Long-read sequencing is employed to generate a high-resolution, isoform-level transcriptomic atlas from a cohort of 14 NPC tumor samples and four immortalized nasopharyngeal epithelial cell lines, and a new class of noncanonical neoantigens with significant potential for developing personalized cancer vaccines for NPC are identified.
Abstract
The widespread transcriptomic diversity driven by alternative splicing (AS) contributes to all hallmarks of cancer and represents a critical source of neoantigens for personalized immunotherapy. However, unlike other major malignancies, the full repertoire of AS in nasopharyngeal carcinoma (NPC) remains underexplored. Here, we employ long-read sequencing (LR-seq) to generate a high-resolution, isoform-level transcriptomic atlas from a cohort of 14 NPC tumor samples and four immortalized nasopharyngeal epithelial cell lines. We identify a substantial number of full-length novel transcripts (22,687; ∼44.38%), which reveal diverse splicing patterns and previously unannotated splicing events. By integrating short-read RNA-seq data to quantify isoform expression, we discover a subset of novel transcripts that are differentially expressed between tumor samples and immortalized nasopharyngeal epithelial cell lines. Furthermore, LR-seq enables precise identification of chimeric readthrough fusion transcripts, such as CLDN15-FIS1 and FOXRED2-TXN2. Finally, we develop a computational framework, tumor-specific splicing neoantigen detection (TS-SNAD), to predict neoantigens originating from novel exon–exon junctions (neojunctions) in tumor-specific novel transcripts. Using this framework, we identify neojunction-derived neoantigens and experimentally validate the immunogenicity of selected HLA-B*40:01-restricted neoantigens. These neojunction-derived peptides constitute a new class of noncanonical neoantigens with significant potential for developing personalized cancer vaccines for NPC.
RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in induced pluripotent stem cell-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that autism risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between alternative transcript processing events, including transcription start sites, exon splicing, and polyadenylation. Finally, our atlas enables variant reinterpretation in autism, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment. Alternative splicing expands neuronal diversity, but many brain RNA isoforms remain uncharacterized. Here, the authors generate a high‑resolution proteogenomic atlas of human neuron development, uncovering thousands of novel isoforms and revealing dynamic splicing changes in autism risk genes.
N. Xu, Katherine M. Rynard, E. Radley et al.· Nature Communications· 0 citations
BACKGROUND
Recurrent IDH-mutant gliomas frequently acquire increased radioresistance, leading to poorer outcomes. Their underlying mechanisms, however, remain largely unknown. We hypothesize that dysregulated RNA alternative splicing (AS) during IDH-mutant glioma recurrence contributes to the enhanced radioresistance by influencing critical cellular pathways.
METHODS
RNA sequencing of paired primary and recurrent IDH-mutant gliomas were analyzed to identify recurrence-associated AS events. Functional effects were assessed in patient-derived glioma stem cells using RNA interference and CRISPR-dCas13-mediated isoform switching. Candidate upstream RNA-binding proteins and antisense oligonucleotide (ASO)-based therapeutic strategies were evaluated in vitro and in vivo.
RESULTS
We identified differentially spliced MutS homolog 5 (MSH5) isoforms between primary and recurrent IDH-mutant gliomas. Primary gliomas predominantly expressed an exon 11/12-skipped MSH5 transcript, whereas recurrent tumors largely retained the full-length isoform. Exon 11/12 skipping introduced a premature termination codon, leading to nonsense-mediated decay and diminished MSH5 expression in primary tumors. Further analyses identified elongation factor Tu GTP binding domain containing 2 (EFTUD2) as an upstream splicing regulator that was upregulated in recurrent tumors and promoted exon 11/12 inclusion, thereby maintaining MSH5 expression and enhancing the repair of radiation-induced DNA double-strand breaks. Inducing MSH5 exon 11/12 skipping with CRISPR-dCas13 or inhibiting EFTUD2 with ASOs reduced MSH5 expression, impaired DNA repair, and sensitized recurrent IDH-mutant glioma to radiotherapy in vitro and in vivo.
CONCLUSIONS
These findings identify an EFTUD2-MSH5 splicing axis that contributes to radioresistance in recurrent IDH-mutant glioma. Therapeutic disruption of this splicing program may represent a strategy to enhance the radiation response in recurrent IDH-mutant glioma.
Aberrant pre-mRNA splicing in cancer generates protein sequences that are rare or absent in normal tissues, creating a rich source of tumor-specific neoantigens for immunotherapy. These splicing-derived neoantigens arise through diverse mechanisms, including recurrent somatic mutations in core spliceosome components (SF3B1, SRSF2, U2AF1, and ZRSR2), epigenetic derepression of transposable elements that give rise to chimeric exon-TE junctions, and coordinated dysregulation of splicing regulatory networks in cancers lacking spliceosome coding mutations. These processes produce two major classes of immunotherapeutic targets: 1) MHC class I-restricted neopeptides that can be recognized by T-cell-based therapies, and 2) extracellular neoepitopes (ExNeoEpitopes) within transmembrane proteins that are accessible to HLA-independent antibody-based modalities, including monoclonal antibodies (mAbs), bispecific engagers (BiTEs), antibody-drug conjugates (ADCs), and chimeric antigen receptor (CAR)-T or CAR-NK cells. Despite their strong immunogenic potential, effective therapeutic exploitation requires overcoming key immunological barriers, including T-cell exhaustion, impaired antigen presentation through MHC-I downregulation, and suppression within the tumor microenvironment. Recent advances in computational neoantigen prediction, immunopeptidomics, surface proteomics, long-read and single-cell isoform sequencing, and AI-guided therapeutic design are enabling more systematic discovery and validation of splicing-derived targets. This review integrates current understanding of the biological origins, immunological barriers, target classes of splicing neoantigens, and the technologies that enable their advancement in cancer immunotherapy.
