Targeted radiotherapy is integral to the increasing survival of cancer patients; however, it has significant side effects, the underlying cellular and molecular mechanisms of which are ill-defined. It is well documented that targeted radiotherapy induces epigenetic changes in neoplastic tissue, which impacts tumour evolution; however, whether epigenetic deregulation also occurs in the surrounding non-neoplastic tissue and contributes to the occurrence of side effects is unknown. We characterized the DNA methylome in a unique cohort of irradiated peri-lesional brain tissue samples and integrated it with gene expression analysis at the spatial level. We show differences in DNA methylation patterns in irradiated brain tissue and identify specific inflammatory micro-environmental niches and their regulatory neuropeptides after irradiation. Finally, we show in a cerebral organoid model, that the same neuropeptides are upregulated as well as similar DNA methylation alterations and disruption of the DNA methylation machinery, in keeping with the interpretation that epigenetic dysregulation plays a role in neurotoxicity, hence raising the possibility it could represent a novel target for the reduction of radiotherapy side effects.

BackgroundGlioblastoma is the most common and aggressive malignant brain tumour in the adult population and its prognosis is dismal. The heterogeneous nature of the tumour, to which epigenetic dysregulation significantly contributes, is among the main therapeutic challenges of the disease.ResultsWe have leveraged SYNGN, an experimental pipeline enabling the syngeneic comparison of glioblastoma stem cells and expanded potential stem cell (EPSC)-derived neural stem cells to identify regulatory features driven by chromatin remodelling specifically in glioblastoma stem cells.ConclusionsWe show epigenetic regulation of the expression of genes and related signalling pathways contributing to glioblastoma development. We also identify novel epigenetically regulated druggable target genes on a patient-specific level, including SMOX and GABBR2.

Abstract Disclosure: K. Laycock: None. C. Cabrera: None. E. Wozniak: None. E. Ng: None. X. Wu: None. E. Goodchild: None. E.A. Azizan: None. J. Boot: None. C. Mein: None. J. MacFarlane: None. M. Gurnell: None. W. Drake: None. M.J. Brown: None. In a recent trial, adrenalectomy achieved complete clinical success at 6 months in only 30% of 78 patients with unilateral primary aldosteronism (PA) (Nat Med 2023 29:190). We have now compared influence of common APA genotypes on success at 2 years, and sought different cells of origin to explain the result. Methods: Home BP, plasma aldosterone and renin were re-measured 2 years after surgery. APA genotype and transcriptome were analysed by RNAseq. Single-nucleus (Sn) RNAs from 17 fresh-frozen adrenals and 9 APAs (3 each with CACNA1D or KCNJ5-mutation), were quantified by 10x Chromium, and cells with mutations identified by targeted amplicon analysis. Unusual cells were confirmed by immunofluorescence (IF), and their origin sought in published datasets for other tissues. Results: 14/18 patients with KCNJ5-mutation, but 0/20 with CACNA1D, had BP <135/85 mmHg off treatment at 2 years (p=0.03, logistic regression on genotype, age, gender, ethnicity). Biochemical success rate was also lower in CACNA1D-mutant patients. The most upregulated gene in CACNAID vs KCNJ5-mutant APAs was the endothelial-progenitor CCM2L (33-fold, p=10-26), followed by neuronal and adrenomedullary genes e.g. PTPRZ1, SLC35F1, UNC79, by 13-17-fold, p=10-[1]0-[1]2). Transcripts of these genes, in Sn analyses of 85203 nuclei from 17 non-tumour samples, clustered in CYP11B2-expressing cells, along with genes characteristic of aldosterone-producing micronodules (APM). The APM cluster was downstream of medulla on pseudotime analysis. In Sn analysis of 9 APAs, CCM2L was highly differentiated in CYP11B2+ cells from 3 CACNA1D- vs 3 KCNJ5-mutants (p=10-[1]06) along with PTPRZ1 (p=10-232), SLC35F1 (p=0) and many other neuronal/medullary transcripts. UMAPs of APAs and adjacent adrenal both revealed a cluster of hybrid cells expressing CCM2L or other endothelial genes, plus CYP11B2 and other zona glomerulosa (ZG) or APM-selective transcripts. The proportion of hybrid cells was higher in CACNA1D-mutant APAs (236/803) and adjacent adrenal (37/946) than KCNJ5-mutant (101/954) and adjacent tissue (8/766). By contrast, CYP11B2+ cells from KCNJ5-mutant APAs were enriched for transcripts in the CYP11B2- ZG-cell cluster of adjacent adrenal. Amplicon analysis of APAs detected CACNA1D mutation in multiple cells of the endothelial cluster. IF confirmed cells expressing CYP11B2 and endothelial transcripts. Mining of GTEX, and Human Cell Atlas led to a cluster of CD34+RENBP+ endothelial progenitors in spleen as likely source of CCM2L+ cells (Gen Biol 2019 21:1). Conclusion: Somatic genotype influences long-term outcome after adrenalectomy for PA, reflecting origin of CACNA1D-, but not KCNJ5-, mutant APAs from APMs. Bilateral APMs have been inferred from their occurrence in most PA adrenals (Nat Rev Neph 2023 19:788). This is supported by our finding of CCM2L+CYP11B2+ hybrid cells, with a likely circulatory origin from spleen. Presentation: 6/3/2024

