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Glioblastoma multiforme (GBM) are lethal tumors that are highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, EGFRvIII, confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSBs), the most lethal lesion inflicted by IR. The EGFR inhibitor Gefitinib (Iressa) and the PI3K inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated-Akt-1 accelerates repair, implicating the PI3K-Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-PKcs. Enhanced radioresistance is abrogated by the DNA-PKcs-specific inhibitor NU7026 and EGFRvIII fails to confer radioresistance in DNA-PKcs-deficient cells. In vivo, orthotopic U87-EGFRvIII-derived tumors display faster rates of DSB repair following whole brain radiotherapy (WBRT) compared to U87-derived tumors. Consequently, EGFRVIII-expressing tumors are radioresistant and continue to grow following WBRT with little impact on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII-expression promotes DNA-PKcs activation and DSB repair, perhaps, as a consequence of hyperactivated PI3K-Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas.
Glioblastoma multiforme (GBM) are solid tumors of the brain with extremely poor prognosis (1). Effective treatment of GBM is complicated by the diffuse infiltrative nature of the disease and extreme radioresistance of these tumors. However, a clear survival advantage of post-resection radiation has been established by randomized trails, showing that the median survival of GBM patients was improved, from approximately 6 months to 10–12 months, following near-maximal brain-tolerated doses of ionizing radiation (IR; 50–60 Gy). The central role that radiation plays in treating GBM is also illustrated by the landmark Stupp study (2) showing that concurrent radiation and Temozolomide, an alkylating agent, can further increase median survival from 12.1 to 14.6 months. It is clear that an improved molecular understanding of GBM radioresistance is needed for the development of rational, tumor-selective radiosensitizing drugs.
As a first step towards understanding the genetic basis of GBM radioresistance, we focused on EGFRvIII, the most commonly amplified/mutated gene in GBMs (1, 3, 4). Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase (RTK) whose ligands include epidermal growth factor (EGF) and transforming growth factor-alpha (TGFα) (5, 6). The EGFR gene is amplified in approximately 50% of GBMs and, of these, about half express a truncated version of the receptor, EGFRvIII (1, 3, 4). Although EGFRvIII lacks the ligand-binding domain (7), it is constitutively active (8), stimulating downstream signaling effectors that include phosphatidylinositol 3-kinase (PI3K), Akt-1, Ras, and mitogen-activated protein kinase (MAPK). Several studies have demonstrated that EGFRvIII promotes malignant growth (9) and is associated with poor prognosis (10) (11).
Previous in vitro studies using established glioma cell lines have shown that EGFRvIII confers resistance to IR (12–14). Xenograft studies have demonstrated that EGFR-specific inhibitors (small molecule as well as α-EGFR antibodies) significantly enhance the efficacy of radiotherapy (15, 16). However, the signaling pathways directly involved in EGFRvIII-mediated radiation resistance have not been completely elucidated. We report here that EGFRvIII expression enhances radioresistance in tumor suppressor-deficient primary mouse astrocytes and in U87 human glioma cell lines by promoting the rapid repair of radiation-induced DNA double-strand breaks (DSBs). Proficient DSB repair is facilitated by hyperactivation of the DSB repair enzyme, DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) (17, 18). We extend these in vitro mechanistic studies to an orthotopic model and show that, following whole brain radiotherapy (WBRT), intracranial U87-EGFRvIII tumors show proficient DSB repair compared to U87-parental tumors. The possible clinical impact of hyperactivating such DSB repair pathways due to EGFRvIII expression was assessed by Kaplan-Meier survival analysis. Nude mice bearing U87-EGFRvIII intracranial tumors receiving WBRT showed no evidence of improved survival, while mice bearing U87-parental tumors showed a remarkable increase in survival following radiation. Taken together, our results suggest that DNA-PKcs inhibitors and/or EGFR inhibitors administered concurrently with radiation may be an effective therapeutic strategy for radiosensitizing these recalcitrant tumors.
Primary astrocytes were isolated from wild type or Ink4a/Arf−/− 5 day old pups as described previously (19). Primary wild type astrocytes were immortalized by retroviral expression of the SV40-large T antigen (SV40-LT). Plasmid construction, virus production and infection protocols have been described in detail previously (20). Transfection of astrocytes with retroviruses expressing mutant, constitutively active EGFR (EGFRvIII), wild type EGFR (EGFRwt), or kinase-dead EGFR (EGFRkd) was carried out as described (19).
