Research Summary/Abstract The sheer genetic heterogeneity and complexity of cancer has been laid bare by next-generation sequencing studies of cancer genomes. A major challenge in the post-genomic era of cancer biology is to move beyond descriptive analyses of genomic data and derive functional insights into the complex multigenic interactions that produce complicated phenotypes associated with cancer evolution. Available experimental systems are poorly-suited to address this problem in a systematic and expeditious manner as they generally lack scale, throughput, economy, or biological relevance. We therefore propose the development of a next-generation functional cancer genomics assay that overcomes these limitations and will be employed here to define complicated genotype-to-phenotype relationships in contexts related to prostate cancer initiation and progression. The assay combines the 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 sequencing analysis to enumerate lentiviral barcodes for the massively parallel association of genotype with phenotype. The major goal of this project is to apply and advance this pioneering strategy to broadly advance our understanding of prostate cancer biology by characterizing compound genetic interactions that (1) transform benign prostate epithelial cells to cancer and establish distinct cancer subtypes, (2) induce neuroendocrine transdifferentiation of prostate cancer, and (3) promote metastatic dissemination. If successful, these studies will characterize and prioritize the functional contributions of multigenic networks to clinically relevant prostate cancer phenotypes. We will also establish multiple new, genetically-defined mouse models of prostate cancer that will better recapitulate the genetic complexity of the human disease. Finally, we anticipate that the next-generation functional cancer genomics strategy will be a flexible and widely applicable experimental tool to rapidly interrogate complex genetic interactions and their impacts on phenotype in many other cancers.