ESTRO 2022 - Abstract Book

S220

Abstract book

ESTRO 2022

1 Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Dept. Radiation Biology, Oslo, Norway; 2 Rikshospitalet, Oslo University Hospital, Dept. Pathology, Oslo, Norway; 3 Institute for Basic Medical Sciences, University of Oslo, Hybrid Technology Hub - Centre of Excellence, Oslo, Norway Purpose or Objective 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. Materials and Methods 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. Results 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. Conclusion 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. Purpose or Objective Localized radiotherapy (RT) can cause T cell-mediated abscopal effects on non-irradiated metastases, particularly in combination with immune checkpoint blockade (ICB). However, results of prospective clinical trials have not met the expectations. We study whether chemotherapeutics can enhance the abscopal effect. Oxaliplatin (Oxa) has been considered as immunogenic, but cisplatin (Cis) and carboplatin (Carbo) have not. We compared these three platinum derivatives in two different abscopal mouse models. Materials and Methods In mice bearing bilateral tumors, the primary tumor was irradiated with 2 × 12 Gy (B16 melanoma model) or 2 × 8 Gy (C51 colon carcinoma model); Cis, Carbo, or Oxa were given once together with RT; anti-PD1 was given weekly. Tumor growth and survival of mice were determined (5–15 mice per group). The dependence of the therapeutic effects on CD8+ T cells and on extracellular ATP (eATP) was determined by using T cell-depleting antibodies and PPADS (a P2 purinergic antagonist), respectively. Frequencies and functionality (differentiation and exhaustion state) of tumor-specific CD8+ T cells were determined by FACS using MHC tetramers and various antibodies. In vitro , chemosensitivity of the tumor cell lines and their production of extracellular ATP were determined by CellTiter-Glo ® 2.0. Ethidium bromide and 2 ′ ,3 ′ - dideoxycytidine incubation was used to deplete mitochondrial DNA (mtDNA) of tumor cells. Results The tumor control compared to RT/ α PD-1 was as follows: RT/ α PD-1/Cis ( p < 0.01) > RT/ α PD-1/Oxa ( p < 0.01) >> RT/ α PD- 1/Carbo ( p > 0.05) (B16 melanoma); RT/ α PD-1/Cis ( p < 0.001) ≈ RT/ α PD-1/Carbo ( p < 0.01) >> RT/ α PD-1/Oxa ( p > 0.05) (C51 model). Triple therapy with Cis resulted in complete abscopal regression in 7/15 and 8/9 mice bearing B16 or C51 tumors, respectively. Triple therapy with Cis was also significantly better than the Cis/ α PD-1 double combination in both tumor models (B16 model, p < 0.01; C51 model, p < 0.05). In vitro , the chemosensitivity was as follows: Cis ≈ Oxa > Carbo (B16 cells); Cis ≈ Carbo > Oxa (C51 cells). In the C51 model, Cis induced more eATP and more cytosolic mtDNA leakage than Oxa. Cis-induced IFNb1 secretion was decreased in mtDNA-depleted compared to wild-type C51 cells. In the C51 model, triple therapy with Cis induced more mature dendritic cells ( p < 0.05), more Ki67+ CD8+ T cells ( p < 0.05), and more tumor- specific T cells ( p < 0.01) compared to triple therapy with Oxa. The enhanced abscopal effect was abrogated when CD8 + T cells were depleted, extracellular ATP signaling was blocked, or mtDNA-depleted C51 cells were injected as primary (irradiated) tumor. OC-0262 Factors determining the potency of platinum derivatives to enhance the abscopal effect R. Luo 1 , K. Onyshchenko 1 , L. Wang 1 , A. Grosu 1 , E. Firat 1 , G. Niedermann 1 1 Faculty of Medicine, Department of Radiation Oncology, Freiburg im Breisgau, Germany