技术资料

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of pathological proteins and synaptic dysfunction. This study aims to investigate the molecular and functional differences between human induced pluripotent stem cells (hiPSCs) derived from patients with sporadic AD (sAD) and age-matched controls (healthy subjects,HS),focusing on their neuronal differentiation and synaptic properties in order to better understand the cellular and molecular mechanisms underlying AD pathology. Skin fibroblasts from sAD patients ( n = 5) and HS subjects ( n = 5) were reprogrammed into hiPSCs using non-integrating Sendai virus vectors. Through karyotyping,we assessed pluripotency markers (OCT4,SOX2,TRA-1–60) and genomic integrity. Neuronal differentiation was evaluated by immunostaining for MAP2 and NEUN. Electrophysiological properties were measured using whole-cell patch-clamp,while protein expression of Aβ,phosphorylated tau,Synapsin-1,Synaptophysin,PSD95,and GluA1 was quantified by western blot. We then focused on PAK1-LIMK1-Cofilin signaling,which plays a key role in regulating synaptic structure and function,both of which are disrupted in neurodegenerative diseases such as AD. sAD and HS hiPSCs displayed similar stemness features and genomic stability. However,they differed in neuronal differentiation and function. sAD-derived neurons (sAD-hNs) displayed increased levels of AD-related proteins,including Aβ and phosphorylated tau. Electrophysiological analyses revealed that while both sAD- and HS-hNs generated action potentials,sAD-hNs exhibited decreased spontaneous synaptic activity. Significant reductions in the expression of synaptic proteins such as Synapsin-1,Synaptophysin,PSD95,and GluA1 were found in sAD-hNs,which are also characterized by reduced neurite length,indicating impaired differentiation. Notably,sAD-hNs demonstrated a marked reduction in LIMK1 phosphorylation,which could be the underlying cause for the changes in cytoskeletal dynamics that we found,leading to the morphological and functional modifications observed in sAD-hNs. To further investigate the involvement of the LIMK1 pathway in the morphological and functional changes observed in sAD neurons,we conducted perturbation experiments using the specific LIMK1 inhibitor,BMS-5. Neurons obtained from healthy subjects treated with the inhibitor showed similar morphological changes to those observed in sAD neurons,confirming that LIMK1 activity is crucial for maintaining normal neuronal structure. Furthermore,administration of the inhibitor to sAD neurons did not exacerbate the morphological alterations,suggesting that LIMK1 activity is already compromised in these cells. Our findings demonstrate that although sAD- and HS-hiPSCs are similar in their stemness and genomic stability,sAD-hNs exhibit distinct functional and structural anomalies mirroring AD pathology. These anomalies include synaptic dysfunction,altered cytoskeletal organization,and accumulation of AD-related proteins. Our study underscores the usefulness of hiPSCs in modeling AD and provides insights into the disease's molecular underpinnings,thus highlighting potential therapeutic targets. The online version contains supplementary material available at 10.1186/s13195-024-01632-3. View Publication

Cancer-associated fibroblasts (CAFs) play pivotal roles in solid tumor initiation,growth,and immune evasion. However,the optimal biomimetic modeling conditions remain elusive. In this study,we investigated the effects of 2D and 3D culturing conditions on human primary CAFs integrated into a modular tumor microenvironment (TME). Using single-nucleus RNA sequencing (snRNAseq) and Proteomics’ Proximity Extension Assays,we characterized CAF transcriptomic profiles and cytokine levels. Remarkably,when cultured in 2D,CAFs exhibited a myofibroblast (myCAF) subtype,whereas in 3D tumor spheroid cultures,CAFs displayed a more inflammatory (iCAF) pathological state. By integrating single-cell gene expression data with functional interrogations of critical TME-related processes [natural killer (NK)-mediated tumor killing,monocyte migration,and macrophage differentiation],we were able to reconcile form with function. In 3D TME spheroid models,CAFs enhance cancer cell growth and immunologically shield cells from NK cell-mediated cytotoxicity,in striking contrast with their 2D TME counterparts. Notably,3D CAF-secreted proteins manifest a more immunosuppressive profile by enhancing monocyte transendothelial migration and differentiation into M2-like tumor-associated macrophages (TAMs). Our findings reveal a more immunosuppressive and clinically relevant desmoplastic TME model that can be employed in industrial drug discovery campaigns to expand the cellular target range of chemotherapeutics. View Publication

