Session Item

The serine/threonine protein kinase ATR is a central regulator of the G2 cell cycle checkpoint and homologous recombination repair after irradiation. When ATR inhibitors are combined with irradiation, cancer cells will enter mitosis with unrepaired DNA lesions, resulting in micronucleus formation and cell death. Interestingly, recent studies suggest that ATR inhibitors, besides their effects on cell cycle checkpoints and DNA repair, may also increase radiation-induced antitumor immune responses. Yet, the underlying mechanisms and their impact in human cancers remain scarcely understood. Notably, the DNA sensing protein cyclic GMP-AMP synthase (cGAS) can bind to cytosolic DNA from ruptured micronuclei, thereby promoting type I interferon (IFN) gene expression. We aimed to assess whether ATR inhibitors, by abrogating the G2 checkpoint, increase cGAS-mediated IFN signaling after irradiation of human cancers.

The human lung cancer cell lines SW900, H1975, A549 and H460 and osteosarcoma cell line U2OS were treated with radiation (2-20 Gy) and two different ATR inhibitors (VE822 at 50/250 nM; AZD6738 at 250/1250 nM). Cell cycle checkpoint abrogation was assayed by flow cytometry. IFN signaling following cGAS-detected cytosolic DNA was measured by phospho-STAT1 immunoblotting and IFN-β ELISA. cGAS depletion was achieved by siRNA transfection, and cGAS localization was assessed through immunofluorescence microscopy. Levels of the exonuclease TREX1 were measured by immunoblotting.

Supporting that the G2 checkpoint prevents IFN induction, we observed a reduction of radiation-induced increase in phospho-STAT1 levels after high radiation doses, correlating with prolonged G2 checkpoint arrest. In contrast, we found no radiation-induced increase in TREX1 levels previously reported to correlate with reduced IFN responses at high radiation doses. Co-treatment with radiation (5 Gy) and ATR inhibitors abrogated the G2 checkpoint in all cell lines, accompanied by increased radiation-induced IFN signaling in U2OS, SW900 and A549, and weakly in H1975. Checkpoint abrogation and IFN signaling similarly depended on ATR inhibitor concentration. cGAS co-localized with micronuclei, and depletion of cGAS abolished IFN responses, indicating its dependency on cGAS-detection of cytosolic DNA from ruptured micronuclei in these cell lines. Contrastingly, H460 cells showed no increase in IFN signaling or detectable cGAS foci. H460 presented higher baseline levels of TREX1 than the other cell lines, suggesting TREX1 to prevent cGAS signaling in H460.

Co-treatment with irradiation and ATR inhibition can increase cGAS-dependent IFN signaling in some, but not all, cancer cell lines. High baseline TREX1 expression warrants further consideration as a possible predictive marker for lack of IFN signaling.