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BACKGROUND AND AIMS The liver plays a central role in all metabolic processes in the body. However, precise characterization of liver metabolism is often obscured by its inherent complexity. Phosphorylated metabolites occupy a prominent position in all anabolic and catabolic pathways. Here, we develop a 31 P nuclear magnetic resonance (NMR)-based method to study the liver "phosphorome" through the simultaneous identification and quantification of multiple hydrophilic and hydrophobic phosphorylated metabolites. APPROACH AND RESULTS We applied this technique to define the metabolic landscape in livers from a mouse model of the rare disease disorder congenital erythropoietic porphyria (CEP) as well as two well-known murine models of nonalcoholic steatohepatitis: one genetic, methionine adenosyltransferase 1A knockout mice, and the other dietary, mice fed a high-fat choline-deficient diet. We report alterations in the concentrations of phosphorylated metabolites that are readouts of the balance between glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid cycle, and oxidative phosphorylation and of phospholipid metabolism and apoptosis. Moreover, these changes correlate with the main histological features: steatosis, apoptosis, iron deposits, and fibrosis. Strikingly, treatment with the repurposed drug ciclopirox improves the phosphoromic profile of CEP mice, an effect that was mirrored by the normalization of liver histology. CONCLUSIONS In conclusion, these findings indicate that NMR-based phosphoromics may be used to unravel metabolic phenotypes of liver injury and to identify the mechanism of drug action.

Purpose: Immune checkpoint inhibitors combined with antiangiogenic agents produce benefits in the treatment of advanced hepatocellular carcinoma (HCC). We investigated the efficacy and immunomodulatory activity of cabozantinib alone and combined with anti-PD1 in experimental models of HCC, and explored the potential target population that might benefit from this combination. Experimental Design: C57BL/6J mice bearing subcutaneous Hepa1-6 or Hep53.4 tumors received cabozantinib, anti-PD1, their combination, or placebo. Tumor and blood samples were analyzed by flow cytometry, IHC, transcriptome, and cytokine profiling. Cabozantinib-related effects were validated in a colorectal cancer patient-derived xenograft model. Transcriptomic data from three human HCC cohorts (cohort 1: n = 167, cohort 2: n = 57, The Cancer Genome Atlas: n = 319) were used to cluster patients according to neutrophil features, and assess their impact on survival. Results: The combination of cabozantinib and anti-PD1 showed increased antitumor efficacy compared with monotherapy and placebo (P < 0.05). Cabozantinib alone significantly increased neutrophil infiltration and reduced intratumor CD8+PD1+ T-cell proportions, while the combination with anti-PD1 further stimulated both effects and significantly decreased regulatory T cell (Treg) infiltration (all P < 0.05). In blood, cabozantinib and especially combination increased the proportions of overall T cells (P < 0.01) and memory/effector T cells (P < 0.05), while lowering the neutrophil-to-lymphocyte ratio (P < 0.001 for combination). Unsupervised clustering of human HCCs revealed that high tumor enrichment in neutrophil features observed with the treatment combination was linked to less aggressive tumors with more differentiated and less proliferative phenotypes. Conclusions: Cabozantinib in combination with anti-PD1 enhanced antitumor immunity by bringing together innate neutrophil-driven and adaptive immune responses, a mechanism of action which favors this approach for HCC treatment.

Background and aims: Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. Approach and results: We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. Conclusions: Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. Lay summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., This article was possible thanks to the inputs from the Instituto de Salud Carlos III, ISCIII (PI09/00751, PI10/02082, PI13/02340). The project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668596 (ChiLTERN) and grant agreement No 826121 (iPC). JCR is supported by the Catalan Agency for Management of University and Research Grants (AGAUR, 2019 FI_B01024). LT is supported by an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813). DS is supported by the Gilead Research Scholar in Liver Disease. JML is supported by the European Union's Horizon 2020 research and innovation programme (HEPCAR, 667273-2), Institucio Catalana de Recerca i Estudis Avancats (ICREA), U.S. Department of Defense (CA150272P3), an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813), National Cancer Institute, Tisch Cancer Institute (P30-CA196521), Samuel Waxman Cancer Research Foundation, Spanish National Health Institute (SAF2016-76390) and AGAUR (SGR-1358). CA and MRS were supported by Ramon y Cajal (RYC-2010-07249) and Miguel Servet (CPII14/00021) programs of the Ministry of Science and Innovation of Spain and ISCIII, respectively. CA, MRS and MS received funding from CIBERehd (CB06/04/0033) and AGAUR (2017-SGR-490). IGTP is a member of the CERCA network of institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.