2-3 pages summary

mRNAs are the molecular templates for the synthesis of proteins. In eukaryotic organisms, the primary gene transcripts, pre- mRNAs, are typically not functional for protein synthesis until internal sequences (introns) are removed and the remaining fragments (exons) are spliced together to generate mature mRNAs (Fig. 1). Indeed, pre- mRNA splicing is essential for the expres- sion of >95% of human genes1,2. The process can also be regulated to generate alternatively spliced mRNAs (Fig. 2) that encode distinct protein variants, which is a mecha- nism often used to maintain cellular homeostasis and to regulate cell differentiation and development1,2.

The splicing process and its regulation are highly relevant for understanding every hallmark of cancer (Fig. 2, Supplementary Table 1), to the point that splic- ing alterations constitute another cancer hallmark3–8. For example, analyses of >8,000 tumours across 32 can- cer types revealed thousands of splicing variants not present in non- malignant tissues, which are likely to generate cancer- specific markers and neoantigens9,10 that could potentially be used as mRNA vaccines11. As another example, the generation of splicing var- iants is frequently responsible for the acquisition of resistance to androgen receptor- targeted therapies in prostate cancer and for resistance to vemurafenib in melanoma12–14. In addition, results indicate that cancer cells impose special demands on the splicing machin- ery such that they become particularly vulnerable to splicing perturbations, a feature that is beginning to be

exploited pharmacologically15–18. In this Review, we outline in detail the splicing process and its alterations in cancer before highlighting opportunities for the development of innovative therapeutic approaches in clinical oncology.

The splicing machinery and cancer The removal of introns involves a chemical mechanism by which specific phosphodiester bonds in the polynu- cleotide chains of RNA are excised, and new ones are formed. This process occurs in two consecutive steps and involves the formation of an unusual 2′–5′ phos- phodiester bond between the 5′ nucleotide of the intron and a key internal adenosine residue (the branch site) located 15–30 nucleotides upstream of the 3′ end of the intron (Fig. 1). This chemical mechanism is identical to that of group II self- catalytic RNAs, possibly reveal- ing the ancestral origin of pre- mRNA introns19. Whereas the sequence of the entire group II intron shapes the elaborate 3D structure that enables their excision in the absence of cofactors, introns in pre- mRNAs har- bour only short consensus sequences at the exon–intron boundaries, known as 5′ and 3′ splice sites, and the removal of these introns relies on one of the most sophis- ticated macromolecular complexes of eukaryotic cells: the spliceosome20,21. The splicing process is an essential step in eukaryotic gene expression and, unsurprisingly, hereditary cancer genes are particularly susceptible to inactivating mutations in splice sites22.

Roles and mechanisms of alternative splicing in cancer — implications for care Sophie C. Bonnal 1,2, Irene López- Oreja 1,2,3 and Juan Valcárcel1,2,4 ✉

Abstract | Removal of introns from messenger RNA precursors (pre- mRNA splicing) is an essential step for the expression of most eukaryotic genes. Alternative splicing enables the regulated generation of multiple mRNA and protein products from a single gene. Cancer cells have general as well as cancer type- specific and subtype- specific alterations in the splicing process that can have prognostic value and contribute to every hallmark of cancer progression, including cancer immune responses. These splicing alterations are often linked to the occurrence of cancer driver mutations in genes encoding either core components or regulators of the splicing machinery. Of therapeutic relevance, the transcriptomic landscape of cancer cells makes them particularly vulnerable to pharmacological inhibition of splicing. Small- molecule splicing modulators are currently in clinical trials and, in addition to splice site- switching antisense oligonucleotides, offer the promise of novel and personalized approaches to cancer treatment.

1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain. 2Universitat Pompeu Fabra, Barcelona, Spain. 3Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.

✉e- mail: juan.valcarcel@ crg.eu

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The spliceosome. The spliceosome consists of five small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5 and U6, which is tightly bound to U4), each of which is composed of one specific small nuclear RNA (snRNA) and a number of associated proteins, as well as >150 additional polypeptides not directly bound to the snRNPs20. The catalytic core of the spliceosome is assembled anew on each intron substrate from separate subcomplexes and is shaped by RNA–RNA interactions between U2 and U6 snRNAs as well as between snRNAs and the intron splice sites20,21 (Fig. 1). Protein components of the spliceosome help to shape the RNA- based active site of this enzymatic complex and are also essential for its function23–28.

Spliceosome assembly. Spliceosome assembly starts with the recognition of the 5′ splice site by U1 snRNP through base pairing interactions involving 6–8 nucleo- tides of U1 snRNA and the 5′ end of the intron (Fig. 1a). Mutations in this region of U1 snRNA induce predict- able changes in 5′ splice site utilization (Table 1), which affect known cancer driver genes across multiple can- cers and confer an adverse prognosis in patients with chronic lymphocytic leukaemia (CLL)29. In Sonic hedge- hog medulloblastomas, these mutations also inactivate tumour suppressors, such as Patched homologue 1, as well as activating proto- oncoproteins, such as GLI2 and cyclin D2 (reF.30).

Initial identification of the 3′ splice site region by the spliceosome involves three separate sequence elements recognized by three interacting proteins, U2AF1, U2AF2 and SF1 (Fig. 1), which are recurrently mutated in can- cer31. U2AF1 binds to the conserved AG dinucleotide at the 3′ end of the intron20 (Fig. 1). U2AF1 is frequently mutated in myeloid malignancies and lung adenocarci- nomas31–33 (Table 1), but how mutation of a functionally conserved factor that recognizes a conserved sequence at the 3′ splice site contributes to cancer progression remains to be fully understood. Mice expressing mutant U2AF1 have altered haematopoiesis, an altered prefer- ence for the nucleotide preceding the 3′ splice site AG motif (Fig. 3a), and splicing changes in haematopoietic

precursor cells in transcripts encoding RNA- processing factors, ribosomal proteins and mRNAs of genes that are mutated in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), such as BCOR or KMT2D34. Such splicing changes might contribute to disease pro- gression; consistent with this concept, mutant U2AF1 leads to abnormal processing of autophagy- related fac- tor 7 (Atg7) pre- mRNA (which, surprisingly, is related to the selection of a distal cleavage and polyadenylation site; Fig. 3a) and to an autophagy defect that favours the transformation of mouse pro- B cells35. However, given the limited conservation of alternative splicing events between humans and mice, the effects of splicing alter- ations observed in mouse models might be different to those in human tumours. In fact, contrary to the expectation for driver mutations, mouse models have shown that splicing factor- mutant cells have a compro- mised competitive repopulation capacity compared with that of splicing factor- wild- type cells15,34,36–41. Finally, a non- canonical role of U2AF1 in translational repres- sion via direct binding near initiation codons is altered in human cell lines harbouring the common U2AF1S34F mutation, leading to increased expression of the secreted chemokine IL-8, which in turn can contribute to inflammation and tumour progression42.

The next step in spliceosome assembly involves ATP- dependent stable binding of the U2 snRNP around the branch site (Fig. 1). As is the case for recognition of the 5′ splice site by U1 snRNA, recognition of the branch site by U2 snRNP involves base pairing interactions, this time between six nucleotides of U2 snRNA and nucleotides flanking the branch- site adenosine in the pre- mRNA intron20. SF3B1 is a key protein component of U2 snRNP that recognizes the branch site and the U2 snRNA–pre- mRNA helix and, through a confor- mational change induced by binding of the pre- mRNA, facilitates the approximation of the branch- site adeno- sine to the 5′ splice site and the first step of the splicing reaction43,44 (as detailed below). Mutations in SF3B1 are among the most frequent in a variety of cancers31, with a prevalence ranging from 5% in breast cancer to 81% in a class of MDS with ring sideroblasts (Table 1). For exam- ple, SF3B1 mutations are detected in ~10% of patients with CLL, with the K700E mutation being among the most frequent single amino acid change observed in any gene in this disease, and are associated with rapid disease progression and unfavourable overall survival45,46.

