Published OnlineFirst October 14, 2019; DOI: 10.1158/2159-8290.CD-19-0399 REVIEW Altered RNA Processing in Cancer Pathogenesis and Therapy Esther A. Obeng1, Connor Stewart2, and Omar Abdel-Wahab2 ABSTRACT Major advances in our understanding of cancer pathogenesis and therapy have come from efforts to catalog genomic alterations in cancer. A growing number of large-scale genomic studies have uncovered mutations that drive cancer by perturbing cotranscrip- tional and post-transcriptional regulation of gene expression. These include alterations that affect each phase of RNA processing, including splicing, transport, editing, and decay of messenger RNA. The discovery of these events illuminates a number of novel therapeutic vulnerabilities generated by aber- rant RNA processing in cancer, several of which have progressed to clinical development. Significance: There is increased recognition that genetic alterations affecting RNA splicing and poly- adenylation are common in cancer and may generate novel therapeutic opportunities. Such mutations may occur within an individual gene or in RNA processing factors themselves, thereby influencing splic- ing of many downstream target genes. This review discusses the biological impact of these mutations on tumorigenesis and the therapeutic approaches targeting cells bearing these mutations. INTRODUCTION RNA splicing and polyadenylation are altered in cancer and functionally drive cancer initiation and maintenance. This The advent of high-throughput transcriptome sequencing is notably only a subset of the means by which RNA process- (RNA-seq) has provided a wealth of information on RNA ing goes awry in cancer. Cancer-associated changes in RNA splicing on a genome-wide scale. It is now understood from editing, RNA modifications, and expression of noncoding RNA-seq that >95% of human genes are subject to alternative RNA species including micro-RNAs and long noncoding splicing (AS), the enzymatic process by which a single gene RNAs are covered in recent excellent publications (1–4). has the potential to produce multiple, potentially function- Finally, motivated by recent data illustrating individual ally distinct pre-mRNA and protein isoforms. Splicing is con- splicing alterations as well as mutations in RNA-splicing sidered to be a major mediator of proteome diversity through factors as therapeutic vulnerabilities in cancer, we discuss its ability to generate multiple transcripts with differing ongoing and future efforts to target RNA splicing for cancer amino acid sequences from a single gene. Moreover, due to therapy. the link between splicing and nonsense-mediated mRNA decay (NMD), splicing also provides a means to regulate gene expression. BASIC MECHANISMS OF RNA Systematic application of RNA-seq to an ever-expanding SPLICING CATALYSIS number of human tumors and matched normal tissues has now identified numerous means by which RNA processing RNA splicing is a nuclear enzymatic process accomplished is dysregulated in cancer. In this review, we focus on how by a macromolecular machine composed of a large constella- tion of RNA binding proteins (RBP) and additional splicing proteins combined with five small nuclear RNAs (snRNA) in complexes known as small nuclear ribonucleoproteins 1Department of Oncology, St. Jude Children’s Research Hospital, Mem- (snRNP; reviewed recently; refs. 5–7). snRNAs base pair with phis, Tennessee. 2Human Oncology and Pathogenesis Program and Leuke- sequences within pre-mRNA that are critical in delineat- mia Service, Department of Medicine, Memorial Sloan Kettering Cancer ing exons from introns. These include the dinucleotides at Center, New York, New York. the first two and last two positions of an intron [known as Corresponding Authors: Omar Abdel-Wahab, Memorial Sloan Kettering Cancer Center, Zuckerman Research Building, 415 E 68th Street, New the 5′ and 3′ splice sites (ss), respectively] and a poorly con- York, NY 10065. Phone: 347-821-1769; Fax: 646-422-0856; E-mail: served sequence within the intron known as the branchpoint [email protected]; and Esther Obeng, 262 Danny Thomas Place, Mail (Fig. 1A). The branchpoint is typically close to the 3′ ss and Stop 354, Memphis, TN 38105. E-mail: [email protected] is usually an adenosine nucleotide. A stretch of pyrimidine Cancer Discov 2019;9:1–18 nucleotides (the polypyrimidine tract) is often adjacent to doi: 10.1158/2159-8290.CD-19-0399 the branchpoint and promotes the spliceosome’s recognition ©2019 American Association for Cancer Research. of the branchpoint. Deletion or mutation of these intronic NOVEMBER 2019 CANCER DISCOVERY | OF1 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 