Abstract PS3-12-16: Reprogramming the Tumor Microenvironment in TNBC: A Novel Strategy Combining Notch Inhibition with Radiotherapy and Checkpoint Blockade

Abstract Background: High-dose radiotherapy (HDRT) is immunogenic, promoting tumor antigen release and enhancing immune checkpoint inhibitor (ICI) efficacy. However, HDRT also induces an immunosuppressive tumor microenvironment (TME), limiting the therapeutic response of combined RT + ICI. The Notch signaling pathway is a critical regulator of immune and stromal cell behavior. We hypothesized that inhibiting Notch signaling using a γ-secretase inhibitor (GSI) AL101 would overcome radiation-induced immunosuppression and synergize with HDRT and αPD-1 therapy in triple-negative breast cancer (TNBC). Methods: EO771 murine TNBC tumors were established in syngeneic 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 αPD-1 antibody (200 µg, Days 0, 3, and 6). A group of mice were sacrificed on Day 10 for mechanistic analyses; others were followed for survival and euthanized when tumors reached 1.5 cm3. Tumors were analyzed by spectral flow cytometry for immune cell subsets. Lung metastasis were evaluated by histology image analysis for metastasis/total lung area using HALO image analysis. Results: Neither GSI nor αPD-1 monotherapy inhibited EO771 tumor growth (median survival: control 14d vs GSI 18d and αPD-1 15d, p>0.05). GSI + HDRT modestly prolonged survival compared to RT alone (41d vs 35d, p < 0.05). Triple therapy (GSI + HDRT + αPD-1) resulted in durable tumor growth control and improved survival (98.5d, p <0.0001). Triple therapy also led to a marked reduction in lung metastases at day 10 (mean metastasis/lung area GSI + HDRT + αPD-1, 5.04±5.011%, vs Ctrl 26.85±18.91%, p<0.05) and at the survival endpoint (mean metastasis area/lung area GSI + HDRT + αPD-1 5.04±5.011%, vs Ctrl 26.85±18.91%, p<0.05). No survival benefit or decrease in lung metastasis was observed with triple therapy in athymic nude mice compared to HDRT alone (19d vs 17 d, p>0.05), confirming an immune-dependent mechanism. Spectral flowcytometry revealed that HDRT alone increased regulatory T cells (Tregs), exhausted T cells, and M2-like suppressive macrophages, while depleting M1-immunostimulatory populations. Triple therapy reversed these effects: Tregs and exhausted T cells were reduced, while CD8+ effector T cells and M1-like macrophages were increased. Importantly, CD103+ dendritic cells (DC), critical for antigen cross-presentation, were restored, suggesting that that GSI reshapes the TME toward an immunostimulatory state. Conclusions: Notch inhibition reprograms the radiation-altered TME to enhance responsiveness to checkpoint blockade in this TNBC model. Triple therapy prolongs survival and reduces metastasis by restoring CD103+ dendritic cells, activating CD8+ T cells, and suppressing immunosuppressive cell populations. These data support the translational potential of integrating Notch inhibition into breast cancer immuno-radiotherapy regimens. Citation Format: Q. Wang, D. Banerjee, E. C. Connolly, D. Yamashiro, E. P. Connolly. Reprogramming the Tumor Microenvironment in TNBC: A Novel Strategy Combining Notch Inhibition with Radiotherapy and Checkpoint Blockade [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-16.