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Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for over 80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. And with the recent success of immunotherapies in HCC, mouse models that better recapitulate the human disease and antitumor immune response are needed. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and syngeneic or xenogeneic tumor implantation strategies. We established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells, characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (sorafenib) and immunotherapy (anti PD-1, anti-TLR) was also assessed and show moderate to high efficiency. Moreover, a panel of xenograft models including Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. But as xenograft models are not completely mimicking the human situation of both immune and liver microenvironment, we have recently initiated the development of a double humanized (immune and liver) transgenic mouse as a better host for human tumor engraftment. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed a model induced by a low dose of streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome NASH and fibrosis within 12 weeks and HCC within 16 weeks. Of interest, 100% of male mice develop HCC within 16 weeks, in accordance with studies showing that men had a 2-to 7-fold higher risk of developing HCC in human. Treatment of mice with lenvatinib alone or in combination with anti-PD1 increases survival and reduces tumor burden as shown with reduced liver weight/body weight ratio at 16 weeks. Altogether, these results demonstrate the usefulness of this comprehensive platform of preclinical in vivo HCC models to discover and identify novel therapeutic strategies that could circumvent the progression of liver cancers. Citation Format: Olivier Duchamp, Gael Krysa, Loic Morgand, Hugo Quillery, Robin Artus, Maxime Ramelet, Jeremy Odillard, Peggy Provent, Sylvie Maubant, Caroline Mignard, Fabrice Viviani, Samira Benhamouche-Trouillet. Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1654.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for >80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited and only a few available (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and implantation strategies. We first established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells. Hepa1.6-derived tumor growth was characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (Sorafenib) and immunotherapy (anti PD-1) was also assessed. Moreover, a panel of Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed two models of non- alcoholic steato-hepatitis (NASH); The first model involves chronic administration of hepatotoxic CCL4 associated with a high-fat and high-fructose diet. Mice develop steatohepatitis and fibrosis within 4 to 8 weeks as assessed with biochemistry parameters (AST, ALT…), gene expression levels of inflammatory and fibrotic genes as well as histological scores and MRI. The effect of obeticholic acid was successfully evaluated in this model. The second model is the STAM model induced by a low dose of Streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome (increased body weight gain, hyperglycemia and hyperlipidemia), NASH (steatosis, inflammation, fibrosis) within 12 weeks and HCC within 16 weeks. The responses to standard of care and to immunotherapy together with the characterization of the immune cell populations are currently under investigation. Altogether, these results demonstrate the usefulness of this liver diseases development program to discover and identify new treatments that could circumvent the progression of liver cancer. Citation Format: Olivier Duchamp, Gael Krysa, Robin Artus, Hugo Quillery, Maxime Ramelet, Valerie Boullay, Laure Levenez, Adelaide Ferment, Jeremy Odillard, Anne-Benedict Boullay, Peggy Provent, Edwige Nicodeme, Samira Benhamouche-Trouillet, Anne Lazzari, Loic Morgand. Liver preclinical models - acute, chronic and cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5638.

Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.

Immunotherapies prime or activate patient’s immune system to fight disease and has recently been a source of registered and promising new cancer treatments with major beneficial clinical outcomes in several cancers. However, by increasing the activity of the immune system, therapies as such targeting the immune checkpoint blockade can have profound inflammatory side effects, termed immune-related adverse events, with particular organs affected such as gastrointestinal tract, endocrine glands, skin and liver. In patients treated with the anti-CTLA-4 antibody, Ipilimumab, the overall incidence of diarrhea and colitis has been reported as 32.8%. This side effect is also observed in patients treated with anti-PD-1 therapy. The reproduction of such aspects of immune checkpoint inhibitors side effects in preclinical animal models would serve to dig into deciphering mechanisms involved, but also to evaluate combination therapies and modalities to reduce and manage such incidence with potential beneficial translation in clinical practice. We thus first ought to establish a checkpoint blockade-related autoimmune mouse model of colitis, using female C57BL/6 mice which were tested to determine their response to orally administered dextran sulfate sodium (DSS) combined to multiple injections of anti-CTLA-4 antibody or an isotype control antibody. After administration of DSS in drinking water for 7 days, mice that received an anti-CTLA-4 antibody showed no difference in body weight loss and Disease Activity Index (composite of body weight loss, stool consistency and presence of blood in stools), when compared to mice that received DSS plus the isotype control antibody. Histological sections analysis from the colons of mice culled at Day 10 confirmed that the combined treatment did not change the colitis score.However, when looking at later time points corresponding to the usual recovery phase of colitis, the effect of anti-CTLA4 treatment was clearly revealed by a sustained body weight loss and the persistence of a high Disease Activity Index, while the isotype control-treated animals slowly returned to baseline values similar to untreated animals. At sacrifice on Day 19, these in life findings were confirmed by a decrease in colon length (- 0.94 cm), an increase in colon weight (+ 231 mg) and a high mucus colon production in the anti-CTLA4-treated group versus the isotype control-treated group. Histological analysis of colons, and a more in depth characterization of the immune infiltrates will be presented. Citation Format: Edwige Nicodeme, Florence Blandel, Valerie Boullay, Yannick Saintillan, Gael Krysa, Anne-Benedicte Boullay, Jean-Jacques Tousaint, Robin Artus, Laure Levenez, Jeremy Odillard, Ingrid Jacquet, Olivier Duchamp, Fabrice Viviani. Immune checkpoint blockade and autoimmune diseases: Development of a mouse model of colitis induced by anti-CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3228.

