New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study.

BACKGROUND AND AIMS: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. METHODS: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. RESULTS: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. CONCLUSIONS: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy.

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

Maresin 1 activates brown adipose tissue and promotes browning of white adipose tissue in mice.

OBJECTIVE: Maresin 1 (MaR1) is a docosahexaenoic acid-derived proresolving lipid mediator with insulin-sensitizing and anti-steatosis properties. Here, we aim to unravel MaR1 actions on brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning. METHODS: MaR1 actions were tested in cultured murine brown adipocytes and in human mesenchymal stem cells (hMSC)-derived adipocytes. In vivo effects of MaR1 were tested in diet-induced obese (DIO) mice and lean WT and Il6 knockout (Il6-/-) mice. RESULTS: In cultured differentiated murine brown adipocytes, MaR1 reduces the expression of inflammatory genes, while stimulates glucose uptake, fatty acid utilization and oxygen consumption rate, along with the upregulation of mitochondrial mass and genes involved in mitochondrial biogenesis and function and the thermogenic program. In Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)-depleted brown adipocytes using siRNA, the stimulatory effect of MaR1 on thermogenic genes was abrogated. In DIO mice, MaR1 promotes BAT remodeling, characterized by higher expression of genes encoding for master regulators of mitochondrial biogenesis and function and iBAT thermogenic activation, together with increased M2 macrophage markers. In addition, MaR1-treated DIO mice exhibit a better response to cold-induced BAT activation. Moreover, MaR1 induces a beige adipocyte signature in inguinal WAT of DIO mice and in hMSC-derived adipocytes. MaR1 potentiates Il6 expression in brown adipocytes and BAT of cold exposed lean WT mice. Interestingly, the thermogenic properties of MaR1 were abrogated in Il6-/- mice. CONCLUSIONS: These data reveal MaR1 as a novel agent that promotes BAT activation and WAT browning by regulating thermogenic program in adipocytes and M2 polarization of macrophages. Moreover, our data suggest that LGR6 receptor is mediating MaR1 actions on brown adipocytes, and that IL-6 is required for the thermogenic effects of MaR1.

FOXP3 expression diversifies the metabolic capacity and enhances the efficacy of CD8 T cells in adoptive immunotherapy of melanoma.

Regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a FOXP3-driven metabolic program that allows them to engage different metabolic pathways. Using a melanoma model of adoptive T cell therapy (ACT), we show that FOXP3 overexpression in mature CD8 T cells improved their antitumor efficacy, favoring their tumor recruitment, proliferation, and cytotoxicity. FOXP3-overexpressing (Foxp3UP) CD8 T cells exhibited features of tissue-resident memory-like and effector T cells, but not suppressor activity. Transcriptomic analysis of tumor-infiltrating Foxp3UP CD8 T cells showed positive enrichment in a wide variety of metabolic pathways, such as glycolysis, fatty acid (FA) metabolism, and oxidative phosphorylation (OXPHOS). Intratumoral Foxp3UP CD8 T cells exhibited an enhanced capacity for glucose and FA uptake as well as accumulation of intracellular lipids. Interestingly, Foxp3UP CD8 T cells compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energy demands. Importantly, their ability to couple glycolysis and OXPHOS allowed them to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role for FOXP3 in the adaptation of CD8 T cells to TME that may enhance their efficacy in ACT.

A new animal model of atrophy-hypertrophy complex and liver damage following Yttrium-90 lobar selective internal radiation therapy in rabbits.

Lobar selective internal radiation therapy (SIRT) is widely used to treat liver tumors inducing atrophy of the treated lobe and contralateral hypertrophy. The lack of animal model has precluded further investigations to improve this treatment. We developed an animal model of liver damage and atrophy-hypertrophy complex after SIRT. Three groups of 5-8 rabbits received transportal SIRT with Yttrium 90 resin microspheres of the cranial lobes with different activities (0.3, 0.6 and 1.2 GBq), corresponding to predicted absorbed radiation dose of 200, 400 and 800 Gy, respectively. Another group received non-loaded microspheres (sham group). Cranial and caudal lobes volumes were assessed using CT volumetry before, 15 and 30 days after SIRT. Liver biochemistry, histopathology and gene expression were evaluated. Four untreated rabbits were used as controls for gene expression studies. All animals receiving 1.2 GBq were euthanized due to clinical deterioration. Cranial SIRT with 0.6 GBq induced caudal lobe hypertrophy after 15 days (median increase 34% -ns-) but produced significant toxicity. Cranial SIRT with 0.3 GBq induced caudal lobe hypertrophy after 30 days (median increase 82%, p = 0.04). No volumetric changes were detected in sham group. Transient increase in serum transaminases was detected in all treated groups returning to normal values at 15 days. There was dose-dependent liver dysfunction with bilirubin elevation and albumin decrease. Histologically, 1.2 GBq group developed permanent severe liver damage with massive necrosis, 0.6 and 0.3 GBq groups developed moderate damage with inflammation and portal fibrosis at 15 days, partially recovering at 30 days. There was no difference in the expression of hepatocyte function and differentiation genes between 0.3 GBq and control groups. Cranial SIRT with 0.3 GBq of 90Y resin microspheres in rabbits is a reliable animal model to analyse the atrophy-hypertrophy complex and liver damage without toxicity.

mRNA-based therapy in a rabbit model of variegate porphyria offers new insights into the pathogenesis of acute attacks.

