BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

BACKGROUND AND AIMS: Chronic HBV infection is the leading cause of HCC and a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade in HBV-related HCC. APPROACH AND RESULTS: A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg+ hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine-induced HCC, TGF-β-activated kinase 1 knockout-induced HCC, HCC in a stelic animal model, or NASH-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8+ T cells, whereas the production of IL-2, TNF-α, and interferon-gamma (IFN-γ) by intrahepatic CD8+ T cells was decreased. Increased expression of TIGIT on CD8+ T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8+ T cells with increased TNF-α and IFN-γ production and an increased number of CD8+ T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade. CONCLUSIONS: Blockade of TIGIT alone enhanced the antitumor activity of CD8+ T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1)S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. APPROACH AND RESULTS: Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis-regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. CONCLUSIONS: Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

BACKGROUND AND AIMS: NAFLD is the most prevalent chronic liver disease worldwide and has emerged as a serious public health issue with no approved treatment. The development of NAFLD is strongly associated with hepatic lipid content, and patients with NAFLD have significantly higher rates of hepatic de novo lipogenesis (DNL) than lean individuals. Leukotriene B4 (LTB4), a metabolite of arachidonic acid, is dramatically increased in obesity and plays important role in proinflammatory cytokine production and insulin resistance. But the role of liver LTB4/LTB4 receptor 1 (Ltb4r1) in lipid metabolism is unclear. APPROACH AND RESULTS: Hepatocyte-specific knockout (HKO) of Ltb4r1 improved hepatic steatosis and systemic insulin resistance in both diet-induced and genetically induced obese mice. The mRNA level of key enzymes involved in DNL and fatty acid esterification decreased in Ltb4r1 HKO obese mice. LTB4/Ltb4r1 directly promoted lipogenesis in HepG2 cells and primary hepatocytes. Mechanically, LTB4/Ltb4r1 promoted lipogenesis by activating the cAMP-protein kinase A (PKA)-inositol-requiring enzyme 1α (IRE1α)-spliced X-box-binding protein 1 (XBP1s) axis in hepatocytes, which in turn promoted the expression of lipogenesis genes regulated by XBP1s. In addition, Ltb4r1 suppression through the Ltb4r1 inhibitor or lentivirus-short hairpin RNA delivery alleviated the fatty liver phenotype in obese mice. CONCLUSIONS: LTB4/Ltb4r1 promotes hepatocyte lipogenesis directly by activating PKA-IRE1α-XBP1s to promote lipogenic gene expression. Inhibition of hepatocyte Ltb4r1 improved hepatic steatosis and insulin resistance. Ltb4r1 is a potential therapeutic target for NAFLD.

BACKGROUND AND AIMS: Gain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs. APPROACH AND RESULTS: The requirement of YAP/TAZ in c-Met/β-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreERT2 KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo. Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/β-Catenin HCCs. YAP is the major Hippo effector in c-Met/β-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model. CONCLUSIONS: Our studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.

BACKGROUND AND AIMS: The NADPH oxidase NOX4 plays a tumor-suppressor function in HCC. Silencing NOX4 confers higher proliferative and migratory capacity to HCC cells and increases their in vivo tumorigenic potential in xenografts in mice. NOX4 gene deletions are frequent in HCC, correlating with higher tumor grade and worse recurrence-free and overall survival rates. However, despite the accumulating evidence of a protective regulatory role in HCC, the cellular processes governed by NOX4 are not yet understood. Accordingly, the aim of this work was to better understand the molecular mechanisms regulated by NOX4 in HCC in order to explain its tumor-suppressor action. APPROACH AND RESULTS: Experimental models: cell-based loss or gain of NOX4 function experiments, in vivo hepatocarcinogenesis induced by diethylnitrosamine in Nox4-deficient mice, and analyses in human HCC samples. Methods include cellular and molecular biology analyses, proteomics, transcriptomics, and metabolomics, as well as histological and immunohistochemical analyses in tissues. Results identified MYC as being negatively regulated by NOX4. MYC mediated mitochondrial dynamics and a transcriptional program leading to increased oxidative metabolism, enhanced use of both glucose and fatty acids, and an overall higher energetic capacity and ATP level. NOX4 deletion induced a redox imbalance that augmented nuclear factor erythroid 2-related factor 2 (Nrf2) activity and was responsible for MYC up-regulation. CONCLUSIONS: Loss of NOX4 in HCC tumor cells induces metabolic reprogramming in a Nrf2/MYC-dependent manner to promote HCC progression.

