DNA methylation of super-enhancers is crucial for the regulation of oncogene and adhesion molecule expression in cancer (55). Aberrant methylation of super-enhancer regions can result in the dysregulation of gene expression, affecting oncogenic pathways and adhesion molecules. Choudhury et al. (55) described that DNA methylation of adhesion-related genes is dynamically variable in a variety of myeloma (MM) subgroups according to solidly established methylation-expression correlations, suggesting that gene-level analyses would be needed to elucidate how DNA methylation operates in concert with other epigenetic modulators of gene expression. In the present study, oncogenic cMAF expression was found to modulate Integrin Subunit Beta 7 (ITGB7) function, specifically within the t (14, 16) MM subgroup. Choudhury et al. (55) showed that in primary B cells, ITGB7 is down-regulated by H3K27me3 enrichment at the transcription start site and upstream promoter. Of note, MM subgroups involve a complex interaction between DNA methylation and chromatin modification, such as in the t (4, 14) subgroup, where the repressive H3K27me3 mark is substituted by the activating marks H3K4me3 and H3K27ac. This change indicates that MM SET in the t (4, 14) subgroup induces global demethylation of the activating mark H3K36 simultaneously with decreasing trimethylation of the repressive mark H3K27 (56, 57). The t (4, 14) subgroup exhibited de novo enrichment of activating histone marks, which was further supported by intermediate DNA methylation levels at the corresponding differentially methylated regions (DMRs) (55). A marked reduction in DNA methylation at enhancer-associated intragenic DMRs, together with increased DHS intensity and enrichment of activating histones and transcription factors, underscores enhanced chromatin accessibility, with a 23 kb CCCTC-binding factor (CTCF)-marked region forming an activation loop that specifically upregulates ITGB7 expression without affecting neighboring genes. Choudhury et al. (55) identified a super-enhancer network on ITGB7 intragenic DMRs in the t (14, 16) subgroup, marked by H3K36me3, H3K4me1, H3K4me3, and H3K27ac. Targeted DNA methylation in MM.1S cells mirrored patient t (14, 16) patterns and increased ITGB7 expression, revealing a nuanced link between methylation and gene expression. sgRNA-2 raised methylation at 5 of 7 CpGs, with CpG-5/6 acting as potential epigenetic switches. Changes in regions 1 and 3 may involve H3K36me3-mediated transcriptional elongation. Choudhury et al. (55) demonstrated that the perturbation of BRD4 in the REN of ITGB7 influences the underlying gene expression, given the functional dependency of epigenetic regulators in these RENs. The results indicated that BRD4 inhibition via JQ1 treatment did not affect endogenous cMAF expression in MM.1S cells (55). Previous studies demonstrating the overexpression or dominant inhibition of cMAF have established the significance of this transcription factor on ITGB7 expression and cell adhesion in MM (58). Furthermore, several genes such as ITGB7, CCND2, CCR1, and Notch were identified as being regulated by cMAF and MAF candidates (e.g., MAFB) in MM (59). These findings underscore the complexity of super enhancer-driven regulation in cancer, particularly in the context of adhesion molecules such as ITGB7.

The activation of oncogenes is a critical characteristic of cancer, facilitating invasion and metastasis (60). Samples from patients with extensive distant metastases exhibited a reduction in histone H3K9 di- and trimethylation relative to those with regional metastatic cancer (61). A study utilized tumor- and metastasis-derived organoids to investigate the progression of pancreatic ductal adenocarcinoma (PDA), and significant and recurrent alterations in H3K27ac levels were observed predominantly in metastatic organoids, regardless of the metastatic location. Roe et al. (62) also identified the transcription factor FOXA1 as a mediator of enhancer reprogramming that promotes cancer metastasis. These findings indicate a non-genetic mechanism of natural selection in cancer progression, supporting the exploration of epigenetic factors in metastasis. Lysine demethylase 6A (KDM6A) facilitates invasion and liver metastases in Kirsten rat sarcoma virus (KRAS)-mutant PDA mice (54). KDM6A, a member of the COMPASS-like complex, is involved in the demethylation of H3K27me3 and also exhibits functions independent of its enzymatic activity (54). KDM6A induced the mis-localization of the COMPASS-like complex from standard enhancers that regulate cell identity genes to super-enhancers associated with genes governing squamous differentiation and metastasis, such as ΔNp63, MYC, and RUNX3 (54).

