GBM'de temozolomid direncini düzenleyen kromatin ile ilişkili genlerin belirlenmesi

Glioblastoma multiforme (GBM) is the most common and malignant of all primary brain tumors. Unfortunately, less than 5% of patients survive five years post diagnosis. Standard-of-care for GBM consists of surgery, irradiation and chemotherapy with Temozolomide (TMZ) and it has remained unchanged in the last 20 years. Recurrence of tumors in a therapy-resistant state is a major contributing factor to the poor prognosis of GBM patients. In this thesis, new in vitro models of acquired TMZ resistance were generated in order to examine the molecular features associated with therapy resistance in GBMs. Transcriptomic differences between paired TMZ-sensitive and TMZ-resistant cell lines were examined by RNA sequencing, which revealed major alterations in the resistant cell populations. Accordingly, TMZ-resistant models were found to express high levels of several DNA repair pathway components, as part of an adaptive mechanism to resist TMZ-induced DNA damage. Specifically, they were highly dependent on Direct Reversal Repair enzyme MGMT activity, a known prognostic marker in GBM. In addition, key elements of Base Excision Repair and Double Strand Break Repair pathways were upregulated in these cell lines. In parallel, TMZ-resistant cells were also found to activate/inhibit selected cell-cycle regulators, such as CDK4, CDK6, WEE1 and p21. These adaptive changes were most likely key elements to ensure their survival under selective pressure. As the acquired therapy-resistance involves genetic and epigenetic changes in tumor cells, epigenetic vulnerabilities of these resistant cell lines were thoroughly examined. Cancers show aberrant epigenetic regulation, mostly through global changes in DNA methylation and altered histone modification patterns. Yet, the epigenetic mechanisms that cause therapy resistance are not well-known. To examine epigenetic mechanisms of TMZ-resistance and identify new epigenetic inhibitors, we performed chemical screens on the TMZ-resistant cells and their parental controls. Using a comprehensive epigenetic probe library against chromatin modifiers, we identified several histone deacetylase inhibitors (HDACi) as potent agents to target TMZ-resistant cells. We then focused on a specific class of HDACs as a target, namely HDAC6, as it was less studied in GBM and its inhibitors were emerging as new anti-cancer agents. Accumulating evidence suggests that HDAC6 has a role in drug resistance and apoptosis in GBM, however its exact role in the therapy-resistant state has remained elusive. Thus, in this thesis the role of HDAC6 inhibition in TMZ resistance was thoroughly studied. Using chemical inhibitors (Ricolinostat, CAY10603) and CRISPR/Cas9-mediated gene silencing, we showed that HDAC6 inhibition caused marked alterations in the DNA repair pathways, led to cell cycle arrest and ultimately apoptosis in GBM cells. In conclusion, by generating and characterizing new models of TMZ-resistance, we were able to identify HDAC6 as a new therapeutic target for therapy-resistant GBMs.