APC mutations dysregulate alternative polyadenylation in cancer.

Background: Alternative polyadenylation (APA) affects most human genes and is recurrently dysregulated in all studied cancers. However, the mechanistic origins of this dysregulation are incompletely understood.

Results: We describe an unbiased analysis of molecular regulators of poly(A) site selection across The Cancer Genome Atlas and identify that colorectal adenocarcinoma is an outlier relative to all other cancer subtypes.

An autoregulatory poison exon in Smndc1 is conserved across kingdoms and influences organism growth.

Many of the most highly conserved elements in the human genome are "poison exons," alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon in Smndc1 is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms.

Multiplexed screening reveals how cancer-specific alternative polyadenylation shapes tumor growth in vivo.

Alternative polyadenylation (APA) is strikingly dysregulated in many cancers. Although global APA dysregulation is frequently associated with poor prognosis, the importance of most individual APA events is controversial simply because few have been functionally studied. Here, we address this gap by developing a CRISPR-Cas9-based screen to manipulate endogenous polyadenylation and systematically quantify how APA events contribute to tumor growth in vivo.

N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia.

Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes.

Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells.

Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production.

Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome.

CRISPR screens have accelerated the discovery of important cancer vulnerabilities. However, single-gene knockout phenotypes can be masked by redundancy among related genes. Paralogs constitute two-thirds of the human protein-coding genome, so existing methods are likely inadequate for assaying a large portion of gene function.

Characterization of neoantigen-specific T cells in cancer resistant to immune checkpoint therapies.

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti-PD-1 and anti-CTLA-4) and are being targeted for vaccination-based therapies.

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy.

RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons.

While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR-Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention.