JHEP Reports

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.

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: Metabolic adaptation is a recognized feature of therapeutic resistance in hepatocellular carcinoma (HCC), but it is unclear whether transcriptional states exposed during acquired resistance are restricted to drug adaptation or reflect broader aggressive tumor biology. We tested whether metabolic programs derived from a lenvatinib-resistance model identify a clinically adverse transcriptional state in an independent HCC patient cohort. Methods: The discovery framework was based on GSE186191, comprising parental and acquired lenvatinib-resistant Hep3B and Huh7 cells. A pre-specified 33-gene lipid-source ledger served as a biological anchor, and three discovery-derived programs, MYC Targets V2, mTORC1 Signaling, and Fatty Acid Metabolism, were frozen before patient-level evaluation. In TCGA-LIHC, single-sample enrichment scores for the three programs were population-standardized and summed to generate an integrated metabolic score. Overall survival was assessed by Kaplan-Meier and Cox analyses. Whole-transcriptome differences between high- and low-score tumors were characterized by preranked gene set enrichment analysis (GSEA). Results: The survival cohort comprised 282 patients (118 deaths), with 141 patients in each median-defined score group. High-score patients had shorter overall survival (log-rank P=0.000419). The continuous score was associated with mortality in univariable analysis (HR 1.86, 95% CI 1.33-2.61; P=0.000293) and in the frozen model adjusted for age, sex, and stage indicators (HR 1.93, 95% CI 1.35-2.76; P=0.000350; n=277). In 327 primary tumors, Fatty Acid Metabolism was strongly depleted in high-score tumors (NES -2.06; FDR<0.001). MYC Targets V2 (NES 1.18; FDR=0.232) and mTORC1 Signaling (NES 1.11; FDR=0.229) showed positive directional enrichment without FDR significance. Conclusions: A lenvatinib-resistance-derived transcriptional program is associated with an adverse-survival state in HCC. The strongest patient-level pathway feature is depletion of canonical fatty-acid metabolism, accompanied by directional MYC/mTORC1 features rather than statistically established pathway activation. These findings support a testable model of metabolic identity remodeling but do not establish causality or clinical prediction of lenvatinib response.

Background & Aims: Immune-mediated liver injury from immune checkpoint inhibitors (ILICI) is a major immune-related adverse event that limits cancer immunotherapy, yet its tissue-level immunobiology is poorly defined and its management is largely extrapolated from autoimmune hepatitis (AIH). We previously identified a tri-cellular CD8+ T cell-macrophage-hepatocyte injury niche in a murine model of ILICI; here, we tested whether this niche is recapitulated in human disease. Methods: We applied imaging mass cytometry with a 32-marker panel to liver biopsies from patients with ILICI (n = 12), AIH as a disease comparator (n = 14), and healthy controls (n = 2), profiling approximately 297,000 single cells across 144 regions of interest with spatially resolved detection of apoptosis (cleaved caspase-3, cC3) and pyroptosis (cleaved gasdermin D, cGSDMD). Results: We detected histiocyte-rich granulomas in ILICI consisting of macrophages and CD8+ T cells, including activated memory-effector subsets. Permutation-based spatial analysis identified CD8+ T cell-macrophage co-localization as the most frequent significant interaction in ILICI, organizing into integrated innate-adaptive cellular neighborhoods that concentrated cC3- and cGSDMD-positive cells. Descriptively, this contrasted with AIH, in which immune cells and stroma were more spatially compartmentalized. CD8+ T-cell and macrophage densities correlated with Ishak necroinflammation scores, jaundice, and granuloma formation. Conclusions: These findings provide the first single-cell spatial proteomic characterization of human ILICI in situ; they recapitulate the tri-cellular CD8-macrophage-hepatocyte niche we previously defined in a murine model and characterize ILICI as a spatially organized innate-adaptive inflammatory process, nominating myeloid signaling and CD8-macrophage interactions as candidate liver-directed targets to uncouple hepatotoxicity from anti-tumor immunity.

