JHEP Reports

Background & AimsHepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. MethodsAdult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. ResultsHBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. ConclusionsHBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implicationsUnderstanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and disrupts hepatocyte identity, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Background and AimsThe incidence of hepatocellular carcinoma (HCC) is increasingly driven by metabolic risk factors, including obesity and iron overload. Although high-fat and high-iron diets independently promote hepatocarcinogenesis, their shared and distinct molecular effects remain unclear. We sought to define how dietary fat and iron differentially shape HCC development. ApproachMale C3HeB/FeJ mice were exposed to long-term high-fat (HFD), high-iron (HID), or combined (HFD+HID) diets for 16.5 months. Tumor burden, hepatic iron distribution, mTOR signaling, oxidized phospholipid (OxPL) accumulation, and transcriptomic alterations across normal, adjacent non-tumor, and tumor liver tissues were analyzed using biochemical, histological, and RNA sequencing approaches. ResultsAll diets induced HCC with comparable tumor burden. HID increased iron levels in non-tumor liver tissue but resulted in relative iron depletion within tumors, indicating tumor-specific iron utilization. Tumors from all diet groups showed robust mTOR activation and increased OxPL accumulation, with stronger oxidative stress signatures in HFD and HFD+HID tumors. Transcriptomic analyses revealed conserved oncogenic programs alongside diet-specific signatures, with HFD exerting a dominant effect on metabolic reprogramming and gene dysregulation, whereas HID preferentially enhanced immune and inflammatory signaling. Progressive, monotonic changes in gene expression were observed across disease stages. Cross-species analyses linked diet-induced mouse tumors to immunologically "hot" human HCC subtypes. ConclusionsDietary fat and iron promote HCC through overlapping yet distinct molecular pathways, highlighting metabolic and immune mechanisms as key targets in diet-associated liver cancer.

Background and AimsSteatotic liver disease (SLD) is characterized by excessive lipid accumulation in hepatocytes, and alcohol consumption may modify the disease course, but the evidence is inclusive. This systematic review and meta-analysis aimed to holistically evaluate the impact of mild, moderate, and high levels of alcohol consumption on hepatic and extrahepatic outcomes in SLD. MethodsWe systematically searched EMBASE, MEDLINE, Web of Science, and the Cochrane Central Register of Controlled Trials for relevant studies. The study outcomes included liver related events, malignancy, mortality and cardiovascular disease among adults with SLD who consumed alcohol. ResultsOf 2228 records identified, twenty-six studies comprising 466611 adults with SLD were included. High alcohol consumption was associated with an increased risk of liver-related events compared with abstinence (2.97, 95% CI 1.61-5.50; p<0.001), and a similar association was observed among alcohol drinkers overall (HR 1.93, 95% CI 1.60-2.33; p<0.001). Moderate alcohol consumption was associated with a higher incidence of malignancy (HR 1.41, 95% CI 1.13-1.78; p=0.677). In contrast, mild alcohol consumption was associated with lower all-cause mortality compared with abstinence (HR 0.88, 95% CI 0.78-0.98; p=0.001). No association was observed between alcohol consumption and cardiovascular disease incidence or hepatocellular carcinoma ConclusionsAlcohol intake may increase the risk of liver-related complications and cancer risk in individuals with SLD. Mild alcohol consumption was associated with lower all-cause mortality, and alcohol intake showed no association with cardiovascular disease incidence. Further studies are needed to clarify the dose-dependent effects of alcohol on hepatic and extrahepatic outcomes in SLD.