Mouse astrocytes, mouse embryonic fibroblasts (MEFs), and human U87 glioma lines were all maintained in α-MEM media containing 10% FBS in a humidified 37°C incubator in the presence of 5% CO2.
For drug treatments, the DNA-PKcs inhibitor [10 μM NU7026 (Calbiochem)], the EGFR inhibitor [5 μM Gefitinib (Iressa) (Astrazeneca Co.)], or the PI3K inhibitor [50 μM LY294002 (Sigma)] was added to cells (Ink4a/Arf−/− cohort, SV40-LT-cohort, or U87 cohort) one hour (h) before irradiation. Control cells were treated with DMSO.
For γ ray irradiation of cells (Ink4a/Arf−/− cohort, SV40-LT-cohort, U87 cohort, or MEFs), a 137Cs source (JL Shepherd and Associates, CA) was used. Mid-brains of mice were irradiated with an X-ray device (Pantak, 300kV, 12mA, 1.65 mm Al) fitted with a specifically-designed collimator providing a 1 cm-diameter field size iso-dose exposure.
300 cells (Ink4a/Arf−/− cohort, SV40-LT-cohort, U87 cohort, or MEFs) were plated in triplicate under reduced-serum (0.5% FBS) conditions. Cells were irradiated at different doses (0 – 8 Gy) after allowing recovery for 4 h. Media containing 10% serum was added back 24 h later. Colonies were allowed to form for approximately 8 days. Cells were then fixed and stained with crystal violet. Colonies with more than 50 cells were scored and mean values for triplicate counts determined as described (21).
DSB repair rates were assessed by quantifying the rates of dissolution of 53BP1 foci as described (22). Approximately 4 ×104 cells (Ink4a/Arf−/− cohort, SV40-LT-cohort, or U87 cohort) were seeded overnight in glass chamber slides under reduced serum (0.5% FBS) conditions. The following day, cells were irradiated with a total dose of 1 Gy, fixed at the indicated times and immunostained with α-53BP1 primary antibody (Cell Signaling) and FITC-conjugated goat anti-rabbit secondary antibody (Molecular Probes) as described (22). The number of 53BP1 foci was determined for each time (average of 100 nuclei) and, after subtracting background (number of foci in unirradiated population), the percentage foci remaining was plotted against time to obtain DSB repair kinetics.
To detect EGFR, EGFRvIII, Akt-1, and phosphoAkt-1, whole cell exracts were prepared from cells (Ink4a/Arf−/− cohort, SV40-LT-cohort, U87 cohort, or MEFs) grown overnight in media containing reduced serum (0.5%) as described (19), separated by 8% PAGE, blotted onto nitrocellulose and probed with α-EGFR (Santa Cruz), α-phosphoAkt-1(ser473) (Cell Signaling), and α-Akt-1 (Cell Signaling). For detection of DNA-PKcs and phospho-DNA-PKcs (in mock- or γ-irradiated U87 cells) nuclear extracts were prepared as described (23), separated by 8% PAGE (low BIS), blotted onto nitrocellulose and probed with α-DNA-PKcs (Neomarkers), α-phosphoDNA-PKcs (T2647) (kind gift from Dr. Benjamin Chen), and α-actin (Sigma) antibodies.
Orthotopic tumors were generated as described (9, 19). U87-parental or U87-EGFRvIII cells were infected with a puromycin-selectable retrovirus expressing luciferase. Equivalent levels of luciferase expression in the two cell lines was verified by Western blotting with an α-luciferase antibody (Sigma). For intracerebral stereotactic inoculation, 5 × 105 cells were suspended in PBS (5μl) and injected into the right corpus striatum of the brains of 4–5 week-old Nu/Nu nude mice (Charles River). Tumors were allowed to develop and monitored by luciferase bioluminescence imaging. All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of UT Southwestern Medical Center.
