Abstract 5564: Crosstalk between ceramide and prostaglandin signaling mediates resistance to immune checkpoint blockade

Abstract Immunotherapy has revolutionized cancer treatment, yet only a fraction of patients develop durable responses to immune checkpoint blockade (ICB). Identifying tumor-intrinsic mechanisms driving αPD-1/αPD-L1 resistance remains critical to improving patient outcomes. We recently discovered that reduced ceramide synthase 4 (CerS4) activity and the subsequent loss of C18/20 ceramide impairs response to ICB through intracellular PD-L1/Caprin-1 signaling. To investigate this further, we generated an orthotopic, transplantable TNBC (E0771) model of ICB resistance through serial in vivo αPD-L1 exposure, yielding the 2RA cell line. 2RA tumors are refractory to both αPD-L1 and αPD-1 therapy, and transcriptomic signatures derived from this model strongly predict clinical ICB outcomes, supporting its relevance to human disease. Functionally, 2RA tumors display reduced CerS4 expression, diminished C18/20 ceramide, and increased intracellular PD-L1/Caprin-1 interaction. Bulk RNA-seq revealed marked enrichment in prostaglandin E2 (PGE2) signaling, a potent immunosuppressive pathway, in 2RA tumors. Mechanistically, we identified that CerS4 inversely regulates prostaglandin-endoperoxide synthase 2 (Ptgs2, COX-2) expression and PGE2 production through the PD-L1/Caprin-1 complex, whereby ceramide directly interacts with PD-L1 to restrict Caprin-1 binding. Analysis of TCGA and ICB-treated patient datasets substantiated the CerS4/PD-L1/COX-2 axis across multiple solid tumor subtypes. Immune profiling via flow cytometry and snRNA-seq identified dysfunctional progenitor and effector CD8+ T cells as central to impaired ICB response in 2RA tumors. Genetic or pharmacological disruption of the CerS4/PD-L1/PGE2 axis, achieved through CerS4 restoration or TGF-β inhibition (LY2157299), restored ICB sensitivity, prolonged survival, and induced tumor rejection in vivo. Targeting PGE2 production with celecoxib, but not aspirin, further enhanced responses when combined with LY2157299 and αPD-1 therapy. Targeted lipidomics and multiplex immunofluorescence (mIF) confirmed that this triple combination potently blocks the ceramide/PGE2 axis and stimulated CD8+ T cell responses to control tumor growth. Finally, mIF analysis of nivolumab-treated pre-surgical human HNSCC specimens (responders vs. non-responders) corroborated these findings by demonstrating reduced tumor ceramide abundance, elevated PanCK+COX-2+ceramidelo populations, and decreased intratumoral CD8+ T cell density amongst non-responders. Collectively, these studies (1) establish a relevant model of ICB resistance, (2) define a mechanistic framework linking ceramide metabolism to prostaglandin-mediated immune suppression, and (3) highlight therapeutic strategies to target ICB resistance and improve patient outcomes. Citation Format: Wyatt O. Wofford, Elif Percin, Han G. Lee, Odai Darawshi, Bryan Granger, Lucy Mulligan, Natalia V. Oleinik, Mohamed F. Kassir, Chase Walton, Paramita Chakraborty, Stefano Berto, Raymond N. DuBois, Shikhar Mehrotra, Besim Ogretmen. Crosstalk between ceramide and prostaglandin signaling mediates resistance to immune checkpoint blockade abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5564.