Molecular Cancer

MiRNA Epitranscriptomics has placed a new layer of complexity in the cancer field. Despite the fast-growing interest in miRNA editing and shifted miRNA isoforms, a simultaneous study of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modifications, including A-to-I RNA editing and shifted miRNA isoforms, in >13K adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments datasets. We investigated the differences among canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint. The combination of canonical miRNAs/miRNA isoforms boosted the quality of clustering results, outlining unique cohorts clinical-pathological features. We described modified miRNAs showing opposite dysregulation with respect to their canonical counterparts in cancer, potentially impacting their targetome and function. The abundance of expressed miRNA isoforms directly impacted the activation/deactivation of critical carcinogenesis pathways. Finally, we experimentally validated unique targeting for a shifted and edited miRNA isoform. Our findings outlined once more the importance of going beyond the well-established paradigm of one-mature-miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression.

Colorectal cancer (CRC) screening remains limited by patient adherence and sub-optimal sensitivity for early-stage disease. While liquid biopsy has revolutionized cancer diagnostics, cfDNA-based methods often struggle with early-stage detection due to low analyte levels. Here, we present a robust cell-free RNA (cfRNA) platform for the early detection of CRC. Using a retrospective cohort of 255 healthy controls and 250 CRC patients, we implemented an optimized workflow featuring a RUVg-based normalization strategy to remove platelet-driven transcriptomic noise. We identified differentially expressed genes enriched in key CRC-associated biological pathways, including inflammation, EMT, and metabolic dysregulation. An XGBoost classifier trained on these features achieved a mean AUC of 0.92 in cross-validation and 0.89 in a validation cohort, demonstrating 67% sensitivity at 90% specificity. Notably, our platform showed particular efficacy in identifying early stage cancer (stage I and II), achieving 73.7% sensitivity at 90% specificity. These findings suggest that cfRNA profiling offers a powerful, non-invasive orthogonal approach to CRC screening, capable of overcoming the sensitivity limitations of DNA-based assays in early-stage disease.

Pleural mesothelioma (PM) is a rare yet aggressive and heterogeneous cancer type with very poor survival rates. Due to its long latency period and nonspecific symptoms, the disease is usually detected at advanced stages, limiting available treatment options and leading to poor survival rates. So far, the disease can only be confirmed through invasive thoracoscopic biopsy. Moreover, the proposed circulating protein biomarkers lack sensitivity and specificity, highlighting the urgent need for novel approaches. In our previous work, we characterized the transcriptomic profile of extracellular vesicles secreted by PM cells and demonstrated their feasibility as a source of circulating biomarkers. To further investigate the role of circulating RNA in tumor progression and its potential use in non-invasive testing for PM, here we present a detailed characterization of RNA cargo carried by PM exosomes - a distinct subset of extracellular vesicles known to serve as key mediators of intercellular communication. By utilizing primary cell cultures established from tissue samples of 11 PM and 6 non-PM patients, followed by exosome isolation, and total RNA sequencing of RNA from isolated exosomes, matching cells, and tissues, we provide new evidence on exosomal RNA secretion, regulation of PM-exosome cargo genes, and their potential functions in recipient cells. We show that PM-exosomal cargo is enriched in genes associated with proliferation, whose key transcriptional factors are SETDB1, FOXM1 and GATA2. We identified Pcbp2, Srsf1 and Srsf9, RNA-binding proteins, which may be involved in selective cargo sorting into PM-exosomes. Our analysis identified six secreted genes, including GAS5, AL031666.3, RSLD1D1, AC103740.2, ADAM10, and AC020892.1, whose exosomal expression was associated with patient survival, as promising biomarkers for patient diagnosis, stratification, and prognosis assessment. Finally, we identify potential target genes of candidate LncRNAs biomarkers and processes in which they are involved.

