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Background Ewing sarcoma (EWS) is a rare, aggressive bone and soft tissue tumor. Survival is 70-80% for localized disease but drops to 30% in metastatic cases, with no improvement in decades. 1 This highlights the urgent need for new therapies. Tumor metabolism is a key vulnerability,2 with EWS and other cancers relying on glycolysis for growth, survival, and immune evasion.3 Targeting glycolysis may not only suppress tumor growth but also improve the immunosuppressive tumor microenvironment. Tumor-driven acidification, from high glycolytic activity, hinders immune cell trafficking and function.4 5 Reducing acidity could enhance immune-based therapies.6 Here, we investigated whether targeting glycolysis using pramlintide,7 an FDA approved amylin analogue for type II Diabetes, can suppress EWS tumor growth.Methods Using multiple EWS cell lines (TC71, A4573, A673, RD-ES), we evaluated the effect of pramlintide on cell proliferation and apoptosis. Apoptosis was assessed by Annexin V staining and confirmed by western blot analysis for caspase 3/7 and PARP activation. To investigate the impact of pramlintide on cellular metabolism, we performed a Seahorse assay to measure changes in glycolytic capacity and oxygen consumption rate (OCR). The in vivo therapeutic effect of pramlintide was assessed using a subcutaneous xenograft model. Briefly, nude mice were injected subcutaneously with 2 x10 6 EWS cells (TC71or A673).8–11 Seven days post-injection, mice were treated with intratumoral injections of either PBS (control) or pramlintide (30 µg per dose), administered twice a week for total of three weeks.Results Treatment of EWS cells with pramlintide (20 µg/ml, 30 µg/ml and 40 µg/ml) significantly suppressed cell proliferation in a dose-dependent manner. Additionally, pramlintide induced apoptosis, as indicated by increased in phosphotidylserine (PS) exposure on the cell surface, measured by Annexin V staining. This was further confirmed by western blot analysis, which showed increased levels of cleaved caspase 3/7 and PARP following treatment. Seahorse metabolic analysis demonstrated that pramlintide reduced both glycolytic capacity and OCR, indicating inhibition of glycolysis in EWS cells. In vivo, intratumoral administration of pramlintide significantly reduced tumor volume in both TC71 and A673 subcutaneous EWS xenograft models compared to PBS-treated controls.Conclusions Pramlintide inhibits glycolysis, suppresses EWS proliferation, induces apoptosis in vitro, and reduces tumor growth in vivo. While targeting metabolism shows promise, as seen with other anti-diabetic agents, it is unlikely to be curative alone. By reducing tumor acidity and reshaping the metabolic microenvironment, pramlintide may enhance immune cell trafficking and function, supporting its use in combination with immunotherapy.References Setty BA, Gikandi A, DuBois SG. Ewing sarcoma drug therapy: current standard of care and emerging agents. Paediatr Drugs 2023;25(4):389-397.DePeaux K, Delgoffe GM, Metabolic barriers to cancer immunotherapy. Nat Rev Immunol 2021;21(12):785-797.Renner K, et al. Metabolic hallmarks of tumor and immune cells in the tumor microenvironment. Front Immunol 2017;8:248.Harmon C, O’Farrelly C, Robinson MW. The immune consequences of lactate in the tumor microenvironment. Adv Exp Med Biol 2020;1259:113-124.Comito G, et al. Lactate modulates CD4(+) T-cell polarization and induces an immunosuppressive environment, which sustains prostate carcinoma progression via TLR8/miR21 axis. Oncogene 2019;38(19):3681-3695.Husain Z, et al. Tumor-derived lactate modifies antitumor immune response: effect on myeloid-derived suppressor cells and NK cells. J Immunol 2013;191(3):1486-95.Yang Y, et al. Pramlintide: a novel therapeutic approach for osteosarcoma through metabolic reprogramming. Cancers (Basel) 2022;14(17).Zhou Z, Yu L, Kleinerman ES. EWS-FLI-1 regulates the neuronal repressor gene REST, which controls Ewing sarcoma growth and vascular morphology. Cancer 2014;120(4):579-88.Zhou Z, et al. Neuronal repressor REST controls ewing sarcoma growth and metastasis by affecting vascular pericyte coverage and vessel perfusion. Cancers (Basel) 2020;12(6).Schadler KL, et al. Delta-like ligand 4 plays a critical role in pericyte/vascular smooth muscle cell formation during vasculogenesis and tumor vessel expansion in Ewing’s sarcoma. Clin Cancer Res 2010;16(3):848-56.Zhou Z, et al. Bone marrow cells participate in tumor vessel formation that supports the growth of Ewing’s sarcoma in the lung. Angiogenesis 2011;14(2):125-33.