Silencing of KDM2B leads to deregulation of apoptosis related genes in GBM

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer protein that can specifically kill tumor cells while sparing healthy ones. Emerging evidences suggest that TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we aimed to identify novel epigenetic mechanisms regulating TRAIL response in Glioblastoma Multiforme (GBM) by a short-hairpin RNA (shRNA) screen. We employed an shRNA-mediated loss of function approach to interrogate the role of 48 genes in DNA and histone modification pathways. From this we identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of Caspase-8, Caspase-3, Caspase-7, and cleavage of PARP. KDM2B knockdown also accelerated the apoptosis process, as revealed by live cell imaging experiments. Moreover, simultaneous knockdown of the methyltransferases responsible for generating the histone marks removed by KDM2B significantly recovered the cell death phenotype observed with KDM2B inhibition. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing and quantitative PCR upon KDM2B loss, which revealed de-repression of pro-apoptotic genes HRK, caspase-7, and DR4 and repression of anti-apoptotic gene Mcl-1. The apoptosis phenotype was dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. In vivo, KDM2B-silenced tumors exhibited slower growth and reduced angiogenic capacity compared to controls. Taken together, our findings suggest a novel mechanism regulating apoptotic response, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells. Overall design: mRNA profiles of U87MG GBM cells transduced either by control shRNA or shRNA targeting KDM2B were generated by RNA-seq (Illumina HiSeq 2500). 2 biological replicates of shControl and shKDM2B total RNAs were barcoded individually and deep sequenced as 3 technical replicates each in 3 lanes.

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Kurt IC, Sur I, Kaya E, Cingoz A, Kazancioglu S, Kahya Z, Toparlak OD, Senbabaoglu F, Kaya Z, Ozyerli E, Karahüseyinoglu S, Lack NA, Gümüs ZH, Onder TT, Bagci-Onder T