Developmental and MAPK-responsive transcription factors drive distinct malignant subtypes and genetic dependencies in pancreatic cancer

Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of regulatory protein activity in single cells identified three PDA Developmental Lineages (PDLs), coexisting in virtually all tumors, whose transcriptional states are mechanistically driven by aberrant activation of Master Regulator (MR) proteins associated with gastrointestinal lineages (GLS state), morphogen and EMT pathways (MOS state), and acinar-to-ductal metaplasia (ALS state), respectively. Each PDL is further subdivided into sub-states characterized by low vs. high MAPK pathway activity. This taxonomy was remarkably conserved across multiple cohorts, cell lines, and PDX models, and harmonized with bulk profile analyses. Cross-state plasticity and MR essentiality was confirmed by barcode-based lineage tracing and CRISPR/Cas9 assays, respectively, while MR ectopic expression induced PDL transdifferentiation. Together these data provide a mechanistic foundation for PDA heterogeneity and a roadmap for targeting PDA cellular subtypes.