Hepatocellular carcinoma (HCC) is the 6th-most common and the 4th-most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening newly identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report, for the first time, the effectiveness of omacetaxine in HCC. In addition, we newly elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.
Ling Li, Gilad Halpert, Michael G. Lerner, Haijie Hu, Peter Dimitrion, Matthew J. Weiss, Jin He, Benjamin Philosophe, Richard Burkhart, William R. Burns, Russell N. Wesson, Andrew MacGregor Cameron, Christopher L. Wolfgang, Christos Georgiades, Satomi Kawamoto, Nilofer S. Azad, Mark Yarchoan, Stephen J. Meltzer, Kiyoko Oshima, Laura M. Ensign, Joel S. Bader, Florin M. Selaru
BACKGROUND. The chemokine system of ligands and receptors is implicated in the progression of Alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. MOTHODS. The coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in two distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific knockouts were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. RESULTS. Selected C-X-C chemokine members of the Interleukin-8 (IL-8) chemokine family and C-C chemokine CCl20 were highly associated with AH compared to HC, but not in patients with liver diseases of other etiologies (NAFLD or HCV). Our previous studies implicate Macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of wild-type mice; this was prevented in hepatocyte-specific Mif knockout (MifΔHep) mice. CONCLUSIONS. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and hepatocyte-derived MIF might drive this inflammatory response.
Kyle L. Poulsen, Xiude D. Fan, Christopher D. Kibler, Emily Huang, Xiaoqin Wu, Megan R. McMullen, Lin Leng, Richard Bucala, Meritxell Ventura-Cots, Josepmaria Argemi, Ramon Bataller, Laura E. Nagy
Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. Here we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require nicotinamide adenine dinucleotide (NAD) as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here we show that despite their NAD-dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating wild type or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Thus, we provide the first evidence for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.
Sarmistha Mukherjee, James Mo, Lauren M. Paolella, Caroline E. Perry, Jade Toth, Mindy M. Hugo, Qingwei Chu, Qiang Tong, Karthikeyani Chellappa, Joseph A. Baur
Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral and structural markers in liver biopsies from patients with HBeAg+ chronic hepatitis B, we provide novel comprehensive visualization, quantitation and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.
Daniel Traum, Yue J. Wang, Kathleen B. Schwarz, Jonathan Schug, David K.H. Wong, Harry L.A. Janssen, Norah A. Terrault, Mandana Khalili, Abdus S. Wahed, Karen F. Murray, Philip Rosenthal, Simon C. Ling, Norberto Rodriguez-Baez, Richard K. Sterling, Daryl T.Y. Lau, Timothy M. Block, Michael D. Feldman, Emma E. Furth, William M. Lee, David E. Kleiner, Anna S. Lok, Klaus H. Kaestner, Kyong-Mi Chang
Hepatocellular death contributes to progression of alcohol–associated (ALD-associated) and non–alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain–like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl–/– mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.
Tatsunori Miyata, Xiaoqin Wu, Xiude Fan, Emily Huang, Carlos Sanz-Garcia, Christina K. Cajigas-Du Ross, Sanjoy Roychowdhury, Annette Bellar, Megan R. McMullen, Jaividhya Dasarathy, Daniela S. Allende, Joan Caballeria, Pau Sancho-Bru, Craig J. McClain, Mack Mitchell, Arthur J. McCullough, Svetlana Radaeva, Bruce Barton, Gyongyi Szabo, Srinivasan Dasarathy, Laura E. Nagy
The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II-restricted model peptide in hepatocytes by autoreactive CD4 T cells. We find that the hepatic peptide was not expressed in the thymus leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4 T cells. In the liver, autoreactive CD4 effector T cells accumulated within portal ectopic lymphoid structures and maturated towards pathogenic IFNγ and TNF co-producing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs, but not of non-specific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared to non-specific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.
