{
  "abstract": "Background High-dose radiotherapy (HDRT) is highly immunogenic, promoting tumor antigen release and enhancing immune checkpoint inhibitor (ICI) efficacy. However, HDRT also induces an immunosuppressive tumor microenvironment (TME), which limits therapeutic benefit. The Notch signaling pathway is a key regulator of immune and stromal cell function. We hypothesized that pharmacologic inhibition of Notch signaling with a γ-secretase inhibitor (GSI), AL101, would overcome radiation-induced immunosuppression and synergistically enhance antitumor efficacy when combined with HDRT and anti-PD-1 therapy.Methods Syngeneic neuroblastoma (9464D) and triple-negative breast cancer (EO771) tumors were established in C57BL/6 mice and athymic nude mice. Mice received GSI AL101 (6.5 mg/kg daily ×10), a single 12 Gy dose of HDRT (Day 3), and anti-PD-1 antibody (200 µg, Days 0, 3, and 6). A subset was sacrificed on Day 10 for mechanistic studies; the remainder were followed for survival and were euthanized when tumors reached 1.5 cm³. Tumors were analyzed by spectral flow cytometry and single-cell RNA sequencing (scRNA-seq) of CD45 + cells using the 10x Genomics platform. Lung metastasis were evaluated by histology image analysis for metastasis/total lung area using HALO image analysis.Results Neither AL101 nor anti-PD-1 treatment alone impacted tumor growth. Dual therapy with GSI and HDRT modestly improved survival compared to HDRT alone. In contrast, the triple combination of GSI+ HDRT + anti-PD-1 resulted in synergistic and durable tumor growth inhibition and significantly prolonged survival in both 9464D and EO771 models ( figure 1a). This regimen also markedly reduced lung metastases at the survival endpoint in EO771-bearing mice. These effects were abrogated in athymic nude mice (figure 1b), confirming an immune-dependent mechanism. scRNA-seq revealed that HDRT alone increased regulatory T cells (Tregs), exhausted T cells, and immunosuppressive M2-like macrophages. Triple therapy reversed these effects, significantly reducing M2-like macrophages (figure 2d), Tregs (figure 2a) and exhausted T cells while increasing CD8+ effector T cells and M1-like macrophages (figure 2c). Importantly, this combination also restored CD103+ dendritic cells (figure 2d) -specialized for antigen cross-presentation—within the TME. Spectral flow cytometry confirmed these findings, demonstrating that GSI treatment reprograms the TME toward an immunostimulatory state.Conclusions This study demonstrates the novel repurposing of GSI (AL101) to remodel the immunosuppressive TME induced by radiation and enhance the efficacy of HDRT combined with ICI. Triple therapy activates CD8 + T cells, reduces Tregs and exhausted T cells, restores cross-presenting dendritic cells, and reverses myeloid-driven suppression, supporting Notch blockade as a promising immuno-radiotherapeutic strategy with strong translational potential.Abstract 678 Figure 1E0771 Tumor cells were implanted into the mammary fat pad of C57BL/6 mice (A), athymic nude mice (B) enrolled when tumors reached 1.5cm3, and treated per the schema. Mice were sacrificed when tumors reached a size 1.5 m3Abstract 678 Figure 2The combination of GSI with HDRT and anti-PD-1 decrease Tregs (CD4+FoxP3+CD25+) and M2 (CD206+) and increases M1 (CD86+) and CD103+ DCs at day 10. Each dot=mouse. *P",
  "authors": [
    {
      "affiliations": [
        "New York Presbyterian - Columbia University Irving Medical Center, New York, NY"
      ],
      "name": "Qi Wang"
    },
    {
      "affiliations": [
        "Emory University School of Medicine, Atlanta, GA"
      ],
      "name": "Erin C Connolly"
    },
    {
      "affiliations": [
        "New York Presbyterian - Columbia University Irving Medical Center, New York, NY"
      ],
      "name": "Debarshi Banerjee"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY"
      ],
      "name": "Claire I Vapouile-Box"
    },
    {
      "affiliations": [
        "New York University Grossman School of Medicine, New York, NY"
      ],
      "name": "Rami Vanguri"
    },
    {
      "affiliations": [
        "New York Presbyterian - Columbia University Irving Medical Center, New York, NY"
      ],
      "name": "Darrell J Yamashiro"
    },
    {
      "affiliations": [
        "New York Presbyterian - Columbia University Irving Medical Center, New York, NY"
      ],
      "name": "Eileen P Connolly"
    }
  ],
  "title": "678 A novel therapeutic approach: notch inhibition enhances radiotherapy and checkpoint blockade via reprogramming of the tumor microenvironment",
  "uid": "6904bef9-64da-5929-bfe2-157fa3be6324"
}