Enhanced radiosensitisation of human glioma cells by combining inhibition of PARP with inhibition of Hsp90

Glioblastoma multiforme are the commonest primary brain tumour and are resistant to standard therapies. The non-dividing nature of normal brain provides an opportunity to enhance the therapeutic ratio by combining radiation with inhibitors of replication-specific DNA repair pathways. Based on our previous findings that inhibition of poly(ADP-ribose) polymerase (PARP) increases radiosensitivity of human glioma cells in a replication-dependent manner and generates excess DNA breaks that are repaired by homologous recombination (HR), we hypothesised that inhibition of HR would amplify the replication-specific radiosensitizing effects of PARP inhibition. Specific inhibitors of HR are not available, but the Heat Shock Protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been reported to inhibit HR function. The radiosensitizing effects of 17-AAG and the PARP inhibitor olaparib were assessed and the underlying mechanisms explored. 17-AAG downregulated Rad51 and BRCA2 protein levels, abrogated induction of Rad51 foci by radiation and inhibited HR measured by the I- Sce 1 assay. Individually, 17-AAG and olaparib had modest, replication-dependent radiosensitising effects on T98G glioma cells. Additive radiosensitisation was observed with combination treatment, mirrored by increases in γH2AX foci in G2 phase cells. Unlike olaparib, 17-AAG did not increase radiation sensitivity of CHO cells, indicating tumour specificity. However, 17-AAG also enhanced radiosensitivity in HR-deficient cells, indicating that its effects were only partially mediated by HR inhibition. Additional mechanisms are likely to include destabilization of oncoproteins that are upregulated in GBM. 17-AAG is therefore a tumour-specific, replication-dependent radiosensitizer that enhances the effects of PARP inhibition. This combination has therapeutic potential in the management of GBM.