P17.17 * INHIBITION OF DNA DAMAGE RESPONSE ABROGATES GLIOBLASTOMA CANCER STEM CELL RADIORESISTANCE

GBM recurrence following radiotherapy is due to a subset of tumour propagating cells with stem like characteristics (cancer stem cells, CSC). CSCs have the ability to recapitulate the tumour of origin whereas the majority of tumour cells (tumour bulk) do not. CSCs in GBM have been reported to exhibit an upregulated DNA damage response (DDR) and a radioresistant phenotype. However, literature and opinion regarding GBM CSC radiosensitivity is conflicting. The prospect of influencing GBM CSC radiosensitivity may provide important clinical benefits, and DDR is an appealing therapeutic target. We examined the radiosensitivity of paired CSC and tumour bulk cultures of primary GBM cell lines. We report that GBM CSC are radioresistant by clonogenic assay, and display upregulation of key DDR elements (PARP-1, pATM, pChk1, pChk2) at baseline and post ionising radiation (IR). GBM CSCs display an enhanced activation of the G2/M checkpoint by FACS analysis. We report a detailed analysis of DSB repair by gamma H2AX foci quantification showing enhanced resolution of DSBs by GBM CSC's at late time points (24 hours post IR). We investigated the effects of inhibition of the key DDR elements ATM, ATR and PARP on GBM CSC radiosensitivity. Inhibition of ATM by KU-55933 provided potent radiosensitisation of GBM CSC's, and reversed the enhanced DSB resolution observed in these populations. However, ATM inhibition only provided partial abrogation of G2/M checkpoint activation in GBM CSC's. Inhibition of ATR by VE-821 provided modest radiosensitisation of CSC's however proved to effectively abrogate G2/M checkpoint responses. The PARP inhibitor olaparib increased DSB burden at 24 hours post IR in GBM CSC's and provided modest radiosensitisation. Given the dependence of GBM CSC's on both proficient G2/M checkpoint activation and DSB repair we combined the checkpoint abrogating qualities of ATR inhibition with the repair deficit produced by PARP inhibition. This combination produced enhanced radiosensitisation of GBM CSC populations. In conclusion we confirm radioresistance of GBM CSC populations using the gold standard, clinically relevant endpoint of clonogenic survival. Strategies which affect both DSB burden at 24 hours post IR and G2/M checkpoint produce enhanced radiosensitisation of GBM CSC's. Our investigations suggest that DDR inhibition is an effective GBM CSC targeted radiosensitisation strategy which may provide important clinical benefits for patients.