Background Gallstones are one of the most common gastrointestinal conditions closely associated with metabolic dysfunction. The metabolic dysfunction - associated fibrosis - 5 (MAF -5) score has been established as a non - invasive indicator for assessing liver fibrosis in individuals with metabolic abnormalities. However, comprehensive large - scale research examining the correlation between the MAF-5 score and gallstone occurrence remains limited. This study seeks to clarify the link between the MAF-5 score and gallstone prevalence using nationally representative data from the National Health and Nutrition Examination Survey (NHANES). Methods This study examined data from 15,560 NHANES 2017-2020 participants aged 20 years or older, ensuring complete records for MAF-5 scores and gallstone status. Gallstone presence was identified through self-reported physician diagnoses. To assess the relationship between MAF-5 scores and gallstone prevalence, weighted logistic regression models were applied, adjusting for demographic characteristics, lifestyle factors, and health conditions. Subgroup analyses were conducted to evaluate the stability of this association and detect possible interactions. Sensitivity analyses were performed by excluding extreme values ({+/-}3SD) to assess result robustness. Furthermore, MAF-5 scores were divided into quartiles to investigate gallstone prevalence trends, and a restricted cubic spline (RCS) model was employed to visualize response patterns. Results A total of 15,560 participants met the inclusion criteria, with 747 in the gallstone group and 6,367 in the non-gallstone group. MAF-5 scores were significantly higher in the gallstone group (P < 0.001). After adjusting for multiple covariates, each unit increase in MAF-5 score correlated with a 14% higher gallstone prevalence (OR = 1.14, 95% CI: 1.06-1.23). Quartile-based analysis indicated that individuals in the highest MAF-5 quartile had a 2.12-fold higher prevalence of gallstones than those in the lowest quartile (OR = 2.12, 95% CI: 1.13-3.98). RCS analysis confirmed a linear association between MAF-5 scores and gallstone prevalence. Subgroup analyses showed this association remained stable across age, sex, and racial/ethnic groups, with no significant interactions. Sensitivity analyses, excluding extreme values ({+/-}3SD), reinforced the reliability of these findings (OR = 1.15, 95% CI: 1.04-1.28). Conclusion The MAF-5 score is significantly and positively associated with gallstone prevalence, independent of demographic and lifestyle confounders. These findings indicate that the MAF-5 score may be a useful tool for assessing gallstone prevalence in individuals with metabolic dysfunction, offering valuable insights for early screening and targeted health management strategies. Keywords: Gallstones, MAF-5 score, Metabolic dysfunction, NHANES, Liver fibrosis.

Background: Focal liver lesions (FLLs) encompass a wide spectrum of benign and malignant pathologies, and accurate diagnosis is essential for appropriate management. Although advances in imaging have improved lesion characterization, histopathologic assessment remains the diagnostic gold standard for indeterminate lesions. Data on the histopathologic spectrum and diagnostic utility of ultrasound-guided percutaneous liver biopsy (US-PLB) in sub-Saharan Africa (SSA) are limited. This study aimed to characterize the histopathologic findings of US-PLB performed for FLLs at a tertiary referral center in SSA and to identify factors associated with hepatocellular carcinoma (HCC). Methods: We conducted a retrospective observational study of adult patients ([&ge;]18 years) who underwent US-PLB for FLL between January 2021 and December 2024 at Adera Medical and Surgical Center. Patients with indeterminate pathology results, incomplete records, biopsies performed for diffuse liver disease, or lesions classified as LI-RADS 1, 2, or 5 were excluded. Demographic, clinical, laboratory, imaging, histopathologic, and outcome data were extracted from medical records. Descriptive statistics were used to summarize patient characteristics and histopathologic diagnoses. Logistic regression analysis was performed to identify factors associated with HCC. Results: A total of 119 were included in the final analysis. The median age was 56 years (IQR 45-65), and 59.7% were male. No major biopsy-related complications were reported. HCC was the most common histopathologic diagnosis, accounting for 42.9% of cases, followed by secondary metastatic tumors (15.9%) and regenerative nodules (15.9%). Other diagnoses included chronic hepatitis (8.4%), cholangiocarcinoma (5.9%), and hepatic abscess (3.4%). Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections were present in 14.3% and 12.4% of patients, respectively. On multivariate analysis, HBV infection (AOR 7.85, 95% CI 1.45-42.60; p=0.017), HCV infection (AOR 9.03, 95% CI 1.41-57.76; p=0.020), and larger tumor size (AOR 1.27, 95% CI 1.11-1.46; p<0.01) were significantly associated with HCC. Conclusion: Ultrasound-guided percutaneous liver biopsy demonstrated a favorable safety profile for the evaluation of FLL. HCC was the predominant histopathologic diagnosis, reflecting the substantial burden of primary liver cancer in this setting. Chronic viral hepatitis and larger tumor size were significantly associated with HCC. These findings support the continued role of US-PLB in the diagnostic evaluation of indeterminate focal liver lesions and underscores the importance of viral hepatitis prevention, surveillance, and early detection strategies in sub-Saharan Africa.