Max Preti, Lena Schlott, David Lübbering, Daria Krzikalla, Anna-Lena Müller, Fenja A. Schuran, Tobias Poch, Miriam Schakat, Sören Weidemann, Ansgar W. Lohse, Christina Weiler-Normann, Marcial Sebode, Dorothee Schwinge, Christoph Schramm, Antonella Carambia, Johannes Herkel
Activation of Farnesoid-X-Receptor (FXR) by obeticholic acid (OCA) reduces hepatic inflammation and fibrosis in patients with primary biliary cholangitis (PBC), a life-threatening cholestatic liver failure. Inhibition of bromodomain-containing protein-4 (BRD4) also has anti-inflammatory, anti-fibrotic effects in mice. We determined the role of BRD4 in FXR function in bile acid (BA) regulation and examined whether the known beneficial effects of OCA are enhanced by inhibiting BRD4 in cholestatic mice. Liver-specific downregulation of BRD4 disrupted BA homeostasis in mice, and FXR-mediated regulation of BA-related genes, including Shp and Cyp7a1, was BRD4-dependent. In cholestatic mice, JQ1 or OCA treatment ameliorated hepatotoxicity, inflammation, and fibrosis, but surprisingly, was antagonistic in combination. Mechanistically, OCA increased binding of FXR and the corepressor SMRT, decreased NF-κB binding at inflammatory genes, and repressed the genes in a BRD4-dependent manner. In PBC patients, hepatic expression of FXR and BRD4 was significantly reduced. In conclusion, BRD4 is a novel cofactor of FXR for maintaining BA homeostasis and hepatoprotection. While BRD4 promotes hepatic inflammation and fibrosis in cholestasis, paradoxically, BRD4 is required for the anti-inflammatory, anti-fibrotic actions of OCA-activated FXR. Co-treatment with OCA and JQ1, individually beneficial, may be antagonistic in treatment of liver disease patients with inflammation and fibrosis complications.
Hyunkyung Jung, Jinjing Chen, Xiangming Hu, Hao Sun, Shwu-Yuan Wu, Cheng-Ming Chiang, Byron Kemper, Lin-Feng Chen, Jongsook Kemper
Adult liver has enormous regenerative capacity; it can regenerate after losing two-thirds of its mass while sustaining essential metabolic functions. How the liver balances dual demands for increased proliferative activity with maintenance of organ function is unknown but essential to prevent liver failure. Using partial hepatectomy (PHx) in mice to model liver regeneration, we integrated single-cell RNA- and ATAC-Seq to map state transitions in approximately 13,000 hepatocytes at single-cell resolution as livers regenerated, and validated key findings with IHC, to uncover how the organ regenerates hepatocytes while simultaneously fulfilling its vital tissue-specific functions. After PHx, hepatocytes rapidly and transiently diversified into multiple distinct populations with distinct functional bifurcation: some retained the chromatin landscapes and transcriptomes of hepatocytes in undamaged adult livers, whereas others transitioned to acquire chromatin landscapes and transcriptomes of fetal hepatocytes. Injury-related signaling pathways known to be critical for regeneration were activated in transitioning hepatocytes, and the most fetal-like hepatocytes exhibited chromatin landscapes that were enriched with transcription factors regulated by those pathways.
Tianyi Chen, Sehhoon Oh, Simon Gregory, Xiling Shen, Anna Mae Diehl
Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation. The transmembrane 6 superfamily member 2 (TM6SF2) E167K genetic variant associates with NAFLD and with reduced plasma triglyceride levels in humans. However, the molecular mechanisms underlying these associations remain unclear. We hypothesized that TM6SF2 E167K affects hepatic very low-density lipoprotein (VLDL) secretion, and studied the kinetics of apolipoprotein B100 (apoB100) and triglyceride metabolism in VLDL in homozygous subjects. In 10 homozygote TM6SF2 E167K carriers and 10 matched controls, we employed stable-isotope tracer and compartmental modeling techniques to determine apoB100 and triglyceride kinetics in the two major VLDL subfractions: large triglyceride-rich VLDL1 and smaller, less triglyceride-rich VLDL2. VLDL1-apoB100 production was markedly reduced in homozygote TM6SF2 E167K carriers compared to controls. Likewise, VLDL1-triglyceride production was 35% lower in the TM6SF2 E167K carriers. In contrast, the direct production rates for VLDL2-apoB100 and triglyceride were not different between carriers and controls. In conclusion, the TM6SF2 E167K genetic variant was linked to a specific reduction in hepatic secretion of large triglyceride-rich VLDL1. The impaired secretion of VLDL1 explains the reduced plasma triglyceride concentration, and provides a basis for understanding the lower risk of cardiovascular disease associated with the TM6SF2 E167K genetic variant. Trial registration: Clinical Trials NCT04209816
Jan Borén, Martin Adiels, Elias Björnson, Niina Matikainen, Sanni Söderlund, Joel T. Rämo, Marcus Ståhlman, Pietari Ripatti, Samuli Ripatti, Aarno Palotie, Rosellina M. Mancina, Antti Hakkarainen, Stefano Romeo, Chris J. Packard, Marja-Riitta Taskinen
In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
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