As discussed above for U2AF1, it is remarkable that mutations in key core components of the splicing appara- tus contribute to cancer progression rather than causing a general defect in intron removal that would probably compromise cell survival. Mutations affecting SF3B1 disrupt the interaction of this protein with the splicing factor SUGP1 (reF.47) and result in the use of cryptic 3′ splice sites typically within a window of 30 nucleotides upstream of the 3′ splice sites used in wild- type cells48–50 (Fig. 3b). These changes probably contribute to cancer progression by affecting the expression or function of specific genes. For example, a variety of SF3B1 muta- tions result in the use of a cryptic 3′ splice site and in inclusion of an associated cryptic exon in transcripts of BRD9, which introduces premature stop codons into the

Key points

• Alternative splicing enables the generation of distinct mRNA and protein isoforms from a single gene. splicing is carried out by the spliceosome, one of the most complex molecular machineries of eukaryotic cells.

• splicing perturbations are common in cancer and are associated with mutations in and/or altered expression of the components of the splicing machinery.

• splicing perturbations contribute to every hallmark of cancer and can generate neoantigens relevant to the design of cancer vaccines and other immunotherapies.

• Cancer cells generate advantageous splicing variants, at the cost of reducing the efficiency or fidelity of the splicing process, thus conferring a special susceptibility to splicing inhibitors and providing a therapeutic window for targeting the splicing process.

• small- molecule modulators of the spliceosome have demonstrated antitumour effects and are particularly active against cancer cells harbouring mutations in spliceosomal components.

• Antisense oligonucleotides offer promise to modulate cancer- relevant alternative splicing decisions, with proof of concept for this type of therapy demonstrated by Nusinersen, a first- in- class treatment for patients with spinal muscular atrophy.

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BRD9 open reading frame that lead to mRNA degra- dation by the process of nonsense- mediated decay51. This process, in turn, results in reduced levels of BRD9 protein, a core component of the non- canonical chro- matin remodelling complex BAF and a potent tumour suppressor, thus promoting uveal melanomagenesis52.

Alternative 3′ splice site usage induced by SF3B1 muta- tions could also be a source of neoantigens for per- sonalized vaccine or adoptive T cell- based therapies53. Surprisingly, a low- level but widespread reduction of intron retention isoforms (that is, enhanced splicing of regulated introns) seems to be the most frequent

Exon

5′ splice site GURAGU

GURAGU

YNURAY YYY AG

Exon Intron

First catalytic step

Second catalytic step

U2AF1U2AF2

U2AF1

PRMT1

U2AF2

SF1U1 YNURAY YYY AG

GU RA GU

GU RA GU

YNURAY YYY AG

GU RA GU

YNURAY YYY AG

3′ splice site

U1 U4

U2 U5

U6

U2

U5 U6

U2 U5 U6C, C*

Bact, B*

B

A

E

U6 snRNA

U2 snRNA

U2 snRNA

U2 snRNA

U2 snRNA

U4 snRNA

Key RNA–RNA interactions occurring at the different stages of spliceosome assemblysnRNP/protein composition

a

b

YNUR YA

YNUR YA

SF3B1

NCT02841540

NCT03666988

YYY AG

RBM15

RBM39

RBM10

AG

YY Y

U5 snRNA

U5 snRNA

U6 snRNA

U6 snRNA

YNUR YA

YNUR YA

U1 snRNA

U1 snRNA GU RA GU

GU RA GU A G

AG

YY Y

U5 snRNA

Fig. 1 | Pre-mRNA splicing and the spliceosome assembly pathway. a | The chemical process of intron removal. Two successive transesterifica- tion reactions, involving the breakage and formation of phosphodiester bonds, result in the removal of introns and in the splicing together of exons. The first step generates two reaction intermediates: the 5′ exon and a lariat- shaped structure harbouring an unusual 2′–5′ phosphodiester bond involving the 5′ nucleotide of the intron and an internal adenosine residue (the branch point, indicated in red) located 15–30 nucleotides upstream of the 3′ end of the intron. In the second step, the two exons are spliced together and the intron is released as a lariat product. b | Spliceosome assembly. The spliceosome, composed of five small nuclear ribonucleo- proteins (snRNPs) and numerous additional proteins, assembles anew on each pre- mRNA molecule through sequential steps involving the dynamic remodelling of its composition and conformation mediated by changes in protein–protein, protein–RNA and RNA–RNA interactions (the latter summarized in the right- hand panels). U1 snRNP is recruited through base

pairing between the 5′ splice site and U1 small nuclear RNA (snRNA), whereas SF1, U2AF2 and U2AF1 recognize the branch- site sequence, the polypyrimidine tract and the AG dinucleotide of the 3′ splice site, respectively , leading to the formation of complex E. U2 snRNP is then recruited to the branch site through base pairing interactions with U2 snRNA , leading to complex A , in which the U2 snRNP component SF3B1  facilitates the recognition of the intron branch- site by U2 snRNA. Other protein components of complex A , such as RBM10, RBM39 and RBM15,  modulate 3′ splice site recognition and RBM15 is itself regulated by PRMT1.  The subsequent recruitment of the U4/U6–U5 tri- snRNP complex to form complex B and the remodelling of numerous interactions within complex B  lead to the formation of the catalytically active conformations of the spliceosome (referred to as Bact, B*, C and C* complexes). ClinicalTrials.gov  identifiers (NCTs) for trials of inhibitors of PRMT1 and SF3B1 (GSK3368715  and H3B-8800, respectively) are indicated. R , purine; Y, pyrimidine;  N, any nucleotide.

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SR SF

1/ SR

SF 2

U2AF1U2AF2SF1

GURAGU YNURAY YYY AG

U1 snRNA

Splicing silencer

Exons

Intron

Splicing enhancer

SMN complex

SR, hnRNP, RBM proteins

Sm proteins

Sm site of U1 snRNA

Arginine dimethylation

U2 snRNA

a

b

c

SF3B1

ESS ESE ESSESEISS

PRMT5

ISE

+++ ––

–

• NCT03573310 • NCT02783300 • NCT03614728 • NCT03854227

D3

D1 B

D1 B

E D2

F G

D3

U1 snRNA

PKM

Bcl-x

MCL1

AR

VEGF165

HIF3α

Constitutive splicing

Intron retention/detained intron (when the intron-containing pre-mRNA remains in the nucleus)

Cassette exons

HIF3α4: tumour suppressor

Mutually exclusive exons

Alternative 3′ splice site

Alternative 5′ splice site

HIF3α: regulator of hypoxia- inducible gene expression

MCL-1S: pro-apoptotic

MCL-1L: anti-apoptotic

PKM1: adult isoform

PKM2: embryonic and tumoural isoform

VEGF-165: angiogenic

VEGF-165b: anti-angiogenic

AR-V7: constitutively active; resistance to enzalutamide

AR: sensitivity to enzalutamide

Bcl-x(L): anti-apoptotic

Bcl-x(S): pro-apoptotic

7

1 2 3 1 2 3

1 3

8

3 4

3

CE3

3 4

3 4

2 3

2 3

CE3

8a7 8b

7 8b

8 9 10 11 8 9 11

8 10 11

7

7 8

8

F

D2 D1

G E

B

D3

8a 8b7

2

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splicing alteration detected in bone marrow samples of SF3B1- mutated MDS54. Remarkably, SF3B1 is the target of several families of natural and synthetic compounds with antitumour activity in animal models (see below).

