Abstract 3978: The downregulation of OGT1 reprograms tumor-associated macrophages and CD4 T cells fate in pancreatic adenocarcinoma tumor model

Abstract O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification that plays a crucial role in development, normal physiology, and the pathophysiology of various diseases, including pancreatic cancer. This process is catalyzed by the enzyme O-GlcNAc transferase (OGT). The hexose biosynthesis pathway provides the necessary substrate, O-GlcNAc, for this reaction. However, the role of O-GlcNAcylation in tumor-associated macrophages has not been extensively studied. The hypoxic tumor microenvironment leads to increased glucose uptake and enhanced glycolysis. Additionally, macrophages undergo significant metabolic changes in this environment. The immunosuppressive nature of the tumor microenvironment promotes the polarization of macrophages toward the non-inflammatory M2-like phenotype, which is characterized by increased CD206, and Arg1 expression. To investigate the role of OGT1 in tumor-associated macrophages, we created a mouse strain, LysM-Cre; Ogtfl/fl, which features OGT1 loss specifically in macrophages. We isolated bone marrow mononuclear cells from both mutant and control animals and differentiated them into bone marrow-derived macrophages (BMDM) in vitro. Following this, we cultured the BMDMs with pancreatic tumor cell-conditioned media (TCM) from pancreatic cancer cells and assessed arginase 1 (Arg1) levels using Western blotting. The BMDMs isolated from LysM-Cre; Ogtfl/fl mice expressed lower levels of arginase 1 compared to control mice after being cultured in TCM. Next, we investigated the effect of pharmacological inhibition of OGT1 using OSMI-1 on the BMDMs, which were treated with TCM or control media, and how these macrophages influenced CD4 T-cell subtypes. The BMDMs were cultured for 72 h in a medium containing 100 µM OSMI-1 under hypoxic conditions (1% O2). Subsequently, the total CD4 T cells were co-cultured with the BMDMs for 62 h in hypoxia and stained to identify markers of Th1 and Th2 cell populations. We observed that macrophages stimulated with TCM reduced the number of IFNy+/IL-12+ CD4+ T cells compared to those treated with control media. In contrast, the inhibition of OGT1 in BMDMs resulted in an increase in the number of IFNy+/IL-12+ CD4+ T cells upon incubation with TCM. These findings suggest a potential role of OGT1 and O-GlcNAcylation in the interplay between macrophages and lymphocytes within the pancreatic adenocarcinoma TME. The data indicate that OGT1 may inhibit the immune components of the tumor microenvironment. This knowledge could pave the way for innovative strategies aimed at shifting the balance towards a more effective anti-tumor immune response. Citation Format: Vira Chumak, Kuldeep Singh Attri, Pankaj Kumar Singh, Kamiya Mehla. The downregulation of OGT1 reprograms tumor-associated macrophages and CD4 T cells fate in pancreatic adenocarcinoma tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3978.