Drug induced liver injury (DILI) continues to be a significant challenge in drug development. Just one or two cases of severe hepatotoxicity in a clinical trial can be enough to raise drug approvability concerns. Though great strides were made in DILI risk assessment and mitigation in clinical trials during the early 2000s, drugs continue to fail in development due to DILI, while severe cases still occur in late phase clinical trials and post-market. Also, changes in drug development will challenge the US Food and Drug Administration's (FDA) current paradigms for DILI risk assessment. Such changes include the rise in biologic agents which may cause hepatotoxicity distinctly different from small molecule drugs, the increasing reliance on smaller underpowered clinical trials for rare diseases, and the growing interest in drugs for acute and chronic liver diseases. We highlight eight key topics that may benefit from review, clarification, research, and discussion between industry, academia, and regulators as the FDA works to address these issues.
BACKGROUND AND AIMS:The American Association for the Study of Liver Diseases (AASLD) recommends that a decline in liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) can be used as a non-invasive endpoint for treatment response in metabolic dysfunction-associated steatohepatitis (MASH), but there are limited data supporting this statement. We examined the association between a ≥30% relative decline in LSM and fibrosis regression. APPROACH AND RESULTS:This prospective study included 160 adults (64% female) with biopsy-proven MASH and stage 2-3 fibrosis from a randomized, phase 2b, multicenter, placebo-controlled trial of the fibroblast growth factor 21 analog pegozafermin. All participants underwent contemporaneous VCTE assessments and liver biopsy at two time-points. The primary endpoint was fibrosis regression without worsening MASH. The median (IQR) age and body mass index of participants were 56.0 (49.0-62.0) years and 36.5 (32.2-40.4) kg/m². The area under the receiver operating curve (AUC) of a ≥30% relative decline in LSM by VCTE for detecting fibrosis regression was 0.68 (95% CI 0.58-0.77). In multivariable analyses adjusted for age, sex, type 2 diabetes, BMI, and ethnicity, a ≥30% relative decline in LSM was independently associated with fibrosis regression (adjusted OR 4.23, 95% CI 1.79-10.38, p=0.001). In a distinct validation cohort (n=48) from U.S. and Singapore, a ≥30% decline in LSM for detecting fibrosis regression yielded an AUC of 0.62 (95% CI 0.48-0.76). CONCLUSION:A ≥30% relative decline in LSM by VCTE had modest accuracy to detect fibrosis regression without worsening MASH. More effective biomarkers for treatment response are required.
BACKGROUND:Alternative polyadenylation (APA) is a key post-transcriptional mechanism that regulates gene expression by modulating 3'UTR length, its dysregulation has been implicated in carcinogenesis. How genetic variants influence APA to affect hepatocellular carcinoma (HCC) prognosis remains unclear. METHODS:Prognosis-APA quantitative trait loci (apaQTL) were performed using genotype and APA profiling from TCGA data. A two-stage survival analysis in 848 Chinese and 369 TCGA LIHC patients and functional validation were used to identify prognostic apaQTL in HCC progression. RESULTS:A total of 2,025 and 817 significant APA events were identified in Chinese and TCGA cohort, respectively. Besides, 859 events were associated with poor prognosis in HCC and enriched in RNA splicing / metabolism pathways. We detected 32,034 significant apaQTLs, predominantly enriched in 3'UTRs and RBP-binding regions. CPEB3 was prioritized as a key APA regulator RBP; its low expression correlated with poor patient survival and promoted proliferation, migration, and invasion in HCC cells. Notably, a functional apaQTL variant rs2037547, located in GSK3B and mediated by CPEB3, demonstrated a poor survival of HCC patients in both cohort (pooled HR=1.29, p=0.016). Mechanistically, rs2037547 promoted aberrant APA at proximal poly(A) sites of GSK3B through CPEB3, leading to increased expression of short 3'UTR isoform. This regulatory alteration enhanced HCC cell proliferation, invasion, and migration, and contributed to HCC progression. CONCLUSION:These findings elucidated the distinct role of apaQTL-mediated APA dysregulation in HCC prognosis, providing insights for prognostic stratification and potential targets for personalized therapy in HCC.
