Note improved survival of IR+BEZ mice.C. Zibotentan (ZD4054) Experimental design == The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic GBM models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation (IR) and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth. == Results == NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of Zibotentan (ZD4054) IR-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared to normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with IR for the treatment of GBM. == Conclusions == These results demonstrate that it may be possible to significantly improve GBM FLN therapy by combining IR with potent and bioavailable DNA repair inhibitors like NVP-BEZ235. Keywords:Glioblastoma, DNA double-strand break, Radiosensitization, NVP-BEZ235, Temozolomide == Introduction == Glioblastomas (GBM) are deadly brain tumors with very poor prognosis (1). Patients with GBM exhibit median survival times of only about 15 months and a 5-12 months survival of less than10% even with aggressive treatment regimens (2). Currently, the standard care consists of surgical resection followed by radiation therapy. Concomitant and adjuvant administration of the chemotherapeutic agent temozolomide (TMZ) was recently added to the standard-of-care and is the only regimen that has improved survival, albeit minimally (2,3). Despite aggressive treatment, these tumors usually recur due to their infiltrative nature and extreme radioresistance (1,4,5). As radiation and TMZ remain the mainstay of GBM therapy, novel radio- and chemo-sensitizing strategies that work with the current therapeutic modality are urgently needed. Ionizing radiation (IR) induces DNA double-strand breaks (DSB), the most deleterious of all types of DNA lesions, that can lead to cell death if left unrepaired (6). Abrogating the DNA damage response (DDR) to these breaks is usually, in principle, a stylish strategy to sensitize cancers to radiation and chemotherapy (7-9). DSBs can be repaired either through the error-prone non-homologous end joining (NHEJ) pathway or the error-free homologous recombination (HR) pathway, in which the PI3K-like kinases, DNA-PKcs and ATM, respectively, are centrally involved (10,11). Therefore, these two apical kinases are very attractive targets for radiosensitization of GBM and other tumors. Research over the past decade has led to the development of specific DNA-PKcs and ATM inhibitors, some of which are quite potent Zibotentan (ZD4054) (12). Unfortunately, potent ATM or DNA-PKcs inhibitors that have good bioavailability for pre-clinical tumor studies and that can also cross the blood-brain-barrier (BBB) have not yet been successfully developed (12). NVP-BEZ235 is usually a Zibotentan (ZD4054) small molecule inhibitor that was originally identified as a dual PI3K/mTOR inhibitor (IC50=4-75nM, IC50=20nM, respectively) (13); reviewed in (14). We reported earlier that NVP-BEZ235 also potently inhibits both ATM and DNA-PKcs, thereby attenuating both HR and NHEJ and resulting in unprecedented radiosensitization in a panel of GBM cell lines (15). NVP-BEZ235 was also found to inhibit ATR, another member of the PI3K-like family (16). Unlike specific inhibitors of PI3K-like kinases (12), NVP-BEZ235 is currently in Phase I/II clinical trials as an mTOR inhibitor and has shown great promise in controlling solid tumors in pre-clinical mouse models (17). Thus, this drug provides us with the unique opportunity of carrying out proof-of-principle experiments in pre-clinical mouse models to test the possibility of improving GBM therapy by blocking both DNA-PKcs and ATM. In this study, we examined whether NVP-BEZ235 could act as a DDR inhibitor in pre-clinical mouse models and, if so, whether combining this drug with IR could be a viable strategy for improving GBM therapy. We find that NVP-BEZ235 can block both DNA-PKcs and ATM and abrogate the repair of IR-induced DSBs in tumorsin vivo, thereby controlling brain tumor growth and significantly prolonging survival of brain tumor-bearing mice. Our results indicate that GBM therapy could possibly be improved by using a combination of IR and specific inhibitors of DNA-PKcs and/or ATM that are potent and bioavailable. == Materials and Methods == == Cell culture and drug treatment == U87MG cells.