Matias Antonio Avila

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 generated 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, and normal bile ducts, healthy liver-derived organoids, and CCA tumoroids, 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% O ₂ , 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (TAZ/Akt and NICD1/Akt) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in TAZ/Akt 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.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy characterized by poor prognosis, limited therapeutic options, and a predominantly immunosuppressive tumor microenvironment. Protein arginine methyltransferase 1 (PRMT1), the major mediator of asymmetric arginine dimethylation, has been implicated in multiple oncogenic processes, although its role in CCA remains unknown. Here, we demonstrate that PRMT1 is frequently overexpressed in human CCA and is associated with aggressive molecular subtypes and immune-desert tumors. Genetic dependency analyses and pharmacological inhibition using type I PRMT inhibitors markedly impaired CCA cell proliferation, clonogenicity, and tumoroid growth. Transcriptomic profiling revealed that PRMT1 inhibition induces broad alterations in gene expression and alternative splicing, affecting pathways involved in proliferation, apoptosis, DNA damage response, metabolism, and immune signaling. Mechanistically, PRMT1 targeting promoted genomic stress, accumulation of cytosolic double-stranded DNA, and activation of the cGAS-STING-TBK1-IRF3 signaling axis, resulting in enhanced interferon signaling and increased expression of T cell-recruiting chemokines, including CXCL9 and CXCL10. PRMT1 inhibition also synergized with cisplatin, poly-ADP-ribose polymerase (PARP) inhibition, and PRMT5 blockade in vitro and in patient-derived tumoroids. Importantly, in an aggressive orthotopic murine model of intrahepatic CCA, combined treatment with the PRMT1 inhibitor GSK3368715 and anti-PD-1 antibodies significantly reduced tumor burden and increased CD4+ and CD8+ T-cell infiltration compared with monotherapies. Collectively, these findings identify PRMT1 as a critical regulator of CCA growth and immune evasion and support the therapeutic potential of PRMT1 inhibition, particularly in combination with immunotherapy.

Background: Liver and lymph node endothelial cell C-type lectin (LSECtin) loss in liver sinusoidal endothelial cells (LSECs) during cirrhosis favors hepatic pro-inflammatory T-cell infiltration. We characterize histone protein post-translational modifications and DNA methylation in LSECs that may control LSECtin expression and evaluate methyltransferase inhibitors' capacity to retrieve LSECtin. Methods: DNA-methylation levels and histone post-translational modifications were studied by methylation array and ChIP-Seq experiments carried out on LSECs isolated by FACS from control and cirrhotic mice. Immortalized LSECs were treated with epigenetic effector inhibitory drugs (OTS186935-Suv39h2i, UNC0642-G9a/Glpi, UNC1999-EZH2/1i, 5-Aza-2’-deoxycitidine-DNMTi), alone and with IL-4, an LSECtin expression inducer. Drugs with regulatory potential were tested in vivo. LSECtin gene and protein expression, lymphocyte differentiation, and Th17-subpopulation proliferation were studied. Results: H3K9me3 and H3K27Me3 were reduced in LSECs of cirrhotic versus control mice. Suv39h2 and Ezh2/1 methyltransferase inhibitors OTS186935 and UNC1999, respectively, reduced Clec4g /LSECtin expression in immortalized LSECs. Murine primary LSECs showed H3K27me3 and H3K9me3 significantly enriched (controls) and reduced (cirrhotics) regions located upstream of the Clec4g promoter. OTS186935 and UNC1999 treated immortalized LSECs reduced H3K9me3 and H3K27me3 enrichment in these regions. Clec4g /LSECtin expression was also controlled by DNA methylation, as confirmed by Clec4g /LSECtin induction after 5-Aza-2’-deoxycitidine-DNMTi treatment in vitro and in vivo. Epigenetic drugs induced LSECtin in LSECs, contracted the hepatic Th17 compartment, attenuated hepatic structural damage, and improved liver function in cirrhosis. Conclusion: H3K9me3 and H3K27me3, and DNA-methylation regulate Clec4g expression in LSECs during cirrhosis. Inhibitory epigenetic-enzyme drugs increase Clec4g /LSECtin expression and provide evidence on relevant genomic regions for Clec4g reprogramming. The LSECtin-modulated hepatic Th17 compartment is contracted in cirrhosis by targeting these modifications.

