Experimental & Molecular Medicine

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

Skeletal muscle possesses remarkable regenerative capacity. However, in limb-girdle muscular dystrophy-2F (LGMD2F), this capacity is compromised by persistent innate immune activation, whose transcriptional landscape remains unexplored. In parallel, (-)-Epicatechin has emerged as a promising compound with beneficial effects on muscle and notable anti-inflammatory properties. We therefore used (-)-Epicatechin treatment to test whether it can alleviate LGMD2F-associated transcriptional and immune dysregulation. Here we provide the first transcriptomic characterization of LGMD2F using the Sgcd-/- mouse model, along with the first RNA-sequencing-based evaluation of (-)-Epicatechin treatment. We profiled two functionally distinct muscles -- the soleus and EDL -- through bulk RNA-sequencing coupled with immune cell-deconvolution. Sgcd-/- muscles exhibited marked transcriptional dysregulation, more pronounced in the soleus and associated with enhanced innate immune signaling. (-)-Epicatechin induced a muscle- and genotype-dependent transcriptional response: in wild-type animals, the EDL displayed the highest number of differentially expressed transcripts, whereas in Sgcd-/- mice, the soleus showed the most prominent response. This shift was accompanied by downregulation of Toll-like receptor and RIG-I-like receptor pathways, along with suppression of NF-{kappa}B2 and interferon-stimulated genes. Together, these findings identify innate immune overactivation as a central feature of LGMD2F and reveal (-)-Epicatechin as a context-dependent modulator of muscle-specific transcriptional responses.

Stimulator of interferon genes (STING) agonists and derivative molecules have been extensively developed for tumor immunotherapy. However, systemic exposure toxicity risks have constrained clinical trial progression and even threatened patient lives. Currently, systematic toxicity assessments for STING agonists remain lacking, with the mode of action for major organ injury yet to be elucidated. Here, we focused on STING agonist-induced lung injury, revealing that systemic administration of STING agonists caused pulmonary hemorrhage, inflammatory alterations, and respiratory dysfunction. Through single-cell RNA sequencing and immune deletion studies, we found that lung endothelial cells could be stimulated by STING agonists and then secreted chemokines and IL-15 to recruit and activate NK cells. NK cells could induce endothelial cell apoptosis via IFN-{gamma}. Tbx21+ NK subpopulations, which activated by endothelial cells, could produce chemokines to recruit neutrophils. Neutrophils secreted IL-1{beta} through positive feedback pathways and form neutrophil extracellular traps during lung injury. This study elucidates the critical role of the endothelial cell-NK cell-neutrophil axis in mediating STING agonist-associated pneumonia, offering insights for developing intervention strategies for STING agonist toxicity.

Aminoglycoside (AG) antibiotics are indispensable for treating severe infections but frequently cause irreversible hearing loss, with no approved preventive therapies. Using in vivo zebrafish lateral line screening combined with computational scaffold-hopping, we identified a novel class of otoprotective compounds. Starting from the ion channel modulator MR16728, we discovered compound 28510 as a potent lead compound. Compound 28510 provided robust, dose-dependent protection against AG-induced hair cell damage, restoring neuromast hair cell integrity to near-control levels in acute assays and demonstrating broad efficacy across clinically relevant AGs (gentamicin, tobramycin, amikacin, streptomycin) in chronic exposures. Importantly, 28510 exhibited a favorable therapeutic window, with low micromolar 50% hair cell protection concentration (HC50) values consistently below toxicity thresholds. Mechanistically, FM1-43 and Texas Red-conjugated gentamicin uptake assays revealed that 28510 does not inhibit mechanotransduction (MET) channel-mediated AG entry, distinguishing it from current clinical candidates and pointing to a novel intracellular protective mechanism. 28510 preserved AG antibacterial activity in E. coli assays, supporting its translational compatibility as a co-therapeutic agent. Combinations of 28510 with related analogs did not yield synergistic protection; 28510 alone remained the most effective compound. In silico absorption, distribution, metabolism, and excretion (ADME) predictions further confirmed its highly favorable drug-like properties, including excellent intestinal and oral absorption. Together, these findings establish 28510 as a first-in-class, non-MET-mediated otoprotective lead with broad efficacy and a favorable therapeutic profile, highlighting a new strategy for preventing AG-induced hearing loss.