BACKGROUND:Alpha-1 antitrypsin deficiency (AATD) may cause chronic lung and liver disease, yet data on major adverse liver outcomes (MALO) across heterozygous and homozygous Z-allele genotypes remains limited. We assessed the risk of MALO across AATD Pi*MZ, Pi*SZ, and Pi*ZZ genotypes compared to the wildtype Pi*MM. METHODS:This retrospective cohort study utilized the Veterans Analysis of Liver Disease cohort (January 2000-April 2025). Genotypes were identified using validated natural language processing (κ=0.89). Multivariable Fine-Gray models estimated the hazard of MALO. RESULTS:Among 22,537 participants with genotype testing (19,665 Pi*MM, 1,656 Pi*MZ, 281 Pi*SZ, and 935 Pi*ZZ) and 352,612 person-years of follow up, MALO risk increased sequentially with allele burden: Pi*MZ (aHR 1.25, 1.11-1.40), Pi*SZ (aHR 1.51, 1.17-1.94), and Pi*ZZ (aHR 1.80, 1.57-2.07), versus Pi*MM. MALO incidence rates for Pi*MM, Pi*MZ, Pi*SZ, and Pi*ZZ, were 11.3, 13.0, 14.6, and 19.2 for 1,000 person-years respectively, while five-year MALO probability was 3.5%, 5.5%, 5.3%, and 8.1% respectively. Pi*ZZ was associated with significantly increased risk of all individual MALO components: decompensation, HCC, liver transplantation (LT), and liver-related death (LRD). Pi*SZ and Pi*MZ were associated with higher risk of decompensation, LT, and LRD, but not HCC. A sensitivity analysis restricted to participants with MASLD showed consistent findings. CONCLUSION:In this national longitudinal cohort of veterans with documented AATD genotype testing and median follow-up of 15.9 years, we observed an increased risk of MALO in both homozygous and heterozygous Z-allele carriers, underscoring the need for timely diagnosis and enhanced clinical surveillance among veterans with known AATD genotypes.
BACKGROUND AND AIMS:The role of shear wave elastography (SWE) in two-step MASLD risk stratification remains unclear. We compared SWE with vibration-controlled transient elastography (VCTE) using American Gastroenterological Association (AGA) and European Association for the Study of the Liver (EASL) algorithms. APPROACH AND RESULTS:Patients with MASLD who underwent both VCTE and SWE during the same session between 2019 and 2025 were included. Risk stratification was performed using AGA-based and EASL-based two-step approaches. The primary endpoint was liver-related events (LREs). Among 2817 patients, the AGA-SWE algorithm classified 2196, 140, and 481 patients as low-risk, intermediate-risk, and high-risk, respectively. Using the AGA-SWE algorithm, both intermediate-risk and high-risk groups showed increased risks of LREs compared with the low-risk group [subdistribution hazard ratio (sHR) 7.60 and 9.86, respectively]. The EASL-SWE algorithm also stratified LRE risk relative to the low-risk group, with higher sHRs observed for the intermediate-low, intermediate-high, and high-risk groups (sHR 2.99, 10.83, and 18.87, respectively). The integrated time-dependent area under the curve through 60 months was 0.770 [95% confidence interval (CI): 0.709, 0.827] and 0.775 (95% CI: 0.712, 0.835) for AGA-SWE and AGA-VCTE, respectively, and 0.804 (95% CI: 0.753, 0.849) and 0.805 (95% CI: 0.750, 0.852) for EASL-SWE and EASL-VCTE, respectively, with no significant differences between SWE and VCTE in either algorithm. No significant net reclassification improvement was observed between SWE-based and VCTE-based AGA algorithms. SWE and VCTE demonstrated a strong correlation (Spearman ρ = 0.713, 95% CI: 0.691-0.735, p <0.001). CONCLUSIONS:SWE-based two-step risk stratification showed similar predictive performance to VCTE-based models for predicting LREs in MASLD, although these findings do not establish formal equivalence.
Liver-on-a-chip (LOC) systems are tunable, microscale environments that can incorporate additional layers of physiologic complexity in comparison to 2D cell culture. LOCs are an in vitro adjunct and potential alternative to in vivo animal models that utilize human-derived cells, typically embedded in a synthetic device containing natural or engineered materials. This enables LOC models to exhibit a high level of customization per organ, disease, or condition. LOCs typically contain dynamic flow to cells embedded in a series of microchannels within the device. This perturbation aims to recreate mechanobiological forces seen in vivo and encourage cell-to-cell interactions in a 3D environment. These models have proven to be particularly advantageous in modeling organs with complex spatial zonation, such as the liver, as oxygen and nutrient gradients can more accurately be represented under these dynamic conditions. LOC systems have also been proposed to improve the accuracy of drug toxicity screening for clinical applications because they employ human-derived cells in more physiologically relevant conditions. This review will first discuss fabrication techniques and considerations when recreating biological microenvironments. Next, the advances of LOC systems and applications for modeling liver disease and infection will be explored. Finally, current limitations and advances for implementing LOC devices into clinical and commercial settings will be examined.
