Abstract PR005: Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations

Abstract It is clear from the high mortality rate in advanced cancers that we need a more complete understanding of the many sources of oncogenic dysregulation in tumors. The importance of the non-coding genome in disease has recently begun to be revealed through expanded use of whole genome sequencing. In particular, 3’ untranslated region (3’UTR) somatic mutations represent an important but largely unexplored avenue of alternative oncogenic gene dysregulation. Individual instances of 3’UTR-mediated oncogenicity are known, including oncogenic RNA binding proteins and microRNAs. However, a comprehensive, high-throughput study of patient-based 3’UTR mutations and their effect on post-transcriptional gene regulation in cancer has yet to be undertaken. To determine the significance of 3’UTR mutations in advanced disease, we identify 3’UTR somatic variants across 185 metastatic castration-resistant prostate tumors, discovering 14,497 single-nucleotide mutations, which are enriched in oncogenic pathways and 3’UTR regulatory elements. We develop two complementary massively parallel reporter assays (MPRAs) to measure how thousands of these patient-based mutations affect two distinct levels of post-transcriptional gene regulation: mRNA translation and stability. These MPRAs identify hundreds of functional variants that allow us to define three determinants of mutation significance: sequence conservation, known regulatory elements, and RNA structure. Furthermore, we demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 base editing of distinct patient 3’UTR mutations into endogenous cellular loci increases oncogenic mRNA translation and cellular stress resistance. Finally, we go back to patients, illustrating that those harboring oncogenic 3’UTR mutations discovered by our methods display particularly poor prognosis. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions. Citation Format: Samantha L. Schuster, Sonali Arora, Cynthia L. Wladyka, Lukas Corey, Bethany L. Stackhouse, Lori Kollath, Eva Corey, Lawrence D. True, Dave Young, Patrick J. Paddison, Andrew C. Hsieh. Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR005.