Epigenetic context defines the transcriptional activity of canonical and noncanonical NF-κB signaling in pancreatic cancer.

NF-κB signaling can be subdivided into canonical and noncanonical pathways, culminating in the transcriptional activity of RELA and RELB, respectively. However, the upstream signals that activate these transcription factors and their specific regulatory roles in pancreatic ductal adenocarcinoma (PDAC) remain incompletely understood. We investigated the differential activation and function of RELA and RELB in PDAC using transcriptome-wide gene expression profiling, genome-wide occupancy mapping, and epigenomic analysis. Temporal activation patterns were assessed following TNFα or TWEAK stimulation. Single-cell RNA sequencing and multiplex immunofluorescence staining were used to characterize activity in primary PDAC tissues. Motif enrichment and chromatin accessibility were evaluated to determine transcription factor binding dynamics and co-regulatory associations. We demonstrate that TNFα is the primary activator of canonical NF-κB signaling via RELA, while TWEAK selectively engages noncanonical signaling through RELB in PDAC. RELA and RELB display distinct temporal dynamics and regulatory activity. RELA binds to both open and closed chromatin and drives a broad transcriptional program, while RELB exclusively occupies pre-accessible chromatin regions co-enriched for AP1 motifs. Motif analysis reveals a particularly strong association of RELB with AP1 elements, suggesting selective co-regulation. Single-cell transcriptomic analysis and multiplex staining in primary tumors reveal distinct spatial and cellular distribution patterns, with RELA and RELB active in separate tumor and microenvironmental compartments. These findings underscore the distinct and complementary roles of TNFα and TWEAK in regulating NF-κB signaling in PDAC. TNFα engages a broader transcriptional program via RELA, whereas TWEAK targets a more selective set of genes marked by chromatin accessibility and AP1 co-binding through RELB. This study provides critical insight into the regulatory dynamics of NF-κB signaling in pancreatic cancer and highlights the specialized functions of RELA and RELB in modulating gene expression and tumor-microenvironment interactions.