技术资料

Anti-CD117 monoclonal antibody (mAb) agents have emerged as exciting alternative conditioning strategies to traditional genotoxic irradiation or chemotherapy for both allogeneic and autologous gene-modified hematopoietic stem cell transplantation. Furthermore,these agents are concurrently being explored in the treatment of mast cell disorders. Despite promising results in animal models and more recently in patients,the short- and long-term effects of these treatments have not been fully explored. We conducted rigorous assessments to evaluate the effects of an antagonistic anti-mCD117 mAb,ACK2,on hematopoiesis in wild-type and Fanconi anemia (FA) mice. Importantly,we found no evidence of short-term DNA damage in either setting following this treatment,suggesting that ACK2 does not induce immediate genotoxicity,providing crucial insights into its safety profile. Surprisingly,FA mice exhibited an increase in colony formation after ACK2 treatment,indicating a potential targeting of hematopoietic stem cells and expansion of hematopoietic progenitor cells. Moreover,the long-term phenotypic and functional changes in hematopoietic stem and progenitor cells did not differ significantly between the ACK2-treated and control groups,in either setting,suggesting that ACK2 does not adversely affect hematopoietic capacity. These findings underscore the safety of these agents when utilized as a short-course treatment in the context of conditioning,as they did not induce significant DNA damage in hematopoietic stem or progenitor cells. However,single-cell RNA sequencing,used to compare gene expression between untreated and treated mice,revealed that the ACK2 mAb,via c-Kit downregulation,effectively modulated the MAPK pathway with Fos downregulation in wild-type and FA mice. Importantly,this modulation was achieved without causing prolonged disruptions. These findings validate the safety of anti-CD117 mAb treatment and also enhance our understanding of its intricate mode of action at the molecular level. View Publication

The etiopathology of Parkinson’s disease has been associated with mitochondrial defects at genetic,laboratory,epidemiological,and clinical levels. These converging lines of evidence suggest that mitochondrial defects are systemic and causative factors in the pathophysiology of PD,rather than being mere correlates. Understanding mitochondrial biology in PD at a granular level is therefore crucial from both basic science and translational perspectives. In a recent study,we investigated mitochondrial alterations in fibroblasts obtained from PD patients assessing mitochondrial function in relation to clinical measures. Our findings demonstrated that the magnitude of mitochondrial alterations parallels disease severity. In this study,we extend these investigations to blood cells and dopamine neurons derived from induced pluripotent stem cells reprogrammed from PD patients. To overcome the inherent metabolic heterogeneity of blood cells,we focused our analyses on metabolically homogeneous,accessible,and expandable erythroblasts. Our results confirm the presence of mitochondrial anomalies in erythroblasts and induced dopamine neurons. Consistent with our previous findings in fibroblasts,we observed that mitochondrial alterations are reversible,as evidenced by enhanced mitochondrial respiration when PD erythroblasts were cultured in a galactose medium that restricts glycolysis. This observation indicates that suppression of mitochondrial respiration may constitute a protective,adaptive response in PD pathogenesis. Notably,this effect was not observed in induced dopamine neurons,suggesting their distinct bioenergetic behavior. In summary,we provide additional evidence for the involvement of mitochondria in the disease process by demonstrating mitochondrial abnormalities in additional cell types relevant to PD. These findings contribute to our understanding of PD pathophysiology and may have implications for the development of novel biomarkers and therapeutic strategies. Subject terms: Energy metabolism,Parkinson's disease View Publication

Cisplatin resistance poses a major challenge in the treatment of oral squamous cell carcinoma (OSCC). Deeper investigations into the mechanisms underlying this drug resistance is of great importance. Here,we used cellular assays and clinical immunohistochemistry to examine molecular pathways involved in both innate and acquired cisplatin resistance. We demonstrated that the p62-mTORC1 signaling complex plays a pivotal role,and is driven by the EGFR signaling network,specifically through the PI3K-Akt axis and the transcription factor C/EBP-β. Elevated p -mTOR expression was associated with cancer relapse and poor prognosis among oral cancer patients. Additionally,we illustrated that mTOR inhibitors enhance the cytotoxic effect of cisplatin,by employing cancer stem cell characteristics. Our work unveils fundamental mechanisms for cisplatin resistance,thereby presenting therapeutic implications for OSCC. View Publication