14, 2019; DOI: 10.1158/2159-8290.CD-19-0399 REVIEW Obeng et al. A Exonic splicing enhancer Exonic splicing suppressor 3 splice site Intronic splicing enhancer 5 splice site Branch point ′ ′ Intronic splicing suppressor B A RBM39 PHF5A SRSF2 U2AF A A OH SF3B1 C Exon Intron Exon Intron Exon Normal splicing event Exon skipping Intron retention Splice-site creating mutation D Cassette RefSeq GenCode exon Alternative 5′ splice site Alternative Ensembl 3′ splice site Cassette exon Alternative 5′ or 3′ ss Retained intron Intron Other retention Mutually exclusive exon OF2 | CANCER DISCOVERY NOVEMBER 2019 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 14, 2019; DOI: 10.1158/2159-8290.CD-19-0399 Splicing in Cancer REVIEW sequences typically greatly abrogates splicing usage at these average, harbor ∼20% more AS events than normal tissues. As sites. In addition, sequences throughout the exon and intron, such, many splicing changes identified in cancer cells can be referred to as splicing enhancers and silencers, are bound by linked to single-nucleotide variants (SNV) within that gene RBPs that recruit or repel spliceosome assembly to promote that disrupt mRNA sequences required for normal splic- or inhibit splicing, respectively. ing (Fig. 1C). Heterozygous cis-acting splicing alterations The enzymatic process of splicing consists of two sequen- act in an allele-specific manner such that the mutant allele tial transesterification reactions (Fig. 1B). In the first step, shows abnormal splicing, whereas the wild-type allele sup- the 2′-hydroxyl group of the branchpoint performs a nucleo- ports normal splicing. Interestingly, prior work from Supek philic attack on the phosphorous atom of the 5′ ss to generate and colleagues and Jung and colleagues found that SNVs a linear left exon and a right intron–exon branched sequence resulting in frameshifts in mRNA sequence due to intron known as a lariat. In the second step, the 2′-hydroxyl group retention (IR) occur most commonly in tumor suppressors of the linear left exon attacks the phosphorous atom of the while sparing oncogenes (13, 14). In contrast, in-frame exonic 3′ ss, thereby concatenating two exons. An excised intron is SNVs disrupting exon usage are most commonly enriched in simultaneously released within the lariat and later degraded oncogenes. by RNA debranching enzymes. Although most studies evaluating the effects of SNVs Over the last four years there have been major advances on splicing have focused on mutations abolishing splice in the mechanistic understanding of spliceosome function, sites, recent reanalysis by Jayasinghe and colleagues of 8,000 with the publication of at least 18 independent structures tumor samples from 33 TCGA cancer types identified many of the spliceosome at a resolution of 3.3 to 9.9 Å (reviewed examples of mutations creating novel splice sites (Fig. 1C; recently; refs. 8–10). Although these initial efforts were ref. 15). These splice site–creating mutations affect many mostly carried out using cryoelectron microscopy (cryo- known cancer genes. Intriguingly, tumors bearing these EM) of yeast spliceosomes, initial structures of the human mutations also appeared to be associated with increased spliceosome followed in 2017. It is almost certain that high- expression of T cell–associated genes and mRNAs encod- resolution structures of the human spliceosome in each ing the immune-checkpoint blockade molecules PD-1 and step of the splicing reaction will be elucidated in the very PD-L1. These data suggest the potential for splicing to gen- near future. erate novel, immunogenic peptides (a topic discussed later in this review). ALTERATIONS IN RNA SPLICING IN CANCER It is important to note that most studies evaluating the impact of cis-acting mutations on splicing have utilized WES Splicing Changes in Cancer Relative data and focused on mutations that occur at 5′ or 3′ exon– to Normal Tissues intron boundaries. However, in vitro screening of sequence Prior to the advent of RNA-seq, evaluation of expressed variants across every nucleotide within model exons has sequence tag libraries from human cancer cells and other shown that >50% of nucleotide substitutions in exons can tissue types revealed that cancer cells have an elevated rate of induce splicing changes with similar effects expected from stop codons relative to noncancer cells (11). These data sug- coding and synonymous (or “silent”) mutations (16, 17). gested increased missplicing in cancer transcriptomes relative Consistent with this, analysis of >3,000 cancer exomes and to normal tissues; the basis for which was not entirely clear. >300 cancer genomes by Supek and colleagues