| Objective: Myelodysplastic syndromes(MDS)are a highly heterogeneous group of myeloid malignancies.About 30% of MDS patients may progress to acute myeloid leukemia(AML,also called sAML),once occurred,the prognosis is extremely poor.So far,the mechanism of disease transformation has not been fully clarified.Our study was aimed to explore the whole-genome methylation alterations and identified epigenetic drivers during MDS progression,and further determined their clinical significance.Methods: Reduced representation bisulfite sequencing(RRBS)was used to analyze the methylation pattern in 4 controls and 4 matched MDS/sAML patients at different stages.At the same time,bioinformatics analysis was applied to screen the prognosis-related genes with potential biological function.Real-time quantitative methylation-specific PCR(RQ-MSP),bisulfite sequencing PCR(BSP)and MethylTarget targeted sequencing were used to further verify and determine the methylation changes of selected gene.Gene expression was determined by real-time quantitative PCR(RQ-PCR)and western blot.Clinical significance and prognosis analysis were based on clinical specimen parameters.Functional studies were evaluated by cell cycle,cell proliferation and cell apoptosis experiments.Results: The whole-genome methylation level was significantly increased during disease progression in 4 matched MDS/sAML patients.In the MDS phase compared to the control,a total of 1090 differential methylation fragments(DMFs)were screened,including 441 hypermethylated fragments and 649 hypomethylated fragments.In the sAML phase compared to the MDS phase,a total of 103 DMFs were screened,including 96 hypermethylated fragments and 7 hypomethylated fragments.For 4 matched MDS/sAML patients compared independently,we screened 5803,3735,4043,and 2,748 differentially methylated genes(DMGs).Combined with preliminary screening and bioinformatics analysis,it was found that the potential molecules ID4 and DLX5 are not only closely related to the prognosis of AML,but also involved in tumorigenesis.In addition,we further expanded to determine the methylation of ID4 and DLX5 in MDS progression using RQ-MSP.The level of ID4 methylation in patients with MDS and AML was significantly higher than that in the control group(P<0.001).In addition,the level of ID4 methylation in patients with Int-2/High risks was significantly higher than patients with Low/Int-1 IPSS risk(P=0.004).ID4 methylation level of sAML patients was also significantly higher than that of primary AML(pAML)patients(P=0.003).Kaplan-Meier analyses revealed that ID4-hypermethyated MDS patients had significantly shorter overall survival(OS)time than ID4-hypomethyated MDS patients(P=0.038).Cox regression multivariate analyses demonstrated that ID4 methylation was not an independent prognostic factor in MDS(HR=1.643,P=0.433).Among total AML and non-M3 AML,there were no significant differences in complete remission(CR)rate among ID4-hypermethylated and-hypomethylated patients(P=0.629 and 0.202).However,ID4-hypermethylated patients had significantly lower CR rate than ID4-hypomethylated in AML patients(P=0.030).Moreover,ID4 methylation level was significantly decreased after post-CR(P<0.001).Kaplan-Meier analyses revealed that ID4-hypermethyated non-M3 AML patients had significantly shorter OS and leukemia-free survival(LFS)time than ID4-hypomethyated non-M3 AML patients(P=0.002 and 0.001).Cox regression multivariate analyses demonstrated that ID4 methylation was an independent prognostic factor in cytogenetically normal(CN-AML)and a trend to significant difference was presented in non-M3 AML(HR=2.483,P=0.004 and HR=1.507,P=0.081).ID4 expression in AML was significantly dowregulated(P=0.003),and was negatively associated with ID4 methylation(R=-0.275,P=0.001).ID4 expression was significantly increased after DAC treatment in K562 cells.Functional studies showed that ID4 transfection overexpression presented pro-apoptotic and anti-proliferative effects in K562 and HL60 cells with cell arrest in G1.ID4 was further identified as a direct target