Clinical Significance And The Roles Of M~6A RNA Methylation Modifiers In Acute Myeloid Leukemia

Background and Objective:m6A is the most prevalent internal epigenetic modification of mammalian mRNA.The m6A RNA methylation modifiers mainly include methyltransferase complex,demethylase and m6A specific binding proteins.Researches shown that m6A methylation modifiers were involved in the regulation of a variety of important physiological processes and tumorigenesis.Their expression levels,prognostic value and the roles varied in different solid tumors.m6A RNA methylation modifiers also played important roles in AML.Recent studies of m6A RNA methylation modifiers in AML mainly focused on the m6A methyltransferase METTL3,METTL14,WTAP and demethylase FTO,ALKBH5.They all could promote proliferation,maintain the undifferentiated state of AML cells,but their expression levels and prognostic value in AML remained unclear.Their molecular mechanism in AML also needs to be further studied.We collected the specimen of AML patients treated in West China hospital,then we detected the mRNA and protein expressions and analyzed the clinical significance and prognostic value of METTL3,METTL14,WTAP and FTO,ALKBH5 in primary AML patients.The molecular mechanism of the m6A RNA methylation modifiers with prognostic value was studied,as well as the anti-leukemia effect targeted on m6A RNA methylation modifiers.Chapter 1 Expression and clinical significance of m6A methyltransferase and demethylase in primary acute myeloid leukemiaMethodsWe collected the bone marrow or peripheral blood specimen of 174 AML patients treated in the Department of Hematology in West China Hospital from September 2018 to April 2020,as well as 16 normal control samples.The Western Blot and qPCR technologies were used to detected the protein and mRNA expressions of METTL3,METTL14,WTAP and FTO,ALKBH5,respectively.Their expressions and clinical significances in AML were studied.Results1.The positive rates of m6A modification enzymes METTL3,METTL14,WTAP,FTO and ALBH5 in primary AML were 80.4%(90/112),65.5%(73/112),83%(93/112),and 80.4%(90/112),58%(65/112),respectively,significantly higher than that in normal controls in which were 0%,0%,0%and 6.3%(1/16),0(p<0.0.001).2.The positive rates of m6A modification enzymes METTL3,METTL14,WTAP,FTO and ALBH5 in relapsed/refractory AML were 83.3%(20/24),62.5%(15/24),79.2%(19/24)and 75%(18/24),58.3%(14/24),similar to that in primary AML.The positive rates of them in AML patients in complete remission were 13.2%(5/38),5.3%(2/38),18.4%(7/38)and 26.3%(10/38),7.9%(3/38),lower than that in in primary AML.3.The five m6A modification enzymes were positive relative in primary AML patients.4.The m6A levels between positive group in which patients positively expression the methyltransferase and demethylase and negative group in which patients negatively expression the methyltransferase and demethylase had no difference.5.Compared with the METTL3-negative group,METTL3-positive AML patients were more common in M1 type and less common in M3 type,with higher WBC count at onset(65.63±86.34×109/L vs 33.70±50.69×109/L,P=0.028),and more accompanied by DNMT3A mutation(19.2%vs 0%,P=0.036).Compared with the METTL 14-negative group,METTL14-positive AML were more common in M1 type and less common in M3 type,with higher WBC count at onset(70.11±92.90×109/L vs 39.23±48.60,P=0.023),and less accompanied by ASXL1 mutation(3.5%vs 21.4%,P=0.014).Compared with the WTAP-negative group,WTAP-positive AML were more common in M1 type,less common in M3 type,more common in the high-risk group(29.6%vs 5.6%,P=0.037).FTO-positive AML were less common in the M3 type than the negative group.Compared with the ALKBH5-negative group,ALKBH5-positive AML were more common in M1 and M4 types,less common in M2 and M3 types,more accompanied by TET2 mutation(14.6%vs 0%,P=0.038),less accompanied by ASXL1 mutation(2.1%vs 18.8%,P=0.015).6.The overall survival time of METTL3-positive AML was shorter than that of the negative group(median OS 13.8months vs undefined,P=0.0344),which was an independent adverse prognostic factor for AML patients(HR:5.635,95%CI:1.147-27.694,P=0.033).The overall survival time and event-free survival time of patients who underwent HSCT were both longer than those who did not in METTL3-positive AML(median OS undefined vs 12 months,P=0.0137;Median EFS 15.7 Months vs 7.2 Months,P=0.0137).The overall survival time of WTAP positive AML was also