技术资料

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However,targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here,we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF,albeit at low biomass,secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH + breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation,thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs. View Publication

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor,while the other two,WDR33v2 (V2) and WDR33v3 (V3),have not been studied. Here,we report that V2 and V3 are generated by alternative polyadenylation,and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly,V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING),the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy,likely by recruiting WIPI2 isoforms. V3,on the other hand,functions to ncrease STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated,indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated. View Publication

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-TCRα expression. Low circulating naïve αβ T cell counts at birth persisted over time,with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased,suggesting that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick,with infection,lymphoproliferation,and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naïve αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells,autoimmune conditions were more frequent in these patients than in the general population. View Publication

Current anticancer therapies cannot eliminate all cancer cells,which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N -methyltransferase 9 (PRMT9),whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML),eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response,suppressing cell survival. Notably,PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase,which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall,we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy. Subject terms: Cancer,Tumour immunology View Publication

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway,suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however,their role in the hematopoietic development in vivo remains unknown. Here,we identify a subpopulation of NG2 + Runx1 + perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2 + cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs,endothelial cells and hematopoietic cells in the murine AGM. Importantly,this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental,as NG2 + Runx1 + cells were not detected in the adult bone marrow,demonstrating the existence of a specialised pericyte population in the HSC-generating niche,unique to the embryo. Subject terms: Cell biology,Haematopoiesis,Cardiovascular biology View Publication

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

The delicate “Excitatory/Inhibitory balance” between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain,in this study,we investigated the critical factor of heterogeneity,focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro,a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation,revealing that the 100E configuration responded reliably,while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition,while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity,offering insights for potential therapeutic interventions in neurological conditions. Overall,this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions,emphasizing the pivotal role of neuron diversity in neural network dynamics. View Publication

Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells,especially relapsed and refractory (R/R)-AML,present high GPX4 levels and enzyme activities,pharmacological inhibition of GPX4 alone has limited application in AML. Thus,whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. Lipid reactive oxygen species (ROS),malondialdehyde (MDA),and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC),ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3),or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally,we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells,especially those with MLL rearrangements,than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects,low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis,and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically,DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK,suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition,DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally,DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients,especially for R/R-AML. The online version contains supplementary material available at 10.1186/s40164-024-00489-4. View Publication

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study,we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype,IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI),a specific LATS inhibitor,or shRNA targeting LATS1/2 significantly reduced clonogenicity,increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore,depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together,our results suggest that LATS could be one of the key factors contributing to the rapid proliferation,reduced apoptosis,and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM. View Publication

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids,organoids,tumoroids,or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies,vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints,we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids,as well as blood vessel organoids generated from pluripotent stem cells,cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids,as they successfully provide intravascular perfusion to these structures. We find that organoid growth,maturation,and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics. Subject terms: Stem-cell biotechnology,Tissue engineering,Biomedical engineering,Induced pluripotent stem cells,Microfluidics View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS,accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic,which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach,we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila . Consistent with a conserved role in disease pathogenesis,pharmacological inhibition of both proteins rescued disease-relevant phenotypes,including mitochondrial trafficking defects and neuromuscular junction failure,in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies,mice,and human iPSC-sMNs,we observed reduced GSK3 inhibitory phosphorylation,suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore,we found that PP2A acts upstream of GSK3,affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs,and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover,increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity,confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers,and reveal an unexplored mechanistic link between PP2A,GSK3,and kinesin-1,that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease. The online version contains supplementary material available at 10.1007/s00401-024-02689-y. View Publication

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer’s disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology,we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein,we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM,however,these compounds lack selectivity and this limits their utility as chemical tools. View Publication