Aims Chemotherapy resistance remains a major challenge in breast cancer (BC) treatment. This study aimed to investigate the role of DNA methylation in this complex process and evaluate the potential of the DNA methyltransferase inhibitor decitabine (DAC) in restoring chemosensitivity. Methods Paclitaxel (PAC)- and doxorubicin (DOX)- resistant BC cell lines were derived from luminal A (T-47D), triple-negative (MDA-MB-231), and HER2-positive (JIMT-1) models and characterized by molecular profiling and functional assays. The therapeutic effects of DAC and DOX were assessed in MDA-MB-231 xenografts, and integrative analyses of DNA methylation and gene expression identified pathways associated with resistance. Follow-up analyses were performed in PAC-resistant MAS98.12 patient-derived xenografts (PDX) and in clinical samples from the NeoAva trial (NCT00773695). Results Resistant cells exhibited a slow-cycling phenotype, reduced tumorigenicity, and widespread genomic alterations. PAC-resistant xenografts showed extensive methylation and transcriptomic reprogramming, partly restored by DAC, which increased Ki-67 expression and enhanced DOX responsiveness. In contrast, PDX tumors displayed less pronounced changes, predominantly hypomethylation, indicating distinct resistance mechanisms. Importantly, xenograft-derived CpG signatures stratified NeoAva patients by treatment response. Conclusions Chemoresistance in BC involves extensive genomic and epigenetic remodeling. Although DAC can modulate methylation and tumor phenotype, rational drug combinations will be required to overcome resistance.

Unique features of noble metal nanomaterials make them indispensable in advanced technologies, smart devices, and, in particular, biomedical applications. However, their prolonged persistence in the body, resulting from limited solubility and inefficient excretion, raises concerns about potential adverse health effects. Despite their widespread use, studies on the long-term biological impact in vivo remain limited, particularly regarding chronic exposure and delayed physiological responses. To elucidate the biological processes triggered by noble metal nanomaterials, we investigated immune re-sponses and gene expression dynamics up to 28 days in rats exposed to polyethylene glycol-coated gold nano-particles (GNPs, similar to 10 nm). GNPs were administered as a single intravenous (iv.) dose of 0.7 mg/kg, and the acquired biological data were related to the amount of GNPs accumulated in individual tissues. Immunosup-pressive effects emerged within 24 h of exposure. Gene expression alterations were organ-specific, time-dependent, and related to GNP levels, with the liver exhibiting the most pronounced changes, followed by the lungs and kidneys. At 24 h post-exposure, deregulated genes were predominantly related to inflammatory, im-mune, and detoxification pathways, suggesting the effective activation of defense mechanisms. By days 7 and 28, aberrant gene expression expanded to pathways involved in cell migration, adhesion, lipid metabolism, and fibrosis, all of which are implicated in long-term health outcomes. These findings underscore the necessity for more advanced and precise methodologies to assess the biological safety of nanoparticles with prolonged retention. Understanding nano-bio interactions, especially long-term ef-fects, is essential for identifying potential risks and developing safer-by-design nanomaterials for biomedical and environmental applications.

The extreme lethality and limited treatment options for pancreatic ductal adenocarcinoma (PDAC) underscore the urgent need for innovative therapeutic strategies. This study presents the first preclinical investigation of a cell-free gene-directed enzyme prodrug therapy (GDEPT) based on conditioned medium (CM) from placenta-derived mesenchymal stem cells (PlacMSCs) engineered to express the yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT) fusion enzyme. The CM was concentrated tenfold (cCM) and characterized by proteomics, transmission electron microscopy, and Western blotting, confirming extracellular vesicle (EV) enrichment and UPRT expression. Therapeutic efficacy was evaluated in coculture models comprising PDAC cell lines (BxPC-3, MIA PaCa-2, SU.86.86), patient-derived xenograft organoids (PDXOs), and cancer-associated fibroblasts (PCAFs). Immunocytochemistry and Western blot analyses revealed a predominant myofibroblastic CAF phenotype, characterized by strong alpha-smooth muscle actin (αSMA) expression and low interleukin-6 levels. Treatment with yCD::UPRT-PlacMSC-cCM in the presence of 5-fluorocytosine (5-FC) enabled efficient enzymatic conversion to 5-fluorouracil (5-FU), yielding 10 μg/mL from an initial 100 μg/mL of 5-FC. This resulted in robust, dose-dependent cytotoxicity (50 % to 80 % reduction in viability) across monocultures and stromal-rich cocultures, effectively overcoming PCAF-mediated drug resistance. Therapeutic response was governed primarily by tumor cell characteristics rather than PCAF heterogeneity. In PDXOs derived from two early-stage (IA, IIB) primary tumors and one metastatic lesion, 100 µL of yCD::UPRT-PlacMSC-cCM induced cytotoxicity comparable to 1 μg/mL of 5-FU, while 25 µL was insufficient to significantly reduce viability. Collectively, these findings demonstrate that yCD::UPRT-PlacMSC-cCM delivers potent, stromal-bypassing cytotoxicity in PDAC models and represents a promising cell-free therapeutic approach for this treatment-refractory cancer.