BACKGROUND AIMS:Liver fibrosis is a global health issue with unclear unified drivers. The RNA-binding protein hnRNPK is critical for hepatic homeostasis, but its role in chronic fibrosis remains undefined. This study investigated hnRNPK-mediated splicing dysregulation in liver fibrosis and its therapeutic potential. APPROACH RESULTS:We mapped hepatocellular hnRNPK expression across a broad spectrum of human and murine fibrotic etiologies. Mechanistic insights were obtained by integrating snRNA-seq, hepatocyte-specific alternative splicing profiling, untargeted and spatial metabolomics, and 3D mitochondrial reconstruction using AT-SEM. We identified reduced hepatocellular hnRNPK abundance as a conserved hallmark of liver fibrosis across distinct pathogenic insults, including MASH, ALD, HBV, and DILI. Hepatocyte-specific Hnrnpk ablation in mice induced aberrant Opa1 splicing, specifically promoting exon 4b inclusion. This disrupted the balance of OPA1 isoforms, depleting L-OPA1 and driving severe mitochondrial fragmentation characterized by distinct "spheroid" ultrastructures. This loss of mitochondrial integrity triggered HSC activation through the cytosolic mtDNA-cGAS-STING pro-inflammatory signaling cascade and the mitochondria-containing pellet. Notably, restoring the hnRNPK-Opa1 axis via AAV-mediated overexpression of Hnrnpk or the Opa1-Δ4b isoform (lacking exon 4b), together with pharmacological promotion of mitochondrial fusion, effectively arrested and regressed established liver fibrosis in multiple murine models. CONCLUSIONS:Our findings establish the loss of hnRNPK as a central, unifying mechanism in liver fibrogenesis. By governing the Opa1 splicing axis, hnRNPK maintains mitochondrial integrity and prevents the pro-inflammatory signaling and secretome that fuel HSC activation. Targeting this RNA-mitochondria axis represents a potent, etiology-independent therapeutic strategy for chronic liver disease.
BACKGROUND:Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality. Optimal treatment decisions are challenging, and existing treatment allocation frameworks may not fully reflect the evolving therapeutic landscape or contemporary clinical practices in North America. METHODS:Using a modified Delphi process, a multidisciplinary panel of 20 North American experts in hepatology, medical oncology, surgery, radiology, and radiation oncology developed a consensus-based treatment framework for HCC, termed the BEACON-HCC system. The framework was informed by current evidence and expert opinion through iterative discussion and voting. In a pilot study using 29 real-world patient cases, we assessed concordance between external expert recommendations and BEACON-HCC recommendations and Barcelona Clinic Liver Cancer (BCLC) 2025 treatment recommendations. RESULTS:The BEACON-HCC treatment allocation framework diverges from prior frameworks by incorporating nuanced clinical features such as degree of intrahepatic tumor burden and vascular invasion, and adverse tumor prognostic markers, to align treatment allocation with tumor biology and therapeutic potential. Key innovations include the incorporation of emerging modalities such as external beam radiation therapy (EBRT), transarterial radioembolization (TARE), and systemic-locoregional combination therapies. In the pilot exercise, expert treatment decisions showed 96.6% concordance with BEACON-HCC recommendations and 72.4% concordance with BCLC recommendations. CONCLUSIONS:BEACON-HCC is a consensus-based framework for treatment allocation that incorporates the expanding range of therapeutic options available to patients in the North American HCC population. Expert treatment decisions showed a high concordance with the BEACON-HCC system; further validation using empiric clinical data is planned through the HCC-Live Consortium.
