技术资料

RNA 5-methylcytosine (m 5 C),a prevalent epitranscriptomic modification that critically regulates gene expression and cellular homeostasis. While its roles in solid tumors have been increasingly recognized,the functional landscape of m 5 C in acute myeloid leukemia (AML) remains unexplored. Here,we identified NSUN2,the principal RNA m 5 C methyltransferase,as a key regulator of AML progression. NSUN2 was aberrantly upregulated in AML patient samples and correlated with poor prognosis. Functional studies demonstrated that NSUN2 promoted leukemic cell proliferation,enhanced tumor growth in xenograft models,and conferred resistance to ferroptosis—a regulated cell death process driven by lipid peroxidation. Mechanistically,NSUN2 catalyzed m⁵C deposition on the 3’UTR of FSP1 (ferroptosis suppressor protein 1) mRNA,facilitating its recognition and stabilization by the m 5 C reader protein YBX1. This NSUN2-YBX1-FSP1 axis protected AML cells from ferroptotic stress by suppressing lipid peroxidation and oxidative damage. Depletion of NSUN2 or FSP1 induced mitochondrial remodeling,which primed cells for ferroptosis. Reconstitution of wild-type NSUN2 or FSP1 rescued ferroptosis resistance,whereas catalytically inactive NSUN2 (C271A/C321A) or non-functional FSP1 mutants (G2A/E156A) failed to reverse this phenotype. Pharmacological inhibition of NSUN2 with MY-1B or targeting FSP1 with iFSP1 exhibited potent anti-leukemic effects,synergizing robustly with ferroptosis inducers,standard chemotherapy,and the BCL-2 inhibitor venetoclax. Our study unveils NSUN2 and FSP1 as prognostic biomarkers and therapeutic targets in AML. We highlight a novel epitranscriptomic mechanism linking RNA methylation to ferroptosis evasion,providing a dual-strategy approach to overcome AML treatment resistance. The online version contains supplementary material available at 10.1186/s12943-025-02394-8. View Publication

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro,we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7,MDA-MB-231,and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study,we showed that stromal cells promoted tumor cell aggregation into spheroids,regardless of the initial proliferation rates,with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids,whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions. View Publication

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve,demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses,therapeutic treatments,and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic,with new subvariants continuously emerging,many harboring spike protein mutations linked to immune evasion. In this study,we characterized a panel of live SARS-CoV-2 strains,including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants,particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC),we established both monoinfections and coinfections involving SARS-CoV-2,Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably,Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020,Washington strain). Furthermore,the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead,it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6,IFN-β,and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together,our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants,as well as their interactions with other respiratory viruses,carrying important implications for therapeutic development and public health preparedness. View Publication

Accumulating evidence suggests that mitogenic signaling during cell cycle arrest can lead to severe cytotoxic outcomes,such as senescence,though the underlying mechanisms remain poorly understood. Here,we explored the link between cell cycle dynamics and the formation of PML-nuclear bodies (PML-NBs),intranuclear structures known to mediate cellular stress responses. Our findings demonstrate that PML-NBs increase their number during interphase arrest. Moreover,the activation of mitogenic ERK signaling by all-trans retinoic acid (ATRA) during CDK4/6 inhibitor-induced cell cycle arrest synergistically enhances the formation of larger PML-NBs by associating with SUMO. This enlargement,triggered by the simultaneous engagement of opposing cell cycle signals,leads to potent cytotoxicity accompanied by either terminal differentiation or apoptosis,depending on the cell type,across multiple acute myeloid leukemia (AML) cell lines. Importantly,in an AML mouse model,this combination treatment significantly improved therapeutic efficacy with minimal effects on normal hematopoiesis. Our results introduce conflicting cell cycle signal-induced cytotoxicity as a promising therapeutic strategy for AML. Subject terms: PML bodies,Apoptosis View Publication

Advancing research in oncology highlights the inverse correlation between antibiotic treatment and the positive outcomes of immune checkpoint inhibitor (ICI) administration,confirming once more the importance of microbiota and microbiota-derived compounds as complementary tools for treating cancer. Among the immune checkpoints,the CD200 cell surface glycoprotein has gained attention for its role in promoting self-tolerance and potentially facilitating tumor growth through interaction with the CD200R1 receptor. We developed a robust AlphaLISA-based screening to identify human gut microbiota-derived proteins that may interact with CD200R1 and screened a library of 10,966 gut bacterial proteins. The antitumor activity of BOC1 was investigated in vitro by cytokine analysis,mixed lymphocyte reactions,and myeloid-derived suppressor cell (MDSC)–T-cell suppression assay. AlphaFold modeling was used to predict potential interaction points between BOC1 and CD200R1. We successfully identified BOC1,a protein from the Bacteroides genus,showing better affinity than the natural ligand,CD200,toward the CD200R1 receptor. BOC1 induces cytokine secretion by monocyte-derived dendritic cells (MoDCs) and enhances CD8 + /CD4 + T-cell populations and IFNγ production,highlighting its potent immunostimulatory properties. BOC1 also negatively impacts the differentiation of MDSCs,maintaining an immature monocytic profile (high CD14 and HLA-DR expression) and restoring T-cell proliferation even at low (10 nM) concentration. Mutation of amino acids within the N-terminal region of BOC1 reduces binding to CD200R1,supporting the importance of this region for a possible interaction with CD200R1. The immunostimulatory properties of BOC1 observed in vitro are compatible with an ICI-like behavior of this bacterial protein. Given that neither the CD200 protein nor the anti-CD200 antibody is able to compete with BOC1 for binding to CD200R1,and as supported by AlphaFold modeling predictions,CD200 and BOC1 might target different regions of CD200R1. View Publication

Despite the success of antiretroviral therapy in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1),persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells,such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses,demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report,we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1β secretion in macrophages and microglia in an RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1β secretion,which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1β secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV-infected macrophages,overexpression of NLRP1 significantly enhanced IL-1β secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA,but not multiply-spliced HIV-1 RNA,with NLRP1,suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together,these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV-infected myeloid cells. View Publication

If iPS cells can be established easily and efficiently using freshly collected blood cells,it will enhance regenerative and personalized medicine. While reports of iPS derivation from blood-derived endothelial progenitor cells using RNA have been documented,none have been reported from peripheral blood-derived mononuclear cells (PBMCs). In this study,we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4,which suppresses p53,improved reprogramming efficiency. Subject terms: Reprogramming,Induced pluripotent stem cells View Publication

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Hsp90 is a molecular chaperone that is often overexpressed across multiple cancer types and has a potential value as a prognostic marker as well as a therapeutic target. Given the high interest in Hsp90 therapies,positron emission tomography or PET imaging of Hsp90 can be a valuable tool for patient selection. The limitations of the previously developed Hsp90 tracers prompted us to evaluate the recently developed brain-permeable [ 11 C]HSP990 PET probe to advance the development of Hsp90-targeted therapeutics. Given the brain accumulation of [ 11 C]HSP990 probe,application for glioblastoma imaging of this tracer is of particular interest. In vitro [ 11 C]HSP990 binding was assessed in breast cancer and glioma cell lines including patient-derived cells using Hsp90 inhibitors and RNA interference knockdown of Hsp90 isoforms. Saturation binding studies were conducted on these cells and tumour tissue homogenates,and autoradiography was performed on tissue sections. Ex vivo biodistribution and in vivo dynamic µPET/CT studies were performed in healthy mice and tumour-bearing mice,including immunocompromised subcutaneous human U87 and MDA-MB-231models and immunocompetent intracranial murine NS/CT-2A models at baseline and following a pre-treatment with Hsp90 inhibitors. High Hsp90-specific tracer uptake was observed in breast cancer and glioma cells,with Hsp90β inhibition resulting in the most substantial reduction in uptake. In vivo uptake was high in U87 tumours but low in MDA-MB-231,presumably due to the differences in Hsp90 expression in tumour tissue versus cultured cells. Differences in maximum binding capacity or B max across cell and tissue types support this hypothesis,especially given that the affinity measured as dissociation constant K d remained similar across all tissue types. Despite high NS/CT-2A tumour uptake in vitro,no contrast between the healthy brain tissue and the NS/CT-2A glioma was observed in vivo due to the high uptake by the healthy brain. [ 11 C]HSP990 is a promising tracer for identifying Hsp90-overexpressing tumours and may hold potential for patient stratification,prognosis,and therapy monitoring of novel Hsp90 therapeutics. High healthy brain uptake of this tracer precluded the differentiation of the tumour in the intracranial NS/CT-2A tumour model,therefore [ 11 C]HSP990 might not be a suitable tracer for the glioblastoma imaging. Tracer with a longer half-life might be needed to compare the washout of the tracer from the brain and the tumour tissue over several hours to identify a suitable imaging window. The online version contains supplementary material available at 10.1186/s41181-025-00386-z. View Publication

Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies,yet the benefits of ICBs are limited to a subset of patients. Therefore,it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs,a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally,CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8 + T cell survival,both in vivo and in vitro. Mechanistically,helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L,controlling its expression through phosphorylation of serine at position 398. Therapeutically,the AMPK agonist AICAR (acadesine) as an inducer of CD137L,exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary,our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity. Subject terms: Tumour immunology,Cancer immunotherapy View Publication

Cost-effective,practical,and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However,the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover,we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly,the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. We formulated B8 with commercially available,growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made,weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry,and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability,genome editing efficiency,single-cell cloning,and differentiation in both monolayer and organoids. Finally,we validated key findings using male (H1) and female (H9) human embryonic stem cells. hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast,cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time,B8+ led to gene expression changes indicative of marked lineage priming,reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state,consistent with intermittent media starvation. Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8,integrating some features of B8 while resembling cE8,emerges as a robust and practical option with limited compromises. B8+,though advantageous in some contexts,warrants caution due to lineage priming effects that may impact differentiation outcomes. View Publication

Natural killer (NK) cells play a crucial role in immune surveillance by recognizing and eliminating tumor cells. However,tumors employ various mechanisms to evade NK cell-mediated immunity. NKp30 is a potent activating receptor on NK cells,but its function can be inhibited by specific ligands secreted by cancer cells. Here,we identified dipeptidase 1 (DPEP1) as a novel ligand for NKp30 in KM12C colon cancer cells,using co-immunoprecipitation,confocal microscopy,and flow cytometry. We examined how the DPEP1–NKp30 interaction affects NK cell activity and found that NK cytotoxicity increased in KM12C cells with DPEP1 knockdown but was significantly reduced in HCT116 cells overexpressing DPEP1. We further demonstrated that DPEP1 is secreted via extracellular vesicles and that its interaction with NKp30 suppressed the expression and secretion of perforin 1,granzyme B,CD107a,and interferon-γ in NK92 cells. In a xenograft mouse model treated with NK92 cells,tumors derived from HCT116/DPEP1 cells were significantly larger than those from HCT116/mock cells. Using peripheral blood-derived human NK cells,we confirmed that DPEP1 inhibited both cytotoxicity and granzyme B secretion. These findings suggest that disrupting the DPEP1–NKp30 interaction may enhance NK cell-mediated cytotoxicity and represent a novel therapeutic strategy for cancer immunotherapy. The online version contains supplementary material available at 10.1038/s41598-025-18475-z. View Publication