An atlas of the human liver diurnal transcriptome and its perturbation by hepatitis C virus infection.

Chronic liver disease and cancer are global health challenges. The role of the circadian clock as a regulator of liver physiology and disease is well established in rodents, however, the identity and epigenetic regulation of rhythmically expressed genes in human disease is less well studied. Here we unravel the rhythmic transcriptome and epigenome of human hepatocytes using male human liver chimeric mice. We identify a large number of rhythmically expressed protein coding genes in human hepatocytes of male chimeric mice, which includes key transcription factors, chromatin modifiers, and critical enzymes. We show that hepatitis C virus (HCV) infection, a major cause of liver disease and cancer, perturbs the transcriptome by altering the rhythmicity of the expression of more than 1000 genes, and affects the epigenome, leading to an activation of critical pathways mediating metabolic alterations, fibrosis, and cancer. HCV-perturbed rhythmic pathways remain dysregulated in patients with advanced liver disease. Collectively, these data support a role for virus-induced perturbation of the hepatic rhythmic transcriptome and pathways in cancer development and may provide opportunities for cancer prevention and biomarkers to predict HCC risk.

Targeting the liver clock improves fibrosis by restoring TGF-ß signaling.

BACKGROUND & AIMS: Liver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC-oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis - a rising global unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

Prognostic benefit of preoperative transarterial chemoembolization in upfront resectable large hepatocellular carcinoma: a multicentric propensity score based analysis of European high-volume centers.

BACKGROUND: Hepatocellular carcinoma (HCC) have a dismal prognosis and any effective neoadjuvant treatment has been validated to date. We aimed to investigate the role of neoadjuvant transarterial chemoembolization (TACE) in upfront resectable HCC larger than 5 cm. METHODS: This is a multicentric retrospective study comparing outcomes of large HCC undergoing TACE followed by surgery or liver resection alone before and after propensity-score matching (PSM). RESULTS: A total of 384 patients were included of whom 60 (15.6%) received TACE. This group did not differ from upfront resected cases neither in terms of disease-free survival (p = 0.246) nor in overall survival (p = 0.276). After PSM, TACE still did not influence long-term outcomes (p = 0.935 and p = 0.172, for DFS and OS respectively). In subgroup analysis, TACE improved OS only in HCC ≥10 cm (p = 0.045), with a borderline significance after portal vein embolization/ligation (p = 0.087) and in single HCC (p = 0.052). CONCLUSIONS: TACE should not be systematically performed in all resectable large HCC. Selected cases could however potentially benefit from this procedure, as patients with huge and single tumors or those necessitating of a PVE.

JAK1 promotes HDV replication and is a potential target for antiviral therapy.

BACKGROUND & AIMS: Chronic co-infection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. To date, no treatment induces efficient viral clearance, and a better characterization of virus-host interactions is required to develop new therapeutic strategies. METHODS: Using loss-of-function strategies, we validated the unexpected proviral activity of Janus kinase 1 (JAK1) - a key player in innate immunity - in the HDV life cycle and determined its mechanism of action on HDV through various functional analyses including co-immunoprecipitation assays. RESULTS: We confirmed the key role of JAK1 kinase activity in HDV infection. Moreover, our results suggest that JAK1 inhibition is associated with a modulation of ERK1/2 activation and S-HDAg phosphorylation, which is crucial for viral replication. Finally, we showed that FDA-approved JAK1-specific inhibitors are efficient antivirals in relevant in vitro models including primary human hepatocytes. CONCLUSIONS: Taken together, we uncovered JAK1 as a key host factor for HDV replication and a potential target for new antiviral treatment. IMPACT AND IMPLICATIONS: Chronic hepatitis D is the most aggressive form of chronic viral hepatitis. As no curative treatment is currently available, new therapeutic strategies based on host-targeting agents are urgently needed. Here, using loss-of-function strategies, we uncover an unexpected interaction between JAK1, a major player in the innate antiviral response, and HDV infection. We demonstrated that JAK1 kinase activity is crucial for both the phosphorylation of the delta antigen and the replication of the virus. By demonstrating the antiviral potential of several FDA-approved JAK1 inhibitors, our results could pave the way for the development of innovative therapeutic strategies to tackle this global health threat.

Inflammation-related prognostic markers in resected hepatocellular carcinoma.

Hepatocellular carcinoma is usually detected late and therapeutic options are unsatisfactory. Despite marked progress in patient care, HCC remains among the deadliest cancers world-wide. While surgical resection remains a key option for early-stage HCC, the 5-year survival rates after surgical resection are limited. One reason for limited outcomes is the lack of reliable prognostic biomarkers to predict HCC recurrence. HCC prognosis has been shown to correlate with different systemic and pathological markers which are associated with patient survival and HCC recurrence. Liver inflammatory processes offer a large variety of systemic and pathological markers which may be exploited to improve the reliability of prognosis and decision making of liver surgeons and hepatologists. The following review aims to dissect the potential tools, targets and prognostic meaning of inflammatory markers in patients with resectable HCC. We analyze changes in circulant cellular populations and assess inflammatory biomarkers as a surrogate of impaired outcomes and provide an overview on predictive gene expression signatures including inflammatory transcriptional patterns, which are representative of poor survival in these patients.