Recognition of splice sites by U1 and U2 snRNPs is assisted and modulated by a number of other factors, some of which have become paradigmatic examples of splicing regulators, including members of various families of RNA- binding proteins, such as arginine– serine- rich (SR), heterogeneous nuclear ribonucleo- protein (hnRNP) and RNA- binding motif (RBM) proteins55. These factors recognize specific regulatory sequences located in introns or exons and facilitate or inhibit, in a position- dependent manner, the recogni- tion of neighbouring splice sites by the core splicing machinery55,56 (Fig. 2a). Synonymous mutations that alter splicing regulatory sequences frequently act as driver mutations in cancer by inducing changes in alternative splicing of pre- mRNAs encoded by proto- oncogenes or tumour suppressor genes, leading either to func- tionally distinct protein isoforms or to frameshifts and nonsense- mediated decay51,57–60. A classic example of such regulation is provided by SRSF2, an SR protein that binds to specific exonic splicing enhancers and stimulates recognition of the flanking splice site by U1 or U2 snRNPs (Fig. 2a). SRSF2 is frequently mutated in a variety of myeloid neoplasms (Table 2), including chronic myelomonocytic leukaemia (CMML); at least one of these mutations modifies the relative affinity of SRSF2 for different binding sites in pre- mRNAs, thus altering the consensus of exonic splicing enhancers that are activated, in turn leading to misregulation of exon inclusion (Fig. 3c) and ultimately to anaemia, leukopenia and morphological dysplasia40,61. Mutations in either SF3B1 or SRSF2 have also been shown to gen- erate isoforms, for example, of MAPKKK7 or caspase 8, that activate nuclear factor- κB signalling18. Another example is presented by RBM10, which promotes skip- ping of exon 9 in pre- mRNA encoding the Notch reg- ulator NUMB, leading to increased expression of the anti- proliferative isoform62 (Fig. 4). The gene encoding RBM10 is frequently mutated in a number of solid tumours, including lung adenocarcinomas, and at least one of the mutant forms fails to regulate the splicing of NUMB, probably by affecting its interactions with other

spliceosomal components (this mutant does not have altered RNA- binding properties) and thereby increasing the expression of the pro- proliferative isoform62–64.

In addition to the major spliceosome pathway, a subset of introns harbouring a distinct configuration of splice- site sequences are excised by the minor spliceo- some. This machinery includes U11 and U12 snRNPs, which have roles equivalent to those of U1 and U2 snRNPs in the recognition of the 5′ and 3′ splice sites, respectively65. ZRSR2 is a component of the U11–U12 di- snRNP complex and has a domain organization sim- ilar to that of U2AF1 and, like U2AF1 itself, is involved in 3′ splice site recognition66. Mutations in ZRSR2 are frequently found in MDS31–33. The mutations are dis- tributed throughout the gene, suggesting that loss of ZRSR2 function contributes to MDS, in contrast to the frequent clustering of mutations in U2AF1, SF3B1 or SRSF2 within specific domains or even residues of these proteins31–33. The presence of ZRSR2 mutations is asso- ciated with a global increase in the retention of U12- type introns67 (Fig. 3d), some of which reside within genes with important functions in cell- cycle control68.

Of relevance, U2AF1, SF3B1 and ZRSR2 have direct roles in 3′ splice site recognition, and SRSF2 can also promote this recognition through its association with exonic enhancers20,21. Whereas all of these proteins are functionally linked to U2 snRNP (or, in the case of ZRSR2, its equivalent in the minor spliceosome), mutations in the genes encoding these factors occur in a mutually exclusive manner (with the exception of SF3B1 and SRSF2 mutations in a minority of patients with MDS)18,31,33. This finding suggests that the muta- tions act on a common pathway and, therefore, once the pathway becomes altered by one mutation, other mutations become redundant and are not positively selected within the tumour. Another prominent feature is that the mutations are heterozygous18, suggesting that at least one functional allele is required for cancer cell survival. Collectively, these observations indicate that a delicate balance is established in cancer cells between the generation of splicing variants favourable for tumour growth and preserving a basic gene- expression process generally required for cellular function. As elaborated on in a later section of this Review, these considera- tions might have important implications for the design of splicing- based cancer therapies. Nevertheless, we emphasize that the mechanisms behind the possible carcinogenic effects of splicing- factor mutations remain to be rigorously proven. One outstanding question is whether these effects rely on alterations in the splic- ing of one, or a few, key target genes or whether they reflect the collective effect of a relatively large number of alterations in splicing, or even whether other mech- anistic explanations exist that are not directly related to splicing. For example, augmented formation of R- loops (RNA–DNA hybrids generated during transcription) has been proposed as a unifying mechanism for the oncogenic role of splicing- factor mutations in MDS69. This phenomenon can lead to replication stress and acti- vation of the ATR pathway, suggesting that ATR inhib- itors can provide therapeutic opportunities for splicing factor- mutated MDS70.

Fig. 2 | Alternative splicing. a | Classical mechanisms of alternative splicing regulation. RNA- binding motif (RBM) proteins, arginine–serine- rich (SR) proteins (including SRSF2)  and heterogeneous nuclear ribonucleoproteins (hnRNPs) bound to exonic or intronic regulatory elements can promote or prevent the recognition of the 5′ splice site by the U1 small nuclear ribonucleoprotein (snRNP) or of the 3′ splice site by SF1, U2AF2, U2AF1 or U2 snRNP, thus affecting splice site choices and therefore alternative splicing decisions. b | The maturation of snRNPs includes the assembly of the Sm proteins B, D1,  D2, D3, E, F and G on specific sequences (Sm sites) of small nuclear RNAs (snRNAs), for  example, U1 snRNA; methylation of Sm B, D1 and D3 by PRMT5 facilitates this process,  which influences the levels of snRNPs and, consequently , alternative splicing decisions. ClinicalTrials.gov identifiers (NCTs) for trials of inhibitors of PRMT5 are indicated.  c | Alternative splicing and effects on cancer. Differential selection of intronic and exonic  sequences as well as differential use of alternative promoters and 3′- end formation sites result is the generation of alternative mRNA isoforms. The figure displays different classes of alternative mRNA- processing events and examples of alternatively spliced products with distinct functions in cancer progression. ESE, exonic splicing enhancer; ESS, exonic  splicing silencer; ISE, intronic splicing enhancer; ISS, intronic splicing silencer.

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After initial recognition of the splice sites by U1 and U2 snRNPs, the tri- snRNP U4–U6–U5 joins the com- plex, triggering remarkable conformational changes and protein exchanges that enable the formation of the cata- lytic core of the spliceosome and thus completion of the splicing reaction71,72. Mutations affecting factors involved in late stages of spliceosome formation and catalytic acti- vation have also been associated with cancer, including PRPF8, the most evolutionarily conserved spliceosomal component and involved in chaperoning the RNA- based catalytic core28. PRPF8 mutations detected in myeloid neoplasms are associated with defects resulting in mis- splicing, and equivalent mutations in yeast cause defects in the proofreading of splicing at the second

catalytic step73. These observations are consistent with results from various studies indicating that the sophis- ticated conformational dynamics of the catalytic core, occurring at late steps of spliceosome assembly and catalysis, can also be the target of splicing regulation21, with consequences for oncogenic transformation.

Altered expression of splicing factors in cancer. Analysis of The Cancer Genome Atlas data across 33 cancer types indicates that putative cancer driver mutations occur in 119 genes encoding core splicing factors and regulators (about 60% of the components of this machinery)33. In addition to mutations, changes in the levels of expres- sion of splicing factors — often associated with genomic

Table 1 | Recurrent splicing- factor mutations in cancer and associated prognosis

Splicing factor

Cancer type Prevalence (%)

Effect on prognosisa

SF3B1 CLL 5–31 (reFs186,187)

Shorter OS45,186, PFS186 and TTT45,188,189 (when clonal: variant allele frequency >12%)190; or no effect on OS190, PFS or ORR187

MDS 7–81 Lower cumulative incidence of disease progression191, longer LFS192,193 (u)194, EFS195 and OS191,193,196–200 (u)194; no effect on OS201

MDS without RS 7 No effect on PFS, AML transformation or OS202

MDS with RS 16–77 Longer LFS203 and OS203,204

Primary myelofibrosis 6.5 No effect on OS205

De novo AML 2.4 Shorter OS and DFS, and lower complete remission rates206

Primary orbital melanoma 36 Tendency for longer OS (in a small cohort of patients)207

Mucosal melanoma 22 Shorter PFS and OS208

Uveal melanoma 15–22 (reFs33,209–211)

