The brain penetrant ATM inhibitor, AZD1390, protects neural stem cells from radiation

Purpose or Objective : Radiotherapy (RT) is a fundamental for the treatment of glioblastoma (GBM). Due to the infiltrative nature of GBM tumors, relatively large volumes of the brain are irradiated at high doses. This frequently causes irreversible neurotoxicity, with devastating effects on cognitive function and quality of life. DNA damage within neural stem cells (NSC) is a key contributing factor in the pathogenesis of radiation-induced cognitive dysfunction.

ATM (ataxia telangiectasia mutated) is a central component of the DNA damage response and a critical determinant of tumour cell survival after radiation. ATM inhibition potently radiosensitises preclinical models of GBM in vitro and in vivo and a novel, brain penetrant ATM inhibitor, AZD1390, is currently in early phase clinical evaluation in combination with RT. In marked contrast to observations in tumour models, genetic knockdown of ATM has minimal impact on radiosensitivity of astrocytes, indeed radioprotective effects have been observed in NSC.

To investigate the exciting hypothesis that ATM inhibition might ameliorate RT-associated normal brain toxicity, we investigated mode of cell death and survival responses of NSCs to combined RT and AZD1390 in vitro and compared them with effects on human GBM cell lines.

Material and Methods: NSCs were derived from the telencephalon of E13 mouse embryos and primary human GBM cell lines (G1/G7 stem) were generated from surgical resection specimens. For all experiments, cells were treated with AZD1390 (0.1-10 nM) 1 hour prior to ionizing radiation exposure (IR; 0-5 Gy). ATM inhibition was assessed by Western blotting of downstream protein targets (KAP1 and p53). Cell viability, neurosphere formation and clonogenic assays were used to measure survival. Mode and timing of cell death was interrogated using IncuCyte live cell analysis to measure proliferation, cytotoxicity and apoptosis up to 72 hours post-IR.

Results : AZD1390 at 1 nM inhibited ATM kinase function by abrogation of KAP1 and p53 phosphorylation within 1 hour of treatment. Critically, NSC integrity was not adversely affected by the combined drug and IR treatment. IR alone reduced NSC survival, which was overcome by AZD1390 treatment, and dose response analysis revealed that AZD1390 enhanced NSC viability at 1 nM, while the clonogenicity of GBM cells was sensitised to IR. NSCs primarily undergo apoptosis in response to IR. AZD1390 at 1 nM enhanced cell proliferation, reduced cytotoxicity and limited the onset of apoptosis in irradiated NSCs, when compared to controls and other inhibitors of the DNA damage response.

Conclusion : We demonstrate in vitro that at likely clinically achievable concentrations, AZD1390 has radioprotective effects on NSCs in marked contrast to its radiosensitising effects on GBM cells. These data provide further pre-clinical evidence to support clinical evaluation of AZD1390 in combination with radiotherapy in GBM.