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Additional file 7.

Additional file 6.

Additional file 8.

Additional file 7: Figure S2. Evaluation of the IC50 values of TAE226 and SOR on HCC cell viability after 2 and 5 days. Cell viability, measured by XTT assay, after 2 and 5 days from treatment with TAE226 or SOR, in HepG2 and Huh7 cells. Values are the mean OD �� SD of three independent experiments repeated at least in duplicate. Data were analysed by ANOVA. ***p

Additional file 6: Figure S1. FAK phosphorylation in HCC cells after IC50 values of treatments. Quantitative analysis of pTyr397FAK expression after 48 h of treatment with the different drugs, in HepG2 and Huh7 cells. Values are the mean arbitrary units �� SD of at least three independent experiments. Data were analysed by 2-tailed Student���s t test. *p

Additional file 10: Figure S5. Analysis of FAK nuclear interaction with specific epigenetics regulators. (A) Coomassie-stained 1D SDS-PAGE gels used for the in-gel digestions of nuclear immunoprecipitates of FAK in HCC cells. The photographs showed gel lanes excision into 10 bands for mass spectrometry characterization of FAK nuclear interactors. (B) Pipeline employed for the analysis of proteomics data.

Additional file 2: Table S2. In vivo antitumor efficacy of TAE226>SOR combination on orthotopic liver tumour.

Additional file 9: Figure S4. Pilot study for quantitative setting of TAE226 concentration in a heterotopic HepG2 xenograft model of HCC. (A) Quantitative analysis of tumour weight at various time points in NT animals or after TAE226 treatments. P values were calculated at day 12 using an unpaired two-tailed t-test, between treated and untreated tumours. **** p

Additional file 3: Table S3. Common up- and down-regulated genes.

Additional file 5: Table S5. GO annotations.

Additional file 8: Figure S3. IGF-1R in HCC cells after treatment with TAE226 > SOR compared to single treatments. Representative immunoblot and quantitative analysis of phosphorylated IGF-1R after 48 h from treatment with the different drugs, in HepG2 (A) and Huh7 cells (B). ��Tubulin served as loading control. Values are the mean arbitrary units �� SD of at least three independent experiments. Data were analysed by 2-tailed Student���s t test. **p

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. Significance: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gas chromatography-mass spectrometry. The α-diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and β-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanone compared to CTRLs. Conclusion: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively. (Hepatology 2017;65:451-464)

Non-alcoholic fatty liver disease(NAFLD) is the most common cause of chronic liver disease worldwide as a result of the increasing prevalence of obesity, starting from early life stages. It is characterized by a spectrum of liver diseases ranging from simple fatty liver(NAFL) to steatohepatitis(NASH), with a possible progression to fibrosis, thus increasing liver-related morbidity and mortality. NAFLD development is driven by the co-action of several risk factors, including obesity and metabolic syndrome, which may be both genetically induced and diet-related. Recently, particular attention has been paid to the gut-liver axis, which may play a physio-pathological role in the onset and progression of the disease. The gut microbiota is intended to act as a bioreactor that can guarantee autonomous metabolic and immunological functions and that can drive functional strategies within the environment of the body in response to external stimuli. The complexity of the gut microbiota suggests that it behaves as an organ. Therefore, the concept of the gut-liver axis must be complemented with the gut-microbiota-liver network due to the high intricacy of the microbiota components and metabolic activities; these activities form the active diet-driven power plant of the host. Such complexity can only be revealed using systems biology, which can integrate clinical phenomics and gut microbiota data. © 2014 by the authors; licensee MDPI, Basel, Switzerland.