Efficient and cell-specific gene delivery to cochlear inner hair cells (IHCs) remains a major challenge for inner ear gene therapy. Here, we identify and characterize a novel AAV2-derived capsid, AAV-WM04, that enables highly efficient and selective IHC transduction at low doses. Using an in vivo-directed evolution strategy, we generated a randomized AAV2 capsid library with 9-amino acid insertions and performed iterative selection in the adult mouse cochlea. Next-generation sequencing revealed enrichment of several variants, among which AAV-WM04 exhibited superior packaging efficiency and pronounced IHC tropism. AAV-WM04 achieved near-complete IHC transduction throughout the cochlear axis in adult mice, outperforming clinically relevant vectors with minimal off-target expression and no detectable ototoxicity. Robust and exclusive IHC transduction was further validated in non-human primates following round window membrane delivery, underscoring translational potential. Therapeutically, AAV-WM04 enabled efficient dual-AAV trans-splicing delivery of the large OTOF gene, resulting in uniform full-length otoferlin expression in IHCs. In a humanized Otof Q829X/Q829X mouse model, AAV-WM04 restored auditory function across a broad frequency range at relatively low doses and achieved durable hearing recovery. Collectively, these findings establish AAV-WM04 as a next-generation IHC-targeted vector with high efficiency, safety, and cross-species applicability for precision gene therapy of hereditary hearing loss.

Primary ovarian insufficiency (POI) and related infertility, early menopause, and endocrine disorders due to hormonal deficiency are major side effects in young female cancer patients undergoing cancer therapy. Current strategies preserving the fertility and hormonal functions of the ovary remain imperfect due to concerns of feasibility, efficacy, or safety. Herein, we identified c-Jun N-terminal kinase (JNK) as a pivotal regulator of the DNA damage response (DDR) signaling in oocytes of primordial follicles in response to DNA-damaging cancer therapy. Using pharmacological JNK inhibition and a genetically modified mouse model with oocyte-specific JNK deletion, together with histological, bioinformatic, and molecular approaches, we demonstrated that JNK inhibition prevented chemotherapy-induced oocyte apoptosis and POI, and preserved long-term reproductive cycles and fertility. Mechanistically, JNK was activated in response to chemotherapy-induced DNA damage in oocytes of primordial follicles, causing activation of transcription factor TAp63 and subsequent oocyte apoptosis, ultimately resulting in diminished ovarian reserve and POI. A more clinically relevant breast cancer-bearing mouse model revealed that JNK inhibition preserved the ovarian reserve without compromising anti-cancer efficacy of chemotherapy. Together, our study identifies oocyte-intrinsic JNK as a promising target for developing ovarian protectants and safeguarding reproductive health and fertility in young female cancer survivors.

The evolutionary trajectory of lung squamous cell carcinoma (LUSC) remains poorly defined, hindering the development of effective therapies. By integrating genomic and transcriptomic sequencing from human LUSC specimens, we delineated a polyclonal-to-monoclonal evolutionary trajectory during LUSC progression. This evolutionary pattern was corroborated by single-cell RNA sequencing, which revealed consistent tumor cell heterogeneity. Specifically, the SBS5 mutational signature was enriched and correlated with poor prognosis independent of tumor stage. By further utilizing the spontaneous LUSC mouse model to identify key genomic and genetic events in LUSC progression, we observed that the JNK pathway was inhibited and that cytoskeleton-related pathways were dysregulated during LUSC development, and identified the mutations in the JNK pathway (e.g., DACT1) and cytoskeletal regulators (e.g., KIF26A). Collectively, these findings established a polyclonal-monoclonal evolution paradigm for LUSC, potentially regulated by JNK pathways, which could benefit LUSC precision therapeutics.