Purpose Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of chronic liver disease and liver-related morbidity worldwide. Although dietary factors may influence MASLD progression, the long-term liver-specific implications of artificially sweetened beverage (ASB) intake remain unclear. We aimed to examine the association between ASB intake and the risk of liver-related adverse events and liver-related death among individuals with MASLD. Methods This prospective cohort study included 50,562 participants with MASLD from the UK Biobank. ASB intake was assessed using 24-hour dietary recalls and categorized as 0, >0-1, and >1 serving/day. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for liver-related adverse events and liver-related death. Restricted cubic spline models were used to assess dose-response patterns, and competing-risk analyses were performed by treating liver-related death as a competing event for liver-related adverse events. Additional substitution, subgroup and sensitivity analyses were conducted to evaluate the robustness of the findings. Results During a median follow-up of 12.8 years, 292 liver-related adverse events and 91 liver-related deaths occurred. Compared with participants reporting no ASB intake, those consuming >1 serving/day had a higher risk of liver-related adverse events in the fully adjusted model (HR 1.40, 95% CI 1.02-1.93; P = 0.039), whereas the association for >0-1 serving/day was not statistically significant (HR 1.26, 95% CI 0.92-1.71; P = 0.149). The risk of liver-related adverse events increased across ASB intake categories (P for trend = 0.023). Restricted cubic spline analysis indicated a positive linear association between ASB intake and liver-related adverse events (P-overall <0.001; P-nonlinearity = 0.72). In competing-risk analysis, the association for >1 serving/day remained consistent after accounting for liver-related death as a competing event (sub-HR 1.40, 95% CI 1.02-1.93; P = 0.038; Gray test P = 0.006). The association was robust in sensitivity analyses. ASB intake was not significantly associated with liver-related death, and beverage substitution analyses showed no significant associations. Conclusion Among individuals with MASLD, high ASB intake, particularly >1 serving/day, was associated with an increased risk of liver-related adverse events, but not liver-related death. This association was consistent across dose-response, competing-risk, and sensitivity analyses, suggesting that high ASB intake may represent a potential dietary risk marker for adverse liver outcomes in MASLD.

Background Liver fibrosis (LF) represents a pivotal pathological phase in the advancement of chronic liver disorders toward cirrhosis. Amino acid metabolism reprogramming plays a pivotal role in its pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Methods Integrating three public datasets (GSE14323, GSE84044, and GSE136103) with amino acid metabolism-related gene sets, we performed consensus clustering, machine learning algorithms, functional enrichment analysis, immune microenvironment composition, regulatory network construction, and drug prediction. Results Fibrotic samples were classified into two amino acid metabolism-related subtypes with distinct immune landscapes and functional phenotypes. Through integrated analysis of differentially expressed genes (DEGs) common to both subtypes, fibrotic versus control comparisons, and amino acid metabolism-related gene sets, four biomarkers, GSTP1, LDHB, OXCT1, and PTGDS, were identified. These biomarkers were enriched in pathways related to epithelial-mesenchymal transition, interferon responses, and TNF/NF-{kappa}B signaling. Notably, GSTP1 and LDHB positively correlated with M1 macrophage infiltration and negatively with regulatory T cell abundance. Single-cell transcriptomic analysis revealed that cholangiocytes expressed all four biomarkers with elevated levels in fibrosis and interacted with macrophages/mesenchymal cells via MIF-CD74/CXCR4. Regulatory network analysis highlighted key modulators, including MALAT1, hsa-miR-3163, OXCT1, SMAD4, and RELA. Furthermore, 5-fluorouracil was predicted as a multi-target compound, with the strongest predicted binding affinity for OXCT1. In vitro validation confirmed the upregulation of GSTP1 and LDHB, aligning with the bioinformatics findings. Conclusion This study identified four amino acid metabolism-related biomarkers, revealing immune heterogeneity and cholangiocyte-centered intercellular communication in LF. These findings establish a foundation for biomarker-based diagnosis, subtype-guided patient stratification, and the development of cell-type-specific therapeutic strategies in LF.

