Molecular Cancer

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. MotivationWhile 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.

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

Neuroblastoma is a neural crest-derived pediatric malignancy characterized by marked cellular heterogeneity and variable differentiation status. Undifferentiated tumors are associated with aggressive clinical behavior, treatment resistance and poor outcome, highlighting the need to identify molecular mechanisms that sustain tumor cell plasticity and prevent differentiation. Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase involved in cell-cycle progression, DNA-damage responses and transcriptional regulation, and has previously been associated with neuroblastoma progression. However, its role in the control of neuroblastoma differentiation remains unclear. Here, we investigated the relationship between VRK1 expression, tumor differentiation and stem-like properties in human neuroblastoma. Analysis of patient tumor datasets and tissue microarrays showed that VRK1 expression is enriched in undifferentiated neuroblastoma and stage 4 tumors, and inversely correlates with established differentiation markers, including DDC, NCAM1 and S100B. This association was maintained in MYCN-non-amplified tumors, indicating that the relationship between VRK1 and differentiation is not dependent on MYCN status. Single-cell transcriptomic analyses further demonstrated elevated VRK1 expression in developmentally immature neural crest progenitor and Schwann cell precursor-like populations. Induction of neuronal or mesenchymal differentiation consistently reduced VRK1 expression in neuroblastoma cell lines and patient-derived cells. Conversely, VRK1 silencing promoted differentiation-marker expression, reduced nestin and Ki67 expression, and produced sustained differentiation-associated changes in xenograft tumors. VRK1 was also enriched in tumorsphere cultures that select for undifferentiated stem-like neuroblastoma cells. VRK1 depletion impaired tumorsphere growth, reduced intratumoral proliferation and altered the balance between undifferentiated cells and differentiated progeny, supporting a role for VRK1 in self-renewal and maintenance of progenitor-like tumor cells. Mechanistically, VRK1 expression positively correlated with the core stemness transcription factor SOX2 in neuroblastoma tumor cells. VRK1 knockdown reduced nuclear SOX2 abundance, whereas VRK1 overexpression increased SOX2 protein levels. In addition, analysis of the VRK1 locus identified an active chromatin configuration and potential SOX2-binding sites, consistent with a regulatory relationship between these factors. Together, these findings identify VRK1 as a regulator of the undifferentiated, proliferative and stem-like state in neuroblastoma. The VRK1-SOX2 axis may contribute to stabilizing tumor-cell immaturity and represents a potential target for differentiation based therapeutic strategies in high-risk neuroblastoma.

Purpose Long non-coding RNAs (lncRNAs) are important regulators of tumor biology, but their dependence on major oncogenic drivers and cancer specificity remain poorly characterized. We aimed to identify lncRNAs regulated by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer and to determine their functional relevance and molecular mechanism. Methods We performed an integrative analysis of lncRNA expression profiles driven by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer cell lines. Candidate lncRNAs were functionally characterized using assays of cancer cell viability, migration, and clonogenicity. Expression in patient-derived tumor and normal tissues and downstream molecular pathways were investigated. Results MYC was associated with the most extensive lncRNA regulation, with expression patterns largely independent of tissue origin. MYC-dependent LINC00997 and the previously uncharacterized AC104447.1, as well as specifically KRAS-dependent RAKRAR (RASSF3 antisense KRAS regulated lncRNA) promoted cancer cell viability, migration, and clonogenicity. LINC00997 and AC104447.1 expression was elevated in specific tumor types compared to normal tissues. We identified CAMOR (CARNMT1 Antisense Multiple-Oncoprotein Regulated lncRNA), a pan-cancer lncRNA upregulated by all three oncogenic drivers. CAMOR promoted viability and migration specifically in cancer cells across analyzed cell lines and tumor types. Mechanistically, CAMOR and neighboring CARNMT1 form a negative feedback regulatory loop affecting CAMOR oncogenic activity. Distinct downstream gene expression signatures suggested that the studied lncRNAs act through different regulatory processes. Conclusion Our findings expand the repertoire of oncogenic lncRNAs and demonstrate that integrating multiple oncogenic contexts enables identification of both driver-specific and broadly acting lncRNAs, providing a framework for identifying lncRNAs that may contribute to cancer development and represent potential diagnostic or therapeutic targets.