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of programmed cell death and is implicated in various pathological conditions, particularly in mediating tumor resistance to immune checkpoint inhibitors (ICBs). In this study, we have pioneered the development of a novel cereblon (CRBN)-recruiting RIPK1 degrader, LD5095, through systematic optimization of linker and CRBN ligand portion. LD5095 demonstrates potent and selective RIPK1 degradation across cell lines, with rapid kinetics and sustained degradation over 72h post-washout. Functionally, RIPK1 degradation by LD5095 significantly sensitized Jurkat cells to TNF-induced apoptosis. Furthermore, LD5095 exhibited favorable pharmacokinetics, including metabolic stability and an extended half-life. Strikingly, in vivo, a single dose of LD5095 achieved durable RIPK1 degradation in xenograft tumors over 6 days. These findings underscore the potential of LD5095 as a chemical probe for studying RIPK1 biology and a promising candidate for cancer treatment.

Epigenetic enzymes, writers, readers and erasers regulate chromatin landscapes and participate in tumor heterogeneity. While therapeutic targeting of these enzymes has shown clinical promise, the comparative efficacy of mono-versus dual-inhibitor strategies remain unclear. Here, we introduce a multi-modal platform that uses NicE-viewSeq and integrates automated deep learning based spatially resolved chromatin accessibility profiling with high-throughput sequencing following epigenetic inhibitor application. Accessible chromatin landscapes were altered along with nucleosome positioning following inhibition of either LSD1 or HDACs alone, or both together. Coordinated modulation of histone marks and the CoREST complex on chromatin was observed across inhibitory conditions. Transcription factor binding analysis identified three predominant families, ETS, RUNT, and bZIP with enhanced chromatin association upon treatments. Mechanistically, a CoREST-RUNX regulatory axis was uncovered wherein JunB, a member of bZIP family displaces CoREST-RUNX at differentially accessible regions, triggering apoptotic pathways. Therefore, JunB-mediated mechanism reveals a convergent therapeutic vulnerability, offering new avenues for optimizing different combinatorial epigenetic therapy in cancer.

Single-cell expression quantitative trait loci (sc-eQTL) analyses are powerful in identifying context-specific susceptibility genes from genome-wide association studies (GWAS) loci. However, few studies have comprehensively investigated cells of lung cancer origin in non-European populations. Here, we built a lung sc-eQTL dataset from 129 Korean women never-smokers with epithelial cell enrichment. eQTL mapping identified 2,229 genes with an eQTL in 33 cell types, including East Asian-specific findings when compared to predominantly European datasets. Integration with single-cell chromatin accessibility data demonstrated an enrichment of cell-type specific eQTLs in cell-type matched candidate enhancers, while shared eQTLs were more frequently found near promoters. Colocalization and transcriptome-wide association study unveiled 36 susceptibility genes from 22 cell types in 22 lung cancer loci, including 10 loci not achieving genome-wide significance in prior GWAS. Around 47% of these genes were from cells of the alveoli, underscoring their importance, especially in lung adenocarcinoma (LUAD) susceptibility. Focusing on the trajectory of alveolar epithelial cell regeneration, we detected 785 cell-state-interacting QTLs, which overlapped with 28% (10) of the identified susceptibility genes. Finally, we experimentally validated East Asian-and alveolar type 2 cell-specific eQTL of TCF7L2 underlying East Asian LUAD locus, 10q25.2. Consistent with its role as a Wnt/{beta}-catenin effector, TCF7L2 displayed significant effect on lung adenocarcinoma cell growth. Our data highlighted context-specific susceptibility genes, especially from alveolar cells of lung, contributing to lung cancer etiology.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is an attractive therapeutic target for metabolic disorders, with GIPR antagonism emerging as a promising strategy for obesity and type 2 diabetes. However, developing functional antibodies against GPCRs remains challenging due to their complex architecture and conformational dynamics. Here, we employed AlfaBodY, an iterative active learning platform integrating structural and sequence information, to in silico design human anti-GIPR antibodies. Through four rounds of optimization, we generated antibodies with high binding affinities. Lead candidates AB106-131 (KD 1.2 nM) and AB106-156 (KD 1.7 nM) exhibited 7 to 10-fold higher affinity than 2G10 (KD 12 nM) while maintaining comparable antagonistic activity in a cAMP reporter assay (IC50 4[~]5 nM). In diet-induced obese mice, AB106-156 alone induced weight loss comparable to that of semaglutide ([~] -15%), while preserving lean mass and achieving sustained weight control after treatment withdrawal. Co-administration with the GLP-1 receptor agonist semaglutide produced synergistic weight reduction (-25.4%) and markedly attenuated the fat-mass rebound observed with semaglutide alone. Our results demonstrate that AI-driven design can generate potent anti-GIPR antibodies with favourable in vivo efficacy, supporting further development of GIPR antagonist for obesity and related metabolic disorders. The AlfaBodY platform enables faster development of more efficacious biologic drugs.