Variegate porphyria (VP) results from haploinsufficiency of protoporphyrinogen oxidase (PPOX), the seventh enzyme in the heme synthesis pathway. There is no VP model that recapitulates the clinical manifestations of acute attacks. Combined administrations of 2-allyl-2-isopropylacetamide and rifampicin in rabbits halved hepatic PPOX activity, resulting in increased accumulation of a potentially neurotoxic heme precursor, lipid peroxidation, inflammation, and hepatocyte cytoplasmic stress. Rabbits also showed hypertension, motor impairment, reduced activity of critical mitochondrial hemoprotein functions, and altered glucose homeostasis. Hemin treatment only resulted in a slight drop in heme precursor accumulation but further increased hepatic heme catabolism, inflammation, and cytoplasmic stress. Hemin replenishment did protect against hypertension, but it failed to restore action potentials in the sciatic nerve or glucose homeostasis. Systemic porphobilinogen deaminase (PBGD) mRNA administration increased hepatic PBGD activity, the third enzyme of the pathway, and rapidly normalized serum and urine porphyrin precursor levels. All features studied were improved, including those related to critical hemoprotein functions. In conclusion, the VP model recapitulates the biochemical characteristics and some clinical manifestations associated with severe acute attacks in humans. Systemic PBGD mRNA provided successful protection against the acute attack, indicating that PBGD, and not PPOX, was the critical enzyme for hepatic heme synthesis in VP rabbits.

An Experimental DUAL Model of Advanced Liver Damage.

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol-associated and metabolic-associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol-associated liver disease plus metabolic-associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA-sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets.

Rupatadine Oral Solution Titration by Body Weight in Paediatric Patients Suffering from Allergic Rhinitis: A Population Pharmacokinetic Study.

BACKGROUND: Allergic rhinitis (AR) and chronic urticaria, both are treated in children with doses of second generation of antihistamines that have been mostly based on extrapolation of data obtained in adults. The objectives of this work were to develop a model to explain the pharmacokinetics (PK) of rupatadine, a second generation antihistamine, administered to children 2-11 years old and to calculate the non-compartmental PK parameters for two groups of age (2-5 and 6-11 years old) based on the individual Bayesian estimates from the selected model. METHODS: Data from two PK studies with rupatadine oral solution (1 mg/mL) were pooled: Study A, an extensive blood sampling study performed in 11 children (6-11 years old) who received a single oral dose of rupatadine; and Study B, a sparse blood sampling study in 40 children (2-5 years old) receiving multiple oral doses. A simultaneous population PK model was developed using data available for all children. Using individual Bayesian estimates from the selected model, steady-state plasma concentrations for both studies were simulated and the non-parametric PK parameters were calculated for two age groups: 2-5 years (subgroup I) and 6-11 years (subgroup II). RESULTS: A two-compartment model with first-order absorption and elimination with clearance depending on body weight, better described the PK of rupatadine for 2-11 year old children. The plasma clearance dependence on weight was linear. The mean (SD) non-compartment PK parameters calculated using simulated plasma profiles at steady state were: Cmax, 2.54 (1.26) vs 1.96 (0.52) ng/mL; AUC0-24h, 10.74 (3.09) vs 10.38 (4.31) ng/mL/h; and t1/2, 12.28 (3.09) vs 15.94 (4.09) h, for children 6-11 and 2-5 years old, respectively. CONCLUSIONS: The PK of rupatadine depends on the weight of paediatric patients but not on their age. The dosage strategy adjusted by body weight in children 2-11 years old (2.5 mL if weight 10-25 kg, and 5 mL if ≥ 25 kg) provides similar exposure between the two groups of age, and to that obtained in adults with the 10 mg dose tablet formulation.

ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity.

ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult.

Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis.

OBJECTIVE: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis. DESIGN: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-ß1 (TGFß1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology. RESULTS: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFß1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFß1 fibrogenic responses in hHSC. TGFß1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity. CONCLUSIONS: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Profound Reprogramming towards Stemness in Pancreatic Cancer Cells as Adaptation to AKT Inhibition.