Across gastrointestinal (GI) cancers, shared malignant programs are layered onto strong anatomical, lineage, and microenvironmental variation, making it difficult to distinguish disease-relevant long noncoding RNAs (lncRNAs) from context-dependent transcriptional signals. We developed a pan-GI integrative framework to classify lncRNAs across colorectal adenocarcinoma, gastric adenocarcinoma, and esophageal cancer using bulk and single-cell transcriptomic resources. This framework evaluates lncRNAs across four complementary dimensions: recurrent tumor-associated expression, clinical association with disease progression and overall survival, co-expression network context, and malignant epithelial expression at single-cell resolution. Paired tumor-normal RNA-seq analyses identified extensive tumor-associated lncRNA dysregulation and defined recurrent pan-GI lncRNAs consistently upregulated across cancer types. Clinical analyses further nominated transcripts linked to tumor extension, nodal involvement, metastatic dissemination, progression-linked expression, and adverse overall survival. Co-expression network analysis identified lncRNAs embedded within disease-associated transcriptional modules, providing functional context for otherwise poorly annotated transcripts. In parallel, single-cell-derived metacell analysis nominated malignant epithelial-associated and detection-supported lncRNAs, helping distinguish tumor-compartment-associated signals from stromal, immune, endothelial, and other microenvironmental contributions. Together, this study establishes an evidence-structured pan-GI lncRNA resource and a generalizable prioritization strategy for nominating disease-associated noncoding transcripts. More broadly, the framework provides a transferable strategy for systematic lncRNA prioritization across other cancers and heterogeneous disease contexts.

BackgroundMulti-analyte liquid biopsies represents an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), a novel multi-analytes liquid biopsy approach for the isolation of extracellular vesicles (EVs) and cell-free DNA (cfDNA) from a single aliquot of blood. MethodsWe assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining RNA and DNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we investigated EVs-derived RNA (EV-RNA) and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Additionally, we utilized imaging flow cytometry to evaluate EVs as potential carriers of the HER2 protein. ResultsWestern blot analysis and immunocapture assay revealed that EVs-enriched proteins were detected at similar levels among the HER2+ and HER2- subtypes. Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, we observed HER2 protein on the surface of EVs isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EVs as companion analyte to cfDNA. ConclusionsThis data confirms the relevance of combining cfDNA and EV-RNA analytes for cancer assessment and supports the ONCE approach as a valuable tool for multi-analytes liquid biopsies clinical implementation.

H19, a lnc-pri-miRNA that encodes miR-675, is dysregulated in numerous cancers. However, the specific mechanisms underlying H19 processing, particularly miR-675 formation, remain unclear. Our study reveals that H19 is highly expressed and m6A modified in a METTL3-dependent manner in glioblastoma (GBM) and glioma stem cells (GSCs). Silencing METTL3 reduced both H19 and miR-675 levels, whereas overexpressing METTL3 promoted miR-675 processing without affecting H19 levels. Further, miR-675 derived from exogenously expressed H19 was affected considerably more in METTL3 silenced glioma cells compared to H19 levels, suggesting differential requirements in the processing of m6A modified H19 transcript. We demonstrate that H19 interacts with m6A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either reduced H19 and miR-675 levels. However, a high level of miR-675 seen in METTL3 overexpressing cells is severely affected in HNRNPA2B1-silenced compared to IGF2BP2-silenced glioma cells. Interestingly, IGF2BP2 silencing more significantly affected H19 stability from exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Site-directed mutagenesis confirmed the presence of two m6A sites in the first exon of H19, with site #1 facilitating HNRNPA2B1 interaction to promote miR-675 processing. In contrast, the IGF2BP2 interaction is promoted by site #2, resulting in enhanced H19 stability. H19-METTL3-HNRNPA2B1-miR675 axis inhibited Calneuron 1 (CALN1), a known target of miR-675, to promote glioma cell migration. Notably, a low CALN1/high H19 predicted a poor prognosis in GBM patients and was further exacerbated by a high METTL3 or HNRNPA2B1 but not IGF2BP2 transcript levels. Thus, we found that the H19 transcript is highly expressed in GBM and m6A modified, and the m6A reader proteins, IGF2BP2 and HNRNPA2B1, regulate the H19 processing differently to promote glioma cell migration.

