Assessing the epigenetic modifiers of drug resistance in human astrocyte and glioblastoma co-cultures

Glioblastoma (GBM) is the most common and malignant of all primary CNS tumours. Unfortunately, GBM has very low survival. The standard treatment regimen consists of surgery, ionizing radiation, and chemotherapy. The most common chemotherapeutic used for the treatment of GBM is temozolomide, followed by carmustine, which could not change the overall survival rates in the last 25 years. Unfortunately, most drug trials fail at the initial phases. For this failure in progress, tumour-microenvironment interactions play an important role, beside others causes. Therefore, understanding the nature of therapy resistance in relation to tumour-microenvironment interactions is essential. In this thesis, a new co-culture model was established to examine temozolomide response of tumour cells in the context of astrocyte microenvironment. Contact-dependent and contact-independent co-culture models were examined with cell viability and imaging methods. Contact-dependent astrocyte-U87MG co-cultures presented temozolomide resistance. Transcriptomic differences between U87MG cells alone and in co-cultures with astrocytes were examined by cell sorting, followed by RNA sequencing. As a testament to increased cell-to-cell interaction, co-culture models were found to express high levels of several cell-extracellular matrix interaction pathway components. Specifically, increased levels of collagen (COL), matrix-metalloproteins (MMP), and tubulin (TUB) families were observed. These adaptive changes were most likely vital elements of drug resistance in co-cultures. As contact-dependent temozolomide resistance may involve genetic and epigenetic changes, epigenetic vulnerabilities of tumour cells in co-cultures were examined. Cancers show altered epigenetic regulation; global changes in DNA and histone methylation. Yet, the epigenetic regulation of temozolomide response in co-cultures is not established. We performed a small molecule epigenetic inhibitor probe library screen to examine and identify epigenetic regulators of temozolomide response in co-cultures. We identified several histone demethylase inhibitors, in combination with temozolomide, as potent agents to target U87MG cells cultured with astrocytes. We then focused on GSK-J4, a histone demethylase inhibitor, as a potent agent that could affect GBM cells that are grown in co-cultures. GSK-J4 is a known agent that inhibits KDM6A and KDM6B, whose roles have not been studied in the context of temozolomide response in GBM. To understand the mechanisms of GSK-J4 in co-cultures, cell viability assays with chemical inhibition and CRISPR/Cas9-targeted gene silencing of KDM6A or KDM6B were applied. Further CITE-single cell sequencing was performed in U87MG-astrocyte co-cultures in the presence and absence of GSK-J4. We showed that both cell compartments were differentially represented at their transcriptome levels. Hypoxia and glycolysis-related genes MT-CYB, MT-CO2, MT-ND2, and MTRNR2L10 were upregulated and apoptosis-related genes were down-regulated in both tumour cells and astrocytes in response to GSK-J4. In conclusion, by generating and characterising co-culture models of GBM, we were able to find epigenetic regulators of temozolomide response and present transcriptomic differences that may serve as potential therapeutic intervention points for GBM in the future.