Infusion of T cells modified with a chimeric antigen receptor (CAR) targeting CD19 has achieved exceptional responses in patients with non-Hodgkin’s lymphoma (NHL),which led to the approval of CAR targeting CD19 (CART19) (Axi-cel and Liso-cel) as second line of treatment for adult patients with relapsed/refractory NHL. Unfortunately,60% of patients still relapse after CART19 due to either a loss of expression of the target antigen (CD19) in the tumor cell,observed in 27% of relapsed patients,a limited CAR-T persistence,and additional mechanisms,including the suppression of the tumor microenvironment. Clinic strategies to prevent target antigen loss include sequential treatment with CARs directed at CD20 or CD22,which have caused loss of the second antigen,suggesting targeting other antigens less prone to disappear. CD79b,expressed in NHL,is a target in patients treated with antibody-drug conjugates (ADC). However,the limited efficacy of ADC suggests that a CAR therapy targeting CD79b might improve results. We designed three new CARs against CD79b termed CAR for Lymphoma (CARLY)1,2 and 3. We compared their efficacy,phenotype,and inflammatory profiles with CART19 (ARI0001) and CARTBCMA (ARI0002h),which can treat NHL. We also analyzed the target antigen’s expression loss (CD79b,CD19,and B-cell maturation antigen(BCMA)). We found that CARLY2 and CARLY3 had high affinity and specificity towards CD79b on B cells. In vitro,all CAR-T cells had similar anti-NHL efficacy,which was retained in an NHL model of CD19 − relapse. In vivo,CARLY3 showed the highest efficacy. Analysis of the loss of the target antigen demonstrated that CARLY cells induced CD79b and CD19 downregulation on NHL cells with concomitant trogocytosis of these antigens to T cells,being most notorious in CARLY2,which had the highest affinity towards CD79b and CD19,and supporting the selection of CARLY3 to design a new treatment for patients with NHL. Finally,we created a CAR treatment based on dual targeting of CD79b and BCMA to avoid losing the target antigen. This treatment showed the highest efficacy and did not cause loss of the target antigen. Based on specificity,efficacy,and loss of the target antigen,CARLY3 represents a potential novel CAR treatment for NHL. View Publication

Inflammation in cystic fibrosis (CF) airways is difficult to treat with well-established regimens often including azithromycin (AZ) as an immunomodulatory drug. As AZ has been reported to require CF transmembrane conductance regulator (CFTR) to be able to reduce interleukin (IL)-8 and given the emergence of highly effective CFTR “triple” modulator therapy (elexacaftor/tezacaftor/ivacaftor; ETI),the aim of this study was to investigate the effect of AZ and ETI,singly and in combination,on ion channel activity and to assess the potential anti-inflammatory effects. Electrophysiological assessment of ETI and AZ was performed on three-dimensional cultures of primary CF human bronchial epithelial (HBE) cells using a Multi Trans-Epithelial Current Clamp. IL-8 from NuLi-1 (non-CF) and CuFi-1 (CF) cells treated with AZ was measured by ELISA. Inflammatory mediators from primary CF HBE cells exposed to tumour necrosis factor-α in the presence of AZ,ETI and their combination,were screened using the Proteome Profiler™ Human Cytokine Array Kit,with selected targets validated by ELISA. AZ did not alter CFTR chloride efflux,nor did it have any synergistic/antagonistic effect in combination with ETI. AZ reduced IL-8 in NuLi-1 but not CuFi-1 cells. The Proteome Profiler™ screen identified several disease-relevant cytokines that were modulated by treatment. Subsequent analysis by ELISA showed IL-8,IL-6,CXCL1 and granulocyte–macrophage colony-stimulating factor to be significantly reduced by treatment with ETI,but not by AZ. Incorporating ETI into the standard of CF care provides an opportunity to re-evaluate therapeutic regimens to reduce treatment burden and safely discontinue chronic treatments such as AZ,without loss of clinical benefit. Identification of redundant treatments in the era of CFTR modulation may improve medication adherence and overcome potential adverse effects associated with the chronic use AZ and other drugs. View Publication

