High-throughput functional characterization of cancer specific alternative polyadenylation

Abstract

RNA dysregulation is a ubiquitous feature of human cancers and misexpression of even a single RNA isoform is sufficient to drive cancer. Among types of RNA processing, alternative cleavage and polyadenylation (APA), a process whereby a messenger RNA is cleaved at a polyadenylation signal sequence and a poly(A) tail is added, stands out as poorly functionally studied yet broadly dysregulated in all cancer subtypes studied. Global patterns in APA are associated with patient outcomes, response to chemotherapy, and patterns of immune infiltration, all of which point to a functional role of APA in human cancers. An absence of molecular tools has led to a large number of cataloged APA events that have no functional understanding and a reliance on a sufficient number of adjacent healthy control tissues has led to an outsized focused on a small number of human cancer subtypes. In this dissertation, I address these problems and describe a computational framework to assess global patterns in poly(A) site selection that allows for intra and inter dataset comparisons without normalization to a healthy tissue control and a novel, CRISPR/Cas9 based method to functionally screen APA events in a live, immunocompetent host. I demonstrated that 3′ UTR lengthening in a single gene, ATG7, reduces melanoma tumor engraftment and boosts tumor immune evasion in both mouse and human melanoma. I then further characterize pan-cancer alterations in APA and identify that colorectal adenocarcinoma displays significantly distinct patterns in poly(A) site selection compared to all other datasets analyzed. I provide compelling evidence from human colorectal adenocarcinoma samples and genetically modified human colon organoid samples that adenomatous polyposis coli (APC) is an RNA binding protein that regulates poly(A) site selection. Together, this work demonstrates that APA is pervasive in human cancers and represent a novel source of potential targets for clinical therapy development.