ID #818 ATR inhibition overrides radiation-induced G2 arrest to drive post-mitotic death in medulloblastoma cells
Jacob Byrne, Raelene Endersby, Jacqueline Whitehouse, Nicholas Gottardo, Annabel Short, Chloe Buckingham, Hetal Dholaria, Jessica Buck, Brooke CarlineAbstract
Background
Survival outcomes for children with high-risk medulloblastoma remain poor, and existing treatments are inadequate. Ionizing radiation (IR) is a critical therapeutic component but is associated with debilitating late effects. High-throughput drug screening has identified DNA damage response kinases as promising therapeutic targets, with ATR inhibitor elimusertib demonstrating radiosensitising activity in preclinical models. Defining the mechanistic basis of ATR inhibitor–mediated radiosensitisation is essential for informing scheduling strategies and the safe advancement of this combination into clinical evaluation.
Aim
Define how ATR inhibition alters cell-cycle progression and DNA damage responses following irradiation in medulloblastoma models to support the optimization and future clinical translation of this combination.
Methods
Medulloblastoma cells were treated with ATR inhibitors (elimusertib, ceralasertib) and IR, alone and in combination. Matrix-based combination assays with mathematical modelling quantified drug–radiation interactions. Cell-cycle kinetics and checkpoint integrity were examined using FUCCI live-cell time lapse imaging and flow cytometry to quantify S-phase arrest, mitotic entry, and apoptosis. DNA damage response signaling and apoptotic markers were evaluated by western blotting.
Results
ATR inhibitors and IR exhibited robust synergy across wide dose ranges. FUCCI imaging revealed that IR induced a sustained G2 arrest (42.5h vs 15.8h in controls), with most cell death occurring prior to, and not after, mitosis. ATR inhibition accelerated S/G2 transit (9.9h) with limited cytotoxicity, particularly when administered in G1. In contrast, combined treatment abrogated the IR-induced G2 checkpoint (12.2h), promoting premature mitotic entry and resulting in substantial post-mitotic death in G1. These findings were validated by flow cytometry. Western blot analysis demonstrated suppression of ATR signaling, increased DNA damage accumulation, and elevated apoptotic marker expression.
Conclusions
Taken together, these data indicate that ATR inhibition converts radiation-induced checkpoint arrest into a lethal mitotic transition, providing a mechanistic basis for its potent radiosensitising activity and rationale for clinical evaluation.