Cell-free DNA (cfDNA) methylation is a valuable biomarker in various cancers including colorectal cancer (CRC), but marker for both early diagnosis and prognostic prediction remain a critical unmet need. Here, we report the development of a 27-DMR (differentially methylated regions) plasma panel with dual diagnostic and prognostic functions. We first identified CRC-specific methylation features from 119 tissue samples and 161 plasma samples. Machine learning algorithms were then applied to develop diagnosis and prognosis models using the plasma samples in training cohort. In the tissue external validation cohort (GSE48684), the cfDNA methylation diagnosis model conducted with the panel, achieved an area under the curve (AUC) of 0.983, and for the plasma cfDNA model in the external validation cohort, the sensitivities for NAA, AA and CRC 0 - II are 48.4%, 52.2% and 66.7% respectively, with a specificity of 88%. Beyond its diagnostic capabilities, the methylation panel was also a powerful predictor of distant metastasis (AUC = 0.955) and patient prognosis (AUC = 0.867). Using normal samples as control, the changes in methylation score in both tissue and plasma were consistent across different lesions, although the degree of alterations varied with severity. The methylation scores vary between paired tissue and blood samples, suggesting distinct mechanisms of migration from tumor tissue to blood for the 27 DMRs. Together, our cfDNA methylation models based on 27 DMRs can identify different stages of CRC and predict metastasis and prognosis, ultimately enabling early intervention and risk stratification for CRC patients.

While recent therapeutic advances have extended the survival of patients with non-small cell lung cancer (NSCLC), overcoming metastatic progression in the CNS remains a significant challenge. Some patients with NSCLC may require concurrent management of CNS and extracranial metastases, while others develop isolated brain metastasis or leptomeningeal disease. These heterogenous clinical outcomes are difficult to predict and diagnose for early intervention with current surveillance modalities. Herein, we comprehensively analyzed gene mutations, copy number variations, and DNA methylation of NSCLC brain metastasis tissue collected at the time of craniotomy, combined with ctDNA sequencing of paired plasma and CSF liquid biopsies. We confirmed a high concordance between the molecular features of brain metastasis tissue with ctDNA from CSF which were largely distinct from ctDNA alterations in paired plasma samples. Plasma ctDNA tumor fraction and ctDNA hypermethylation were most significantly associated with extracranial metastasis and overall survival. Alternatively, we identified specific hypermethylated DNA loci in brain metastasis tissue and CSF ctDNA as significant correlates of brain metastasis progression and risk of leptomeningeal disease. Our findings support the utility of integrating ctDNA testing from CSF and plasma, while revealing distinct epigenetic features and biomarkers of brain metastasis or leptomeningeal disease.

The role of red blood cells (RBCs) in tumorigenesis is poorly understood. We previously identified RBC-microRNAs with aberrations linked to lung cancer, including miR-93-5p. Here we find that miR-93-5p levels are elevated in RBC-derived exosomes among lung cancer patients and are associated with their shorter survivals. RBC-derived miR-93-5p transfers to cancer cells primarily through the exosomal pathway. The transferred RBC-miR-93-5p can target PTEN in cancer cells, and hence increase cell proliferation, invasion, and migration. RBC-derived miR-93-5p accelerates, whereas targeting miR-93-5p diminishes tumor growth in xenograft models. These findings reveal a novel biological function of RBCs in tumorigenesis, where they facilitate cancer progression by transferring the oncomiR via exosomes, thereby offering new diagnostic and treatment strategies for lung cancer.

