Engineering Large Chromosomal Deletions in Mice to Advance Precision Oncology

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Project Summary/Abstract Genomic deletions, including both focal and large arm-level chromosomal deletions, are frequent genetic events that promote cancer initiation and progression. Focal deletions have yielded significant insights into mechanisms underlying tumorigenesis by helping identify tumor suppressor genes (TSGs) residing in the deleted loci. In contrast, little is known about the biological and phenotypic impacts of large chromosomal deletions in cancer despite their occurrence 30 times more frequently than focal deletions. We and other researchers have shown that such deletions impact the activities of multiple neighboring TSGs and constitute a driving force in tumorigenesis. Additionally, concomitant loss of multiple genes triggered by broad-scale deletions can create phenotypes that differ fundamentally from those arising from the loss of a single TSG, and may offer unanticipated therapeutic opportunities. These losses should therefore be considered as distinct genetic events and their analysis should be focused on the integrated effects of chromosomal deletion rather than the impact of a single critical TSG. Genetically engineered mouse (GEM) models provide an ideal tool for investigating the consequences of genetic aberrations in tumor biology. However, due to the limited cloning capacity of targeting vectors (~300 Kb) and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions in mice has proven highly challenging, and the in vivo role of such lesions in tumorigenesis has therefore been significantly understudied. To fill this critical gap, we have developed an innovative approach to chromosomal engineering over large genetic distances through CRISPR/Cas9 technology in mouse embryonic stem (ES) cells. The resulting ES clones carrying the desired genome edits make possible the creation of conditional knockout mice that accurately mimic cancer-associated large deletions. Given the prevalence and significance of large chromosomal deletions in prostate cancer, here we propose to utilize our innovative approach to generate and fully characterize two novel GEM models that each harbor a common but distinct prostate cancer-associated large deletion, with a goal of validating and credentialing these models as genetically and biologically robust representations of human prostate cancer. In Aim 1, we will develop conditional mouse lines using CRISPR/Cas9 technology to target commonly deleted large chromosomal loci in human prostate cancer. In Aim 2, we will develop mouse models of prostate cancer harboring large chromosomal deletions to analyze their biological and phenotypic impacts on prostate cancer development. Taken together, these models and their characterization will meet multiple goals of this grant opportunity. Successful completion of these investigations will not only contribute greatly to the implementation of precision oncology research but also enhance the applicability of animal models to translational research.