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The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown in Vitro and As Orthotopic Xenografts Cindy R.Timme, Barbara H

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The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown in Vitro and As Orthotopic Xenografts Cindy R.Timme, Barbara H Published OnlineFirst March 16, 2018; DOI: 10.1158/1535-7163.MCT-17-1267 Small Molecule Therapeutics Molecular Cancer Therapeutics The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and as Orthotopic Xenografts Cindy R.Timme, Barbara H. Rath, John W. O'Neill, Kevin Camphausen, and Philip J. Tofilon Abstract Radiotherapy is a primary treatment modality for glioblasto- sensitization is mediated by an inhibition of DNA repair. Extend- mas (GBM). Because DNA-PKcs is a critical factor in the repair of ing these results to an in vivo model, treatment of mice with VX- radiation-induced double strand breaks (DSB), this study evalu- 984 inhibited radiation-induced DNA-PKcs phosphorylation in ated the potential of VX-984, a new DNA-PKcs inhibitor, to orthotopic brain tumor xenografts, indicating that this compound enhance the radiosensitivity of GBM cells. Treatment of the crosses the blood–brain tumor barrier at sufficient concentra- established GBM cell line U251 and the GBM stem-like cell (GSC) tions. For mice bearing U251 or NSC11 brain tumors, VX-984 line NSC11 with VX-984 under in vitro conditions resulted in a treatment alone had no significant effect on overall survival; concentration-dependent inhibition of radiation-induced DNA- radiation alone increased survival. The survival of mice receiv- PKcs phosphorylation. In a similar concentration-dependent ing the combination protocol was significantly increased as manner, VX-984 treatment enhanced the radiosensitivity of each compared with control and as compared with radiation alone. GBM cell line as defined by clonogenic analysis. As determined by These results indicate that VX-984 enhances the radiosensitivity gH2AX expression and neutral comet analyses, VX-984 inhibited of brain tumor xenografts and suggest that it may be of benefit the repair of radiation-induced DNA double-strand break in U251 in the therapeutic management of GBM. Mol Cancer Ther; 17(6); and NSC11 GBM cells, suggesting that the VX-984-induced radio- 1207–16. Ó2018 AACR. Introduction recruitment of DNA-PKcs, which results in activation of its kinase activity and subsequent autophosphorylation. This pro- A primary treatment modality for glioblastoma (GBM) is cess leads to the recruitment of the other NHEJ components radiotherapy, which is typically used in combination with surgery and ultimately DSB ligation (4). Cells deficient in Ku or DNA- and temozolomide. Despite this combined treatment, median PKcs have significantly diminished DSB repair capacity and are survival rates for GBM remain at 12 to 15 months after diagnosis radiosensitive (5). DNA-PKcs was initially linked to GBM (1). Although they can be extensively invasive, GBMs usually recur radiosensitivity by studies on the M059J and M059K in the initial radiation treatment volume (1–3). This local failure cell lines, which were isolated from different areas of a human pattern is indicative of radioresistance and suggests that the GBM biopsy specimen (6). The DNA-PKcs–deficient M059J therapeutic response of GBM could be improved by the addition cells are highly radiosensitive compared with the DNA-PK– of a radiosensitizer. A source of potential targets for GBM radio- proficient M059K cell line. Introduction of a fragment of sensitization are molecules participating in the repair of DNA human chromosome 8 that contains the DNA-PKcs gene double-strand breaks (DSB), the lesion primarily responsible for reduced the radiosensitivity of M059J (7); treatment with the radiation-induced cell death. DNA-PKcs inhibitor NU7026 increased the radiosensitivity of In solid tumor cells, a critical process mediating DSB repair is M059K but not M059J cells (8, 9). Combined with its estab- nonhomologous end-joining (NHEJ), which results in the lished role in NHEJ, these data implicate DNA-PKcs as a direct ligation of broken DNA strands and operates throughout potential target for GBM radiosensitization. the cell cycle (except during mitosis; ref. 4). An essential A number of small molecule inhibitors of DNA-PKcs have been component of NHEJ is DNA-dependent protein kinase developed and shown to be effective radiosensitizers in vitro (10). (DNA-PK), which is composed of a catalytic serine/threonine However, in general, they suffer from poor in vivo pharmacoki- protein kinase (DNA-PKcs/PRKDC) and a Ku heterodimer netics and bioavailability, which limits their clinical applicability (Ku70 and Ku80; refs. 4, 5). NHEJ is initiated by binding of (10). Moreover, as compared with other tumor sites, drugs target- the Ku heterodimer to the exposed ends of a DSB followed by