Longer EFS and cancer- specific survival210; late onset metastases,  intermediate risk of metastases, but worse DFS in disomy 3  group212; tendency for longer OS (in a small cohort of patients)209

Breast cancer 5–10 Shorter OS (luminal B and progesterone receptor- negative  disease)213

SRSF2 MDS 4–18 Shorter OS214–216; higher risk of AML transformation215

MDS without RS 10 Shorter PFS202

CMML 25–47 No effect on OS214,217

Primary myelofibrosis 8.5–17 Shorter LFS218,219 and OS219–221

Secondary myelofibrosis 1.0–4.2 Shorter OS (u)222

Secondary (myeloproliferative neoplasm) AML

16–18 Shorter OS223,224

De novo AML 5 Shorter OS and lower complete remission rates206

U1 Sonic hedgehog medulloblastoma

8.8 Increased risk of relapse but no effect on OS30

Hepatocellular carcinoma 5.8 No effect on OS29

CLL 3.8 Shorter TTT but no effect on OS29

U2AF1 MDS 7–17 Increased risk of secondary AML225,226 (including in young, low- risk patients227) and shorter OS226–228 (u)216,229; no effect on OS230

MDS without RS 7 Shorter PFS202

Primary myelofibrosis 16 (65% Q157)

Shorter OS (Q157 mutation)231 (u)232; no effect on LFS231

De novo AML 3 Lower complete remission rates, shorter DFS and OS206

Lung adenocarcinoma 3 Shorter PFS63

AML , acute myeloid leukaemia; CLL , chronic lymphocytic leukaemia; CMML , chronic myelomonocytic leukaemia; DFS, disease-  free survival; EFS, event- free survival; LFS, leukaemia- free survival; MDS, myelodysplastic syndrome; OS, overall survival; ORR ,  objective response rate; PFS, progression- free survival; RS, ring sideroblasts; TTT, time to first treatment; (u), studies demonstrating  noted effect on univariate analysis only. aIn comparison with cancers without splicing factor mutations.

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rearrangements — have been associated with oncogene- sis or loss of tumour suppression: 84% of RNA- binding proteins and >70% of splicing factors have been found to be dysregulated at the level of mRNA expression in cancers74–76. Proteomic analyses also indicate a subtype- independent signature of spliceosome dysregulation in CLL77. Furthermore, expression of the SR protein SRSF1 is frequently upregulated in various solid tumours, including breast and lung cancers; SRSF1 is a direct target of MYC and its elevated expression correlates

with increased tumour grade, decreased survival and resistance to chemotherapy78–81. These features are asso- ciated with alterations of alternative splicing in genes controlling cell growth, apoptosis or motility6. Notably, titration of SRSF1 activity using decoy oligonucleotides containing SRSF1- binding sites results in the inhibi- tion of cancer cell growth and apoptosis in vitro and in mouse xenograft models82.

Chromosomal rearrangements resulting in fusions between transcription factors and the splicing regulatory

SR SF

1/ SR

SF 2

GURAGU AGYYYYNURAY

U2AF1 S34F/Y

U2AF1 Q157P/R

Cassette exons

GURAGU AG YYYYNUR YA YNUR YA

SF3B1 WT

SF3B1 K700E

d ZRSR2

c SRSF2

SRSF2 WT

SRSF2 P95H/L/R

Cassette exons

U2AF1 WT

U2AF1 WT

Alternative 3′ splice site

Alternative 3′ splice site

U2AF1 S34F

Alternative polyadenylation

pA pA

U2AF1 WT

U2AF1 S34F

pA

pA pA

Canonical junction

Alternative junction

Canonical junction Alternative junction

Intron retention

SF3B1 WT

SF3B1 K700E

Regulated intron

a U2AF1

C T/

AGT C/

AGC A/

G A/

U2AF1U2AF2 U2AF1U2AF2SF1

U2AF1U2AF2SF1

Intron retention

ZRSR2 WT

ZRSR2 MUT

U12 type intron

b SF3B1

AG SF3B1SF3B1

YNUR YA

ZRSR2

ACAT SF3B1

GURAGU YYY

CCNG GGNG

YNURAY AG

CCNG, GGNG

CCNG GGNG

AG

-CAG -TAG

-AGG -AGA

C T/ G A/

Fig. 3 | Effect of cancer-associated mutations in splicing factors on alternative splice site selection. a | U2AF1 mutations are associated with differential inclusion of cassette exons harbouring specific nucleotide sequences at positions − 3 and + 1 at the preceding 3′ splice site. Processing patterns favoured by the mutations are shown by thicker lines. The U2AF1S34F mutation is also associated with alternative 3′ splice site usage and alternative polyadenylation. b | SF3B1 mutations are associated with activation of cryptic 3′ splice sites linked to usage of a different branch point. SF3B1 mutations are also associated with enhanced splicing of regulated introns. c | SRSF2 mutations are associated with differential inclusion of cassette exons via preferential recognition of specific exonic splicing enhancer sequences. Processing patterns favoured by the mutations are shown by thicker lines. d | ZRSR2 mutations (ZRSR2 MUT) are  associated with retention of U12 introns; these introns are normally removed by the minor spliceosome, of which  ZRSR2 is a component. WT, wild type.

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Table 2 | Splicing- based modulation of drug responses

Gene or protein

Isoforms conferring resistance Isoforms conferring sensitivity Tools to revert resistance or promote sensitivity

Refs

Mechanism: change in splicing isoform expression

AIMP2 AIMP2- DX2: paclitaxel NA BC- DXI01 to reduce the expression  of AIMP2- DX2

233,234

AR AR- V7: enzalutamide and abiraterone NA Downregulation of hnRNP A1 by  siRNA , quercetin; BRD4 inhibitor to  prevent AR- V7–ZFX pathway

235–238

Bax2 NA BaxΔ2: chemotherapeutic agents (adriamycin)

NA 239

BCL2L1 NA Bcl- xS: chemotherapeutic drugs (including cisplatin and 5FdU) and radiation

Antisense to promote Bcl- xS  isoform

240

BCL2L11/BIM Isoform lacking exon 4: TKI (imatinib) NA Antisense to block inclusion of exon 3 and enhance inclusion of exon 4

241

BCR- ABL BCR- ABL135INS: imatinib NA NA 242

BRAF BRAF3-9: vemurafenib NA SSA and meayamycin B to decrease  the expression of BRAF3–9

129

BRCA1 BRCA1- Δ11q: PARP inhibitor and cisplatin NA Pladienolide B to reduce the level of  BRCA1- Δ11q

243

BRCA2 BRCA2ΔE5 + 7: crosslinking agents (mitomycin C)

NA NA 244

Caspase 2 (CASP2)

CASP-2L- Pro: etoposide, camptothecin and death receptor agonists

NA NA 245

Caspase 3 (CASP3)

Caspase-3s: chemotherapeutic drugs (etoposide and methotrexate)

NA siRNA depletion of caspase-3s 246

CD19 CD19 Δex2: anti- CD19 CAR T cells NA NA 120

Cyclin D1 (CCND1)

Cyclin D1b: oestrogen antagonists NA NA 247

EGFR EGFRvIII: radiation, reversible EGFR inhibitor (gefitinib and erlotinib)

EGFRvIII: irreversible EGFR inhibitor (HKI-272)

NA 248,249

ER ERα36: tamoxifen NA NA 250

FPGS Aberrant splice variants: antifolate NA NA 251

HER2 HER2Δ16: trastuzumab NA Quinones to inhibit HER2Δ16 action

252,253

HLA- G HLA- G protein isoforms but HLA- G1: NK cell- mediated lysis

NA NA 254

IG20 MADD and DENN- SV: TRAIL NA shRNA depletion of MADD 255,256

IKZF1 IK6: TKI (imatinib and dasatinib) NA NA 257

MCL1 Mcl-1(L): Bcl- x(L) inhibitor ABT-737 NA Meayamycin B to enhance Mcl-1(S)  expression

97

MET NA Exon 14 skipping: MET inhibitors NA 258

MKNK2 MNK2b: gemcitabine MNK2a: chemotherapeutic drugs (doxorubicin, cisplatin and temozolomide)

Antisense to promote MNK2a isoform

180,259

PIK3CD PI3KCD- S: PI3Kδ inhibitor (idelalisib) NA NA 260

RON NA RONΔ160: wortmannin NA 261

Survivin (BIRC5)

NA Survivin 2B: antitumour activity in taxane- resistant ovarian cancer

NA 262

TP53 ∆133p53: 5- fluorouracil NA siRNA depletion of ∆133p53 263

Mechanism: change in splicing factor expression

SRSF1 NA NA siRNA to decrease SRSF1 expression enhances growth inhibition in response to cisplatin and topotecan

264

5FdU; 5- fluorodeoxyuridine; CAR , chimeric antigen receptor; hnRNP, heterogenous nuclear ribonucleoproteins; NA , not available; NK , natural killer; PARP,  poly(ADP- ribose) polymerase; shRNA , short hairpin RNA; siRNA , short interfering RNA; SSA , spliceostatin A; TKI, tyrosine kinase inhibitor; TRAIL , TNF- related  apoptosis inducing ligand.

