Alternative mRNA splicing in cancer immunother.pdf

The advent of immunotherapy has revolutionized the treatment of many forms of cancer.It is now well estab-lished that Tcells have the ability to reject tumours upon binding to antigenic peptides,derived from endogenous cellular proteins or exogenous viral proteins,presented by the major histocompatibility complex(MHC)on the surface of tumour cells.Several promising immuno-therapeutic anticancer approaches,such as therapeutic vaccines and Tcell receptor engineered Tcells(TCR-T cells)for adoptive cell therapy,rely on the identification of suitable target antigens1.Historically,the focus has been on three classes of tumour antigens:tumour-specific somatic non-synonymous mutation-derived neoantigens;cancer germline antigens;and antigens derived from viral genes that are expressed by virally infected tumour cells(for example,E6/E7 from human papilloma virus)2.Clinical studies have revealed remarkable outcomes both for TCR-T cell therapy targeting cancer germline anti-gens and for neoantigen-based vaccines1.Forinstance,TCR-T cells targeting NY-ESO-1,a tumour-specific shared germline antigen,have been shown to mediate sustained antigen-specific antitumour effects in patients with multiple myeloma,as well as several other can-cer types35.Further,personalized vaccines targeting mutation-derived neoantigens have been shown to elicit strong neoepitope-specific Tcell responses in patients with melanoma(an immunologically hot tumour with a high tumour mutational burden(TMB)and glioblas-toma(an immunologically cold tumour with a relatively low TMB)610.Despite the unprecedented durable response rates observed with cancer immunotherapies in some patients,one of the major obstacles for the broader appli-cability of such therapies is the lack of currently known targetabletumour-specific antigens(TSAs)for many cancer types1.The selection of appropriate antigens is critical to ensure the safety and efficacy of immuno-therapy.Melanoma-associated antigen 3(MAGE-A3)and melanoma antigen recognized by Tcells 1(MART-1)have been two leading target antigens for TCR-T cell-based cancer therapies due to their frequent expression in several tumour types and their restricted/low expres-sion in normal tissues.However,several clinical cases with unexpected severe off-target toxicities have been reported1113.For example,in patients with melanoma,immunotherapy with Tcells engineered with a high-affinity Tcell receptor(F5-TCR)targeting MART-1 showed higher clinical efficacy compared with treatment with Tcells engineered with a relatively low-affinity TCR(F4-TCR)but also caused uveitis,vitiligo and hearing loss due to MART-1 expression on melanocytes in the eye,skin and middle ear14.TCR-T cells and vaccines that target neoantigens may enable safer and more durable antitumour effects15,although mutational loads vary widely across different tumour types and identifying suitable targets remains a problem16.In focusing on somatic mutation-derived neoanti-gens in tumour cells,possible neoepitopes derived from mRNA processing events are often overlooked.With respect to cancer,the most well-studied mRNA process-ing event,and the focus of this review,is mRNA splicing.Nonetheless,processing events such as mRNA poly-adenylation and mRNA editing have also been shown to play a role in tumour development and can result in an increased immunotherapy target space(Box1).The advent of next-generation sequencing technologies has allowed for a wealth of transcriptomic data to be generated.Such data have helped to illuminate the widespread nature of alternative processing in cancer17 and have the potential to be used to identify neoepitopes derived from tumour-specific mRNA processing events,thereby expanding the repertoire of suitable targets for Major histocompatibility complex(MHC).A set of genes that code for cell surface proteins(most notably the MHC class I and class II glycoproteins)that are responsible for presenting antigens to lymphocytes.Adoptive cell therapyA type of immunotherapy approach that uses antigen-specific Tcells to treat patients with chronic viral infections or various malignancies.Non-synonymous mutationA nucleotide mutation that changes the amino acid sequence of a protein.Alternative mRNA splicing in cancer immunotherapyLukeFrankiw1,DavidBaltimore 1*and GuidengLi 2,3*Abstract|Immunotherapies are yielding effective treatments for several previously untreatable cancers.Still,the identification of suitable antigens specific to the tumour that can be targets for cancer vaccines and Tcell therapies is a challenge.Alternative processing of mRNA,a phenomenon that has been shown to alter the proteomic diversity of many cancers,may offer the potential of a broadened target space.Here,we discuss the promise of analysing mRNA processing events in cancer cells,with an emphasis on mRNA splicing,for the identification of potential new targets for cancer immunotherapy.Further,we highlight the challenges that must be overcome for this new avenue to have clinical applicability.1Division of Biology and Biological Engineering,California Institute of Technology,Pasadena,CA,USA.2Center of Systems Medicine,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing,China.3Suzhou Institute of Systems Medicine,Suzhou,China.