Purpose: High linear energy transfer (LET) protons are more densely ionizing than photons and low LET protons. Therefore, high LET protons create more clustered DNA damage and double strand breaks (DSBs). The double stranded DNA fragments generated in the form of micronuclei are the key activators of the cGAS-STING pathway, thereby potentiating anti-tumor immunity. The cGAS-STING pathway results in downstream activation of pro-inflammatory cytokines and signals that can lead to destruction of tumor cells. Here we show that the combination of high LET protons with an inhibitor of ATR (ATRi), a key protein in DNA damage response, amplify the production of precursors to antitumor immune activation, specifically cGAS-positive micronuclei (MN-cGAS+), more so than photons+ATRi.
Methods: H1299 and PANC-1 cell lines were treated with 6 MV x-rays and 9.9 keV/m (dose-weighted LET in water) protons alone or with an ATRi (1 µM, AZD6738). We then assessed MN snd MN-cGAS+ 24 and 72 h after radiation+ATRi. The Micronuclei were assessed as the proportion of micronuclei or cGAS+ relative to the quantity of nuclei counted for each treatment tested. This was done using a custom auto-counting macro generated using ImageJ.
Results: ATRi radiosensitized both cell lines to photons and protons with sensitizations relative to photons alone of 1.126±0.049/1.003±0.025 (H1299/PANC-1) (photons+ATRi) and 1.547±0.029/1.448±0.043 (H1299/PANC-1) (protons+ATRi). At 72 h and 5 Gy for H1299 cells, protons+ATRi (0.5326±0.015 MN/nucleus and 0.2426±0.044 MN-cGAS+/nucleus) amplified MN and MN-cGAS+ compared to photons alone (0.3184±0.033 MN/nucleus and 0.1716±0.042 MN-cGAS+/nucleus), protons alone (0.4529±0.007 MN/nucleus and 0.2444±0.018 MN-cGAS+/nucleus) and photons+ATRi. Although of lower magnitude, the same trends were observed at 24 h/5 Gy and 72 h/2 Gy. Results for PANC-1 were also similar.
Conclusion: In addition to radiosensitization from combining protons+ATRi, high LET protons+ATRi amplify MN-cGAS+, which has been reported to be a precursor of antitumor immune signaling.
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.