Combining Radiofrequency Ablation with Hepatic Resection for Liver-Only Colorectal Metastases: A Propensity-Score Based Analysis of Long-Term Outcomes.

BACKGROUND: Combining liver resection (LR) with radiofrequency ablation (RFA) is nowadays an accepted option for treating colorectal liver metastases (CRLMs), but the number of lesions ablated is regularly described as a recurrence risk factor. In this study, we report our experience and determine the impact of RFA on long-term outcomes. METHOD: This is a retrospective study including patients undergoing LR with or without RFA for CRLM. All variables influencing disease-free survival (DFS) and disease-specific survival (DSS) were examined through a Cox regression analysis before and after propensity-score matching (PSM). RESULTS: Among the 128 patients included, 71 (55.5%) underwent LR alone and 57 (44.5%) underwent LR+RFA. With univariate analysis, LR+RFA showed a significantly worse DFS than LR alone (p = 0.028), which was not confirmed after PSM (p = 0.064). Thermal ablation did not influence DSS before or after matching (p = 0.282 and p = 0.189). When analyzing the subgroups of patients according to number of RFAs performed, no difference in long-term outcomes was observed (after PSM: p = 0.192 for DFS and p = 0.624 for DSS). Analysis of site of recurrence revealed that neither performing an RFA (p = 0.893) nor the number of lesions ablated (p = 0.093, p = 0.550, and p = 0.087 for 1, 2, and ≥ 2 RFAs) were associated with an increased risk of liver-only relapse. DISCUSSION: In the setting of a parenchymal sparing strategy, combining RFA with LR is safe in terms of oncological outcomes. Tumor burden, rather than RFA performed, independently influences risk of recurrence and patient survival.

Paradigm shift: should preoperative 3D reconstruction models become mandatory before hepatectomy for hepatocellular carcinoma (HCC)? Results of a multicenter prospective trial.

BACKGROUND: A preoperative surgical strategy before hepatectomy for hepatocellular carcinoma is fundamental to minimize postoperative morbidity and mortality and to reach the best oncologic outcomes. Preoperative 3D reconstruction models may help to better choose the type of procedure to perform and possibly change the initially established plan based on conventional 2D imaging. METHODS: A non-randomized multicenter prospective trial with 136 patients presenting with a resectable hepatocellular carcinoma who underwent open or minimally invasive liver resection. Measurement was based on the modification rate analysis between conventional 2D imaging (named "Plan A") and 3D model analysis ("Plan B"), and from Plan B to the final procedure performed (named "Plan C"). RESULTS: The modification rate from Plan B to Plan C (18%) was less frequent than the modification from Plan A to Plan B (35%) (OR = 0.32 [0.15; 0.64]). Concerning secondary objectives, resection margins were underestimated in Plan B as compared to Plan C (-3.10 mm [-5.04; -1.15]). CONCLUSION: Preoperative 3D imaging is associated with a better prediction of the performed surgical procedure for liver resections in HCC, as compared to classical 2D imaging.

Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

A stable hepatitis D virus-producing cell line for host target and drug discovery.

Chronic hepatitis D is the most aggressive form of chronic viral hepatitis. It is caused by super-infection of hepatitis B virus (HBV)-infected hepatocytes with hepatitis D virus (HDV). While the recent conditional approval of bulevirtide for HDV treatment offers a new therapeutic modality in Europe, there is an unmet medical need to further improve therapy. A more detailed characterization of virus-host interactions is needed for the identification of novel therapeutic targets. Addressing this need, we engineered a new stably-transformed cell line, named HuH7-2C8D, producing high titer recombinant HDV and allowing the study of viral particles morphogenesis and infectivity. Using this culture system, where viral propagation by re-infection is limited, we observed an increased accumulation of edited version of the viral genomes within secreted HDV viral particles over time that is accompanied with a decrease in viral particle infectivity. We confirmed the interaction of HDV proteins with a previously described host factor in HuH7-2C8D cells and additionally showed that these cells are suitable for co-culture assays with other cell types such as macrophages. Finally, the use of HuH7-2C8D cells allowed to confirm the dual antiviral activity of farnesyl transferase inhibitors, including the clinical candidate lonafarnib, against HDV. In conclusion, we have established an easy-to-handle cell culture model to investigate HDV replication, morphogenesis, and host interactions. HuH7-2C8D cells are also suitable for high-throughput antiviral screening assays for the development of new therapeutic strategies.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

Hyperspectral Imaging in Major Hepatectomies: Preliminary Results from the Ex-Machyna Trial.

Ischemia-reperfusion injury during major hepatic resections is associated with high rates of post-operative complications and liver failure. Real-time intra-operative detection of liver dysfunction could provide great insight into clinical outcomes. In the present study, we demonstrate the intra-operative application of a novel optical technology, hyperspectral imaging (HSI), to predict short-term post-operative outcomes after major hepatectomy. We considered fifteen consecutive patients undergoing major hepatic resection for malignant liver lesions from January 2020 to June 2021. HSI measures included tissue water index (TWI), organ hemoglobin index (OHI), tissue oxygenation (StO2%), and near infrared (NIR). Pre-operative, intra-operative, and post-operative serum and clinical outcomes were collected. NIR values were higher in unhealthy liver tissue (p = 0.003). StO2% negatively correlated with post-operative serum ALT values (r = -0.602), while ΔStO2% positively correlated with ALP (r = 0.594). TWI significantly correlated with post-operative reintervention and OHI with post-operative sepsis and liver failure. In conclusion, the HSI imaging system is accurate and precise in translating from pre-clinical to human studies in this first clinical trial. HSI indices are related to serum and outcome metrics. Further experimental and clinical studies are necessary to determine clinical value of this technology.

Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

Safety and Antiviral Activity of EGFR Inhibition by Erlotinib in Chronic Hepatitis C Patients: A Phase Ib Randomized Controlled Trial.

INTRODUCTION: Significant hepatocellular carcinoma (HCC) risk persists after chronic hepatitis C (CHC) cure. Preclinical studies have shown that erlotinib, an oral epidermal growth factor receptor (EGFR) inhibitor, has an antiviral activity and HCC chemopreventive effect. Erlotinib is metabolized in the liver, and its safety in patients with CHC is unknown. This study aimed to assess the safety and antiviral activity of erlotinib in patients with CHC. METHODS: In this investigator-initiated dose-escalation phase Ib prospective randomized double-blind placebo-controlled study, noncirrhotic hepatitis C virus (HCV) patients received placebo or erlotinib (50 or 100 mg/d) for 14 days with a placebo-erlotinib ratio of 1:3. Primary end points were safety and viral load reduction at the end of treatment (EOT). The secondary end point was viral load reduction 14 days after EOT. RESULTS: This study analyzed data of 3 patients receiving placebo, 3 patients receiving erlotinib 50 mg/d, and 3 patients receiving erlotinib 100 mg/d. One grade 3 adverse event was reported in the placebo group (liver enzymes elevation), leading to treatment discontinuation and patient replacement, and 1 in the erlotinib 100 mg/d group (pericarditis), which was not considered to be treatment-related. Grade 2 skin rash was observed in 1 erlotinib 100 mg/d patient. No significant HCV-RNA level reduction was noted during treatment, but 2 of the 3 patients in the erlotinib 100 mg/d group showed a decrease of >0.5 log HCV-RNA 14 days after EOT. DISCUSSION: Erlotinib demonstrated to be safe in noncirrhotic CHC patients. An antiviral activity at 100 mg/d confirms a functional role of EGFR as an HCV host factor in patients. These results provide perspectives to further study erlotinib as an HCC chemopreventive agent in patients with CHC.

Cell-based cccDNA reporter assay combined with functional genomics identifies YBX1 as HBV cccDNA host factor and antiviral candidate target.

OBJECTIVES: Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation. One major challenge to address this urgent question is the absence of robust models for the study of cccDNA biology. DESIGN: We established a cell-based HBV cccDNA reporter assay and performed a loss-of-function screen targeting 239 genes encoding the human DNA damage response machinery. RESULTS: Overcoming the limitations of current models, the reporter assay enables to quantity cccDNA levels using a robust ELISA as a readout. A loss-of-function screen identified 27 candidate cccDNA host factors, including Y box binding protein 1 (YBX1), a DNA binding protein regulating transcription and translation. Validation studies in authentic infection models revealed a robust decrease in HBV cccDNA levels following silencing, providing proof-of-concept for the importance of YBX1 in the early steps of the HBV life cycle. In patients, YBX1 expression robustly correlates with both HBV load and liver disease progression. CONCLUSION: Our cell-based reporter assay enables the discovery of HBV cccDNA host factors including YBX1 and is suitable for the characterisation of cccDNA-related host factors, antiviral targets and compounds.

Automatic Liver Viability Scoring with Deep Learning and Hyperspectral Imaging.

Hyperspectral imaging (HSI) is a non-invasive imaging modality already applied to evaluate hepatic oxygenation and to discriminate different models of hepatic ischemia. Nevertheless, the ability of HSI to detect and predict the reperfusion damage intraoperatively was not yet assessed. Hypoxia caused by hepatic artery occlusion (HAO) in the liver brings about dreadful vascular complications known as ischemia-reperfusion injury (IRI). Here, we show the evaluation of liver viability in an HAO model with an artificial intelligence-based analysis of HSI. We have combined the potential of HSI to extract quantitative optical tissue properties with a deep learning-based model using convolutional neural networks. The artificial intelligence (AI) score of liver viability showed a significant correlation with capillary lactate from the liver surface (r = -0.78, p = 0.0320) and Suzuki's score (r = -0.96, p = 0.0012). CD31 immunostaining confirmed the microvascular damage accordingly with the AI score. Our results ultimately show the potential of an HSI-AI-based analysis to predict liver viability, thereby prompting for intraoperative tool development to explore its application in a clinical setting.

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells.

The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.