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Purpose: To evaluate glutamine uptake inhibition (GI) in combination with radiotherapy as a potential strategy for improving treatment response in triple negative breast cancer (TNBC). Methods: Two TNBC cell lines were utilized for this study, MDA-MB-231 (human) and 4T1 (murine). Prior dose response curves demonstrated 2 Gy and 4 Gy to be appropriate single fraction radiation doses, respectively. Cells were treated with one of four treatments: no radiation (RT) and no GI, GI alone, RT alone or combination RT+GI. GI was accomplished using a commercially available inhibitor, CB-839. Clonogenic assay was performed to evaluate response to treatment. In addition, extracellular flux analysis consisting of glycostress and mitostress tests were performed via Seahorse XFp analyzer. Flow cytometry was performed to evaluate apoptosis and cell cycle arrest. Western blotting for ɣ-H2AX and cyclin-dependent kinases was performed. In vivo experiments utilized subcutaneous 4T1 tumor implants, which were treated with either drug vehicle, RT alone, GI alone or combination GI+RT. Tumor volumes were measured three times per week and collected at 2000 mm3. Results: Clonogenic assay demonstrated significant reduction in both 4T1 and MDA-MB-231 cells exposed to the combination GI+RT as compared to either therapy alone. Glutamine uptake inhibition significantly reduced glycolysis in both MDA-MB-231 (33.8% compared with control) and 4T1 cells (62.8% compared with control). The addition of radiation to GI in MDA-MB-231 cells further reduced glycolysis by 54.2% and 79.9% in 4T1 cells (p-value < 0.01). ATP production was reduced by 36.1% with GI alone in MDA-MB-231 cells and 67.2% when combined with radiotherapy. A similar effect was observed in 4T1 cells with ATP production decreasing by 50% (p-value 0.03) with GI alone and 87.2% (p-value < 0.001) with combination therapy. Molecular analysis demonstrated an increase in ɣ-H2AX expression post radiation in those groups receiving combination therapy as compared with single modality therapy and control. The in vivo model demonstrated an initial synergistic response to GI+RT with respect to tumor growth; however, this response was diminished with conclusion of drug therapy. Conclusion: In the present study, we have shown that glutamine uptake inhibition sensitizes murine TNBC cells (4T1) and human TNBC cells (MDA-MB-231) to radiation. Molecularly, we demonstrate that GI coupled with RT significantly impacts DNA double strand break repair. In vivo data corroborates the synergistic relationship between GI and RT seen in the in vitro experiments. Future studies will focus on optimization of the dose and delivery timing of CB-839 in combination with RT in a preclinical model of TNBC with the intent to translate the concept into the clinic. Citation Format: Bassel Bashjawish, Mohammad Kamran, Brittany A. Simone. Glutamine uptake inhibition as a strategy for radiosensitization in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2410.