Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells

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 ; 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, 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 These data identify RS as a cancer stem cell-specific target with significant clinical potential. 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. .

DNA repair genes in astrocytoma tumorigenesis, progression and therapy resistance

“…Recently, Carruthers and coworkers showed that GBM CSCs present constitutively high levels of replicative stress, both in vitro and in vivo, and provided evidence that this phenotype underlies the activation of DDR and consequent radiation resistance (Carruthers et al, 2018). In agreement, recent studies have demonstrated that GBM CSCs exhibit greater efficiency in the activation of DNA damage sensors, as ATM, 53BP1 and H2AX, and are more resistant to CT.…”

“…Paradoxically, DDR and the repair machinery act as a double-edged sword during tumorigenesis and cancer progression. Once the tumor has been established, replicative stress can promote the aberrant constitutive activation of DDR and repair execution (Bartkova et al, 2010;Carruthers et al, 2018), allowing tumor progression and driving treatment resistance. In turn, the exacerbated activity of these pathways defend malignantly transformed cells from replicative stress, high mutation rates, and the rampant genome instability (Kauffmann et al, 2008;Turner et al, 2015).…”

Drug resistance mechanisms of cancer stem-like cells and their therapeutic potential as drug targets

“…Bao et al [3] reported that once patients were irradiated in therapeutic regimen, survived glioblastoma cells became more resistant to irradiation, suggesting that CSCs were not eradicated and started to self-renew after therapy. Very recently, Carruthers et al [45] shed light on how glioblastoma CSCs possess higher resistance to radiotherapy. They found that DNA damage response is constitutively activated in CSCs, because of higher level of replicative stress generated in those cells.…”

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition

“… 40 In radiotherapy of glioblastoma, replication stress leading to constitutive activation of the DNA damage response pathway under irradiation is thought to mediate radioresistance. 41 , 42 Radioresistance in CD133-expressing glioma cells, is mediated via activation of the DNA damage pathway, making its inhibition a promising opportunity to circumvent this tumor escape mechanism. 43 Yet, a combination of AZD6738 with radiation in vivo revealed no prolonged survival in this model.…”