技术资料

PU.1 ( SPI1 ) is pivotal in hematopoiesis,yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis,we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells,respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically,KLF1 overexpression restores myeloid lineage potential,while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together,our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT,potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences,Molecular biology,Molecular interaction,Cell biology; View Publication

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity,research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast,cancer cell-intrinsic PD-1 functional regulation is not well understood. Here,we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling,increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer,and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors. Subject terms: Melanoma,Cancer immunotherapy,Tumour immunology View Publication

Despite advances in breast cancer screening and treatment,prognosis for metastatic disease remains dismal at 30% five-year survival. This is due,in large,to the failure of current therapeutics to target properties unique to metastatic cells. One of the drivers of metastasis is miR-10b,a small noncoding RNA implicated in cancer cell invasion,migration,viability,and proliferation. We have developed a nanodrug,termed MN-anti-miR10b,that delivers anti-miR-10b antisense oligomers to cancer cells. In mouse models of metastatic triple-negative breast cancer,MN-anti-miR10b has been shown to prevent onset of metastasis and eliminate existing metastases in combination with chemotherapy,even after treatment has been stopped. Recent studies have implicated miR-10b in conferring stem cell-like properties onto cancer cells,such as chemoresistance. In this study,we show transcriptional evidence that inhibition of miR-10b with MN-anti-miR10b activates developmental processes in cancer cells and that stem-like cancer cells have increased miR-10b expression. We then demonstrate that treatment of breast cancer cells with MN-anti-miR10b reduces their stemness,confirming that these properties make metastatic cells susceptible to the nanodrug actions. Collectively,these findings indicate that inhibition of miR-10b functions to impair breast cancer cell stemness,positioning MN-anti-miR10b as an effective treatment option for stem-like breast cancer subtypes. View Publication

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies,although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein ( TOPORS ),which encodes a ubiquitin/SUMO E3 ligase,augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis,suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks,which undergo SUMOylation,followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain,which mediates ubiquitination,is responsible for HMA resistance. In TOPORS knockout cells,DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination,resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1,thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently,the ubiquitination inhibitor,TAK-243,and the SUMOylation inhibitor,TAK-981,show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks. Subject terms: Myelodysplastic syndrome,Myelodysplastic syndrome View Publication

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut,brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1,designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another,through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage,including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However,the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined. The online version contains supplementary material available at 10.1186/s12864-024-10723-2. View Publication

Astrocytes are essential for the formation and maintenance of neural networks. However,a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques,primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing,we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons,consistent with their further maturation. In coculture with human neurons,multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min −1 (hPSC-derived),2.86 ± 0.64 min −1 (rat); p = 0.19]. In comparison,we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived),24.77 ± 4.04 Hz (rat); p < 0.01],and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived),1.07 ± 0.14 Hz (rat); p < 0.001],consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived),8.39 ± 0.63/100 μm 2 (rat); p < 0.001]. Taken together,we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation,providing a fully human platform for investigating astrocyte function and neuronal-glial interactions. View Publication

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion,governing progression,and leukemic transformation of hematological clonal disorders. However,our ability to interrogate the niche in pre-malignant conditions has been limited,as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here,we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity,cell viability,and HSC-supportive functions of the stromal cells,suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2 + /− and its healthy counterpart,showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably,stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context. View Publication

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD),contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153),a key epigenetic regulator of NSC stemness,characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism,nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration,mitochondrial dysfunction,proteasomal processing and RNA degradation. Furthermore,recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors,marked by BrdU/DCX and BrdU/PSANCAM positivity and,later,the integration of differentiating neurons in the cell granule layer (BrdU/NeuN + cells) compared with GFP-injected AD mice. Consistently,Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. The online version contains supplementary material available at 10.1186/s13287-024-03805-1. View Publication

High-grade serous ovarian cancer remains a poorly understood disease with a high mortality rate. Although most patients respond to cytotoxic therapies,a majority will experience recurrence. This may be due to a minority of drug-resistant cancer stem-like cells (CSC) that survive chemotherapy and are capable of repopulating heterogeneous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor-associated macrophages (TAM) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs,we studied the effects of carboplatin on macrophage phenotypes using both THP1- and peripheral blood mononuclear cell (PBMC)–derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like proinflammatory phenotype that promotes SOX2 expression,spheroid formation,and CD117 + ovarian CSCs,and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient proinflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for high-grade serous ovarian cancer. Significance: We show that chemotherapy enhances proinflammatory macrophage phenotypes that correlate with ovarian cancer progression. Given that macrophages are the most prominent immune cell within these tumors,this work provides the foundation for future translational studies targeting specific macrophage populations during chemotherapy,a promising approach to prevent relapse in ovarian cancer. 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

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

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance,they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors,which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT),a well-known checkpoint receptor involved in T-cell exhaustion,has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here,we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability,are rapidly and efficiently taken up by cells,and can be easily incorporated into cell product manufacturing. INTASYL PH-804,which targets TIGIT,suppresses TIGIT mRNA and protein expression in NK cells,resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition,thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy,particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x. View Publication