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proteins EWS and RBM15 are characteristic of Ewing sarcomas and paediatric acute megakaryocytic leu- kaemia, respectively83,84. Owing to the RNA- binding function of these proteins, these genetic lesions can also alter alternative splicing of pre- mRNAs of multi- ple cancer- relevant genes, including those encoding the RNA–DNA helicases DHX9 and ARID1A, with oncogenic effects85,86. Finally, substantial differences in the expression of snRNAs across cancer samples have been detected, and genes affected by snRNA levels in a breast cancer cell line were found to be preferentially mis- spliced in a cohort of patients with invasive breast ductal carcinomas87, suggesting that changes in snRNA levels can also contribute to cancer progression.

Splicing programmes in oncogenesis Extensive alternative splicing programmes contribute to the regulation of cell differentiation and organ devel- opment. Classical examples include sex determination in Drosophila and cell pluripotency, epithelial– mesenchymal transition or neuronal synapse formation in vertebrates1,2. Disruption of such programmes in cancer cells can contribute to virtually every aspect of tumour progression3–8,88,89 (Fig. 4, Supplementary Table 1).

For example, retention of a number of so- called detained introns, which leads to accumulation of pre- mRNA in the nucleus until a stimulus triggers splicing and conse- quently rapid expression of gene products, in transcripts associated with proliferation, senescence and apopto- sis contributes to neurogenesis. Programmed intron retention is disrupted in glioblastomas and inhibition of PRMT5, which is an arginine N- methyltransferase important for snRNP biogenesis and detained intron splicing, potently inhibits glioblastoma progression in mouse models, including patient- derived xenograft models16 (Fig. 2b). Interestingly, increased splicing of regulated introns is a major effect of SF3B1 mutations in MDS54, while intron retention is a widespread mecha- nism of tumour- suppressor inactivation by single nucleotide mutations in cancer90.

Reversal of splicing patterns of adult or differenti- ated cells is a common theme in cancer. A paradigmatic example involves the switch from adult to embryonic splicing patterns for a pair of mutually exclusive exons encoded by the pyruvate kinase PKM gene, which con- fers a growth advantage to cancer cells by enabling rapid energy generation through aerobic glycolysis (the Warburg effect) and by shunting glucose towards

Proliferation

NUMB-PRR(L) (↑) <--> NUMB-PRR(S) (↓)

Metabolism

PKM1 (↓) <--> PKM2 (↑)

Cell cycle

p120-1A (↑) <--> p120-3A

Apoptosis

Bcl-x(S) (↑) <--> Bcl-x(L) (↓)

Genomic instability DNA damage Mutations

RAC1b (↑) <---> RAC1

Motility

RONΔ165 (↑) <---> RON

EMT

FGFR2 IIIc (↑) <---> FGFR2 IIIb (↓)  

Stress

HIF1αL <---> HIF1αS (↑)

Angiogenesis

VEGF165b (↓) <---> VEGF165 (↑)

Invasion

KLF6-SV1 (↑) <---> KLF6-FL

Signalling

MAP2K4 <---> MAP2K4Δ (↑)

Immune destruction

CEACAM1(L) <---> CEACAM1(S) (↑)  

Fig. 4 | Effect of alternative splicing dysregulation on cancer progression. The diagram illustrates different hallmarks of cancer265 along with examples of alternative splicing events that contribute to the regulation of the different processes of tumorigenesis. Arrows up and down indicate the isoforms contributing the most and the least, respectively , to each process. A non- exhaustive list of additional examples is provided in Supplementary Table 1. EMT, epithelial–mesenchymal transition.

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other biosynthesis processes91 (Figs 2c,4, Supplementary Table 1). In another example of developmental repro- gramming during oncogenesis, repression of an alter- natively spliced exon that is enriched in neurons results in inactivation of the tumour suppressor annexin A7 in glioblastoma precursor cells, enabling lineage- specific activation of EGFR signalling92 (Supplementary Table 1).

A number of apoptosis- regulatory genes gener- ate alternatively spliced protein isoforms with oppo- site activities, which is a physiological programme often subverted in tumours to enable cancer cells to escape from intrinsic programmed cell death as well as radiotherapy- induced or chemotherapy- induced cyto- toxicity93. For example, the use of alternative 5′ splice sites in the Bcl- x pre- mRNA generates the anti- apoptotic Bcl- x(L) and pro- apoptotic Bcl- x(S) protein isoforms94 (Figs 2c,4, Supplementary Table 1). Bcl- x(L) is tran- scriptionally upregulated in many cancers and is asso- ciated with chemoresistance and with RAS- induced expression of stemness regulators and maintenance of a cancer- initiating cell phenotype94,95. In addition, inclusion or skipping of the single internal exon respec- tively generate the anti- apoptotic and pro- apoptotic isoforms of MCL1 (reF.96) (Fig. 2c). The switch towards the pro- apoptotic, exon- skipping isoform of MCL1 mediates the cytotoxic effects of splicing- inhibitory anti- tumour drugs (such as gossypol or obatoclax) as well as (re)sensitizing cancer cells to Bcl- x(L) inhibitors97,98.

Numerous examples exist of the effect that alter- ations in the relative expression of particular mRNA isoforms have on almost every hallmark of tumour progression, including cell invasion, angiogenesis, cell metabolism3–6,99 (Fig. 4, Supplementary Table 1) and, of particular relevance for clinical oncology, responses to anticancer therapy and the development of drug resist- ance (reviewed elsewhere7,100) (Table 2). For example, on the one hand, widespread disruption of splicing- factor expression and alternative splicing has been observed in therapy- resistant secondary AML stem cells and MDS progenitor cells101, which, on the other hand, makes them particularly sensitive to splicing- inhibitory drugs (see below) (Fig. 5a).

Further general perturbations of splicing in cancer, revealing thousands of cancer- specific variants, have been profusely reported. These studies reveal patterns of altered splicing both across cancers (affecting cell cycle, cell adhesion and migration, and the insulin sig- nalling pathway102) as well as in cancer type- specific and subtype- specific profiles (with potential prognos- tic value9,58,103–113) and even evidence of intratumoural splicing heterogeneity114.

The generation of neoantigens through altered splic- ing (including intron retention), intron polyadenylation or the generation of fusion transcripts offers important opportunities for the design of cancer vaccines and for chimeric antigen receptor and T cell receptor- engineered T  cell- based adoptive cell therapies9–11,18,58,110,115,116. Transcriptome and proteome analyses of breast and ovarian cancers indicate that splicing- derived neoanti- gens are at least twice as prevalent as those created by single amino acid mutations9,58,116. Profiling of splicing changes in tumours might therefore provide biomarkers

for the use of immune- checkpoint inhibitors, such as anti- PD-1 and/or anti- cytotoxic T lymphocyte protein 4 (CTLA-4) antibodies10, and possibly for the design of personalized vaccines117,118. For example, expression of an alternatively spliced form of CD20 in B cell lympho- mas generates immunogenic epitopes that are presented by both major histocompatibility complexes I and II and can thus be recognized by T cells, resulting in the kill- ing of lymphoma cells119. Conversely, the selection of pre- existing alternatively spliced variants of CD19 on malignant B cells leads to escape from anti- CD19 chi- meric antigen receptor T cell immunotherapy, which occurs in 10–20% of paediatric patients with B cell acute lymphoblastic leukaemia120 (Fig. 5b, Table 2).