shorter than that of the negative group(median OS 15months vs undefined,P=0.0394),but WTAP was not an independent adverse prognostic factor for AML.In the WTAP positive AML,the OS and EFS of those having HSCT was significantly longer than that who not(median OS undefined vs 12 months,P=0.0081;median EFS undefined vs 7.1 months,P=0.0107).METTL14,FTO and ALKBH5 had no prognostic value in AML.Conclusions1.m6A modification enzymes METTL3,METTL14,WTAP,FTO and ALKBH5 had higher positive rates in both primary AML and relapsed/refractory AML.2.There was no difference in m6A level between the positive and negative m6A RNA methylation modifiers groups.3.AML patients with methyltransferase METTL3 positive were more likely to accompanied by DNMT3A mutation,and AML patients with demethylase ALKBH5 positive were more likely to accompanied by TET2 mutation.4.The OS of METTL3-positive AML was shorter than that of the negative group.METTL3 was an independent adverse prognostic factor for AML patients.Therefore,HSCT was recommended for METTL3-positive AML patients.Chapter 2 METTL3 played a role in acute myeloid leukemia through the PGC-1α/MAPK pathwayMethodsWe knocked down the METTL3 expression in K562 and MV4-11 cell lines using the short hairpin RNA(shRNA).The RNA-Seq data of K562-shCTR and K562shMETTL3 cells were analyzed by KEGG enrichment using GSEA software to search for differentially expressed signaling pathway gene sets,then we got the downstream gene of METTLL3.We knocked down the target gene by using the lentiviral system to study its effects on the proliferation,apoptosis,cycle and differentiation of AML cells,so as to further study the mechanism of METTL3 in AML.Results1.KEGG enrichment analysis of RNA-Seq showed that the differential gene sets of K562-shCTR and K562-shMETTL3 cells were enriched into MAPK signaling pathway,and MAPK signaling pathway was activated after METTL3 knocked down.2.After METTL3 knockdown,the mRNA levels of mitochondrial biosynthesis gene PGC-1α and oxidative phosphorylation related genes ATP5F1A,ATP5MC1,COX5a and CYCs in K562 and MV4-11 cells were all decreased,and the protein expression level of PGC-1α was also decreased.3.Correlation analysis of PGC-1α and METTL3 mRNA expression levels in 105 primary patients showed that PGC-1α and METTL3 were positively correlated(r=0.3303,P=0.0006).4.Once METTL3 was knocked down,the total mRNA m6A level of K562 and MV411 cells was decreased(P=0.0172,P<0.0001),as well as the mRNA m6A level of PGC-1α in K562 cells(P=0.0245).5.The mRNA and protein levels of METTL3 kept unchanged while PGC-1α was knocked down in K562.6.While PGC-1α expression was down regulated in K562,the proliferation and apoptosis were inhibited,the rates of CD14 and CD11b positive cells was increased,but the cell cycle was not affected.7.The phosphorylation levels of P38,c-Jun and Erk1/2 protein,which were important molecules of MAPK signaling pathway,were increased in K562 cells when when PGC-1α expression was down regulated.8.After knockdown of PGC-1α,mRNA levels of ROS detoxification system genes such as reactive oxygen species SOD1,SOD2,GPX1,Catalase and UCP2 in K562 cells were decreased.9.The mRNA levels of ROS detoxification systems SOD1,SOD2,GPx1,Catalase and UCP2 in K562 and MV4-11 cells were decreased after METTL3 knockdown.10.METTL3 was highly expressed in leukemic stem cells(LSCs),while PGC-1αshowed no difference between LSCs and non-LSCs.11.The sensitivity of AML cells to azacytidine was decreased once METTL3 knocked down,and the protein expression level of METTL3 was decreased after azacytidine was applied to K562 and MV4-11 cells.Conclusions1.The MAPK signaling pathway was activated after METTL3 knockdown.The expression of PGC-1α was decreased after METTL3 knockdown in AML cells,and there was a positive correlation between METTL3 and PGC-1α expression in AML patients.2.The proliferation was inhibited,apoptosis and differentiation were increased in AML cells while PGC-1α knockdown,which also activated the MAPK signaling pathway.All of these were the same as the knockdown of METTL3.3.The mRNA level of gene of ROS detoxification system decreased after METTL3 or PGC-1α knockdown.4.METTL3 was highly expressed in LSCs,while PGC-1α showed no difference between LSCs and non-LSCs.5.METTL3 positive AML cells were more sensitive to azacytidine than negative cells.Azacytidine could reduce the expression level of METTL3 protein in AML cells.Chapter 3 The expression and