OP01 * MISMATCH REPAIR DEFICIENCY CONFERS INCREASED SENSITIVITY TO IONISING RADIATION (XR) IN GLIOBLASTOMA

Abstract

INTRODUCTION: Combination chemo-radiotherapy, including the alkylating agent temozolomide (TMZ) is standard of care for patients with glioma. Increased TMZ use has lead to sustained interest in the mechanisms underlying acquired resistance. Amongst these, deficiency in mismatch repair (MMR) is best described, with TMZ treatment promoting loss of functional MMR proteins most frequently through point mutations in MSH2/6, which may then lead to a hyper-mutation phenotype. Since patients are increasingly exposed to TMZ as first-line treatment with subsequent radiotherapy we set out to investigate how MMR status may influence response to XR and/or radiosensitising agents. METHOD: Effects of MMR protein deficiency on response to XR in paired isogenic glioma cell lines; U251 (MMR proficient) and U251.TR3 (MMR deficient, TMZ-induced MSH6mut) lines determined by clonogenic assay and immunofluorescence to detect γH2AX foci. MMR proteins were down-regulated by siRNA transfection. MMR protein expression was determined by western blotting and MMR deficient cell lines were further characterised by sequencing. RESULTS: We have shown that U251.TR3 cells (MMR deficient) are more sensitive to radiation-induced cell death than MMR proficient U251 cells in clonogenic assays (surviving fraction at 2Gy 0.41 ± 0.03 and 0.71 ± 0.04 respectively, p < 0.001). This sensitisation is accompanied by an increase in gH2AX foci (28 ± 3 and 50 ± 6 foci per cell in U251 and U251.TR3 cells respectively). On-going work is investigating whether this effect is due to MMR deficiency per se or secondary mutations driven by a hyper-mutation phenotype. CONCLUSION: Detection of MMR proteins following TMZ exposure may serve as a tool to stratify patients for treatment.