Mu-Tian Tang, H. L. Grimes, Nathan Salomonis· Frontiers in Immunology· 0 citations
Circular RNAs (circRNAs) are an emerging class of RNAs with biomarker potential, but their detection in liquid biopsies is challenging due to low abundance. We developed Ouro-seq, a novel long-read sequencing protocol optimized for full-length circRNA recovery. Applied to urine, cervico-vaginal self-samples from cervical cancer patients, and plasma from lung cancer patients and controls, Ouro-seq recovered 2-5 times more and substantially longer circRNA molecules than conventional methods. Plasma contained predominantly exonic circRNAs, while urine and cervico-vaginal samples were dominated by previously undercharacterized intergenic circRNAs. We also identified extensive alternative circularization and splicing events. Functional analysis revealed distinct specialization patterns: exonic circRNAs showed enhanced miRNA sponging potential, while circRNAs from unplaced genomic scaffolds demonstrated greater peptide-coding capacity. This study establishes Ouro-seq as a valuable tool for comprehensive circRNA characterization in low-yield clinical samples and advances circRNA biology understanding with potential biomarker discovery and disease monitoring applications. Motivation While circular RNAs (circRNAs) constitute a minor fraction of total RNA, they may play critical roles in cancer development. CircRNA concentrations are typically too low for detection by Oxford Nanopore Long-Read Sequencing (LRS), particularly in samples with limited RNA content, such as liquid biopsies. Consequently, LRS-based circRNA analysis from liquid biopsies remains unexplored. To overcome these technical limitations, we developed an optimized circRNA enrichment method utilizing short-amplicon suppression, enabling circRNA profiling from urine, plasma, and cervico-vaginal samples.
B. Wever, Yara van den Burgt, F. Moulière et al.· bioRxiv· 0 citations
A specific protein (SRSF2) acts like a rogue editor, altering the genetic instructions of another molecule (hnRNPD) and creates a “shield” (PD-L1) on the surface of the cancer cell, effectively blinding the immune system and allowing the tumor to grow unchecked.
Zhao Cheng, Lin Jiang, Ming-yang Wang et al.· Oncogene· 0 citations
BACKGROUND
Cancer-associated chimeric RNAs represent an important class of molecular alterations with diagnostic and prognostic potential. However, cis-spliced fusion RNAs generated by read-through transcription between adjacent same-strand genes remain insufficiently characterized because conventional short-read RNA sequencing and fusion-calling pipelines often deprioritize adjacent-gene junctions. Defining the cancer-associated cis-spliced fusion RNA landscape may expand the molecular genetic repertoire used for tumor classification, clinical correlation, and biomarker development.
METHODS
We integrated Oxford Nanopore long-read direct RNA sequencing of five human cancer cell lines with large-scale tumor, normal-tissue, molecular, and clinical datasets. Adjacent same-strand gene pairs were screened for cis-spliced fusion RNA junctions and filtered using mapping-quality and canonical splice-motif criteria. Candidate junctions were then evaluated by short-read reanalysis of 30,562 TCGA and GTEx RNA-seq samples using Snaptron. Tumor enrichment, driver-mutation association, pathway co-expression, pathologic stage, overall survival, biomarker performance, and biofluid detectability were assessed, with parental gene expression a covariate in the driver, pathway, stage and survival models and local splice coverage in the within-patient and stage models.
RESULTS
We identified 461 quality-controlled cis-spliced fusion RNA junctions, 431 of them absent from four major chimeric-RNA databases. We then tested this long-read panel against a wider short-read universe. Sixty-eight junctions, 27 of them panel members, were enriched in matched TCGA tumor cohorts, and 34 of the 68 were highly tumor-enriched relative to matched normal tissues. Within patients, junction detection remained tumor-associated after adjustment for 5' and 3' parental gene expression, library depth, and local splice coverage (odds ratio 1.59, p = 2.1 × 10⁻⁵). These junctions marked lineage-specific molecular programs, including TP53-mutated basal-like breast cancer and the NFE2L2-KEAP1 cytoprotective axis in lung squamous carcinoma. Fifty-eight candidate-cohort pairs changed monotonically across pathologic stage in cross-section, 35 rising and 23 falling, within a 363-candidate scan, 317 of them panel members. Across a 996-candidate survival screen, 300 of them panel members, six junctions stratified TCGA overall survival at hazard ratios of 2.2 to 4.5, and 2.2 to 4.8 after adjustment for 5' parental gene expression (6/6 significant with the raw log-rank threshold, 1/6 with a Benjamini-Hochberg q below 0.05 across all 1,228 tests). Two of the six are panel members and four showed high specificity against matched normal tissues. Biofluid detection did not distinguish cancer from control plasma cell-free RNA cohorts.
CONCLUSIONS
This study defines cis-spliced fusion RNAs as a previously underappreciated class of cancer-associated transcript alterations, linked here to driver-associated molecular programs, pathologic stage, and survival across a screening universe wider than the 461-candidate long-read panel. That evidence is consistent with a tumor association not fully explained by measured parental gene expression. Discovery, selection and modeling of the six survival candidates took place within TCGA, and we present them for confirmation in an independent junction-resolved cohort. This work provides a discovery framework and a prioritized set of candidate markers for cancer classification and prognosis, ready for evaluation in independent cohorts.
Han-Ying Jhuang, Li-Tzu Wang· Cancer Genetics· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
