Diversity Supplement: Uncovering diverse genotype-phenotype relationships in prostate cancer

项目总结

Project Summary A significant challenge in the field of functional cancer genomics is to establish experimental systems with biologic relevance, economy, and scale to derive insights into the interactions of diverse genetic events in cancer initiation and progression. We developed a next-generation functional cancer genomics assay that combines efficient delivery of random, compound genetic perturbations from barcoded lentiviral libraries encoding gain-of-function and/or loss-of-function events; biological selection for a cancer phenotype; and single-cell DNA amplicon sequencing analysis to enumerate lentiviral barcodes for the massively parallel association of genotype with phenotype (histology). Here we will substantially improve upon this prototype assay by modifying the lentiviral constructs such that they incorporate and transcribe DNA barcodes that can be captured by single-cell RNA sequencing, thereby enabling simultaneous single-cell determination of lentiviral barcodes (genotype) and transcriptomes (phenotype). To accomplish this, we will engineer DNA barcodes and a simian virus 40 polyadenylation signal sequence into the 3’ untranslated region of a green fluorescent protein (GFP) expressed from an internal cytomegalovirus (CMV) promoter. We will also enhance overall GFP transcription and mitigate CMV promoter silencing by placing a ubiquitous chromatin opening element (UCOE) upstream of the CMV promoter. Further, we will determine technical parameters related to single-cell RNA sequencing to determine the relative read depths necessary to accurately capture compound barcodes and discriminate cell populations based on transcriptional profiles from a defined cell mixture. Once optimized, this technology will be incorporated into an established mouse prostate epithelial transformation assay to demonstrate proof-of-principle of this approach and define how oncogenic events may cooperate to impart distinct prostate cancer cell states. This diversity supplement will support Gerardo Javier, a graduate student at the University of Washington who is conducting research under the primary mentorship of John K. Lee, MD, PhD at Fred Hutch. He will also receive co-mentorship from McGarry Houghton, MD who is a senior faculty member at Fred Hutch. A training plan has been crafted to ensure Gerardo’s enrichment related to scientific productivity, education, communication skills, professional development, networking, and promotion of underrepresented minorities in biomedical sciences. This support will be beneficial to his training as a graduate student focused on cancer biology and his ultimate transition to a post-doctoral fellowship as he continues his career trajectory of becoming an impactful independent cancer researcher.