Novel strategies for the treatment of malignant gliomas are urgently needed. They are characterised by an inherent resistance to both chemo- and radiotherapeutics resulting in unrelenting tumour progression. While the exact mechanisms of treatment resistance remain undefined, it is now recognized that multiple components within the apoptotic pathway are heavily dysregulated in glioma cells and that the over-expression of anti-apoptotic proteins in patient samples correlates with inferior patient survival. The Inhibitor of Apoptosis Proteins (lAPs) represent the final molecular blockade preventing cellular apoptosis and have been identified as a potential rational therapeutic target in gliomas. The work described herein was focused on the development of novel therapeutic strategies that target the lAPs in malignant gliomas, that are readily translatable to the clinic, and that have the potential to improve patient outcomes. The first series of studies examined the hypothesis that targeting the lAPs in conjunction with other conventional and targeted therapies would overcome treatment resistance, and enhance anti-tumour activity. The novel, small molecule, lAP inhibitor LBW242 was shown to successfully target the lAPs in glioma cells and inhibit their ability to bind to and inactivate caspases. However when tested as a single agent in vitro, no stand alone anti-glioma activity of LBW242 was demonstrated. A screen of the activity of LBW242 in combination other pro-apoptotic compounds led to the discovery that lAP inhibition applied in combination with receptor tyrosine kinase (RTK) inhibition led to enhanced caspase activation and induction of apoptosis with a subsequent synergistic anti-glioma effect. The most profound effect was demonstrated with the specific combination of PDGFR and lAP inhibition both in vitro and in vivo as well as in primary patient derived glioma tumourspheres. While multiple RTKs have previously been validated as rational therapeutic targets, the clinical failure of RTK inhibitors in glioma patients has to date remained unexplained. The results in this thesis provide a novel explanation for the resistance of glioma cells to these targeted therapies, and more importantly offer a clinically tractable strategy of overcoming that resistance and improving patient outcomes. The second series of studies investigated the mechanism of synergy between lAP and RTK inhibition. The results showed that PDGFR inhibition does not stimulate apoptosis in glioma cells by previously described pathways. A screen of the entire apoptotic pathway revealed that treatment with imatinib modulates the expression of the anti-apoptotic protein NOL3/ARC. The results showed that imatinib treatment leads to down-regulation of NOL3 and that this effect is critical to the synergy between lAP and PDGFR inhibition. Further analysis suggested a critical role for NOL3 in gliomagenesis and treatment resistance NOL3 was found to be highly expressed in malignant gliomas and with expression levels that are inversely correlated with patient outcomes. A role for NOL3 has not previously been described in malignant gliomas. Finally, a series of studies were undertaken that tested the use of LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide. In vitro assays demonstrated that LBW242 enhanced the pro-apoptotic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple glioma cell lines. Athymic mice bearing established human malignant glioma tumour xenografts treated with LBW242 plus radiation and temozolomide demonstrated a profound and synergistic suppression of tumour growth. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in highly resistant glioma stem cells with a corresponding inhibition of tumour growth. The results indicate a potentially powerful strategy to enhance the therapeutic activity of standard-of-care therapies in glioma patients. Collectively, the findings of the studies in this thesis contribute to a better understanding of the mechanisms of treatment resistance in malignant gliomas, and demonstrate that the pro-apoptotic and anti-glioma effects of radiotherapy, chemotherapy and specific targeted therapies can be enhanced by the addition of a novel, small molecule lAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for glioma patients.
| Identifer | oai:union.ndltd.org:ADTP/258678 |
| Date | January 2009 |
| Creators | Zielger, David, Women's & Children's Health, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. Women's & Children's Health |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Zielger David., http://unsworks.unsw.edu.au/copyright |
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