Thus, both the dysregulation of developmental splice site switches and the generation of cancer- specific splic- ing isoforms contribute transcriptome changes relevant for tumour biology, and these changes are linked to alterations in the activity of splicing factors and regula- tors. Cancer cells therefore have characteristic splicing landscapes.

Splicing addiction of cancer cells Several lines of evidence are consistent with the concept that cancer cells are particularly vulnerable to pertur- bations in the splicing process. As a consequence, their survival depends on certain requirements of splicing activity121 (Fig. 5, box 1). First, as mentioned earlier, can- cer driver mutations in splicing factors are heterozygous, and thus one wild- type allele is required to support cell growth18. Furthermore, the mutations are mostly mutu- ally exclusive31,33, suggesting that each mutation, while conferring some advantage for tumour progression, also imposes a splicing- related burden that, when combined with the effects of other mutations, results in the syn- thetic lethality of cancer cells. Indeed, the co- expression of mutant forms of SF3B1 and SRSF2 in haematopoietic progenitors has been shown to cause cell death18 (box 1). By contrast, the co- expression of mutants of SRSF2 and the epigenetic modifier IDH2 result in more profound splicing changes than each mutation alone and have coordinated effects on the epigenome and RNA splic- ing that promote leukaemogenesis122. More generally, in genome- wide screens, spliceosome- related genes (including SF3B1) are the most enriched category of genes for which reductions in copy number compromise cancer cell survival123,124, again suggesting that limited levels of splicing factors or splicing activity in cancer cells can result in lethality upon further stress in the splicing machinery.

Second, general splicing inhibitors exert stronger, or even more selective, effects on cancer cells than on non- transformed cells101,125,126. Furthermore, the effects of drugs targeting either SF3B1, arginine methylases (which are important for proper snRNP assembly) or other splicing factors are particularly deleterious in cells and tumours harbouring mutations in spliceoso- mal components15,17,18,127,128. Another, possibly related, observation is that melanoma cells that acquire resist- ance to vemurafenib through the generation of splicing variants in BRAF, the target of this drug, revert to the normal pattern of splicing upon treatment with splicing

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inhibitors129 (Fig. 5c, Table 2), perhaps owing to the nega- tive selection of cells generating splicing variants because of their particular sensitivity to these drugs. These find- ings might be explained in terms similar to the synthetic lethal interactions discussed above.

Third, MYC oncogene- activated tumour cells are particularly vulnerable to the depletion or mutation of splicing factors or to treatment with splicing inhib- itors130–132. These observations have been interpreted as a consequence of the high demands imposed on the splicing machinery by the widespread activation of gene expression induced by MYC. This situation might be akin to the global modulation of splicing efficiency in yeast depending upon the expression status of the numerous and abundantly expressed ribosomal protein genes133. These observations again underline the con- cept of the lethal accumulation of splicing stresses in cancer cells. Notably, MYC also drives the expression of a number of splicing regulators, including hnRNP

proteins such as PTB3,131, which have been shown to modulate the alternative splicing of transcripts encoding proteins important for tumour progression, including PKM (explaining the Warburg effect) or the tumour suppressor annexin A7 (explaining lineage- specific enhancement of EGFR signalling)3,92. A number of other networks weaving circuits of transcriptional and post- transcriptional regulation relevant in cancer have been reported75,134,135.

A pervading concept is that alterations in the splicing machinery confer advantages to tumour cells (for exam- ple, through the production of abnormal protein iso- forms or changes in normal cellular isoform ratios) at the cost of reducing the efficiency or fidelity of the splicing process. This precarious equilibrium can be broken by further perturbations of splicing activity (for example, by mutations, inhibitors or excess demand), leading to cytotoxicity and thus revealing splicing as a potential Achilles’ heel of cancer cells (Fig. 5, box 1).

H3B-8800

SF3B1 mutations a

Topotecan

SRSF1 downregulation

Spliceostatin

BRAF c

AR

BRAF3–9 (resistant to vemurafenib)

AR-V7 (resistant to enzalutamide and abiraterone)

• CD19 • HER2

• AR • ER

b

Quercetin

Sy nth

eti c l

eth ali

ty Resistance

Reverting resistance

• CD19 Δex2 splice variant (resistant to anti-CD19 CAR T cells)

• HER2Δ16 (resistant to trastuzumab)

Splicing pattern

Cell receptor

Alternatively spliced receptor

Altered SF expression

SF mutation

• Anti-CD19 CAR T cells

• Trastuzumab

• Enzalutamide or abiraterone

• Tamoxifen

• AR-V7 (resistant to enzalutamide and abiraterone)

• ERα36 (resistant to tamoxifen)

Fig. 5 | Influence of alternative splicing on cancer drug vulnerability and resistance. Mutations or changes in  expression of splicing factors can make cancer cells particularly vulnerable to antitumour drugs (synthetic lethality) (part a). Changes in the profile of alternatively spliced isoforms can also generate (part b) or revert resistance to chemotherapy or immunotherapies (part c). The figure provides examples of these three conditions and an extended list is provided in Table 2. AR , androgen receptor; CAR , chimeric antigen receptor; ER , oestrogen receptor; HER , human  epidermal growth factor receptor; SF, splicing factor.

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Splicing- based therapeutics in oncology Substantial preclinical work has identified a variety of small- molecule compounds as well as genetic and other approaches to target the spliceosome or its products with potential therapeutic effects (Fig. 6a, Supplementary Table 2). Herein, we focus on two novel approaches with emerging clinical applicability: small- molecule splicing modulators, several of which are currently being tested in clinical trials, and splicing- modifying antisense oligonucleotides.

Small- molecule splicing modulators. Three chemically distinct families of bacterial fermentation pro ducts and synthetic derivatives, FR901464 (including spliceo- statin A, meayamycin and thailanstatins), pladieno- lide B (including E7107, H3B-8800 and FD-895) and GEX1 (including herboxidiene), each sharing a com- mon pharmacophore, have splicing modulatory and anti proliferative or pro- apoptotic activities in vitro and antitumour effects in various mouse models of cancer (reviewed elsewhere7,136–138) (Fig. 6). These com- pounds bind to the HEAT repeats domain of SF3B1 and lock the protein in an open conformation, preventing the transition to a closed conformation that recog- nizes the branch site and flanking pre- mRNA–snRNA helix139,140 (Fig. 6). The establishment of this closed conformation is essential for the first step of catalysis because it helps to bring together the branch- site adeno- sine and the 5′ splice site. How inhibitors of such a fun- damental step can lead to antitumour effects instead of causing general splicing inhibition and cellular toxicity remains an open question. Available evidence indicates that, at concentrations at which the drugs exert anti- tumour effects, these compounds do not induce wide- spread and massive inhibition of the splicing process, but rather retention of particular introns and changes in

alternative splicing in a number of transcripts of genes with functions connected with the control of cell- cycle progression and apoptosis141–144. Understanding the molecular basis for these selective effects could help in the design of antitumour drugs with high potency and specificity towards key target transcripts, thereby limiting potential adverse effects.

