P13.23RADIOSENSITISATION OF PRIMARY GLIOBLASTOMA MULTIFORME CELLS FOLLOWING INHIBITION OF THE METABOLIC AUTOPHAGY PATHWAY

BACKGROUND: Glioblastoma multiforme (GBM) remains a challenge for clinicians due to its heterogeneity and resistance to conventional treatment with temozolomide and radiation. This may be due to GBM stem cells which have been implicated in both tumour recurrence and progression. Autophagy is an evolutionarily conserved metabolic process with dual role in carcinogenesis. It acts as a tumour suppressive mechanism in normal tissues, but promotes tumour survival upon genotoxic stress. Our previous work showed that an autophagy gene expression signature is enriched in GBM tumours belonging to the mesenchymal subtype, an association that is maintained in primary glioblastoma cells. The main objective of the present work is to investigate the role of autophagy in primary GBM cells, following a fractionated course of radiotherapy and combination treatment (TMZ and fractionated RT). METHOD: We used primary GBM cells from fully consented patients supplied by local biobanks. Autophagy was inhibited both by pharmacological means, chloroquine (CQ) and hydroxychloroquine (HCQ) and by the stable knockdown of the essential autophagy gene ATG7. Cells were irradiated using the HS X- ray systems Varian NDI- 32 .Temozolomide was used at the clinically relevant dose of 100uM. Using the cell viability SRB assay and InCucyte derived growth curves response to radiotherapy was assessed following either a single 5 Gy fraction or a fractionated schedule of 10Gy in 5 fractions. RESULTS: Inhibition of autophagy using CQ/HCQ/knockdown did not significantly alter growth characteristics of primary GBM cells at steady state, as demonstrated by growth curves. Using clinically relevant schedules of fractionated radiotherapy, we demonstrated a statistically significant radiosensitisation upon autophagy inhibition, which was not seen following a single 5Gy treatment. This effect was seen regardless of the method of autophagy inhibition and persisted when cells co-treated with TMZ underwent fractionated radiotherapy. CONCLUSION: There are currently a large number of clinical trials based on the inhibition of the metabolic autophagy pathway, using CQ and HCQ. Our work implicates autophagy in resistance to radiotherapy in primary GBM cells and suggests that autophagy inhibition will be an effective means of improving outcome of radiotherapy. A randomised phase II study investigating the role of HCQ to inhibit autophagy in addition to short course radiotherapy in patients aged 70years and older w