Melanoma being one of the most common and deadliest skin cancers, has been rising since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. Nowadays the standard-of-care of advanced melanoma is resection followed by immune checkpoint inhibition based immunotherapy. However, a substantial proportion of patients either do not respond or develop resistances. This underscores a need for novel approaches and therapeutic targets as well as a better understanding of the mechanisms of melanoma pathogenesis. Long non-coding RNAs (lncRNAs) comprise a poorly characterized class of functional players and promising targets in promoting malignancy. Certain lncRNAs have been identified to play integral roles in melanoma progression and drug resistances, however systematic screens to uncover novel functional lncRNAs are scarce. Here, we profile differentially expressed lncRNAs in patient derived short-term metastatic cultures and BRAF-MEK-inhibition resistant cells. We conduct a focused growth-related CRISPR-inhibition screen of overexpressed lncRNAs, validate and functionally characterize lncRNA hits with respect to cellular growth, invasive capacities and apoptosis in vitro as well as the transcriptomic impact of our lead candidate the novel lncRNA XLOC_030781. In sum, we extend the current knowledge of ncRNAs and their potential relevance on melanoma. SignificancePreviously considered as transcriptional noise, lncRNAs have emerged as novel players in regulating many cellular aspects in health and disease including melanoma. However, the number and as well as the extent of functional significance of most lncRNAs remains elusive. We provide a comprehensive strategy to identify functionally relevant lncRNAs in melanoma by combining expression profiling with CRISPR-inhibition growths screens lowering the experimental effort. We also provide a larger resource of differentially expressed lncRNAs with potential implications in melanoma growth and invasion. Our results broaden the characterized of lncRNAs as potential targets for future therapeutic applications.

Recent studies have shown that the cell surface undergoes post-transcriptional modification by N-linked glycosylation. However, the question of whether RNA can be glycosylated by O-glycans remains to be explored. The presence of O-glycosylation in cells is indirectly revealed by the presence of O-glycans on RNAs following treatment with O-glycoproteases. To identify RNA O-glycosylation, we have developed a chemoenzymatic method for capturing and enriching O-glycosylated RNA (O-glycoRNA) using covalent immobilization on a solid support. GalNAcEXO selectively releases Tn-containing O-glycosylated RNAs (TnORNA). Using this method and SPCgRNA, we compared the expression of O-glycoRNAs and N-glycoRNAs in pancreatic cancer cell lines and tissues. We found that glycosylated miR-103a-3p, miR-122-5p, and miR-4492 regulate pancreatic cancer cell growth and proliferation through the PI3K-Akt pathway. In vitro assays and PDAC tissue analysis confirmed the potential regulatory roles of Tn-O-glycosylated miRNAs in pancreatic tumor growth and metastasis. Furthermore, a significant number (131) of miRNAs carrying both N- and Tn-O-glycosylation were identified, indicating the co-occurrence of N-linked and O-linked glycosylation on small RNAs. We have also developed PONglyRNA, an online bioinformatic tool for the site-specific prediction of RNA glycosylation. PONglyRNA identifies glycosylation motifs based on RNA sequence and has been validated using our glycoRNA data. In conclusion, this study establishes robust experimental and computational tools for identifying O-linked glycoRNAs. Additionally, it uncovers the novel role of glycosylation in PDAC development and progression through altered glycosylation of oncogenic miRNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/599663v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@199511corg.highwire.dtl.DTLVardef@9c3338org.highwire.dtl.DTLVardef@e6d315org.highwire.dtl.DTLVardef@2c1fc1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Resistance to cancer treatment remains the leading cause of cancer-related deaths. In tumors with low mutational burden such as pediatric cancers, alternative transcripts, including circular RNAs (circRNAs), have been identified as involved in treatment resistance. However, their isoforms are often missed by commonly used short-read sequencing. Here, we employ long-read sequencing to identify full-length circRNA isoforms associated with resistance in ALK -driven pediatric cancers. Using cell models and a cohort of ALK -translocated anaplastic large-cell lymphoma (ALK+ ALCL) patients, two circRNAs were detected as specifically upregulated in resistant cases and associated with worse disease outcomes. Similar findings were observed in the pediatric cancer neuroblastoma. These circRNAs were also more abundant in liquid biopsies from ALKi-resistant ALK+ ALCL and neuroblastoma patients. This demonstrates that long-read sequencing allows for uncovering disease-relevant circRNA isoforms that could serve as biomarkers for resistance detection in a clinical setting.

