AbstractIntroduction: Childhood cancer patients are often treated with chemotherapy regimens developed in adults which can result in life lasting side effects such as, organ failure, infertility and learning difficulties. This is due to potent traditional chemotherapy drugs in use targeting rapidly proliferating cells indiscriminately, which is problematic in growing children. There is now a recognised need for improvement of paediatric treatments through use of more selective approaches to reduce unwanted side effects. Here, altered metabolism is the focus of investigating novel opportunities to produce potent and selective treatments for some of the most prevalent paediatric cancer types through use of a representative cell line panel.
Method: Investigations into the metabolic phenotype of the cell line panel under normal growth conditions and when metabolically stressed were undertaken using a real-time metabolic Seahorse XFp analyser. Chemosensitivity assays were performed using inhibitors of key targets to assess potency, selectivity and chemopotentiation when combined with standard chemotherapeutic agents, and under varying oxygen tensions. Mechanistic studies were conducted through utilisation of NAD(H) assays, image cytometry, migration assays, immunoblotting, qPCR mRNA analysis and microscopy.
Results: Paediatric cancer cell lines showed utilisation of both OXPHOS and glycolysis with a general trend of increased rates of both pathways compared to non-cancer cell models. Across the seven neuroblastoma cell lines analysed, there was considerable heterogeneity in overall metabolic activity and variation in extent of any metabolic bias towards one pathway or another. In many of the paediatric cancer cell lines, metabolic bias compared to non-cancer cell models was towards OXPHOS. Interestingly, paediatric cancer cell lines showed very limited glycolytic or OXPHOS reserve compared to non-cancer cells suggesting limited scope to metabolically compensate for targeting of either pathway. Analyses of NAD+ re-generation pathways indicated common deficiency in enzyme NAPRT and cancer cell selective chemosensitivity to inhibition of NAD+-salvage enzyme NAMPT in all of the paediatric cancer cell lines. NAMPT inhibition was able to selectively chemopotentiate DNA alkylating agent temozolomide against selective paediatric cancer cell lines, including under hypoxic (0.1% O2) conditions. This contrasted with non-selective chemopotentiation by PARP inhibitor rucaparib. Cancer selective chemopotentiation by NAMPT inhibition correlated with preferential NAD+ depletion and decreased PARP activity although other mechanisms may be contributing to effects. Inhibition of NAD+-dependent deacetylase SIRT1 induced a neuronal-like morphology in some cell lines with some evidence of increased mRNA expression of neuronal marker MAP2 and decreased expression of neural CSC marker nestin. Similarly, preliminary investigation of acetate as a potential epigenetic differentiation agent also caused decreased nestin and induced neurite-like cell projections.
Discussion and Conclusions: Proof-of-principle was provided that NAMPT inhibition could selectively chemopotentiate clinical alkylating agent temozolomide in vitro both under normoxia and hypoxic conditions, however, this was only observed in a subset of the cell lines which is not yet fully understood. Preliminary mechanistic studies suggested that this chemopotentiation correlates with PARP activity inhibition via NAD+ depletion. Preliminary investigations also suggested the potential of metabo-epigenetic approaches, for example to induce epigenetic alterations and a change in paediatric cancer cell stemness or differentiation status. Overall, several novel metabolic approaches for the potential therapeutic targeting of paediatric cancers are indicated with further preclinical investigation and confirmation of some preliminary results required.
|Date of Award
|3 Oct 2022
|Simon Allison (Main Supervisor) & Roger Phillips (Co-Supervisor)