Background:Heterozygous variants in CTNND2, encoding the brain-specific protein δ-catenin, are associated with a broad spectrum of neurodevelopmental disorders, including dyslexia, attention deficit hyperactivity disorder, intellectual disability, and autism. Despite its clinical significance, the full phenotypic spectrum of CTNND2-associated disorders and the neurodevelopmental role of δ-catenin, a key component of the cadherin-catenin cell adhesion complex, remain poorly defined. Methods:Through international collaboration, we assembled the phenotypic and molecular information for 57 individuals, 42 previously unpublished, carrying heterozygous CTNND2 variants. All individuals were evaluated by local clinicians, and the variants were identified through exome or genome sequencing, clinical microarray, or karyotyping. To investigate the effects of δ-catenin loss on early neurogenesis, we performed neural differentiation and transcriptomic profiling in three patient-derived neural stem cell lines and three CRISPR-Cas9-generated CTNND2 knockout lines. In one patient-derived line, we further analyzed cerebral organoid development and performed pathway modulation to assess phenotypic rescue. Results:The 41 CTNND2 variants included 12 previously reported loss-of-function- and one missense variant, and 28 novel variants comprising 10 missense and 18 predicted loss-of-function changes. Eight of the novel variants occurred de novo, and 12 were inherited from a parent with a neurodevelopmental phenotype. The most common clinical features were developmental delay (90%), intellectual disability (74%), and behavioral abnormalities (79%). Functional studies revealed impaired early neurogenesis in one patient-derived line, characterized by aberrant neural rosette formation. Transcriptome analysis showed dysregulated WNT signaling, and partial rescue of these defects was achieved by modulating the WNT pathway, highlighting δ-catenin's role in early neural development. Conclusions:This study defines the clinical symptoms of CTNND2-related neurodevelopmental disorders, outlining a recognizable yet variable phenotype that overlaps with other forms of intellectual disability and autism. Our findings provide preliminary evidence of genotype-phenotype correlations and highlight δ-catenin's critical role in modulating WNT signaling during early neural development. These insights advance our understanding of CTNND2-associated disorders and support the importance of mechanistic studies to inform personalized diagnostics and therapies.

Aims Chemotherapy resistance remains a major challenge in breast cancer (BC) treatment. This study investigated whether resistance development is associated with DNA methylation changes and assessed the potential of the DNA methyltransferase inhibitor decitabine (DAC) to reverse these alterations and enhance chemosensitivity. Methods Molecular profiling and functional assays were used to characterize paclitaxel-(PAC) and doxorubicin-(DOX) resistant BC cell lines derived from luminal A (T-47D), triple-negative (MDA-MB-231), and HER2-positive trastuzumab-resistant (JIMT-1) models. Therapeutic responses to DAC and DOX, alone and in combination, were evaluated in MDA-MB-231 xenografts. DNA methylation–associated gene expression changes were analyzed through integrative approaches. Results Chemoresistant cells exhibited a slow-cycling phenotype, reduced tumorigenicity, and extensive genomic alterations. Upregulation of RELB and downregulation of PPARG were observed across several resistant cell lines, while CDA expression was uniformly elevated in all DOX-resistant models. PAC-resistant xenografts displayed widespread methylation and transcriptomic reprogramming. DAC treatment partially restored aberrant methylation patterns and increased Ki-67 expression, potentially enhancing DOX responsiveness. Conclusions Chemoresistance in BC involves extensive genomic and epigenetic reprogramming. DAC modulates methylation and tumor phenotype but is insufficient to overcome resistance, highlighting the need for rational combination strategies. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Ministry of Education, Science, Research and Sport of the Slovak Republic European Commission, https://ror.org/00k4n6c32 European Union French National Research Agency [1]: pending:yes

