Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes. Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes

The poor correlation of mutational landscapes with phenotypes limits our understanding of pancreatic ductal adenocarcinoma (PDAC) pathogenesis and metastasis. Here we show a critical role of oncogenic dosage-variation in PDAC biology and phenotypic diversification. We found gene-dosage increase of mutant KRASMUT in human PDAC precursors, driving both early tumorigenesis and metastasis, thus rationalizing early PDAC dissemination. To overcome limitations posed to gene-dosage studies by PDAC´s stroma-richness we developed large cell culture resources of metastatic mouse PDAC. Integration of their genomes, transcriptomes and tumor phenotypes with functional studies and human data, revealed additional widespread effects of oncogenic dosage-variation on cell morphology/plasticity, histopathology and clinical outcome, with highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, yet with lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumor-suppressor alterations (Cdkn2a, Trp53, Tgfb-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive early progression and shape downstream PDAC biology. Our study uncovers universal principles in Ras-driven oncogenesis with potential relevance beyond pancreatic cancer. Sub-studies: *PRJEB23116: Whole exome sequencing of KrasG12D-driven mouse pancreatic cancer cell cultures derived from primary pancreatic tumors (n=38) and liver or lung metastases (n=5). *PRJEB23110: Whole genome sequencing of KrasG12D-driven mouse primary pancreatic cancer cell culture for bioinformatic inference of chromothripsis. *PRJEB23183: Bulk 3-prime transcript end RNA-Sequencing of KrasG12D-driven mouse pancreatic cancer cell cultures derived from primary pancreatic tumors (n=38). *PRJEB23378: Bulk 3-prime transcript end RNA-Sequencing of epithelial/mesenchymal primary pancreatic cancer cell cultures enriched from a CRISPR/Cas9-edited, KrasG12D-driven mouse model of pancreatic cancer. *PRJEB23186: Bulk 3-prime transcript end RNA-Sequencing of KRASG12D- and GFP-overexpressing human pancreatic cancer cell lines.