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T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione–disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S -glutathionylation of the NAD ⁺-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S -glutathionylation to vertebrate development and successful embryonic angiogenesis.

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of theVegfbgene in mice lacks an overt phenotype. Here we show that knockdown ofVegfba,but notVegfbb,in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain.Vegfbamorpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects,Vegfba,but notVegfaa,mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection ofNeuropilin1(Nrp1) mRNA showed an overlapping expression pattern withVegfba,and knockdown ofNrp1produced a nearly identically lethal phenotype asVegfbaknockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued theVegfbamorpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defectiveVhl-mutant zebrafish strain, which again rescued theVegfbamorpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.

Cutaneous malignant melanoma (CMM) is the deadliest form of skin cancer and clinically challenging due to its propensity to develop therapy resistance. Reactive oxygen species (ROS) can induce DNA damage and play a significant role in CMM. MTH1 protein protects from ROS damage and is often overexpressed in different cancer types including CMM. Herein, we report that MTH1 inhibitor TH1579 induced ROS levels, increased DNA damage responses, caused mitotic arrest and suppressed CMM proliferation leading to cell death both in vitro and in an in vivo xenograft CMM zebrafish disease model. TH1579 was more potent in abrogating cell proliferation and inducing cell death in a heterogeneous co-culture setting when compared with CMM standard treatments, vemurafenib or trametinib, showing its broad anticancer activity. Silencing MTH1 alone exhibited similar cytotoxic effects with concomitant induction of mitotic arrest and ROS induction culminating in cell death in most CMM cell lines tested, further emphasizing the importance of MTH1 in CMM cells. Furthermore, overexpression of receptor tyrosine kinase AXL, previously demonstrated to contribute to BRAF inhibitor resistance, sensitized BRAF mutant and BRAF/NRAS wildtype CMM cells to TH1579. AXL overexpression culminated in increased ROS levels in CMM cells. Moreover, silencing of a protein that has shown opposing effects on cell proliferation, CAV-1, decreased sensitivity to TH1579 in a BRAF inhibitor resistant cell line. AXL-MTH1 and CAV-1-MTH1 mRNA expressions were correlated as seen in CMM clinical samples. Finally, TH1579 in combination with BRAF inhibitor exhibited a more potent cell killing effect in BRAF mutant cells both in vitro and in vivo. In summary, we show that TH1579-mediated efficacy is independent of BRAF/NRAS mutational status but dependent on the expression of AXL and CAV-1.

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.

The cell cycle is a key regulator of endothelial cell specification into tip and stalk cell phenotypes, which are essential for angiogenesis in both normal development and pathological conditions. While the tumor suppressor p53 is known to regulate the cell cycle and influence cell fate, its role in modulating the cell fate of these phenotypes remains unclear. Using non-genotoxic small molecule and stapled peptide compounds to pharmacologically activate p53 via MDM2 inhibition, we demonstrate that graded levels of p53 induce distinct cellular fates in normal endothelial cells. Low levels of p53 induce reversible cell cycle arrest by reducing DNA replication, while high levels induce senescence and cell death. Surprisingly, all tested levels of p53 activation reduced the growth of venous blood vessels in vitro and in zebrafish embryo models. This reduction in sprouting may stem from distinct cellular responses in tip-like and non-tip-like cells to pharmacological p53 activation: low p53 levels primarily reduced proliferation in non-tip-like cells, whereas high levels decreased the frequency of tip-like cells and the expression of genes associated with tip and stalk cell identities. Our findings show for the first time that pharmacological p53 activation modulates endothelial cell fate in a dose-dependent manner during sprouting angiogenesis. They also highlight the potential of using graded p53 modulation as a therapeutic strategy to target abnormal tip or stalk cell development in pathological angiogenesis, such as in cancer.

The gut microbiome produces metabolites that interact with the aryl hydrocarbon receptor (AhR), a key regulator of immune homoeostasis in the gut 1 , 2 . Here we show that oral exposure to graphene oxide (GO) modulates the composition of the gut microbiome in adult zebrafish, with significant differences in wild-type versus ahr2 -deficient animals. Furthermore, GO was found to elicit AhR-dependent induction of cyp1a and homing of lck + cells to the gut in germ-free zebrafish larvae when combined with the short-chain fatty acid butyrate. To obtain further insights into the immune responses to GO, we used single-cell RNA sequencing to profile cells from whole germ-free embryos as well as cells enriched for lck . These studies provided evidence for the existence of innate lymphoid cell (ILC)-like cells 3 in germ-free zebrafish. Moreover, GO endowed with a ‘corona’ of microbial butyrate triggered the induction of ILC2-like cells with attributes of regulatory cells. Taken together, this study shows that a nanomaterial can influence the crosstalk between the microbiome and immune system in an AhR-dependent manner. Although the toxicity of graphene‐based nanomaterials on human health has been extensively studied, their impact on the microbiome remains poorly understood. Using zebrafish as a model, we show that graphene oxide modulates the immune system in a microbiome‐dependent manner through a mechanism mediated by the aryl hydrocarbon receptor. The study suggests an interplay among graphene‐based nanomaterials, microbiome and innate immune system.

[...]the authors describe regulatory thiol switches in nuclear factor kappa B (NFκB), a disintegrin and metalloproteinase 17 (ADAM17), and high mobility group box 1 protein (HMGB1) as well as in pathways related to inflammatory signaling including the TLR cascades.The work entitled “Cold Atmospheric Plasma Induces Apoptosis and Oxidative Stress Pathway Regulation in T-Lymphoblastoid Leukemia Cells” by E. Turrini et al. aimed to mechanistically analyze the impact of cold atmospheric plasma and the induction of reactive oxygen and nitrogen species (ROS/RNS) on apoptosis, DNA damage, and the consecutive upregulation of redox-related enzymes, such as, superoxide dismutase, catalase, and glutathione reductase, thereby connecting the short-lived plasma-generated species to central cellular redox signaling pathways.Among the statistically significant genes associated with cancer initiation and progression, the authors found Forkhead box M1 (FoxM1) and thioredoxin reductase 1 (TrxR1) to be critical for the regulation of oxidative stress levels in all analyzed tumor types.[...]A. Leone et al. discuss how the identified signaling networks correlate to cancer stem cell signatures and provide by that knowledge on which redox pathways could be prioritized for the development of novel therapies.

ObjectiveTo better comprehend transcriptional phenotypes of cancer cells, we globally characterised RNA-binding proteins (RBPs) to identify altered RNAs, including long non-coding RNAs (lncRNAs).DesignTo unravel RBP-lncRNA interactions in cancer, we curated a list of ~2300 highly expressed RBPs in human cells, tested effects of RBPs and lncRNAs on patient survival in multiple cohorts, altered expression levels, integrated various sequencing, molecular and cell-based data.ResultsHigh expression of RBPs negatively affected patient survival in 21 cancer types, especially hepatocellular carcinoma (HCC). After knockdown of the top 10 upregulated RBPs and subsequent transcriptome analysis, we identified 88 differentially expressed lncRNAs, including 34 novel transcripts. CRISPRa-mediated overexpression of four lncRNAs had major effects on the HCC cell phenotype and transcriptome. Further investigation of four RBP-lncRNA pairs revealed involvement in distinct regulatory processes. The most noticeable RBP-lncRNA connection affected lipid metabolism, whereby the non-canonical RBP CCT3 regulated LINC00326 in a chaperonin-independent manner. Perturbation of the CCT3-LINC00326 regulatory network led to decreased lipid accumulation and increased lipid degradation in cellulo as well as diminished tumour growth in vivo.ConclusionsWe revealed that RBP gene expression is perturbed in HCC and identified that RBPs exerted additional functions beyond their tasks under normal physiological conditions, which can be stimulated or intensified via lncRNAs and affected tumour growth.

Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.