Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma.

Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, in newly generated, clinically-relevant somatic female HCC mouse models, we identify cancer genetics' specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. Mitogen-activated protein kinase (MAPK)-activated, NrasG12D-driven tumors exhibit a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME. Mechanistically, we report a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbs the accumulation of these cells, reduces inflammation, induces cancer cell death and prolongs animal survival. Furthermore, GM-CSF neutralization synergizes with a vascular endothelial growth factor (VEGF) inhibitor to restrain HCC outgrowth. These findings underscore the profound alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.

Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Re-purposing the pro-senescence properties of doxorubicin to introduce immunotherapy in breast cancer brain metastasis.

An increasing number of breast cancer patients develop brain metastases (BM). Standard-of-care treatments are largely inefficient, and breast cancer brain metastasis (BCBM) patients are considered untreatable. Immunotherapies are not successfully employed in BCBM, in part because breast cancer is a "cold" tumor and also because the brain tissue has a unique immune landscape. Here, we generate and characterize immunocompetent models of BCBM derived from PyMT and Neu mammary tumors to test how harnessing the pro-senescence properties of doxorubicin can be used to prime the specific immune BCBM microenvironment. We reveal that BCBM senescent cells, induced by doxorubicin, trigger the recruitment of PD1-expressing T cells to the brain. Importantly, we demonstrate that induction of senescence with doxorubicin improves the efficacy of immunotherapy with anti-PD1 in BCBM in a CD8 T cell-dependent manner, thereby providing an optimized strategy to introduce immune-based treatments in this lethal disease. In addition, our BCBM models can be used for pre-clinical testing of other therapeutic strategies in the future.

EGFR activation limits the response of liver cancer to lenvatinib.

Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR)  by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.

Multiparametric Analyses of Hepatocellular Carcinoma Somatic Mouse Models and Their Associated Tumor Microenvironment.

The rising incidence and increasing mortality of hepatocellular carcinoma (HCC), combined with its high tumor heterogeneity, lack of druggable targets, and tendency to develop resistance to chemotherapeutics, make the development of better models for this cancer an urgent challenge. To better mimic the high diversity within the HCC genetic landscape, versatile somatic murine models have recently been developed using the hydrodynamic tail vein injection (HDTVi) system. These represent novel in vivo tools to interrogate HCC phenotype and response to therapy, and importantly, allow further analyses of the associated tumor microenvironment (TME) shaped by distinct genetic backgrounds. Here, we describe several optimized protocols to generate, collect, and experimentally utilize various samples obtained from HCC somatic mouse models generated by HDTVi. More specifically, we focus on techniques relevant to ex vivo analyses of the complex liver TME using multiparameter flow cytometric analyses of over 21 markers, immunohistochemistry, immunofluorescence, and histochemistry. We describe the transcriptional assessment of whole tissue, or of isolated immune subsets by flow-cytometry-based cell sorting, and other protein-oriented analyses. Together, these streamlined protocols allow the optimal use of each HCC murine model of interest and will assist researchers in deciphering the relations between cancer cell genetics and systemic and local changes in immune cell landscapes in the context of HCC progression. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of HCC mouse models by hydrodynamic tail vein injection Basic Protocol 2: Assessment of HCC tumor progression by magnetic resonance imaging Basic Protocol 3: Mouse sacrifice and sample collection in HCC mouse models Support Protocol 1: Preparation of serum or plasma from blood Basic Protocol 4: Single-cell preparation and HCC immune landscape phenotyping by flow cytometry Alternate Protocol 1: Flow cytometric analysis of circulating immune cells Support Protocol 2: Generation, maintenance, and characterization of HCC cell lines Support Protocol 3: Fluorescence-activated cell sorting of liver single-cell preparation Basic Protocol 5: Preparation and immunohistochemical analysis of tumor tissues from HCC-bearing liver Alternate Protocol 2: Preparation and analyses for immunofluorescence staining of HCC-bearing liver Support Protocol 4: Liver-specific phenotypic analyses of liver sections Support Protocol 5: Immunohistochemical quantification in liver sections Basic Protocol 6: Preparation of snap-frozen tumor tissue from extracted liver and transcriptional analyses of bulk tumor or sorted cells Alternate Protocol 3: Protein analyses from HCC samples and serum or plasma.

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.

Understanding the Origin and Diversity of Macrophages to Tailor Their Targeting in Solid Cancers.

Tumor-associated macrophages (TAMs) are a major component of the tumor immune microenvironment (TIME) and are associated with a poor prognostic factor in several cancers. TAMs promote tumor growth by facilitating immunosuppression, angiogenesis, and inflammation, and can promote tumor recurrence post-therapeutic intervention. Major TAM-targeted therapies include depletion, reprogramming, as well as disrupting the balance of macrophage recruitment and their effector functions. However, intervention-targeting macrophages have been challenging, since TAM populations are highly plastic and adaptation or resistance to these approaches often arise. Defining a roadmap of macrophage dynamics in the TIME related to tissue and tumor type could represent exploitable vulnerabilities related to their altered functions in cancer malignancy. Here, we review multiple macrophage-targeting strategies in brain, liver, and lung cancers, which all emerge in tissues rich in resident macrophages. We discuss the successes and failures of these therapeutic approaches as well as the potential of personalized macrophage-targeting treatments in combination therapies.