Liver cancer remains a major global health burden with high mortality and limited treatment response prediction tools. Patient-derived cancer organoids have emerged as promising preclinical models that recapitulate tumor heterogeneity; however, the biological significance of morphological diversity within established organoids remains poorly characterized in hepatocellular carcinoma (HCC). In this exploratory study, we investigated whether distinct organoid growth phenotypes reflect underlying tumor biology and correlate with clinical outcomes. We established liver cancer organoids from resected tumor tissues of 27 patients and analyzed their clinical, histological, and genomic characteristics. Organoids were classified morphologically into cystic and solid types. Whole exome sequencing (WES) was conducted on six matched tumor-organoid pairs to assess genomic fidelity. Associations between organoid establishment, growth characteristics, and clinical parameters were statistically evaluated. Progression-free survival (PFS) was analyzed using the Kaplan-Meier method and univariate Cox proportional hazards regression. Organoids were successfully established in 13 of 27 cases (48.1%). Solid-type organoids were significantly associated with shorter PFS compared to cystic types (HR = 13.91; p = 0.0039, log-rank test). Organoid establishment was more frequent in older patients (>70 years), those with HBV infection, and tumors with positive {beta}-catenin expression. WES analysis demonstrated high concordance in somatic mutation profiles and variant allele frequency distributions between tissues and corresponding organoids. In univariate Cox regression, organoid growth pattern (solid vs. cystic) showed a significant association with PFS within this exploratory cohort (p = 0.0207). Patient-derived liver cancer organoids preserved the genomic and histopathological features of the original tumors. Notably, solid morphology was associated with shorter PFS, suggesting that organoid growth phenotype may serve as a supplementary indicator of tumor biological behavior in HCC. Given the modest cohort size and the absence of multivariate analysis, these preliminary findings should be interpreted with caution and warrant further large-scale validation.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Alpha-1 antitrypsin deficiency (AATD) caused by the Z variant leads to hepatic accumulation of misfolded AAT polymers and liver disease. Although proteotoxic stress is well established, its impact on lipid metabolism, mitochondrial function, and organelle homeostasis remains incompletely understood. The effects of Z-AAT accumulation were investigated in Z-HepG2 cells and 3D patient-derived ZZ hepatic organoids through protein aggregation, lipid storage, mitochondrial structure and function, peroxisomal dynamics, and comprehensive transcriptomic and proteomic analyses. Z-AAT expression led to intracellular polymer accumulation and reduced secretion, together with lipid accumulation, mitochondrial structural abnormalities, increased mitochondrial number but impaired respiratory capacity. Metabolic profiling revealed reduced oxidative phosphorylation and partial reliance on glucose metabolism. Peroxisomes displayed increased mass, consistent with altered lipid handling. Multi-omics analysis demonstrated widespread transcriptional and proteomic reprogramming related to protein synthesis, lipid metabolism, and mitochondrial function. Proteomic analysis confirmed proteotoxic stress-induced mitochondrial dysfunction, impaired lipid handling, and activation of stress response, inflammatory and vesicular trafficking pathways. Importantly, lipid supplementation elicited adaptive mitochondrial transcriptional responses in control cells, whereas Z-HepG2 cells showed a blunted response to lipid challenge. In conclusion, Z-AAT accumulation disrupts hepatic lipid processing and impaired mitochondrial and peroxisomal homeostasis, producing diminished metabolic flexibility likely contributing to AATD-associated liver disease.