Squamous cell carcinoma (SCC) remains difficult to treat, particularly in recurrent or metastatic disease. Although EGFR signaling is a well-established driver of SCC pathogenesis, mechanisms regulating its activity remain incompletely understood. CD109, a GPI-anchored glycoprotein frequently overexpressed in SCC, has been implicated in EGFR signaling, but its mechanisms remain poorly defined. Here, we show that CD109 is elevated in head and neck SCC (HNSCC) in the TCGA PanCancer Atlas and significantly correlates with reduced disease-free survival. Integrative transcriptomic (TCGA PanCancer Atlas) and proteomic (LinkedOmics) analyses revealed strong positive correlations between CD109, EGFR, and SMURF2. These findings were further validated by immunohistochemistry in HNSCC patient samples. Mechanistically, CD109 overexpression impaired EGFR degradation, suppressed degradation-associated EGFR pY1045 phosphorylation, and enhanced signaling-associated pY1068 phosphorylation, resulting in increased STAT3, AKT, and ERK signaling. We identify SMURF2 as a mediator of these effects, with CD109 promoting EGFR-SMURF2 complex formation. SMURF2 gain- and loss-of-function studies in SCC cells, demonstrated that SMURF2 stabilizes EGFR by reducing EGFR interaction with the ubiquitin ligase c-Cbl, thereby limiting EGFR turnover in a CD109-dependent manner. Functionally, the CD109-SMURF2-EGFR axis promotes EGF-induced stemness, invasion, and proliferation in SCC cells. Together, these findings identify CD109 as a molecular switch controlling EGFR fate by redirecting EGFR from c-Cbl-mediated degradation toward SMURF2-dependent stabilization, sustaining oncogenic signaling. This previously unrecognized regulatory pathway reveals the CD109-SMURF2 axis as a vulnerability with potential for targeted intervention in EGFR-driven SCCs.

Background: Long non-coding RNAs (lncRNAs) are emerging regulators of tumor initiation and progression through their effects on stemness, lineage commitment, and transcriptional plasticity. The contribution of lncRNA reprogramming during early p53-deficient gliomagenesis remains poorly understood. Methods: Human induced pluripotent stem cells (iPSCs) carrying a complete TP53 knockout were differentiated into neural progenitor cells (NPCs) and cerebral organoids. Bulk RNA sequencing was performed at multiple differentiation stages and compared with established glioblastoma cell lines (U87 and U118) to identify lncRNA expression changes associated with p53 loss and tumor-like transformation. Results: p53 deficiency induced a progressive and stage-specific remodeling of the lncRNA landscape, with the most pronounced transcriptional shift observed in 40-day NPCs. This late-stage profile partially overlapped with glioblastoma-like cell lines and was characterized by the upregulation of five lncRNAs enriched in both p53-deficient neural models and glioma cells. Principal component analysis showed that these lncRNAs clustered with tumor-associated samples and were strongly co-expressed with mitotic cell-cycle genes. In particular, LINC00973 and LINC01583 were highly expressed in primary glioblastoma datasets, while RP5-875H18.9 and RP11-1094H24.4 emerged as previously unrecognized glioma-associated transcripts. Conclusion: Loss of p53 drives stage-dependent lncRNA reprogramming during neural differentiation and promotes the emergence of glioma-associated transcriptional features. These findings identify candidate lncRNAs that may contribute to early gliomagenesis and represent potential biomarkers or therapeutic targets in glioblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood. Its clinical behavior ranges from spontaneous regression to lethal, treatment-refractory disease. Aberrant 2,6-sialylation contributes to aggressive phenotypes in many cancers, but the role of ST6Gal2, a neural-enriched 2,6-sialyltransferase, in neuroblastoma is largely unexplored. Here, we examine the clinical and functional significance of ST6Gal2 in neuroblastoma. In two independent public cohorts (SEQC, n=498; Kocak, n=649), high ST6GAL2 expression was associated with significantly worse overall and event-free survival. In the SEQC cohort, ST6GAL2 expression was higher in high-risk and MYCN-amplified tumors, varied across International Neuroblastoma Staging System stages, and correlated positively with a mesenchymal transcriptional signature (Spearman {rho}=0.181). The mesenchymal correlation was reproduced in the Kocak cohort ({rho}=0.204). Stable shRNA-mediated knockdown of ST6GAL2 in SK-N-AS and SK-N-BE(2) cells reduced proliferation and viability, impaired wound closure, and decreased migration and invasion. In preliminary experiments in SK-N-AS cells, ST6GAL2 knockdown reduced binding of Sambucus nigra agglutinin, consistent with a role for ST6Gal2 in 2,6-sialylation. Together, these findings link ST6Gal2 expression to aggressive clinical and transcriptional features and pro-tumorigenic phenotypes in neuroblastoma and nominate ST6Gal2-mediated sialylation as a candidate pathway for mechanistic study.