Serial bioluminescence images (BLI) of tumor-bearing mice were obtained using the IVIS Lumina System (Xenogen Corp., Alameda CA) coupled to Living Image data acquisition software (Xenogen Corp.). During imaging, mice were anaesthetized with isoflurane (Baxter International Inc., Deerfield, IL) and a solution of D-luciferin (450 mg/kg in PBS; total volume: 250 μl; Biosynthesis, Naperville, IL) was administered subcutaneously in the neck region. Images were acquired between 10 and 20 minutes post-luciferin administration and peak luminescence signals were recorded. The BLI signals emanating from the tumors were quantified by measuring photon flux within a region of interest (ROI) using the Living Image software package.
For pathological analyses and immunohistochemistry, brains were fixed in 10% formaldehyde and processed for hematoxylin and eosin (H&E) staining by standard techniques. Entire brains were sectioned in 1–2 mm coronal blocks and submitted in one cassette for paraffin embedding and sectioning. Sections (10 μm) were treated with xylene and washed with ethanol. Antigen retrieval was performed by sodium citrate (10 mM, 20 min) treatment. Sections were then permeabilized in Triton X-100 and blocked with 5% goat serum. After incubation with α-53BP1 antibody (Cell Signaling) (at 4°C, overnight) cells were treated (at room temperature, 2 h) with FITC-conjugated goat anti-rabbit antibody (Molecular Probes). Sections were washed and mounted in Vectashield containing DAPI (Vector Labs).
We examined the contribution of EGFRvIII to radioresistance in primary astrocytes, the presumptive cells of origin of GBMs (19). For analyses of IR resistance, we used either Ink4a/Arf-null primary mouse astrocytes (henceforth referred to as Ink4a/Arf−/− astrocytes) or astrocytes in which p53/Rb tumor suppressors were inactivated by retroviral expression of SV40-LT (henceforth referred to as SV40-LT-astrocytes). Ink4a/Arf (p16/p19) and p53/Rb tumor suppressors are frequently lost in GBM (1, 3, 4). Therefore, these two cell lines, with two distinct, GBM-relevant tumor suppressor backgrounds, are most appropriate for this study. To assess the impact of EGFRvIII on IR resistance, we compared EGFRvIII to equivalently-expressed levels of wild type EGFR (EGFRwt) or kinase-dead EGFR (EGFRkd) (Fig 1a). Resistance to IR was quantified by standard colony formation assays. Expression of EGFRvIII, but not EGFRwt or EGFRkd, resulted in dramatically increased survival of both Ink4a/Arf−/− astrocytes and SV40-LT-astrocytes (Fig 1b). Radioresistance conferred by EGFRvIII was abrogated by the small-molecule inhibitor Gefitinib (Iressa) that binds to the ATP-binding pocket of EGFR (6) (Figs 1c). These data suggest that EGFRvIII signaling is involved in the increased resistance to IR irrespective of the tumor suppressor background.
The major mechanism by which IR induces lethality is by introducing DSBs. We examined whether the increased resistance to IR conferred by EGFRvIII expression might be due to enhanced DSB repair. SV40-LT and Ink4a/Arf−/− parental and EGFRvIII-expressing lines were irradiated with a total dose of 1 Gy and induction and rate of repair of DSBs were visualized by immunofluorescence staining for 53BP1 foci as described (22) (Fig 2a). Compared to parental lines, the EGFRvIII-expressing lines displayed significantly faster DSB repair kinetics, completing repair by 4 h (Fig 2b). Pre-treatment of these cells with Gefitinib (Iressa), which inhibits EGFRvIII signaling (6), slowed down repair to the extent observed in parental lines. These data clearly link improved repair kinetics with signaling from the EGFRvIII receptor.
DNA-PKcs is a critical component of the predominant DSB repair pathway in mammalian cells – non-homologous end joining (NHEJ) (17, 18). We tested the hypothesis that EGFR signaling promotes DSB repair via efficient activation of DNA-PKcs (24–26). To investigate a possible link between EGFRvIII and DNA-PKcs activity in glioblastomas, we pre-treated EGFRvIII-expressing astrocytes with NU7026, a potent and specific DNA-PKcs inhibitor (27). Pre-treatment with NU7026 blocked fast repair kinetics observed in EGFRvIII-expressing astrocytes (Fig 2b). The slower kinetics of DSB repair, in turn, correlated with increased radiation sensitivity in survival assays (Fig 2c), underscoring the potential usefulness of small-molecule inhibitors of DNA-PKcs as radiosensitizers for GBM treatment.
