技术资料

Hematopoietic stem and progenitor cells (HSPCs) that have impaired differentiation can transform into leukemic blasts. However,the mechanism that controls differentiation remains elusive. Here,we show that the genetic elimination of Proteinase 3 (PRTN3) in mice led to spontaneous myeloid differentiation. Mechanistically,our findings indicate that PRTN3 interacts with the N-terminal of STAT3,serving as a negative regulator of STAT3-dependent myeloid differentiation. Specifically,PRTN3 promotes STAT3 ubiquitination and degradation,while simultaneously reducing STAT3 phosphorylation and nuclear translocation during G-CSF-stimulated myeloid differentiation. Strikingly,pharmacological inhibition of STAT3 (Stattic) partially counteracted the effects of PRTN3 deficiency on myeloid differentiation. Moreover,the deficiency of PRTN3 in primary AML blasts promotes the differentiation of those cells into functional neutrophils capable of chemotaxis and phagocytosis,ultimately resulting in improved overall survival rates for recipients. These findings indicate PRTN3 exerts an inhibitory effect on STAT3-dependent myeloid differentiation and could be a promising therapeutic target for the treatment of acute myeloid leukemia. Subject terms: Signal transduction,Haematological diseases View Publication

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF),which is not expressed in guided cortical organoids,we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP,LIFR,and HOPX,closely matching human fetal oRG. Finally,incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely. View Publication

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic,but pericellular oxygen levels,which are affected by oxygen diffusivity and consumption,are rarely reported. Here,we provide evidence that several cell types in culture actually experience local hypoxia,with important implications for cell metabolism and function. We focused initially on adipocytes,as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions,cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria,lowered HIF1α activity,and resulted in widespread transcriptional rewiring. Functionally,adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli,recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages,hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types,and that considering pericellular oxygen improves the quality,reproducibility and translatability of culture models. View Publication

Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet,since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling,it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Serial modifications to an existing iMGL protocol were made,including but not limited to changes in growth factor combination to drive microglial differentiation,until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines,the quality of the new iMGL protocol was validated through cell yield assessment,measurement of microglia marker expression,transcriptomic comparison to primary microglia,and evaluation of inflammatory and phagocytic activities. Similarly,molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol,decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally,ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression,a heightened capacity to internalize myelin,as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency,as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL,and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. We optimized a pre-existing iMGL protocol,generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol,we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant,with preliminary characterization pointing toward functional alterations in migratory,phagocytic and inflammatory activities. The online version contains supplementary material available at 10.1186/s13024-024-00723-x. View Publication

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

Cancer remains one of the leading causes of mortality worldwide,leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade,CD103 + T cells have been associated with better clinical prognosis in patients with cancer. However,the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here,we show an unexpected and transient CD61 expression,which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling,improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically,the presence of CD61 + tumor-infiltrating T lymphocytes is associated with improved clinical outcomes,mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion,this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells,which potentiates a new target for immune-based cellular therapies. Subject terms: T cells,Tumour immunology,Lymphocyte activation 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

Prostate cancer (PCa) is one of the most prevalent cancers in men and is associated with high mortality and disability rates. β-hydroxybutyrate (BHB),a ketone body,has received increasing attention for its role in cancer. However,its role in PCa remains unclear. This study aimed to explore the mechanism and feasibility of BHB as a treatment alternative for PCa. Colony formation assay,flow cytometry,western blot assay,and transwell assays were performed to determine the effect of BHB on the proliferation and metastasis of PCa cells. Tumor sphere formation and aldehyde dehydrogenase assays were used to identify the impact of BHB or indoleacetamide-N-methyltransferase (INMT) on the stemness of PCa cells. N6-methyladenosine (m6A)–meRIP real-time reverse transcription polymerase chain reaction and dual luciferase assays were conducted to confirm INMT upregulation via the METTL3–m6A pathway. Co-IP assay was used to detect the epigenetic modification of INMT by BHB-mediated β-hydroxybutyrylation (kbhb) and screen enzymes that regulate INMT kbhb. Mouse xenograft experiments demonstrated the antitumor effects of BHB in vivo. BHB can inhibit the proliferation,migration,and invasion of PCa cells by suppressing their stemness. Mechanistically,INMT,whose expression is upregulated by the METTL3–m6A pathway,was demonstrated to be an oncogenic gene that promotes the stem-like characteristics of PCa cells. BHB can suppress the malignant phenotypes of PCa by kbhb of INMT,which in turn inhibits INMT expression. Our findings indicate a role of BHB in PCa metabolic therapy,thereby suggesting an epigenetic therapeutic strategy to target INMT in aggressive PCa. Not applicable. The online version contains supplementary material available at 10.1186/s12935-024-03277-6. 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

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system,catalysing the formation of reactive oxygen species that are important in anti‐microbial action,but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti‐cancer drugs,but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy‐related leukaemia by protecting haematopoietic cells from TOP2 poison‐mediated genotoxic damage while preserving the anti‐cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide‐induced TOP2B‐DNA covalent complexes and resulting DNA double‐strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti‐inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons. View Publication

Genetic deletion of Dusp1 eliminates CSF3R-induced leukemia. Inhibition of Dusp1 induces the expression of Bim and p53 in oncogenic context,resulting in selective demise of leukemic cells. View Publication