Mechanisms of ATM Inhibitor AZD1390-Mediated Radiosensitization by Comparing DNA DSB Formation and Repair in 4T1 Cells

Radioresistant cancers often exhibit upregulated DNA damage response (DDR) proteins including ataxia telangiectasia mutated (ATM), recovering more readily from radiation-induced DNA double-strand breaks (DSBs). ATM inhibitors (ATMi) are therefore being explored as adjuvants in radiotherapy to both enhance radiosensitivity and minimise normal tissue complications. The molecule AZD1390 has been developed as an inhibitor of ATM and is undergoing assessment in clinical trials. However, traditional markers of DNA DSB repair, such as {gamma}H2AX, represent stages of DDR downstream from the action of ATM. The ATMi AZD1390, developed by AstraZeneca, ultimately prevents phosphorylation of the H2AX histone variant. In order to quantify ATMi AZD1390 action, the novel and highly complementary assay SensiTive Recognition of Individual DNA Ends (STRIDE) was employed to directly quantify DSBs in comparison to {gamma}H2AX after X-ray irradiation of 4T1 cells. Findings revealed that ATM inhibition via AZD1390 delays DSB repair initiation and appears to play a greater role in suppressing DDR beyond ATM inhibition alone. In X-ray irradiated conditions, obfuscation of DSBs commenced between 30- to 45-minutes post-irradiation without AZD1390 versus 45 to 60-minutes for cells pretreated with AZD1390 and entirely prevented {gamma}H2AX in the majority of cells. STRIDE and {gamma}H2AX exhibited almost no co-localization indicating that they provide distinct and complementary information. DSB formation was also assessed in cells fixed pre-insult to minimise any biological response, providing unprecedented assessment of DSB formation. These results highlight the potential of STRIDE to accurately measure DNA damage response kinetics, paving the way for more precise mechanistic studies into the role of ATM in radiotherapy.