Zhang et al. (63) used H3K37ac ChIP-seq data to characterize AJUBA LIM protein (AJUBA) as a gene associated with super-enhancers in hepatocellular carcinoma (HCC). The quantity of lung metastasis nodules markedly rose in mice exhibiting elevated AJUBA expression, while it diminished with reduced expression (63). TCF4 interacted with AJBUBA-associated super-enhancers, indicating a potential regulatory role of TCF4 in the oncogenic expression of AJUBA and the metastatic progression in HCC (63). In another study, Kim et al. (64) investigated DNA methylation patterns in gastric cancer to identify novel epigenetic targets. Their study employed RLGS to identify DCBLD2 as a novel epigenetic target in gastric cancer. Overexpression of DCBLD2 results in reduced cell proliferation in 293T and vascular smooth muscle cells (65). Existing evidence indicates that DCBLD2 may significantly influence cancer cell proliferation and metastasis. Kim et al. (64) found that DCBLD2 was often silenced through epigenetic mechanisms in gastric cancer, highlighting its role in suppressing cell proliferation and invasion in this context. Besides, Toyota et al. (66) proposed a novel molecular phenotype characterized by promoter CpG hypermethylation in CRC. This epigenetic silencing correlates with reduced tumor proliferation and invasiveness, underscoring the functional significance of DNA methylation in cancer metastasis. Collectively, these findings highlight the critical role of super-enhancers and DNA methylation in modulating cancer progression and metastasis. Understanding these epigenetic mechanisms offers insights into the metastatic cascade and provides a foundation for developing targeted therapies aimed at disrupting super-enhancer-mediated oncogene activation.

Immune modulation by DNA methylation in cancer involves epigenetic changes in super-enhancers and enhancers that regulate genes controlling immune response, immune checkpoint molecules, and inflammation (67). Dysregulated DNA methylation at these enhancer regions can lead to either immune suppression or immune activation, which significantly influences cancer progression and immune escape (68). Chronic lymphocytic leukemia (CLL) is known by the relentless accumulation of CD19+ B cells, leading to its designation as a malignancy that currently lacks a definitive cure (69). The challenges in obtaining curative treatments for CLL are partially influenced by the adaptability of the transcriptional response governed by epigenetic mechanisms. In a study conducted by Shul et al. (69), the researchers aimed to unravel the complexities of the transcriptional landscape in CLL through an integrative approach. Their analysis included B cell enhancer and super-enhancer signatures identified from H3K27Ac ChIP-seq data (CD19+ B cells, GM12878, and MEC1), DNA methylation profiles derived from reduced-representation bisulfite sequencing of samples from CLL patients and healthy donors, and expression patterns obtained via RNA sequencing of CLL and healthy donor samples. Shul et al. (69) identified super-enhancers in each ChIP-seq profile, accounting for approximately 4% of the total enhancers detected. Specifically, they found 741 super-enhancers in GM12878, 374 super-enhancers in MEC1, and 523 super-enhancers in the CD19+ B cells (69). Molecular Signatures Database (MSigDB) gene ontology analysis indicated that numerous genes associated with super-enhancers participate in pathways that regulate immune signaling activation, such as TNF-α via NF-KB and inflammatory response, as well as metabolic homeostasis, including MTORC1 and fatty acid metabolism (69). Additional analysis of the expression levels of super-enhancer-associated genes in CLL patients revealed 190 transcripts that were significantly overexpressed in CLL patient B cells. This overexpressed subset of super-enhancer-associated transcripts was enriched in genes related to immune signaling (such as FCER2 and LCK) and metabolic regulation (such as ENO2 and LSR). Shul et al. (69) analyzed differential DNA methylation from reduced-representation bisulfite sequencing samples, revealing 744 DNA methylation CpG sites that overlapped with identified B cell enhancers. The majority of the DNA methylation CpG sites in CLL exhibited significant hypomethylation (69). Hypomethylated enhancers included super-enhancers associated with overexpressed transcripts SEPT9, ENO2, RXRA, and CCR7, along with a typical enhancer linked to the overexpressed transcript Programmed cell death protein 1 (PDCD1). Shul et al. (69) investigated the impact of targeting enhancer-driven gene expression in CLL by comparing the effects of the BET bromodomain inhibitor JQ1 and the cyclin-dependent kinase-7 (CDK7) inhibitor THZ1 ( Figure 2 ). Using insights from integrative analyses of B cell enhancers, they performed in vitro assays and RNA sequencing on CLL cell lines MEC1 and MEC2 treated with JQ1 or THZ1. The results showed that JQ1 inhibits proliferation in CLL cell lines, suppresses IgM-induced proliferation in primary CLL cells, and modulates the transcription of genes involved in immune signaling pathways. Conversely, THZ1 exhibited distinct effects by reducing cell viability, inducing apoptosis, and selectively downregulating genes linked to metabolic regulation. These findings suggest that DNA hypomethylation in B cell enhancers influences immune signaling and metabolic gene expression in CLL, with JQ1 and THZ1 exerting differential impacts on these pathways through BET bromodomain or CDK7 inhibition.