Purpose: Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, with prognosis significantly influenced by genetic and epigenetic factors. Reliable and cost-effective methods to detect chromosomal aberrations and DNA methylation changes are essential for improving prognostication and informing treatment strategies in UM. This study evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting UM-specific copy number variations (CNVs) and promoter methylation changes across 25 tumor suppressor genes (TSGs). Methods: DNA from 58 UM tissues was analyzed with the SALSA MLPA Probemix P027 Uveal melanoma kit, and a subset of 18 samples was further assessed using the SALSA MLPA Probemix ME002-C1 Tumour suppressor mix 2 kit to identify key CNVs and methylation alterations linked to poor prognosis. Validation was carried out with a high-resolution comparative genomic hybridization (CGH) array on 10 samples and the Illumina Infinium Methylation EPIC v1.0 BeadChip array on 25 samples. Results: Our findings indicate that MLPA is a versatile and robust method for detecting CNVs, showing strong correlations with CGH data and highlighting specific CNV patterns linked to clinical outcomes in UM. However, the ME002-C1 kit showed limited utility for comprehensive methylation analysis, as differential methylation was not observed in the studied TSG loci. Conclusions: Although MLPA effectively identifies CNVs relevant to UM prognosis, integrating additional methylation-specific approaches could broaden the scope of DNA methylation analysis, offering a more comprehensive molecular understanding of UM that may enhance prognostication and personalized treatment.

Developmental Delay with Gastrointestinal, Cardiovascular, Genitourinary, and Skeletal Abnormalities syndrome (DEGCAGS, MIM #619488) is caused by biallelic, loss-of-function (LoF) ZNF699 variants, and is characterized by variable neurodevelopmental disability, discordant organ anomalies among full siblings and infant mortality. ZNF699 encodes a KRAB zinc finger protein of unknown function. We aimed to investigate the genotype-phenotype spectrum of DEGCAGS and the possibility of a diagnostic DNA methylation episignature, to facilitate the diagnosis of a highly variable condition lacking pathognomonic clinical findings. We collected data on 30 affected individuals (12 new). GestaltMatcher analyzed fifty-three facial photographs from five individuals. In nine individuals, methylation profiling of blood-DNA was performed, and a classification model was constructed to differentiate DEGCAGS from controls. We expand the ZNF699-related molecular spectrum and show that biallelic, LoF, ZNF699 variants cause unique clinical findings with age-related presentation and a similar facial gestalt. We also identified a robust episignature for DEGCAGS syndrome. DEGCAGS syndrome is a clinically variable recessive syndrome even among siblings with a distinct methylation episignature which can be used as a screening, diagnostic and classification tool for ZNF699 variants. Analysis of differentially methylated regions suggested an effect on genes potentially implicated in the syndrome’s pathogenesis.

AIM:Circular DNA segments TREC (T-cell receptor excision circles) formed during T-lymphocyte maturation in the thymus, are a sensitive marker of thymic lymphocyte production in a broader manner. Quantification using qPCR is proposed as a surrogate marker of T cell malfunction in various primary and secondary conditions in a non-SCID selected risk newborn population. METHODS:We collected 207 dry blood spot samples during the years 2015-2018, from newly admitted risk newborns. TREC values calculated per 106 cells were determined and a cut-off values of 5th percentile was set. The positive control group consisted of patients (n=13) with genetically confirmed SCID. RESULTS:The median TREC value was 34,591.56 (18,074.08-60,228.58) for girls resp. 28,391.20 (13,835.01-51,835.93) per 106 cells for boys, P=0.046. Neonates born by C-section have been found to have higher TREC levels compared to neonates born by spontaneous delivery (P=0.018). In the group of preterm newborns (n=104), 3.8% had TREC value < 5th percentile, half of them died due to sepsis as opposed to no fatalities in preterm newborns with sepsis and TREC value > 5th percentile. In the group of term newborns (n=103) 9 children (8.7%) had TREC < 5th percentile, half of them were treated for asphyxia, with no fatal complications. CONCLUSION:TREC levels calculated for the 5th percentile of a risk neonatal group is suggested as a surrogate marker for increased risk of fatal septic complication. Early recognition of these newborns within a risk scoring system using TREC levels could lead to potentially lifesaving interventions.

Suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) ER-associated degradation (ERAD) plays a critical role in many physiological processes in mice, including immunity, water homeostasis, and energy metabolism; however, its relevance and importance in humans remain unclear, as no disease variant has been identified. Here, we report a biallelic SEL1L variant (p. Cys141Tyr) in 5 patients from a consanguineous Slovakian family. These patients presented with not only ERAD-associated neurodevelopmental disorders with onset in infancy (ENDI) syndromes, but infantile-onset agammaglobulinemia with no mature B cells, resulting in frequent infections and early death. This variant disrupted the formation of a disulfide bond in the luminal fibronectin II domain of SEL1L, largely abolishing the function of the SEL1L-HRD1 ERAD complex in part via proteasomal-mediated self destruction by HRD1. This study reports a disease entity termed ENDI-agammaglobulinemia (ENDI-A) syndrome and establishes an inverse correlation between SEL1L-HRD1 ERAD functionality and disease severity in humans.

The 3-mercaptopyruvate sulfurtransferase (MPST) is a protein persulfidase, occurring mainly in mitochondria. Although function of this protein in cancer cells has been already studied, no clear outcome can be postulated up to now. Therefore, we focused on the determination of function of MPST in colon (HCT116 cells)/colorectal (DLD1 cells) cancers. In silico analysis revealed that in gastrointestinal cancers, MPST together with its binding partners can be either of a high risk or might have a protective effect. Silencing of MPST gene resulted in decreased ATP, while acetyl-CoA levels were elevated. Increased apoptosis was detected in cells with silenced MPST gene, which was accompanied by decrease in mitochondrial membrane potential, but no changes in IP3 receptor’s protein. Mitochondria underwent activation of fission and elevated DRP1 expression after MPST silencing. Proliferation and migration of DLD1 and HCT116 cells were markedly affected, showing the importance of MPST protein in colon/colorectal cancer development.

Conventional toxicological methods and integrated transcriptomic analysis were used to comprehensively assess the potential health hazard of residual metal nanoparticles accumulated in the body due to poor solubility.

Aims/Purpose: It has been observed that large UM lesions with significant infiltration of macrophages and CD8+ T cells exhibit specific genetic profiles that increase the risk of liver metastasis. Additionally, recent discoveries of DNA methylation changes in Class 2 tumours undergoing metastasis suggest a potential role of epigenetic alterations in UM progression. Methods: To identify factors involved in UM metastasis, we implanted 25 viable UM tumours (consisting of 11 Class 1 tumours with a good prognosis and 14 Class 2 tumours with a poor prognosis) subcutaneously into immunodeficient NSG mice. From these, we successfully established ten patient‐derived xenografts (PDXs) using Class 2 tumour tissues. Two metastatic PDXs were derived from liver (UM50) and peritoneal (UM58) metastases spontaneously developed on mice. Results: Comprehensive characterization of both primary tumours and PDX tissues was performed using bulk transcriptomics and methylomics techniques. Among the enriched metabolic pathways identified in the liver metastasis‐derived PDX tissue of sample UM50 compared to the PDX tissue from the primary tumour, four were related to epigenetic regulation, including the activation of rRNA expression by ERCC6 and EHMT2, methylation of histones and DNA by Polycomb Repressive Complex 2 (PRC2), regulation of rRNA expression by NAD‐dependent Deacetylase Sirtuin‐1, and the DNA methylation pathway. Conclusions: These findings highlight the potential importance of epigenetic deregulation in UM progression and provide a foundation for future investigations. Further analysis using the Chromium Single Cell ATAC platform to examine chromatin accessibility at the single‐cell level in primary and metastatic PDX tissues of UM50 and UM58 that are ongoing can contribute to unravelling the precise role of epigenetic alterations in UM metastasis. Continued research in this area may lead to the development of novel therapeutic strategies targeting epigenetic mechanisms and improving outcomes for patients with metastatic UM.