BACKGROUND AND AIMS: Recent studies suggest that mitochondrial dysfunction promotes progression to NASH by aggravating the gut-liver status. However, the underlying mechanism remains unclear. Herein, we hypothesized that enhanced mitochondrial activity might reshape a specific microbiota signature that, when transferred to germ-free (GF) mice, could delay NASH progression. APPROACH AND RESULTS: Wild-type and methylation-controlled J protein knockout (MCJ-KO) mice were fed for 6 weeks with either control or a choline-deficient, L-amino acid-defined, high-fat diet (CDA-HFD). One mouse of each group acted as a donor of cecal microbiota to GF mice, who also underwent the CDA-HFD model for 3 weeks. Hepatic injury, intestinal barrier, gut microbiome, and the associated fecal metabolome were then studied. Following 6 weeks of CDA-HFD, the absence of methylation-controlled J protein, an inhibitor of mitochondrial complex I activity, reduced hepatic injury and improved gut-liver axis in an aggressive NASH dietary model. This effect was transferred to GF mice through cecal microbiota transplantation. We suggest that the specific microbiota profile of MCJ-KO, characterized by an increase in the fecal relative abundance of Dorea and Oscillospira genera and a reduction in AF12, Allboaculum, and [Ruminococcus], exerted protective actions through enhancing short-chain fatty acids, nicotinamide adenine dinucleotide (NAD+ ) metabolism, and sirtuin activity, subsequently increasing fatty acid oxidation in GF mice. Importantly, we identified Dorea genus as one of the main modulators of this microbiota-dependent protective phenotype. CONCLUSIONS: Overall, we provide evidence for the relevance of mitochondria-microbiota interplay during NASH and that targeting it could be a valuable therapeutic approach.

BACKGROUND AND AIMS: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or perizygotic embryos. These approaches are time-consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a rapid in vivo multiplexed editing (RIME) method and provide proof of concept of this system. APPROACH AND RESULTS: RIME, a system wherein CRISPR/caspase 9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1 month from conceptualization to knockout, and highly efficient, enabling editing in >95% of target cells. To highlight its use, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation. CONCLUSIONS: RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo.

BACKGROUND AND AIMS: The etiology of liver diseases has changed in recent years, but its impact on the comparative burden of liver cancer between males and females is unclear. We estimated sex differences in the burden of liver cancer across 204 countries and territories from 2010 to 2019. APPROACH AND RESULTS: We analyzed temporal trends in the burden of liver cancer using the methodology framework of the 2019 Global Burden of Disease study. We estimated annual frequencies and age-standardized rates (ASRs) of liver cancer incidence, death, and disability-adjusted life-years (DALYs) by sex, country, region, and etiology of liver disease. Globally in 2019, the frequency of incident cases, deaths, and DALYs due to liver cancer were 376,483, 333,672, and 9,048,723 in males, versus 157,881, 150,904, and 3,479,699 in females. From 2010 to 2019, the incidence ASRs in males increased while death and DALY ASRs remained stable; incidence, death, and DALY ASRs in females decreased. Death ASRs for both sexes increased only in the Americas and remained stable or declined in remaining regions. In 2019, hepatitis B was the leading cause of liver cancer death in males, and hepatitis C in females. From 2010 to 2019, NASH had the fastest growing death ASRs in males and females. The ratio of female-to-male death ASRs in 2019 was lowest in hepatitis B (0.2) and highest in NASH (0.9). CONCLUSIONS: The overall burden of liver cancer is higher in males, although incidence and death ASRs from NASH-associated liver cancer in females approach that of males.