BackgroundThe mA modification is the most abundant RNA epigenetic mark in colorectal cancer (CRC). In this study, we focused on Hakai, a methyltransferase writer-associated protein whose molecular function within the complex and link to CRC immune regulation remain poorly characterized. MethodsWe performed RNA-seq to assess transcriptomic changes after Hakai silencing in CRC models and MeRIP-seq to profile methylation alterations. Validation was carried out by Western blot, flow cytometry, ELISA, and RT-qPCR. Protein interactions within the writer complex were analysed by co-immunoprecipitation, and changes in protein localization by cell fractionation assays. Peripheral blood from healthy donors and lamina propria mononuclear cells from CRC patients were incubated with supernatants from Hakai-silenced CRC cells to evaluate effects on immune responses. ResultsHakai silencing induced phenotypic changes in both monolayer and 3D CRC cultures, while its impact on the global transcriptome was limited. However, significant alterations in the methylation of immune response-related genes and reduced total mA levels were observed. Hakai interacted with the writer-associated protein VIRMA, and its silencing altered the subcellular localization of METTL3. Moreover, conditioned media from Hakai-silenced CRC cells modulated the expression of immune response markers in patient-derived gut immune cells. ConclusionsHakai acts as a component of the mA writer complex in CRC cells, influencing RNA methylation and the expression of immune-related genes. These findings suggest that Hakai silencing may enhance antitumour immune activity and represent a potential strategy to boost cancer immunity in colorectal cancer.

A key limitation in cancer transcriptomics is the lack of accompanying genomic profiling such as whole-genome sequencing (WGS) or copy number alteration (CNA) data. Here we address this by showing that RNA-seq alone can be used to infer chromosomal aberrations and identify biologically meaningful patterns in lung cancer. Through a large-scale meta-analysis of publicly available RNA-seq datasets from non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and matched controls, we reconstructed large scale CNA profiles and identified deletions in 3p, 9p, and 17p as the most frequent genomic events. Validation against paired WGS data confirmed a high degree of accuracy for RNA-seq-based inference. Our analyses revealed that while deletion-associated transcriptional heterogeneity exists, approximately 25% of differentially expressed genes were shared across all three deletion classes, indicating a conserved oncogenic program in lung cancer. Enrichment analysis linked these shared genes to pathways governing cell division, DNA replication, and extracellular matrix organization, while deletion-specific effects reflected disruption of tumor suppressor pathways, notably p53 signaling in 17p-deleted tumors, leading to deregulation of NOS2 and PLOD2. Integrating gene-level expression data, we identified both shared and deletion-specific biomarkers: TPX2 was consistently overexpressed across all deletion groups, while PTPRZ1 and CLDN9 were uniquely associated with del3p and del9p, respectively. Experimental validation in lung cancer cell lines confirmed these predictions, particularly the upregulation of CLDN9 in del9p carriers. By demonstrating that RNA-seq data can capture large-scale chromosomal events and reveal their transcriptional consequences, this study establishes an efficient framework for genomic inference and biomarker discovery, introducing CLDN9 as a novel, deletion-specific marker with potential prognostic and therapeutic value for 9p-deleted lung cancers.