Since pharmacological agents like NU7026 may have non-specific effects beyond simply blocking their target protein, a genetic strategy was used to establish a mechanistic link between EGFRvIII expression and DNA-PKcs activation. We expressed EGFRvIII in mouse embryonic fibroblasts (MEFs) from DNA-PKcs knockout mice (28) (Fig 3a) and assayed for radiation sensitivity by colony formation assays. Expression of EGFRvIII in DNA-PKcs+/+ MEFs led to a considerable increase in radiation resistance (Fig 3b). Significantly, expression of EGFRvIII in DNA-PKcs−/− cells failed to confer any increase in radioresistance. These results strongly suggest that DNA-PKcs is required for the radiation resistance conferred by EGFRvIII.
EGFRvIII preferentially signals through the PI3K-Akt-1 pathway (29, 30). Pharmacological inhibition of this pathway can block DSB repair and radiosensitize mammalian cells (31, 32). Conversely, activation of this pathway, due to loss of PTEN, results in proficient DSB repair and radioresistance (33). Moreover, Akt-1 is reported to translocate into the nucleus upon irradiation and interact and co-localize with DNA-PKcs at DSBs (34–36). Therefore, we investigated whether EGFRvIII expression directly affected DSB repair via the PI3K-Akt-1 pathway. Towards this end, we ectopically expressed constitutively active, myristylated-Akt-1 in SV40-LT astrocytes (Fig 4a). Expression of myristylated-Akt-1 resulted in efficient DSB repair similar to that noted upon EGFRvIII expression (Fig 4b). Conversely, treatment of EGFRvIII-expressing SV40-LT-astrocytes with LY294002, a specific inhibitor of PI3K (37), resulted in slower DSB repair, similar to that seen in parental cells (Fig 4b). These results raise the possibility that EGFRvIII expression might influence DSB repair via the PI3K-Akt-1 pathway.
Having established a genetic link between EGFRvIII and DNA-PKcs, we investigated whether EGFRvIII over-expression resulted in hyperactivation of DNA-PKcs in response to IR. Activation of DNA-PKcs in response to IR involves its autophosphorylation at defined serine/threonine residues (18). The extent of phosphorylation can be quantified by Western blotting with phospho-specific antibodies and provides an accurate measure of DNA-PKcs activation in vivo (38). Since these phospho-specific antibodies recognize only human DNA-PKcs, we used a human glioma cell line, U87-MG, to examine the effects of EGFRvIII over-expression on DNA-PKcs activation. Expression of EGFRvIII, EGFRwt and EGFRkd in these lines has been described (39) and was confirmed by Western blotting (Fig 5a). Expression of EGFRvIII, but not EGFRwt, in U87 cells significantly increased radiation survival (Fig 5b). As with mouse astrocytes, U87 cells expressing EGFRvIII were significantly sensitized to IR by pre-treatment with the DNA-PKcs inhibitor, NU7026. Interestingly, expression of EGFRkd also resulted in a certain degree of radiosensitization as reported (13), possibly due to a dominant-negative effect. Most importantly, EGFRvIII expression resulted in faster and improved DSB repair kinetics compared to parental U87 lines, recapitulating results obtained with mouse astrocytes (Fig 5c).
Having established that EGFRvIII over-expression in U87-MG cells recapitulates results obtained with mouse astrocytes, we examined the extent of DNA-PKcs activation after IR in U87 cells. Phosphorylation of DNA-PKcs was quantified by Western blotting of nuclear extracts from mock-irradiated or irradiated cells with a phospho-DNA-PKcs antibody (pT2647) (18, 23) (Fig 5d). Although total levels of DNA-PKcs (bottom panel) were the same in all four cell lines, DNA-PKcs activation, as evidenced by its phosphorylation at T2647, was remarkably higher in cells expressing EGFRvIII (top panel), providing a mechanistic link between EGFRvIII over-expression and improved DSB repair in these cells.