Introduction: The overlap between chronic hepatitis B (CHB) and metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging global health challenge. We investigated the impact of MASLD and metabolic comorbidity in a diverse London viral hepatitis clinic. Methods: This retrospective cross-sectional study (May 2018-Feb 2024) included adults with CHB having controlled attenuation parameter (CAP) measurements. MASLD was defined as CAP >264 dB/m plus [&ge;]1 cardiometabolic factor (CMF). We used univariable and multivariable models to examine MASLD's relationship with liver stiffness and hepatitis B viral load (HBV VL). Results: Among 323 individuals (67% male, median age 36), most were from Black (35%) or non-white British/Irish (29%) backgrounds. Overall, 64% had [&ge;]1 CMF, and 20% had MASLD. The CHB/MASLD group was significantly older (median 43 vs 35 years, p<0.001) with higher median alanine transaminase (35 vs 30 IU/L, p=0.02) and liver stiffness (5.3 vs 4.7 kPa, p<0.001). Following adjustment for covariates, MASLD remained significantly associated with liver stiffness ({beta} = 0.48 kPa, p=0.03). While univariable analysis showed significantly lower HBV VL in people with MASLD (median 54 vs 417 IU/ml, p=0.004), adjusted multivariable analysis revealed no significant association between MASLD and log10 HBV VL (p=0.2). Conclusions: Although adjusted analysis does not support an independent association between MASLD and HBV VL, the data highlight a substantial cardiometabolic burden in this CHB population and clearly link MASLD to more severe liver disease. Holistic consideration of metabolic comorbidities is crucial in comprehensive CHB management.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease for which the mechanisms linking lipid dysregulation to fibrosis remain poorly defined. Hepatic phosphatidylcholine (PC) content is reduced in MASH, but how this alteration drives disease progression is unclear. Here, we identify a role for copper (Cu) homeostasis as a downstream effector of impaired PC biosynthesis. Using single-nucleus RNA sequencing in complementary genetic and dietary mouse models, we found that reduced hepatic PC is associated with marked depletion of hepatic Cu and a concomitant increase in circulating Cu, indicating disrupted Cu distribution. Mechanistically, PC depletion impaired plasma membrane localization of the high-affinity Cu transporter CTR1 (SLC31A1) in hepatocytes, limiting Cu uptake. In human hepatic stellate cells, Cu promoted fibrogenic activation, whereas suppression of Cu import or pharmacologic inhibition of MAPK signaling attenuated fibronectin deposition. In vivo, liver-directed Cu supplementation restored hepatic Cu levels and reduced steatosis but failed to improve fibrosis. In contrast, pharmacologic Cu chelation with bathocuproinedisulfonic acid (BCS) reduced fibrosis without affecting inflammation. Together, these findings identify Cu redistribution as a consequence of impaired PC biosynthesis and implicate Cu-dependent signaling in stellate cell activation, fibrogenesis and MASH pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/723926v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@144d748org.highwire.dtl.DTLVardef@91dd8corg.highwire.dtl.DTLVardef@683686org.highwire.dtl.DTLVardef@1d3a0da_HPS_FORMAT_FIGEXP M_FIG C_FIG

The mitochondrial membrane protein phosphoglycerate mutase 5 (PGAM5) is a protein of interest in the complex transition from hepatic steatosis to hepatocellular carcinoma. PGAM5 is a serine/threonine/histidine phosphatase that plays a role in mitochondrial biogenesis, mitophagy, and multiple cell death pathways. Increased expression of PGAM5 in hepatocellular carcinoma is correlated with reduced patient survival. In this study, we demonstrate that loss of PGAM5 alters the bioenergetic landscape of liver cancer by promoting mitochondrial oxidant injury and suppressing the glycerophospholipid and lysophospholipid pathways, leading to accumulation of the bioactive phospholipid lysophosphatidylcholine. Additionally, PGAM5 deletion downregulates fatty acid biosynthesis, resulting in reduced cellular diacylglycerol concentrations through two probable mechanisms: attenuated long chain fatty acid uptake and suppressed de novo synthesis. These findings underscore the broad impact of a single phosphatase on mitochondrial function and provide a rationale for therapeutically targeting PGAM5 to disrupt lipid metabolism in hepatocellular carcinoma.