E7107, an SF3B1 inhibitor derived from pladieno- lide B, entered clinical testing in 2007 as a first- in- class molecule in open label, phase I dose- escalation studies involving patients with advanced- stage solid tumours not responding to approved therapies (NCT00499499 and NCT00459823). The compound had previously shown promising effects in reducing the growth of human xenograft tumours in mice, without apparent toxicity145. In one clinical study, 8 of 26 patients (31%) had disease stabilization; however, two patients experi- enced optical neuritis and loss of vision (irreversible in one patient) at the maximum tolerated dose (4.3 mg/m2) or at a sub- maximum dose (3.2 mg/m2), leading to dis- continuation of the study146. Another study enrolled 40 patients and, although disease control for 3 months was observed in eight patients (20%), one patient devel- oped bilateral optical neuritis at a dose of 4.0 mg/m2 (reF.147). Why should a general inhibitor of splicing cause specifically ocular adverse effects? One plausible expla- nation is that eye tissues might be particularly sensi- tive to a reduction in splicing activity, perhaps owing to the high proliferative requirements of retinal tissue and/or alterations in the patterns of splicing of genes essential for retinal cell differentiation. Conceivably, these adverse effects could be mechanistically related to the consequences of mutations in splicing factors or in retina- specific alternative exons that have been associated with ocular pathologies, including retinitis pigmentosa148. These mutations affect genes encoding factors such as PRP8, PRP31 or PRP3, which have been implicated in fundamental functions required for the activation of splicing catalysis149. Contrary to expecta- tion, the mutations do not compromise overall organism viability but rather cause ocular pathologies, once again highlighting the functional plasticity of the core splicing machinery150.

H3B-8800 is another pladienolide B- derived SF3B1 inhibitor that has been developed with the specific aim of targeting cancer cells harbouring splicing- factor mutations151. H3B-8800 potently inhibits splicing but preferentially kills epithelial and haematological cancer cells harbouring spliceosomal mutations, possibly by inducing the retention of GC- rich introns in pre- mRNAs encoding other splicing factors151; thus, in this context, H3B-8800 might have synergistic effects on splicing activity that compromise cancer cell viability according to the synthetic lethality paradigm. H3B-8800 entered phase I clinical trials in 2016, with a focus on patients with MDS, AML and CMML (NCT02841540). Initial results revealed dose- dependent target engagement, a predictable pharmacokinetics profile and a favourable safety profile, even with prolonged dosing. Although objective therapeutic responses have not been achieved to date, 14% of patients had reduced requirements for red blood cell or platelet transfusions152.

Box 1 | Splicing-factor mutations and the activity of splicing- based drugs

Sensitization • sF3B1 K700e mutation causes sensitivity to e7107 (reF.15)

• sRsF2 P95H mutation causes sensitivity to e7107 (reF.266)

• u2AF1 s34F mutation causes sensitivity to sudemycins and e7107 (reF.17)

• sF3B1 K700e and sFsR2 P95H mutations cause sensitivity to H3B-8800 (reF.151)

• sF3B1 K700e, sF3B1 K666N, sF3B1 H662Q, sRsF2 P95H and u2AF1 s34F mutations cause sensitivity to e7820 (reF.127)

• sF3B1 Y765C, sF3B1 K700e, u2AF1 s34F, u2AF1 Q157P, sRsF2 P95H and sRsF2 P95l mutations cause sensitivity to inhibition of PRmts128

• several sF3B1 mutations cause sensitivity to sudemycin and ibrutinib, and various mutations in components for the RNA- processing machinery cause sensitivity to sudemycins267

Synthetic lethality • sF3B1 K700e and sRsF2 P95H mutations are synthetically lethal18

• mYC activation is synthetically lethal with inhibition of core components of the spliceosome (by depletion of BuD31, sF3B1, u2AF1 and eFtDu2) and with treatment with sudemycin D6 (reFs130,268)

Resistance • sF3B1 R1074H, sF3B1 v1078A, sF3B1 v1078I and PHF5A Y36C mutations confer

different levels of resistance to H3B-8800, herboxidiene and pladienolide143

• sF3B1 R1074H mutation confers resistance to pladienolide and e7107 (reF.269)

• sF3B1 R1074H and PHF5A Y36C mutations confer resistance to H3B-8800 (reF.151)

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An entirely different mechanism operates in the case of the arylsulfonamides E7820, indisulam, tasi- sulam and chloroquinoxaline, which are experimental anticancer drugs that promote recruitment of the splic- ing factor RBM39 to the E3 ligase substrate receptor

DCAF15, leading to RBM39 ubiquitylation and degra- dation153,154 (Supplementary Table 2). RBM39 is related to the 3′ splice site- recognizing protein U2AF2 and was originally described as a cofactor of steroid- mediated transcriptional and post- transcriptional responses155.

Cellular signalling

AKT ERK

Spliceosome

snRNPs

Splicing

, WNT, JNK

Phosphorylation

Splice-site switch

SF3B1 targeting

Nucleus

Cytoplasm

Kinase inhibitors

Inhibitors of epigenetic regulators SF/RBM

inhibitors

SRPK CLK

Core spliceosome inhibitors

Splice-site switch

mRNA isoform- specific targeting

Inhibition of protein isoforms

ISE ESEISS ESS

Translation

Splicing enhancer Steps targeted

Antisense oligonucleotides

Degraded splicing factor

Splicing silencer

Exons

SR, hnRNP or RBM proteins

Antibody

Protein

SF/RBM activity, cellular localization, degradation and expression

Target Inhibitor Clinical trial NCT03901469 NCT02705469

NCT02711956 MK-8628 NCT02259114

BET ZEN003694

Target Inhibitor Clinical trial

SF3B1 H3B-8800 NCT02841540 PRMT1 GSK3368715 NCT03666988

JNJ-6461978 NCT03573310 NCT03614728 NCT02783300

PF06939999 NCT03854227

PRMT5 GSK3326595

P

e13 e14b

e13 e14a e14b

Mnk2b

Mnk2a

Mnk2

e14a e14be13

• Resensitization to chemotherapy

• Inhibition of glioblastoma development

U2 snRNP

Intron

U2 snRNP

In the presence of drug

In the absence of drug

YYY AG

SF3B1 PHF5A

YNUR YA

YYY AGYNUR Y A

YNURY

SF3B1

PHF5A

U1 U4

U2U5

U6

Pro-oncogenic

P

Fig. 6 | Approaches to modulate cancer-relevant splicing events. Tools have been implemented to induce splicing changes at various levels, including modulators of signalling pathways regulating RNA- binding motif (RBM) proteins and splicing factors (SFs) involved in splicing; compounds  directly targeting spliceosomal components, including antitumour drugs that bind in the interface between SF3B1 and PHF5A components of the U2  small nuclear ribonucleoprotein (snRNP) (the top right inset illustrates how U2 snRNP can be redirected to a decoy , unproductive branch site in the presence of the drug); drugs that target protein–protein or protein–RNA 

interactions that affect splice- site accessibility; antisense oligonucleotides  that induce switches in splice- site utilization (illustrated in the bottom right inset) or that target specific mRNA isoforms; and isoform- specific  antibodies that inhibit protein function. An extended list of examples is provided in Supplementary Table 2. ClinicalTrials.gov identifiers (NCTs) for trials of small- molecule modulators of these steps are indicated as well as their targets. ESE, exonic splicing enhancer; ESS, exonic splicing silencer;  hnRNP, heterogeneous nuclear ribonucleoprotein; ISE, intronic splicing  enhancer; ISS, intronic splicing silencer; SR , arginine–serine- rich.

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Degradation of RBM39 results in altered cassette exon inclusion and/or skipping and thus in cytotoxicity in a number of cancer cell lines153. Interestingly, haemato- poietic or lymphoid malignancies harbouring splicing factor mutations are particularly sensitive to indisulam cytotoxity127, again supporting the idea of synthetic lethality between different splicing deficiencies. RBM39 regulates a splicing programme that is crucial for the survival of AML cells, owing to the involvement of key leukaemogenesis genes, including HOXA9, BMI1 and GATA2 (reF.127). A phase II study in patients with relapsed and/or refractory AML or high- risk MDS revealed the beneficial effects of indisulam adminis- tration along with chemotherapy in patients stratified according to DCAF15 expression, with an estimated 1- year overall survival of 51% in responders com- pared with 8% in non- responders156. The more general implication of these observations is that, on the basis of structural insights into the RBM39 RNA recognition motif domain and DCAF15 complexes in the presence of existing arylsulfonamide drugs157–160, other arylsulfo- namide derivatives might be generated to specifically target other RNA recognition motif domain- containing RNA- binding proteins.

