DNA replication stress generates radiation resistance in glioblastoma stem like cells


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Ross Carruthers1
1University of Glasgow

Abstract

Background

The mechanisms determining upregulated DNA damage response (DDR) and radiation resistance in glioblastoma stem-like cells (GSC) are unclear, despite having broad implications for understanding of GSC phenotype, gliomagenesis and glioblastoma (GBM) treatments. We investigated DNA replication stress (RS) as a determinant of GSC radioresistance and as a therapeutic target.

Method

DNA fibre assay was utilised in primary GBM cultures to compare RS in CD133+ and CD133- populations. Immunofluorescence quantified expression of GSC markers and RS response proteins in human GBMs. RNA sequencing quantified expression of long genes (>800kbp) in GSC enriched and deplete cell cultures. Finally we targeted GSC RS response with PARP (olaparib) and ATR (VE-821) inhibition in vitro.

Results

GSC show increased expression of the RS markers pCHK1, pATR and RPA versus non-GSC. GSC show reduced DNA replication velocity relative to non-GSC by DNA fibre assay. Gamma H2AX foci in S phase GSCs colocalise with foci of BrDU incorporation. We show that long neural genes are preferentially expressed in GSCs relative to non-GSCs, consistent with replication/transcription collisions as the cause of elevated RS in GSCs. Slowing of DNA replication forks with aphidicolin generates radiation resistance in non-GSCs, linking RS to radioresistance. Finally, dual combined inhibition of PARP and ATR preferentially reduces cell viability in GSC compared to non-GSC and induces gamma H2AX foci preferentially in GSCs. This combination dramatically reduces neurosphere generation from and potently radiosensitises GSC.

Conclusion

We propose RS as a novel determinant of radiation resistance and the underlying mechanism of preferential DDR activation in GSC. We hypothesise GSCs exhibit elevated RS due to constitutive expression of long neural genes generating activation of DDR via replication/transcription collisions and subsequent enhanced DNA repair. Our findings shed new light on GSC biology, and identify novel therapeutics with potential to improve clinical outcomes.