BACKGROUND AND AIMS:Liver fibrosis and cirrhosis are major global health burdens with limited treatments. Activated HSCs drive fibrosis through increased proliferation, migration, collagen deposition and glycolysis. The aim of this study is to elucidate the signaling events that lead to enhanced glycolysis, subsequent HSC activation, and liver fibrosis. APPROACH AND RESULTS:Utilizing a phospho-kinase array, we identified with no lysine kinase 1 (WNK1) as a new kinase in primary human HSCs activated by the profibrotic PDGF-B. PDGF-mediated glycolysis in HSCs was attenuated by the WNK1 selective inhibitor, WNK-IN-11, as measured by live-cell metabolic assay, Glifon-300 fluorescence, Glucose-Glo assay, and glucose transporter 1 immunofluorescence. To understand how WNK1 promotes glycolysis, we employed chemical genetics where an engineered WNK1 carrying threonine 301 to glycine mutation (WNK1 T301G ) mutant with a modified ATP pocket accommodated a specific ATP-γ-S analogue to thio-phosphorylate its substrates. Thio-phosphorylated proteins were analyzed by mass spectrometry, which identified the glycolytic protein, triosephosphate isomerase 1 (TPI1), as a novel WNK1 direct substrate. TPI1 interacted with WNK1 in condensates, membrane-less phase separation structures. Condensate disruption and knockdown of TPI1 or WNK1 reduced HSC proliferation and migration in vitro. In vivo, pharmacological or genetic WNK1 inhibition significantly attenuated liver fibrosis in carbon tetrachloride and high-fat, choline-deficient, L -amino acid diet mouse models. Finally, WNK1 inhibition reduced fibrogenesis ex vivo in human precision-cut liver slices. CONCLUSIONS:These findings identify WNK1 as a previously unrecognized kinase that promotes HSC activation and liver fibrosis via TPI1-mediated glycolysis, highlighting the WNK1-TPI1 axis as a potential therapeutic target.
BACKGROUND AND AIMS:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of HCC, particularly in obesity, yet mechanisms linking hepatocyte dysfunction to tumorigenesis remain unclear. Mixed lineage kinase domain-like protein (MLKL), the effector of necroptosis, is elevated in MASLD, but its hepatocyte-intrinsic role in obesity-driven MASLD-HCC is unknown. APPROACH AND RESULTS:Using a long-term western diet (WD)-induced MASLD-HCC model in hepatocyte-specific MLKL knockout ( MlklHepKO ) mice, we defined MLKL's hepatocyte-intrinsic function. WD increased hepatocyte MLKL protein expression without detectable necroptosis activation, indicating a necroptosis-independent role. MLKL deficiency did not alter WD-induced inflammation, fibrosis, or liver injury but increased hepatic lipid accumulation while reducing lipotoxic lipid species and preserving mitochondrial function. WD-fed MlklHepKO mice developed fewer and smaller tumors with reduced incidence, multiplicity, proliferation, and stemness. Transcriptomic analysis revealed upregulation of mitochondrial oxidative phosphorylation pathways in MlklHepKO livers. WD suppressed the mitochondrial fusion protein and tumor suppressor mitofusin 2 (MFN2), whereas MLKL deficiency restored MFN2 expression post-translationally. In HCC cells, MLKL deletion reduced proliferation, improved mitochondrial respiration, and decreased glycolysis; these effects were reversed by MFN2 deletion. MLKL is localized to nuclear and mitochondrial compartments, consistent with organelle-intrinsic functions. The human MLKL inhibitor necrosulfonamide (NSA) suppressed HepG2 xenograft growth, and elevated MLKL expression in human HCC correlated with poorer overall survival. CONCLUSIONS:Hepatocyte MLKL promotes MASLD-associated HCC through a non-necroptotic mechanism involving MFN2 suppression, impaired mitochondrial function, and increased tumor proliferation and stemness. These findings identify MLKL as a potential therapeutic target in MASLD-associated HCC.
BACKGROUND:Finite therapy resulting in sustained hepatitis B surface antigen (HBsAg) loss for patients with chronic HBV infection (CHB) is an important therapeutic goal. In patients with HBeAg-negative infection, nucleos(t)ide analog (NA) withdrawal may achieve HBsAg loss in 5%-20% of patients after 3 years. Pegylated interferon (PEG-IFNα) is a recognized treatment for CHB. METHODS:NUC-B was a randomized, multicenter trial in NA-treated non-cirrhotic HBeAg-negative patients with CHB. Patients were allocated to either NA withdrawal alone (control) or NA withdrawal followed by a 16-week course of PEG-IFNα 180 μg weekly commencing 4 weeks after NA cessation (PEG-IFNα). The primary endpoint was HBsAg loss at 3 years. RESULTS:The target recruitment of 240 patients was not achieved. In all, 156 patients, 82 to the control arm, 74 to the PEG-IFNα arm, were recruited between 2017 and 2021; median age 45 years, 24% female, HBV genotypes-A 16%, B 6%, C 4%, D 24%, E 22%, other 1%, ānd unknown 26%. At 3 years, 3% of patients in the control arm and 14% of patients in the PEG-IFNα arm lost HBsAg (OR 5.39; 95% CI (1.11, 26.19); p =0.037). In the control arm, 34.9% of patients returned to NA therapy compared with 28.4% in the PEG-IFNα arm. Exaggerated flares occurred in 27.9% of patients in the control arm compared with 13.4% in the PEG-IFNα arm. CONCLUSIONS:The use of adjuvant PEG-IFNα therapy after withdrawal of NA therapy increases the rate of HBsAg loss while simultaneously reducing the number of exaggerated flares.
