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, Tgfβ-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.

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf AVKA allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf AVKA . Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.