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.

Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demonstrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1 as a suitable target for therapeutic intervention specifically in PDAC.

Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon/ transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed the unique qualities of PiggyBac for genome-wide mutagenesis and discovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 20 mouse lines to be compatible with both transposases in constitutive, tissue-or temporal-specific mutagenesis. Mice with different transposon types, copy numbers and chromosomal locations support wide applicability.Genetic screening in higher organisms has been hampered for decades by the lack of efficient insertional mutagenesis tools. Retroviruses have been used for cancer gene discovery in mice, but their application has been limited to the study of hematopoietic and mammary tumors due to viral tropism for these tissues (1). DNA transposons, which are the key insertional mutagens in lower organisms, were inactivated in vertebrate genomes millions of years ago. Only recently have new transposons been engineered to be active in mammalian cells, a development that provides opportunities for their use as genetic tools in higher organisms (2). Sleeping Beauty (SB), a TC1/mariner transposon, was reconstructed from dormant elements in fish genomes and optimized to transpose in multiple cell types (3), including mouse embryonic stem cells (4). Further improvements of SB led to its successful application for somatic mutagenesis in mice (5, 6). Another transposon, PiggyBac (PB) from the cabbage looper moth, was recently engineered to be highly active in mammalian cells (7) and has been shown to have biological properties distinct from those of SB (2, 7-9). PB can move larger DNA fragments (allowing complex transgene designs to be incorporated into the transposon) and it has a weaker tendency for local hopping in vitro (which makes it an attractive candidate for genome-wide mutagenesis). Furthermore, in contrast to SB, PB does not leave undesired footprint mutations after transposition. Finally, PB and SB have different integration preferences. (10)). All transposons possess PB and SB inverted terminal repeats (ITRs), allowing mobilization with both transposases. Promoter/enhancer elements, a splice donor, bidirectional SV40 polyAs and two splice acceptors were introduced in between the ITRs to allow gain or loss of function mutations, depending on the transposon orientation and its spatial relation to genes. Transgenic mice were generated with three va...

SummaryWe show that BRAFV600E initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic BrafV600E expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.

Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis, whose largest subgroup is characterized by somatic mutations in NPM1, the gene for Nucleophosmin1. These mutations, termed NPM1c, result in cytoplasmic dislocation of Nucleophosmin1 and are associated with distinctive transcriptional signatures2, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in murine hemopoietic stem cells causes Hox overexpression, enhanced self-renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty3-4 transposon which caused rapid-onset AML in 80% of Npm1c+ mice, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. Recurrent integrations were also identified in known and novel leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results give new pathogenetic insights and identify therapeutic targets in NPM1c+ AML.

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The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.