Abstract Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between -0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, DNA methylation values of nine genes, HTR2B , AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2 , and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUC ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining A HNAK2, CALHM2, and IL12RB and Signature 2 A HNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (HR = 4.38, 95% CI 1.30-16.41, HR = 5.59, 95% CI 1.30-16.41; HR = 3.43, 95% CI 1.30-16.41, HR = 4.61, 95% CI 1.30-16.41 and HR = 4.95, 95% CI 1.39-17.58, respectively). Conclusions: Our results demonstrate a significant role of DNA methylation aberrancy in UM progression. The advantages of DNA as a biological material and excellent prediction accuracies of methylation markers open the perspective for their more extensive clinical use.

DEAR EDITOR Uveal melanoma (UM) is a rare, aggressive cancer with limited treatment options. Despite significant advancements in understanding its genetic background,1, 2 the precise contribution of epigenomic alterations to the pathogenesis and progression of the disease remain elusive. In this study, we utilized a carefully curated set of UM samples to define the epigenomic and transcriptomic landscapes of high-risk tumours and identify novel, clinically relevant methylation markers and therapeutic targets. We stratified UM patients into risk groups based on UM-specific chromosomal rearrangements, particularly monosomy 3 (M3) and BAP1 mutations in tumour tissues (Figure 1A). Please refer to the Supporting information for a detailed description of patient clinical characteristics and methods (Additional file 1: Tables S1-S3). Specifically, we identified 25 low-risk and 33 high-risk patients, of which 21 (63.6%) carried the BAP1 mutation (Figure 1B, C). We observed extensive gene expression reprogramming in high-risk UMs, resulting in 2262 differentially expressed genes (DEGs) including 60 epigenetic regulators, histone modifiers and chromatin remodelers. Furthermore, we identified 44 398 differentially methylated CpGs, with hypermethylation more frequent in TSS1500 and CpG shores (Supporting Information Additional file 1: Figures S1, S2; Additional file 2: Tables S1-4). Integrative analysis revealed 635 hypomethylated upregulated and 309 hypermethylated downregulated genes in high-risk tumours (Figure 2A,B). A significant proportion of methylation-regulated DEGs belong to specific functional groups, including epigenetic modifiers, transcription factors, tumour suppressor genes and oncogenes (Figure 2C), demonstrating the critical role of DNA methylation in controlling cell fate. The median β values of differentially methylated CpGs, were lower in the high-risk UMs, suggesting that epigenetic gene activation can be more common than repression (Figure 2D). BAP1 expression negatively correlated with cg01493712 DNA methylation β value (r = −.496; p = .014), implying epigenetic control of BAP1 itself (Figure 2E). Aberrant DNA methylation, distributed relatively uniformly across the entire genome, was associated with the dysregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signalling, or ECM-receptor interaction (Figure 2F,G; Supporting Information Additional file 2: Table S5). These findings highlight the critical role of DNA methylation aberrancy in driving transcriptomic changes associated with poor prognosis. Based on integrative analysis findings, we selected nine candidate genes, three upregulated and six downregulated. The selection was guided by fold change (FC), Δβ values and the number of CpGs inversely correlated with gene expression. The upregulated genes were HTR2B (FC = 191.4), AHNAK2 (FC = 12.6) and CALHM2 (FC = 7.8), while the downregulated genes were SLC25A38 (FC = −4.6), EDNRB (FC = −4.7), TLR1 (FC = −8.6), RNF43 (FC = −10.8), IL12RB2 (FC = −18.1) and MEGF10 (FC = −25.2). Their expression (Supporting Information Additional file 1: Figure S3) was significantly associated with UM overall survival (OS) data, available in The Cancer Genome Atlas dataset (Supporting Information Additional file 1: Figure S4).3 The correlation between DNA methylation percentage measured by pyrosequencing in 58 UM tumours and β values were highly significant (Supporting Information Additional file 1: Table S4). In addition, individual DNA methylation values showed minimal overlap between high- and low-risk tissues (p < .001) (Figure 3A), indicating the potential use of these markers for predicting risk groups with excellent diagnostic accuracy. AUC values ranged from .870 to .956 (p < .001) (Figure 3B; Supporting Information Additional file 1: Table S5). By combining methylation values of hypomethylated AHNAK2 and CALHM2 genes with values of hypermethylated IL12RB2 (Signature 1) or SLC25A38 (Signature 2) genes, we achieved AUC values of .999 and .994, respectively (p < .001), demonstrating the robustness and