The circadian clock maintains temporal control of metabolic processes and exerts a key role in adipocyte development. Discovery of clock-modulatory compounds may provide new avenues for metabolic disease therapy. Here we report the identification of flavonoid compounds, Quercetin and Fisetin, as clock-activating molecules with direct inhibitory action on adipogenesis and adipocyte lipid metabolism. Quercetin and Fisetin displayed robust ROR agonism that promoted clock oscillation with induction of clock genes. Treating preadipocytes with these compounds blocked their adipogenic differentiation. In mature adipocytes, Quercetin and Fisetin suppressed lipid accumulation by inhibiting lipogenic enzymes. Furthermore, activation of ROR by a synthetic agonist or ectopic expression were sufficient to inhibit adipogenesis. In mice treated with Quercetin or Fisetin, ROR was markedly induced in adipose depots with strong suppression of the adipogenic and lipogenic programs. While quercetin significantly attenuated lipid storage in adipose tissue in vivo accompanied with lowering of free fatty acids and improved insulin sensitivity, fisetin displayed a less robust effect with differential regulation of lipolytic pathway. Collectively, these findings uncovered the clock-activating properties of quercetin and fisetin that prevent adipocyte maturation and hypertrophy to limit adipose tissue expansion. These actions contribute, at least in part, to their beneficial effects on metabolic disorders.

Methylmalonic acidemia (MMA) is a recessive genetic disease caused by variants in the MMUT (mitochondrial enzyme methylmalonyl-CoA mutase) gene or by defects in transport or metabolism of MMUT cofactor (5 deoxyadenosylcobalamin), including variants in the MMAB gene. For the most recurrent pathogenic MMAB variant, c.556C>T (R186W), we identified a corrective editing strategy using adenine base editing. Deploying an adenine base editor mRNA and optimized hybrid guide RNA with lipid nanoparticles, we observed efficient in vitro corrective editing of the variant to wild-type, with minimized bystander editing and off-target editing in hepatocytes. These observations lay the groundwork for a gene editing therapy for patients with MMA resulting from at least one copy of the MMAB c.556C>T (R186W) variant, as well as a platform of similar therapies for patients with MMA caused by other variants amenable to adenine base editing.

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohns disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

Pain perception is initiated upon activation of nociceptors of the dorsal root ganglia (DRG) and trigeminal ganglia. We identified G protein-coupled receptors (GPCRs) expressed in CGRP+ mouse and human nociceptors and found that agonists of several identified Gi/o-coupled and orphan GPCRs attenuated neuronal excitability. Experiments focusing on the Gi/o-coupled serotonin receptor Htr1b, which is expressed in mouse and human CGRP+ DRG neurons, revealed that Htr1b/1d agonists, the triptans sumatriptan and zolmitriptan, attenuated CGRP+ neuron excitability in vitro and exhibited analgesia across several pain models, including neuropathic pain. Conditional genetic deletion experiments showed that triptan-induced analgesia is mediated by Htr1b expressed in A-fiber mechanonociceptors. Also, triptan-associated adverse effects are partially mediated by Htr1b-independent targets. Further testing identified the GPCR Gpr19 as an additional promising target for treating pain. These findings establish a preclinical screening platform for identifying novel analgesics and reveal nociceptor GPCRs that may be targeted to treat pain.