Neuroblastoma is a heterogeneous pediatric malignancy characterized by distinct genomic subgroups and cell-state plasticity between adrenergic (ADRN) and mesenchymal (MES) phenotypes. While small extracellular vesicles (sEVs) mediate pre-metastatic niche priming, the degree to which sEV proteomes reflect parental genomic and phenotypic identity remains unresolved. Here, we isolated sEVs from six neuroblastoma cell models (IMR-5/75, BE(2)-C, GI-ME-N, CLB-GA, LAN-6, and SK-N-FI) representing three genomic subgroups (MYCN-amplified, TERT-rearranged, and unaltered) using OptiPrep density gradient centrifugation followed by size exclusion chromatography and ultrafiltration. High-resolution mass spectrometry of sEV proteomes revealed that sEVs carry highly reproducible protein signatures that tightly correlate with the parental genomic subgroups. Analyzing paired cell/sEV samples, we demonstrate cell-to-EV concordance of lineage-specific markers: ADRN sEVs selectively enriched L1CAM and dopamine {beta}-hydroxylase (DBH), whereas MES sEVs enriched SERPINE1 and type III collagen (COL3A1). These findings indicate that neuroblastoma-derived sEVs systematically preserve both the genomic landscape and the lineage transdifferentiation states of their cells of origin. This study establishes a robust molecular foundation for utilizing sEV-based proteomics as a surrogate to track cellular plasticity and for studying sEV-mediated microenvironmental remodeling.

SummaryA catch-all intronic guide RNA pair excises the KIAA1549--BRAF oncofusion across its major variants, with productive junction excision confirmed by gain-of-function PCR in patient-derived glioma cells. An allele-specific guide selectively disrupts BRAF V600E, in patient-derived pediatric low-grade glioma cells. Pediatric low-grade glioma (pLGG) is the most common brain tumor of childhood, accounting for 30--50% of all pediatric central nervous system malignancies1. The disease is almost universally driven by activating mutations in the BRAF serine/threonine kinase: a chromosomal tandem duplication generating the KIAA1549--BRAF oncofusion in approximately 70% of cases, or the BRAF V600E gain-of-function point mutation in approximately 15%2. Current targeted pharmacotherapies, including the RAF inhibitor tovorafenib, require continuous dosing, are not allele-specific, and carry risks of long-term toxicity in children. A one-time genomic intervention that permanently disables the oncogenic BRAF alteration while preserving wild-type BRAF signaling represents a compelling therapeutic alternative. In this study, we describe the design and experimental validation of allele-specific CRISPR guide RNAs targeting both the KIAA1549--BRAF oncofusion and the BRAF V600E point mutation. For the oncofusion, we developed a double-cut intronic excision strategy in which a guide RNA targeting KIAA1549 intron 14 is paired with a guide RNA targeting BRAF intron 11. Because the genomic breakpoints of all four major fusion variants (KB 16:9, 15:9, 16:11, and 15:11) fall within these introns, a single guide pair can address the full landscape of fusion heterogeneity in a single intervention. For BRAF V600E, we exploited a unique PAM sequence created by the pathogenic TBA transversion at codon 600, enabling allele-specific SpCas9 and AsCas12a guide designs that distinguish the mutant from the wild-type allele at single-nucleotide resolution. We screened guide RNA candidates by ribonucleoprotein (RNP) nucleofection in A375 human melanoma cells (BRAF V600E homozygous) and in patient-derived 3635 PXA glioma cells (BRAF V600E heterozygous). The top KIAA1549 intron 14 guide, K9_i14_A_Cas9, achieved 66% indel frequency in A375 cells. The top BRAF intron 11 guides, B_i11_A_Cas9 and B_i11_D_Cas9, achieved 84% and 85% indel frequency, respectively. For BRAF V600E, the best allele-specific SpCas9 guide achieved l57% editing in A375 cells and l74% editing in 3635 PXA patient-derived glioma cells. Dual-cut excision of the KIAA1549--BRAF junction was confirmed by a gain-of-function PCR assay designed to detect the excision junction amplicon ([~]191 bp) produced by NHEJ-mediated rejoining of the KIAA1549 intron 14 and BRAF intron 11 cut ends.

