ESTRO 2023 - Abstract Book

S2015

Digital Posters

ESTRO 2023

1 Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Purpose or Objective Inhibition of the ATR kinase abrogates the G2 checkpoint and suppresses DNA repair after irradiation, resulting in micronucleus formation and increased cell death. Recent studies suggest that ATR inhibition may also increase antitumor immune responses, but the underlying mechanisms remain poorly understood. Inhibitors of apoptotic caspases could potentially also be used to increase antitumor immune effects, but their roles in radiation-induced immunogenicity are not well understood. We have examined whether ATR inhibition can potentiate radiation-induced immunogenic cell death and interferon signaling in human cancer cells, and whether caspase activation contributes to regulate these responses. Materials and Methods Human lung cancer and osteosarcoma cell lines (SW900, H1975, A549, H460, U2OS) were treated with radiation and ATR inhibitors (VE822, AZD6738) in the absence and presence of a pan-caspase inhibitor (Q-VD-OPh). Caspase activation and IFN signaling were measured by immunoblotting of cell extracts at 1-6 days after treatment. IFN- β secretion was measured by ELISA. Transfection with siRNAs ( CGAS , STING1 ) was used to explore the hypothesis that cytosolic DNA from ruptured micronuclei activates the cGAS-STING-IFN pathway. Hallmark factors of immunogenic cell death were assessed by immunoblotting of cell growth medium (HMGB1 release), the CellTiter-Glo assay (ATP secretion) and flow cytometry (surface-presentation of calreticulin). Results Co-treatment with radiation (5 Gy) and ATR inhibitors abrogated the G2 checkpoint in all cell lines, accompanied by micronucleus formation. In absence of the caspase inhibitor, the radiation-induced IFN response was moderately increased by ATR inhibition in four out of five cell lines. Consistent with activation of the cGAS-STING-IFN pathway, depletion of cGAS or STING abolished the induction of IFN response. Addition of the caspase inhibitor to cells treated with ATR inhibitor and radiation resulted in markedly increased IFN response in all cell lines. The response was dependent on the co-treatment, as no or only a small increase in IFN was found when caspase inhibition was combined with either ATR inhibition or irradiation alone. The ATR inhibitors also increased the release of HMGB1 after irradiation, but this effect was suppressed upon caspase inhibition. Secretion of ATP was increased by ATR inhibition to a variable extent in the different cell lines, and slightly further increased by caspase inhibition. Surface-presentation of calreticulin was increased after irradiation, but not further increased by the ATR or caspase inhibitors. Conclusion ATR inhibition can increase IFN signaling and two hallmark factors of immunogenic cell death in irradiated human cancer cells, but the responses vary between cell lines. Treatment-induced caspase activity counteracts the IFN response, but promotes HMGB1 release. Caspase activation thus appears to play both suppressive and stimulative roles in radiation- induced immunogenicity. 1 Radboudumc, Radiotherapy & Oncoimmunology laboratory, Department of Radiation Oncology, Nijmegen, The Netherlands Purpose or Objective Radiotherapy (RT) is part of standard care anti-tumor treatment in the clinic. The cGAS (cyclic GMP-AMP synthase)/STING (stimulator of IFN genes) pathway is key for the recognition of cytoplasmic DNA, released upon RT, and for the induction of immune responses following RT. cGAS was first described as a cytosolic DNA sensor, however, an increasing amount of literature describes a variety of cGAS localization patterns (membrane bound, nuclear or specific foci including localization in micronuclei or droplets), which are cell-type and condition dependent. To this date, the role of cGAS localization and activation following distinct RT schedules and how this relates to immune activation remains largely unclear. Here, we aim to study the cGAS/STING pathway in tumor cells and its effect on immune cells after distinct tumor ablative regimens. Materials and Methods We established murine tumor cell clones (mouse colon tumor line MC38) using CRISPR-cas9 technology stably expressing cGAS fluorescent protein from its endogenous chromosomal location. These clones were subjected to thorough validation, assessing cGAS integration in the genome yielding at least 45 positive clones, followed by analysis of cGAS (fluorescent protein) expression on mRNA and protein level. Using these cells, we have now explored the effect of different RT schedules (ranging from 2 Gy to 20 Gy, single dose or fractionated) on cGAS activation and localization patterns using elaborate microscopic analysis in vitro & in vivo. Results We successfully developed MC38 tumor cell clones expressing endogenous levels of a cGAS fluorescent protein. In addition, the cGAS fluorescent fusion protein was fully functional as indicated by similar 1) cGAMP production 2) STING phosphorylation and 3) ISG expression upon RT as compared to wild-type cGAS. Upon RT treatment, cGAS localization strongly shifted towards micronuclear localization in vitro (figure 1) as well as following RT in vivo, resulting in a 4-fold increase (p<0.01) (figure 2) and are linked to cGAS activation. Moreover, RT induced cGAS re-localization was associated with the recruitment of immune cells into the tumor microenvironment. cGAS re-localization in tumors following different RT treatments are now quantified and related to gene expression changes in purified tumor infiltrating immune cells isolated from these same tumors. PO-2241 Understanding cGAS activation: the key to unravelling the full potential of ablative therapies? V. Mekers 1 , M. Looman 1 , L. van den Boogaard 1 , J. Bussink 1 , M. Ansems 1 , G. Adema 1