Me-BAF155 interacts directly with BRD4 to control the expression of oncogenes driven by super-enhancers in Triple-negative breast cancer (TNBC) (70). Furthermore, me-BAF155 inhibits interferon-stimulated gene (ISG) in cancer cells and prevents T-cell infiltration to metastatic locations (71). Kim et al. (70) identified the genomic localization of me-BAF155 at super-enhancers, consistent with the established function of the SWI/SNF complex in regulating super-enhancers across various biological systems. In alignment with the findings of Kim et al. (70), CARM1i significantly suppressed the expression of various super-enhancer-regulated oncogenes, including MYC, similar to the effects observed with JQ1. The clinical application of BET inhibitors has encountered significant challenges, including limited efficacy, pronounced adverse effects, and the frequent emergence of drug resistance in solid tumors (72). Specifically, JQ1 repressed the expression of ISGs, whereas the CARM1 inhibitor activated their expression (73). JQ1 likely promotes metastasis in the 4T1.2 model by suppressing ISG expression in tumor cells. Conversely, EZM2302 enhances ISG activation, increases CD8+ T cell tumor infiltration, and strengthens their cytotoxic function, while leaving CD4+ T cell and macrophage populations unchanged. Kumar et al. (74) discovered CARM1 as a negative regulator of T-cell immunity using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) screen. Genetic knockout or pharmacological inhibition of CARM1 in T cells was shown to enhance their anti-tumor activity. These findings suggest that inhibiting CARM1 strengthens the type 1 IFN response within tumors, thereby boosting T cell-mediated immunity and improving anti-metastatic effects. Kim et al. (70) revealed that BAF155 methylation plays a crucial role in the activation of ISGs. In immunocompromised PDX models, CARM1i exhibited pronounced anti-growth and anti-metastatic effects, suggesting that inhibiting CARM1 might also impact the functions of other immune cell types (75).

CXCL13 signaling is recognized for its interference with chemotherapeutic responses, and increased CXCL13 expression by cancer cells enhances autocrine and paracrine effects within the tumor microenvironment (TME), resulting in various outcomes (76). CXCL13 produced by MM cells stimulates the secretion of CXCL13 in adjacent macrophages through Bruton’s tyrosine kinase (BTK) signaling (76). Macrophages subsequently promote CXCL13 expression in MM via transforming growth-factor beta (TGF-β) signaling (77). The CXCR5-CXCL13 axis demonstrates resistance to chemotherapeutic agents, such as bortezomib in MM or 5-fluorouracil in CRC and mantle cell lymphoma (78, 79). The CXCR5-CXCL13 axis facilitates metastasis in breast cancer by regulating the epithelial-to-mesenchymal transition (EMT) (80). CXCR5-CXCL13 signaling facilitates tumorigenesis in phosphatase and tensin homolog (PTEN)-deficient cancers through protein kinase C (PKC) signaling (81). The loss of PTEN can lead to the expression of CXCL13 through NF-kB signaling (81). PTEN is a gene regulated by p53, indicating that CXCR5-CXCL13 signaling may disrupt p53 regulation. Although CXCL13 is commonly expressed in both hematological and solid tumors, the mechanisms underlying its abnormal expression in cancer cells remain unclear. TGF-β-induced SOX4 enhances CXCL13 expression during Th2 cell differentiation, while retinoic acid and neuronal signaling also stimulate CXCL13 expression in murine embryonic stromal cells (82). Enhancer tethering has been proposed as a critical mechanism for the expression of oncogenes in cancer (83). Gothwal et al. (84) identified a super-enhancer near the CXCL13 locus in cancer cells, linking aberrant CXCL13 expression to impaired GCDBL cell migration and suppression of p53 target genes in B-lymphomas, CRC, and HCC (84). The findings highlight the epigenetic regulation of CXCL13 expression and enhancer-promoter interactions, linking disrupted regulatory mechanisms to the diverse roles of CXCR5-CXCL13 signaling in both hematological and solid tumors.

