Dysregulation and therapeutic targeting of RNA splicing in cancer

Abstract

High-throughput sequencing and functional characterization of the cancer transcriptome have uncovered cancer-specific dysregulation of RNA splicing across a variety of cancers. Alterations in the cancer genome and dysregulation of RNA splicing factors lead to missplicing, splicing alteration-dependent gene expression and, in some cases, generation of novel splicing-derived proteins. Here, we review recent advances in our understanding of aberrant splicing in cancer pathogenesis and present strategies to harness cancer-specific aberrant splicing for therapeutic intent.

Fig. 1 |. Mechanisms of RNA splicing and dysregulation in cancer.

a, Sequential transesterification reactions involved in removal of an intron with resultant splicing product. During splicing catalysis, the branch site adenosine (A) nucleotide carries out nucleophilic attack of the 5′ss, forming a lariat, and the 3′ OH of the released 5′ exon performs a second nucleophilic attack on the last nucleotide of the intron at the 3′ss, joining the exons and releasing the intron lariat. b, Key sequence features and early splicing factors that govern splicing are shown. Sequence elements required for spliceosome assembly include the 5′ss and 3′ss, the polypyrimidine (poly(Y)) tract and the branch site residue that often follows the illustrated consensus motifs. The U1 snRNP (green) initiates splicing by recognizing the 5′ss consensus sequence. The U2 snRNP complex (brown) consisting of SF3B1 and other proteins is recruited to the branch site residue by the U2AF heterodimer (orange), which recognizes the 3′ss. Enhancers (ESE and ISE (intronic splicing enhancer)) and silencers (ESS (exonic splicing silencer) and ISS (intronic splicing silencer)) are recognized by specific trans-acting RNA-binding proteins, including SR proteins and hnRNPs. SR proteins commonly serve as enhancers of splicing, whereas hnRNPs commonly repress splicing. c, The following are mechanisms by which RNA splicing is altered in cancer: (1) cis-acting mutations affecting splicing regulatory sequences, (2) trans-acting mutations in the U1 snRNA, (3) mutations in RNA splicing factors and (4) changes in splicing factor expression. d, Alternative splicing of BCL-2 family of cell death factors BCL2L1 and MCL1 and alternative transcripts with resultant protein domain structures. Alternative splicing of BCL2L1 leads to generation of proapoptotic BCL-xS (green), which inhibits antiapoptotic BCL-xL (red) function, allowing BAX/BAK activation of mitochondrial outer membrane permeabilization (MOMP) and induction of apoptosis. Similarly, alternative splicing of MCL1 leads to generation of proapoptotic MCL1-S (green), inducing apoptosis through inhibition of antiapoptotic MCL1-L (green) function. Ex, exon. e, Protein diagrams (colored regions) of four splicing factors (SF3B1, U2AF1, SRSF2 and ZRSR2) and secondary RNA structure of the U1 and U2 snRNAs, with depictions of the most frequently reported hotspot mutations in red. ZnF, zinc-finger; RS, serine/arginine-rich domain; RRM, RNA recognition motif; UHM, U2AF homology motif.

Fig. 2 |. Recent advances in genomic analysis of RNA splicing.

