Background

Glioblastoma (GBM) is a radioresistant tumour associated with local recurrence following chemoradiotherapy. Tumour recurrence is due to a subset of tumour propagating cells with stem like characteristics (cancer stem cells CSC). CSCs in GBM have been suggested to have an upregulated DNA damage response (DDR), and a radioresistant phenotype. However, literature and opinion regarding GBM CSC radiosensitivity is conflicting.

DDR inhibitor drugs may enable inhibition of upregulated DDR seen in CSC's. If DDR inhibitors can be combined with radiotherapy this could produce clinical benefits such as improved local control and survival. We investigated GBM CSC radioresistance and explored the effects of inhibition of the DDR protein ATM.

Method

CSC enriched cultures from primary GBM cell lines were cultured in media without foetal calf serum (FCS) with the addition of growth factors. Cancer bulk cells (CBC) were cultured in medium containing FCS. Western blotting was used to quantify phosphorylated DDR proteins in response to radiation plus or minus the ATM inhibitor Ku55933. FACS analysis of phosphorylated Histone H3 was used to investigate G2/M checkpoint function. We utilised immunofluorescence to quantify gamma H2AX foci as a marker of DNA double strand breaks (DSB). Clonogenic survival assays were performed as a clinically relevant measure of radiation sensitivity.

Results

Data from two primary cell cultures show that GBM CSC are radioresistant compared to CBC's and show upregulation of phosphorylated DDR proteins after irradiation, enhanced G2/M checkpoint activation and enhanced repair 24 hours after irradiation. All of these radioresistance features were abrogated by chemical inhibition of ATM, which radiosensitised GBM CSC's to a greater extent than CBC's. The mechanism of radiosensitisation by Ku55933 involves inhibition of the G2/M checkpoint and delayed DNA DSB resolution in GBM CSC's.

Conclusion

ATM inhibition may be a CSC targeted strategy to produce clinical benefit in glioblastoma.