Rationale: Immune checkpoint inhibitors (ICIs) have demonstrated significant efficacy against head and neck squamous cell carcinoma (HNSCC),but their overall response rate (ORR) remains limited. Previous studies have highlighted the crucial role of sphingosine kinases (SPHKs) in the tumor microenvironment (TME); however,their function in immunotherapy remains unclear. Methods: We conducted comprehensive bioinformatics analysis,functional studies,and clinical validation,to investigate the role of SPHK1 in the immunology of HNSCC. Results: Functionally,SPHK1 significantly promoted tumor growth by inhibiting anti-tumor immunity in immune-competent HNSCC mouse models and tumor-T cell co-cultures. Mechanistic analysis revealed that SPHK1 regulated matrix metalloproteinase-1 (MMP1) expression via the MAPK1 pathway,which subsequently influenced tumor programmed cell death ligand 1 (PD-L1) expression. Furthermore,SPHK1 and MMP1 could predict the efficacy of programmed cell death 1 monoclonal antibody (PD-1 mAb) immunotherapy in HNSCC and were independent risk factors for survival in patients with HNSCC. Conclusion: Our study reveals a novel role for SPHK1 in mediating immune evasion in HNSCC through the regulation of the MMP1-PD-L1 axis. We identified SPHK1 and MMP1 as predictive biomarkers for the therapeutic response to PD-1 mAb and provided new therapeutic targets for patients with HNSCC. View Publication

Erythropoiesis is a crucial process in hematopoiesis,yet it remains highly susceptible to disruption by various diseases,which significantly contribute to the global challenges of anemia and blood shortages. Current treatments like erythropoietin (EPO) or glucocorticoids often fall short,especially for hereditary anemias such as Diamond-Blackfan anemia (DBA). To uncover new erythropoiesis-stimulating agents,we devised a screening system using primary human hematopoietic stem and progenitor cells (HSPCs). We discovered that BRAF inhibitors (BRAFi),commonly used to treat BRAF V600E melanoma,can unexpectedly and effectively promote progenitor cell proliferation by temporarily delaying erythroid differentiation. Notably,these inhibitors exhibited pronounced efficacy even under cytokine-restricted conditions and in patient samples of DBA. Mechanistically,although these BRAFi inhibit the MAPK cascade in BRAF V600E mutant cells,they paradoxically act as amplifiers in wild-type BRAF cells,potently enhancing the cascade. Furthermore,we found that while the oncogenic BRAF V600E mutation disrupts hematopoiesis and erythropoiesis through AP-1 hyperactivation,BRAFi minimally impact HSPC self-renewal and differentiation. In vivo studies have shown that BRAFi can enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia in the Rpl11 haploinsufficiency DBA model,as well as other relevant anemia models. This discovery underscores the role of the MAPK pathway in hematopoiesis and positions BRAFi as a promising therapeutic option for improving hematopoietic reconstitution and treating anemias,including DBA. Subject terms: Drug screening,Molecular medicine View Publication

Breast cancer is a heterogeneous disease comprising multiple molecularly distinct subtypes with varied prevalence,prognostics,and treatment strategies. Among them,triple-negative breast cancer,though the least prevalent,is the most aggressive subtype,with limited therapeutic options. Recent emergence of competing endogenous RNA (ceRNA) networks has highlighted how long noncoding RNAs (lncRNAs),microRNAs (miRs),and mRNA orchestrate a complex interplay meticulously modulating mRNA functionality. Focusing on TNBC,this study aimed to construct a ceRNA network using differentially expressed lncRNAs,miRs,and mRNAs. We queried the differentially expressed lncRNAs (DElncRNAs) between TNBC and luminal samples and found 389 upregulated and 386 downregulated lncRNAs,including novel transcripts in TNBC. DElncRNAs were further evaluated for their clinical,functional,and mechanistic relevance to TNBCs using the lnc2cancer 3.0 database,which presented LUCAT1 (lung cancer-associated transcript 1) as a putative node. Next,the ceRNA network (lncRNA–miRNA–mRNA) of LUCAT1 was established. Several miRNA–mRNA connections of LUCAT1 implicated in regulating stemness (LUCAT1-miR-375-Yap1,LUCAT1-miR181-5p-Wnt,LUCAT1-miR-199a-5p-ZEB1),apoptosis (LUCAT1-miR-181c-5p-Bcl2),drug efflux (LUCAT1-miR-200c-ABCB1,LRP1,MRP5,MDR1),and sheddase activities (LUCAT1-miR-493-5p-ADAM10) were identified,indicating an intricate regulatory mechanism of LUCAT1 in TNBC. Indeed,LUCAT1 silencing led to mitigated cell growth,migration,and stem-like features in TNBC. This work sheds light on the LUCAT1 ceRNA network in TNBC and implies its involvement in TNBC growth and progression. View Publication