Cancer cells acquire resistance to cytotoxic therapies targeting major survival pathways by adapting their metabolism. The AKT pathway is a major regulator of human pancreatic adenocarcinoma progression and a key pharmacological target. The mechanisms of adaptation to long-term silencing of AKT isoforms of human and mouse pancreatic adenocarcinoma cancer cells were studied. Following silencing, cancer cells remained quiescent for long periods of time, after which they recovered proliferative capacities. Adaptation caused profound proteomic changes largely affecting mitochondrial biogenesis, energy metabolism and acquisition of a number of distinct cancer stem cell (CSC) characteristics depending on the AKT isoform that was silenced. The adaptation to AKT1 silencing drove most de-differentiation and acquisition of stemness through C-MYC down-modulation and NANOG upregulation, which were required for survival of adapted CSCs. The changes associated to adaptation sensitized cancer cells to inhibitors targeting regulators of oxidative respiration and mitochondrial biogenesis. In vivo pharmacological co-inhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models.

Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-expression with Plastid Thioredoxin m.

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein's overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.

Cardiotrophin-1 is an anti-inflammatory cytokine and promotes IL-4-induced M2 macrophage polarization.

Macrophages play a central role in tissue remodeling, repair, and resolution of inflammation. Macrophage polarization to M1 or M2 activation status may determine the progression or resolution of the inflammatory response. We have previously reported that cardiotrophin-1 (CT-1) displays both cytoprotective and metabolic activities. The role of CT-1 in inflammation remains poorly understood. Here, we employed recombinant CT-1 (rCT-1) and used CT-1-null mice and myeloid-specific CT-1 transgenic mice to investigate whether CT-1 might play a role in the modulation of the inflammatory response. We observed that CT-1 deficiency was associated with enhanced release of inflammatory mediators and with stronger activation of NF-κB in response to LPS, whereas the inflammatory response was attenuated in CT-1 transgenic mice or by administering rCT-1 to wild-type animals prior to LPS challenge. We found that CT-1 promoted IL-6 expression only by nonhematopoietic cells, whereas LPS up-regulated IL-6 expression in both hematopoietic and nonhematopoietic cells. Notably, rCT-1 inhibited LPS-mediated soluble IL-6R induction. Using IL-6-/- mice, we showed that rCT-1 inhibited LPS-induced TNF-α and IFN-γ response in an IL-6-independent manner. Importantly, we demonstrated that CT-1 primes macrophages for IL-4-dependent M2 polarization by inducing IL-4 receptor expression. Mechanistic analyses showed that the signal transducer and activator of transcription 3-suppressor of cytokine signaling 3 axis mediates this effect. Our findings support the notion that CT-1 is a critical regulator of inflammation and suggest that rCT-1 could be a molecule with potential therapeutic application in inflammatory conditions.-Carneros, D., Santamaría, E. M., Larequi, E., Vélez-Ortiz, J. M., Reboredo, M., Mancheño, U., Perugorria, M. J., Navas, P., Romero-Gómez, M., Prieto, J., Hervás-Stubbs, S., Bustos, M. Cardiotrophin-1 is an anti-inflammatory cytokine and promotes IL-4-induced M2 macrophage polarization.

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins.

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.

Dual Targeting of Histone Methyltransferase G9a and DNA-Methyltransferase 1 for the Treatment of Experimental Hepatocellular Carcinoma.

Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.

The Epidermal Growth Factor Receptor Ligand Amphiregulin Protects From Cholestatic Liver Injury and Regulates Bile Acids Synthesis.

Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Rupatadine oral solution for 2-5-year-old children with allergic rhinitis: a safety, open-label, prospective study.

BACKGROUND: There are few clinical trials that assess the efficacy of antihistamines in very young children. Rupatadine is a second-generation antihistamine indicated for the treatment of allergic rhinitis (AR) and urticaria. In this study, AR symptoms were evaluated before and after daily 1 mg/mL rupatadine oral solution administration in 2-5-year-old children. METHODS: A multicenter open-label study was carried out in 2-5-year-old children with AR. Safety assessments were collected during the study including spontaneous adverse events, vital signs, and electrocardiogram (QTc interval). Additionally, evaluations of Total Five Symptoms Score (T5SS, including: nasal congestion; sneezing; rhinorrhoea; itchy nose, mouth, throat, and/or ears; and itchy, watery, and red eyes) were analyzed. Symptoms were evaluated by parents/legal guardian before and after 4 weeks of rupatadine administration, dosed according to body weight. RESULTS: A total of 44 children received the study treatment. Only 15 adverse events were reported. All of them were of mild intensity and considered not related to the study treatment. No patient exceeded the standard parameter of >450 ms in the last visit, for the QTc interval on their electrocardiograms. From a maximum score value of 15, T5SS values at Day 14 (6.35) and Day 28 (5.42) were both statistically significant different (p<0.001) from the baseline T5SS value (mean 8.65), with a reduction of 26.6% and 37.4%, respectively. All individual symptoms, including nasal congestion, showed also a decrease from baseline at both 14 and 28 days. CONCLUSION: Rupatadine 1 mg/mL oral solution was found to be safe in 2-5-year-old children, correlating with an improvement of AR symptoms, overall and each individually, after a daily dose administration. With this study, we enlarge the available information in this very young pediatric patients' group, in which there is a general lack of clinical evidence.

Mitochondrial Morphological and Functional Reprogramming Following CD137 (4-1BB) Costimulation.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.