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Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient’s quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently,there are no approved preventative measures or treatment options for CIPN,highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study,we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine,a chemotherapeutic used for the treatment of breast cancers,osteosarcomas,and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen,we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles – AZD7762,A-674563,Blebbistatin,Glesatinib,KW-2449,and Pelitinib,all novel neuroprotectants against vincristine toxicity to neurons. In addition,four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study,we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent,vincristine which could have therapeutic potential in the clinic. The online version contains supplementary material available at 10.1007/s00018-024-05340-x. View Publication

Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and myeloid skewing. We report that culture of bone marrow (BM) HSCs from aged mice with epidermal growth factor (EGF) suppressed myeloid skewing,increased multipotent colony formation,and increased HSC repopulation in primary and secondary transplantation assays. Mice transplanted with aged,EGF-treated HSCs displayed increased donor cell engraftment within BM HSCs and systemic administration of EGF to aged mice increased HSC self-renewal capacity in primary and secondary transplantation assays. Expression of a dominant negative EGFR in Scl/Tal1 + hematopoietic cells caused increased myeloid skewing and depletion of long term-HSCs in 15-month-old mice. EGF treatment decreased DNA damage in aged HSCs and shifted the transcriptome of aged HSCs from genes regulating cell death to genes involved in HSC self-renewal and DNA repair but had no effect on HSC senescence. These data suggest that EGFR signaling regulates the repopulating capacity of aged HSCs. Subject areas: Human physiology,cellular physiology,molecular medicine,stem cells research,functional aspects of cell biology View Publication

Olanzapine (OLZ) reverses chronic stress-induced anxiety. Chronic stress promotes cancer development via abnormal neuro-endocrine activation. However,how intervention of brain-body interaction reverses chronic stress-induced tumorigenesis remains elusive. Kras LSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model were used to study the effect of OLZ on cancer stemness and anxiety-like behaviors. Cancer stemness was evaluated by qPCR,western-blotting,immunohistology staining and flow-cytometry analysis of stemness markers,and cancer stem-like function was assessed by serial dilution tumorigenesis in mice and extreme limiting dilution analysis in primary tumor cells. Anxiety-like behaviors in mice were detected by elevated plus maze and open field test. Depression-like behaviors in mice were detected by tail suspension test. Anxiety and depression states in human were assessed by Hospital Anxiety and Depression Scale (HADS). Chemo-sensitivity of lung cancer was assessed by in vivo syngeneic tumor model and in vitro CCK-8 assay in lung cancer cell lines. In this study,we found that OLZ reversed chronic stress-enhanced lung tumorigenesis in both Kras LSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model. OLZ relieved anxiety and depression-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing under chronic stress. NE activated ADRB2-cAMP-PKA-CREB pathway to promote CLOCK transcription,leading to cancer stem-like traits. As such,CLOCK-deficiency or OLZ reverses NE/chronic stress-induced gemcitabine (GEM) resistance in lung cancer. Of note,tumoral CLOCK expression is positively associated with stress status,serum NE level and poor prognosis in lung cancer patients. We identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ suppresses mPFC-NE-CLOCK axis to reverse chronic stress-induced anxiety-like behaviors and lung cancer stemness. Decreased NE-releasing prevents activation of ADRB2-cAMP-PKA-CREB pathway to inhibit CLOCK transcription,thus reversing lung cancer stem-like traits and chemoresistance under chronic stress. The online version contains supplementary material available at 10.1186/s12964-024-01747-y. View Publication

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

Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins,in particular albumin,which is prone to quality variations and presents a substantial cost factor,hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations,we have developed chemically defined,entirely protein-free media based on RPMI,supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). Both media compositions enable the efficient,directed differentiation of embryonic and induced hPSCs,matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture,producing > 1.3 × 10 9 cardiomyocytes in a single process run. Transcriptome analysis,flow cytometry,electrophysiology,and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. This study promotes the robustness and upscaling of the cardiomyogenic differentiation process,substantially reduces media costs,and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration. The online version contains supplementary material available at 10.1186/s13287-024-03826-w. View Publication

Mucus stasis is a pathologic hallmark of muco-obstructive diseases,including cystic fibrosis (CF). Mucins,the principal component of mucus,are extensively modified with hydroxyl (O)-linked glycans,which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however,the consequences of reduced sialylation on mucus clearance have not been fully determined. Here,we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin,MUC5B,and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways,and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally,we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall,this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases. Subject terms: Biophysical chemistry,Glycobiology,Respiration 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