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Glioblastoma is one of the most lethal tumours. However, current standard of care therapy is ineffective at eradicating the entire tumour cell population. This fractional killing occurs as glioblastoma cells possess great intertumour (patient-to-patient) and intratumour (cell-to-cell) heterogeneities, leading to tumour recurrence. Microtubules are required for proliferation and other integral cell processes, thus one proposed therapeutic approach to glioblastoma is the use of microtubule-targeting agents (MTAs). It is postulated that this ‘non-targeted’ approach would kill all tumour cells. However, while classical MTAs such as taxanes and Vinca alkaloids are clinically successful anti-cancer drugs and are effective at killing glioblastoma cells in vitro, the polarity and large molecular mass of these drugs render them useless for the treatment brain tumours, as they cannot cross the blood-brain barrier. Our laboratory has been developing small-molecule MTAs, based on the lead inhibitor CMPD1, which are able to cross the blood-brain barrier and effectively kill glioblastoma cells in vivo. While the drug development of small-molecule MTAs for glioblastoma therapy is commercial-in-confidence, the overarching aim of this PhD candidature was to assess the translational potential of microtubule-targeting agents for glioblastoma therapy. We first questioned whether microtubule heterogeneity, resulting from numerous tubulin isoforms and their post-translational modifications impacts on sensitivity of glioblastoma cells to MTAs. Using a panel of 12 genetically diverse glioblastoma stem cell lines and per-division growth rate inhibition metrics we established that the total α- and β-tubulin levels impact on MTA sensitivity. The baseline levels of α- and β-tubulin were up to 40% lower in cells that were not effectively killed by MTAs. Further, low α/β-tubulin expression was associated with higher degree of stemness. Importantly, we discovered that in every glioblastoma cell line, regardless of tubulin expression levels and sensitivity to MTA, a small subpopulation of cells survived MTA treatment via reversible non-mutational dormancy. The cells that survived the treatment, known as drug-tolerant persister (DTP) cells, resumed proliferation in ‘drug holidays’ and displayed the same sensitivity to MTAs as their treatment-naïve parental population. Hence, the drug-tolerant state is a survival mechanism mediated by reversible epigenetic processes, often via changes to the histone H3 subunit of the nucleosome. We used SWATH-MS, a technique emerging as a gold-standard in large-scale proteomics, to assess changes in histone H3 post-translational modifications in DTP cells and compared these to modifications in treatment-naïve parental cells. The analysis revealed that DTP cells exhibit a global decrease in histone lysine acetylation and an increase in histone lysine methylation, which is consistent with a genetically repressive chromatin state. Assessment of transcript levels of histone lysine methyltransferases (KMTs) and demethylases (KDMs) demonstrated more increases in KMT than KDM transcripts in DTP cells relative to their treatment-naïve parental counterparts, supporting SWATH-MS findings. A screen of a library of epigenetic probes and a series of pharmacological assays using disease-relevant cell models discovered that DTP cell recovery and return to a proliferative state was hampered when treated with CMPD1 in combination with inhibitors targeting KDM4 or KDM6. Taken together, the research presented in this thesis suggests that small-molecule MTA are promising drugs to treat glioblastoma patients. However, in order to achieve complete killing of all glioblastoma cells within a tumour population, MTAs must be combined with drugs targeting DTPs. It is hypothesised that KDM inhibitors prevent the demethylation of methylated lysine residues acquired in drug-tolerant cells, and hence, prevent recovery. Further, we identified KDM4 as a potential novel target in treatment-naïve glioblastoma cells. Given the lack of orthogonal and cell-permeable KDM4 inhibitors to validate KDM4 as a target, we established a high-throughput AlphaScreen KDM4 inhibition assay to begin the drug discovery process. Using this assay, we screened a small chemical library and identified two hit molecules that offer excellent starting points for future hit-to-lead optimisation and the development of KDM4 inhibitors. |
