Clinical Significance Of PCDH17 Expression In Acute Myeloid Leukemia And Its Regulatory Mechanisms

Objective Leukemia stem cell（LSC）-enriched genes have been shown to be highly prognostic in acute myeloid leukemia（AML）.However,these genes are mainly specifically up-regulated in LSCs,whereas the prognostic value of tumor suppressor genes（TSGs）that are repressed early in LSC remains largely unknown.In this study,we aimed to identify significantly dysregulated TSGs in LSCs using bioinformatic methods,and to further explore the expression level,clinical significance,and epigenetic regulatory mechanisms of the gene with potential prognostic value.Methods1.By comparing mRNA expression profiling and DNA methylation profiling data of LSCs and hematopoietic stem cells（HSCs）,candidate TSGs that were down-regulated and hypermethylated in LSCs were screened.2.The candidate genes were checked in the tumor suppressor gene database,and Cox regression survival analysis was used to identify genes with potential prognostic value;the PCDH17 gene was screened for further study.3.Real-time quantitative PCR（RQ-PCR）was carried out to assess the expression of PCDH17.The RQ-PCR results,combined with data from public repository,were used to analyze and validate the expression status and clinical significance of PCDH17.4.Real-time quantitative methylation-specific PCR（RQ-MSP）and MethylTarget targeted bisulfite sequencing were used to detect the methylation status of PCDH17.5.Hypomethylating agent 5-aza-2’-deoxycytidine（5-aza-dC）was used to treat leukemia cell line HL60 for evaluating whether repression of PCDH17 is mediated by its promoter methylation.Results The down-regulated and hypermethylated genes in LSCs showed significant enrichment of adhesion molecules and members of the cadherin family.The transcript level of PCDH17 in LSCs was significantly lower than that in HSCs(P=6.757×10^-5),while its methylation level was significantly up-regulated(P=3.764×10^-5).Reduced expression of PCDH17 was observed in four AML microarray data sets;our RQ-PCR data also confirmed its down-regulation in AML patients（P=0.009）.Moreover,the expression level of PCDH17 showed a dynamic trend during the differentiation of hematopoietic cells.In the TCGA cohort,patients with low PCDH17 expression were more likely to be female（P=0.0001）,had higher white blood cell（WBC）counts,higher percentage of bone marrow（BM）and peripheral blood（PB）blasts（P<0.0001,P=0.04,and P<0.001）than patients with high PCDH17 expression.There were no other significant differences in presenting clinical characteristics between these two groups including age（P=0.41）and karyotype classification（P=0.343）.In addition,patients with low PCDH17 expression had more FLT3-ITD and NPM1 mutations（P=0.002 and P=0.020）and less TP53mutations（P=0.005）than patients with high PCDH17 expression.In our cohort,we confirmed the association between low PCDH17 expression and higher percentage of BM blasts（P=0.017）.Besides,low PCDH17 expression was more often observed in intermediate-risk patients（P<0.001）or in cytogenetically normal AML patients（P=0.030）.Compared with patients with high PCDH17 expression,those with low PCDH17 expression had an inferior complete remission（CR）rate（P=0.020）.Furthermore,PCDH17 expression was significantly up-regulated in patients achieved CR after induction therapy（P<0.001）.Low PCDH17 expression predicted worse overall survival（OS）in four independent cohorts,which was also confirmed in both the whole AML and the CN-AML patients.Moreover,in the TCGA cohort,dichotomous stratification of PCDH17 expression was still able to discriminate between shorter and longer OS in the FLT3-ITD absent,NPM1 mutated,and TP53wild-type patients（P=0.0074,0.017,and 0.0015,respectively）.Multivariate analysis showed that the low PCDH17 expression was an independent predictor of poor prognosis in both whole AML and CN-AML patients.Through bioinformatics analysis of TCGA data,it was found that PCDH17 expression-associated gene signatures were characterized by deregulations of EMT-and Wnt pathway-related genes.MethylTarget methylation detection showed that PCDH17 methylation level in AML was significantly higher than controls(P=3.176×10^-11).Moreover,there was a significant negative correlation between PCDH17 expression and its promoter methylation in AML patients（R=-0.30,P=0.036）.After 5-aza-dC treatment of HL60 cell line,PCDH17 expression was increased,with a concomitant reduction of PCDH17 promoter methylation.Further analysis of public data showed that the expression of PCDH17 could also be regulated by EZH2-mediated epigenetic suppression.Conclusions Transcriptional silencing of PCDH17 is a common feature in AML patients and is related to specific clinical parameters and gene mutations.Low PCDH17 expression predicts poor prognosis in AML patients;it further dichotomizes survival in the molecularly defined subgroups of AML patients.PCDH17 expression was mainly regulated by its promoter,which is hypermethylated in AML patients..