As alluded to earlier, EPZ015666 (also known as GSK3235025), an inhibitor of the arginine N- methyltransferase PRMT5, prevents splicing of cer- tain introns and reduces the growth of patient- derived glioblastoma xenografts16, as well as mantle- cell lym- phoma xenografts161. Other inhibitors of PRMT5 and of the related enzyme PRMT1 are in phase I clinical trials involving patients with a variety of advanced- stage solid tumours and haematological malignan- cies (NCT03573310, NCT02783300, NCT03614728, NCT03666988 and NCT03854227) (Fig. 6). PRMT5 catalyses symmetric dimethylation of the terminal amino group of the side chain of arginine, whereas PRMT1 catalyses asymmetric dimethylation162. Inhibitors of these enzymes display synergistic effects163, consistent with each having distinct sub strates within the splice- osome. Indeed, PRMT5 modifies snRNP- associated Sm proteins, and PRMT1 modifies the splicing regula- tor RBM15 (reFs164,165). Whether the antitumour effects of these inhibitors occur through these targets, other targets in the spliceosome or proteins of other cellular machiner- ies (such as those involved in transcription or chromatin remodelling) remains to be formally proven.

A number of other drugs targeting splicing factors have shown encouraging preclinical effects in mouse models of cancer. These drugs include inhibitors of SRPK and CLK protein kinases that phosphorylate SR proteins and thereby inhibit angiogenesis by inducing changes in the alternative splicing of VEGF166,167 (Fig. 6). Other splicing inhibitors targeting a variety of spliceo- somal components also reduce cancer cell proliferation in vitro168–171, but their effects in animal models of cancer are not yet known.

Antisense oligonucleotides. Antisense oligonucleotides provide an entirely different therapeutic approach to splicing modulation (Fig. 6, Supplementary Table 2). These molecules bind to pre- mRNA sequences (splice

site or splicing regulatory motifs), thereby preventing their recognition by spliceosomal or regulatory factors and causing splice site switching172. The main advantage of this approach is the selectivity provided by the recog- nition of specific target sequences; the main difficulty is delivering oligonucleotides to particular tissues, as they often accumulate in the liver and kidney172. The use of nusinersen in the treatment of patients with spinal muscular atrophy is paradigmatic of this therapeutic approach. Nusinersen is an antisense oligo nucleotide directed against an intronic silencer in SMN2, which, by enhancing the inclusion of exon 7 in SMN2 mRNAs, restores the levels of functional SMN proteins in patients with spinal muscular atrophy who have inactivating mutations in SMN1 (reFs173,174). Maintenance treatments (consisting of quarterly intrathecal lumbar injections of 12 mg nusinersen) have remarkable therapeutic effects that have improved the quality of life and life expectancy in these patients175. Interestingly, orally bioavailable small- molecule modulators that enhance the recogni- tion of the 5′ splice site of SMN2 exon 7 with considera- ble specificity are under development176; branaplam and risdiplam are currently in clinical trials (NCT02268552, NCT02908685, NCT03779334, NCT03988907, NCT03920865, NCT02913482 and NCT03032172), although — as was the case for E7107 — eye toxicity has been reported in one of these trials.

Preclinical work suggests that antisense oligonucleo- tides might also have therapeutic value in oncology (reviewed elsewhere7) (Supplementary Table 2). For example, antisense oligonucleotide- mediated redirection of Bcl- x pre- mRNA splicing in favour of the Bcl- x(S) isoform induces apoptosis in breast or prostate cancer cells177,178. In mouse models, the administration of such oligonucleotides using lipid nanoparticles resulted in the modification of Bcl- x pre- mRNA splicing in lung metastases of melanoma xenografts and reduced the tumour burden179. Another example is provided by the antisense oligonucleotide- mediated switch between alternatively spliced isoforms of the kinase MKNK2 with antagonistic oncogenic or tumour- suppressor proper- ties (Fig. 6); use of this therapeutic approach activates the p38 MAPK- signalling pathway and inhibits the onco- genicity of glioblastoma cells, re- sensitizing these cells to chemotherapy, and inhibits the growth of a glioblastoma cell line in a mouse xenograft model180. As mentioned earlier, antisense oligonucleotides can also be used as decoys to titrate away tumour- promoting RNA- binding factors as illustrated by the induction of apoptosis and inhibition of cancer growth with antisense oligonucle- otides containing binding sites for transcripts of the SRSF1 oncogene82.

Conclusions In this Review, we have highlighted the functional effect of perturbations in the splicing process and of mutations or changes in the activity of splicing factors in cancer. In addition to changes in other steps of RNA processing, including 3′ end formation, RNA editing and RNA mod- ifications that also contribute to cancer biology (reviewed elsewhere7,181,182), the monitoring of splicing alterations can provide abundant and effective biomarkers for use in

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the diagnosis, prognostication, therapy and monitoring of patients with cancer. For this promise to be realized, an urgent need exists for the development of highly sen- sitive, specific and cost- effective assays for the detection of alternatively spliced isoforms, including solutions to the challenges of detecting transcript variants in single cells and of implementing cost- effective next- generation sequencing methods in the clinic183. Systems biology approaches are also needed to provide an integrated view of the gene- expression landscape of cancers and identify key regulatory networks and hubs75,134,184 that might present particularly important therapeutic vul- nerabilities. However, crucial to our understanding is whether reversal of a pathogenic state can be reached through targeting a small number of key transcriptome changes or whether therapies will need to revert wider regulatory programmes. Related to this concept is the open question of whether cancer- relevant mutations in splicing factors act through unifying mechanisms and common targets69, which could help to identify general therapeutic approaches.

Given the relatively limited conservation of mecha- nisms regulating alternative splicing between mouse and humans, the development of humanized animal models

as well as human organoids will be essential to model cancer initiation and progression as well as to test novel, splicing- based therapeutic approaches. Developing more effective therapeutic agents, including small molecules that target alternative splicing events controlling cell pro- liferation, apoptosis or other key hallmarks with high specificity, is another key challenge. The combination of structural and transcriptomic approaches should help to reveal the molecular basis of the activities of these mol- ecules on spliceosome function and to rationalize their design. The identification of small- molecule modulators of RNA structure able to induce splice site- selection switches is another priority area for future studies185. Further research into chemical modifications that improve the stability, specificity and delivery of antisense oligonucleotides as well as a better understanding of the mechanisms of their cellular uptake will help advance these agents to clinical stages of testing. Conceivably, future personalized therapies will exploit the specificity and versatility of such agents through the use of ‘cock- tails’ of antisense oligonucleotides targeting the specific profile of splicing alterations of each tumour or patient.

Published online 17 April 2020

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Acknowledgements The authors thank Dolors Colomer, Armando López- Guillermo and members of their laboratory for critical reading of the manuscript, and Adrian Krainer, Omar Abdel- Wahab and Rotem Karni for their constructive critique during the review process. I.L.- O. is a recipient of a Severo Ochoa PhD4MD Program Fellowship. The work of the authors is supported by the European Research Council, Worldwide Cancer Research, the Spanish Ministry of Economy and Competitiveness, the Agència de Gestió d’Ajuts Universitaris i de Recerca, and the Centre of Excellence Severo Ochoa Award (to the Centre for Genomic Regulation of the Barcelona Institute of Science and Technology).

Author contributions All authors made a substantial contribution to all aspects of the manuscript.

Competing interests J.V. is a member of the Scientific Advisory Boards of Remix Therapeutics and Stoke Therapeutics. The other authors declare no competing interests.

Reviewer information Nature Reviews Clinical Oncology thanks Seishi Ogawa, Rotem Karni and another, anonymous, reviewer for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information Supplementary information is available for this paper at https://doi.org/10.1038/s41571-020-0350- x.

RelaTed linkS ClinicalTrials.gov: https://www.clinicaltrials.gov

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