技术资料

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

Targeted therapy interventions using tyrosine kinase inhibitors (TKIs) provide encouraging treatment responses in patients with ALK -rearranged lung adenocarcinomas,yet resistance occurs almost inevitably. In addition to tumor cell-intrinsic resistance mechanisms,accumulating evidence suggests that cancer-associated fibroblasts (CAFs) within the tumor microenvironment contribute to therapy resistance. This study aimed to investigate CAF-driven molecular networks that shape the therapeutic susceptibility of ALK -driven lung adenocarcinoma cells. Three-dimensional (3D) spheroid co-cultures comprising ALK -rearranged lung adenocarcinoma cells and CAFs were utilized to model the tumor microenvironment. Single-cell RNA sequencing was performed to uncover transcriptional differences between TKI-treated homotypic and heterotypic spheroids. Functional assays assessed the effects of CAF-conditioned medium and CAF-secreted factors on tumor cell survival,proliferation,lipid metabolism,and downstream AKT signaling. The therapeutic potential of targeting metabolic vulnerabilities was evaluated using pharmacological inhibition of lipid metabolism and by ferroptosis induction. CAFs significantly diminished the apoptotic response of lung tumor cells to ALK inhibitors while simultaneously enhancing their proliferative capacity. Single-cell RNA sequencing identified lipogenesis-associated genes as a key transcriptional difference between TKI-treated homotypic and heterotypic lung tumor spheroids. CAF-conditioned medium and the CAF-secreted factors HGF and NRG1 activated AKT signaling in 3D-cultured ALK-rearranged lung tumor cells,leading to increased de novo lipogenesis and suppression of lipid peroxidation. These metabolic adaptations were critical for promoting tumor cell survival and fostering therapy resistance. Notably,both dual inhibition of ALK and the lipid-regulatory factor SREBP-1,as well as co-treatment with ferroptosis inducers such as erastin or RSL3,effectively disrupted the CAF-driven metabolic-supportive niche and restored sensitivity of resistant lung tumor spheroids to ALK inhibition. This study highlights a critical role for CAFs in mediating resistance to ALK-TKIs by reprogramming lipid metabolism in ALK-rearranged lung cancer cells. It suggests that targeting these metabolic vulnerabilities,particularly through inhibition of lipid metabolism or induction of ferroptosis,could provide a novel therapeutic approach to overcome resistance and improve patient outcomes. The online version contains supplementary material available at 10.1186/s40170-025-00400-7. View Publication

Severe cutaneous adverse drug reactions (SCARs) are life-threatening diseases,which are associated with human leukocyte antigen ( HLA ) risk variants. However,the low positive predictive values of HLA variants suggest additional factors influence disease susceptibility. Using dapsone hypersensitivity syndrome (DHS) as a paradigm for SCARs,we show that the DHS patients harbor a sex-related global reduction in blood NK cells,contributing to the higher incidence of reactions in females. Single-cell RNA sequencing revealed a decrease in the immunoregulatory CD56 low XCL1/2 low NK cell subset and an expansion of CD56 high XCL1/2 high NK cell subsets with an effector phenotype in DHS patients compared to dapsone-tolerant individuals. Functionally,interleukin-15 superagonist-induced activation of NK cells exacerbated SCARs-like symptoms in a murine model. Mechanistically,TSC22 domain family member 3 (TSC22D3) deficiency enhanced NK cell effector function,shifting the immune response from CD4+ T cell to CD8+ T cell function. These results demonstrate that TSC22D3-regulated NK cells play a critical role in predisposing to drug hypersensitivity reactions,bridging innate and adaptive immune dysregulation in SCARs pathogenesis. Our study highlights the importance of NK cell heterogeneity and TSC22D3 in immune-mediated hypersensitivity disorders,offering potential therapeutic targets for SCARs and related conditions. Subject terms: Innate immunity,Innate immunity View Publication

Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse,yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. Both leukemic cells and MSCs produced TGF-β,with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity,adhesion,and migration,supporting its immunosuppressive role. In functional assays,TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids,particularly in models incorporating MSCs. Additionally,MSCs themselves provided a protective environment for leukemic cells,further reducing NK cell effectiveness in 2D co-cultures. TGF-β suppresses both NK cell cytotoxicity and migration,limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β–mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials. View Publication