BACKGROUND AND AIMS: Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. APPROACH AND RESULTS: Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3-null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1-associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. CONCLUSIONS: Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

BACKGROUND AND AIMS: Janus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo. APPROACH AND RESULTS: Transcriptome analyses of JAK2 in human livers and other targets of pacritinib have been shown to correlate with profibrotic factors. Although transcription of JAK2 correlated significantly with type I collagen expression and other profibrotic genes, no correlation was observed for interleukin-1 receptor-associated kinase and colony-stimulating factor 1 receptor. Pacritinib decreased gene expression of fibrosis markers in mouse primary and human-derived HSCs in vitro. Moreover, pacritinib decreased the proliferation, contraction, and migration of HSCs. C57 BL/6J mice received ethanol in drinking water (16%) or Western diet in combination with carbon tetrachloride intoxication for 7 weeks to induce alcoholic or nonalcoholic fatty liver disease. Pacritinib significantly reduced liver fibrosis assessed by gene expression and Sirius red staining, as well as HSC activation assessed by alpha-smooth muscle actin immunostaining in fibrotic mice. Furthermore, pacritinib decreased the gene expression of hepatic steatosis markers in experimental alcoholic liver disease. Additionally, pacritinib protected against liver injury as assessed by aminotransferase levels. CONCLUSIONS: This study demonstrates that the JAK2 inhibitor pacritinib may be promising for the treatment of alcoholic and nonalcoholic liver fibrosis and may be therefore relevant for human pathology.

BACKGROUND AND AIMS: BFKB8488A is a bispecific antibody targeting fibroblast growth factor receptor 1c and Klothoβ. This phase 1b study assessed safety, tolerability, pharmacokinetics, immunogenicity, and pharmacodynamics of BFKB8488A in patients with type 2 diabetes mellitus (T2DM) or NAFLD. APPROACH AND RESULTS: Patients were randomized to receive multiple doses of BFKB8488A at various dose levels and dosing intervals (weekly, every 2 weeks, or every 4 weeks) or placebo for 12 weeks. The primary outcome was the safety of BFKB8488A. Overall, 153 patients (T2DM: 91; NAFLD: 62) were enrolled and received at least one dose of treatment. Of these, 102 patients (62.7%) reported at least one adverse event (BFKB8488A: 83 [68.6%]; placebo: 19 [59.4%]). BFKB8488A exhibited nonlinear pharmacokinetics, with greater than dose-proportional increases in exposure. The treatment-emergent antidrug antibody incidence was 22.7%. Overall, trends in exposure-dependent increases in high-density lipoprotein (HDL) and decreases in triglyceride levels were observed. Decreases in alanine aminotransferase and aspartate aminotransferase were 0.7% and 9.2% for medium exposure and 7.3% and 11.2% for high-exposure tertiles, compared with increases of 7.5% and 17% in the placebo group, respectively, at Day 85. In patients with NAFLD, the mean decrease from baseline liver fat was 13.0%, 34.5%, and 49.0% in the low-, medium-, and high-exposure tertiles, respectively, compared with 0.1% with placebo at Day 85. CONCLUSIONS: BFKB8488A was adequately tolerated in patients with T2DM or NAFLD, leading to triglyceride reduction, HDL improvements, and trends in improvement in markers of liver health for both populations and marked liver fat reduction in patients with NAFLD. (ClinicalTrials.gov: NCT03060538).

The syndemic of hazardous alcohol consumption, opioid use, and obesity has led to important changes in liver disease epidemiology that have exacerbated health disparities. Health disparities occur when plausibly avoidable health differences are experienced by socially disadvantaged populations. Highlighting health disparities, their sources, and consequences in chronic liver disease is fundamental to improving liver health outcomes. There have been large increases in alcohol use disorder in women, racial and ethnic minorities, and those experiencing poverty in the context of poor access to alcohol treatment, leading to increasing rates of alcohol-associated liver diseases. Rising rates of NAFLD and associated fibrosis have been observed in Hispanic persons, women aged > 50, and individuals experiencing food insecurity. Access to viral hepatitis screening and linkage to treatment are suboptimal for racial and ethnic minorities and individuals who are uninsured or underinsured, resulting in greater liver-related mortality and later-stage diagnoses of HCC. Data from more diverse cohorts on autoimmune and cholestatic liver diseases are lacking, supporting the need to study the contemporary epidemiology of these disorders in greater detail. Herein, we review the existing literature on racial and ethnic, gender, and socioeconomic disparities in chronic liver diseases using a social determinants of health framework to better understand how social and structural factors cause health disparities and affect chronic liver disease outcomes. We also propose potential solutions to eliminate disparities, outlining health-policy, health-system, community, and individual solutions to promote equity and improve health outcomes.