Background:Platelet function is driven by the expression of specialized surface markers. The concept of distinct circulating subpopulations of platelets has emerged in recent years, but their exact nature remains debatable. Objectives:To design a spectral flow cytometry-based phenotyping workflow to provide a more comprehensive characterization, at a global and individual level, of surface markers in resting and activated healthy platelets, and to apply this workflow to investigate how responses differ according to platelet age. Methods:A 14-marker flow cytometry panel was developed and applied to vehicle- or agonist-stimulated platelet-rich plasma and whole blood samples obtained from healthy volunteers, or to platelets sorted according to SYTO-13 (Thermo Fisher Scientific) staining intensity as an indicator of platelet age. Data were analyzed using both user-led and independent approaches incorporating novel machine learning-based algorithms. Results:The assay detected differences in marker expression in healthy platelets, at rest and on agonist activation, in both platelet-rich plasma and whole blood samples, that are consistent with the literature. Machine learning identified stimulated populations of platelets with high accuracy (>80%). Similarly, machine learning differentiation between young and old platelet populations achieved 76% accuracy, primarily weighted by forward scatter, cluster of differentiation (CD) 41, side scatter, glycoprotein VI, CD61, and CD42b expression patterns. Conclusion:Our approach provides a powerful phenotypic assay coupled with robust bioinformatic and machine learning workflows for deep analysis of platelet subpopulations. Cleavable receptors, glycoprotein VI and CD42b, contribute to defining shared and unique subpopulations. This adoptable, low-volume approach will be valuable in deep characterization of platelets in disease.

Introduction: Intergenerational inheritance of stress is the transmission of acquired exposures down to the next generation. Complete parental rejection of newborns in captive primate colonies is considered a severe form of early life stress and widespread across globe. Intergenerational effects associated with parental rejection are uncharacterised but could have significant impact on progeny, affecting their welfare. Methods: We investigated intergenerational inheritance of rejection in a species where parental rejections occurs spontaneously in captivity (Aotus nancymaae), and its effects across 2 generations. We analysed demographic records (n=1389), observed stress and affiliative behaviours (n=65, 10 families, 2 generations), and profiled miRNAs expression in blood (48 from 10 families, 2 generations). Results: Rejected individuals showed significantly reduced lifespan, and a lower probability of reaching reproductive age and increased rates of clinic visits throughout life. Remarkably, well-reared descendants of rejected animals showed a similar reduction in surviving to reproductive age and lifespan as seen in their parents. Behavioural analysis revealed persistent alterations in stress reactivity and social behaviour, with rejected adults showing significant increase in fear aggression and reduced infant care, and infants showing reduced locomotion and biting, but also positive stress coping. Blood miRNA analysis revealed no significant differences between rejected and controls, but rejected female offspring, showed a significant upregulation of mml-miR-30a-5p an miRNA upregulated in the plasma of humans who after exposure to early life stress, suffer from behavioural disorders. Discussion: Our findings demonstrate that parental rejection is associated intergenerational effects on stress and affiliative behaviours, reduced survival, and miRNA expression, suggesting intergenerational inheritance of stress. This report contributing to understanding developmental origins of ill health and mortality in captive primates. ### Competing Interest Statement The authors have declared no competing interest.