*e-mail:baltimocaltech.edu;https:/doi.org/10.1038/s41577-019-0195-7REVIEwSNature reviews|ImmunologyBox 1|Beyond RnA splicing:non-canonical neoepitopesrNa splicing is just one of the processing steps that occurs in a pre-messenger rNa transcript(pre-mrNa)before the formation of a mature transcript.although splicing is the best-studied process with respect to cancer,dysregulation of other processing steps is known to occur.in particular,polyadenylation(pa)and RNA editing have the potential to alter the proteome of a cancer cell and,thus,like rNa splicing,the identification of such events might broaden the immunogenic target space.Polyadenylation involves the cleavage and addition of a stretch of adenosines,termed the poly(a)tail,to the 3 end of the vast majority of eukaryotic mrNas.Polyadenylation is complicated by the fact that the majority of human genes contain more than one pa site and that mrNa transcripts are frequently alternatively polyadenylated156.the majority of alternative polyadenylation(aPa)sites are in the 3 untranslated region(utr)and can alter the stability,localization and translation of a given transcript157.However,there are many aPa events that are located in intronic regions upstream of the last exon which act to generate either non-coding transcripts or transcripts with truncated coding regions158.the classic intronic polyadenylation(iPa)event occurs in the igM heavy chain mrNa wherein,upon activation,a proximal iPa site is used,resulting in a shift to the secreted form of the antibody from the membrane-bound form159(see the figure,part a).the increased transcriptome complexity created through aPa carries with it the risk of gene dysregulation,and it is perhaps no surprise that aPa has been associated with tumorigenesis160163.recent work has focused on iPa,which has been shown to be a common mechanism of tumour-suppressor inactivation in chronic lymphocytic leukaemia162.Further,it was shown that the kinase CDK12,which is a key regulator of transcription elongation,also has a role in regulating genes involved in DNa repair by suppressing iPa163.in CDK12 mutant tumours,loss of suppression of iPa leads to impaired production of full-length(FL)gene products for several genes involved in DNa repair.with respect to immunotherapy,the identification of iPa events is exciting due to the potential discovery of tumour-specific peptides.when cancer-specific iPa events occur in the coding region,sequences downstream of the nearest 5 splice site(ss)and upstream of the new polyadenylation site will be translated,creating peptides that might be presented on major histocompatibility complex(MHC)molecules and recognized by the immune system.these iPa events commonly occur in genes that are important for disease progression162,163,which makes such peptides excellent immunotherapy targets.However,peptides derived from tumour-specific iPa events that bind to MHC molecules have yet to be identified and it is uncertain how immunogenic such peptides would be.as more data from methods such as 3 seq164,which is used to identify and quantify polyadenylation site usage,become available,we will better understand the extent to which iPa events can alter the immunotherapeutic target space.another step in processing of pre-mrNa is rNa editing(see the figure,part b).the most common form of rNa editing involves the conversion of adenosine to inosine(a-to-i),a process catalysed by the adenosine deaminases acting on rNa(aDars)165,166.Because most cellular machinery interprets inosine as guanine167,a-to-i editing can alter the amino acid sequence coded by a given transcript.Like splicing and polyadenylation,rNa editing has been shown to be dysregulated in many types of cancer168171,and it was recently reported that peptides derived from over-edited transcripts are presented by MHC molecules in a subset of tumour samples172.Most prevalent in ovarian cancer,breast cancer and melanoma,it was further shown that effector CD8+Tcells specific for such peptides were present in the respective tumours,indicating that the peptides are indeed immunogenic.it is important to note that these peptides cannot be considered tumour specific.as editing still occurs in healthy tissue,and peptides derived from editing events are present on MHC molecules of healthy tissues,these over-edited peptides can be classified as tumour-associated shared self-antigens.as such,the therapeutic window that offers efficacy with limited toxic effects would first need to be defined for potential therapies targeted at such peptides.extending the focus beyond rNa processing,recent work has uncovered several other non-canonical neoantigens that promise