Purpose: ATR is a DNA damage response protein essential to double strand break (DSB) repair and cell cycle checkpoint regulation. Here we investigate the influence an ATR inhibitor (ATRi) has on DSB repair and radiosensitization in response to photons and protons.
Methods: We exposed H1299 cancer cells to 6 MeV x-rays and 9.9 keV/μm protons (dose-weighted LET) with and without an ATRi (0.1-5 μM, AZD6738). We then performed immunofluorescent staining of 53BP1, a DNA repair protein involved in double stand break repair and fixed cells at 2, 4, and 24 h timepoints post-irradiation. Clonogenic cell survival assay was also performed.
Results: ATRi radiosensitized to both photons and protons and increased the RBE of protons. At 4 h post-irradiation, increasing ATRi concentration suppressed the number of 53BP1 foci. Cells exposed to 5 Gy of photons alone had an average of 28.55±0.17 foci/nucleus while the photons + ATRi (1 μM ) had a significantly lower average of 21.7±0.2 foci/nucleus at 4 h post-irradiation. However, at 24 h post-irradiation, increasing ATRi concentration resulted in a significant increase in 53BP1 foci (p<0.0001). Photons alone had an average of 10.61±0.11 foci/nucleus while the photons + ATRi (1 μM) had a significant increase at 16.43±0.15 foci/nucleus. Additionally, we observed a threshold on the ATRi concentration in the 24 h timepoint. Below a concentration of 0.5 μM, ATRi had very little effect on 53BP1 foci number for all timepoints. This suggests that ATR inhibition has an effective time window relating to the stage of DNA repair.
Conclusion: ATR inhibition sensitizes cancer cells to both photons and protons. The sensitization is due in part to the role of ATR in DSB repair as the number of persistent 53BP1 foci at 24 h post-irradiation was much higher relative to radiation alone.
Funding Support, Disclosures, and Conflict of Interest: This research was supported in part by RP170040, 1R21CA252411-01, Emerson Collective, and MD Anderson Cancer Center. Dr. Sawakuchi has research agreements with Alpha Tau Medical and Artios Pharma. Dr. Shaitelman has a research agreement with Exact Sciences.