potential clinical utility of these epigenetic markers in UM risk stratification. Kaplan–Meier survival curves were generated with the log-rank test, and univariate Cox regression analysis was performed to confirm that DNA methylation of CALHM2 and MEGF10 genes and both methylation signatures were sufficient to stratify patients reasonably well as the standard risk groups based on chromosomal rearrangements and mutation profiling (Figure 3C, Table 1). The DNA methylation repatterning in UM was initially attributed to the loss of BAP1, a gene coding for a deubiquitinating hydrolase that exerts diverse functions such as cell cycle regulation, DNA damage repair, chromatin remodelling and gene expression control.4 Although UM is considered poorly immunogenic due to its immune-privileged site of origin, it has been proposed that BAP1 loss may promote the immunosuppressive tumour microenvironment (TME).5 UM is a unique tumour type in which a high density of tumour-infiltrating lymphocytes and tumour-associated macrophages paradoxically correlates with a worse prognosis, highlighting the complex interaction between the TME and the immune response. Epigenetic regulations play a critical role in shaping these intricate relations.6 Accordingly, six of the top nine methylation-regulated genes have been previously linked to immune functions. Specifically, EDNRB, IL12RB2, CALHM2 and RNF43, were identified among UM prognostic genes that interact with immune and stromal cells in the TME.7 IL-12Rβ2, a subunit of the IL-12 receptor, generates high-affinity binding sites for IL-12, one of the most potent antitumor cytokines.8 The prognostic significance of CALHM2 and RNF43 is further reinforced by their listing among the most important DEGs related to UM survival.3 Additionally, AHNAK2, shown to promote UM cell proliferation and migration,9 was found to correlate with infiltration of immune cell subpopulations such as CD8+ and CD4+.10 Our findings align with a recent report by Figueiredo et al.,5 which revealed that the downregulation of TLR1, a gene responsible for immune activation, is correlated with M3 status but not BAP1 expression, indicating epigenomic reprogramming independent of BAP1 mutations. These results highlight the importance of further exploring epigenetic regulation of the unique immune landscape of UM, which presents both challenges and opportunities for developing effective treatments for high-risk patients. Overall, our study provides compelling evidence for the substantial role of DNA methylation in UM progression by regulating the expression of genes involved in critical biological processes such as immune evasion, calcium homeostasis, adhesion and migration. Importantly, we demonstrate that the DNA methylation status of carefully selected CpG sites has the potential to serve as reliable prognostic biomarkers, underscoring the clinical relevance of DNA methylation analysis in UM. By leveraging the power of epigenetic profiling, we can gain a powerful tool for patient stratification, which can aid in personalized therapy and ultimately lead to improved outcomes. Foremost, we express our gratitude to all patients who consented to participate in this study. We would also like to thank Dr. Andrea Štanclova (Department of Molecular Biology and Genomics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Slovakia) for her technical assistance and valuable contributions. Finally, we thank the Slovak Cancer Research Foundation for their enduring assistance and support. The authors declare that they have no competing interests. This research was funded by the Slovak Research and Development Agency Grant number APVV-17-0369, The Ministry of Education, Science, Research and Sport of the Slovak Republic, Grant number VEGA 1/0395/21 and LISPER (ITMS 313011V446: Integrative strategy in the development of personalized medicine of selected malignant tumors and its impact on quality of life. Operational program integrated infrastructure 2014−2020) projects. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. A total of 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between -0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, the DNA methylation values of nine genes, HTR2B, AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2, and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUCs ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining AHNAK2, CALHM2, and IL12RB and Signature 2 AHNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (HR = 4.38, 95% CI 1.30-16.41, HR = 5.59, 95% CI 1.30-16.41; HR = 3.43, 95% CI 1.30-16.41, HR = 4.61, 95% CI 1.30-16.41 and HR = 4.95, 95% CI 1.39-17.58, respectively). Conclusions: Our results demonstrate a significant role of DNA methylation aberrancy in UM progression. The advantages of DNA as a biological material and the excellent prediction accuracies of methylation markers open the perspective for their more extensive clinical use.