Aplastic anemia (AA) is a life-threatening bone marrow (BM) failure syndrome characterized by pancytopenia. Recent studies revealed that dysfunctional endothelial progenitor cells (EPCs),critical components of the BM microenvironment,are involved in hematopoietic-dysfunction-related diseases,including AA. However,the mechanism underlying EPC damage in AA remains unknown. Here we find that transforming growth factor-β (TGF-β) signaling is hyperactive in dysfunctional AA EPCs with impaired hematopoietic support and immune regulatory ability,and TGF-β inhibition promotes hematopoiesis and immune rebalance by repairing dysfunctional EPCs. Through impaired EPC and AA murine models,we validated that TGF-β inhibition restores EPC dysfunction to improve hematopoiesis and immune status in vitro and in vivo. RNA sequencing and real-time quantitative polymerase chain reaction provided further validation. These results indicate that dysfunctional BM EPCs with hyperactive TGF-β signaling are involved in AA. TGF-β inhibition promotes multilineage hematopoiesis recovery and immune balance by repairing dysfunctional EPCs,providing a potential therapeutic strategy for AA. Subject terms: Experimental models of disease,Translational research View Publication

The hematopoietic stem cell and multipotent progenitor (HSC/MPP) pool dynamically responds to stress to adapt blood output to specific physiological demands. In β-thalassemia (Bthal),severe anemia and ineffective erythropoiesis generate expansion of erythroid precursors and a chronic stress status in the bone marrow (BM) microenvironment. However,the response to the BM altered status at the level of the HSC/MPP compartment in terms of lineage commitment has not been investigated. Bulk and single-cell RNA-sequencing reveal that Bthal HSCs/MPPs are expanded and activated with enhanced priming along the whole Ery differentiation trajectory. Consistently,HSC/MPP showed an altered TGFβ expression and autophagy transcriptional signatures along with a declined dormancy state. We discovered that the altered TGFβ signaling fosters the Ery potential of HSCs by reducing their autophagic levels,and in vivo stimulation of autophagy is sufficient to rescue the imbalance of the HSC compartment. Our findings identify the interplay between TGFβ and HSC autophagy as a key driver in the context of non-malignant hematopoiesis. Subject terms: Haematopoietic stem cells,Haematological diseases,Autophagy View Publication

Prenatal nicotine exposure impairs fetal cortical grey matter volume,but the precise cellular mechanisms remain poorly understood. This study elucidates the role of nicotinic acetylcholine receptors (nAChRs) in progenitor cells and radial glia (RG) during human cortical development. We identify two nAChR subunits—CHRNA7 and the human-specific CHRFAM7A—expressed in SOX2+ progenitors and neurons,with CHRFAM7A particularly enriched along RG endfeet. nAChR activation in organotypic slices and dissociated cultures increases RG proliferation while decreasing neuronal differentiation,whereas nAChR knockdown reduces RG and increases neurons. Single-cell RNA sequencing reveals that nicotine exposure downregulates key genes in excitatory neurons (ENs),with CHRNA7 or CHRFAM7A selectively modulating these changes,suggesting an evolutionary divergence in regulatory pathways. Furthermore,we identify YAP1 as a critical downstream effector of nAChR signaling,and inhibiting YAP1 reverses nicotine-induced phenotypic alterations in oRG cells,highlighting its role in nicotine-induced neurodevelopmental pathophysiology. Subject terms: Neuronal development,Developmental neurogenesis,Neural stem cells View Publication

In search for broad-spectrum antivirals,we discover a small molecule inhibitor,RMC-113,that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrate selective inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Lipidomics analysis reveals alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and links its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We identify PIP4K2C’s roles in SARS-CoV-2 entry,RNA replication,and assembly/egress,validating it as a druggable antiviral target. Integrating proteomics,single-cell transcriptomics,and functional assays,reveals that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced autophagic flux impairment. Promoting viral protein degradation by reversing autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C,an understudied kinase,and propose dual PIP4K2C and PIKfyve inhibition as a candidate strategy to combat emerging viruses. Subject terms: SARS-CoV-2,Target identification,Autophagy View Publication

Chronic myeloid leukemia (CML) remains a therapeutic challenge,particularly in patients who develop resistance to standard tyrosine kinase inhibitors (TKIs) such as imatinib. Here,we present the first demonstration of the potent anti-leukemic activity of the histone deacetylase (HDAC) inhibitor martinostat in both TKI-sensitive and TKI-resistant CML. Structural and biochemical analyses confirmed the efficient and selective binding of martinostat to HDAC isoenzyme ligand-binding pockets,resulting in histone and tubulin hyperacetylation in both imatinib-sensitive and resistant CML cells,outperforming vorinostat,a clinically used HDAC inhibitor (HDACi). It selectively impaired CML cell proliferation and viability and induced apoptosis across various CML models,including resistant cell models and patient blasts,with minimal toxicity to healthy cells and low developmental toxicity in zebrafish. In addition to its single-agent efficacy,martinostat demonstrated enhanced anticancer effects when combined with imatinib,both in vitro and in vivo,significantly reducing tumor growth in resistant CML xenograft models. Mechanistically,mRNA-seq data showed that martinostat disrupted key survival signaling pathways and amplified apoptotic responses,contributing to its anticancer activity. These findings highlight the potential of martinostat as a selective,low-toxicity HDACi that,combined with TKIs,could provide an effective strategy to overcome drug resistance in CML and improve therapeutic outcomes. The online version contains supplementary material available at 10.1186/s13148-025-01921-0. View Publication

The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST,its efficiency remains low. Here,we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST -positive hPSCs from ~ 5 to ~ 43.7%,providing a robust method for stabilizing XCI in female hPSCs for diverse applications. The online version contains supplementary material available at 10.1186/s13287-025-04501-4. View Publication

The hypoxic tumor microenvironment,particularly hypoxia-conditioned cancer-associated fibroblasts (CAFs),drives breast cancer (BC) progression and therapy resistance. However,the molecular mechanisms linking hypoxic CAFs to BC plasticity and chemoresistance remain elusive. Primary CAFs were isolated from high-grade BC tissues (Grade III) and characterized (α-SMA⁺/CD34⁻/pan-CK⁻),with normal fibroblasts (NFs) from reduction mammoplasty as controls. Hypoxic CAF-derived exosomal circSTAT3 stability was validated using RNase R resistance and actinomycin D assays. Exosomes were characterized via transmission electron microscopy (TEM),dynamic light scattering (DLS),and marker profiling (CD63⁺/TSG101⁺/Alix⁺,calnexin⁻). Functional effects of hypoxic CAF exosomes on TNBC cells (MDA-MB-231,SUM159) were assessed through proliferation/migration assays,stemness/epithelial-mesenchymal transition (EMT) marker analysis,and siRNA-mediated circSTAT3 knockdown. Mechanistic studies employed luciferase assays and RNA immunoprecipitation (RIP). Chemoresistance was evaluated by cisplatin half-maximal inhibitory concentration (IC₅₀). In vivo tumor growth and stemness enrichment were analyzed in xenografts. Clinical validation used BC tissues (n = 60) and plasma exosomes from BC patients (n = 40) versus healthy controls (n = 25). Hypoxic CAF-derived exosomes efficiently transferred circSTAT3 to TNBC cells,promoting proliferation,migration,EMT,and stemness marker expression. SiRNA-mediated circSTAT3 knockdown reversed these effects. Mechanistically,circSTAT3 acted as a competitive endogenous RNA (ceRNA),sponging miR-671-5p to derepress NOTCH1. Hypoxic CAF exosomes increased cisplatin IC₅₀ in TNBC cells,while circSTAT3 depletion restored chemosensitivity. In vivo,hypoxic CAF exosomes accelerated tumor growth,enriched CD44⁺/NOTCH1⁺ populations,and elevated circulating exosomal circSTAT3. Clinically,circSTAT3 was significantly upregulated in advanced BC tissues (p < 0.01) and patient plasma exosomes (p < 0.01),correlating with lymph node metastasis. This study identifies a hypoxia-driven feedforward loop wherein CAF-derived exosomal circSTAT3 promotes TNBC stemness and chemoresistance via miR-671-5p/NOTCH1 signaling. CircSTAT3 redefines stromal-tumor crosstalk as a circRNA-driven process and serves as both a circulating non-invasive biomarker and a promising therapeutic target to disrupt stromal-mediated resistance in aggressive TNBC. The online version contains supplementary material available at 10.1186/s12967-025-06794-8. View Publication