技术资料

Super enhancers (SEs),characterized by clusters of enhancers,are instrumental in shaping cellular identity and function. Given this crucial involvement of SEs in cell lineage commitment,and considering the pivotal position of surface ectoderm in differentiating into a wide array of cell types,the study of these SEs holds immense promise for advancing cell-based therapeutic applications. In this study,we profiled the SE landscape in surface ectoderm cells derived from pluripotent stem cell differentiation. By leveraging 3D genomic data,we discerned active histone modifications and frequent chromatin interactions of SEs with target genes. Notably,perturbing specific SE using a CRISPR-dCas9-mediated approach resulted in decreased expression of the connected gene. Subsequently,we constructed a regulatory network of core transcription factors (TFs) operating on SEs and uncovered their control over the differentiation process by forming regulatory network with key TFs,such as TEAD1. Knocking down TEADs attenuated the differentiation process and target gene activation,whereas YAP-TEAD activation expedited the differentiation process by promoting the early establishment of SEs. Collectively,our findings shed light on the crucial role of SEs and identify YAP-TEAD as vital regulators controlling surface ectoderm commitment,thereby providing a novel insight into lineage commitment and stem cell-based epithelial regeneration. View Publication

Human induced pluripotent stem cells (hiPSCs) offer significant potential for differentiation and research applications in cardiovascular diseases. When induced differentiated hiPSC-derived cardiomyocytes (hiPSC-CMs) are transplanted into the infarcted myocardial region,they exhibit extremely low survival rates and unsatisfactory therapeutic effects due to ischemia,hypoxia,and immune inflammation in the surrounding environment. To address this issue,we used Panax notoginseng saponin R1 (NGR1),which has demonstrated significant protective effects in prior research,to pretreat hiPSC-CMs before transplantation. Utilizing an in vitro H2O2 oxidative stress model and a nude mouse myocardial infarction (MI) model,we investigated the mechanism through which NGR1 pretreatment enhances the therapeutic efficacy of hiPSC-CM transplantation. The results revealed that the hiPSC-CMs expressed cTnT. NGR1 did not promote the proliferation of hiPSC-CMs but instead induced elevated levels of p-Akt protein in these cells. Compared to hiPSC-CM transplantation alone,transplantation of hiPSC-CMs pretreated with NGR1 exhibited higher ejection fraction (EF) and fractional shortening (FS) values,along with reduced infarct size and collagen deposition. Additionally,there were more HNA-positive cardiomyocytes in the cardiac tissue,fewer TUNEL-positive signals,and increased VWF-positive and Lyve1-positive signals. Furthermore,the gene expression levels of VEGFC,IGF-1,and SDF-1 were higher. Therefore,NGR1 pretreatment improves the survival of transplanted hiPSC-CMs in tissues,reduces myocardial apoptosis,enhances cardiac function,decreases infarct size and collagen deposition,promotes angiogenesis and lymphangiogenesis,and stimulates paracrine secretion. View Publication

Ferroptosis,an iron-dependent,lipid peroxidation-driven programmed cell death,holds substantial promise for cancer therapy,yet its translational potential is hindered by widespread intrinsic resistance. While glutathione peroxidase 4 (GPX4) is a well-established ferroptosis suppressor,the epigenetic circuitry coordinating GPX4-related mechanisms remains elusive. Here,via genome-wide screening,we identify ten-eleven translocation 1 (TET1)—a key mediator of DNA 5-hydroxymethylation—as a master controller of cancer cell ferroptosis susceptibility. In acute myeloid leukemia (AML),TET1 enhances 5hmC deposition at the glutamate-cysteine ligase catalytic subunit (GCLC) promoter to activate glutathione/γ-glutamyl-peptide metabolism,fortifying GPX4-dependent defense. Concurrently,TET1 activates NFκB signaling to upregulate GTP cyclohydrolase-1 (GCH1),conferring GPX4-independent ferroptosis resistance. Critically,co-targeting TET1/GCLC/GCH1 with low-dose ferroptosis inducers exhibits potent therapeutic effects against both ferroptosis-sensitive and -resistant AML. Our work positions TET1 as a pivotal epigenetic hub governing ferroptosis surveillance,and provides a translatable strategy to overcome ferroptosis resistance in cancer,with AML as a paradigm. DNA demethylation enzyme TET1 is a known oncogene in leukemia. Here,the authors discover that TET1 is involved in GPX4-dependent and -independent ferroptosis in acute myeloid leukemia via the regulation of GSH synthesis enzyme GCLC and BH4 synthesis enzyme GCH1. View Publication

Chimeric antigen receptor (CAR)-T cell therapy targeting antigens shared with normal T cells requires genetic modifications to prevent fratricide. This phase 1 trial evaluates autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ hematologic malignancies. The overall response rate is 85.7%,including four complete responses. All patients experience cytokine release syndrome (six grade 1–2,one grade 3),and two patients develop immune effector cell-associated neurotoxicity syndrome. The most common grade ≥3 adverse events are cytopenia and infection,with unique observations of rash and autoimmune-related events. Post-infusion immunophenotyping shows persistent depletion of CD5+ T cells and CD19+ B cells,with reduced CD4/CD8 ratios. The human CD5 knockin murine model reveals skin lesions without significant vital organ involvement. These findings demonstrate CT125A’s therapeutic potential in CD5+ malignancies while highlighting the need for safety optimization. The trial has been registered at ClinicalTrials.gov (NCT04767308). Graphical abstract Highlights•CT125A achieves 85.7% response rate in relapsed/refractory CD5+ malignancies•CD5 gene deletion prevents fratricide and enhances CAR-T cell persistence•Prolonged CD5+ T cell aplasia associates with infections and autoimmune events•Mouse model reveals on-target,off-tumor effects primarily affecting skin tissue Cheng et al. report a phase 1 trial of autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ malignancies. CT125A achieves an 85.7% response rate but causes prolonged immunosuppression,infections,and autoimmune events,highlighting the need for safety optimization strategies. View Publication

Metastatic melanoma is an aggressive malignancy with limited long-term treatment success due to therapeutic resistance and immune evasion. The transient receptor potential melastatin 8 (TRPM8) ion channel is overexpressed in melanoma but its role as therapeutic target remains unexplored. We investigated the anti-tumor effects of novel TRPM8 modulators in metastatic melanoma cells using viability assays,apoptosis markers,mitochondrial function analyses,reactive oxygen species (ROS) measurements and gene silencing. Their functional impact was further assessed in 3D melanoma organoids,clonogenic survival assays,and natural killer (NK) cell co-culture systems. TRPM8 is significantly overexpressed in metastatic melanoma,as compared with the normal counterparts. Its pharmacological inhibition with novel modulators selectively induces calcium-independent mitochondrial apoptosis characterized by ROS accumulation,mitochondrial membrane depolarization,cytochrome c release,and caspase-3 activation. This process involves activation of the ATM/p53 pathway and upregulation of pro-apoptotic proteins. Additionally,TRPM8 modulators increase expression of the NK cell-activating ligand ULBP1,enhancing melanoma susceptibility to NK-mediated cytotoxicity. Our study identifies TRPM8 as a promising biomarker in melanoma. Its targeting triggers mitochondrial cell death and simultaneously boosts NK cell recognition via ULBP1/NKG2D engagement. TRPM8 targeting in combination with immunotherapy might be,hence,further explored in clinical setting of advanced melanoma. View Publication

Gestational transfer of brain‐reactive antibodies is a risk factor for neurodevelopmental disorders. Contactin‐associated protein‐like 2 (CASPR2) is a known target for pathogenic maternal autoantibodies which have been proposed to interfere with fetal neurodevelopment. However,the impact of CASPR2 antibodies on human brain development remains largely unknown. Here,to better understand the neurophysiological changes that occur in the presence of these pathogenic autoantibodies,we cultured unguided human neural organoids for a period of 6‐months in media containing anti‐CASPR2 antibodies. We then performed neurophysiological characterization via whole‐cell patch‐clamp and calcium imaging in acute organoid slices. Our results reveal that CASPR2 antibody exposure increased spontaneous synaptic activity,enhanced the maximal frequency of action potential firing and of spontaneous network activity. These findings are consistent with a state of neuronal hyperexcitability,a phenotype which is observed in several models of neurodevelopmental disorders. Mechanistically,the alterations observed in action potential waveform are in accordance with a role for CASPR2 in the regulation of voltage‐gated potassium channels and a pathological role for CASPR2 autoantibodies in driving neuronal hyperexcitability. Maternal antibodies targeting CASPR2 are a known risk factor for neurodevelopmental disorders,yet their impact on early human brain development remains unclear. We modeled this exposure using human neural organoids treated with patient‐derived CASPR2 antibodies up to the age of 6 months. Our study reveals that these antibodies drive neurons into a state of pathological hyperexcitability by specifically impairing action potential repolarization and enhancing excitatory synaptic transmission. These findings provide novel mechanistic evidence linking maternal autoimmunity to the excitation/inhibition imbalance characteristic of autism,highlighting a potential biological origin for antibody‐mediated neurodevelopmental conditions. View Publication

Understanding of the factors governing immune responses in cancer remains incomplete,limiting patient benefit. In this study,we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time,with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges,including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context,whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection,and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade,revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment. View Publication

Neural cells in the adult human central nervous system (CNS) display extensive transcriptional heterogeneity. How different layers of epigenetic regulation underpin this heterogeneity is poorly understood. Here we profile,at the single-nuclei epigenomic level,distinct regions of the adult human CNS,for chromatin accessibility and simultaneously for the histone modifications H3K27me3 and H3K27ac. We unveil a putative SOX10 enhancer and primed chromatin signatures at HOX loci in spinal-cord-derived human oligodendroglia (OLG) and astrocytes,but not microglia. These signatures in adult OLG were reminiscent of developmental profiles but were decoupled from robust gene expression. Moreover,using high-resolution Micro-C,we show that induced pluripotent stem-cell-derived human OLGs exhibit a HOX chromatin architecture compatible with the primed chromatin in adult OLGs,bearing a strong resemblance not only to OLG developmental architecture but also to high-grade pontine gliomas. Thus,epigenetic memory from developmental states in adult OLG not only enables them to promptly transcribe Hox family genes during regeneration but also makes them susceptible to gliomagenesis. Single-nucleus epigenomic maps of the adult human brain and spinal cord reveal that adult oligodendroglia retain developmental chromatin patterns,suggesting a molecular memory that may shape repair processes and cancer vulnerability. View Publication

PF-956980 is a selective inhibitor of JAK3,related in structure to CP-690550,a compound being evaluated in clinical trials for rheumatoid arthritis and prevention of allograft rejection. PF-956980 has been evaluated against a panel of 30 kinases,and found to have nanomolar potency against only JAK3. Cellular and whole blood activity of this compound parallels its potency and selectivity in enzyme assays. It was effective in vivo at inhibiting the delayed type hypersensivity reaction in mice. We compared 2 commercially available JAK3 inhibitors (WHI-P131 and WHI-P154) in the same panel of biochemical and cellular assays and found them to be neither potent nor selective for JAK3. Both were found to be nanomolar inhibitors of the EGF receptor family of kinases. As these compounds have been used in numerous publications in the transplant and autoimmune disease literature,their specificity should be considered when interpreting these results. View Publication

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells,we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model,donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly,highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer,accumulated in vivo at advanced proliferative cycles,and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted,apoptosis-resistant phenotype,including: 1) reduced apoptosis after staurosporine addition,serum starvation,or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection,we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4,IL-10,perforin,or Fas ligand; 2) could not be reversed by IL-2,IL-7,or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis,persist in vivo,and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells. View Publication

Platelets are an abundant source of CD40 ligand (CD154),an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases,including systemic lupus erythematosus (SLE),diabetes,and cardiovascular disease. Heretofore considered largely restricted to activated T cells,we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells,megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore,using several established megakaryocyte-like cells lines,we performed promoter analysis of the CD154 gene and found that NFAT,a calcium-dependent transcriptional regulator associated with activated T cells,mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall,these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated. View Publication

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system,receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however,an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects,whereby LPA receptor-expressing cells exposed to,or primed� View Publication