The liver is the sixth most common site of primary cancer in humans and the fourth leading cause of cancer-related death in the world. Hepatocellular carcinoma (HCC) accounts for 90% of liver cancers. HCC is a prevalent disease with a progression that is modulated by the immune system. Half of the patients with HCC receive systemic therapies, traditionally sorafenib or lenvatinib, as a first-line therapy. In the last few years, immune-checkpoint inhibitors (ICIs) have revolutionized cancer therapy and have gained an increased interest in the treatment of HCC. In 2020, the combination of atezolizumab (anti-programmed death-ligand 1) and bevacizumab (anti-vascular endothelial growth factor) improved overall survival over sorafenib, resulting in Food and Drug Administration (FDA) approval as a first-line treatment for patients with advanced HCC. Despite these major advances, a better molecular and cellular characterization of the tumor microenvironment is still needed because it has a crucial role in the development and progression of HCC. Inflamed (hot) and noninflamed (cold) HCC tumors and genomic signatures have been associated with response to ICIs. However, there are no additional biomarkers to guide clinical decision-making. Other immune-targeting strategies, such as adoptive T-cell transfer, vaccination, and virotherapy, are currently under development. This review provides an overview on the HCC immune microenvironment, different cellular players, current available immunotherapies, and potential immunotherapy modalities.

As the third most common cause of cancer-related death worldwide with significant mortality rates in the United States, hepatocellular carcinoma has strong association with cirrhosis and chronic hepatitis B virus (HBV) with a growing at-risk population from the rise in chronic liver disease from alcohol use and nonalcoholic fatty liver disease. Despite this, progress in identifying at-risk individuals and early detection of HCC in these populations have lagged behind treatment advances.The lack of consensus may undermine widespread adoption of surveillance programs, thus preventing HCC detection at a curable stage. This public policy corner piece focuses on opportunities for prevention of HCC by focusing on its principal risk factors: viral hepatitis, NAFLD, and alcohol-related liver disease, and three key action points to reverse the course of this public health crisis: 1) Awareness and education; 2) Screening and diagnosis, and 3) Partnerships and advocacy.

BACKGROUND AND AIMS: Increasing evidence suggests that mesenchymal stem cells (MSCs) home to injured local tissues and the tumor microenvironment in the liver. Chronic inflammation is regarded as the major trait of primary liver cancer. However, the characteristics of endogenous MSCs in the inflammatory environment and their role in the occurrence of liver cancer remain obscure. APPROACH AND RESULTS: Using single-cell RNA sequencing, we identified a distinct inflammation-associated subset of MSCs, namely AIF1+ CSF1R+ MSCs, which existed in the microenvironment before the occurrence of liver cancer. Furthermore, we found that this MSC subgroup is likely to be induced by TNF-α stimulation through the TNFR1/SIRT1 (sirtuin 1) pathway. In a rat primary liver cancer model, we showed that MSCs with high SIRT1 expression (Ad-Sirt1-MSCs) promoted macrophage recruitment and synergistically facilitated liver cancer occurrence by secreting C-C motif chemokine ligand (CCL) 5. Interestingly, depletion of macrophages or knockdown of CCL5 expression in Ad-Sirt1-MSCs attenuated the promotive effect of Ad-Sirt1-MSCs on liver inflammation and hepatocarcinogenesis (HCG). Finally, we demonstrated that SIRT1 up-regulated CCL5 expression through activation of the AKT/HIF1α signaling axis in MSCs. CONCLUSIONS: Together, our results show that MSCs, which are mobilized to the injured site, can be educated by macrophages. In turn, the educated MSCs are involved in generating a chronic inflammatory microenvironment and promoting HCG.

