技术资料

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

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

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

The molecular mechanisms regulating CD8 + cytotoxic T lymphocytes (CTL) are not fully understood. Here,we show that the peroxisome proliferator–activated receptor δ (PPARδ) suppresses CTL cytotoxicity by inhibiting RelA DNA binding. Treatment of Apc Min/+ mice with the PPARδ agonist GW501516 reduced the activation of normal and tumor-associated intestinal CD8 + T cells and increased intestinal adenoma burden. PPARδ knockout or knockdown in CTLs increased their cytotoxicity against colorectal cancer cells,whereas overexpression of PPARδ or agonist treatment decreased it. Correspondingly,perforin,granzyme B,and IFNγ protein and mRNA levels were higher in PPARδ knockout or knockdown CTLs and lower in PPARδ overexpressing or agonist-treated CTLs. Mechanistically,we found that PPARδ binds to RelA,interfering with RelA–p50 heterodimer formation in the nucleus,thereby inhibiting its DNA binding in CTLs. Thus,PPARδ is a critical regulator of CTL effector function. Significance: Here,we provide the first direct evidence that PPARδ plays a critical role in suppressing the immune response against tumors by downregulating RelA DNA-binding activity. This results in decreased expression of perforin,granzyme B,and IFNγ. Thus,PPARδ may serve as a valuable target for developing future cancer immunotherapies. View Publication

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD,even before symptom onset. Because these early disease phenotypes remain incompletely understood,we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients,presymptomatic C9ORF72-HRE (C9-HRE) carriers,and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition,single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids,including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further,molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly,organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology,whereas the extent to which more downstream cellular defects,as found in C9-ALS/FTD models,were detected varied for the different presymptomatic C9-HRE cases. Together,these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies. The online version contains supplementary material available at 10.1186/s40478-024-01857-1. View Publication

Hematopoietic stem cells (HSCs) react to various stress conditions. However,it is unclear whether and how HSCs respond to severe anemia. Here,we demonstrate that upon induction of acute anemia,HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia,lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs,transcription levels of lipid metabolism-related genes,such as very low-density lipoprotein receptor ( Vldlr ),are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential,whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential,which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility,and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions. Subject terms: Haematopoietic stem cells,Fat metabolism,Chromatin,Anaemia View Publication