Multiplexed functional genomic analysis of somatic 5’ untranslated region mutations across the spectrum of human prostate cancer (Exome-Seq)

The functional consequences of cancer patient-derived genetic variants within the 5’ untranslated regions (UTRs) on a genome-wide scale and their effects on mRNA transcript and translation levels are poorly understood. To systematically interrogate the mutational landscape of 5’ UTRs across cancer patients with localized to metastatic disease, we analyzed the genomes of 226 prostate cancer patients and observed thousands of mutations, many of which impact known cis-regulatory elements. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to simultaneously quantify the effects of 545 5’ UTR somatic mutations across recurrently mutated or cancer-related genes from our patient cohort. Our method enabled unprecedented insights into how 5’ UTR mutations can control multiple levels of gene expression simultaneously. In particular, we identified 190 mutations that significantly altered 5’ UTR function, either by creating new DNA binding elements, disrupting known translation regulatory motifs, or simultaneously impacting both transcript levels and mRNA translation. Furthermore, we also determined that 5’ UTR mutations to the MAP kinase signaling pathway are significantly associated with early metastasis. This study is the first to comprehensively interrogate the functional 5’ UTR mutational landscape of a human cancer revealing the importance of untranslated regions in regulating multiple levels of oncogenic gene expression and provides a high-throughput functional genomics solution applicable to many genetically driven diseases. We searched for somatic 5’ UTR single nucleotide variants in a cohort of five primary mCRPC patient-derived xenografts (PDX) belonging to the LuCaP series23, which encompass major genomic and phenotypic features of human prostate cancer, including adenocarcinoma (LuCaP 78, LuCaP 81, LuCaP 92), neuroendocrine prostate cancer (LuCaP 145.2), and a hypermutated prostate cancer (LuCaP 147). The five LuCaP series of prostate cancer xenografts used in this study (LuCaPs 78, 81, 92, 145.2 and 147) were obtained from the University of Washington Prostate Cancer Biorepository and generated from advanced prostate cancer patients Processed data are produced for each matched pair of cancer tissue and matched normal sample. VCF files for the paired samples are included on the prostate cancer sample.