Head and neck squamous cell carcinoma (HNSCC) remains clinically challenging, with limited molecularly targeted options and a strong dependence on the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are major stromal regulators that shape tumor progression, extracellular matrix remodeling, invasion, and therapeutic response. However, how CAF heterogeneity and plasticity translate into distinct tumor-promoting functions and targetable vulnerabilities remains insufficiently defined. Here, we integrated patient-matched primary CAFs and normal fibroblasts with 3D functional assays, tumor-stroma co-culture models, quantitative extracellular matrix analysis, whole-proteome profiling, and pharmacological perturbation. Primary fibroblast populations displayed marked interpatient heterogeneity and context-dependent plasticity in invasion, contractility, and responsiveness to tumor-derived signals, whereas enhanced fibronectin-rich matrix deposition and disorganization emerged as a conserved CAF-associated feature. Both normal fibroblasts and CAFs promoted HNSCC cell invasion in a population-dependent manner, whereas CAFs consistently induced less compact and more dispersed tumor nest architectures. Integrative functional analyses identified distinct CAF phenotypes characterized by either invasive and matrix-remodeling activity or high responsiveness to tumor-derived cues. Proteomic profiling revealed recurrent enrichment of adhesion, cytoskeletal, and extracellular matrix programs and guided the selection of pharmacological inhibitors aimed at modulating specific CAF-mediated pro-tumoral functions. Pharmacological targeting selectively altered these functions: CHI3L1 inhibition disrupted fibronectin matrix deposition, broad phosphodiesterase inhibition increased matrix alignment, and FZD7 inhibition consistently blocked tumor-induced CAF invasion across all tested populations. These findings define functionally distinct and pharmacologically targetable CAF programs in HNSCC and support stromal-directed interventions as a rational component of future combination treatment strategies.

High-risk neuroblastoma remains a major clinical challenge despite intensive multimodal treatment. Alterations in the anaplastic lymphoma kinase (ALK) gene are frequent in NB and correlate with poor outcome. ALK-F1174L and ALK-R1275Q are the most prevalent activating mutations, but their specific effects on tumor behavior remain unclear. Here, we compared the impacts of ALK-wild-type (wt), ALK-F1174L and ALK-R1275Q in several NB xenograft models. In the SK-N-Be2c model, ALK-F1174L mediated numerous transcriptomic alterations without affecting tumor behavior. In GIMEN cells, only ALK-F1174L and ALK-R1275Q conferred tumorigenicity with distinct growth kinetics and transcriptional programs. In the mesenchymal SH-EP model, ALK variants induced distinct phenotypes, with ALK-wt and ALK-F1174L promoting partial transition toward neuroblastic identity. All ALK variants induced lung metastases, but with allele-specific dissemination patterns. Transcriptomic profiling of SH-EP tumors and corresponding metastases revealed partly opposing pathway regulation between ALK-F1174L and ALK-R1275Q. Overall, ALK variants differentially modulate NB behavior in a context-dependent manner.

Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.

With increased lung cancer screening, early-stage diagnoses and recurrences are expected to rise. Identifying the ~20% of patients with early-stage non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), who relapse after curative-intent surgery remains a major clinical challenge. Here, we identify a COX-2-associated pro-tumorigenic inflammatory signature (PTI) in resected tumors as an independent predictor of disease relapse in both LUAD and LUSC, with particular utility within one year after surgery in stage I NSCLC. We developed a clinically compatible workflow for PTI scoring in tumor resections and validated its predictive performance in real-world samples from routine care and screening programs. Spatial immune profiling revealed that PTIhigh tumors, which swiftly recur, exhibit markedly reduced tumor cell content alongside expanded neutrophil-rich immune-stromal compartments. These findings link COX-2-driven inflammation to early post-surgical recurrence in NSCLC and indicate that PTI may serve as a biomarker for risk stratification to guide imaging surveillance and adjuvant therapy decisions.