BackgroundFontan-associated liver disease (FALD) is a universal complication of the Fontan palliation characterized by chronic congestion and progressive hepatic fibrosis. Current diagnostics rely on invasive biopsies or non-specific biochemical and imaging biomarkers that fail to capture early fibrogenesis, creating a critical need for non-invasive biomarkers to stratify disease severity. MethodsWe utilized a translational ovine Fontan model (n = 19) to investigate circulating serum extracellular vesicles (sEVs) as reporters of hepatic pathology. Longitudinal serum samples paired with liver elastography were collected, and sEVs were subjected to multi-omic profiling including small RNA sequencing and proteomics. Regularized regression was used to identify transcriptomic predictors, which were integrated with time post-surgery into an ordinal logistic regression framework to construct the Fontan EV Score (FES). Model performance was evaluated on a held-out test cohort and benchmarked against established serological fibrosis indices. To validate the biological relevance of the FES panel, TGF-{beta}-treated human liver organoids were generated and scored miRNA expression was assessed. ResultsThe sEV proteome exhibited robust separation by surgical physiology, while the small RNA cargo was primarily stratified by fibrotic status. Bioinformatic analysis confirmed a high hepatic origin for these transcripts and identified enrichment of inflammatory pathways including Toll-like receptor and Interleukin-17 cascades in fibrotic subjects. The FES, incorporating time post-surgery and eleven small RNA biomarkers, demonstrated high predictive accuracy in the independent testing cohort with an AUC of 0.876 for moderate and 0.963 for severe fibrosis, substantially outperforming APRI (AUC = 0.618) and FIB-4 (AUC = 0.731). In TGF-{beta}-treated human liver organoids, several scoring miRNAs, including miR-125a-5p and miR-193b-5p, were directionally responsive to profibrotic stimulation. ConclusionsCirculating sEVs carry a liver-associated molecular cargo that can be leveraged for the non-invasive prediction of FALD severity. The FES provides a biologically validated scoring system that substantially outperforms existing serological indices and offers a new avenue for early detection and risk stratification of FALD Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIFontan-associated liver disease (FALD) is a nearly universal consequence of the Fontan circulation, driven by chronic venous hypertension and reduced cardiac output. C_LIO_LICurrent surveillance tools, including transaminases, composite serological indices (APRI, FIB-4), and elastography, have limited sensitivity and specificity for detecting and staging hepatic fibrosis in the Fontan population. C_LIO_LICirculating small extracellular vesicles (sEVs) carry tissue-derived molecular cargo and have shown diagnostic potential in other liver diseases, but their utility in FALD has not been explored. C_LI What New Information Does This Article Contribute?O_LIMulti-omic profiling of circulating sEVs in a translational ovine Fontan model reveals that the small RNA cargo is stratified by fibrotic status and enriched for inflammatory pathways associated with hepatic stellate cell activation. C_LIO_LIThe Fontan EV Score (FES), integrating time post-surgery with eleven circulating small RNA biomarkers, predicts FALD severity with substantially greater accuracy than APRI and FIB-4. C_LIO_LITGF-{beta}-treated human liver organoids confirm that several FES-associated miRNAs are directly responsive to profibrotic stimulation, providing biological validation independent of Fontan hemodynamics. C_LI This study demonstrates that circulating sEVs function as non-invasive reporters of hepatic fibrogenesis in the Fontan circulation and introduces the first EV-based scoring system for FALD risk stratification. The FES achieved an AUC of 0.876 for moderate and 0.963 for severe fibrosis in an independent test cohort, outperforming established serological indices that were originally developed for viral hepatitis but which perform poorly in congestive hepatopathy. By combining molecular biomarker discovery with in vitro functional validation, this work establishes a foundation for developing targeted, non-invasive diagnostics to guide surveillance and clinical decision-making in the growing Fontan patient population.

