Abstract 929: Altered lipid metabolism drives immunotherapy resistance via an EGFR/CERS4/PD-L1/PGE2 signaling axis in solid tumors

Abstract Immunotherapy has transformed cancer treatment, yet durable responses to immune checkpoint blockade (ICB) are limited to a subset of patients. Identifying tumor-intrinsic resistance mechanisms is critical to improving ICB efficacy and outcomes. We recently identified crosstalk between lipid metabolism via the downregulation of ceramide synthase 4 (CerS4) and internalized programmed death-ligand 1 (PD-L1) signaling, which promotes metastasis and impairs ICB response. Our data also demonstrated that αPD-L1 therapy enhanced internalized PD-L1 signaling, suggesting a role in adaptive ICB resistance. To investigate the underlying mechanism, we established an E0771-allograft-based triple-negative breast cancer (TNBC) model of ICB resistance, E0771-2RA, through serial αPD-L1 treatment and subculturing of resistant tumors. The 2RA model is resistant to both αPD-L1 and αPD-1 therapy in-vivo, with reduced CerS4/ceramide levels and increased PD-L1 internalization, consistent with our prior findings. Whole transcriptomic RNA sequencing of E0771 and 2RA tumors revealed enrichment in epidermal growth factor receptor (EGFR) and prostaglandin E2 (PGE2) signaling in 2RA tumors. Mechanistically, we found that (1) EGFR signaling negatively regulates CerS4 expression; (2) CerS4 suppression enhances PD-L1 internalization and subsequent association with Caprin-1; and (3) the intracellular PD-L1-Caprin-1 complex stabilizes prostaglandin-endoperoxide synthase 2 (Ptgs2, Cox2) mRNA to promote PGE2 production. Immune profiling of E0771 and 2RA tumors using flow cytometry and single-nuclei RNA sequencing revealed M2 macrophage polarization, PMN-MDSC recruitment, and T cell dysfunction in 2RA tumors. However, pharmacological targeting of PGE2 signaling and exogenous CerS4/ceramide restoration in ICB-resistant 2RA tumors primarily alleviated T cell dysfunction, suggesting the T cell compartment is the primary target of this signaling axis. Single-nuclei RNA sequencing and CellChat analyses revealed abundant TGF-β signaling in 2RA tumors, which we found to regulate PD-L1 internalization in-vitro, presenting a possible therapeutic vulnerability. Thus, to target this mechanism, we combined αPD-1 therapy with TGF-β inhibition in-vivo, which restored ICB sensitivity and prolonged survival. Using TCGA and human ICB resistance datasets, we validated the link between EGFR, CerS4, PD-L1, and PGE2 signaling in TNBC, head and neck squamous cell carcinoma (HNSCC), and melanoma. Based on our mechanistic and sequencing data, we additionally curated a gene signature to predict ICB response vs. resistance in solid tumors. Our findings uncover a novel mechanistic link between altered CerS4-dependent lipid metabolism, EGFR, PD-L1, and PGE2 signaling and introduce mechanism-based therapeutic strategies for overcoming ICB resistance in solid tumors. Citation Format: Wyatt O. Wofford, Han Gyul Lee, Bryan Granger, Elif Percin, Mohamed Faisal Kassir, Lucy Mulligan, Odai Darawshi, Paramita Chakarborty, Salih Gencer, Natalia Oleinik, Stefano Berto, Shikhar Mehrotra, Besim Ogretmen. Altered lipid metabolism drives immunotherapy resistance via an EGFR/CERS4/PD-L1/PGE2 signaling axis in solid tumors [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 929.