Analysis of chromatin remodelling in neoplastic stem cells as compared to ontogenetically related neural stem cells, reveals multifactorial epigenetic regulation of signalling pathways known to contribute to glioblastoma development. It also identifies novel epigenetically regulated druggable target genes on a patient-specific level, including SMOX and GABBR2 which could be further developed for future translational approaches to more effectively treat this neoplasm.

Introduction Non-alcoholic steatohepatitis (NASH) is a common, progressive inflammatory liver condition with no approved therapies. Hepatocyte lipotoxicity results in inflammation, immune (T cell) infiltration and stellate cell activation leading to fibrosis. Hepatic Toll-like receptors (TLR) sense gut-derived inflammatory signals such as lipopolysaccharide (via TLR4) and flagellin (TLR5) and may represent therapeutic targets in NASH. Flagellin and TLR5 have been implicated in murine NASH models and here we test the hypothesis that this pathway plays a pathogenic role in human disease. Methods Plasma TLR5 binding capacity and flagellin, stool Flic gene load (shotgun metagenomics sequencing) and hepatic TLR5 gene expression were measured in samples from 139 patients and 24 controls recruited from outpatient clinics and elective bariatric surgery. Lipotoxicity was modelled in vitro using oleic (1mM) and palmitic (0.5mM) acid in human hepatocyte-like (HepG2) and stellate (LX2) cells. Results Compared to controls, plasma TLR5-binding capacity was increased in advanced NASH fibrosis but not earlier stages of disease (TLR4-binding increased at all disease stages) as was flagellin concentration (538.3v 745.8pg/ml,p=0.004) which normalised in samples taken median 83 days following bariatric surgery (n=20, p=0.005). Stool Flic gene expression and hepatic TLR5 (but not TLR2 or TLR4) expression were increased in NASH, along with markers of intestinal permeability (FABP2, D-lactate). In vitro, TLR5 inhibition attenuated toxic lipid-mediated IL8 protein expression (all p<0.001, vs control) in HepG2. TLR5 inhibition also attenuated both flagellin- and toxic lipid-induced IL8 production in LX2 cells by 1.9-fold (p=0.019) and 2.1-fold (p=0.032) respectively. Although neither flagellin nor toxic lipids directly induced pro-collagen 1a1 production in LX2 cells, lipid-injured HepG2-conditioned media induced 3.2-fold increase in pro-collagen 1a1 production compared to control (p=0.0015). TLR5 inhibition in HepG2 cells prior to media transfer led to a reduction in LX2 pro-collagen 1a1 production (p=0.030). Similarly, lipid-injured HepG2-conditioned media induced Th1 differentiation of naïve T cells from healthy donors in a TLR5-dependent manner (p<0.001). Conclusions TLR5 signalling is activated in human NASH, reverses following bariatric surgery and is associated with mechanisms of lipid-mediated inflammation and stellate cell activation. This pathway has potential as a novel therapeutic target in NASH.