Liver sinusoidal endothelial cells (LSECs) separate the blood from the hepatic parenchyma and thus are at the frontline as scavengers of blood-borne waste, pathogens and metabolic stimuli. LSECs are also critical for sensing systemic iron availability by controlling the synthesis of bone morphogenetic protein (BMP) 6, which is essential for hepcidin expression in hepatocytes. Hepcidin maintains systemic iron homeostasis by inhibiting dietary iron uptake and iron release from iron recycling macrophages. Hepcidin is also an acute-phase protein and its activation by inflammation requires active BMP signaling. It is incompletely understood how signals derived from inflammation, cellular damage and iron are integrated by the liver to assure adequate hepcidin expression. Here, we show that Bmp6 expression is activated in primary LSEC cultures upon their exposure to danger-associated molecular patterns (DAMPs), such as heme and myoglobin, pathogen-associated molecular pattern (PAMPs), such as lipopolysaccharide (LPS) and Fibroblast-Stimulating Lipopeptide-1 (FSL1), or oxidative stress inducers (H2O2). Interestingly, all regulatory cues converge at the MAPK signaling pathway, although the specific signaling branches involved are stimulus-specific. Of note, Bmp6 upregulation in LSECs in response to all signals tested is strongly enhanced by the hepatocyte secretome. As hepatocytes critically depend on active BMP/SMAD signaling to control hepcidin activation, our results reveal that multiple sources of signaling input activating Bmp6 in LSECs and hepcidin in hepatocytes serve to determine BMP/SMAD signaling strength. Furthermore, our findings identify hypoferremia (low plasma iron levels), the result of high hepcidin levels due to elevated Bmp6, as a convergent response in conditions of inflammation, oxidative stress and cellular damage. HighlightsO_LIDAMPs (heme and myoglobin), PAMPs (LPS) and oxidative stress activate Bmp6 mRNA expression via the MAPK signaling pathway C_LIO_LIThe TLR/MAPK/BMP6 regulatory axis integrates inflammatory and iron signals C_LIO_LIOur work uncovers a novel connection between innate immune sensing, oxidative stress and hepatic iron homeostasis C_LI

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by excessive hepatic lipid accumulation driven by increased de novo lipogenesis (DNL) and impaired lipid oxidation. p107, a member of the retinoblastoma (Rb) family, extensively studied in the context of cell cycle regulation and adipocyte differentiation recently has been identified as a metabolic regulator controlling thermogenic activity. However, its role in hepatic lipid homeostasis remains poorly understood. Here, we identify the cell cycle regulator p107 as a key modulator of hepatic lipid metabolism. p107 expression is increased in patients with MASLD and correlates with disease severity. In mouse models, global and liver-specific p107 deficiency protect against high-fat diet-induced steatosis without affecting body weight. This is associated with reduced expression of lipogenic enzymes including fatty acid synthase (FASN), and enhanced mitochondrial oxidative pathways. Conversely, hepatic restoration of p107 reversed these effects and promoted lipid accumulation and endoplasmic reticulum stress. Consistent with this in human hepatocytes, p107 silencing reduces lipid accumulation, decreases DNL and enhances mitochondrial respiration, whereas p107 overexpression induces the opposite phenotype. Notably, FASN knockdown attenuates the pro-steatotic effects of p107, indicating that it is a critical downstream mediator of p107. Together, these findings establish p107 as a physiological regulator of hepatic lipid metabolism, with its dysregulation contributing to the development of MASLD.