The introduction of antibody–drug conjugates represents a significant advancement in targeted therapy of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our study aims to investigate the role of the DNA damage response pathway and the impact of PARP1 inhibition,utilizing talazoparib,on the response of AML and ALL cells to Gemtuzumab ozogamicin (GO) and Inotuzumab ozogamicin (INO),respectively. AML and ALL cells were treated with GO,INO and γ-calicheamicin in order to induce severe DNA damage and activate the G2/M cell-cycle checkpoint in a dose- and time-dependent manner. The efficacy of PARP1 inhibitors and,in particular,talazoparib in enhancing INO or GO against ALL or AML cells was assessed through measurements of cell viability,cell death,cell cycle progression,DNA damage repair,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. We observed that both ALL and AML cell lines activate the G2/M cell-cycle checkpoint in response to γ-calicheamicin-induced DNA damage,highlighting a shared cellular response mechanism. Talazoparib significantly enhanced the efficacy of INO against ALL cell lines,resulting in reduced cell viability,increased cell death,G2/M cell-cycle checkpoint override,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. Strong synergism was observed in primary ALL cells treated with the combination. In contrast,AML cells exhibited a heterogeneous response to talazoparib in combination with GO. Our findings suggest a potential link between the differential responses of ALL and AML cells to the drug combinations and the ability of talazoparibto override G2/M cell-cycle arrest induced by antibody–drug conjugates. PARP1 emerges as a key player in the response of ALL cells to INO and represents a promising target for therapeutic intervention in this leukemia setting. Our study sheds light on the intricate interplay between the DNA damage response pathway,PARP1 inhibition,and response of γ-calicheamicin-induced DNA damages in AML and ALL. These findings underscore the importance of targeted therapeutic strategies and pave the way for future research aimed at optimizing leukemia treatment approaches. The online version contains supplementary material available at 10.1186/s12967-024-05838-9. View Publication

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 −/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence,we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice,and in lineage specific,regulated and robust transgene expression. This approach,which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells,may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases. Subject terms: Targeted gene repair,Haematopoietic stem cells,Microglial cells View Publication

Embryonic stem cells possess the remarkable ability to self-organize into blastocyst-like structures upon induction. These stem cell-based embryo models serve as invaluable platforms for studying embryogenesis and therapeutic developments. Nevertheless,the specific intrinsic regulators that govern this potential for blastoid formation remain unknown. Here we demonstrate an intrinsic program that plays a crucial role in both blastoids and blastocysts across multiple species. We first establish metrics for grading the resemblance of blastoids to mouse blastocysts,and identify the differential activation of gene regulons involved in lineage specification among various blastoid grades. Notably,abrogation of nuclear receptor subfamily 1,group H,member 2 (Nr1h2) drastically reduces blastoid formation. Nr1h2 activation alone is sufficient to rewire conventional ESC into a distinct pluripotency state,enabling them to form blastoids with enhanced implantation capacity in the uterus and contribute to both embryonic and extraembryonic lineages in vivo. Through integrative multi-omics analyses,we uncover the broad regulatory role of Nr1h2 in the transcriptome,chromatin accessibility and epigenome,targeting genes associated with embryonic lineage and the transposable element SINE-B1. The Nr1h2-centred intrinsic program governs and drives the development of both blastoids and early embryos. Subject terms: Embryonic stem cells,Pluripotency,Epigenomics View Publication