Abrogation of radioresistance in glioblastoma stem‐like cells by inhibition of ATM kinase

Abstract

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU-55933. We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC's display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU-55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6-3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU-55933 in GBM CSCs. ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.

Selective Inhibition of Parallel DNA Damage Response Pathways Optimizes Radiosensitization of Glioblastoma Stem-like Cells

“…Several preclinical studies have successfully used DDRi to increase chemoand radiosensitivity in glioblastoma models, but these results were not substantiated in the treatment-resistant GSC population (30)(31)(32)(33)(34). More recently, we have shown the ATM inhibitor KU55933 to be a potent radiosensitizer of GSC (27), and inhibition of PARP has also been shown to overcome radioresistance of GSCs (35). These findings corroborate and extend the landmark study by Bao and colleagues in which inhibition of CHK1 and CHK2 was shown to enhance the radiosensitivity of GSCs (10).…”

“…Neurosphere assay was performed as described previously (27) and in Supplementary materials and Methods.…”

“…Using the same techniques, three additional primary glioblastoma cell lines (R10, R15, and R24) were expanded by culturing under selective media to enrich for or deplete the GSC population. In immunodeficient mice, GSCenriched populations of E2, G7 (27,37), R10, and R15 cells generated orthotopic tumors that recapitulated the human disease whereas GSC-depleted tumor bulk populations did not ( Supplementary Fig. S1).…”

“…Such discrepancies may reflect methodologic differences or comparisons between nonisogenic cell lines. Indeed, we have recently demonstrated that radioresistance of GSC populations is associated with enhanced ATMdependent DSB repair proficiency (27). Currently, there is intense research into the development of small-molecule inhibitors of DDR proteins (DDRi), one aim of which is to increase the therapeutic index of standard treatments (28).…”

Science in Focus: Combining Radiotherapy with Inhibitors of the DNA Damage Response

“…Nonetheless, several research groups have demonstrated potent radiosensitising effects of ATM inhibitors including KU-55933 and KU-60019 both in vitro and in vivo, with particularly pronounced effects observed in glioblastoma (GBM) models [12]. The ability of ATM inhibition to overcome the innate and striking radioresistance of GBM stem-like cells has been documented by a number of authors and supports the potential therapeutic value of DDR inhibition in this currently incurable cancer [13]. The observation that p53 wild type cells appear to be relatively unresponsive to ATM inhibition indicates likely tumour specificity and also provides opportunities for patient selection [12].…”