Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and non-invasively measures ATM kinase activity in living cells and subjects.
Methods and Materials
Using the split luciferase technology we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence.
Treatment of ATMR expressing cells with ATM inhibitors resulted in a dose dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, while a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and siRNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models which correlated in a time-dependent fashion with changes in Chk2 activity.
We describe the development and validation of a novel, specific, non-invasive bioluminescent reporter that enables monitoring of ATM activity in real-time in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens, and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in pre-clinical models.