significance of PGC-1α in adult acute myeloid leukemiaMethodsWe collected bone marrow or peripheral blood samples of 108 newly diagnosed adult AML patients admitted to the Department of Hematology,West China Hospital,from September 2018 to December 2019,as well as 16 normal control samples.The relative mRNA levels of PGC-1α were determined by qPCR.The mRNA levels of PGC-1α in AML patients were analyzed in TCGA database,GSE13159 dataset and the primary AML in West China Hospital,and compared with normal controls.The AML patients were divided into PGC-1α high expression group and PGC-1α low expression group according to the median method.The expression and clinical significance of PGC-1αin AML patients were analyzed.Results1.The expression level of PGC-1α was higher than that of normal controls by analysis of TCGA data set,GSE13159 data set and patients with newly diagnosed AML in West China Hospital(P=0.0011,P=0.0011,P=0.0477).2.By analysis of TCGA data sets,it was found that the high expression of PGC-1αwas more common in M4 and M5,less common in M0 and M2,more accompanied by FLT3-ITD mutation,more often with 11q23 karyotype,less with t(8;21),t(16;16)/inv(16)karyotype.By analyzing AML patients in West China Hospital,we found that patients with high PGC-1α expression were more common in M1 type,with low platelet count at onset(106.68±79.417×109/L vs 264.18±223.801×109/L,P=0.000).3.By analyzing the TCGA data set,we found that the OS of AML in the high PGC1α expression group was shorter than that in the low expression group(median OS:11.2Months vs 19.2Months,P=0.0294),and there was no statistical difference in EFS between the two groups(median EFS:12.0Months vs 22.2 Months,P=0.0515).Survival analysis of patients in West China Hospital showed that the median OS of PGC-1α with high expression of AML was shorter than that of the low expression group(median OS:12.7months vs undefined,P=0.0275),and there was no statistically significant difference in EFS(median EFS:6.3 months vs 10.0 months,P=0.1168).Conclusions1.PGC-1α was highly expressed in primary AML.2.Primary AML patients with high PGC-1α expression had shorter OS than those with low PGC-1α expression.Chapter 4 Study of the anti-leukemia effects of the WTAP small molecular inhibitor ZR239MethodsWe used the technologies of MTT,flow cytometry,Western Blot,qPCR and mass spectrometry to study the effects of ZR239,a small molecule WTAP inhibitor designed and developed by the State Key Laboratory of Sichuan University,on the proliferation,apoptosis,cycle,differentiation and m6A level of AML cells,as well as the mechanism of its anti-leukemia effect.Results1.The WTAP inhibitor ZR239 could inhibit the proliferation of MV4-11 and MOLM-13 cells,with IC50 values of 4.662 and 3.055uM at the 72 hour,respectively.2.The apoptosis of MV4-11 and MOLM-13 cells was increased after ZR239 treatment compared with the control group.3.After treated with ZR239 on MV4-11 and MOLM-13 cells,the number of cells in G0/G1 phase increased(P=0.0047,P=0.0248),and the number of cells in G2/M phase decreased(P=0.0256,P=0.0444).4.The proportion of CD 14 and CD11b positive cells increased after ZR239 treated in MV4-11 and MOLM-13 cells5.The mRNA m6A level in MV4-11 and MOLM-13 cells were significantly lower after ZR239 treatment than that of the control group.6.ZR239 did not change the mRNA and protein expression levels of WTAP in MV411 and MOLM-13 cells.7.ZR239 could increase the phosphorylation levels of Akt and mTOR proteins in MV4-11 and MOLM-13 cells,as well as the apoptotic protein Caspase-3,while the expression level of anti-apoptotic protein Bcl-2 was decreased.Conclusions1.The WTAP inhibitor ZR239 can inhibit the proliferation,promote apoptosis and differentiation,and block the cell cycle of AML cells.2.ZR239 did not affect the expression level of WTAP in AML cells,but reduced the mRNA m6A level and inhibited the Akt/mTOR pathway.Conclusions1.The m6A RNA methylation modifiers METTL3,METTL14,WTAP,FTO and ALKBH5 have higher positive rates in AML patients.METTL3-positive AML patients are always accompanied by DNMT3A mutation,and METTL3 is an independent poor prognostic factor in AML.2.PGC-1α is highly expressed in AML and predicts poor prognosis.MELTL3 plays a role in AML by regulating the expression of PGC-1α,thus further regulates the expression of ROS detoxification enzyme,and negatively regulates the MAPK signaling pathway.3.Inhibition of WTAP has an anti-leukemia effect.