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. 2014 Dec;8(8):1603-15.
doi: 10.1016/j.molonc.2014.06.012. Epub 2014 Jun 27.

Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells

Yi Chieh Lim  1 , Tara L Roberts  1 , Bryan W Day  2 , Brett W Stringer  2 , Sergei Kozlov  2 , Shazrul Fazry  2 , Zara C Bruce  2 , Kathleen S Ensbey  2 , David G Walker  3 , Andrew W Boyd  2 , Martin F Lavin  4
Affiliations

Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells

Yi Chieh Lim et al. Mol Oncol. 2014 Dec.

Abstract

Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G1/S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival.

Keywords: ATM inhibitor; DNA damage; DNA double strand break repair; Glioma initiating cell; Homologous recombination; Neural progenitor cell.

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Figures

Figure 1
Figure 1
The effect of ATM inhibition on glioma initiating cells. (A) Cell viability was measured by trypan blue exclusion assay. The difference in cell count at each time point was normalised back to its own individual unirradiated sample. ATMi concentration was chosen based on the least cytotoxic effect. Data show the mean from a combination of three independent experiments and error bars display the standard deviation (±SD). (B) 5 μM ATMi was validated by examining the activation of ATM as measured by autophosphorylation of Serine 1981 and the ATM substrate Chk2 at Serine516 in response to IR. Cells were treated with or without inhibitor for 1 h prior to irradiation and harvested post‐IR 1 h later. (C) Cells were harvested at 16 h and stained with PI to measure cell‐cycle progression. X‐axis indicates fluorescence intensity of PI and Y‐axis measures the total cell number. (D) Asynchronous cells were pre‐treated with 5 μM ATMi prior to IR. The cells were fixed, harvested and immunostained for Ser10Histone H3 and PI dye. X‐axis represents mean fluorescence intensity of PI and the Y‐axis Ser10Histone H3 Alexa 488 nm fluorescence. (E) Quantification of the total number of mitotic cells as determined by Ser10Histone H3 staining. Data are pooled from two independent experiments and represented as mean ± SD.
Figure 2
Figure 2
Rad51 foci accumulation post‐IR is enhanced by ATMi treatment of GICs and NPCs. Cells were treated with (A) DMSO, (C) ATMi or (E) DNA‐PKi prior to 2Gy IR, harvested at the indicated times and then immunolabelled to detect Rad51 foci (green). Representative images for each time point show Rad51 foci and Hoechst nuclear stain (blue). (B), (D) and (F) Quantification of Rad51 positive cells was determined by a nucleus with >5 foci. Data show the mean from a combination of three independent experiments and error bars display the ±SD (*p < 0.05, **p < 0.01).
Figure 3
Figure 3
ATMi‐treated GICs are unable to compensate for IR‐induced DNA damage through the NHEJ pathway. Cells were treated with (A) DMSO, (C) DNA‐PKi or (E) ATMi for 1 h prior to 2Gy IR, harvested and immunolabelled at indicated time points. Representative images of NPCs and GICs with γH2AX foci formation (green) and Hoechst nuclear stain (blue). (B), (D) and (F) Each time point, the average foci number was scored from approximately 40 nuclei per experiment. Data show the mean from a combination of three independent experiments and error bars display the ±SD (*p < 0.05, **p < 0.01).
Figure 4
Figure 4
GICs are dependent on HR‐mediated repair for survival. (A) Cells were pre‐treated with DMSO, ATMi or DNA‐PKi for 1 h prior to IR. Survival of cells was determined by trypan blue exclusion assay at the indicated time points. Data are presented as mean from three independent experiments. (B) Clonogenic assay was performed at 96 h post‐treatment with the indicated inhibitors followed by 2Gy IR. Cells were stained with crystal violet to visualise colonies. (C) Cell density is proportionate to the intensity of crystal violet stain. Cell survival was determined by measuring the absorbance of crystal violet stain from individual wells. Data show the mean from a combination of three independent experiments and error bars display the ±SD.
Figure 5
Figure 5
ATMi treatment prior to ionizing radiation increases the survival of tumour bearing animals. (A) Wk1‐luciferase tagged cells were pre‐treated with DMSO, DNA‐PKi or ATMi for 1 h prior to 2Gy IR. Live cells were determined by trypan blue exclusion assay 48 h later followed by intracranial injection of equal numbers of cells. Image shows the progression of tumour growth in Nod/Scid mice. (B) Kaplan–Meier survival plot showing survival of mice from the individual treated groups (n = 5) (*p < 0.05). (C) Coronal plane sections of mouse brains injected with Wk1‐luciferase tagged cells that received DMSO, DNA‐PKi or ATMi in combination with 2Gy IR treatment. Photomicrographs (10× magnification) of H&E stained sections showed dense cellularity of neoplastic cells with invasive growth. White line with indicated arrows (far right panel) shows partial indistinctive border of early tumour progression in 2Gy + ATMi treated group.
Figure 6
Figure 6
A simplified model for the effect of ATM inhibitor on GICs which have limited NHEJ repair. Post‐surgical radiotherapy triggers ATM and DNA‐PKcs activation during early DNA DSB responses and initiates a signalling cascade. While NPC relies primarily NHEJ to repair DNA DSB, GICs employ HR through ATM activation. The presence of ATMi renders G2‐checkpoint activation ineffective and prevents HR repair in GICs. Whilst allowing NPCs to compensate through NHEJ repair. GICs relatively low NHEJ activity prevents them from DNA DSB repair compensation. Consequently the ATMi treatment results in the increased cell death of GICs.
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