Human in vitro hepatic models that accurately recapitulate liver function are essential for fundamental and translational research; however, currently utilised models for disease modelling and drug discovery lack physiological fidelity and require prolonged culture time. Here, we present a streamlined 10-day protocol for efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte like cells (HLCs) and hepatic liver organoids (HLOs). Both models exhibited mature hepatocyte differentiation, as evidenced by albumin secretion and CYP3A4 metabolic activity. Interestingly, HLOs display enhanced multicellular complexity, incorporating endothelial, stellate, and macrophage populations along with hepatocytes, thereby more closely recapitulating the native liver microenvironment than HLCs. Here, steatosis was induced in both platforms, which resulted in triglyceride accumulation and upregulated lipogenic markers (DGAT1, DGAT2). However, only HLOs recapitulated advanced disease characteristics, including inflammatory (IL-10) and fibrotic (alpha SMA, COL1A1) responses. Resmetirom, a thyroid hormone receptor-{beta} agonist, significantly reduced steatosis and restored molecular signatures in both models. Additionally, transplanted organoids demonstrated prolonged survival and displayed host-derived vascularization, thereby validating in vivo maturation. Collectively, this platform provides a rapid and physiologically relevant liver model, with 3D HLOs offering superior utility for disease modelling, therapeutic evaluation, and regenerative applications due to their enhanced functional and physiological relevance.

Background and aimsAlcohol-associated hepatitis (AH) is characterized by excessive inflammation and blunted antiviral interferon (IFN) responses. We hypothesized that specific gut microbiome-derived metabolites could selectively enhance interferon signaling while limiting NF-{kappa}B mediated inflammation, thereby restoring immune balance in AH. Our goal is to identify microbiome-derived metabolites that differentially regulate the NF-{kappa}B and IFN signaling pathways. Methods and resultsWe used human monocytic THP1-Dual cells, which secrete reporters for NF-{kappa}B and IFN signaling, to model innate immune responses and screened a library of 152 gut microbiome-derived metabolites. From the metabolite screen, 4-hydroxyphenylacetic acid (4-HPAA) emerged as a unique immunomodulator: in LPS-challenged cells, 4-HPAA selectively increased IFN signaling with minimal NF-{kappa}B activation. 4-HPAA was evaluated in vivo using a NIAAA-model, with 4-HPAA supplementation (0.4mg/ml) added to the diet. In the NIAAA-model, dietary 4-HPAA did not induce liver injury and was associated with enhanced interferon-stimulated gene expression. Simultaneously, 4-HPAA reduced pro-inflammatory markers such as Il1{beta}, Ly6g and F4/80 compared to the group exposed to ethanol alone. Metabolomic profiling of mouse cecal contents revealed 4-HPAA supplementation counteracted ethanols metabolic effects, selectively reducing triglyceride-associated lipids that had accumulated with ethanol feeding. Conclusions4-HPAA enhances interferon signaling and antiviral gene induction while dampening NF-{kappa}B-driven inflammation in the presence of LPS, both in vitro and in vivo. In an acute-on-chronic alcohol injury model, 4-HPAA attenuated hepatic inflammation, reduced immune cell recruitment, and activated antioxidant defenses, reflecting a shift toward a more hepatoprotective effect. 4-HPAA treatment was associated with reduced pro-inflammatory markers and modest attenuation of ethanol-induced liver injury. Additionally, 4-HPAA reversed ethanol-induced lipid-dysregulation, particularly triglyceride accumulation, highlighting its metabolic benefit in alcohol-fed mice. In conclusion, 4-HPAA rebalances immune and metabolic pathways by enhancing IFN signaling, suppressing NF-{kappa}B inflammation, and reversing alcohol-induced hepatic injury and lipid accumulation.

Background and AimsAcute-on-chronic liver failure (ACLF) is associated with high short-term mortality, but substantial heterogeneity among existing diagnostic and prognostic models results in inconsistent patient identification and risk assessment. We conducted a systematic head-to-head comparison of major ACLF diagnostic and prognostic models to evaluate concordance, short-term mortality prediction and clinical utility, with the goal of informing harmonization of ACLF assessment. MethodsWe analysed 3,370 patients with acute decompensation of cirrhosis in the COSSH cohort, with external validation in an independent Ambi-Spective cohort from India (n=2,055). Five ACLF diagnostic models were evaluated for identification of patients at risk of 28-day mortality. Reclassification was assessed using net reclassification improvement. Prognostic scores were compared using concordance index, integrated discrimination improvement, calibration, and decision-curve analysis. ResultsDiagnostic frameworks identified markedly different proportions of ACLF. A-TANGO and COSSH-ACLF classified the largest high-risk populations while maintaining substantial short-term mortality and balanced sensitivity-specificity profiles. Compared with COSSH-ACLF, A-TANGO improved net reclassification by 7.7%, with further gains versus EASL-CLIF (11.8%), APASL-ACLF (36.4%), and NACSELD-ACLF (45.9%). In the external cohort, A-TANGO and COSSH-ACLF showed similar discrimination and identified comparable proportions of patients. Combined application of the two models delineated three clinically meaningful strata, identifying a discordant intermediate-risk group with approximately 11% 28-day mortality. Among prognostic scores, COSSH-ACLF II and A-TANGO OF scores demonstrated strong and complementary performance across cohorts. ConclusionsOutcome-anchored ACLF definitions converge in identifying patients at highest short-term risk across diverse populations. Alignment between A-TANGO and COSSH-ACLF, together with identification of an intermediate-risk phenotype, supports a data-driven framework for improving consistency and advancing global harmonization of ACLF diagnosis and risk stratification.

Abstract textO_ST_ABSBackgroundC_ST_ABSAs hepatitis A (HA) incidence declines in Europe and infections occur later in life, clinical presentations may worsen, particularly during outbreaks involving adults. AimWe analysed temporal trends and factors associated with severe disease and mortality among patients hospitalised for HA in France between 2013 and 2024. MethodsICD-10 codes B150 or B159 as primary discharge diagnosis were used to identify HA cases from the National Discharge Data Set. Severity (hepatic and/or extrahepatic organ failure within 12 weeks post-admission) and mortality were analysed using adjusted odds ratios in original and propensity-matched samples. Trends were assessed across five periods covering the 2017 epidemic and COVID-19, with 2013-2016 as reference. ResultsAmong 7,928 cases (60.6% male; median age 30) 29.1% developed severe HA, and 1.43% died. Risk of severe HA increased with age (+17% of risk per decade, p < 0.001), male sex (+39%, p < 0.001), smoking (+25%, p=0.024), liver risk factors (+32%, p=0.026), and cirrhosis (+48%, p = 0.024). Risk of death increased with cirrhosis (3.55-fold, p < 0.001) and high Charlson Comorbidity Index (CCI) (9.95-fold, p < 0.001), but not with advanced age. Compared with 2013-2016, severe HA increased by 60% (p<0.001) and case fatality increased 2.22-fold (p=0.003) in 2021-2024. ConclusionsHA severity and mortality have increased in France over the last decade, with advanced age and male sex increasing severity but not mortality, and high CCI limiting access to organ support, thereby increasing mortality in frail patients. Our findings highlight the need for targeted prevention and optimized care strategies for high-risk groups.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide. Current treatments offer limited efficacy and no definitive cure, underscoring the urgent need for more selective and effective therapeutic strategies. This study investigated the synthetic lethality caused by co-targeting two metabolic genes, ATP citrate lyase (ACLY) and oxoglutarate dehydrogenase (OGDH), in HCC cells. Using valproic acid (VPA) and bempedoic acid (BA) as pharmacological inhibitors of OGDH and ACLY, respectively, we observed a strong synergistic effect in inhibiting the proliferation of HCC cell lines (Hep3B and Huh7), compared to using these drugs individually. Importantly, this combination treatment exhibited little increased cytotoxicity in the non-cancerous liver cell line THLE-2, indicating a degree of selectivity. Our findings are consistent with previous reports implicating USP13 as a metabolic regulator of ACLY and OGDH in various cancers, suggesting that the inhibition of USP13 may prevent HCC cell proliferation primarily through its downstream effects on ACLY and OGDH. By directly co-targeting ACLY and OGDH, our approach may offer a more precise and safer alternative to USP13 inhibition. Additionally, while both VPA and BA have been individually associated with beneficial effects in liver disease, their combined application in the context of HCC has not been previously investigated. Limitations include the reliance on cell line models, highlighting the need for validation in more physiologically relevant systems such as human organoids and animal models. Overall, this study provides a compelling rationale for further investigation into ACLY and OGDH as a synthetic lethal pair and the therapeutic potential of the VPA-BA combination treatment in HCC.