1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF-beta), a well-known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI-1 and collagens. The identification of GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF-beta pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF-beta signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF-beta-overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)-induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC(50) of 140 nM and in a cellular assay inhibited TGF-beta-induced transcription of PAI-1 (IC(50): 500 nM). In vivo, GW6604 (40 mg kg(-1) p.o.) increased liver regeneration in TGF-beta-overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN-induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg(-1) p.o., b.i.d.), mortality was prevented and DMN-induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP-1 and TGF-beta were reduced by 50-75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.

Optimization of the screening hit 1 led to the identification of novel 1,5-naphthyridine aminothiazole and pyrazole derivatives, which are potent and selective inhibitors of the transforming growth factor-beta type I receptor, ALK5. Compounds 15 and 19, which inhibited ALK5 autophosphorylation with IC50 = 6 and 4 nM, respectively, showed potent activities in both binding and cellular assays and exhibited selectivity over p38 mitogen-activated protein kinase. The X-ray crystal structure of 19 in complex with human ALK5 is described, confirming the binding mode proposed from docking studies.

Bromodomains (BRDs) are small protein domains found in a variety of proteins that recognize and bind to acetylated histone tails. This binding affects chromatin structure and facilitates the localisation of transcriptional complexes to specific genes, thereby regulating epigenetically controlled processes including gene transcription and mRNA elongation. Inhibitors of the bromodomain and extra-terminal (BET) proteins BRD2-4 and T, which prevent bromodomain binding to acetyl-modified histone tails, have shown therapeutic promise in several diseases. We report here the discovery of 1,5-naphthyridine derivatives as potent inhibitors of the BET bromodomain family with good cell activity and oral pharmacokinetic parameters. X-ray crystal structures of naphthyridine isomers have been solved and quantum mechanical calculations have been used to explain the higher affinity of the 1,5-isomer over the others. The best compounds were progressed in a mouse model of inflammation and exhibited dose-dependent anti-inflammatory pharmacology.

A novel series of quinoline isoxazole BET family bromodomain inhibitors are discussed. Crystallography is used to illustrate binding modes and rationalize their SAR. One member, I-BET151 (GSK1210151A), shows good oral bioavailability in both the rat and minipig as well as demonstrating efficient suppression of bacterial induced inflammation and sepsis in a murine in vivo endotoxaemia model.

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.

Starting from quinazoline 3a, we designed potent and selective ALK5 inhibitors over p38MAP kinase from a rational drug design approach based on co-crystal structures in the human ALK5 kinase domain. The quinazoline 3d exhibited also in vivo activity in an acute rat model of DMN-induced liver fibrosis when administered orally at 5 mg/kg (bid).

1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF-beta), a well-known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI-1 and collagens. The identification of GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF-beta pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF-beta signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF-beta-overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)-induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC(50) of 140 nM and in a cellular assay inhibited TGF-beta-induced transcription of PAI-1 (IC(50): 500 nM). In vivo, GW6604 (40 mg kg(-1) p.o.) increased liver regeneration in TGF-beta-overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN-induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg(-1) p.o., b.i.d.), mortality was prevented and DMN-induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP-1 and TGF-beta were reduced by 50-75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.