BackgroundInterleukin-32 (IL-32) presents a long-standing paradox in liver disease, with markedly elevated expression in hepatocellular carcinoma (HCC) yet a protective role against hepatic steatosis. The absence of a canonical receptor or defined secretory pathway has obscured its biological function. This study aimed to resolve this paradox by delineating the regulatory mechanisms that govern IL-32 activity during hepatocarcinogenesis. MethodsWe analyzed two in-house prospective cohorts, including a MASLD cohort and a MASLD-associated HCC cohort, integrating matched transcriptomic and metabolomic data. Targeted lipidomics and multi-omics analyses were combined with single-cell and spatial transcriptomics. Key findings were validated using functional assays and gene perturbation models. ResultsWe identified a disease stage-specific transcriptional switch in which noncanonical NF-{kappa}B signaling (NFKB2/RELB) replaces canonical NF-{kappa}B as the primary activator of IL-32, forming an auto-amplifying inflammatory loop. This switch is enabled by FOXO1, which acts as a pioneer factor to maintain chromatin accessibility at IL32 and NF-{kappa}B loci. Functionally, IL-32 is coupled to lipid metabolism through DGAT2; however, this axis becomes uncoupled in HCC, where DGAT2 loss rewires NF-{kappa}B/ERK signaling without recapitulating global metabolic remodeling, thereby sensitizing cells to inflammatory activation. ConclusionsThese findings resolve the functional paradox of IL-32 by revealing a multi-layered regulatory network that reprograms its activity during liver disease progression, and define IL-32 as a context-dependent integrator of metabolic and inflammatory signaling, whose regulatory network is rewired during hepatocarcinogenesis to promote a sustained pro-inflammatory state. HighlightsO_LINoncanonical NF-{kappa}B (NFKB2/RELB) drives a self-amplifying IL-32 loop in HCC. C_LIO_LIFOXO1 licenses this switch by maintaining chromatin accessibility at IL32 and NF-{kappa}B loci. C_LIO_LIIL-32 shifts from a metabolic regulator in MASLD to an inflammatory driver in HCC. C_LI

