Molecular Cancer

Head and neck squamous cell carcinoma (HNSCC) remains associated with substantial morbidity and a 5-year overall survival rate of approximately 60%, reflecting persistent radio- and chemo-resistance and the lack of effective precision medicine strategies. Patient-Derived Tumor Organoids (PDTO) constitute promising functional models that may predict individual treatment response. In this study, we generated PDTO from surgically resected HNSCC of the oral cavity, oropharynx, larynx, and hypopharynx. A total of 20 long-term PDTO lines were established, maintaining growth over seven passages and successfully cryopreserved, capturing the molecular and clinical diversity of the patient cohort. These PDTO faithfully recapitulated histological features, major tumor marker expression, and the genomic and transcriptomic landscapes of their tumors of origin, with stability over time. Functional assays revealed heterogeneous responses to cisplatin and X-rays. Importantly, in vitro sensitivity of PDTO was associated with clinical outcome of patients at 24 months. Cisplatin response of PDTO predicted prognosis with 66.7% sensitivity and 100% specificity, while X-ray response showed 91.7% sensitivity and 75% specificity. Notably, all patients whose PDTO were classified as resistant to both cisplatin and X-rays experienced relapse and/or death within 24 months. Collectively, the successful long-term expansion and cryopreservation of HNSCC PDTO establish a stable and scalable preclinical resource that captures the molecular and clinical heterogeneity of the disease. This biobank provides a valuable platform for mechanistic studies and for the evaluation of innovative therapeutic strategies. This cohort represents one of the largest clinically annotated HNSCC PDTO collections to date, demonstrating a robust association between PDTO response to cisplatin and X-rays and patient prognosis. These findings support the predictive potential of PDTO-based functional assays and argue for their integration into standardized, rapid, and miniaturized precision oncology workflows for HNSCC.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein WDR77 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are the small nuclear ribonucleoproteins SNRPB, SNRPD1 and SNRPD3, which are critical for spliceosome assembly and RNA splicing fidelity. MTAP-deleted cancers exhibit increased sensitivity to PRMT5 inhibition due to elevated levels of methylthioadenosine (MTA), a natural inhibitor of PRMT5. This vulnerability is exploited by MTA-cooperative PRMT5 inhibitors, exemplified by TNG908 and TNG462 which selectively target PRMT5 in MTAP-deleted cells while sparing MTAP-wildtype (WT) cells. Consistent with this mechanism, treatment with TNG908 in preclinical studies induces widespread splicing alterations in MTAP-deleted cancer models, with minimal effects in MTAP-WT cells. These splicing changes are consistent across diverse MTAP-deleted tumor types, including glioblastoma, pancreatic, and non-small cell lung cancer, indicating a histology-agnostic response to PRMT5 inhibition. Moreover, treatment of MTAP-WT cells with exogenous MTA mimics the splicing alterations observed with PRMT5 inhibition, as does pharmacologic inhibition of MTAP further supporting a mechanistic link between MTA accumulation, PRMT5 modulation, and aberrant splicing. Given that MTAP deletions occur in approximately 10-15% of human cancers, the identification of a robust RNA splicing signature offers a valuable pharmacodynamic biomarker for monitoring the activity of PRMT5 inhibitors. This splicing-based readout may also serve as a predictive biomarker of therapeutic response, offering greater specificity than global SDMA levels. Collectively these data suggest that a PRMT5-dependent RNA splicing signature can monitor the pharmacodynamic activity of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells.

The clinical translation of RNA interference (RNAi) therapeutics remains limited by inefficient delivery and cancer-target accumulation. Here, we report the development of a new cationic liposome (CLP) nanocarrier engineered for delivery and controlled-release of small interfering RNA (siRNA) targeting the epidermal growth factor receptor (EGFR) in human colorectal cancer. CLPs were synthesized from ethylphosphocholine-based lipids and PEGylated components, with folic acid (FA) tissue-specific ligand and fluorophore labelling. These nanocarriers exhibited robust physicochemical stability across a broad pH and temperature range, efficient siRNA complexation, and nuclease-protection of siRNA. Functional studies revealed that CLP-siEGFR achieved effective cytosolic siRNA cargo release and EGFR silencing in vitro, proving to be more effective than conventional lipid-based transfection systems. In human xenograft models, intravenously administered CLP-siEGFR showed enhanced tumor localization, prolonged siRNA retention, and significant tumor growth suppression, accompanied by marked downregulation of EGFR. Importantly, systemic dosing was well-tolerated, with no evidence of hepatotoxicity, nephrotoxicity, or hematological abnormalities. These results position CLP nanocarriers as an effective platform for targeted RNAi therapeutics, offering translational potential for precision oncology applications.

The high heterogeneity of pediatric cancers presents significant diagnostic challenges, underscoring the need for accurate classification. Although molecular profiling supports first-line diagnostics and guides treatment, it can delay final diagnosis. While Nanopore-based methylation analysis has enabled rapid CNS tumor diagnosis, its application to pediatric solid tumors and lymphomas has remained largely unexplored. We developed Tucan, a deep-learning classifier trained on 3,818 methylation array profiles representing 84 subtypes, designed to classify tumors from sparse Nanopore methylation data. In retrospective validation (n=514), Tucan generated confident predictions (CFT[≥] 0.7) within 30 minutes of sequencing in 385 cases, achieving 372 correct diagnoses (F1-score: 0.98). In prospective testing (n=74; 63 classifiable), 52 samples reached the confidence threshold with 96% accuracy, confirming the original diagnosis in 47 cases and correctly refining or revising it in three. Together, Tucan enables rapid, high-confidence molecular classification of pediatric solid tumors and lymphomas.

Microsatellites are abundant genomic elements that contribute to genetic diversity and disease-associated regulatory variation. Although long-read sequencing enables accurate resolution of repetitive regions, computational methods for fully resolved microsatellite genotyping remain limited. Here, we introduce variant motif where (vmwhere), a computational framework for identifying, genotyping, decomposing, and visualizing complex tetrameric microsatellites from long-read sequencing data. Using simulated error-free reads, vmwhere accurately measures several genotyping metrics, including allele length, repeat length, maximum consecutive repeat length, and motif density. Applied to long-read whole-genome sequencing data, vmwhere identified sequence interruptions, motif-specific differences in repeat architecture, and ancestry-associated allele variation, including long repeat alleles that exceed short-read sequencing limitations. We applied vmwhere to GGAA microsatellites in Ewing sarcoma, an aggressive pediatric cancer driven by EWS-FLI1 fusion oncoprotein, which binds to microsatellites and remodels chromatin. Genome-wide integration of long-read-defined microsatellite architecture with chromatin accessibility and EWS-FLI1 binding revealed that GGAA repeat structure was associated with chromatin state, with longer consecutive repeat microsatellites exhibiting increased EWS-FLI1 binding and chromatin accessibility. Cell line-specific expansions and contractions of GGAA microsatellite repeat length were associated with gains and losses of chromatin accessibility. Further, we identified haplotype-specific chromatin states, with preferential binding and accessibility at longer alleles. Together, these results establish vmwhere as a scalable framework for resolving population-level microsatellite variation and linking repeat architecture to chromatin state. Repeat structure and length characteristics provides insights into genotype-function relationships at microsatellite repeats in cancer.

Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

Classical and basal-like transcriptional subtypes of pancreatic ductal adenocarcinoma (PDAC) are prognostic and may predict response to different chemotherapy regimens and RAS inhibitors. Current subtyping methods rely on tissue biopsies and remain challenging to integrate into clinical workflows. Herein, we present a novel approach for non-invasive subtyping of PDAC based on epigenomic profiling of circulating tumor DNA (ctDNA). In a multi-omics cohort of patient-derived xenografts, we identify highly recurrent regulatory elements associated with classical and basal-like PDAC. We then demonstrate that these epigenomic signatures can identify PDAC subtype from plasma epigenomic profiling in a multi-institutional cohort of patients with metastatic PDAC and integrate information from circulating histone modifications and DNA methylation to develop the Pancreatic Integrated Epigenomic Score (PIES). PIES is concordant with tissue-based labels and captures transcriptional subtype heterogeneity observed within biopsies. Furthermore, it improves prognostication over tissue-based subtyping suggestive of the recovery of ground truth tumor biology from plasma ctDNA. Our work provides a proof-of-concept for a circulating biomarker that enables transcriptional subtyping and informs therapeutic decisions in pancreatic cancer.

BackgroundLong non-coding RNAs (lncRNAs) have emerged as key regulators of tumor biology, however, thus far none have translated to cancer therapies. The lncRNA MALAT1 is overexpressed in more than 20 cancers, including breast cancer and has been shown to function via various mechanisms in a context-dependent manner, in 2D cell lines and mouse models. However, its functional role and therapeutic potential have not been evaluated in clinically relevant patient-derived models. MethodsWe investigated the therapeutic potential of a MALAT1-targeting antisense oligonucleotide (ASO) for breast cancer, using clinically relevant 3D human patient-derived organoids (PDOs) and PDO-xenograft (PDO-X) models. We systematically evaluated the efficiency of MALAT1-targeting ASOs using a biobank of 28 PDO models. Using three independent PDO-X models of triple negative breast cancer (TNBC), we targeted MALAT1 in vivo to study its impact on transcription, alternative splicing, stromal remodeling and metastasis. ResultsAcross PDO-X models, MALAT1 depletion reproducibly drove widespread alternative splicing changes across all event types, particularly intron retention events, accompanied by modest gene expression alterations. Differentially spliced transcripts were enriched for targets of shared cancer-associated transcription factors, and MALAT1 knockdown altered the relative abundance of previously unannotated splicing isoforms. Beyond tumor-intrinsic effects, tumor-specific MALAT1 depletion induced a consistent reduction in macrophage-associated gene signatures and reduced lung metastatic burden. ConclusionsOur data define MALAT1s multifaceted role in TNBC, coordinating alternative splicing, transcriptional fine-tuning, tumor-stroma crosstalk, and metastatic progression. Our study provides strong preclinical evidence supporting MALAT1-targeted ASO therapy and establishes PDO-X models as a clinically relevant platform for functional interrogation of TNBC therapies.

Cancer cell evasion of therapy is a highly adaptive process that undermines the efficacy of many treatment strategies. A significant milestone in the study of these mechanisms has been the advent of pooled CRISPR knockout screens, which enable high-throughput, genome-wide interrogations of tumor dependencies and synthetic lethal interactions, advancing our understanding of how cancer cells adapt to and evade therapies. However, the utility of this approach diminishes when applied to dynamic biological contexts, where processes are transient and sensitivity to routine cell culture manipulations that introduce noise and limit meaningful discoveries. To overcome these limitations, we present RESTRICT-seq, a next-generation pooled screening methodology that restricts Cas9 nuclear activation in controlled, repeated cycles. By confining Cas9 catalytic activity to strict temporal windows, RESTRICT-seq mitigates undesired fitness penalties that routinely accumulate throughout pooled screens. When benchmarked against conventional pooled screens and standard inducible protocols, RESTRICT-seq revealed significantly fewer divergent cell clones and increased signal-to-noise ratio, overcoming a key limitation of traditional methods. Leveraging RESTRICT-seq, we conducted a comprehensive functional survey of the druggable mammalian epigenome, uncovering several elusive epigenetic drivers of treatment resistance in cutaneous squamous cell carcinoma (cSCC). This revealed PAK1 as a previously unrecognized mediator of cSCC resistance in human and mouse SCC, offering new insights into a prognostic marker and therapeutic target of high clinical significance. Our findings establish RESTRICT-seq as a powerful tool for extending the applicability of pooled CRISPR screens to dynamic and previously intractable biological contexts.

Osteosarcoma is a malignant bone tumor with a high risk of metastatic relapse and poor outcomes due to primary and acquired chemoresistance. This highlights the medical need to develop effective targeted approaches to overcome chemoresistance. Recent studies have revealed the roles of metabolic reprogramming and mitochondria-nucleus crosstalk in osteosarcoma progression, indicating the potential of these cellular processes as therapeutic targets. The complex formed by mitochondrial apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) orchestrates the import and oxidative folding of cysteine-rich, nuclear-encoded proteins, thereby regulating key mitochondrial functions and metabolism. Here, we identified mitoxantrone as an inhibitor of the AIF/CHCHD4 mitochondrial import machinery and revealed a new mitoxantrone-induced metabolic vulnerability in some osteosarcoma cell line models, characterized by intracellular glutamine accumulation and an increase in nucleotide synthesis. As a result, synergy was found between mitoxantrone and the glutaminase inhibitor telaglenastat in both in vitro and in vivo osteosarcoma models. Collectively, our findings position the AIF/CHCHD4 complex as a druggable therapeutic target and provide a combination strategy for mitoxantrone/telaglenastat treatment to overcome metabolic adaptations and chemoresistance in osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/716303v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1229e58org.highwire.dtl.DTLVardef@1c9af45org.highwire.dtl.DTLVardef@120d2borg.highwire.dtl.DTLVardef@11e8216_HPS_FORMAT_FIGEXP M_FIG C_FIG

For mRNA-based cancer gene therapy, we engineered a membrane-bound fusion protein combining interferon-{gamma} (IFN{gamma}) with the Fas intracellular domain (FasICD) to couple local IFN{gamma} signaling with Fas-driven apoptotic tumor cell death. IFN{gamma}-FasICD was robustly expressed on the plasma membrane after mRNA transfection. In murine cancer cell lines, IFN{gamma}-FasICD mRNA reduced viability within 24 h, resulting in [~]50% cell death in MC38 cells and [~]75% in B16OVA cells, exceeding the cytotoxicity of the FasICD-deleted control (IFN{gamma}-Fas{Delta}). Mechanistically, IFN{gamma}-FasICD induced predominantly apoptotic rather than necrotic cell death. IFN{gamma}-FasICD also activated IFN{gamma} receptor signaling in both cancer and the immune cells, inducing IFN{gamma}-responsive genes in IFN{gamma}R-high B16OVA cells and triggering STAT1 phosphorylation in co-cultured splenocytes. For in vivo delivery, IFN{gamma}-FasICD mRNA was formulated in lipid nanoparticles (LNPs), enabling strong intratumoral expression that peaked at [~]3 h and persisted for more than 48 h. Repeated intratumoral injections of LNP-formulated IFN{gamma}-FasICD mRNA suppressed the growth of established B16OVA and MC38 tumors and improved survival, with [~]40% and [~]20% of mice surviving beyond 30 days, respectively. IFN{gamma}-FasICD treatment remodeled the tumor microenvironment by increasing tumor-infiltrating CD45+ cells and CD8+ T cells, while further reducing FOXP3+ regulatory T cells. Moreover, NK/NKT cells and cDC1/cDC2 populations were increased, and their activation was enhanced. In tumor-draining lymph nodes, IFN{gamma}-FasICD mRNA promoted dendritic cell migration and increased priming and differentiation of CD8+ T cells toward effector and memory phenotypes, accompanied by enhanced functional activation of IFN{gamma}-producing CD8+ T cells and highly cytotoxic NK cells in peripheral blood. Overall, our findings provide a mechanistic foundation for cytokine-death receptor fusion proteins as an in vivo antitumor strategy that can reprogram tumor cells into localized sources of both apoptotic signals and immune-activating cues.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer characterized by a high abundance of cancer-associated fibroblasts (CAFs), which influence therapy response, tumor biology and tumor aggressiveness. CAFs are a heterogeneous cell type and previous single-cell RNA sequencing (scRNAseq) of PDAC tumors identified three main CAF subtypes: myofibroblastic, inflammatory and antigen-presenting CAFs (myCAF, iCAF, apCAF, respectively). However, scRNAseq on large patient cohorts is often not feasible due to costs and technical constraints. Therefore, bulk RNAseq deconvolution can be used to identify cell types within the heterogeneous tumor microenvironment. Here, Statescope deconvolution was used to identify different cell types of the tumor microenvironment within an early onset PDAC cohort, comprising 74 patients aged under 60. Three CAF populations were identified (iCAFs, myCAFs and desmoplastic CAFs), and their correlations with tumor microenvironment components, mutational signatures and survival were examined. iCAFs were associated with classical-like tumor cells, whereas myCAFs and desmoplastic CAFs correlated with basal-like tumor cells. Desmoplastic CAFs are associated with inflammatory granulocytes/neutrophils, while negatively associating with monocyte-derived macrophages and immature/transitional B cells. No associations were observed between mutational signatures and the abundance of CAF and epithelial tumor subtypes. Interestingly, a high abundance of CAFs, and specifically increased iCAF abundance, was associated with improved survival. This iCAF-mediated survival effect was predominantly apparent in female patients. All in all, deconvolution of bulk RNA sequencing data, followed by its integration with clinical and biological parameters, reveals the heterogeneity and prognostic implications of CAF subpopulations in the tumor microenvironment of early onset PDAC patients.

