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Background: Triple-negative breast cancers (TNBCs) typically have a greater immune cell infiltrate and are more likely to respond to immune checkpoint inhibition (ICI) than ER+ or HER2+ breast cancers. However,there is a crucial need to optimize combining chemotherapy strategies with ICI to enhance overall survival in TNBC. Methods: Therefore,we developed a high-throughput co-culture screening assay to identify compounds that enhance CD8+ T-cell-mediated tumor cell cytotoxicity. Over 400 FDA-approved compounds or agents under investigation for oncology indications were included in the screening library. Results: Four chemotherapy agents were chosen as priority hits for mechanistic follow-up due to their ability to enhance T-cell-mediated cytotoxicity at multiple doses and multiple time points: paclitaxel,bleomycin sulfate,ispinesib,and etoposide. Lead compounds affected the expression of MHCI,MHCII,and PD-L1 and induced markers of immunogenic cell death (extracellular ATP or HMGB1). Conclusions: Based on the ability to increase tumor cell susceptibility to T-cell-mediated cytotoxicity while minimizing T-cell toxicity,bleomycin was identified as the most promising lead candidate. Overall,the results of these studies provide mechanistic insight into potential new chemotherapy partners to enhance anti-PD-1 efficacy in TNBC patients. View Publication

SummaryThe comprehensive genomic impact of ionizing radiation (IR),a carcinogen,on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells,we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs),including balanced inversions,translocations,composite SVs (deletion-insertion,deletion-inversion,and deletion-translocation composites),and complex genomic rearrangements (CGRs),including chromoplexy,chromothripsis,and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome,comprising 2.15 indels,0.17 SVs,and 0.01 CGRs,despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose,regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall,our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes. Graphical abstract Highlights•Single-cell-resolution DNA sequencing reveals IR-associated mutational signatures•IR-associated mutations include short deletions and simple and complex rearrangements•Simultaneous multiple DSBs cause chromothripsis,chromoplexy,and SVs by BFB cycles•1 Gy IR exposure generates ∼2.33 mutations per Gb in mammalian cells Youk,Kwon,Lim,Kim,Kim et al. used single-cell-resolution DNA sequencing to investigate the quantitative and qualitative impact of ionizing radiation (IR) on the mammalian genome. They found characteristic mutational signatures,comprised of specific short deletions and simple and complex structural variations. Complex genomic rearrangements,prevalent in cancer,were induced in post-irradiated cells,suggesting their role in tumor development. The study reveals mutational rates proportional to IR dose and consistent across cell types,underscoring IR’s universal mutagenic impact on mammalian cells. View Publication

Unlike most cancer types,the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS),we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly,gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed,syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8+ T cells into the tumor microenvironment. Consistently,FOXM1 suppresses CD8+ T cell chemotaxis in vitro and antigen-dependent CD8+ T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response. View Publication

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs,such as the heart,is crucial for a normal pregnancy. However,the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT),which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells,promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover,in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis,including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo. The genetic link between placenta function and congenital heart defects has been established,though the cellular mechanisms underlying this connection is less clear. Here they show that PIBF1 regulates syncytiotrophoblast fusion and that loss of PIBF1 also negatively impacts heart development,providing a potential link between the development of these two organs. View Publication

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here,we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing,mass spectrometry-based proteomics,and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically,myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking,potentially distorting cell population representations and affecting drugs targeting these cells. Additionally,highly granular AML cell numbers decreased after freezing. Secondly,protein expression levels,as well as sensitivity to drugs targeting cell proliferation,metabolism,tyrosine kinases (e.g.,JAK,KIT,FLT3),and BH3 mimetics were notably affected by biobanking. Moreover,drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed,freezing cells had a considerable impact at an individual level,which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility,and where validation of data generated from biobanked cohorts may be particularly important. View Publication

Innate immunity provides the first line of defense through multiple mechanisms,including pyrogen production and cell death. While elevated body temperature during infection is beneficial to clear pathogens,heat stress (HS) can lead to inflammation and pathology. Links between pathogen exposure,HS,cytokine release,and inflammation have been observed,but fundamental innate immune mechanisms driving pathology during pathogen exposure and HS remain unclear. Here,we use multiple genetic approaches to elucidate innate immune pathways in infection or LPS and HS models. Our results show that bacteria and LPS robustly increase inflammatory cell death during HS that is dependent on caspase-1,caspase-11,caspase-8,and RIPK3 through the PANoptosis pathway. Caspase-7 also contributes to PANoptosis in this context. Furthermore,NINJ1 is an important executioner of this cell death to release inflammatory molecules,independent of other pore-forming executioner proteins,gasdermin D,gasdermin E,and MLKL. In an in vivo HS model,mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients. Fevers are known to be both beneficial and detrimental in disease,but the fundamental innate immune mechanisms driving pathology in this context remain unclear. Here,the authors show that a combination of LPS and heat stress induces inflammatory cell death,PANoptosis,that is dependent on the executioner molecule Ninjurin 1 (Ninj1) to release inflammatory molecules and drive pathogenesis. View Publication