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: To date, no precision oncology approach has been established for HCC. Despite the diverse underlying causes, HCC development exhibits a uniform pathophysiology characterised by chronic hyper-proliferation, resulting from hepatocyte apoptosis and compensatory liver regeneration. This chronic hyper-proliferative pressure, termed regeneration stress, drives genomic instability during HCC onset, yet its therapeutic potential remains poorly explored. This study aimed to identify targetable vulnerabilities tied to regeneration stress and establish clinically applicable markers for treatment stratification. Methods: Weighted gene co-expression network analysis (WGCNA) was applied on external bulk RNA-seq datasets to define a LIVer REgeneration Stress Signature (LIVRESS). The signature was functionally validated using HCC patient-derived organoids (HCC-Org), and vulnerabilities were mapped using mid-throughput drug screening, single-molecule and single-cell assays, and multi-omic integration. Results: High LIVRESS scores, characterised by enrichment in replication, mitotic and DNA damage repair pathways, identified a subset of HCC patients with aggressive disease and poorer survival across aetiologies. HCC-Org with high LIVRESS scores displayed exquisite sensitivity to multiple inhibitors of the checkpoint kinase ATR. Although HCC-Org models exhibited a baseline reduction in replication fork speed, sensitivity to ATR inhibitor (ATRi) was decoupled from replication fork dynamics and rather linked to intrinsic mitotic instability. ATR inhibition triggers mitotic failure and apoptosis in LIVRESSHigh HCC-Org. This killing effect was significantly potentiated by combining ATRi with PARPi or WEE1i. Multi-omic integration identified KPNA2 as a surrogate biomarker of ATRi sensitivity. Conclusion: Our findings demonstrate that a subset of HCC-Org, characterised by high liver regeneration-associated stress, is vulnerable to ATRi-based therapies. By focusing on a comprehensive regenerative stress model, we establish a framework to stratify HCC patients and implement biomarker-driven, ATR-based therapies for HCC patients with advanced disease. Impact and implications: Regeneration stress is a key factor that drives genomic instability in HCC, providing a basis for the LIVRESS to identify patients dependent on ATR-mediated checkpoints. These findings reveal a conceptual shift for researchers and trialists: ATRi efficacy is decoupled from replication fork dynamics and instead leverages mitotic fragility. Practically, the LIVRESS and its IHC surrogate marker (KPNA2) offer a scalable roadmap for physicians to improve patient stratification in ATRi-based precision oncology trials. While requiring prospective validation, these results pave the way toward biomarker-driven therapies for advanced HCC.

Alcohol exposure perturbs intracellular calcium (Ca2+) homeostasis in digestive organs, yet whether common or organ-specific mechanisms coordinate this response remains unclear. Using an acute ethanol paradigm in zebrafish, single-cell transcriptomics revealed broad up-regulation of Ca2+-signaling genes in hepatocytes and pancreatic acinar cells. In vivo Ca2+ buffering with SpiCee, a genetically encoded chelator, demonstrated a shared requirement for Ca2+ flux: in hepatocytes, lineage-restricted buffering was associated with pronounced cytoplasmic vacuolation composed of lipid-negative vesicles, consistent with stalled lysosomes or autophagosomes; in pancreatic acinar cells, it was associated with accumulation of aggregated/misfolded protein. Mechanistic experiments using pharmacological inhibitors implicated distinct molecular contributors in each tissue. In hepatocytes, inhibition of Pikfyve or its downstream effector, the lysosomal Ca2+ channel TRPML1, phenocopied Ca2+ buffering. While, in acinar cells, Pick1 inhibition produced analogous associations. These data position Pikfyve and Pick1 as organ-specific components linked to the Ca2+-coupled alcohol response. Notably, pharmacologic activation of TRPML1 in hepatocytes recapitulated alcohol-like Ca2+ dynamics but increased macrophage recruitment and cell death, indicating that Ca2+ signaling is required for the alcohol response yet can be detrimental when amplified. Together, our results support a model in which alcohol elicits a shared Ca2+ dynamics across liver and pancreas, modulated by tissue-specific molecular nodes.

Background & AimsHepatocellular carcinoma (HCC), a predominant form of liver cancer, remains a significant clinical unmet need. Given that 30-50% of HCC cases harbour mutations in the Wnt/{beta}-catenin signalling pathway, targeting this cascade represents a promising therapeutic strategy. However, the clinical translation of Wnt inhibitors has been hindered by severe adverse events observed in preclinical models and early-phase clinical trials, primarily due to the essential role of Wnt signalling in maintaining normal tissues such as the intestine and bone. MethodsWe examined the efficacy of Tegavivint, a first-in-class Wnt pathway inhibitor that targets TBL1, against HCC to elucidate its underlying mechanism of action. We evaluated the dose-response of Tegavivint and its effects on the cell cycle, apoptosis, and Wnt target gene expression using HepG2, HUH6, and HUH7 cell lines in vitro. Furthermore, we employed an orthotopic xenograft transplant model using HepG2 cells in immunodeficient mice to assess the safety profile and on-target anti-cancer efficacy of Tegavivint in vivo. ResultsTegavivint exhibited potent Wnt pathway-suppressing effects in cancer cells with constitutive Wnt pathway activation. Notably, Tegavivint displayed robust anti-tumour activity across a broad range of HCC cell lines, regardless of their Wnt pathway activation status. While Tegavivint inhibited the Wnt pathway and triggered the activation of apoptotic pathways in most cell lines, our findings suggest that it also can induce cell death by activating alternative non-apoptotic pathways in apoptosis-resistant cancer cells. In an orthotopic transplant mouse model, Tegavivint significantly downregulated Wnt pathway target genes, inhibited cell proliferation, induced apoptosis and suppressed growth in tumours. ConclusionsTaken together, our data establish a robust foundation for evaluating Tegavivint as a novel therapeutic option, specifically tailored for HCC patients harbouring Wnt-driven hepatic malignancies.

