Investigation and Therapeutic Targeting of Metabolic Dependencies in Malignant Brain Tumours

Abstract

Malignant brain tumours are highly lethal cancers that affect the adult and paediatric populations. Glioblastoma (GBM), the common high-grade CNS tumour in adulthood is highly lethal with median patient survival of only 14.6 months despite aggressive treatment. Fatty acid oxidation (FAO) was previously established as a metabolic dependency in GBM-stem like cells which can contribute to tumour progression. Targeting this pathway in combination with standard therapy, may offer more effective treatment strategy for GBM. In childhood, medulloblastoma (MB) is the common primary malignant brain tumour and despite advances in its molecular sub-classification and risk-stratification a subset remain difficult to treat. MBGRP3 patients represent around 25% of all medulloblastoma cases. Amplification and elevated expression of MYC is a notable abnormality in this group and also correlates with poorer clinical outcomes. Since direct targeting of MYC remains elusive, understanding and exploiting metabolic dependencies in MYC-amplified MBGRP3 may reveal novel therapeutic opportunities. For this project, in vivo and in vitro metabolic profiling using magnetic resonance imaging and spectroscopy was utilised to evaluate metabolic alterations and associated with the pathobiology of GBM and MB. Metabolic profiling in GBM focused on tumour progression and treatment-induced changes following combination therapy using the FAO inhibitor, etomoxir alongside temozolomide chemotherapy in a clinically relevant model of GBM. To better understand the role of MYC in MB metabolism, we engineered three independent MYC-amplified MBGRP3 cell lines (D425, D283, HDMB03), to each harbour doxycycline-inducible anti-MYC shRNAs (two independent species) or a non silencing shRNA control. We utilised 1H high resolution magic angle spectroscopy (HRMAS) and stable isotope resolved metabolomics to assess changes in intracellular metabolites and pathway dynamics when MYC expression was modulated. Findings from the GBM study supported previous observations suggesting FAO inhibition was a viable strategy for targeting GBM. For MB, MYC knockdown (KD) resulted in a marked reduction in proliferation and cell cycle progression analogous to MYC-dependent cancer phenotypes. Metabolic profiling revealed consistent MYC dependent changes in metabolites across the three MYC-amplified cell lines. Notably, glycine was consistently found to be accumulated following MYC KD cells. 13C-glucose labelling showed a reduction in serine and glycine synthesis following MYC knockdown. In human primary tumours, expression of PHGDH, the rate limiting enzyme in de novo serine synthesis, was associated with MYC amplification and poorer survival outcomes. Furthermore, MYC expressing cells showed greater sensitivity to pharmacological inhibition of PHGDH compared to MYC KD (Group 3) and SHH subgroup cell lines. Together, these findings provide insights into the metabolic vulnerabilities of the most common malignant brain tumours arising in children and adults, and suggest novel therapeutic opportunities. Combination therapy using etomoxir and temozolomide could provide more effective therapy for GBM. Furthermore, metabolic profiling uncovered MYC-dependent metabolic alterations and revealed the de novo serine/glycine synthesis pathway as a novel and clinically relevant therapeutic target in Group 3 MYC-amplified MB.