Metabolic dysfunction-associated steatotic liver disease (MASLD) exhibits marked heterogeneity and sex differences, yet the molecular mechanisms underlying disease progression remain incompletely understood. Here, we present the largest integrative multi-omics study to date combining matched liver tissue and blood profiling in 211 biopsy-confirmed, morbidly obese individuals with MASLD undergoing bariatric surgery. We integrate hepatic transcriptomics, metabolomics, and lipidomics with serum metabolomics to resolve compartment-specific and sex-dependent molecular networks. Across sexes, MASLD is characterized by suppressed hepatic amino acid metabolism and extensive lipid remodeling, accompanied by inverse metabolic signatures in circulation, consistent with systemic spillover. Strikingly, disease progression in men is driven by a streamlined triacylglycerol-centric pathway that mediates transcriptional effects on steatosis and inflammation, whereas women exhibit distributed, multi-layered networks linking lipid, amino acid, and immune pathways. Mediation analyses identify hepatic lipid modules as key intermediates connecting gene expression to histopathology. These findings reveal sex-specific molecular architectures of MASLD, demonstrate that circulating biomarkers do not reflect hepatic metabolism, and provide a framework for sex-specific precision medicine.

Background and AimsThe tumor-stroma interface in hepatocellular carcinoma (HCC) harbors critical intercellular interactions that shape immune evasion and treatment response, yet its spatial architecture remains poorly characterized across etiologies. Whether hepatitis B virus (HBV)-related and non-B non-C (NBNC) HCC share conserved border niche features or exhibit etiology-specific microenvironment programs is unknown. We aimed to spatially resolve the tumor boundary ecosystem and identify etiology-associated signaling networks with translational relevance. Approach and ResultsWe performed 10x Visium spatial transcriptomics on 11 HCC specimens (7 HBV, 4 NBNC) and applied a machine-learning pipeline integrating CancerFinder and SpaceFlow to define tumor, boundary, and stromal domains. Across etiologies, the boundary zone showed a recurrent desmoplastic niche characterized by cancer-associated fibroblast, tumor-associated macrophage, and tumor endothelial cell accumulation with collagen-integrin extracellular matrix remodeling, including COL1A1-ITGA11 and COL4A1-ITGAV. Etiology-associated differences were observed in the organization of border-zone signaling programs. In representative HBV-related sections, CCL19-CCR7 signaling showed a comparatively restricted, endothelial-skewed topology, whereas representative NBNC sections showed broader inflammatory ligand-receptor networks with elevated NF-kB-associated pathway activity. ConclusionsThe HCC tumor-stroma border harbors a recurrent desmoplastic niche upon which etiology-associated immune regulatory programs may be superimposed. These findings generate spatial hypotheses relevant to etiology-informed biomarker development and future therapeutic stratification.

