Abstract 1545: IL-9-mediated glutathione synthetase (GSS) expression modulates anti-oxidant response in cutaneous T cell lymphoma (CTCL)

CTCL is a rare and clinically heterogeneous blood disorder with an increased incidence. Management of the disease poses a significant challenge, and there is an urgent need for novel therapies that can cure the disease. Since CTCL is a rare disease, the pathophysiology is not completely characterized. Inflammation is a crucial driver of CTCL progression, and IL-9, an inflammatory cytokine, is prevalent in the tumor microenvironment. Previously, our lab reported that the Th9 axis reduces oxidative stress, and in this follow-up study, we deduced the signaling mechanism underlying IL-9-mediated antioxidant response to have a comprehensive understanding of the metabolic rewiring in CTCL. High-throughput label-free quantitative LC-MS/MS of IL-9-treated lymphoma cells revealed some of the differentially expressed antioxidant enzymes, including peroxiredoxin-4 (fold change = 2.015, p = 0.018), glutathione synthetase (fold change = 4.381, p = 0.001), and glutathione peroxidase 1 (fold change = 1.88, p = 0.021) among which glutathione synthetase was highly expressed. Further, IH NMR spectroscopy identified the metabolic pathways affected by IL-9 signaling. Pathway analysis using MetaboAnalyst 5.0 depicted enhanced glutathione metabolism and elevated glutathione levels. To substantiate the multi-omics predictions, we checked the expression of GSS in multiple CTCL lymphoma lines (MJ, HuT 78, and Jurkat) and activated T-cells using flow cytometry. We found a high intensity of GSS in MJ (4605.75 ± 641 vs. 3921 ± 1187, p = 0.04), HuT 78 (11305 ± 530 vs. 8912 ± 603.8, p = 0.003), Jurkat (2955 ± 678 vs. 2190 ± 696, p = 0.001) and activated T cells (4593 ± 172 vs. 3657 vs. 206, p = 0.005) under IL-9 exposure. In agreement, the biochemical assay demonstrated elevated glutathione levels in lymphoma cells treated with IL-9. Immunohistochemical analysis revealed that GSS was widely expressed by infiltrating T-cells in skin biopsy specimens from 5 patients with mycosis fungoides (MF). Subsequently, flow cytometry analysis of liquid biopsy specimens from 11 patients demonstrated its strong expression with a 52% (p = 0.005) increase in circulating CD3+ T-cells in patients with MF compared to healthy volunteers. This cumulative analysis asserted its clinical relevance and identified it as a crucial enzyme in IL-9-mediated antioxidant signaling. Further, treating cells with plumbagin depleted GSS levels and increased ROS generation to many folds in all the lymphoma cell lines and activated T cells. In line with this, depleted GSS levels through plumbagin treatment reduced cell growth and potentiated cell death, suggesting that targeting this metabolic pathway sensitizes cancer cells. In conclusion, this study underscores GSS as a critical metabolic vulnerability. Citation Format: Diksha Attrish,Bhavuk Dhamija,Ditipriya Mukherjee,Vinanti Sawant,Neha Sharma,Tanuja Shet,Hasmukh Jain,Rahul Purwar. IL-9-mediated glutathione synthetase (GSS) expression modulates anti-oxidant response in cutaneous T cell lymphoma (CTCL) [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 1545.