ing GBM must confront the additional pharmacokinetic obstacle of the blood–brain tumor barrier (BBTB; refs. 11, 12). Toward identifying an inhibitor appropriate for GBM therapy, we have Radiation Oncology Branch, National Cancer Institute, Bethesda, Maryland. investigated the radiosensitizing potential of VX-984 (13), a new Corresponding Author: Philip J. Tofilon, National Cancer Institute, 10 Center ATP-competitive DNA-PKcs inhibitor. The data presented here Drive-MSC1002, Building 10, B3B69B, Bethesda, MD 20892. Phone: 240-858- show that the VX-984 inhibits radiation-induced DNA-PKcs 3048; Fax: 240-541-4525; E-mail: tofi[email protected] phosphorylation and DSB repair in GBM cells grown under in doi: 10.1158/1535-7163.MCT-17-1267 vitro conditions, effects that are accompanied by an increase in Ó2018 American Association for Cancer Research. radiosensitivity. VX-984 is also shown to inhibit radiation- www.aacrjournals.org 1207 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst March 16, 2018; DOI: 10.1158/1535-7163.MCT-17-1267 Timme et al. induced DNA-PKcs phosphorylation in orthotopic brain tumor anti-mouse horseradish peroxidase-conjugated secondary anti- xenografts. Although VX-984 treatment had no effect on the bodies were purchased from GE Healthcare Life Sciences. Bands survival of mice bearing orthotopic brain tumors, it did enhance were quantified by densitometry analysis using Image Studio Lite the radiation-induced prolongation of survival. These results (LICOR Biosciences). suggest that the combination of VX-984 and radiotherapy may improve GBM therapeutic response. Clonogenic survival assay U251 cells growing in monolayer were disaggregated into a Materials and Methods single-cell suspension and seeded onto tissue culture plates. þ NSC11 CD133 neurospheres were disaggregated into single cells Cell lines and treatments and seeded onto tissue culture plates coated with poly-L-lysine The U251 human GBM cell line was obtained from the Division (Invitrogen) as previously described, which allows for adherent of Cancer Treatment and Diagnosis Tumor Repository (DCTD), colony formation (15). To evaluate radiosensitivity, cells were National Cancer Institute (NCI) grown in Dulbecco's Modified plated at clonal density in 6-well plates and irradiated the next Eagle Medium (DMEM) supplemented with 10% fetal bovine day. Ten to 18 days after irradiation, plates were stained with 0.5% serum (FBS, Invitrogen), and maintained in an atmosphere of 5% crystal violet, the number of colonies determined, and the sur- CO /95% air at 37 C. U251 cells were cultured less than 3 months 2 viving fractions were calculated. Radiation survival curves were after resuscitation and were last authenticated in May 2015 by STR generated after normalizing for the cytotoxicity induced by VX- analysis (Indexx Laboratories). The glioblastoma stem-like cell 984. The data presented are the mean Æ SEM of 3 independent (GSC) line NSC11 (kindly provided by Dr. Frederick Lang, MD experiments. Anderson Cancer Center) was isolated from human GBM surgical specimens as previously described (14). Neurospheres were main- Immunofluorescent analysis of gH2AX foci tained in stem cell medium consisting of DMEM/F-12 (Invitro- To visualize foci, cells grown in chamber slides were fixed with gen), B27 supplement (Invitrogen), and human recombinant 10% neutral buffered formalin (NBF), permeabilized with 0.1% bFGF and EGF (50 ng/mL each, R&D Systems) at 37 C, þ Triton X-100, and blocked with 1% bovine serum albumin in PBS 5%CO /5%O . CD133 NSC11 cells were isolated from neuro- 2 2 containing 5% FBS. The slides were incubated with antibody to sphere cultures by fluorescence-activated cell sorting (FACS) as þ phospho-H2AX (Millipore) followed by incubation with anti- reported previously (15). All CD133 NSC11 cells met the criteria mouse-AlexaFluor488 (Invitrogen) and mounted with Prolong for tumor stem-like cells including self-renewal, differentiation Diamond antifade reagent containing DAPI (Invitrogen) to visu- along glial and neuronal pathways, expression of stem cell related alize nuclei. Images were captured on a Zeiss upright fluorescent genes, and formation of brain tumors when implanted in immu- þ microscope equipped with a 63Â oil immersion lens. Automated nodeficient mice (15). For use in an in vitro experiment, CD133 image analysis to determine the number of foci per nucleus was NSC11 neurosphere cultures were disaggregated into single cells performed using ZEN software with the advanced Image Proces- as described (15) and seeded onto poly-L-ornithine (Invitrogen)/ sing and Analysis module (Zeiss). Foci determination was quan- laminin (Sigma-Aldrich) coated tissue culture dishes in stem cell tified in at least 30 cells per condition. Data presented are the media. Under these conditions, single-cell GSCs attach and grow þ mean Æ SEM of 3 independent
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