Replication stress drives constitutive activation of the DNA damage response and radioresistance in glioblastoma stem-like cells

Carruthers, Ross D. and Ahmed, Shafiq U. and Ramachandran, Shaliny and Strathdee, Karen and Kurian, Kathreena M. and Hedley, Ann and Gomez-Roman, Natividad and Kalna, Gabriela and Neilson, Mathew and Gilmour, Lesley and Stevenson, Katrina H. and Hammond, Ester M. and Chalmers, Anthony J. (2018) Replication stress drives constitutive activation of the DNA damage response and radioresistance in glioblastoma stem-like cells. Cancer Research, 78 (17). pp. 5060-5071. ISSN 0008-5472

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    Abstract

    Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM.