BACKGROUND:Lung adenocarcinoma (LUAD) remains a leading cause of cancer mortality, and current therapies are limited by drug resistance and toxicity. Ferroptosis offers an attractive strategy for cancer therapy, but classical ferroptosis is iron‑dependent. Repurposing approved drugs offers a rapid strategy to identify novel anti‑LUAD agents, yet the mechanism by which 4‑Hydroxytamoxifen (4‑OHT) exerts estrogen receptor-independent anti‑tumour effects remains unclear. METHODS:We performed a drug repurposing screen of 950 endogenous metabolites in LUAD cells. Anti‑tumour activity was validated in vitro and in vivo using LUAD cell lines and mouse models. Target identification and mechanistic studies employed LiP‑MS, pull-down SPR, CETSA, metabolomics, and enzyme activity assays. Clinical relevance was assessed via tissue microarrays and TCGA analysis. Drug synergy was quantified using the Chou-Talalay method. RESULTS:4‑OHT inhibited LUAD cell proliferation with IC50 values of 3.86 ± 0.26 µM to 13.58 ± 0.55 µM independent of estrogen receptor. It triggered noncanonical ferroptosis characterised by GSH depletion (reduced by about 60%), GPX4 downregulation, lipid peroxidation (about 2.5-fold increase), and iron independence. Mechanistically, 4‑OHT directly bound AKR1B10 (KD(M) was 7.98e-06) and inhibited its enzymatic activity, leading to ACC1 degradation, lipid deficiency, and accumulation of toxic lipid‑derived carbonyls. AKR1B10 knockout abolished 4‑OHT‑induced cell death, while rescue restored sensitivity. Nanatinostat similarly targeted AKR1B10 and induced the same cell death modality. AKR1B10 upregulation in LUAD tissues correlated with reduced overall survival rates. CONCLUSION:This study demonstrates that 4‑OHT directly inhibits AKR1B10 enzymatic activity to induce a noncanonical ferroptosis in LUAD. AKR1B10 overexpression correlates with poor prognosis and chemoresistance in LUAD patients. Nonetheless, AKR1B10 inhibition warrants further investigation as a therapeutic strategy. HIGHLIGHTS:4‑OHT directly inhibits AKR1B10 enzymatic activity to induce noncanonical ferroptosis in LUAD. AKR1B10 sustains LUAD survival by stabilising ACC1 and detoxifying reactive carbonyl species (RCSs). Nanatinostat similarly targets AKR1B10 and elicits the same cell death modality. AKR1B10 overexpression correlates with poor prognosis and chemoresistance in LUAD.
BACKGROUND:Oocyte cryopreservation is widely used in assisted reproductive technology, but its effects on embryonic development remain a concern. This study aimed to determine whether oocyte cryopreservation induces transcriptional alterations at the early cleavage and blastocyst stages. METHODS:We performed single-cell RNA sequencing on mouse Metaphase II oocyte and embryos at early/late 1 cell, early/late 2 cell, morula and blastocyst stages derived from cryopreserved (cryo) and fresh (fresh) oocytes. Transcriptomic profiles were compared between the two groups. RESULTS:Global transcriptomes were highly similar between cryo and fresh blastocysts. Oocyte cryopreservation did not alter the sex ratio or the developmental progression through preimplantation development. In blastocysts, cryopreservation affected only a few individual genes. In both male and female blastocysts, although oocyte cryopreservation increased the proportion of low Xist-expressing cells, it did not disrupt X-linked gene dosage. In the inner cell mass (ICM) of blastocysts, a negative correlation between X-linked gene expression and cell cycle progression was identified, and this negative correlation was more pronounced in trophectoderm (TE) cells. Overall, this stage-dependent coordination remained unperturbed by cryopreservation. CellChat analysis revealed that the signalling interaction strength between ICM and TE also showed no significant changes in the cryo group. CONCLUSIONS:Oocyte cryopreservation does not severely disrupt overall transcriptional integrity, developmental potency and X-chromosome dosage compensation of embryos, but it may cause subtle molecular changes. These findings highlight the need for continued refinement of cryopreservation methods and further investigation into the sub-acute safety of assisted reproductive technology procedures.
BACKGROUND:Doxorubicin, an anthracycline chemotherapeutic agent, is widely used in diffuse large B‑cell lymphoma (DLBCL) treatment, yet its clinical efficacy is often compromised by drug resistance. Protein arginine methyltransferase 7 (PRMT7) is a methyltransferase implicated in tumourigenesis and cancer progression. However, its precise role and underlying mechanisms in DLBCL progression and doxorubicin resistance remain unclear. METHODS:We analysed PRMT7 expression in DLBCL cell lines and patient specimens using bioinformatic databases, western blotting, reverse Transcription Polymerase Chain Reactionand immunohistochemistry. Functional studies were performed in DLBCL cell lines through CRISPR/Cas9‑mediated knockout and overexpression systems, combined with in vitro assays for proliferation, apoptosis and doxorubicin sensitivity, as well as in vivo xenograft models. Mechanistically, co‑immunoprecipitation, arginine methylation assays and immunofluorescence were employed to characterise the PRMT7-Forkhead box K (FOXK)1/2-Dishevelled Segment Polarity Protein 2 (DVL2) axis and its modulation of Wnt/ (beta) β‑catenin signalling. In addition, we designed and evaluated FOXK‑methylation‑competitive inhibitory peptides for their capacity to sensitise DLBCL cells to doxorubicin. RESULTS:Our study revealed that PRMT7 is upregulated in DLBCL and promotes both tumour proliferation and doxorubicin resistance. Conversely, knockdown of PRMT7 inhibited DLBCL cell proliferation and enhanced sensitivity to doxorubicin. Mechanistically, PRMT7 catalyses arginine methylation of FOXK1 at arginine (R) 191 and FOXK2 at R144, which markedly increases their binding affinity for DVL2 and facilitates DVL2 nuclear translocation. This event leads to constitutive activation of the Wnt/β‑catenin signalling pathway. Importantly, we developed a FOXK‑derived peptide that competitively inhibits FOXK methylation, suppresses Wnt/β‑catenin signalling and significantly potentiates the antitumour efficacy of doxorubicin in DLBCL. CONCLUSION:Our findings indicate that the PRMT7/FOXK/DVL2/Wnt-β-catenin axis serves as a novel driver of DLBCL progression and doxorubicin resistance. Targeting the PRMT7-FOXK methylation interface may offer a potential therapeutic approach for overcoming doxorubicin resistance in DLBCL, although further validation in clinical settings is warranted. KEY POINTS:Upregulated PRMT7 drives DLBCL progression and doxorubicin resistance PRMT7 methylates FOXK1 at arginine 191 and FOXK2 at arginine 144 FOXK1/2 methylation enhances interaction with DVL2 and promotes nuclear translocation FOXK1/2 methylation dactivate Wnt/ß-catenin signaling through DVL2 nuclear entry A novel peptide inhibitory blocks FOXK1/2 methylation and restores doxorubicin sensitivity.
BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant solid tumors, characterized by strong invasiveness and poor clinical prognosis. Recent studies have revealed that long non-coding RNAs (lncRNAs) can serve as translational templates to produce functional microproteins, which further participate in the regulation of cellular metabolism. OBJECTIVE:To investigate the function and detailed mechanism of the novel protein 1503-91aa encoded by LINC01503 in gemcitabine-resistant PDAC, and explore the possibility of clinical translation. METHODS:The differentially expressed lncRNAs in gemcitabine-resistant PDAC cells with coding potential were screened by whole transcriptome sequencing and coding prediction from database. A series of cellular, molecular and in vivo assays were performed to characterize the novel microprotein 1503-91aa, its upstream transcriptional regulation, and its role in mediating PDAC gemcitabine resistance. A series of molecular assays clarified the interaction between 1503-91aa and carnitine palmitoyl transferase 1A (CPT1A). Finally, the clinical translational potential of the CPT1A inhibitor etomoxir in combination with gemcitabine was validated using our center's gemcitabine-resistant patient-derived xenograft (PDX) model. RESULTS:Here, we revealed that LINC01503 had coding potential in drug-resistant PDAC and encoded a novel 91-amino acid protein, which designated as 1503-91aa. Simultaneously, LINC01503's transcriptional regulation is mediated by CTCF and then 1503-91aa expression levels were significantly upregulated in PDAC. Functionally, 1503-91aa, instead of LINC01503 confers gemcitabine resistance in vitro and in vivo. Mechanistically, 1503-91aa targeted the CPT1A, and up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote fatty acid oxidation (FAO) and thereby facilitate gemcitabine resistance. We discovered that CPT1A functions as a succinyltransferase to regulate the succinylation level of CTCF, thereby controlling its stability. This establishes a positive feedback loop that continuously sustains 1503-91aa protein levels to regulate CPT1A activity. Consequently, this induces persistent FAO in PDAC cells, ensuring a continuous energy source that facilitates gemcitabine resistance in pancreatic cancer. Notably, the combined application of the etomoxir and gemcitabine exerts a synergistic effect on PDAC. CONCLUSIONS:These findings provide novel insights into the molecular mechanisms underlying gemcitabine resistance in PDAC and highlight CTCF/LINC01503-91aa/CPT1A feedback loop as a potential prognostic biomarkers and therapeutic targets. KEY POINTS:We identified that LINC01503 is capable of encoding the protein product 1503-91aa. 1503-91aa competed with MCoA to unleash CPT1A activity for fatty acid oxidation and thereby mediating gemcitabine resistance in PDAC. Etomoxir and gemcitabine have synergistic effects in gemcitabine efficiency.
BACKGROUND:Nailfold capillaroscopy is a non-invasive method to visualize altered microcirculation in pediatric rheumatic disease, with potential to aid in diagnostic differentiation and disease monitoring. We used nailfold video capillaroscopy (NVC) and machine learning to identify patterns of capillaroscopic features in juvenile dermatomyositis (JDM) and associations of capillaroscopic features with clinical data. METHODS:NVC features were quantified using automated neural network-based software in 76 individuals, including 18 controls, 33 JDM, 17 childhood-onset systemic lupus erythematosus (cSLE), and 8 overlap myositis (OM) patients. Unsupervised cluster analysis by capillaroscopic features was performed, and clinical features were characterized by cluster. Differences in capillaroscopic features between disease groups were assessed using the Kruskal-Wallis test. For JDM and OM patients, capillaroscopic and clinical feature associations were assessed using Spearman correlation. In 10 treatment-naïve myositis patients (JDM + OM), changes in capillaroscopic features between diagnosis and 3-month follow-up were evaluated. RESULTS:Cluster analysis identified three patient clusters, characterized by the presence of either branched or enlarged capillaries, or absence of these abnormalities. The "branched" cluster was most frequently assigned among JDM and OM and consisted of no controls. The majority of TIF1y+ JDM (4/5) were in the "branched" cluster. In JDM and OM, microhaemorrhage density correlated with disease duration (r = -.45, p = .0033), physician global assessment score (r = .6, p = .0004), lactate dehydrogenase (r = .43, p = .01), von Willebrand factor antigen (r = .51, p = .043), and neopterin (r = .69, p = .01). In 10 myositis patients, microhaemorrhage density decreased while capillary density increased from diagnosis to three months post-treatment. CONCLUSIONS:A branched capillaroscopic pattern was observed more frequently in JDM and OM, particularly TIF1y+ JDM patients within our cohort. Microhaemorrhage density was the most changeable capillaroscopic feature and associated with markers of increased disease activity.
BACKGROUND:The pathology of intervertebral disc degeneration (IDD) is characterized by metabolic dysregulation within nucleus pulposus (NP) cells. TRIM25 has been implicated in diverse tumors and pathological processes, yet its precise role in mediating mitochondrial function and metabolic alterations during IDD progression remains unclear. METHODS:Transcriptome sequencing was performed to analyze gene expression changes during IDD progression. Molecular biology techniques including co-immunoprecipitation and ubiquitination assays were used to investigate the interaction between TRIM25 and the glycolytic enzyme ENO1 and its regulatory mechanism. Cellular and in vivo animal models were employed to validate the effects of the TRIM25-USP7-ENO1 axis on glycolysis, mitochondrial function, ATP levels, and extracellular matrix degradation. RESULTS:Transcriptome sequencing revealed that glycolysis-related pathways and TRIM25 were significantly upregulated during IDD progression. Mechanistically, TRIM25 interacted with ENO1. Contrary to its typical E3 ligase function, TRIM25 overexpression stabilized ENO1 by reducing its K48-linked polyubiquitination. Furthermore, TRIM25 enhanced the interaction between USP7 and ENO1, leading to USP7-mediated deubiquitination and stabilization of ENO1. Disruption of the TRIM25-USP7-ENO1 axis suppressed glycolysis, improved mitochondrial function, elevated ATP levels, and inhibited extracellular matrix degradation. In vivo, modulation of this axis correspondingly accelerated or ameliorated IDD progression. CONCLUSION:We identified a novel TRIM25-USP7-ENO1 axis, through which TRIM25 stabilizes ENO1 by promoting USP7-ENO1 interaction and subsequent USP7-dependent deubiquitination. This non-canonical function expands the known role of TRIM25 and highlights a promising therapeutic target for restoring mitochondrial dysfunction and metabolic homeostasis in IDD. KEY POINTS:TRIM25 recruits the deubiquitinating enzyme USP7 to collaboratively enhance the deubiquitination and stability of the key glycolytic enzyme ENO1, establishing a new regulatory pathway linking inflammation and metabolism. This regulatory axis exacerbates glycolysis, impairs mitochondrial function, disrupts cellular energy homeostasis, and ultimately leads to extracellular matrix degradation, systematically explaining a new pathological mechanism of IDD. Both cellular and animal models confirm that intervention in the TRIM25/USP7/ENO1 axis effectively reverses metabolic imbalance and degenerative progression, offering a promising new therapeutic strategy for IDD. This study extends TRIM25's role from immune regulation to metabolic processes and is the first to report USP7's involvement in glycolytic enzyme regulation, providing a new paradigm for studying "non-canonical" functions of E3 ligases and deubiquitinating enzymes.