Cho et al. (85) evaluated the relationship between DMR methylation and immune infiltration by analyzing bulk transcriptome data using CIBERSORT and the LM22 signature matrix. They identified infiltration-associated methylation regions (IMRs), where methylation of specific pDMRs and eDMRs correlated with immune cell infiltration in tumors. Alterations in oncogenic pathways, such as the MAPK and WNT-β-catenin (CTNNB1, WNT3A, WNT7B), were linked to changes in the TME (85). Functional analysis of IMRs overlapping with pDMRs and eDMRs revealed that CpG hypermethylation of immune genes in PMDs contributes to immune evasion and suppression (85). Only 727 of 4,915 IMR-pDMRs and 136 of 6,313 IMR-eDMRs overlapped with PMDs. Reactome pathways, such as ‘GPCR signaling’ and ‘keratinization,’ were highly enriched for genes that are controlled by IMR-pDMRs overlapping PMDs, out of which 12 were immune-related (85). In contrast, cancer hallmarks and cancer genes in CancerSEA and CancerMine were more associated with genes controlled by IMR-eDMRs than by IMR-pDMRs that do not overlap with PMDs. In summary, super-enhancer regulation, particularly through DNA methylation, provides a potential therapeutic avenue, offering insights into immune evasion and immune system activation within the TME. These studies illustrate the multifaceted interplay between enhancer methylation, immune signaling, and immune cell infiltration, highlighting innovative strategies for cancer immunotherapy.

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to adapt their energy production and biosynthetic needs to sustain rapid proliferation (86). A pivotal mechanism driving these metabolic changes is the epigenetic regulation of gene expression, particularly through the methylation of super-enhancers. A recent study by Alam et al. (87) revealed that KMT2D acts as an epigenetic regulator in lung adenocarcinoma (LUAD) by promoting the activity of super-enhancers, including the Per2 super-enhancer, which supports the expression of the tumor suppressor gene Per2. Their research showed that KMT2D-driven Per2 expression suppresses genes involved in tumor-promoting glycolysis ( Figure 3 ). Conversely, a deficiency or loss of KMT2D leads to reduced Per2 levels, resulting in the activation of glycolytic genes. Experiments showed that the heightened glycolysis observed in KMT2D-deficient lung cancer cells could be effectively suppressed through pharmacological intervention (87). The glycolysis pathway is notably enriched in the Kras; Kmt2d−/− tumor model compared to the Kras model, as well as in human LUAD tumors exhibiting low or mutant KMT2D relative to those with high wild-type KMT2D (87). Results presented by Alam et al. (87) showed that lung-specific Kmt2d deletion accelerates KRAS-driven tumorigenesis in mice, decreases survival, and indicates that Kmt2d loss cooperates with oncogenic Kras to promote LUAD progression. The tumor-suppressive role of KMT2D in lung cancer is supported by findings that Kmt2d loss: I) upregulates glycolytic genes (Eno1, Pgk1, Pgam1, Ldha, and Gapdh); II) downregulates the tumor suppressor gene Per2; and III) increases spheroid size in a 3D lung cancer cell culture. Recent studies demonstrate that KMT2D functions as a tumor suppressor in melanoma and pancreatic cancer cells (88, 89). Also, other research has shown that the genetic ablation of Kmt2d in B cells increases the development of B cell lymphoma, further supporting the tumor-suppressive role of KMT2D (90, 91). Consequently, the tumorigenic role of KMT2D may vary by cell type, although numerous studies indicate that KMT2D predominantly functions as a tumor suppressor in most tissues.

