Working to exploit intertional mutagenesis for cancer gene discovery since 1996.
Research projects in the Largaespada lab are aimed at identifying mutations and other changes that drive the development of cancer, which must be determined for developing molecularly targeted therapeutics.
The Largaespada lab pioneered the use of a vertebrate-active transposon system, called Sleeping Beauty (SB), for insertional mutagenesis in mouse somatic cells. SB is being used as a tool for forward genetic screens for cancer genes involved in sarcoma, hepatocellular carcinoma, mammary, gastro-intestinal tract and NF1 syndrome-associated nervous system cancers.
Novel Mouse Models
Also, novel mouse models are being used for preclinical evaluation of new drugs and drug combinations for cancer treatment. A large focus is on drugs to treat tumors associated with Neurofibromatosis Type 1 (NF1) syndrome.
The lab is also working to utilize and improve the latest methods in genome engineering, like CRISPR/Cas9, to study cancer gene function and uncover cancer vulnerabilities.
The Largaespada lab is focused on cancer related problems including cancer initiation, progression, metastasis and treatment resistance. Genes and pathways important for cancer relevant phenotypes are being sought using functional genomics approaches. In particular, the lab has invested heavily in the use of Sleeping Beauty (SB) transposon mutagenesis in mice to create various types of cancer and to reveal novel cancer genes at recurrently mutated loci. These models and phenotypically and genetically similar to human cancers, allow meaningful genotype-phenotype correlations to be drawn, and reflect human cancer genetic heterogeneity within and between different tumors. We have applied deep RNA sequencing to transposon induced tumors and can easily find fusion transcripts between the transposon and endogenous genes. These analyses have revealed new cancer genes missed by only studying transposon insertions at the level of genomic DNA. These data also allow genotype-phenotype correlations to be drawn finding downstream gene expression changes for specific gene alterations. Follow-up studies on these genes and pathways involve genetic engineering of normal human cells using targeted nucleases such as CRISPR/Cas9. Genes from our forward cancer genetic screens using SB mice are being knocked out in normal immortalized human cells and cancer cell lines. These studies are revealing what cancer phenotypes these genes control. We are using engineered cell line models, and genetically engineered mouse models, to test the effects of pharmacological interventions, and for genetic synthetic lethal studies. Improved methods for transposon mutagenesis, genome editing and genome manipulation in general are being pursued.
Neurofibromatosis Type 1 and Brain Tumors
Neurofibromatosis Type 1 (NF1) syndrome is an autosomal dominant genetic disorder characterized by a predisposition to peripheral nerve sheath tumors called neurofibromas, optic pathway gliomas and other brain tumors, leukemia, pheochromocytoma, osteosarcoma and various other forms of cancer. We are using multiple model systems to explore genetic and other factors that cause NF1 associated tumors and to test new therapeutic interventions. These include Sleeping Beauty (SB) transposon mutagenesis in mice, genetically engineered mouse models, engineered human Schwann cells, and a new porcine model of NF1. New technologies for modeling brain tumors and new brain tumor genes are also being investigated using similar approaches.
Immunofluorescence images of Schwann cells isolated from the sciatic nerve of a genetically engineered minipig
Green=GFAP (expressed by Schwann cells); Red=actin (stains the cytoskeleton); Blue=DAPI (stains the nucleus)
Immunofluorescent images of human medulloblastoma cells stained for ARHGAP36 expression
Green=cytoskeleton; Red=F-actin; Blue=nuclear DNA (blue)
Immunofluorescent image of mouse neuronal progenitor cells engineered to overexpress ARHGAP36
Green=ARHGAP36 expression; Red=cytoskeleton F-actin; Blue=nuclear DNA
Osteosarcomas are the most common form of primary bone tumor, with a peak incidence in adolescence. While roughly 60% can be cured using surgery and adjuvant chemotherapy, when metastases occur the prognosis is dismal. We lack an understanding of what drives metastatic disease or what causes metastatic osteosarcoma to resist chemotherapy. We have performed a Sleeping Beauty (SB) based forward genetic screen for osteosarcoma development and metastatic progression. This approach is being complemented with whole genome CRISPR/Cas9 library screening in human cells. Insight from these screens is being used to test new therapies using in vitro and in vivo model systems.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. HCC usually occurs in the context of chronic liver damage such as that caused by chronic hepatitis infection, cirrhosis, or fatty liver disease. HCC are genetically complex and druggable drivers are still being sought. We have performed several Sleeping Beauty (SB) based forward genetic screens for HCC development and progression in male and female mice, in various genetic contexts, and in normal or damaged livers. These data have suggested an interaction between specific gene mutations and pathways and the context in which HCC develops. We hope to use this insight for new HCC prevention and treatment strategies.
It has become increasingly clear that the immune system can be leveraged for cancer therapy. This includes new methods for ex vivo genetic engineering of white blood cells for therapy, cancer vaccines and ways to enhance existing anti-tumor immunity. We are combining genetics and immunology for cancer therapy in several ways. This includes new methods for non-viral gene delivery and gene modification of human cells. In addition, we’re investigating methods to make tumor cells more visible to the immune system using drugs to force cancer cells to express “cryptic neoantigens”. Prospectively identified cryptic neoantigens could be used as the basis for personalized cancer vaccines.