GBM'lerin TRAIL'a cevabında kromatin modifiye-edici enzimlerin fonksiyonlarının araştırılması

Glioblastoma multiforme (GBM) is an aggressive brain cancer with very limited treatment options. Average survival rate of the patients diagnosed with GBM is around 12 months. The invasive nature of GBMs and the presence of the blood brain barrier (BBB) that limits the delivery of therapeutic agents are among the obstacles for the poor prognosis of GBMs. Surgical resection of the intracranial tumors are also limited due to safety concerns. Tumor-necrosis-factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic bioagent that can target invasive GBM cells in the brain when delivered locally, and program tumor cells for cell death while leaving healthy cells untouched. However, a major obstacle against TRAIL-based therapies is that different tumor cells of a single tumor tissue or different tumors, exhibit differential sensitivity against TRAIL. This highly versatile behavior of the tumor cells is believed to be due to differential regulation of apoptosis mediators. Emerging evidence suggests that TRAIL-resistance is associated with the aberrant expression of the key components of the apoptotic program. However, how these components are regulated at an epigenetic level is not understood. Since transcription levels of all genes are tightly regulated under the control of master transcription factors in concert with epigenetic modifying enzymes, we conducted a short-hairpin RNA (shRNA) screen to assess the function of chromatin modifying-enzymes and aimed to identify novel chromatin-based mechanisms regulating the TRAIL response. We employed 2-3 shRNAs/gene to interrogate the role of 47 genes in DNA and histone modification pathways. Accordingly, we tested shRNAs targeting DNA-methyltransferases (DNMTs), Histone-Methyltransferases (HMTs), Histone demethylases (HDMs), Methyl-DNA binding proteins, and Polycomb complex members (PrC). Upon infection with individual shRNAs, GBM cells were tested for their TRAIL response using viability assays. As a result, we identified KDM2B and RING1A, an H3K36-specific demethylase and a E3 ubiquitin-protein ligase of H2AK119 respectively, as novel regulators of TRAIL response. We then focused our studies on KDM2B. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, and activated caspase-8, caspase-3, caspase-7. Also, KDM2B knockdown accelerated the apoptosis process, as revealed by live cell imaging and real-time cell analyses. Moreover, simultaneous knockdown of the methyltransferases responsible for catalyzing the reverse modification of KDM2B significantly recovered the cell death phenotype observed with KDM2B inhibition. In order to decipher the downstream molecular pathways regulated by KDM2B, we examined the expression levels of apoptosis-related genes by RNA-seq and quantitative RT-PCR and observed de-repression of pro-apoptotic genes HRK, caspase-7, DR4 and repression of anti-apoptotic gene Mcl-1 upon KDM2B silencing. We also observed that KDM2B-silenced tumors exhibited slower growth compared to controls in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells. Therefore, identifying the direct DNA targets of KDM2B might provide a new understanding of apoptosis response in GBMs and aid in the design of successful pro-apoptotic therapies.