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions,ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis,where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling,as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons,and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However,our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants,we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters,we demonstrate receptor-specific autophagic capture of ER in axons,and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions. Hoyer et al. establish that selective autophagy mechanisms are needed to remodel the ER and its proteome during in vitro neurogenesis across neuronal subcompartments and decode the substrate selectivity of ER-phagy receptors. View Publication

IntroductionConventional foot-and-mouth disease (FMD) vaccines have been developed to enhance their effectiveness; however,several drawbacks remain,such as slow induction of antibody titers,short-lived immune response,and local side effects at the vaccination site. Therefore,we created a novel FMD vaccine that simultaneously induces cellular and humoral immune responses using the Dectin-2 agonist,D-galacto-D-mannan,as an adjuvant.MethodsWe evaluated the innate and adaptive (cellular and humoral) immune responses elicited by the novel FMD vaccine and elucidated the signaling pathway involved both in vitro and in vivo using mice and pigs,as well as immune cells derived from these animals.ResultsD-galacto-D-mannan elicited early,mid-,and long-term immunity via simultaneous induction of cellular and humoral immune responses by promoting the expression of immunoregulatory molecules. D-galacto-D-mannan also enhanced the immune response and coordinated vaccine-mediated immune response by suppressing genes associated with excessive inflammatory responses,such as nuclear factor kappa B,via Sirtuin 1 expression.ConclusionOur findings elucidated the immunological mechanisms induced by D-galacto-D-mannan,suggesting a background for the robust cellular and humoral immune responses induced by FMD vaccines containing D-galacto-D-mannan. Our study will help to facilitate the improvement of conventional FMD vaccines and the design of next-generation FMD vaccines. View Publication

SummaryStreptococcus suis is a zoonotic pathogen that can cause meningitis and septicaemia. The consumption of undercooked pig products is an important risk factor for zoonotic infections,suggesting an oral route of infection. In a human enteroid model,we show that the zoonotic CC1 genotype has a 40% higher translocation frequency than the non-zoonotic CC16 genotype. Translocation occurred without increasing the permeability or disrupting the adherens junctions and tight junctions of the epithelial monolayer. The translocation of zoonotic S. suis was correlated with the presence of Gb3-positive cells,a human glycolipid receptor found on Paneth cells and targeted by multiple enteric pathogens. The virulence factors Streptococcal adhesin Protein and suilysin,known to interact with Gb3,were not essential for translocation in our epithelial model. Thus,the ability to translocate across an enteroid monolayer correlates with S. suis core genome composition and the presence of Gb3-positive cells in the intestinal epithelium. Graphical abstract Highlights•S. suis SS2CC1 translocates more across enteroid monolayers than SS2CC20 and SS9CC16•S. suis translocation occurs without the disruption of the enteroid monolayer•Gb3-positive cells are present within human enteroid monolayers•Zoonotic S. suis translocation correlates with Gb3-positive cell presence Medical microbiology; Microbiology; Molecular microbiology View Publication

The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time,label-free,impedance-based method used to study various cell behaviors such as cell growth,viability,migration,and barrier function in vitro. So far,the ECIS technology has exclusively been performed on cell lines. Organoids,however,are cultured from tissue-specific stem cells,which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper,we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features • We present a protocol that allows the assessment of various cell functions,such as proliferation and barrier formation,with ECIS on organoid-derived monolayers.• The protocol facilitates intestinal barrier research on patient tissue-derived organoids,providing a valuable tool for disease modeling. View Publication

AbstractBreakpoint cluster region‐Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia‐positive (Ph+) B‐cell acute lymphoblastic leukemia (B‐ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients,50% of Ph+ B‐ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B‐ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B‐ALL cells,particularly in combination with other drugs. Here,we present that peroxiredoxin‐1 (PRDX1),one of the enzymes of the TXN system,is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B‐ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib‐treated,PRDX1‐deficient cells revealed that the nonhomologous end‐joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B‐ALL cells. Accordingly,PRDX1‐deficient Ph+ B‐ALL cells were susceptible to NHEJ inhibitors. Finally,we demonstrated the potent efficacy of a novel combination of TKIs,TXN inhibitors,and NHEJ inhibitors against Ph+ B‐ALL cell lines and primary cells,which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B‐ALL. View Publication

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7,which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity,whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation,the sensory neurons start spontaneous action potential firing around day 22,with increasing firing rate until day 40. Here,we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing,and we expect this iPS cell model system to be useful for drug discovery and disease modeling.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00424-024-02945-w. View Publication