Metabolic-associated steatotic liver disease (MASLD) is a prevalent liver disease driven by complex dysregulation of hepatic lipid metabolism. Here we show that copper deficiency is a nutrient vulnerability in steatotic liver disease and that selective liver-targeted copper supplementation can reduce excess lipid accumulation. Analysis of steatotic patient and mouse tissues identify widespread alterations in hepatic copper homeostasis markers. Integrated multi-omics analyses reveal that copper induces lipolysis of PLIN2-containing lipid droplets while lipid importer CD36 is downregulated. We show that copper inhibits cAMP hydrolase activity of PDE3B, thus activating PKA-mediated HSL and AMPK activation upstream of lipolysis. Fatty acids liberated through lipolysis are subsequently degraded via enhanced mitochondrial fatty acid oxidation, supported by energetic rewiring toward oxidative phosphorylation (OXPHOS) with increased copper-dependent complex IV and SOD1 activity. Our findings establish a multi-pronged mechanism by which hepatic copper supplementation coordinately regulates lipid metabolism in response to steatosis and unveils a therapeutic metallomedicine strategy to rewire lipid regulation. SummaryLiver-targeted copper supplementation reduces diet-induced liver steatosis by dual activation of lipolysis and fatty acid degradation pathways. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/725917v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@32d5eorg.highwire.dtl.DTLVardef@97b6f4org.highwire.dtl.DTLVardef@6c4e80org.highwire.dtl.DTLVardef@95d16a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background & Aims: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the leading cause of chronic liver disease in children. However, accurate, noninvasive diagnostic tools remain limited. Current screening methods are invasive or lack sensitivity. Breath-based volatile organic compound (VOC) analysis offers a simple approach with potential for point of care screening. This study aimed to identify and validate breath VOC signatures of pediatric MASLD. Approach & Results: We conducted a prospective IRB approved cohort study at the Childrens Hospital of Philadelphia (CHOP). Children aged between 7 and 20 years with MASLD (n=22), as defined by hepatic steatosis either by liver biopsy or imaging and 1 cardiometabolic risk factor, and a control group without MASLD (n=20) were enrolled. Breath samples were collected using a standardized protocol and analyzed by untargeted comprehensive two-dimensional gas chromatography-mass spectrometry (GCGCMS). Machine learning and unsupervised clustering were applied to identify discriminatory VOCs and assess heterogeneity. Untargeted GCGCMS analysis identified a distinct breath VOC signature in children with MASLD compared with non MASLD controls. A Random Forest model achieved a sensitivity of 73% and specificity of 65%, with AUC of 0.84. The VOC 2,4-dimethyl-1-heptene demonstrated strong diagnostic performance in the discovery cohort with a sensitivity of 85%, specificity of 77% and an AUC of 0.81. Unsupervised clustering revealed four MASLD subgroups with distinct volatile phenotypes associated with differences in liver enzymes and metabolic parameters. External validation in a second pediatric cohort confirmed reproducible reductions in o/p-xylene in subjects with MASLD. Conclusions: Pediatric MASLD is associated with a reproducible breath VOC signature identified by untargeted GCGCMS. These findings support breath analysis as a scalable, noninvasive screening and stratification tool for pediatric MASLD and warrant validation in larger, longitudinal studies.

Hepatocyte growth factor (HGF) is a multifunctional cytokine stored in the extracellular matrix as an inactive precursor and is essential for tissue repair. How HGF activity is dynamically regulated during regeneration remains unclear. Here, we identify extracellular matrix protein 1 (ECM1) as a physiological inhibitor of active HGF during liver regeneration. Following 70% partial hepatectomy, active HGF rapidly increases in parallel with a sharp decline in ECM1, and preventing this downregulation delays liver mass recovery. Mechanistically, ECM1 directly binds the active HGF -subunit through a mechanism dependent on residue R392, thereby suppressing c-MET-ERK-MYC signaling and hepatocyte proliferation. Loss of ECM1 permits activation of this pathway, whereas MYC overexpression rescues ECM1-mediated growth inhibition. In patients, proliferative hepatocytes localize to ECM1-negative regions. Supported by transcriptomic analyses and computational modeling, these findings identify ECM1 as an extracellular gatekeeper whose transient downregulation enables HGF-driven tissue repair. HighlightsO_LIECM1 is temporally downregulated during liver regeneration, while forced ECM1 expression delays liver mass recovery after 70% PHx by suppressing hepatocyte proliferation. C_LIO_LIRapid and transient ECM1 downregulation permits HGF-c-Met-ERK-MYC signaling to drive hepatocyte cell-cycle entry and expansion. C_LIO_LIECM1 directly binds the active HGF -subunit with residue R392 playing a critical role, revealing a novel extracellular mechanism for growth factor inhibition. C_LIO_LILoss of ECM1 marks proliferative hepatocytes in human liver repair, identifying ECM1 as a tunable regulator of regenerative capacity. C_LI