Characterization of Pattern Recognition Receptor Expression and Functionality in Liver Primary Cells and Derived Cell Lines.

Different liver cell types are endowed with immunological properties, including cell-intrinsic innate immune functions that are important to initially control pathogen infections. However, a full landscape of expression and functionality of the innate immune signaling pathways in the major human liver cells is still missing. In order to comparatively characterize these pathways, we purified primary human hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells (LSEC), and Kupffer cells (KC) from human liver resections. We assessed mRNA and protein expression level of the major innate immune sensors, as well as checkpoint-inhibitor ligands in the purified cells, and found Toll-like receptors (TLR), RIG-I-like receptors, as well as several DNA cytosolic sensors to be expressed in the liver microenvironment. Amongst the cells tested, KC were shown to be most broadly active upon stimulation with PRR ligands emphasizing their predominant role in innate immune sensing the liver microenvironment. By KC immortalization, we generated a cell line that retained higher innate immune functionality as compared to THP1 cells, which are routinely used to study monocyte/macrophages functions. Our findings and the establishment of the KC line will help to understand immune mechanisms behind antiviral effects of TLR agonists or checkpoint inhibitors, which are in current preclinical or clinical development.

NOD1 Participates in the Innate Immune Response Triggered by Hepatitis C Virus Polymerase.

UNLABELLED: Hepatitis C virus (HCV) triggers innate immunity signaling in the infected cell. Replication of the viral genome is dispensable for this phenotype, and we along with others have recently shown that NS5B, the viral RNA-dependent RNA polymerase, synthesizes double-stranded RNA (dsRNA) from cellular templates, thus eliciting an inflammatory response, notably via activation of type I interferon and lymphotoxin ß. Here, we investigated intracellular signal transduction pathways involved in this process. Using HepaRG cells, a model that largely recapitulates the in vivo complexities of the innate immunity receptor signaling, we have confirmed that NS5B triggered increased expression of the canonical pattern recognition receptors (PRRs) specific for dsRNA, namely, RIG-I, MDA5, and Toll-like receptor 3 (TLR3). Unexpectedly, intracellular dsRNA also led to accumulation of NOD1, a receptor classically involved in recognition of bacterial peptidoglycans. NOD1 activation, confirmed by analysis of its downstream targets, was likely due to its interaction with dsRNA and was independent of RIG-I and mitochondrial antiviral signaling protein (MAVS/IPS-1/Cardif/VISA) signaling. It is likely to have a functional significance in the cellular response in the context of HCV infection since interference with the NOD1 pathway severely reduced the inflammatory response elicited by NS5B. IMPORTANCE: In this study, we show that NOD1, a PRR that normally senses bacterial peptidoglycans, is activated by HCV viral polymerase, probably through an interaction with dsRNA, suggesting that NOD1 acts as an RNA ligand recognition receptor. In consequence, interference with NOD1-mediated signaling significantly weakens the inflammatory response to dsRNA. These results add a new level of complexity to the understanding of the cross talk between different classes of pattern recognition receptors and may be related to certain complications of chronic hepatitis C virus infection.

Hepatitis C viral proteins perturb metabolic liver zonation.

BACKGROUND & AIMS: The metabolic identity of a hepatocyte is determined by its position along the porto-centrilobular axis of a liver lobule. Altered patterns of metabolic liver zonation are associated with several pathologies. In hepatitis C, although only a minority of hepatocytes harbour the virus, the liver undergoes major systemic metabolic changes. We have investigated the HCV-driven mechanisms that allow the systemic loss of metabolic zonation. METHODS: Transgenic mice with hepatocyte-targeted expression of all HCV proteins (FL-N/35 model) and needle biopsies from hepatitis C patients were studied with respect to patterns of lipid deposition in the context of metabolic zonation of the liver lobule. RESULTS: We report that low levels of viral proteins are sufficient to drive striking alterations of hepatic metabolic zonation. In mice, a major lipogenic enzyme, fatty acid synthase, was redistributed from its normal periportal expression into the midzone of the lobule, coinciding with a highly specific midzone accumulation of lipids. Strikingly, alteration of zonation was not limited to lipogenic enzymes and appeared to be driven by systemic signalling via the Wnt/ß-catenin pathway. Importantly, we show that similarly perturbed metabolic zonation appears to precede steatosis in early stages of human disease associated with HCV infection. CONCLUSIONS: Our results rationalize systemic effects on liver metabolism, triggered by a minority of infected cells, thus opening new perspectives for the investigation of HCV-related pathologies.

Lymphotoxin signaling is initiated by the viral polymerase in HCV-linked tumorigenesis.

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-ß in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKß inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-ß pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.