Background & Aims: Hepatoblastoma (HB) is the most common malignant liver tumour in children. Despite improved survival in low‐risk disease, outcomes for advanced or relapsed HB remain poor, emphasising the need for new therapeutic targets. Hypoxia, a hallmark of aggressive tumours, has recently been implicated in HB pathogenesis, but the molecular mechanisms involved are unclear. This study aimed to characterise the hypoxia‐driven transcriptomic landscape of HB and identify key mediators of tumour progression. Methods: Transcriptomic analyses of HB cell lines cultured under normoxic and hypoxic conditions were combined with bioinformatic interrogation of public HB datasets, immunohistochemistry of human and murine tumours and plasma ELISA assays. Functional roles of trefoil factor 3 (TFF3) were evaluated through overexpression and shRNA‐mediated knockdown in vitro and in a β‐catenin/YAP‐driven mouse model of HB. Results: Hypoxia induced broad transcriptional reprogramming in HB cells, including significant upregulation of TFF3 , a secreted oncogenic peptide. TFF3 expression was elevated in HB tissues and plasma, and colocalized with hypoxia marker carbonic anhydrase 9 (CA9). TFF3 promoted proliferation, anchorage‐independent growth and cisplatin resistance under both normoxia and hypoxia. Knockdown of murine Tff3 suppressed tumour formation and angiogenesis in vivo. Transcriptomic and molecular analyses revealed that TFF3 sustains C‐MYC expression and modulates mTOR/GSK3β signalling. Conclusions: TFF3 is a hypoxia‐inducible factor that enhances HB cell proliferation, survival and chemoresistance. Its tumour‐promoting activity through C‐MYC and mTOR pathways identifies TFF3 as a potential therapeutic target and circulating biomarker in hepatoblastoma.

Background/Objectives: HEBERSaVax is a therapeutic cancer vaccine based on recombinant human VEGF antigen adjuvated with VSSP or Aluminum Phosphate (AP). Clinical trials demonstrated the vaccine’s safety and tolerability, with predominantly mild to moderate (grade 1–2) local adverse events. Initial dose optimization studies using the VSSP (Center of Molecular Immunology (CIM), Havana, Cuba) adjuvant showed that increasing the antigen dose to 800 μg significantly enhanced immunogenicity, as measured by improved seroconversion rates, stronger blockade of VEGF/VEGFR1-2 interactions, and reduced platelet-derived VEGF levels. Methods: The AP adjuvant was used to perform essential preclinical validation in non-human primates to support the transition of the 800 μg antigen dose to Phase II clinical trials (CENTAURO-4 and CENTAURO-6). Results: HEBERSaVax adjuvated with AP induced: (1) robust humoral responses with high-titer anti-VEGF antibodies (peak 1:15,000), (2) functional biological activity, specifically the suppression of VEGF-mediated signal transduction, in 90% (9/10 animals), and (3) measurable cellular immune responses. All immunogenic effects were achieved without evidence of systemic toxicity, confirming the safety profile of this preparation. Conclusions: These findings provide compelling preclinical evidence that the 800 μg HEBERSaVax/AP combination maintains the immunogenic potential previously observed with VSSP while demonstrating an equally favorable safety profile. The results strongly support continued clinical development of this VEGF-targeted immunotherapy for cancer treatment.

In the present study, we investigated the role of the gut-liver crosstalk in the pathogenesis of steatotic liver disease (SLD) induced by the compounding and deleterious effects of alcohol and metabolic risk factors, and explored the potential translational aspects of microbiome-based interventions. The effects of combined exposure to alcohol and a high-fat, high-cholesterol diet (HFHC) Western diet (WD) were tested in a dietary mouse DUAL model and compared to mice fed only with WD. Liver and gut phenotypes were evaluated via histochemistry, flow cytometry, gene expression, proteomic, and metabolomic analyses. The effects on the gut microbiota were studied in both DUAL mice and MASLD patients with a history of alcohol consumption. Antibiotic-induced microbiota depletion (AIMD) and microbiota modulation therapies (probiotics and fecal microbiota transplant (FMT)) were performed in mice. Primary human hepatocytes and HepG2 cells were used to study the underlying mechanisms. Zebrafish larvae exposed to alcohol and a HFHC diet were used as a validation model. Alcohol in combination with WD synergistically exacerbated SLD. DUAL-diet-induced disruption of the intestinal barrier led to LPS leakage into the bloodstream and subsequent TLR4-mediated hepatic inflammation. This, together with enhanced intestinal fat absorption, and impaired intrahepatic lipid oxidation - particularly due to insufficient CPT-1 activity - contributed to prominent steatohepatitis. The DUAL-induced changes in the gut microbiota showed similarities to human dysbiosis in MASLD patients who consumed alcohol, including an increase in Bacteroides and Alistipes. AIMD improved pathology, indicating a causal role of the microbiota in the pathophysiology of DUAL steatohepatitis, whilst early microbiome modulation via FMT induced mild improvements in liver and gut physiology. These results indicated that the microbiota‒gut‒liver axis plays a crucial role in the progression of SLD intensified by alcohol and concurrent metabolic risk factors, thus providing a promising translational target for potential therapeutic interventions.

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.