Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome

The Keap1 (Kelch-like ECH-associated protein 1) protein tightly regulates the functions of Nrf2 (nuclear factor-erythroid 2-related factor 2) which plays a pivotal role in the cellular response to oxidative stress. Initial reports uncovered mutations in the KEAP1 gene in non-small cell lung cancer (NSCLC) leading to a permanent nrf2 activation. Mutations were often associated with loss of heterozigosity at the KEAP1 gene locus and promoter hypermethylation suggesting that biallelic inactivation is a common event in NSCLC. In this study, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas and if promoter aberrant methylation may impact patient's outcome. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Curve Characteristics (ROC) analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman's Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman's Rho -0.656 P=0.0001) and in a cell line before and after treatment with 2-deoxy-5 Azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results indicate that that KEAP1 expression is frequently methylated in malignant gliomas and may represent a novel predictor of patient's outcome.