to greatly expand the immunotherapy target space.For example,a complete response to anti-programmed cell death 1(PD-1)therapy was reported to have been mediated by an immune response targeted at an immunogenic peptide derived from a gene fusion event173.the authors suggest that the immunodominant epitope underlying regression of the tumour is probably derived from a DEKAFF2 fusion expressed in tumour cells.expanding the analysis to 30 different cancer types revealed that 24%of cancers that expressed fusion proteins had a fusion-derived neoepitope predicted to bind to patient-specific MHCs173.Finally,in a cohort of patients with melanoma who responded to anti-PD-1 therapy,it was shown that predicted fusion neoantigens were eliminated,likely due to immune evasion173.this implicates gene fusions as a source of immunogenic neoantigens that could serve as a predictive biomarker for checkpoint inhibitor response.DownstreampA siteIPA siteabIPA isoformFL isoformAAAAAAPotential IPA-derivedneoepitopesAAAAAA5 SS3 SS5 SS3 SSAntigen processingand presentationTranslationRNA editingADARAINeoantigensNewly formed antigens that have not been previously recognized by the immune system.Cancer germline antigensAntigens that are normally exclusively expressed in germline cells but have aberrant expression in tumours,such as NY-ESo-1.Tumour mutational burden(TMB).Also referred to as the tumour mutational load,this is a measurement of mutations carried by tumour tissue taken from a patient.Tumour-specific antigens(TSAs).Antigens that are exclusively presented by tumour cells but not by any other cells.RNA editingA molecular process resulting in alteration of the RNA sequence before translating to protein.Alternative polyadenylation(APA).An RNA-processing event that generates distinct 3 termini on mRNAs and other RNA polymerase II for cancers such as B cell acute lymphoblastic leukaemia,which has a low preva-lence of somatic mutations and copy number variations but displays widespread mRNA splicing aberrations,the expanded target space could lead to the develop-ment of efficient immunotherapies18.Further,because the somatic mutation-derived neoepitope load has been shown to positively correlate with response to immune checkpoint blockade therapy in many cancer types1924,uncovering processing-derived neoepitopes might offer clinical utility as a predictive biomarker.In this Review,we explore emerging evidence suggesting that mRNA processing-derived neoantigens can be suitable TSAs for cancer immunotherapy and discuss the major challenges that lie ahead.Alternative mRNA splicing in cancerThe processing of pre-mRNA transcripts(pre-mRNAs)represents an essential step in the ultimate function-ality of a gene product.The vast majority of human genes contain multiple exons,with adjoining intronic sequences that need to be spliced from a transcribed pre-mRNA to form the mature mRNA.Alternative splicing,a process by which a single pre-mRNA can be variably spliced into unique mature transcripts,can contribute to transcriptomic and proteomic diversity2527(FIg.1a).This process is tightly regulated in different tissues,cell types and differentiation stages2833.Of note,one specific alternative splicing event,intron retention,can be derived from a regulated process affecting select junctions,or from a lack of processing throughout an entire gene30,32.Although the association between dysregulated splicing events with specific cancers has been known for many years34,the recent transcriptomic characterization of cancers has led to the finding that such events are much more frequent than previously predicted18,3539.Although our understanding of the extent to which specific alternative splicing events drive tumorigenesis is still evolving,there are several factors that help to explain the widespread dysregulation of splicing in cancer.First and foremost,a surprising finding from the genomic characterization of different cancers was the recur-rent somatic mutations found in genes encoding core spliceosome components as well as in trans-acting splic-ing factors that are essential to the regulation of alter-native splicing40,41.Frequent mutations in components of the spliceosome were initially detected in patients with myelodysplastic syndrome4244 and chronic lymphocytic leukaemia45,46 but later also found in a wide variety of solid tumours such as breast cancers4749,pancreatic ductal adenocarcinoma50,uveal melanoma51,52 and lung adenocarcinoma53.Mutations that affect spliceosomal components can alter splicing efficiency and splice-site selection.For example,a recent transcriptomic analysis of chronic lymphocytic leukaemia cells revealed that mutations in the small ribonuclear protein(RNP)U2 Alternative splicingA regulated process during gene expression that results in a single gene coding for multiple proteins.Intron retentionA form of alternative splicing that results in inclusion of introns in the final protein product.SpliceosomeThe multi-megadalton r