Supplementary Table 1. Analysis of clonogenics survival curves from Figure 1C and 1D. Supplementary Figure S1. Generation of orthotopic tumours from R10 and R15 GSCs. Supplementary Figure S2. GSCs are more resistant to IR induced cell death than paired bulk populations. Supplementary Figure S3. Cell cycle profile and proliferation rates of GSC and bulk population. Supplementary Figure S4. Enhanced activation of CHK1 in GSCs. Supplementary Figure S5. Absence of G1 cell cycle checkpoint in irradiated GBM cell lines. Supplementary Figure S6. Analysis of G2/M cell cycle checkpoint. Supplementary Figure S7. Rapid activation of G2/M cell cycle checkpoint in E2 CD133+ cells. Supplementary Figure S8. Treatment of G7 GSCs with the CHK1 inhibitor SCH 900776 (SCH). Supplementary Figure S9. Radiosensitisation of E2 and G7 cell lines with the CHK1 inhibitor CHIR 124 (CHIR). Supplementary Figure S10. Enhanced DNA repair attenuates radiosensitisation of E2 GSCs to CHK1 inhibitor (SCH). Supplementary Figure S11. Increase in gamma-H2AX foci in GSCs compared to bulk population following combined ATR and PARP inhibition.
ARTICLE ABSTRACT
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. Cancer Res; 75(20); 4416–28. ©2015 AACR.
