技术资料

Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP,or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk,thus potentially offering a new target for breast cancer treatment. We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration,invasion,and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice,Balb/c mice,and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity,we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. Herein,we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone,named 12G2,which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth,was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions,16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases. The online version contains supplementary material available at 10.1186/s13058-024-01873-y. View Publication

Heme oxygenase-1 (HO-1,HMOX1 ) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions,heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here,we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1 -deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1,the effect that was further enhanced in response to δ-aminolevulinic acid (ALA),a substrate in heme synthesis. This was associated with replication stress,as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1 -deficient patient. Interestingly,in the absence of HO-1,the speed of fork progression was higher,and the response to DNA conformational hindrance less stringent,indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead,we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53,an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin,which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1,presumably contributing to its widely recognized cytoprotective activity. View Publication

Immunodeficiency,Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2,accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family,which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here,we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34 + -blood cells of a patient homozygous for the p.Cys408Gly mutation,the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain,perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency,including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation,ICF2-iPSCs showed decreased vitality and a lower percentage of CD34 + /CD43 + /CD45 + progenitors. Overall,the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype. View Publication

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function,accompanied by impaired antigen receptor downstream calcium,ZAP70,ERK,and C-JUN signaling,compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN,but not other antigen receptor downstream components,maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation,independent of anti-exhaustion. We conduct an open-label,non-randomized,single-arm,phase I trial of C-JUN-overexpressing CAR-T in AML ( NCT04835519 ) with safety and efficacy as primary and secondary endpoints,respectively. Of the four patients treated,one has grade 4 (dose-limiting toxicity) and three have grade 1–2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus,overexpressing C-JUN endows CAR-T efficacy in AML. Subject terms: Translational research,Leukaemia View Publication

Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors,the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children,whereas in adults pre-existing mutant clones are selected by the exposure. However,selective pressures induced by chemotherapy early in life are less well studied. Here,we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome,characterized by a germline TP53 mutation,we find that the t-MN already expands during treatment,suggesting that platinum-induced growth inhibition is TP53- dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN,which is important for the development of more targeted,patient-specific treatment regimens and follow-up. Subject terms: Cancer genomics,Cancer genomics,Haematological cancer,Paediatric cancer View Publication

microRNAs (miRNAs) are small,non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types,but its effect on inducing specific anti-tumor immune responses is unclear. Therefore,we examined the effect of our lipid nanoparticle (LNP) formulated,chemically modified,synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines,even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death. View Publication

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this,the molecular mechanisms that allow H . influenzae to establish persistent infections of human epithelia are not well understood. Here,we have investigated how H . influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H . influenzae infection with an initial,‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently,an apparent tolerance to large extracellular and intraepithelial burdens of H . influenzae developed,with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations,and appears to involve interruption of NFκB signalling. This is the first time that large-scale,persistence-promoting immunomodulatory effects of H . influenzae during infection have been observed,and we were able to demonstrate that only infections with live,but not heat-killed H . influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β,IL-36γ and TNFα. Interestingly,NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection,resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H . influenzae in the host,our data further indicate the presence of infection stage-specific gene expression modules,highlighting fundamental similarities between immune responses in NHNE and canonical immune cells,which merit further investigation. View Publication

Autoimmune thyroid disease (AITD) is a common autoimmune disease. In a GWAS meta-analysis of 110,945 cases and 1,084,290 controls,290 sequence variants at 225 loci are associated with AITD. Of these variants,115 are previously unreported. Multiomics analysis yields 235 candidate genes outside the MHC-region and the findings highlight the importance of genes involved in T-cell regulation. A rare 5’-UTR variant (rs781745126-T,MAF = 0.13% in Iceland) in LAG3 has the largest effect (OR = 3.42,P = 2.2 × 10 −16 ) and generates a novel start codon for an open reading frame upstream of the canonical protein translation initiation site. rs781745126-T reduces mRNA and surface expression of the inhibitory immune checkpoint LAG-3 co-receptor on activated lymphocyte subsets and halves LAG-3 levels in plasma among heterozygotes. All three homozygous carriers of rs781745126-T have AITD,of whom one also has two other T-cell mediated diseases,that is vitiligo and type 1 diabetes. rs781745126-T associates nominally with vitiligo (OR = 5.1,P = 6.5 × 10 −3 ) but not with type 1 diabetes. Thus,the effect of rs781745126-T is akin to drugs that inhibit LAG-3,which unleash immune responses and can have thyroid dysfunction and vitiligo as adverse events. This illustrates how a multiomics approach can reveal potential drug targets and safety concerns. Subject terms: Genetics research,Disease genetics,Thyroid diseases,Genome-wide association studies,Gene expression View Publication

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly,laborious,time-consuming,complex,and require skilled personnel. Hence,utilisation of most diagnostics for AAT is impracticable in rural areas,where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection,without relying on expensive equipment,would facilitate AAT detection. In turn,early management,would reduce disease incidence and severity. Today,several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context,we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T . congolense infections,which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here,originated from a past study. Briefly,the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T . congolense glycosomal fructose-1,6-bisphosphate aldolase ( Tco ALD) was discovered as the cognate Ag of Nb474H. In this study,splenocytes were harvested from a mouse immunized with recombinant Tco ALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on Tco ALD retrieved a lone binder,designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native Tco ALD released during infection by T . congolense parasites. Hitherto,development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) “hybrid” sandwich technology offers prospects for exploration,using the unique specificity of Nb as a key determinant in Ag capturing,while using the versatility of monoclonal Ab to adapt to various detection conditions. View Publication

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise,with a negligible affinity for mismatched substrates,but its low cellular targeting efficiency limits therapeutic use. Here,we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency,knock-in rates,and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally,we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor,highlighting its therapeutic utility. Subject terms: CRISPR-Cas9 genome editing,Molecular medicine,Genetic engineering,CRISPR-Cas9 genome editing View Publication

The development of haematopoiesis involves the coordinated action of numerous genes,some of which are implicated in haematological malignancies. However,the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here,we report a previously uncharacterised gene in haematopoiesis,identified by screening mutant embryonic stem cells. The gene,‘ attenuated haematopoietic development ( Ahed )’,encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward,leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed -deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model,we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways,and published databases present cancer patients with somatic mutations in Ahed . Collectively,our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis,implicating its association with malignancies. Subject terms: Lymphopoiesis,Development,Haematopoietic stem cells,Differentiation View Publication

Hypoxia-activated prodrug (HAP) is a promising candidate for highly tumor-specific chemotherapy. However,the oxygenation heterogeneity and dense extracellular matrix (ECM) of tumor,as well as the potential resistance to chemotherapy,have severely impeded the resulting overall efficacy of HAP. A HAP potentiating strategy is proposed based on ultrasound responsive nanodroplets (PTP@PLGA),which is composed of protoporphyrin (PpIX),perfluoropropane (PFP) and a typical HAP,tirapazamine (TPZ). The intense vaporization of PFP upon ultrasound irradiation can magnify the sonomechanical effect,which loosens the ECM to promote the penetration of TPZ into the deep hypoxic region. Meanwhile,the PpIX enabled sonodynamic effect can further reduce the oxygen level,thus activating the TPZ in the relatively normoxic region as well. Surprisingly,abovementioned ultrasound effect also results in the downregulation of the stemness of cancer cells,which is highly associated with drug-refractoriness. This work manifests an ideal example of ultrasound-based nanotechnology for potentiating HAP and also reveals the potential acoustic effect of intervening cancer stem-like cells. The online version contains supplementary material available at 10.1186/s12951-024-02623-0. View Publication