BACKGROUND AND AIMS: The American Gastroenterological Association (AGA) recently launched the Clinical Care Pathway for the Risk Stratification and Management of Patients with NAFLD to identify adults with significant fibrosis. We aimed to examine this pathway's performance in the US population. APPROACH AND RESULTS: Using the 2017-2018 National Health and Nutrition Examination Survey data, we identified participants aged ≥18 with available Fibrosis-4 (FIB-4) score and liver stiffness measurement (LSM) in the absence of other liver diseases. Based on the AGA clinical pathway, FIB-4 < 1.3 and LSM < 8 kilopascals (kPa) by vibration-controlled transient elastography (VCTE) are associated with low risk of significant fibrosis. Using these cutoffs, we examined the pathway performance using negative predictive value (NPV) and positive predictive value (PPV) and explored alternative risk-stratification strategies. There were 2322 participants with available data (projected to 94.2 million US adults). The NPV of LSM ≥ 8 kPa among those with FIB-4 < 1.3 was 90%, whereas the PPV among those with FIB-4 1.3-2.67 was 13%. As diabetes was a strong predictor of fibrosis, we propose a simple, alternative strategy to eliminate the indeterminate FIB-4 range and perform VCTE in those with FIB-4 ≥ 1.3 and diabetes. This strategy would decrease the number of VCTEs from 14.5 to 4.9 million and increase PPV from 13% to 33% without compromising the NPV among those who did not undergo VCTE. CONCLUSION: The implementation of the current AGA clinical pathway would lead to overutilization of VCTE. An alternative strategy using FIB-4 ≥ 1.3 and diabetes to select adults undergoing second-line testing will improve this pathway's performance and minimize unnecessary VCTEs.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

BACKGROUND AND AIMS: Early identification of modifiable risk factors is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). We aimed to systematically explore the relationships between genetically predicted modifiable risk factors and NAFLD. APPROACH AND RESULTS: We applied univariable and multivariable Mendelian randomization analyses to explore the relationships between 35 modifiable risk factors and NAFLD. We also evaluated the combined results in three independent large genome-wide association studies. Genetically predicted alcohol frequency, elevated serum levels of liver enzymes, triglycerides, C-reactive protein, and obesity traits, including body mass index, waist circumference, and body fat mass, were associated with increased risks of NAFLD (all with p < 0.05). Poor physical condition had a suggestive increased risk for NAFLD (odds ratio [OR] = 2.63, p = 0.042). Genetically instrumented type 2 diabetes (T2DM), hypothyroidism, and hypertension all increased the risk for NAFLD, and the ORs (95% confidence interval) were 1.508 (1.20-1.90), 13.08 (1.53-111.65), and 3.11 (1.33-7.31) for a 1-U increase in log-transformed odds, respectively. The positive associations of T2DM and hypertension with NAFLD remained significant in multivariable analyses. The combined results from the discovery and two replication datasets further confirmed that alcohol frequency, elevated serum liver enzymes, poor physical condition, obesity traits, T2DM, and hypertension significantly increase the risk of NAFLD, whereas higher education and high-density lipoprotein cholesterol (HDL-cholesterol) could lower NAFLD risk. CONCLUSIONS: Genetically predicted alcohol frequency, elevated serum liver enzymes, poor physical condition, obesity traits, T2DM, and hypertension were associated with an increased risk of NAFLD, whereas higher education and HDL-cholesterol were associated with a decreased risk of NAFLD.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection causes oxidative stress (OS) and alters mitochondria in experimental models. Our goal was to investigate whether HBV might alter liver mitochondria also in humans, and the resulting mitochondrial stress might account for the progression of fibrosis in chronic hepatitis B (CHB). APPROACH AND RESULTS: The study included 146 treatment-naïve CHB mono-infected patients. Patients with CHB and advanced fibrosis (AF) or cirrhosis (F3-F4) were compared to patients with no/mild-moderate fibrosis (F0-F2). Patients with CHB were further compared to patients with chronic hepatitis C (CHC; n = 33), nonalcoholic steatohepatatis (NASH; n = 12), and healthy controls (n = 24). We detected oxidative damage to mitochondrial DNA (mtDNA), including mtDNA strand beaks, and identified multiple mtDNA deletions in patients with F3-F4 as compared to patients with F0-F2. Alterations in mitochondrial function, mitochondrial unfolded protein response, biogenesis, mitophagy, and liver inflammation were observed in patients with AF or cirrhosis associated with CHB, CHC, and NASH. In vitro, significant increases of the mitochondrial formation of superoxide and peroxynitrite as well as mtDNA damage, nitration of the mitochondrial respiratory chain complexes, and impairment of complex I occurred in HepG2 cells replicating HBV or transiently expressing hepatitits B virus X protein. mtDNA damage and complex I impairment were prevented with the superoxide-scavenging Mito-Tempo or with inducible nitric oxide synthase (iNOS)-specific inhibitor 1400 W. CONCLUSIONS: Our results emphasized the importance of mitochondrial OS, mtDNA damage, and associated alterations in mitochondrial function and dynamics in AF or cirrhosis in CHB and NASH. Mitochondria might be a target in drug development to stop fibrosis progression.

