations in AML patients,and is closely related to AML relapse and drug resistance.2.Nrf2 leads to genetic instability drug resistance by affecting DNA mismatch repair defects in AML.3.High expression of Nrf2 increases the risk of unstable drug resistance by inhibiting the expression of MSH2 protein.4.Nrf2 inhibits the protein expression of MSH2 in a way that is not completely dependent on ROS.5.Nrf2 overexpression mediates drug resistance by activating JNK/c-Jun signal pathway.