BACKGROUND AND AIMS:Survodutide was associated with improvements in liver-related endpoints in a phase 2 trial in MASH (NCT04771273). This post hoc mediation analysis evaluated the proportion of the direct treatment effect versus the indirect effect mediated by weight reduction. APPROACH AND RESULTS:Data were included for participants with fibrosis stages F2-F3 and paired baseline/end-of-treatment biopsy readings; survodutide dose arms were pooled. The causal mediation analysis model included treatment (exposure) and percentage change in body weight (mediator), with baseline body weight, type 2 diabetes status, and fibrosis stage as covariates. Outcomes included histological endpoints and non-invasive tests (NITs) after 48 weeks' treatment. Total, direct (weight reduction-independent), and indirect (weight reduction-dependent) treatment effects were calculated for survodutide versus placebo. This analysis included 170 participants. For histological endpoints, the percentage of the total treatment effect mediated by weight reduction (indirect effect) was estimated at 66.7% for resolution of MASH without worsening of fibrosis, 71.8% for improvement in MASH without worsening of fibrosis, and 36.3% for improvement in fibrosis without worsening of MASH (suggesting a greater direct effect). Similarly, for NITs related to inflammation/fibrosis, <50% of the total effect was mediated by weight reduction [16.5% (AST)-38.6% (Enhanced Liver Fibrosis)]. For steatosis-related endpoints (MRI-proton density fat fraction and FibroScan Controlled Attenuation Parameter), a higher proportion was mediated by weight reduction (58.2% and 77.4%, respectively). CONCLUSIONS:Endpoints related to improvements in inflammation/fibrosis were predominantly weight reduction-independent, suggestive of a potential role of direct glucagon receptor agonism in the liver.
BACKGROUND AND AIMS:Recompensation of decompensated metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatotic liver disease with alcohol consumption (MetALD) cirrhosis remains unknown. APPROACH:We assessed rates and predictors of recompensation (Baveno VII criteria) and outcomes after modifiable interventions, including alcohol abstinence, dry-weight loss, and cardiometabolic risk factor management, in a cohort of patients with decompensated MASLD/MetALD cirrhosis between October 2019 and March 2025. RESULTS:Among 344 patients with steatotic liver disease-related cirrhosis (mean MELD-Na 14.7±3.6; MASLD-158, MetALD-186), 64(18.6%) achieved recompensation; MASLD (17.7%) and MetALD (19.4%), at a median of 15 months(IQR: 14.5-15.5) over 3.9 years (IQR: 2.8-4) follow-up. Baseline phosphatidylethanol (PEth) was negative in MASLD but positive in 69.4% of MetALD patients ( p =0.001). In all, 61 patients (17.7%) died, 11 (3.2%) underwent liver transplantation, and 5 (1.5%) developed hepatocellular carcinoma. Mortality was lower in MASLD than in MetALD (12.0% vs. 22.6%; p =0.011). In competing-risk regression adjusted for baseline MELD-Na, absence of large esophageal varices (sHR 1.97, 95% CI: 1.22-3.99; p =0.021), weight loss ≥10% (sHR 7.42, 95% CI: 3.15-17.47; p <0.001), alcohol abstinence (sHR 1.87, 95% CI: 1.29-3.32; p =0.003), and glycemic control (sHR 1.43, 95% CI: 1.03-2.79; p =0.038) were associated with recompensation. Higher MELD-Na (sHR 1.13, 95% CI: 1.06-1.2; p <0.001), MetALD etiology (sHR 1.87, 95% CI: 1.09-3.22; p =0.023), lack of recompensation ( p <0.001), and ongoing alcohol consumption (sHR 5.3, 95% CI: 2.85-9.74; p <0.001) were associated with mortality. CONCLUSIONS:PEth improved the ascertainment of alcohol exposure and phenotyping of MASLD and MetALD. Weight loss ≥10%, achievement of glycemic control, and absence of large varices were independently associated with recompensation, which occurred in 18.6% of patients with decompensated MASLD/MetALD cirrhosis.
