Hepatocellular carcinoma (HCC) is the most common primary liver tumor, often arising in the context of chronic liver disease. Despite recent advances in systemic therapies, including the use of immune checkpoint inhibitors (ICIs), clinical outcomes remain suboptimal, with many patients exhibiting primary or acquired resistance. Accumulating evidence indicates that the dysregulation of epigenetic mechanisms contributes to HCC development, and may also play a crucial role in shaping the tumor immune microenvironment, influencing responses to treatments. In this study, we analyzed the expression profiles of a comprehensive set of epigenetic regulators across publicly available transcriptomic datasets of HCC and non-tumoral liver tissues. Our findings reveal a consistent dysregulation of key epigenetic modifiers, particularly those involved in DNA methylation and histone modification. Furthermore, our analysis underscores the need for a deeper understanding of the epigenetic landscape of HCC, as specific epigenetic patterns are directly associated with disease development, the major mutational, immune, and transcriptional subclasses of HCC, and patient clinical outcomes. Our study provides a foundation for integrating epigenetic biomarkers into patient stratification and therapeutic decision-making. A more comprehensive analysis of epigenetic alterations could pave the way for novel predictive markers and combination strategies that could enhance the efficacy of ICIs in HCC.

Background: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterised by inflammation and fibrosis of the bile ducts, conferring an increased risk of cholangiocarcinoma (CCA). However, detecting CCA early in PSC patients remains challenging due to the limited sensitivity of conventional diagnostic methods, including imaging or bile duct brush cytology during endoscopic retrograde cholangiopancreatography (ERCP). This study aims to evaluate the potential of bile cell‐free DNA (cfDNA) mutational analysis, termed the Bilemut assay, as a tool for CCA detection in PSC patients. Methods: Sixty‐three PSC patients undergoing ERCP due to biliary strictures were prospectively recruited. Bile samples were collected, and cfDNA was extracted and analysed using the Oncomine Pan‐Cancer Cell‐Free assay. Twenty healthy liver donors were included for comparison. Samples with a mutant allele frequency (MAF) ≥ 0.1% were considered positive. Correlations between mutational status and clinical characteristics were assessed. Results: cfDNA mutational analysis was successful in all bile samples. Mutations predominantly in KRAS , GNAS , and TP53 were detected in 36.5% (23/63) of PSC patients, compared to 10% (2/20) of healthy donors ( p = 0.0269). The clinical characteristics of Bilemut‐positive and ‐negative patients were comparable, though there was a trend towards a lower prevalence of inflammatory bowel disease in the Bilemut‐positive group. Among PSC patients diagnosed with CCA during follow‐up, 75% were Bilemut‐positive, suggesting an association between mutational status and malignancy risk. Conclusions: Mutational analysis of cfDNA obtained from bile collected from PSC patients undergoing ERCP is feasible. Implementing the Bilemut assay may help identify patients needing closer surveillance and further imaging studies.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20^-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20^-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Alpha-aminoterminal acetyltransferase B (NatB) is a critical enzyme responsible for acetylating the aminoterminal end of proteins, thereby modifying approximately 21% of the proteome. This post-translational modification impacts protein folding, structure, stability, and interactions between proteins which, in turn, play a crucial role in modulating several biological functions. NatB has been widely studied for its role in cytoskeleton function and cell cycle regulation in different organisms, from yeast to human tumor cells. In this study, we aimed to understand the biological importance of this modification by inactivating the catalytic subunit of the NatB enzymatic complex, Naa20, in non-transformed mammal cells. Our findings demonstrate that depletion of NAA20 results in decreased cell cycle progression and DNA replication initiation, ultimately leading to the senescence program. Furthermore, we have identified NatB substrates that play a role in cell cycle progression, and their stability is compromised when NatB is inactivated. These results underscore the significance of N-terminal acetylation by NatB in regulating cell cycle progression and DNA replication.

Background: Epigenetic alterations play an increasingly recognized role in carcinogenesis and in the development of resistance to anticancer therapies. Epigenetic enzymes (writers and erasers) and effectors (readers) are largely influenced by the availability of metabolites grenerated through one-carbon metabolism (OCM), the tricarboxylic acid (TCA) cycle, and acetyl-CoA synthesis (ACS). In this study we examined the expression of epigenetic and metabolic genes to investigate their interplay in cholangiocarcinoma (CCA). Method: We examined 257 epigenetic genes (EpiGs), 96 metabolic genes (MGs), and 189 rate-limiting enzymes (RLEs) in transcriptomic data from iCCA, eCCA, CCA organoids, and normal bile ducts, alongside prognostic signatures. CRISPR-Cas9 DepMap data evaluated the impact of EpiGs and MGs on cell viability. HuCCT-1 iCCA cells were exposed to hypoxia (1% O₂, 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (Akt/TAZ, Akt/NICD) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in Akt/TAZ livers. Results: Several EpiGs were upregulated in iCCA and eCCA, including writers (DNMT1, EZH2, SUZ12), readers (CBX3, PHF20L1, SMARCA4), and erasers (HDAC1, HDAC3, KDM5C). MGs in OCM, TCA, and ACS pathways were dysregulated (up: GART, IDH2, TYMS; down: ALDH1L1, MAT1A, SHMT1). Integrated analyses identified 27 EpiGs and 8 MGs whose overexpression predicted poor survival. Subsets of EpiGs, MGs, and RLEs were linked to proliferative, high-recurrence iCCA subclasses. CRISPR screens highlighted 50 EpiGs and 23 MGs essential for CCA viability. Tumor microenvironment (TME) analyses revealed distinct immune-stromal subclasses with coherent epigenetic-metabolic signatures. In CCA cells, hypoxia induced epigenetic programs that mirrored those in CCA patients. Transcriptomic analyses in human and multi-omic analyses (transcriptomic, metabolomic, and proteomic studies) in mouse CCA livers highlighted rewiring of nucleotide, one-carbon, lipid, and mitochondrial pathways, with evidence of metabolic-epigenetic crosstalk. Conclusion: EpiGs and MGs are markedly altered in both human and experimental CCA, with several changes particularly enriched in aggressive molecular subclasses associated with poor prognosis. We observed substantial rewiring of epigenetic cofactor-related MG expression in CCAs. Functional assays validated new targets among EpiGs (e.g. CBX3, CHD4, DEK, SMARCA4, and TRIM28) and MGs (TYMS and IDH2) in CCA.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB ( Naa20 ^-/- ) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB-mediated acetylation serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20 ^-/- MEFs exhibited reduced responsiveness to extrinsic pro-apoptotic stimuli, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway impacts apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.