{
  "abstract": "Background Radiation therapy (RT) can activate the type I interferon (IFN-I) pathway in cancer cells and the tumor microenvironment (TME) via canonical pathways of viral defense. 1 IFN-I signaling plays a critical role in enhancing RT-induced responses to immune checkpoint inhibitors (ICI) in preclinical triple negative breast cancer (TNBC) models and in some patients.2 3 This activation is mediated by cGAS sensing of cytosolic DNA, cGAMP synthesis, and STING activation. MYC amplification causes epigenetic downregulation of STING and resistance to ICI.4 We used patient-derived tumor organoids (PDOs) with differing MYC status to study functional responses to RT.Methods Two molecularly characterized TNBC PDOs, TNBC#1 (from MYC wild type tumor) and TNBC#2 (from MYC-amplified tumor), were mock treated or irradiated with 1-3 daily doses of 8 Gy and harvested after 24 hours. Gene expression (DESeq2, GSEA), protein (western blot, flow cytometry), and cGAMP (ELISA) were analyzed. Healthy donor CD14+ monocytes were M-CSF matured for three days before co-culture.Results Western blot confirmed expression of cGAS and STING in TNBC#1; TNBC#2 showed robust cGAS but abrogated STING expression. In TNBC#1, RT upregulated IFN-I, TNFα-via-NF-κB, and MHC-I antigen presentation pathways, and CD73 (padj < 0.05). TNBC#2 showed upregulation of TNFα-via-NF-κB (padj < 0.05), but not IFN-I or antigen presentation pathways. Flow cytometry confirmed increased MHC-I, CD73, and PDL1 (padj. < 0.05) in TNBC#1 post-RT. In TNBC#2, only CD73 and PDL1 were induced (padj. < 0.05), suggesting RT may favor immune escape in the latter. Because RT-induced cGAMP can be exported by ABCC1 and trigger IFN-I in myeloid cells, 5 we assessed ABCC1 and found high expression in both PDOs. ENPP1 and ENPP3, reported in breast cancer,6 convert cGAMP to AMP, then to immunosuppressive adenosine via CD73. RT upregulated ENPP1 and ENPP3 in both models. Consistent with ENPP1-mediated degradation, cGAMP was detected in supernatant only when ENPP1 was inhibited.In co-culture, PDOs suppressed MHC-II and CD86 on monocyte-derived macrophages, an effect reversed when TNBC#1 was irradiated prior to co-culture.Conclusions These findings suggest that the balance between immune-activating and immunosuppressive signals induced by RT in TNBC is influenced, at least in part, by STING expression within cancer cells. In MYC-amplified tumors, RT may potentiate immune evasion via cGAMP degradation and adenosine; inhibiting ENPP1/3, and CD73 may restore innate immune activation. PDO models can inform precision RT immunotherapy combinations.Acknowledgements Funding: Supported by BCRF 24-053 and DOD W81XWH-21-2-0034. DS is supported by a Burroughs Wellcome Physician-Scientist Award.References Deng L, et al. STING-dependent cytosolic DNA sensing promotes radiation-induced type I interferon-dependent antitumor immunity. Immunity. 2014;41:843–852.Rudqvist NP, et al. Immunotherapy targeting different immune compartments in combination with radiation therapy induces regression of resistant tumors. Nat Commun. 2023;14:5146.Shiao SL, et al. Single-cell and spatial profiling identify three response trajectories to pembrolizumab and radiation therapy in triple negative breast cancer. Cancer Cell. 2024;42:70–84.Lee KM, et al. Epigenetic repression of STING by MYC promotes immune evasion and resistance to immune checkpoint inhibitors in triple-negative breast cancer. Cancer Cell. 2022;40:36–50.Carozza JA, et al. Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. Nat Cancer. 2020;1:184–196.Mardjuki R, et al. Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint. Cell Rep. 2024;43:114209.Ethics Approval Informed consent and ethics approval was obtained via the DIGNITY Study (De-convoluting interactions between genes, the cancer environment, and the immune system to develop therapies that work for you) IRB Protocol #: 21-06023682",
  "authors": [
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Diego Sialer"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Xiaoxue Deng"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Maud Charpentier"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Neil Rupchand"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Marvin Campos"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Jenna Moyer"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Wen Shen"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Olivier Elemento"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, Institute for Computational Biomedicine, New York, NY, USA"
      ],
      "name": "Doron Betel"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Silvia Formenti"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA",
        "Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, NY, USA"
      ],
      "name": "Sandra Demaria"
    }
  ],
  "title": "905 Regulation of radiation-induced innate immune signaling in patient-derived triple-negative breast cancer organoids",
  "uid": "0eda8031-3bfe-5aa8-b6ea-1366e47a5177"
}