Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3' untranslated region mutations.

3' untranslated region (3' UTR) somatic mutations represent a largely unexplored avenue of alternative oncogenic gene dysregulation. To determine the significance of 3' UTR mutations in disease, we identify 3' UTR somatic variants across 185 advanced prostate tumors, discovering 14,497 single-nucleotide mutations enriched in oncogenic pathways and 3' UTR regulatory elements. By developing two complementary massively parallel reporter assays, we measure how thousands of patient-based mutations affect mRNA translation and stability and identify hundreds of functional variants that allow us to define determinants of mutation significance. We demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 endogenous editing of distinct variants increases cellular stress resistance and that patients harboring oncogenic 3' UTR mutations have a particularly poor prognosis. This work represents an expansive 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.

Multiplexed functional genomic analysis of 5' untranslated region mutations across the spectrum of prostate cancer.

The functional consequences of genetic variants within 5' untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5' UTR mutations in human prostate cancer. We show that 5' UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5' UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5' UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5' UTRs are functional in cancer.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

IFN-I and IL-22 mediate protective effects of intestinal viral infection.

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells, which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition, we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.

The Untranslated Regions of mRNAs in Cancer.

The 5' and 3' untranslated regions (UTRs) regulate crucial aspects of post-transcriptional gene regulation that are necessary for the maintenance of cellular homeostasis. When these processes go awry through mutation or misexpression of certain regulatory elements, the subsequent deregulation of oncogenic gene expression can drive or enhance cancer pathogenesis. Although the number of known cancer-related mutations in UTR regulatory elements has recently increased markedly as a result of advances in whole-genome sequencing, little is known about how the majority of these genetic aberrations contribute functionally to disease. In this review we explore the regulatory functions of UTRs, how they are co-opted in cancer, new technologies to interrogate cancerous UTRs, and potential therapeutic opportunities stemming from these regions.

Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota.

As a conserved pathway that lies at the intersection between host defence and cellular homeostasis, autophagy serves as a rheostat for immune reactions. In particular, autophagy suppresses excess type I interferon (IFN-I) production in response to viral nucleic acids. It is unknown how this function of autophagy relates to the intestinal barrier where host-microbe interactions are pervasive and perpetual. Here, we demonstrate that mice deficient in autophagy proteins are protected from the intestinal bacterial pathogen Citrobacter rodentium in a manner dependent on IFN-I signalling and nucleic acid sensing pathways. Enhanced IFN-stimulated gene expression in intestinal tissue of autophagy-deficient mice in the absence of infection was mediated by the gut microbiota. Additionally, monocytes infiltrating into the autophagy-deficient intestinal microenvironment displayed an enhanced inflammatory profile and were necessary for protection against C. rodentium. Finally, we demonstrate that the microbiota-dependent IFN-I production that occurs in the autophagy-deficient host also protects against chemical injury of the intestine. Thus, autophagy proteins prevent a spontaneous IFN-I response to microbiota that is beneficial in the presence of infectious and non-infectious intestinal hazards. These results identify a role for autophagy proteins in controlling the magnitude of IFN-I signalling at the intestinal barrier.