Chemoresistance is associated with tumor relapse and unfavorable prognosis. Multiple mechanisms underlying chemoresistance have been elucidated,including stemness and DNA damage repair. Here,the involvement of the WNT receptor,FZD5,in ovarian cancer (OC) chemoresistance was investigated. OC cells were analyzed using in vitro techniques including cell transfection,western blot,immunofluorescence and phalloidin staining,CCK8 assay,colony formation,flowcytometry,real-time PCR,and tumorisphere formation. Pearson correlation analysis of the expression levels of relevant genes was conducted using data from the CCLE database. Further,the behavior of OC cells in vivo was assessed by generation of a mouse xenograft model. Functional studies in OC cells showed that FZD5 contributes to epithelial phenotype maintenance,growth,stemness,HR repair,and chemoresistance. Mechanistically,FZD5 modulates the expression of ALDH1A1,a functional marker for cancer stem-like cells,in a β-catenin-dependent manner. ALDH1A1 activates Akt signaling,further upregulating RAD51 and BRCA1,to promote HR repair. Taken together,these findings demonstrate that the FZD5-ALDH1A1-Akt pathway is responsible for OC cell survival,and targeting this pathway can sensitize OC cells to DNA damage-based therapy. The online version contains supplementary material available at 10.1186/s12964-024-01585-y. View Publication

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here,we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABA A receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover,metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03 YEMK . Overall,cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia. Subject areas: Neuroscience,Cellular neuroscience,Stem cells research View Publication

Homology Directed Repair (HDR) enables precise genome editing,but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes,such as Non-Homologous End Joining (NHEJ). In this study,we develop a functional,pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target,53BP1,identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing,synergize with a DNAPK inhibitor molecule,and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes. Subject terms: Targeted gene repair,Homologous recombination,High-throughput screening View Publication

Mutations in mexZ,encoding a negative regulator of the expression of the mexXY efflux pump genes,are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin,mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory,suggesting that their selection during infection may not be necessarily,or only,related to tobramycin therapy. Here,we show that mexZ -mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model,thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA,a quorum sensing-controlled gene,encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump,MexAB,which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways,thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients,something unnoticeable using standard laboratory tests. Subject terms: Antimicrobial resistance,Pathogens,Infection View Publication

Neocortex expansion during evolution is linked to higher numbers of neurons,which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here,we show that EREG,encoding the growth factor EPIREGULIN,is expressed in the human developing neocortex and in gorilla cerebral organoids,but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells,whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation,and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally,we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone. View Publication

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease,the highly prevalent PeV-A1 is not associated with CNS disease. Here,we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively,our findings align with clinical observations and suggest a role for neuroinflammation,rather than viral replication,in PeV-A3 (and E11) infection. Subject terms: Infection,Central nervous system infections,Viral host response,Innate immunity View Publication

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis,erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis 1 to define the anatomy of normal and stress haematopoiesis. In the steady state,across the skeleton,single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels,where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells,which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults,as it was maintained after haemorrhage,systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment,and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF,and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis,define the anatomy of normal and stress responses,identify discrete microanatomical production sites that confer plasticity to haematopoiesis,and uncover unprecedented heterogeneity of stress responses across the skeleton. Subject terms: Bone marrow cells,Haematopoietic stem cells,Ageing,Imaging the immune system,Haematopoietic stem cells View Publication

Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however,the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues,especially in malignant tissues; however,its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung,breast,gastric,colon,and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next,colony formation assay,MTT assay,EdU assay,transwell assay,wound healing assay,flow cytometric analysis,sphere formation assay,western blotting analysis,mouse xenograft model analysis,RNA-sequencing assay,immunofluorescence assay,and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation,invasion,stemness,chemotherapy sensitivity,and integrin/FAK/TAZ/mTOR activation. Further,liquid chromatography–tandem mass spectrometry analysis,GST pull-down assay,and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B,myosin heavy chain 9 (MYH9),and CUL9. TMEM120B expression was elevated in lung,breast,gastric,colon,and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage,lymph node metastasis,and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation,invasion,and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9,which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore,TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely,overexpression of TMEM120B-∆CCD delayed the formation of FAs,suppressed TAZ-mTOR signaling,and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5). Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis,maintaining stemness and accelerating chemotherapy resistance. The online version contains supplementary material available at 10.1186/s13058-024-01802-z. View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology View Publication