Abstract The alteration of the glioblastoma genome by epigenetic mechanisms that share functions with normal developmental processes, such as self-renewal and fate specification of NSC, is a key piece of evidence that links brain cancer pathogenesis with dysregulated stem cell functions. A patient-specific comparison of glioblastoma cells with NSC, a putative cell of origin of at least a proportion of these tumours, is not feasible as patient-matched endogenous NSC are not surgically accessible and all epigenetic studies in glioblastoma have so far compared epigenetic changes of different tumours with each other or to comparators obtained from foetal brains or an unrelated donor. We reasoned that availability of syngeneic GSC and NSC pairs would allow to identify crucial epigenetic differences on a patient-specific basis and would provide essential therapeutic contrast to define disease-and patient-intrinsic biomarkers of drug response that are less confounded by germline variation. We have derived GSC from IDH-wildtype glioblastoma and harnessed state-of-the-art stem cell technologies to generate patient-matched fibroblast-derived EPSC, which were induced to NSC (iNSC). We demonstrate that integrated analysis of the transcriptome and DNA methylome of GSC/iNSC pairs identifies druggable target genes (PTGER4, ALDH3B1, NTRK2) in a proportion of patients and we validate this patient-specific prediction of drug response at pre-clinical level in 3D synGLICO and in in vivo models. Integration of small RNA regulation to the pipeline allows refined in silico prediction of drug responses on a patient-specific basis. Most recently we have added assessment of chromatin remodelling (H3K4me3, H3K27ac, H3K3me3, H3K27me3 and ATACSeq) to the pipeline and shown that redistribution of selected histone marks as well as shifts in chromatin states across the genome identifies known and novel druggable regulatory mechanisms in GSC, which are specific of the neoplastic context. In conclusion, SYNGN is an epigenetic platform which can identify patient-specific druggable targets in glioblastoma.

Introduction The time it takes for food to travel from the oral cavity until it is expelled in stool is known as the whole-gut transit-time (WGTT). The composition of the gut microbiome and its metabolites are major contributors in gut health. Indeed, alterations in the gut microbiome, bile acid levels, and WGTT were reported in various gastrointestinal disorders including irritable bowel syndrome, Parkinson's disease, bile acid diarrhoea and colorectal cancer. However, the impact of the WGTT on the microbiome and bile acid metabolism has yet to be established. Methods Healthy volunteers were administered Loperamide and Senna. Each for a total of 6 days, in random order, separated by an interval of at least 4 weeks. The microbiome and the bile acid composition were analysed in stool samples, and markers of bile acid synthesis were analysed in blood samples. Results Administration of Senna and Loperamide resulted in the desired outcomes: a decrease and increase of the WGTT respectively, as well as alteration to the stool form, the frequency of bowel movements, and the stool weight. The Senna treatment group had a significant increase of both primary (p=0.003) and secondary (p=0.008) bile acids in the stool, while the blood concentration of the ileal hormone fibroblast growth factor 19, which regulates bile acid synthesis, was significantly reduced compared to the Loperamide treatment group. Slowing WGTT by administration of Loperamide increased levels of bacterial bile acid transforming genes namely, bile salt hydrolase genes (p=0.02), as well as bacterial species richness (p=0.04). More specifically, an over 20-fold increase of the probiotic specie Bifidobacterium dentium as well as Bifidobacterium angulatum. By contrast, administration of Senna resulted in a 6-fold elevation of species specifically associated with dysbiosis and bile acid metabolism such as Ruminococcus gnavus, while B. dentium decreased 23-fold. WGTT was negatively correlated with total primary and secondary bile acids (specifically chenodeoxycholic acid, ursodeoxycholic acid and glycochenodeoxycholic acid), the presence of Gemella sanguinis and R. gnavus, while species richness significantly decreased. Thus, changes in WGTT impacted both the composition of the microbiome and bile acid metabolism, however both reverted back to their original condition within 21 days of finishing treatments. Conclusion Our study provides strong evidence that changes in WGTT are a contributing factor to dysbiosis and bile acid imbalance. Our findings suggest that gut transit time should be considered a crucial factor in future microbiome research, as they show changes in gut transit time can have a major effect on microbiome composition and function, independent from gastro-intestinal disease.

We describe a subset of glioblastoma, the most prevalent malignant adult brain tumour, harbouring a bias towards hypomethylation at defined differentially methylated regions. This epigenetic signature correlates with an enrichment for an astrocytic gene signature, which together with the identification of enriched predicted binding sites of transcription factors known to cause demethylation and to be involved in astrocytic/glial lineage specification, point to a shared ontogeny between these glioblastomas and astroglial progenitors. At functional level, increased invasiveness, at least in part mediated by SRPX2, and macrophage infiltration characterise this subset of glioblastoma.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM.