MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network

Rapid Functional Dissection of Genetic Networks Via RNAi In Mouse Embryos

The development and maintenance of epithelial tissues is regulated by a complex array of signal cues from adjacent cells, the extracellular milieu and intracellular signaling cascades. In the mammalian epidermis, these cues instruct the specification and invagination of hair follicles as well as the stratification and turnover of the interfollicular epidermis. These processes rely on a coordinate balance of tissue growth, differentiation and regulation of cell-cell adhesion to maintain the integrity of the epithelium. Understanding the interplay between the various pathways controlling tissue development requires model systems that recapitulate the events that occur in vivo. Genetic studies in Drosophila and other lower eukaryotes have uncovered many of the genes and pathways involved. However, genetic studies in mammals, where the relation to human development and disease is more direct are often hampered by the length of time required to generate mutations in the gene of interest. The ability to probe genetic interactions is even more limited in this system as the generation of double or triple mutants is even more inefficient. To circumvent these limitations, I have developed and optimized a lentivirus-based strategy to achieve rapid manipulation of the mammalian epidermis in vivo. Using ultrasound-guided in utero injections of fluorescently-traceable lentivirus particles carrying shRNA or Cre-recombinase into mouse embryos, I demonstrated a highly efficient, non-invasive, selective transduction of surface epithelium. Epidermal progenitors stably incorporated and propagated the desired genetic alterations. Importantly, I achieved epidermal-specific infection using small generic promoters from existing shRNA libraries, thus enabling rapid assessment of gene function and complex genetic interactions in skin morphogenesis and disease in vivo. Using this technology, I have developed a new quantitative method to ascertain whether a gene confers a growth advantage or disadvantage by measuring relative growth of mutant clones in a mosaic tissue. Taking the adherens junction protein α-catenin as a paradigm, this approach was used to uncover new insights into its role as a widely expressed tumor suppressor and regulator of epithelial integrity. Using simultaneous gene depletion I uncovered physiological interactions between α-catenin and the Ras- MAPK and Trp53 pathways in regulating skin proliferation and apoptosis, respectively. Surprisingly, the apoptotic cells were primarily localized in the suprabasal cells. I found that cells lacking α-catenin showed elevated focal adhesion signaling, and using the lentiviral knockdown approach, I found that co-depletion of focal adhesion signaling components reduced the protection from apoptosis afforded to the basal layer cells in the absence of α-catenin. These studies illustrate the strategy, its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers, and yield new insights into the complex mechanisms of growth regulation in tissues

Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation

Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation