技术资料

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However,whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis,here we combined bioinformatic analysis of the FASN-related immune cell landscape,real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout ( FASN KO ) cell models,and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression,cytolytic activity signatures,and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity,accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy. Subject terms: Cancer metabolism,Oncogenesis View Publication

Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development,metabolism,and reproduction. In mammals,THRα has two major splicing isoforms,THRα1 and THRα2. The better-characterized isoform,THRα1,is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform,THRα2,lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However,the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies,we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3′-end. In all datasets,we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults. View Publication

Drugs work by binding to a specific 3D structure on a protein. Drug discovery has historically been driven by prior knowledge of function,either of a protein or chemical. This knowledge of function then drives investigations to probe chemical/protein interactions. We undertook a different approach. We first identified unique 3D structures,agnostic of function,and investigated whether they could lead us to innovative therapeutics. Using a synchrotron-based X-ray source,we first determined high-resolution structures of hundreds of proteins. With a supercomputer running analytical programs created by us,we identified novel 3D structures and screened for chemicals binding them. We then tested their ability to inhibit cancer growth without damaging normal cells. We identified a potent inhibitor of a deadly cancer,melanoma. It was not toxic to normal cells even at 2100-fold higher doses. It worked by inducing anoikis,a fundamental process of known importance for cancer. Therapeutics that selectively induce anoikis are needed. In summary,we demonstrate the power of using a 3D protein structure as the starting point to discover new biology and drugs. Drug discovery historically starts with an established function,either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function,we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds,we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures,we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast,prostate,colon,or lung cancer,and for toxicity on normal human bone marrow stem cells,both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound,Dxr2-017,exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays,Dxr2-017 had an IC50 of 12 nM against melanoma cells,while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis,a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable. View Publication

Stimulating erythropoiesis is essential in the treatment of various types of anemia. Sheng Xue Ning (SXN) is commonly used in China as an iron supplement to treat iron deficiency anemia,renal anemia,and anemia in pregnancy. This research reports a novel effect of SXN in enhancing the proliferation of hematopoietic stem/progenitor cell (HSPC) to promote erythropoiesis in the bone marrow,which is distinct from conventional iron supplements that primarily aid in the maturation of red blood cells. Employing a model of hematopoietic dysfunction induced by X-ray exposure,we evaluated the efficacy of SXN in restoring hematopoietic function. SXN significantly promoted the recovery of peripheral erythroid cells and enhanced the proliferation and differentiation of Lin − /c-KIT + /Sca-1 + HSPC in mice exposed to X-ray irradiation. Our results showed that SXN elevated the expression of stem cell factor (SCF) and activated the SCF/c-KIT/PI3K/AKT signaling pathway,facilitating the proliferation and differentiation of HSPC. In vitro,SXN markedly enhanced the proliferation of bone marrow nucleated cell (BMNC) and the colony-forming capacity of BFU-E,CFU-E,and CFU-GM,while also elevating the expression of proteins involved in the SCF/c-KIT/PI3K/AKT pathway in BMNC. Additionally,SXN enhanced the proliferation and differentiation of mesenchymal stem cell (MSC) and increased SCF secretion. In conclusion,SXN demonstrates the capacity to enhance erythropoiesis by upregulating SCF expression,thereby promoting HSPC proliferation and differentiation via the SCF/c-KIT/PI3K/AKT pathway. SXN may offer a new strategy for improving the activity of HSPC and promoting erythropoiesis in the treatment of hematopoiesis disorders. View Publication

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (V H ) and light chain germline segment pairs,or parallel lineages,bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct V H lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo. Subject terms: Protein design,Protein design,VDJ recombination,Class switch recombination,Plasma cells View Publication

Epithelial-to-mesenchymal transition (EMT),cancer stem cells (CSCs),and colorectal cancer (CRC) therapy resistance are closely associated. Prior reports have demonstrated that sphingosine-1-phosphate (S1P) supports stem cells and maintains the CSC phenotype. We hypothesized that the EMT inducer SNAI1 drives S1P signaling to amplify CSC self-renewal capacity and chemoresistance. CRC cell lines with or without ectopic expression of SNAI1 were used to study the role of S1P signaling as mediators of cancer stemness and 5-fluorouracil (5FU) chemoresistance. The therapeutic ability of sphingosine kinase 2 (SPHK2) was assessed using siRNA and ABC294640,a SPHK2 inhibitor. CSCs were isolated from patient-derived xenografts (PDXs) and assessed for SPHK2 and SNAI1 expression. Ectopic SNAI1 expressing cell lines demonstrated elevated SPHK2 expression and increased SPHK2 promoter activity. SPHK2 inhibition with siRNA or ABC294640 ablated in vitro self-renewal and sensitized cells to 5FU. CSCs isolated from CRC PDXs express increased SPHK2 relative to the non-CSC population. Combination ABC294640/5FU therapy significantly inhibited tumor growth in mice and enhanced 5FU response in therapy-resistant CRC patient-derived tumor organoids (PDTOs). SNAI1/SPHK2 signaling mediates cancer stemness and 5FU resistance,implicating S1P as a therapeutic target for CRC. The S1P inhibitor ABC294640 holds potential as a therapeutic agent to target CSCs in therapy refractory CRC. View Publication

Down syndrome predisposes individuals to haematological abnormalities,such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood 1 – 3 . Here,to understand dysregulated haematopoiesis in Down syndrome,we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human fetal liver and bone marrow samples from 3 fetuses with disomy and 15 fetuses with trisomy. We found that differences in gene expression in Down syndrome were dependent on both cell type and environment. Furthermore,we found multiple lines of evidence that haematopoietic stem cells (HSCs) in Down syndrome are ‘primed’ to differentiate. We subsequently established a Down syndrome-specific map linking non-coding elements to genes in disomic and trisomic HSCs using 10X multiome data. By integrating this map with genetic variants associated with blood cell counts,we discovered that trisomy restructured regulatory interactions to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Furthermore,as mutations in Down syndrome display a signature of oxidative stress 4,5,we validated both increased mitochondrial mass and oxidative stress in Down syndrome,and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Together,our single-cell,multi-omic resource provides a high-resolution molecular map of fetal haematopoiesis in Down syndrome and indicates significant regulatory restructuring giving rise to co-occurring haematological conditions. Subject terms: Haematopoietic stem cells,Leukaemia,Haematopoiesis,Haematological diseases,Aneuploidy View Publication

KRAS is among the most commonly mutated oncogenes in human malignancies. Although the advent of sotorasib and adagrasib,has lifted the “undruggable” stigma of KRAS,the resistance to KRAS inhibitors quickly becomes a major issue. Here,we reported that aldehyde dehydrogenase 1 family member A1 (ALDH1A1),an enzyme in retinoic acid biosynthesis and redox balance,increases in response to KRAS inhibitors and confers resistance in a range of cancer types. KRAS inhibitors' efficacy is significantly improved in sensitive or drug-resistant cells,patient-derived organoids (PDO),and xenograft models by ALDH1A1 knockout,loss of enzyme function,or inhibitor. Furthermore,we discovered that ALDH1A1 suppresses the efficacy of KRAS inhibitors by counteracting ferroptosis. ALDH1A1 detoxicates deleterious aldehydes,boosts the synthesis of NADH and retinoic acid (RA),and improves RARA function. ALDH1A1 also activates the CREB1/GPX4 pathway,stimulates the production of lipid droplets in a pH-dependent manner,and subsequently prevents ferroptosis induced by KRAS inhibitors. Meanwhile,we established that GTF2I is dephosphorylated at S784 via ERK by KRAS inhibitors,which hinders its nuclear translocation and mediates ALDH1A1's upregulation in response to KRAS inhibitors. In summary,the results offer valuable insights into targeting ALDH1A1 to enhance the effectiveness of KRAS-targeted therapy through ferroptosis in cancer treatment. View Publication

Autosomal recessive osteopetrosis (ARO) is a rare genetic disease,characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation,leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT),but its success is limited by several constraints. Conversely,gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. The Tcirg1 -defective oc/oc mouse model displays a short lifespan and high bone density,closely mirroring the human condition. In this work,we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. First,we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype,allowing long-term survival and reducing extramedullary haematopoiesis. Then,we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood,facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally,pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs,albeit at lower level than conventional total body irradiation,and led to long-term survival and correction of bone phenotype,in the absence of acute toxicity. These results will pave the way to the implementation of an effective GT protocol,reducing the transplant-related complication risks in the very young and severely affected ARO patients. View Publication

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions,no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0,day 13,day 40,and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover,the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together,our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment,displaying promising prospects as biomarkers for monitoring RTT progression. The online version contains supplementary material available at 10.1007/s00018-024-05409-7. View Publication

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However,current QC methods,reliant on molecular staining,pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC),a machine learning-driven,multidimensional,high-content,and high-throughput flow cytometry approach,in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975),T cells and non-T cells in white blood cells (ROC-AUC = 0.969),activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990),and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers,assess viability,identify specific cell subsets or phenotypic states,and remove impurities during cell therapy manufacturing. Thus,LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Subject terms: Biotechnology,Cell biology,Immunology,Biomedical engineering View Publication

Paulownin,a natural compound derived from Paulownia tomentosa wood,exhibits various physiological functions,including anti-bacterial and anti-fungal effects. However,the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study,we investigated the effect of paulownin on NK cell activity both in vitro and in vivo,and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia,human colon,and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules,including granzyme B and perforin in NK-92 cells. Moreover,these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice,resulting in enhanced cytotoxicity against YAC-1 cells. Moreover,the anti-tumor effects of paulownin were reduced upon the depletion of NK cells. Therefore,these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy. View Publication