BACKGROUND:As a pivotal metabolic enzyme, cysteine dioxygenase type 1 (CDO1) exerts tumour-suppressive effects across diverse tumour types, and its expression is strongly correlated with clinical prognosis. However, the molecular mechanisms underlying CDO1-mediated tumour suppression in gastric cancer (GC), its relationship with the tumour-associated immune microenvironment, and pharmacological strategies to restore its expression remain poorly understood. METHODS:CDO1 expression and prognosis were evaluated by multi-omics and tissue microarray analyses. Tumour microenvironment and immune infiltration were analyzed using ESTIMATE and ssGSEA. Downstream pathways and interacting proteins were identified by transcriptomics, co-immunoprecipitation, and GST pull-down. CDO1 function was assessed by proliferation, apoptosis, and migration assays in gain- and loss-of-function models. In vivo tumorigenesis and CDO1-dependent decitabine efficacy were evaluated by subcutaneous xenografts. Patient-derived organoids were used to assess decitabine sensitivity and 5-FU synergy. RESULTS:Compared with normal controls, CDO1 expression was notably decreased in GC tissues, and its low expression was strongly linked to unfavourable prognosis, supporting its utility as a biomarker for prognosis. Elevated CDO1 levels correlated with an immune-active tumour microenvironment and reduced metastatic signatures. Mechanistically, CDO1 directly bound to PI3K p85α, disrupting p85α-p110α dimerization, thereby attenuating PI3K/AKT phosphorylation and downregulating THBS1 expression. CDO1 overexpression led to reduced proliferation, invasiveness, and EMT, accompanied by increased apoptosis. These effects were reversed by PI3K activation or THBS1 co-overexpression. Decitabine was identified as an agent that epigenetically restores CDO1 expression. Critically, CDO1 knockdown significantly attenuated the anti-tumour efficacy of decitabine in vivo, confirming that decitabine acts primarily through CDO1 reactivation. Decitabine synergized with 5-FU in both organoids and xenografts. CONCLUSIONS:Our data identify CDO1 as both a biomarker for prognosis and a tumour suppressor in gastric cancer. They reveal a CDO1-PI3K/AKT-THBS1 signalling axis and support the epigenetic reactivation of CDO1 by decitabine as a translatable therapeutic strategy. KEY POINTS:CDO1 is frequently downregulated in gastric cancer and serves as an independent favourable prognostic biomarker. CDO1 directly binds PI3K p85α, disrupting p85α-p110α dimerization to suppress the PI3K/AKT-THBS1 signalling axis. Decitabine epigenetically restores CDO1 expression, and its anti-tumour activity is critically CDO1-dependent in vivo. Combining decitabine with 5-FU synergistically overcomes gastric cancer growth in patient-derived organoids and subcutaneous xenograft models.
Abstract Background Chronic stress is increasingly recognised as a risk factor for poor prognosis in colorectal cancer (CRC) through sustained activation of the hypothalamic–pituitary–adrenal (HPA) axis and sympathetic nervous system, yet the mechanisms by which psychological stress signals are transmitted from the central nervous system to the peripheral tumour immune microenvironment remain poorly understood. Brain‐derived small extracellular vesicles (BD‐sEVs), which can cross the blood–brain barrier, represent potential mediators of this neuroimmune crosstalk. Methods A chronic restraint stress (CRS) mouse model was established in both subcutaneous (MC38) and orthotopic (CT26) CRC models. BD‐sEVs were enriched from plasma using anti‐L1 cell adhesion molecule (L1CAM) antibodies and characterised by transmission electron microscopy and nanoparticle tracking analysis. Multichannel flow cytometry was used to analyse immune cell populations in the tumour microenvironment. Mechanistic studies included microRNA (miRNA) sequencing, single‐cell RNA sequencing, co‐immunoprecipitation, mass spectrometry, mitochondrial function assessment and mitochondrial DNA (mtDNA) detection. Clinical validation was performed in a retrospective cohort (n = 67) and a prospective cohort (n = 37) of CRC patients, with anxiety levels assessed by the Hamilton Anxiety Rating Scale (HAMA). Results We show that chronic stress‐activated brain regions remodel the miRNA cargo of circulating BD‐sEVs, which are taken up by CD4+ naïve T cells within the tumour microenvironment, promoting regulatory T‐cell (Treg) differentiation and immunosuppressive function. Mechanistically, stress‐responsive miRNAs (miR‐342‐3p, miR‐15a‐5p and miR‐381‐3p) upregulate phorbol‐12‐myristate‐13‐acetate‐induced protein 1 (NOXA), which directly binds the mitochondrial chaperone heat shock protein 60 (HSP60) and inhibits its chaperone activity, triggering selective mtDNA release into the cytoplasm. Cytosolic mtDNA activates the cyclic GMP‒AMP synthase (cGAS)‒stimulator of interferon genes (STING)‒type I interferon (IFN‐I) pathway, driving Treg differentiation independently of the canonical myeloid cell leukaemia 1 (MCL‐1)‐dependent apoptotic pathway. Clinically, intratumoural CD4+NOXA/Forkhead box P3 (FOXP3) expression correlates significantly with patient anxiety scores and predicts adverse survival outcomes. Conclusion Our study reveals an immunosuppressive ‘chronic stress‒BD‐sEV‒NOXA‒HSP60‒Treg’ axis and provides crucial mechanistic insights into the psychoneuroimmunological contributions to cancer progression and novel targets for therapeutic intervention. Key points Chronic stress promotes the differentiation of regulatory T cells (Treg) in the tumour microenvironment through brain‐derived small extracellular vesicles (BD‐sEVs) to inhibit antitumour immunity. Mechanistically, BD‐sEV‐induced phorbol‐12‐myristate‐13‐acetate‐induced protein 1 (NOXA) binds heat shock protein 60 (HSP60) and inhibits its chaperone activity, triggering mitochondrial DNA (mtDNA) release and activation of the cyclic GMP‒AMP synthase (cGAS)‒stimulator of interferon genes (STING) pathway to drive Treg differentiation. The stress‒BD‐sEV‒NOXA‒HSP60‒Treg axis is a key pathway that connects psychological stress with peripheral tumour immunosuppression. The expression of NOXA and Forkhead box P3 (FOXP3) in clinical tumour samples correlates with the level of anxiety and may serve as potential prognostic biomarkers.
Abstract Background Promyelocytic leukaemia protein (PML), a key regulator of nuclear architecture and cellular homeostasis, is increasingly recognised for its tumour‐suppressive functions. In neuroblastoma (NBL), a clinically heterogeneous and aggressive paediatric malignancy, the prognostic role of PML and its post‐translational modifications remains largely unexplored. Methods We investigated the prognostic and predictive significance of PML expression and its site‐specific phosphorylation at serine 518 (S518) in a cohort of 121 NBL patients. Custom‐synthesised antibodies and high‐resolution tissue microarrays were used to quantitatively profile PML and pPML S518 expression via automated immunohistochemistry and digital image analysis. Survival outcomes were assessed using Kaplan‐Meier and Cox regression analyses. Mechanistic insights were obtained using reverse‑engineered phospho‑mutant models enabling selective modulation of S518 phosphorylation. Results Our findings reveal a striking inverse relationship between PML abundance and S518 phosphorylation, with low PML and high pPML S518 strongly associated with advanced disease stage, metastasis, relapse, and therapy resistance. Survival analyses demonstrate that low PML predicts poor overall survival (OS), progression‐free survival (PFS), and relapse‐free survival (RFS), while elevated pPML S518 correlates with significantly worse outcomes across all endpoints. Multivariate Cox regression confirms both markers as independent predictors of survival. Mechanistically, reverse‐engineered phospho‐mutant models that enable selective switch‐on/switch‐off modulation of S518 phosphorylation demonstrated that phosphorylation at this site reduces PML abundance and promotes invasive cellular phenotype. Conclusion PML loss and site‐specific phosphorylation of PML at S518 represent robust prognostic and predictive biomarkers with potential utility in risk stratification. Targeting PML phosphorylation may offer a promising translational strategy to improve therapeutic efficacy in high‑risk NBL. Key points PML depletion is a defining feature of aggressive neuroblastoma and is associated with advanced stage, metastasis and relapse. S518 phosphorylation emerges as the dominant post translational trigger committing PML to ubiquitin mediated degradation. High pPML S518 and low PML form a powerful prognostic axis that independently predicts OS, PFS, and RFS in neuroblastoma. PML loss and S518 phosphorylation define a molecular framework of heightened cellular plasticity underlying aggressive clinical behavior.
Abstract Background The interferon‐induced transmembrane (IFITM) protein family, including IFITM1, IFITM2, IFITM3, and the less‐characterized IFITM5 and IFITM10, has emerged as a recurrent yet mechanistically complex group of players in cancer, reported as oncogenic in one setting and tumor‐restraining in another, immunosensitizing in one context and immunotherapy‐resistance‐promoting in the next. Rather than treating this literature as contradictory, we argue it is unresolved, and we organize it around a four‐axis framework defined by the interaction of member identity, interferon (IFN)‐input quality, cellular compartment, and tumor milieu. Main body We first establish the foundational biology that compels this framework: family members are not interchangeable, occupying distinct subcellular compartments and partner networks; their abundance is set jointly by upstream IFN signaling and tumor‐cell‐intrinsic regulators; and post‐translational state gates their function independently of expression. We then refine the prevailing view of IFN signaling as monolithic, reformulating the direction of IFITM functional output around an acute‐versus‐chronic IFN distinction in which transient signaling drives major histocompatibility complex class I (MHC‐I) induction, antigen presentation, and immunogenicity, whereas sustained signaling promotes programmed death‐ligand 1 (PD‐L1)‐coupled adaptive immune resistance and engagement of the interferon‐stimulated gene resistance signature (ISG.RS). Surveying tumor‐cell‐intrinsic, immune‐cell‐intrinsic, stromal, and microbiota‐conditioned compartments across diverse malignancies, we show that the sign of any IFITM effect is set by where, when, and which member is expressed. The field's most prominent conflicts are reframed as falsifiable hypotheses with defined discriminating experiments. Conclusion We conclude that translational maturity remains limited to biomarker discovery, and that compartment‐resolved, IFN‐quality‐stratified, immune‐competent studies, not further expression surveys, are the necessary next step. Key points Member, IFN quality, compartment, and milieu define IFITM cancer effects. IFITM paralogs engage distinct signaling networks across tumor and immune cells. Acute IFN favors MHC‐I immunogenicity; chronic IFN drives PD‐L1 resistance. Context resolves opposing IFITM effects into experimentally testable predictions. Near‐term translation favors biomarker stratification over direct IFITM targeting.
Abstract Background Diffuse large B‐cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Methods In this study, we analysed a real‐world cohort of 178 newly diagnosed DLBCL patients homogeneously treated with R‐CHOP‐like regimens, integrating RNA sequencing, targeted mutational profiling and digital deconvolution using the EcoTyper algorithm. Results Five malignant B‐cell states (S1–S5) were identified, each reflecting distinct differentiation and transcriptional programs with prognostic relevance, S1 and S5 representing the most favourable and adverse subtypes, respectively. EcoTyper‐defined lymphoma ecotypes (LE1–LE9), capturing co‐associations between malignant and microenvironmental cell states, further refined prognostic classification, with LE1/LE2 and LE9 associated with the worst and most favourable prognosis, respectively. Both B‐cell states and LE retained independent prognostic value in multivariable analyses after adjusting for the cell of origin and the revised international prognostic index. Mutational profiling revealed state‐specific patterns: S1 harboured mutations in genes involved in epigenetic regulation, cytoskeletal organisation and tumour microenvironment (TME) interactions (CREBBP, EZH2 and ACTB), whereas S5 was enriched for mutations promoting survival signalling, immune evasion and differentiation blockade (MYD88, CD79B and TP53). In vitro validation using human five DLBCL cell lines independently recapitulated the S1 and S5 transcriptomic states, supporting the existence of intrinsic, cell‐autonomous biological programs. Pathway analysis indicated that S1 was linked to cell adhesion, extracellular matrix remodelling and inflammatory signalling, while S5 displayed enrichment of cell‐cycle deregulation, metabolic reprogramming and immune evasion. Accordingly, S1 tumours appear reliant on TME interactions, whereas S5 tumours adopt a predominantly TME independent, intrinsically aggressive phenotype. Conclusions Collectively, these findings delineate a biologically coherent framework linking pathway deregulation with cell state and ecotype‐specific mutational landscapes, refining DLBCL risk classification and adding an additional dimension to conventional prognostic models.
