Discovery of a peripheral 5HT2A antagonist as a clinical candidate for metabolic dysfunction-associated steatohepatitis.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is currently the leading cause of chronic liver disease worldwide. Metabolic Dysfunction-Associated Steatohepatitis (MASH), an advanced form of MASLD, can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Based on recent findings by our team that liver 5HT2A knockout male mice suppressed steatosis and reduced fibrosis-related gene expression, we developed a peripheral 5HT2A antagonist, compound 11c for MASH. It shows good in vitro activity, stability, and in vivo pharmacokinetics (PK) in rats and dogs. Compound 11c also shows good in vivo efficacy in a diet-induced obesity (DIO) male mice model and in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) male mice model, effectively improving histologic features of MASH and fibrosis. According to the tissue distribution study using [14C]-labeled 11c, the compound was determined to be a peripheral 5HT2A antagonist. Collectively, first-in-class compound 11c shows promise as a therapeutic agent for the treatment of MASLD and MASH.

Two distinct receptor-binding domains of human glycyl-tRNA synthetase 1 displayed on extracellular vesicles activate M1 polarization and phagocytic bridging of macrophages to cancer cells.

Macrophages play important roles in cancer microenvironment. Human cytosolic glycyl-tRNA synthetase (GARS1) was previously shown to be secreted via extracellular vesicles (EVs) from macrophages to trigger cancer cell death. However, the effects of GARS1-containing EVs (GARS1-EVs) on macrophages as well as on cancer cells and the working mechanisms of GARS1 in cancer microenvironment are not yet understood. Here we show that GARS1-EVs induce M1 polarization and facilitate phagocytosis of macrophages. GARS1-EVs triggers M1 polarization of macrophage via the specific interaction of the extracellular cadherin subdomains 1-4 of the cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2) with the N-terminal WHEP domain containing peptide region of GARS1, and activates the RAF-MEK-ERK pathway for M1 type cytokine production and phagocytosis. Besides, GARS1 interacted with cadherin 6 (CDH6) of cancer cells via its C-terminal tRNA-binding domain to induce cancer cell death. In vivo model, GARS1-EVs showed potent suppressive activity against tumor initiation via M1 type macrophages. GARS1 displayed on macrophage-secreted extracellular vesicles suppressed tumor growth in dual mode, namely through pro-apoptotic effect on cancer cells and M1 polarization effect on macrophages. Collectively, these results elucidate the unique tumor suppressive activity and mechanism of GARS1-EVs by activating M1 macrophage via CELSR2 as well as by direct killing of cancer cells via CDH6.

Identification of drug combinations on the basis of machine learning to maximize anti-aging effects.

Aging is a multifactorial process that involves numerous genetic changes, so identifying anti-aging agents is quite challenging. Age-associated genetic factors must be better understood to search appropriately for anti-aging agents. We utilized an aging-related gene expression pattern-trained machine learning system that can implement reversible changes in aging by linking combinatory drugs. In silico gene expression pattern-based drug repositioning strategies, such as connectivity map, have been developed as a method for unique drug discovery. However, these strategies have limitations such as lists that differ for input and drug-inducing genes or constraints to compare experimental cell lines to target diseases. To address this issue and improve the prediction success rate, we modified the original version of expression profiles with a stepwise-filtered method. We utilized a machine learning system called deep-neural network (DNN). Here we report that combinational drug pairs using differential expressed genes (DEG) had a more enhanced anti-aging effect compared with single independent treatments on leukemia cells. This study shows potential drug combinations to retard the effects of aging with higher efficacy using innovative machine learning techniques.

Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1.

BACKGROUND: The generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed. METHODS: A unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood. RESULTS: Here, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists. CONCLUSIONS: We identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.

Plasma Lysyl-tRNA Synthetase 1 (KARS1) as a Novel Diagnostic and Monitoring Biomarker for Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of world cancer deaths. To improve the survival rate of CRC, diagnosis and post-operative monitoring is necessary. Currently, biomarkers are used for CRC diagnosis and prognosis. However, these biomarkers have limitations of specificity and sensitivity. Levels of plasma lysyl-tRNA synthetase (KARS1), which was reported to be secreted from colon cancer cells by stimuli, along with other secreted aminoacyl-tRNA synthetases (ARSs), were analyzed in CRC and compared with the currently used biomarkers. The KARS1 levels of CRC patients (n = 164) plasma were shown to be higher than those of healthy volunteers (n = 32). The diagnostic values of plasma KARS1 were also evaluated by receiving operating characteristic (ROC) curve. Compared with other biomarkers and ARSs, KARS1 showed the best diagnostic value for CRC. The cancer specificity and burden correlation of plasma KARS1 level were validated using azoxymethane (AOM)/dextran sodium sulfate (DSS) model, and paired pre- and post-surgery CRC patient plasma. In the AOM/DSS model, the plasma level of KARS1 showed high correlation with number of polyps, but not for inflammation. Using paired pre- and post-surgery CRC plasma samples (n = 60), the plasma level of KARS1 was significantly decreased in post-surgery samples. Based on these evidence, KARS1, a surrogate biomarker reflecting CRC burden, can be used as a novel diagnostic and post-operative monitoring biomarker for CRC.

Extracellular vesicles derived from macrophages display glycyl-tRNA synthetase 1 and exhibit anti-cancer activity.

Glycyl-tRNA synthetase 1 (GARS1), a cytosolic enzyme secreted from macrophages, promotes apoptosis in cancer cells. However, the mechanism underlying GARS1 secretion has not been elucidated. Here, we report that GARS1 is secreted through unique extracellular vesicles (EVs) with a hydrodynamic diameter of 20-58 nm (mean diameter: 36.9 nm) and a buoyant density of 1.13-1.17 g/ml. GARS1 was anchored to the surface of these EVs through palmitoylated C390 residue. Proteomic analysis identified 164 proteins that were uniquely enriched in the GARS1-containing EVs (GARS1-EVs). Among the identified factors, insulin-like growth factor II receptor, and vimentin also contributed to the anti-cancer activity of GARS1-EVs. This study identified the unique secretory vesicles containing GARS1 and various intracellular factors that are involved in the immunological defence response against tumorigenesis.

Unique N-terminal extension domain of human asparaginyl-tRNA synthetase elicits CCR3-mediated chemokine activity.

Asparaginyl-tRNA synthetase (NRS) is not only essential in protein translation but also associated with autoimmune diseases. Particularly, patients with antibodies that recognize NRS often develop interstitial lung disease (ILD). However, the underlying mechanism of how NRS is recognized by immune cells and provokes inflammatory responses is not well-understood. Here, we found that the crystal structure of the unique N-terminal extension domain of human NRS (named as UNE-N, where -N denotes NRS) resembles that of the chemotactic N-terminal domain of NRS from a filarial nematode, Brugia malayi, which recruits and activates specific immune cells by interacting with CXC chemokine receptor 1 and 2. UNE-N induced migration of CC chemokine receptor 3 (CCR3)-expressing cells. The chemokine activity of UNE-N was significantly reduced by suppressing CCR3 expression with CCR3-targeting siRNA, and the loop3 region of UNE-N was shown to interact mainly with the extracellular domains of CCR3 in nuclear magnetic resonance perturbation experiments. Based on these results, evolutionarily acquired UNE-N elicits chemokine activities that would promote NRS-CCR3-mediated proinflammatory signaling in ILD.

Caspase-8 controls the secretion of inflammatory lysyl-tRNA synthetase in exosomes from cancer cells.

Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-α and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.

Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.