Neuronal-cancer cell interactions are a key component of brain tumor pathophysiology, yet many fundamental questions remain unanswered, with even more remaining unasked. Here, we present a comprehensive brain tumor reference map generated from bulk RNA-seq data of ~4,300 adult and pediatric brain tumors, including various glioma subtypes, medulloblastomas, ependymomas, and meningiomas, alongside ~1,400 healthy brain samples. By analyzing synaptic processes and synaptic gene expression across tumor types, we reveal the complexity of neuron-tumor crosstalk and its potential impact on patient survival. Our findings identify key synaptic signaling pathways and dysregulated patterns in brain tumors, including HNRNPH2 upregulation, a key factor in RNA processing and metabolism, in gliomas and medulloblastomas. Additionally, we highlight the role of cholinergic signaling, particularly CHRNA9, as a driver of glioma progression, which is also upregulated in WNT and Group 4 medulloblastomas, ependymomas, and meningiomas. Furthermore, P2 purinergic signaling emerges as a key player in glioma progression. These insights lay the groundwork for future studies exploring whether targeting these pathways could reprogram the tumor microenvironment toward an anti-tumor state, ultimately leading to novel therapeutic strategies and improved patient outcomes.

MYCN amplification is associated with poor outcomes in neuroblastoma (NB). MYCN encodes a transcription factor that induces the expression of mitochondrial one-carbon enzymes and chaperones, promoting metabolic reprogramming and aggressiveness in NB tumor cells. While the contribution of MYCN to changes in the mitochondrial proteome is well established, how mitochondrial proteostasis contributes to MYCN activity remains poorly understood. Mitochondrial HSP60 (HSPD1) is a conserved mitochondrial chaperone promoting the mitochondrial one-carbon pathway and mitochondrial translation by folding the one-carbon enzyme MTHFD2 and preventing aggregation of mitochondrial ribosomal proteins. Here, we show that HSPD1 depletion in MYCN-amplified NB cells led to reduced mitochondrial translation, MYCN expression, and tumorigenesis in vitro and in vivo. In addition, HSPD1 mRNA and protein correlate with MYCN expression in NB tumors. These results establish HSPD1 as a promising target for the treatment of MYCN-amplified NB.

Oxford Nanopore Technologies based methylation profiling enables rapid, accurate CNS tumor classification but is currently mostly performed with MinION flow cells (~$1,000 USD). We benchmarked the low cost Flongle flow cell (~$100, 10 fold reduction) for methylation based tumor classification. Across 12 pediatric CNS tumor samples with highly variable sequencing yields (17 to 330 Mbp), both Sturgeon and nanoDx classifiers achieved perfect diagnostic accuracy when operating above significance thresholds, despite extreme data sparsity. Bootstrap analysis determined empirically defined minimum data thresholds. These results demonstrate that Flongle flow cells enable cost effective deployment of rapid, accurate nanopore based CNS diagnostics while maintaining clinical reliability.

