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

Immunotherapy has revolutionized cancer treatment, yet only a fraction of patients develop durable responses to immune checkpoint blockade (ICB). Identifying tumor-intrinsic mechanisms driving alphaPD-1/alphaPD-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 alphaPD-L1 exposure, yielding the 2RA cell line. 2RA tumors are refractory to both alphaPD-L1 and alphaPD-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 E 2 (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-beta inhibition (LY2157299), restored ICB sensitivity, prolonged survival, and induced tumor rejection in vivo . Targeting PGE 2 production with celecoxib, but not aspirin, further enhanced responses when combined with LY2157299 and alphaPD-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 + ceramide lo 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.