Abstract PS4-03-09: Positron emission tomography imaging detected enhanced anti-tumor immunity in triple-negative breast cancer mediated by glutamine antagonism

Abstract OBJECTIVES: Glutaminolysis is a metabolic pathway deployed by aggressive cancers including triple-negative breast cancer (TNBC) to support their growth and progression. Immune checkpoint inhibitors (ICI) are FDA approved treatment modalities for TNBC; however, treatment responses are modest and variable. To overcome the heterogeneity in response, there is a critical need to understand the unique characteristics underlying immune activity responding to ICI. Our prior study has shown that TNBC tumors are enriched with myeloid-derived suppressor cells (MDSCs), which were significantly reduced by a pan-glutamine metabolic inhibitor JHU083, a pool compound of DRP-104/ Sirpiglenastat being evaluated in clinical trials. JHU083/DRP-104 is the prodrug form of DON (6-Diazo-5-oxo-L-norleucine). Here we examined the impact of glutamine antagonism in cancer versus immune cells traversing the tumor microenvironment. PET imaging is an important tool to identify patients most suited to the therapeutic promise of ICI. Leveraging the recent development of a radio-labeled antibody against CD69, a well characterized early-activation cell surface marker, we tested whether this immune-PET marker can detect the enhanced activation of T cells in the TNBC tumor immune microenvironment. METHODS: Murine (4T1, EO771) and human (HCC1806) TNBC and ER+ (MCF7) cell lines obtained from ATCC, T cells and MDSCs isolated from the spleen, were treated with JHU083 (0.1µM) to assess ROS and lipid ROS levels using Dihydroethidium and C11BODIPY assays. The impact of MDSC on T cell activation was examined by co-culturing T cells with MDSCs for one day in culture media containing 0.05 µg/ml PMA and 0.1 µg/ml ionomycin and either 0.1 µM DON or without DON. For in vivo imaging studies, 4T1 cells were inoculated into the mammary fat pad of BALB/c mice. When tumors grew to approx. 100 mm3, mice were randomized and enrolled in control (no treatment), ICI (treated with PD-1 at 250 and CTLA4 at 100 μg/mouse twice/week i.p.) or ICI plus JHU083 (1.82 mg/kg, i.p. every other day from day 0) group. After one week treatment, each mouse received a 100 ∼ 300 µCi of [89Zr]-DFO-H1.2F3 (anti-CD69 mAb) via tail vein injection and PET scan was performed at 48 and 72 hours post-injection. After PET imaging, FACS analysis was performed to profile immune cells in the tumor. Results: 0.1 µM DON does not induce cytotoxicity in TNBC cells, MDSCs or T cells, however it markedly increases cellular reactive oxygen species and lipid peroxidation in cancer cells and MDSCs but not in the activated or naïve T cells. CD69 PET signals (%ID/g) in 4T1 tumors are significantly higher after ICI (13%, p<0.05) or JHU083 plus ICI treatment (15%, p<0.05) compared to controls (9%). FACS analysis of tumors after PET imaging reveals elevated CD69+ cell population in treated tumors compared to controls. Meanwhile the CD45+CD11b+ cell fraction in ICI treated tumors (76%) remains similar to controls (84%) but decreased substantially after JHU083 plus ICI treatment (19%). In co-cultures, the presence of MDSCs reduced the fraction of CD69+ and granzyme B+ CD8+ T cells, confirming MDSC’s immune suppressive activity. However, adding 0.1 µM DON in co-culture reversed the functional competence of T cells. DISCUSSIONS & CONCLUSION: By selectively disrupting metabolism in cancer cells and MDSCs while sparing T cells that provide anti-tumor control, glutamine antagonism has immediate translational relevance for adoptive immunotherapies. Although ICI therapy removes PD-1 and CTLA4 mediated inhibition on T cells, it fails to inhibit tumor growth likely due to predominant presence of MDSCs which suppress CD8+ T cells as demonstrated by co-culture data. Combining JHU083 with ICI reduces MDSC infiltration to the tumor, leading to enhanced anti-tumor immunity detected by [89Zr]CD69 PET. Citation Format: H. Choi, B. Hassan, R. O'Connor, F. Michael, D. Mankoff, R. Zhou. Positron emission tomography imaging detected enhanced anti-tumor immunity in triple-negative breast cancer mediated by glutamine antagonism [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 PS4-03-09.