Ewing Sarcoma (ES) is an aggressive neoplasm arising in bones and soft tissues driven by the oncogenic fusion EWSR1::FLI1. Through epigenetic deregulation, EWSR1::FLI1 generates de novo super-enhancers that control the expression of key genes for tumor cell maintenance. By an integrative in silico analysis, we identified the subunit of the Mediator complex MED13L and RERE, a member of the atrophin family of arginine-glutamic acid dipeptide repeat-containing proteins, as genes regulated by EWSR1::FLI1-bound super-enhancers. We confirmed that EWSR1::FLI1 regulates MED13L and RERE expression in ES cell lines and showed that these proteins are highly expressed in Ewing primary tumors. Besides the well-established role of the Mediator complex in transcriptional regulation given its association with the RNA polymerase II, in ES cells the DNA binding sites of MED13L overlap with those of RERE and EWSR1::FLI1 in genes that control protein translation and alternative splicing (AS). Accordingly, the expression of various spliceosome components is co-regulated by MED13L, RERE and the oncogene, leading to AS in ES cells. We identified RBM39, a splicing factor downregulated after MED13L and RERE depletion, as a direct transcriptional target of EWSR1::FLI1. Consistently, in vitro viability experiments using indisulam, which induces selective DCAF15-dependent proteosome degradation of RBM39, demonstrate ES cells highly and specifically sensitive to RBM39 inhibition. In vivo experiments with mice xenografted with ES cells show complete tumor regression with indisulam, highlighting the potential of this approach as a novel and promising therapeutic strategy for Ewing sarcoma. STATEMENT OF SIGNIFICANCEEwing sarcoma (ES) is characterized by FET::ETS oncoproteins that act as pioneer transcription factors. Here, we identified two genes controlled by EWSR1::FLI1-bound super-enhancers, MED13L and RERE, and characterized the mechanism by which these proteins cooperate with the oncogene to regulate RNA metabolism and ribosomal processes in ES cells. These findings have led to the identification of the splicing factor RBM39 as a vulnerability in ES, as supported by the extraordinary sensitivity of these tumors to monotherapy with RBM39 degrader indisulam.

Abstract Head and neck adenoid cystic carcinoma (HNACC) shows highly variable clinical behavior that histopathological features only partly predict, and prognostic biomarkers for aggressive disease at diagnosis remain limited. Here, we show by integrating bulk, single-cell, and spatial transcriptomes from 25 HNACC tumors that poor survival is marked by profound loss of myoepithelial tumor cells and expansion of luminal tumor states. Both outcome groups share a myoepithelial-like origin, but aggressive tumors converge on an actively dividing, MYC/E2F-driven luminal state concentrated in discrete spatial niches. Aggressiveness therefore reflects where tumors end along a shared developmental path rather than a distinct lineage or altered immune infiltration. A four-gene classifier capturing this state predicted survival in an independent cohort, outperforming larger signatures and flagging aggressive subgroups in two others. These findings define a lineage proliferation axis linked to HNACC outcome, providing a basis for prospective risk stratification and proliferative-directed therapy.

Glioblastoma's pronounced migratory capacity underlies its diffuse invasion, presenting a formidable barrier to successful treatment. Ex vivo characterization of glioblastoma cells isolated from freshly resected clinical samples under physiologically relevant conditions revealed two distinct migratory phenotypes, Fast Migrating (FM) and Slow Migrating (SM). These phenotypes reflect distinct mechanosensitivity profiles and are associated with pharmacological responses that support the motor clutch model of cell migration. Analysis of genes associated with these phenotypes revealed a transcriptomic signature that closely associated with in vitro cell migration, histological invasion in patient specimens, and clinical survival. Single-nucleus RNA sequencing revealed that FM and SM cells coexist within a single glioblastoma, with FM cells enriched at the periphery and SM cells localized to the tumor core. Collectively, our study demonstrates the utility of ex vivo glioblastoma characterization, allowing decoding of tumor heterogeneity and clinical prognostication as well as providing a framework for deconvoluting the complex cancer phenotype.

Mutant-selective and pan-RAS(ON) multi-selective inhibitors have improved clinical outcomes for patients with RAS-driven cancers; however, adaptive resistance limits the depth and durability of response. Using an integrated genetic, transcriptomic, and mass-spectrometry-based quantitative temporal proteomic platform, we profile the adaptive response to KRASG12C inhibitors (KRASi) in KRASG12C-mutant lung adenocarcinoma models. Our analyses reveal remodeling of signaling pathways and the cell surface proteome, including cell surface proteins selectively upregulated across cell lines, tumor models, and treatment durations. Folate receptor alpha (FOLR1) emerged as a therapeutically actionable target that is significantly upregulated with KRASi and is part of a broader acute cellular state transition characterized by acquisition of an epithelial identity. Combining KRASi with mirvetuximab soravtansine-gynx, a clinically approved FOLR1-targeting antibody drug conjugate, enhanced tumor cell cytotoxicity and deepened response and tumor control in cellular and in vivo models. Additionally, this work represents a comprehensive multi-omic resource characterizing non-genetic temporal adaptation to KRASi for future mechanistic and therapeutic investigations.