Abstract 7189: EED inhibitor APG-5918 renders vulnerability to immunotherapy by rewiring sphingolipid metabolism via nSMase2-ceramide pathway in pancreatic cancer

Abstract Background: Pancreatic cancer has a poor prognosis and is primary resistant to immune checkpoint inhibitors (ICIs) partially due to the inhibitory immune microenvironment. To improve the responsiveness of pancreatic cancer to ICIs, researchers have explored various combination therapies, including the use of epigenetic inhibitors. Embryonic Ectoderm Development (EED), a regulatory component of the PRC2 histone modification complex, has emerged as a target of interest. This study aims to investigate the potential effects of combining EED inhibition with PD-1 blockade in treating pancreatic cancer. Methods: Allograft models of murine pancreatic cancer cell lines, KPC and Panc02, were used to evaluate the combined anti-tumor effects of PD-1 antibody and a novel EED inhibitor, APG-5918. To determine how EED inhibition and PD-1 blockade influenced immune responses, in vivo tumor samples underwent single-cell sequencing and flow cytometry to assess immune cell infiltration and gene expression changes. Additionally, lipidomic metabolic sequencing and ELISA were used to measure changes in ceramide levels in vitro following EED or SMPD3 inhibition. Macrophage polarization phenotypes were further characterized using RT-qPCR and flow cytometry in the human monocyte lines THP1 and Raw264.7. Results: Our study demonstrated that the EED inhibitor APG-5918 synergizes with PD-1 antibody to exert anti-tumor effects in immune-competent murine pancreatic cancer allograft models. Single-cell sequencing of mouse tumor tissues revealed, and subsequent flow cytometry confirmed, a significant increase in M1 macrophages and CD8+ T cells in tumors from the combination treatment group compared with other groups. Transcriptomic analysis showed that EED inhibition significantly upregulated SMPD3 expression, which encodes the ceramide-producing enzyme nSMase2, and enriched the sphingolipid metabolic pathway. Quantitative lipidomic analysis revealed an increase in ceramide C16 levels in KPC and Panc1 cells treated with APG-5918, and ELISA confirmed that ceramide levels could be restored following treatment with the SMPD3 inhibitor GW4869. Furthermore, EED knockdown or supplementation with exogenous ceramide significantly promoted macrophage polarization toward the M1 phenotype in the human monocyte lines THP1 and Raw264.7 in vitro. Conclusions: Taken together, our results demonstrate that the novel EED inhibitor APG-5918 can reshape the tumor immune microenvironment by activating the nSMase2-ceramide pathway, overcoming immunotherapy resistance in pancreatic cancer. This promising approach, combining EED inhibition with PD-1 blockade, will be further explored in translational and clinical studies. Citation Format: Jing Yang, Fan Chen, Qiuyun Luo, Mingyu Lai, Jiachen Lu, Dajun Yang, Miaozhen Qiu. EED inhibitor APG-5918 renders vulnerability to immunotherapy by rewiring sphingolipid metabolism via nSMase2-ceramide pathway in pancreatic cancer [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 7189.