Selective inhibition of parallel DNA damage response pathways optimizes radiosensitization of glioblastoma stem-like cells

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Ahmed, Shafiq ; Carruthers, Ross ; Gilmour, Lesley ; Yildirim, Salih ; Watts, Colin ; Chalmers, Anthony (2015)
  • Publisher: American Association for Cancer Research
  • Related identifiers: doi: 10.1158/0008-5472.can-14-3790
  • Subject: top_sciences
    mesheuropmc: endocrine system | biological phenomena, cell phenomena, and immunity

Glioblastoma is the most common form of 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 confirmed by clonogenic survival assays that GSCs were significantly more radioresistant than paired tumor bulk populations. DNA damage response targets ATM, 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 increased mitotic catastrophe and a modest increase in radiation sensitivity. Inhibition of ATM had dual effects on cell-cycle checkpoint regulation and DNA repair 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.
  • References (3)

    2 3 4 T im e ( H r s ) s50 l ls le Supplementary Figure S9. Radiosensitisation of E2 and G7 cells with the CHK1 inhibitor CHIR 124 (CHIR) (A) Dose response of CHK1 inhibition in E2 GSCs following 1 hr pre-treatment with CHIR followed by 120Jcm-2 UV for 1hr. (B) Plot summarising flow cytometry data showing inhibition of IR induced G2/M checkpoint activation in E2 and G7 GSCs treated with 0.3µM CHIR for 1hr followed by 5Gy IR, mean ± SEM from n≥3 experiments, **p<0.01,***p<0.001 relative to IR alone.

    (C-D) Clonogenic survival curves showing radiosensitisation of E2 and G7 GSCs and bulk populations following 0.3µM of CHIR treatment.

    analysis. The number of nuclei analysed for each condition ranged from 65 to 307 (CENPF positive) and 300 to 460 (CENPF negative).

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