Given that cell culture conditions in vitro may not necessarily recapitulate the complex microenvironment within a tumor in vivo, it is possible that the efficient DSB repair observed upon EGFRvIII expression may not hold true in the context of GBMs. Therefore, we used, for the first time, a mouse orthotopic glioma model to visualize DSBs and quantify DNA repair in vivo. U87-parental and U87-EGFRvIII cells (both expressing luciferase reporters) were stereotactically injected into the striatum of a cohort of nude mice (19). Intracranial tumor growth was monitored by serial luciferase imaging. Once tumors reached 50% maximal tolerated size (established in pilot experiments and corresponding to approximately 11–13 days post-implantation for U87-EGFRvIII cells and 16–18 days for U87-parental cells), mice were anaesthetized and received cranial irradiation (total dose: 2 Gy). Mice were anaesthetized and cardiac perfused with fixative at 0.5, 2, 4, or 8 h post-irradiation. Brains were paraffin embedded for routine immunohistochemistry and all tumor sections were H&E stained to determine the size and location of tumors (Fig 6a). Tumor sections were then stained with α-53BP1 antibody to visualize DSBs and quantify repair kinetics (Fig 6a). DSB repair kinetics indicated significantly faster DSB repair rates in tumors derived from U87-EGFRvIII cells (Fig 6b). These results, demonstrating in vivo tumor cell repair kinetics for the first time, suggest that EGFRvIII over-expression in glioma-relevant orthotopic tumors significantly augments the repair of radiation-induced DNA damage.
To test whether proficient DNA repair mechanism(s) observed in EGFRvIII-expressing tumors translates into in vivo radioresistance, we exposed nude mice bearing intracranial U87-parental or U87-EGFRvIII tumors to whole brain radiotherapy (WBRT). As reported previously (40), the rate of U87-EGFRvIII intracranial tumor growth is significantly higher than that of U87-parental tumors. Due to the faster growth rate of U87-EGFRvIII glioma cells, the timing of WBRT was adjusted such that IR (4 Gy, one time dose) was delivered when the tumors were approximately of similar size (at day 6 post-implantation for U87-EGFRvIII tumors and at day 10 for U87-parental tumors) (Fig 6c). Mice were sacrificed when they became moribund or at day 30 post-implantation. Following WBRT, the growth rate of U87-parental tumors was significantly reduced compared to growth of mock-irradiated tumors (Fig 6c). In contrast, there was no difference in the rate of U87-EGFRvIII tumor growth with or without radiation. Correspondingly, following WBRT, there was little improvement in overall survival (Kaplan-Meier analyses) of mice bearing U87-EGFRvIII tumors compared to mock-irradiated mice (Fig 6d). In contrast, all mice with U87-parental intracranial tumors that were irradiated were alive at time of sacrifice. These data indicate that proficient DSB repair in EGFRvIII-expressing tumors contributes to tumor radioresistance. We speculate that the extremely rapid and complete DSB repair observed in EGFRvIII-expressing cells may prevent the initiation of programmed cell death upon IR resulting in tumors that are refractory to radiation.
Our data show that EGFRvIII over-expression results in increased resistance to IR in primary mouse astrocytes that either lack Ink4a/Arf tumor-suppressor genes or have functionally suppressed p53/Rb due to SV40 large-T antigen expression. Similar results are also observed when EGFRvIII is expressed in human U87-MG glioma cells. Increased radioresistance is specific to the constitutively active EGFRvIII receptor and is not seen with equivalent levels of EGFRwt or EGFRkd expression. EGFRvIII-mediated radioresistance is associated with hyperactivation of DNA-PKcs and enhanced DSB repair kinetics that is possibly transduced via the PI3K-Akt-1 pathway. These observations may provide an important mechanistic basis for the radioresistance of GBMs that poses a major obstacle to effective treatment of these tumors. In addition to identifying accelerated DSB repair in EGFRvIII-expressing cells in vitro, we also provide in vivo evidence, using an orthotopic glioma model, that corroborate these findings and show these mechanisms to be operative in the context of the complex tumor microenvironment.
