Tumor and Stem Cell Biology Selective Inhibition of Parallel DNA Damage Response Pathways Optimizes Radiosensitization of Glioblastoma Stem-like Cells

Glioblastoma is themost common formof primary brain tumor in adults and is essentially incurable. Despite aggressive treatment regimens centered on radiotherapy, tumor recurrence is inevitable and is thought to be driven by glioblastoma stem-like cells (GSC) that are highly radioresistant. DNA damage response pathways are key determinants of radiosensitivity but the extent to which these overlapping and parallel signaling components contribute to GSC radioresistance is unclear. Using a panel of primary patient-derived glioblastoma cell lines, we confirmedby clonogenic survival assays thatGSCswere significantlymore radioresistant thanpaired tumor bulkpopulations.DNAdamage response targetsATM,ATR,CHK1, and PARP1 were upregulated in GSCs, and CHK1 was preferentially activated following irradiation. Consequently, GSCs exhibit rapid G2–M cell-cycle checkpoint activation and enhanced DNA repair. Inhibition of CHK1 or ATR successfully abrogated G2–M checkpoint function, leading to increasedmitotic catastropheanda modest increase in radiation sensitivity. Inhibition of ATM had dual effectsoncell-cycle checkpoint regulationandDNArepair that were associated with greater radiosensitizing effects on GSCs than inhibition of CHK1, ATR, or PARP alone. Combined inhibition of PARP and ATR resulted in a profound radiosensitization of GSCs, which was of greater magnitude than in bulk populations and also exceeded the effect of ATM inhibition. These data demonstrate that multiple, parallel DNA damage signaling pathways contribute to GSC radioresistance and that combined inhibition of cell-cycle checkpoint and DNA repair targets provides the most effective means to overcome radioresistance of GSC. Cancer Res; 75(20); 4416–28. 2015 AACR. Introduction Glioblastoma is the most common primary brain tumor in adults. Despite optimal treatment consisting of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide chemotherapy, median survival remains dismal at 12 to 15 months (1). Treatment responses are inevitably followed by relapse, typically within the maximally irradiated volume (2, 3). In glioblastoma, tumorigenic cells display complex clonal dynamics in which genetically distinct subclones have variable serial repopulating activity in vivo (4, 5). This readout is likely to represent activity of self-renewing glioblastoma "stem-like" cells (GSC) whose self-renewal ability varies on the basis of frequency and/or quantitative features and underpins the evolution of resistant disease (6). Consistent with this, GSCs that express stem cell markers such as CD133, SSEA-1 (CD15), nestin, SOX2, and OLIG2 (7–10) are more resistant to radiotherapy and conventional chemotherapy than more differentiated "tumor bulk" cells (10–14). There is an urgent need to develop targeted treatment strategies that will overcome the innate resistance of GSCs, improve local tumor control, and extend patient survival. Radiotherapy is a vital therapeutic modality for glioblastoma that causes singleand double-stranded DNA breaks that evoke a multifaceted DNA damage response (DDR). At the apex of the DDR lie the serine/threonine protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), which maintain genomic integrity by activating cell-cycle checkpoints andDNA repair pathways (15). ATM ismainly activated by DNA double-strand breaks (DSB), whereas ATR responds to single-stranded regions of DNA generated at stalled replication forks and during processing of DSBs by nucleases (16–19). The MRN (MRE11–RAD50–NBS1) complex has key roles in sensing and processing DSBs as well as activating ATM and ATR (20). ATR activates cell-cycle checkpoint kinase proteins, including CHK1, whereas ATM functions primarily through activation of CHK2. These downstream checkpoint kinases activate G1 and G2–Mcellcycle checkpoints through phosphorylation of phosphatases CDC25A andCDC25Cand kinases CDK1 andWEE1 that regulate cell-cycle progression (21). In addition, ATMpromotes repair of a subset of DSBs. PARP facilitates repair of radiation-induced single-strand breaks (SSB), and the radiosensitizing effects of PARP inhibitors are well-characterized (22). Translational Radiation Biology, Institute of Cancer Sciences,Wolfson Wohl CancerResearchCentre, UniversityofGlasgow,Glasgow,United Kingdom. Department of Clinical Neurosciences, Division of Neurosurgery, ED Adrian Building, Forvie Site, Robinson Way, Cambridge University, Cambridge, United Kingdom. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for S. Yildirim: Muhendislik Fak€ ultesi, Karabuk Universitesi, Karabuk, Turkey. Corresponding Authors: Shafiq U. Ahmed and Anthony J. Chalmers, Institute of Cancer Sciences, WolfsonWohl Cancer Research Centre, University of Glasgow, Gascube Estate, Glasgow G61 1QH, United Kingdom. Phone: 0141-330-3984; 0141-330-6426; Fax: 0141-330-4890; E-mail: [email protected]; [email protected] doi: 10.1158/0008-5472.CAN-14-379