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Abstract
Diffuse intrinsic pontine glioma (DIPG) is an aggressive paediatric brainstem tumour with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation, and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. This thesis investigates the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG.
High expression levels of key players of the polyamine pathway were observed in DIPG samples by qPCR and western blotting. Alamar blue cytotoxicity and soft-agar clonogenic assays, showed that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Addition of polyamine transporter inhibitor AMXT 1501 to DFMO led to synergistic inhibition of DIPG proliferation. Western blotting and flow-cytometric analysis of Annexin V-stained cells showed that combination treatment enhanced apoptosis. Consistent with the in vitro results, the combination of DFMO and AMXT 1501 significantly prolonged the survival of mice bearing DIPG orthografts. DIPG tumours contained higher polyamine concentrations compared to healthy brain tissue, treatment with the combination therapy led to significantly lower levels of intratumoral polyamines. This combination therapy significantly extended the survival of DIPG engrafted mice, with 6/9 mice surviving until the humane endpoint of 160 days. Examination of RNA expression levels in a cohort of high-risk childhood cancers showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG and other paediatric high-grade gliomas compared with all other high-risk childhood cancers. Together these results suggest that this strategy of dual polyamine inhibition may be potent novel therapy for these paediatric brain tumours. AMXT 1501 is currently in clinical development and following completion of an adult Phase 1 trial a clinical trial for DIPG patients is planned.
Tumour cells are dependent upon arginine, a semi-essential amino acid, metabolised by arginase enzymes into ornithine, a pivotal precursor to the polyamine pathway. Pegylated arginase (BCT-100) has recently been shown to significantly delay tumour development, prolonging survival of neuroblastoma-prone Th-MYCN mice. Arginine depletion therapy as a single agent and in combination with polyamine pathway inhibitors in DIPG was investigated. We found that ARG2, the gene encoding for arginase II, is significantly over-expressed in DIPG tumours compared to normal brain. Arginine depletion via BCT-100 reduced DIPG cell proliferation and colony formation in patient-derived cell lines. Treatment of orthotopic patient-derived xenograft models of DIPG, with BCT-100 (4x/week) significantly delayed tumour development and increased the survival of the mice (p<0.0001). The combination of BCT-100 with DFMO led to significant enhancement in DIPG survival (p<0.005 compared to single agent treatments). Triple combination therapy with the addition of the polyamine transport inhibitor AMXT 1501 led to a potent and profound improvement in survival. These data show that arginine depletion therapy using BCT-100 combined with dual polyamine inhibitory agents represents a potentially exciting new approach for the treatment of DIPG.
Collectively, this study has provided a firm foundation for future investigations into the important roles of polyamines in DIPG / paediatric brain tumour biology and metabolism and has also led to the development of an international clinical trial for DIPG patients.