Meningiomas are the most common primary central nervous system tumors in adults, posing a significant burden to society. Although a large percentage of lower-grade meningiomas are curable by surgery or radiation alone, high-grade and a subset of low-grade meningiomas demonstrate recurrences and complications from treatment. Systemic therapies for meningioma remain ineffective, and no targeted treatments are approved. Despite the central role of YAP1/TAZ-TEAD signaling in NF2-deficient/mutant tumors, no studies have systematically examined TEAD inhibition across molecularly defined meningioma subtypes or investigated mechanisms of resistance in this disease. We have recently shown that YAP1/TAZ signaling is an oncogenic driver of meningioma. Here, using established and patient-derived meningioma cell lines, we demonstrate that genetic ablation of YAP1/TAZ suppresses growth in both NF2 mutant and NF2 wild type cell lines, establishing YAP1/TAZ-TEAD signaling as a shared oncogenic dependency. Pharmacologic TEAD inhibition suppressed growth of benign NF2 mutant and a subset of higher-grade NF2 mutant meningiomas, whereas NF2 wild type meningiomas were generally more resistant. RNA-Seq and Western Blot analysis identified compensatory activation of MEK-ERK, mTOR-S6, and FAK signaling in resistant lines exhibit. Importantly, co-targeting these pathways was able to overcome resistance to TEADi and was superior to MEK/mTOR/FAK inhibition alone. These studies provide a compelling proof-of-concept that TEADi represents a novel therapeutic vulnerability in meningioma and reveal adaptive signaling responses that can be therapeutically exploited.

Accurate and timely diagnosis is essential for effective lung cancer treatment. However, contemporary diagnostic workflows rely on sequential immunohistochemistry of small biopsy specimens, which can exhaust limited tissue, compromise diagnostic accuracy, and delay treatment decisions with clinical consequences. Here, we demonstrate that multiplexed imaging overcomes these limitations by enabling comprehensive lung cancer diagnosis from a single tissue section. We developed and validated a clinically informed multiplexed antibody panel that integrates tumor diagnosis and classification, predictive biomarker assessment, and tumor immune profiling. In diagnostic biopsies, multiplexed imaging achieved 96% concordance with standard pathological diagnosis, while enabling accurate automated PD-L1 scoring and rapid detection of clinically approved and emerging actionable targets. This approach preserves scarce tissue, supports quantitative computational analysis to streamline diagnosis, and generates research-grade spatial data while accelerating diagnostic workflow. Together, these findings establish multiplexed imaging as a robust, time and tissue-efficient framework for lung cancer diagnostics that bridges clinical care and translational discovery.

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Centrosomes are essential for spindle assembly and accurate chromosome segregation during mitosis. We found that the RNA demethylase FTO localizes to centrosomes in both human and mouse cells. Disruption of FTO function through a newly developed small-molecule inhibitor impaired spindle organization and centrosome function. This was evidenced by chromosome misalignment during mitosis, prometaphase arrest, followed by mitotic slippage or apoptosis. Mechanistically, we found that FTO is essential for intact localization of NuMA and KIFC1 proteins required for chromosome localization during mitosis. Importantly, cancers characterized by chromosomal instability (CIN) showed increased sensitivity to FTO inhibition compared to normal cells. These findings underscore FTO as a promising therapeutic target in CIN cancer subsets.

The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated -omics approach revealed off-target effects for thienopyridine carboxamide compounds upon the translation apparatus. Chemoproteomics and CRISPR-GOF analyses suggested binding of the compound to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting the TP63-FBW7-c-MYC signaling axis, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas arising sporadically or in people with neurofibromatosis type 1 (NF1). Their marked heterogeneity challenges diagnosis and has hampered an integrative view of MPNST molecular pathogenesis. Here, a thorough whole-genome and transcriptome analysis of MPNSTs and the re-analysis of a large independent cohort allowed us to identify three molecular subtypes of MPNSTs (G1-G3) with distinct genomic identities and clinicopathological features. Furthermore, it provided a simple and unifying model of MPNST development, defining a distinct progression path for each group. This work uncovers new genomic aspects of MPNSTs, including the identification of recurrent copy-neutral loss of heterozygosity regions, distinct copy-number profiles among G1-G3, and CDKN2A-inactivating translocations in pre-malignant lesions (ANNUBPs). Altogether, these analyses overcome the dominant influence of PRC2 status in MPNST classification and provide a framework for their differential diagnosis and potential precision oncology treatment. SIGNIFICANCEMPNST is a highly heterogeneous soft-tissue sarcoma with difficult clinical management and no effective systemic therapies. This work defines three molecular subtypes of MPNSTs with distinct development paths and histological and clinical characteristics with potential impact on translational studies and subtype-tailored treatments.