Amplification of the constitutively active EGFRvIII receptor is one of the most significant genetic alterations in GBM (1, 3, 4). Expression of EGFRvIII correlates with poor prognosis in patients (41) and higher tumorigenic capacity in both orthotopic and de novo mouse glioma models (9) (11). Our work and that of Golding et al [“Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells”; Cancer Biology Therapy; In Press (K. Valerie, personal communication)], indicate that a major contributing factor to GBM radioresistance is augmented DSB repair. Our finding, that EGFRvIII stimulates the repair of radiation-induced DNA damage, is not completely unexpected given that EGFR is activated by radiation (42–45) and EGFRvIII displays even higher levels of radiation-induced activation compared to the wild type receptor (12). Our observation, that EGFRvIII promotes the repair of DSBs via DNA-PKcs, is consistent with previous reports showing that, in human bronchial carcinoma cells, radiation induces EGFR nuclear import and direct physical association with, and activation of, DNA-PKcs (24, 46, 47). However, we see no evidence of nuclear translocation of EGFRvIII under basal conditions or under graded doses of IR in primary murine astrocytes and in U87 glioma cells or orthotopic tumors. It is, therefore, plausible that EGFRvIII expression in GBM tumor cells might enhance DSB repair via the canonical PI3K-Akt-1 pathway rather than by direct physical association with DNA-PKcs.
Hyperactivation of the PI3K-Akt-1 pathway due to EGFR amplification (and concomitant PTEN loss) is one of the hallmarks of GBMs (1, 3, 4). Moreover, while ligand-activated EGFR stimulates both the RAS-RAF-MAPK and PI3K-Akt-1 pathways (7), EGFRvIII preferentially activates PI3K-Akt-1 (29, 30). Recent reports have suggested that activated Akt-1 may play a role in the repair of DSBs. Blocking PI3K-Akt-1 signaling using small-molecule inhibitors impairs DSB repair in GBM (33) and breast cancer (31) cells resulting in radiation sensitivity. Conversely, hyperactivation of PI3K-Akt-1 due to PTEN deletion promotes DSB repair and radioresistance (33). We find that a PI3K inhibitor, LY294002 (37), can abrogate the accelerated DSB repair conferred by EGFRvIII over-expression and that hyperactivation of Akt-1 mimics the effects of EGFRvIII expression on DSB repair in astrocytes. A close physical association between Akt-1 and DNA-PKcs at sites of DSBs upon IR has been demonstrated (34–36). Activation of DNA-PKcs by IR involves its phosphorylation at several serine/threonine residues (17, 18). As Akt-1 is a serine/threonine kinase, it is conceivable that the hyperphosphorylation of DNA-PKcs observed by us in EGFRvIII-expressing cells may be potentiated by activated Akt-1. Indeed, siRNA-mediated knockdown of Akt-1 has been recently shown to attenuate DNA-PKcs autophosphorylation upon IR (48). The presence of major components of the PI3K-Akt-1 pathway in the nucleus (49) presages a more intimate link between RTK signaling and nuclear processes like DNA repair than has been previously postulated. In future studies, it will be important to elucidate the precise mechanism by which PI3K-Akt-1 activation by EGFRvIII might promote DNA-PKcs hyperactivation in GBM tumor cells.
Our finding that Gefitinib (Iressa) can abrogate the proficient DSB repair and radiation resistance due to EGFRvIII is consistent with data from GBM clinical trials in which 15–20% of GBM patients experience significant tumor regression in response to EGFR small-molecule inhibitors (50, 51). Our results with a small molecule inhibitor of DNA-PKcs, demonstrating significant radiosensitization of murine astrocytes and human glioma cells expressing EGFRvIII, are important from a therapeutic standpoint. Several DNA-PKcs inhibitors are currently being developed as radiosensitizers (52) and it may be worthwhile in the future to develop formulations of DNA-PKcs inhibitors that are compatible with in vivo testing in preclinical GBM models. In aggregate, our results demonstrate that DNA-PKcs hyperactivation and proficient DSB repair in EGFRvIII-expressing cells and tumors provide a mechanistic basis for the marked radioresistance of GBMs with EGFR amplification and PTEN loss (51). These results suggest that DNA-PKcs and/or EGFR inhibition concurrent with radiation might be an effective strategy for radiosensitizing high-grade gliomas.
This work was supported by grants from NASA (NNA05CS97G to SB), the Goldhirsh Foundation (RMB, FF), NIH (CA122972 to DAB and PO1 CA095616 to WKC and FF). WKC is a fellow of the National Foundation for Cancer Research. This is paper no CSCN046.