Schematic of available tools used to assess RNA splicing alterations. One gene (exons in gray) can produce multiple transcripts (different colors represent different exons) through alternative splicing, which allows for variation in RNA modifications, including m⁶A methylation and alterations in poly(A) tail sequence. While detection of splicing events and isoforms from conventional RNA-seq is the most mature method, short-read lengths (100–200 base pairs) rarely span splice junctions, requiring methods to infer full-length RNA transcripts^–. Importantly, short-read RNA-seq cannot differentiate intermediate splicing products from final splicing products and is unable to accurately quantitate the efficacy of the two-step enzymatic splicing reaction. Two distinct long-read RNA-seq technologies have been commercialized by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). PacBio uses fluorescently labeled dNTPs and DNA polymerase to create average read lengths of >20 kilobases. By contrast, ONT uses biological nanopores within a membrane that translocate nucleic acid under an electric current and has no length limit^–. Furthermore, ONT nanopore sequencing has the ability for direct RNA sequencing and detection of epigenetic modifications, including RNA modifications. Both long-read RNA-seq systems can generate millions of reads, allowing for comprehensive expression profiling. Such third-generation sequencing technologies have been used in characterizing isoforms in organisms with poorly annotated transcriptomes^, as well as for novel isoform discovery^,. There is substantial heterogeneity in RNA splicing and gene expression among individual cells, even within a clonal population, which highlights limitations to the sequencing of bulk cell populations for gaining insight into splicing regulation and function. While single-cell RNA-seq is high throughput, detection and quantification of splicing changes from single cells remain major challenges, as the most widely used platforms for single-cell RNA-seq rely primarily on sequencing of 5′ and 3′ ends of transcripts. However, several studies have recently performed long-read sequencing of RNA from individual cells^,. Further efforts to probe splicing at the single-cell level using similar approaches may be enlightening as well as efforts to merge spatial transcriptomics with analysis of RNA splicing.

Fig. 3 |. Chemical inhibitors of RNA splicing.

Diagram of key sequence features (5′ss and 3′ss) and splicing factors (U1 snRNP, green; U2 snRNP, brown; U2AF heterodimer, orange) involved in splicing catalysis with accompanying splicing targeting mechanisms with natural products and small-molecule inhibitors. Top left, SF3b complex inhibitors bind to the branch site residue-binding pocket of SF3B1, blocking U2 snRNP recognition of the branch site, leading to intron retention and cassette exon skipping. Top right, protein domains of select UHM protein family members and drugs that have been shown to bind and inhibit these proteins. NSC 194308 targets U2AF2 through binding between its RRM domains to enhance U2AF2’s binding to RNA. Alternatively, phenothiazines target U2AF2 through binding of the UHM domains of several different UHM protein-containing proteins, including U2AF1, U2AF2, PUF60 and SPF45. Bottom right, RBM39 degraders induce an interaction between the E3 ubiquitin ligase adaptor protein DCAF15 and RBM39, leading to polyubiquitination and proteasomal degradation of RBM39. Bottom left, several post-translational modifications are known to regulate splicing function, including lysine phosphorylation and arginine methylation. Splicing factors are the most abundant arginine-methylated substrates in cells, and PRMT inhibitors are currently under clinical investigation. CLK, Cdc-like kinase; SRPK, serine/arginine protein kinase; DYRK, dual-specificity tyrosine-regulated kinases.

Fig. 4 |. Novel uses of splicing modulator drugs and synthetic introns responsive to splicing factor mutations.

a, Tumor-specific alternative splicing events are abundant in cancer and produce immunogenic neoantigens. Pharmacologic modulation of splicing using RBM39 degraders or PRMT inhibition induces novel splicing-derived neoepitopes that are presented on MHC class I. Splicing inhibition can improve response to immune checkpoint blockade through increased neoantigen generation. b, Diagram depicting the methodology and concept of synthetic intronic sequences to drive selective gene expression in cells with cancer-associated mutations in the RNA-splicing machinery, as described recently in ref. . In brief, endogenous splicing events, which are differentially used in splicing factor-mutant cells, are used to generate optimized shortened synthetic sequences. These optimized introns are then used to interrupt the protein-coding sequence of a gene of interest such that the gene of interest is only expressed in cells with an altered RNA splicing machinery. Such an approach may eventually be used to discover drugs that specifically regulate mutant splicing activity. c, Diagram depicting a splicing switch element. The switch element allows for precise control of gene replacement or gene editing after exposure to the small molecule LMI070. In the absence of exon 7 (E7) a premature stop codon blocks translation of the gene of interest. LMI070 regulates usage of exon 7, thereby regulating translation of the gene of interest to a protein product (such as erythropoietin, Cas9 or a fluorescent protein, such as green fluorescence protein (GFP)). Diagram reproduced from ref. , Springer Nature.