Detection of reliable markers of therapy response and drug resistance remains a major unmet need in brain cancer, as serial tumor biopsy is often not feasible. This challenge is particularly acute in diffuse midline glioma (DMG), a fatal pediatric brain tumor for which new targeted therapies are entering clinical use, yet tools for real-time molecular analysis of tumor evolution during therapy remain lacking. Here, we demonstrate that plasma tumor-derived extracellular vesicle (EV) profiling provides a minimally invasive and complementary approach for diagnosis and longitudinal molecular monitoring in H3K27M-mutant DMG. Across patient-derived tumor models and clinical plasma samples, EV mRNA levels correlated strongly with parental tumor transcriptomes. EV H3K27M mRNA enabled discrimination of DMG from non-DMG controls, and exploratory EV response-associated mRNAs were linked to radiographic response and progression-free survival in patients receiving ONC201. To enable tumor-selective EV enrichment and multiplexed molecular profiling within a clinically practical workflow, we developed a new integrated platform supporting same-day EV analysis from small plasma volumes. This work represents the first proof-of-concept demonstration of the diagnostic and treatment response relevance of tumor-derived EV RNA in pediatric DMG and establishes a generalizable framework for minimally invasive longitudinal molecular monitoring in diseases where tissue access is inherently limited.

Gliomas are biologically heterogeneous brain tumors with marked differences in clinical behavior based on the IDH1 mutation status. While epigenetic dysregulation is well characterized, the contribution of RNA modifications, particularly N6-methyladenosine (m6A), remains underexplored. Using direct RNA nanopore sequencing of patient-derived gliomas, we generated the first isoform-resolved m6A maps across IDH1-mutant and wild-type tumors. IDH1-mutant gliomas exhibited globally elevated m6A methylation, along with increased expression of methyltransferases (METTL3, METTL14) and stabilizing readers (YTHDF3). In contrast, wild-type glioblastomas showed enhanced expression of m6A erasers (ALKBH5, FTO) and RNA decay factors (YTHDF2). These subtype-specific differences in m6A architecture impacted transcript stability, isoform usage, and gene expression. Isoform-level analyses revealed stronger prognostic associations than gene-level parameter, including for IGF2BP2-202, PUF60-202, and GLUL-203. Our study establishes m6A as a critical, subtype-specific layer of RNA regulation in glioma with clinical and therapeutic implications.

ObjectiveRNA adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a pervasive post-transcriptional mechanism that expands transcriptomic and proteomic diversity. However, the global landscape of RNA A-to-I editing, as well as its functional and clinical significance in esophageal squamous cell carcinoma (ESCC), remains largely unexplored. This study aimed to systematically characterize the RNA editome of ESCC and elucidate its biological and clinical significance. MethodsWhole-transcriptome sequencing was performed on 121 primary ESCC tumors, with or without lymph node metastasis, together with matched normal tissues, to construct a high-resolution RNA editome. ADAR1-regulated RNA editing events were identified, and their functional consequences were investigated using integrated transcriptomic, phosphoproteomic, and RNA immunoprecipitation sequencing (RIP-seq) analyses. Associations between CDK13 editing, cGAS-STING-interferon-stimulated gene (ISG) signaling, and patient survival were further evaluated. ResultsA total of 222,020 high-confidence RNA editing sites were identified, of which approximately 98% were A-to-I events, including 124,486 ESCC-specific edits predominantly enriched in introns, 3' untranslated regions, and non-coding RNAs, highlighting a pervasive post-transcriptional regulatory layer. RNA A-to-I editing was extensively remodeled and globally up-regulated in non-metastatic ESCC, whereas only minimal changes were observed during lymph node metastasis, indicating that RNA editing alterations predominantly occur during early tumorigenesis. CDK13 emerged as a recurrent ADAR1 target, with RNA editing inversely correlated with CDK13 expression. ADAR1-mediated CDK13 editing established a positive feedback loop associated with enhanced interferon-stimulated gene (ISG) signaling and poorer survival in non-metastatic ESCC. Mechanistically, partial attenuation of CDK13 induced chronic, tumor cell-intrinsic activation of the cGAS-STING-ISG pathway. Integrated multi-omics analyses further revealed that CDK13 regulates phosphorylation networks governing cytoskeleton organization, intracellular trafficking, RNA homeostasis, and immune signaling. ConclusionRNA A-to-I editing represents a dynamic regulatory mechanism driving early ESCC progression and remodeling tumor cell-intrinsic immune signaling. ADAR1-mediated editing of CDK13 provides a mechanistic link between RNA editing and cGAS-STING-ISG pathway activation, revealing potential therapeutic vulnerabilities and supporting its utility as an early prognostic biomarker in ESCC.