TMEM135 has been implicated in lipid metabolism, but its role in regulating hepatic lipid homeostasis remains unclear. Here, we investigated how TMEM135 affects hepatic lipid metabolism using Tmem135 mutant mice with liver-specific Pex5 deletion. The Tmem135 mutation induced a lipogenic state characterized by depletion of docosahexaenoic acid (DHA), activation of SREBP-dependent pathways, and increased monounsaturated fatty acids without causing hepatic steatosis. In contrast, loss of PEX5-dependent peroxisomal function in Tmem135 mutant mice resulted in marked hepatic lipid accumulation, indicating that peroxisomal metabolism buffers the elevated lipogenic state. Fish oil supplementation to Tmem135 mutant mice restored DHA levels and suppressed lipogenesis. Proteomics identified distinct DHA-sensitive metabolic programs, including activation of SREBP-dependent lipogenesis. Quantitative malonyl-proteomics revealed increased malonylation of glycolytic enzymes, accompanied by altered glycolytic output. Together, these findings identify TMEM135 as a central regulator of hepatic lipid metabolism and uncover a coordinated mechanism linking lipid availability, lipogenesis, and post-translational metabolic regulation. HighlightTmem135 mutation induces a lipogenic state with increased lipolysis without hepatic steatosis. PEX5-dependent peroxisomal function buffers lipid accumulation in Tmem135 mutant liver. Fish oil supplementation restores DHA and suppresses SREBP-dependent lipogenesis in Tmem135 mutants. DHA-sensitive protein malonylation targets glycolytic enzymes in Tmem135 mutant liver.

Background: Alpha-1 antitrypsin deficiency (AATD) caused by the PI*ZZ mutation (Glu342Lys) results in hepatic accumulation of misfolded AAT-Z protein and reduced circulating AAT levels, leading to progressive liver disease and emphysema. Gene correction therapy represents a potentially curative approach by directly correcting the underlying genetic defect. We report the first case of successful hepatic gene correction with early histological and functional assessment. Methods/Case presentation: We report the case of a 66-year-old male patient with PI*ZZ AATD who underwent gene correction therapy within the YOLT-202 phase I/Ia clinical trial (clinical trial.gov ID NCT07193615). Ten weeks post treatment a liver biopsy was performed to re-evaluate pre-existing F2 liver fibrosis as measured by elastography before entering the study. Serum samples allowed functional assessment of the AAT-mediated elastase inhibition. Results: Liver biopsy did not show signs of hepatic inflammation and demonstrated 54% (Sanger) and 57% (Illumina) gene correction rate of the PI*ZZ variant on the DNA level with no bystander edits or off-target effects. Following a transient elevation of transaminases during the early post-treatment period, liver enzymes normalized. Monthly serum AAT measurements demonstrated biologically active and stable therapeutic levels throughout follow-up. Conclusions: This case demonstrates efficient and precise hepatic gene correction without concerning histological alterations and with substantial improvement of functional parameters, supporting the feasibility and safety of gene editing approaches for AATD.