Vaccine adjuvants are critical for enhancing immune responses and sustaining antibody production. Although their safety profiles are well established, assessments have largely focused on metabolic and excretory organs such as the liver and kidneys, with limited attention to the heart. Here, we systematically evaluated the cardiac effects of five representative adjuvants in mice: alum, MF59, AS03, Sigma Adjuvant Systems, and lipid A. None of the adjuvants impaired baseline cardiac contractile function. Notably, lipid A uniquely enhanced mitochondrial respiratory capacity in rat and human induced pluripotent stem cell-derived cardiomyocytes and promoted mitochondrial membrane hyperpolarization. We next examined its therapeutic potential in a doxorubicin (Dox)-induced heart failure model characterized by mitochondrial dysfunction. Co-administration of lipid A with influenza hemagglutinin (HA) antigen significantly ameliorated cardiac dysfunction. In parallel, lipid A prevented the Dox-induced decline in anti-HA antibody titers, an effect associated with preservation of splenic B cell populations. Collectively, these findings reveal a previously unappreciated cytoprotective dimension of lipid A, demonstrating that it not only potentiates immune responses but also counteracts chemotherapy-induced functional decline by enhancing mitochondrial activity.

Inflammatory bowel disease (IBD), comprising Ulcerative colitis (UC) and Crohns Disease, is a chronic relapsing immune-mediated inflammatory disorder of the gut. The intestinal mucus layer is a protective barrier that safeguards direct exposure of epithelium to luminal microbes and antigens. A prolonged disruption of the mucus layer may contribute to the development of IBD. Loss of mucin-producing goblet cells is a hallmark of UC. The underlying molecular mechanism controlling goblet regulation remains poorly understood. In the current work, we show a key role for NCoR1 (Nuclear corepressor 1) in goblet cell regulation. A specific downregulation of NCoR1 in intestinal crypts and goblet cells was observed in human UC and mice models. While NCoR1 was upregulated during goblet cell differentiation, inflammatory cues downregulated its expression. Experimental loss of NCoR1 resulted in exacerbated disease in a murine model of colitis, whereas its upregulation via Vitamin D led to a rescue. ChIP-seq led to the identification of KLF-16, a transcription factor, as a target of NCoR1. NCoR1 -KLF16 regulatory axis regulated key goblet cell proteins, including MUC2. Mechanistically, the regulation of MUC2 is modulated by the NCoR1-KLF16 axis, via mTOR signalling. In conclusion, this work shows a critical involvement of NCoR1-KLF16 in governing goblet cell function and intestinal homeostasis.

Brain metastases from renal cell carcinoma (RCC) remain a major cause of morbidity and mortality, yet the genomic features associated with metastatic dissemination remain poorly understood. Whole-exome sequencing was performed on 72 RCC brain metastasis samples with matched normal. To identify candidate genomic alterations associated with brain metastasis, the genomic alterations detected in the brain metastases were compared against alterations in extracranial metastases from the MSK-ECM cohort (n=137) and primary RCC tumors from TCGA (n=432) by case-control analyses. Candidate alterations were also identified through matched-pair analyses comparing brain metastases with matched primary tumors or extracranial metastases from the same patient (n=25). A random survival forest model incorporating the candidate CNA events was developed to predict overall survival. The candidate CNAs were further evaluated using functional experimental data from MetMap and DepMap. Survival analyses were conducted to assess the prognostic relevance of these alterations. We identified recurrent CNAs enriched in RCC brain metastases, including 4q loss, 7p gain, 7q gain, 8p loss, 8q gain, 9p21.3 deletion, 12q15 amplification, and 14q loss. These alterations were associated with significantly poorer patient survival among RCC patients. A random survival forest model based on these CNA events stratified TCGA-KIRC patients into prognostically distinct risk groups (C-index = 0.64). Among the recurrent CNAs, 8p loss, 8q gain, 9p21.3 deletion were associated with increased incidence of brain metastases across multiple primary cancer types in xenograft mouse models. These alterations were also strongly associated with metastatic progression and poor prognosis across RCC, lung adenocarcinoma, breast cancer, and melanoma. These findings indicate a shared genomic basis for brain tropism and highlight the potential utility of copy-number alterations as biomarkers for risk stratification and clinical decision-making.