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification, enriched in the CNS yet poorly characterized in glioma. Using long-read RNA sequencing, we mapped m6A in an in vitro glioma model following knockdown (KD) of the reader IGF2BP2, writer METTL3, and eraser ALKBH5, with naive glioma cells and astrocytes as controls. Glioma cells exhibited a two-fold reduction in global m6A, suggesting progressive loss from healthy to malignant states. Integrated analysis revealed that m6A mediated control of gene expression is influenced by modification topology (CDS:3'UTR), transcript biotype, and length. Regulator KD, particularly ALKBH5 induced redistribution of m6A toward 3'UTR with consequent gene upregulation. We also identified m6A-mediated isoform switching, with a higher usage of retained intron and nonsense-mediated decay isoforms. Structural and splicing alterations at the isoform level were identified unique to each KD condition indicating m6A driven aberrant alternative splicing. At the functional level, KD specific remodeling of oncogenic signaling was also observed. ALKBH5 KD suppressed MYC targets and pro-apoptotic signaling while METTL3 KD enhanced mTOR and PI3K-AKT signaling. Collectively, these results demonstrate that m6A mediated regulation in glioma is highly context-dependent, defining distinct clinically relevant phenotypes. This has implications for future biomarker discovery and development of targeted therapeutics.

Two cell identities, noradrenergic and mesenchymal, have been characterized in neuroblastoma cell lines according to their epigenetic landscapes relying on specific circuitries of transcription factors. Yet, their relationship and relative contribution in patient tumors remain poorly defined. Here, we demonstrate that the knock-out of GATA3, but not of PHOX2A or PHOX2B, in noradrenergic cells induces a mesenchymal phenotype. Our results document spontaneous plasticity in several models between both identities and show that plasticity relies on epigenetic reprogramming. We demonstrate that an in vivo microenvironment provides a powerful pressure towards a noradrenergic identity for these models. Consistently, tumor cells with a mesenchymal identity are not detected in a series of PDX models. Further study of the intra-tumor noradrenergic heterogeneity reveals two distinct cell populations exhibiting features of chromaffin-like or sympathoblast-like cells. This work emphasizes that both external cues of the environment and intrinsic factors control plasticity and cell identity in neuroblastoma.

About 50% of poor prognosis neuroblastoma arises due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN amplified (MNA) neuroblastoma. Here we evaluate PRMT5 inhibitors GSK3203591/GSK3326593 as targeted therapeutics for MNA neuroblastoma and show MYCN-dependent growth inhibition and apoptosis. RNAseq revealed dysregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Metabolic tracing showed glutamine metabolism was impeded following GSK3203591 treatment, which disrupted the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Glutaminase (GLS) protein was decreased by GSK3203591 despite unchanged transcript levels, suggesting post-transcriptional regulation. We demonstrate the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following splicing alterations; accordingly, we observed hypomethylation of GLS mRNA and decreased GLS following YTHDF3 knockdown. In vivo efficacy of GSK3326593 was confirmed by increased survival of Th-MYCN mice together with splicing events and protein decreases consistent with in vitro data. Our study supports the spliceosome as a key vulnerability of MNA neuroblastoma and rationalises PRMT5 inhibition as a targeted therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/582087v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ac6b5org.highwire.dtl.DTLVardef@1eb9520org.highwire.dtl.DTLVardef@18b980corg.highwire.dtl.DTLVardef@1ab46e0_HPS_FORMAT_FIGEXP M_FIG C_FIG