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

Hepatocellular carcinomas (HCC) are genetically heterogeneous cancers frequently characterized by MYC gene amplification or hyperactivating {beta}-catenin (CTNNB1) mutations. Analysis of TCGA transcriptomics revealed that MYC-driven HCC tumors have decreased expression of mtDNA-encoded genes, but increased expression of nuclear-encoded mitochondrial genes. To investigate this apparent discrepancy, we generated MYC- and CTNNB1-driven murine HCCs, all of which displayed aberrant mitochondrial metabolism. Notably, MYC-driven tumors exhibited significant reductions in OXPHOS and TCA cycle activity that correlated with increased ROS levels, as well as elevated mitochondrial turnover through mitochondrial fission and mitophagy. MYC induces the expression of nuclear respiratory factor 1 (NRF1), which regulates DRP1 and other genes to promote receptor-mediated mitophagy. Knocking out DRP1 reduced mitophagy and ROS levels and promoted survival of HCC-bearing mice. These results identify elevated mitochondrial turnover as a potential therapeutic target in MYC-driven HCC. SignificanceHepatocellular carcinoma can arise from multiple oncogenes, making targeted therapy more difficult. Here we show that tumors with MYC amplification lose mitochondrial function via fission and mitophagy upregulation. Targeting mitochondrial quality control results in increased survival suggesting a therapeutic window in MYC-driven HCC.

Alcohol-induced toxicity is driven largely by the accumulation of acetaldehyde and disruption of hepatic redox homeostasis during ethanol metabolism. Oxidation of ethanol by alcohol dehydrogenase (ADH) consumes nicotinamide adenine dinucleotide (NAD) while generating NADH, shifting the intracellular redox state toward a highly reduced environment that impairs mitochondrial function, limits endogenous aldehyde dehydrogenase (ALDH)-mediated acetaldehyde clearance, and promotes oxidative stress and tissue injury. We investigated whether UT-018, a novel metabolic intervention, could support endogenous metabolic resilience during acute ethanol exposure using complementary in vitro and in vivo models. Mechanistic in vitro studies evaluated ADH-dependent NADH generation and NAD add-back experiments, while in vivo investigations assessed serum ALDH-associated activity, circulating acetaldehyde concentrations, and gross gastrointestinal and hepatic morphology following acute ethanol challenge. UT-018 reduced ethanol-associated NADH accumulation in a concentration-dependent manner without evidence of irreversible ADH inhibition. Restoration of NADH generation following supplementation with exogenous NAD demonstrated reversible modulation of ethanol-associated redox biology rather than direct enzymatic inhibition. In vivo, UT-018 enhanced serum ALDH-associated activity, reduced circulating acetaldehyde concentrations by approximately 27 to 33% compared with ethanol-treated controls. Metabolic biomarkers were accompanied by preservation of gross colon and liver morphology following acute ethanol exposure. Collectively, these findings support coordinated biological activity across multiple interconnected stages of alcohol metabolism and support a systems-level mechanism in which restoration of redox homeostasis enhances endogenous aldehyde detoxification, reduces acetaldehyde burden, and preserves tissue integrity. These results identify alcohol metabolism restoration as a promising strategy for enhancing physiological resilience to acute alcohol exposure and provide a rationale for further preclinical and clinical evaluation of UT-018. HighlightsO_LIUT-018 restored ethanol-associated redox homeostasis by reducing excessive NADH accumulation without irreversible inhibition of alcohol dehydrogenase in vitro. C_LIO_LIRestoration of redox balance was associated with enhanced endogenous aldehyde dehydrogenase (ALDH)-associated activity following acute ethanol exposure in vivo. C_LIO_LIUT-018 reduced circulating acetaldehyde concentrations by approximately 30%. C_LIO_LIThe metabolic homeostasis was accompanied by preservation of gross gastrointestinal and hepatic morphology in an acute ethanol challenge model. C_LIO_LIThe collective findings support a systems-level mechanism in which modulation of endogenous alcohol related metabolic pathways enhances physiological resilience to acute alcohol exposure. C_LI