of miR-335.Herein,miR-335 expression,independent of its methylation,was significantly increased(P=0.009)and negatively correlated with reduced ID4 expression in AML(R=-0.146,P=0.043).Significant differences in CR rate were observed among three groups(with both/either/none aberrant miR-335/ID4 expression)in both total AML,non-M3 AML,and CN-AML patients(P=0.024,0.029,and 0.018).Moreover,Logistic regression analysis showed that aberrant miR-335/ID4 expression independently affected chemotherapy response in non-M3 AML and CN-AML patients(HR=2.010,P=0.033 and HR=3.224,P=0.009).The OS and LFS were negatively affected by miR-335 overexpresion(P=0.001 and <0.001)and ID4 underexpression(P<0.001 and =0.020)in non-M3 AML,and also in CN-AML(P=0.002 and 0.003;P=0.001 and 0.026).Moreover,significant differences in OS and LFS were also found among three groups(with both/either/none aberrant miR-335/ID4 expression)in non-M3 AML(P<0.001 and 0.001)and CN-AML patients(P<0.001 and =0.003).Cox regression analysis showed that aberrant miR-335/ID4 expression independently affected OS in non-M3 AML and CN-AML patients(HR=1.894,P<0.001 and HR=2.376,P<0.001).Gain-of-function experiments in vitro showed the oncogenic role of miR-335 by affecting cell apoptosis and proliferation in AML,and could be rescued by ID4 restoration.Mechanistically,we identified and verified that miR-335/ID4 contributed to leukemogenesis through activating PI3K/Akt signaling pathway.By targeted sequencing,the level of DLX5 methylation in patients with MDS(n=35)was a little higher than that in the control group(n=25)(P=0.063),whereas the level of DLX5 methylation in patients with AML(n=111)was significantly higher than that in the control group and MDS group(P<0.001 and 0.001).According to the result of RQ-MSP in expanded samples,the level of DLX5 methylation in MDS(n=61),pAML(n=148),and sAML patients(n=11)was significantly higher than that in controls(n=46)(P=0.034,<0.001,and <0.001).In addition,the level of DLX5 methylation in sAML patients was significantly higher than that in pAML patients(P=0.008).RQ-MSP results were positively associated with targeted sequencing results(R=0.566,P<0.001).ROC curve analysis revealed that DLX5 methylation could act as a potential biomarker distinguishing AML/MDS from controls(AUC=0.715,P<0.001;AUC=0.620,P=0.034).DLX5-hypermethylated AML patients showed lower CR rate than DLX5-hypomethylated AML patients in total AML(P=0.043)but not in non-M3 AML(P=0.135)and CN-AML(P=0.081).Logistic regression analysis showed that DLX5 methylation independently affected chemotherapy response in total AML patients(HR=0.452,P=0.067).DLX5-hypermethylated MDS patients showed shorter OS and LFS time than DLX5-hypomethylated MDS patients(P=0.010 and 0.006).Cox regression analysis showed that DLX5 methylation was an independent prognostic factor for OS and LFS in MDS(HR=2.340,P=0.038 and HR=2.394,P=0.030).In total and non-M3 AML,DLX5 hyper-methylated patients showed shorter OS time than DLX5 hypo-methylated MDS patients(P=0.002 and 0.024)but not for LFS(P=0.057 and 0.120).In CN-AML,DLX5 hyper-methylated patients showed shorter OS and LFS time than DLX5 hypo-methylated MDS patients(P=0.003 and 0.009).Cox regression analysis displayed that DLX5 methylation was an independent prognostic factor for OS and LFS in CN-AML(HR=2.345,P=0.025)and a trend in non-M3 AML(HR=1.536,P=0.071).DLX5 expression in AML was significantly dowregulated(P<0.001),and was negatively associated with DLX5 methylation(R=-0.306,P=0.039).ID4 expression was significantly increased after DAC treatment in SKM-1 cells.Functional studies showed that DLX5 presented pro-apoptotic and anti-proliferative effects in SKM-1 cells with cell arrest in G1.Conclusions: Hypermethylation of whole-genome was a frequent event during the progression of MDS,including ID4 and DLX5 methylation.ID4 and DLX5 methylation are involved in the MDS progression through regulating gene expression and may serve as molecular markers of prognosis in patients with MDS and AML. |
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