Chronic hepatitis D infection results in the most severe form of chronic viral hepatitis but currently lacks effective treatment options. Therapy with pegylated interferon alpha is recommended for finite treatment duration by major liver societies. Still, it is plagued by low rates of sustained virologic response (SVR) and frequent relapses even if SVR is achieved. Recently, a wave of investigational therapies has come under evaluation, including bulevirtide, lonafarnib, pegylated interferon lambda, and REP-2139 creating excitement with this viral infection. However, there has been significant variability in the endpoints used to evaluate these therapeutics. One of the recently introduced endpoints is characterized by a decline in HDV RNA by 2 logs, with or without achieving an undetectable serum hepatitis D virus (HDV) RNA, as a marker of virologic response. Furthermore, this measure has been combined with alanine aminotransferase normalization, also known as a biochemical response, to formulate the primary endpoint of several late-stage studies. Per recent guidance by the US Food and Drug Administration, these should be surrogate endpoints that will ultimately portend long-term clinical benefits. These clinical benefits may include reducing the risk of progression to cirrhosis, hepatic decompensation, hepatocellular carcinoma, liver transplantation, and mortality. However, the optimal way to measure success in HDV clinical trials remains unknown and will continue to evolve.

BACKGROUND AND AIMS: The mammalian liver harbors heterogeneous cell types that communicate via local paracrine signaling. Recent studies have delineated the transcriptomic landscape of the liver in NASH that provides insights into liver cell heterogeneity, intercellular crosstalk, and disease-associated reprogramming. However, the nature of intrahepatic signaling and its role in NASH progression remain obscure. APPROACH AND RESULTS: Here, we performed transcriptomic analyses and identified cardiotrophin-like cytokine factor 1 (CLCF1), a member of the IL-6 family cytokines, as a cholangiocyte-derived paracrine factor that was elevated in the liver from diet-induced NASH mice and patients with NASH. Adenovirus-associated virus-mediated overexpression of CLCF1 in the liver ameliorated NASH pathologies in two diet-induced NASH models in mice, illustrating that CLCF1 induction may serve an adaptive and protective role during NASH pathogenesis. Unexpectedly, messenger RNA and protein levels of leukemia inhibitory factor receptor (LIFR), a subunit of the receptor complex for CLCF1, were markedly downregulated in NASH liver. Hepatocyte-specific inactivation of LIFR accelerated NASH progression in mice, supporting an important role of intrahepatic cytokine signaling in maintaining tissue homeostasis under metabolic stress conditions. CONCLUSIONS: Together, this study sheds light on the molecular nature of intrahepatic paracrine signaling during NASH pathogenesis and uncovers potential targets for therapeutic intervention.