Recent findings indicate that in medulloblastoma occurring in brain-specific Kmt2d knockout mice, the loss of Kmt2d significantly enhances Ras signaling pathways through a marked increase in the expression of various Ras activator genes, including Rasgrp1, Rasgrf1, Rasgrf2, Rapgef5, and Rgl1 (92). The study further demonstrates that KMT2D activates the expression of DNA methyltransferase 3A (Dnmt3a), which subsequently represses the expression of Ras activator genes. KMT2D knockdown resulted in a reduction of DNMT3A expression in human lymphoma cells (90). The KMT2D complex has been demonstrated to interact with tissue-specific DNA-binding transcription factors. The KMT2D complex co-localizes with MyoD in myocyte differentiation and interacts with Peroxisome proliferator-activated receptor gamma (PPAR-γ) and C/EBP in adipocyte differentiation (93).

Alam et al. (87) identified multiple oncogenic glycolytic genes, including Eno1, Pgk1, Pgam1, Ldha, Gapdh, and Cdk1, as target genes of PER2. KMT2D-mediated Per2 activation constitutes a novel tumor-suppressive mechanism that connects an epigenetic tumor suppressor with a regulator of circadian rhythms. PER2 translocates to the nucleus in the evening, where it downregulates gene expression by antagonizing the CLOCK: BMAL1 heterodimeric activator of the circadian rhythm. PER2 undergoes gradual phosphorylation, which facilitates its ubiquitination and subsequent proteasomal degradation. These findings indicate a tumor-suppressive mechanism whereby KMT2D indirectly downregulates glycolytic genes through the enhancement of Per2 expression via super-enhancer activation, thereby suppressing LUAD. Understanding these mechanisms may pave the way for novel insights into the mechanisms of super-enhancer methylation, which can further define tumor metabolism.

In hematological malignancies, super-enhancer DNA methylation emerges as a pivotal mechanism in leukemogenesis and disease progression, driving dysregulated transcriptional programs. In this regard, acute myeloid leukemia (AML) clones exhibited aberrant DNA methylation, particularly at CpG sites associated with enhancer regions, characterized by a notable prevalence of hypomethylation (129). AML exhibiting distinct cytogenetic and mutational characteristics demonstrates varying DNA methylation profiles. Notably, mutations in the DNMT3A and IDH genes display opposing patterns of enhancer DNA methylation, indicating that the epigenetic ramifications of these mutations may significantly influence their malignant phenotype (129). AML with CEBPA silencing is an exception, showing promoter hypermethylation with minimal changes at enhancers, reflecting the distinct clinical and biological features of this subtype (129).

Research by Qu et al. (130) indicated that both AML patients with gene mutations and those without have an abnormal DNA methylome when compared to healthy CD34+ cells, with significant alterations observed in enhancer regulatory areas. Genome-wide analysis of these cells has associated alterations in DNA methylation with the deposition of chromatin marks in enhancer regions, demonstrating a substantial association between DNA hypomethylation and active chromatin marks (H3K4me1, H3K4me3, DNase sensitivity, and H3K27ac) (130). Thus, DNA demethylation activates novel and poised enhancers in AML, resulting in a leukemia-associated transcriptome in these cells (130). Significantly, abnormal enhancer DNA methylation in AML has been demonstrated to be independent of the anticipated differentiation-related alterations at these loci, indicating that this atypical DNA methylation profile is distinctive to the pathological condition in AML and may represent a pivotal event in leukemogenesis (131).

A study carried out by Yang et al. (132) uncovered the function of enhancer DNA methylation in AML pathogenesis and its linkage to DNMT3A, a gene that is commonly mutated in myeloid neoplasms. Heterozygous knockout of DNMT3A and FLT3-ITD mutation resulted in de novo AML through DNA methylation deficiency in DNMT3A knockout mice. Enhancer methylation changes were noted on DNMT3A knockdown, with possible sites for transcription factor binding engaging in myeloid differentiation. The same enhancer hypomethylation signatures were found to be present in AML patient samples with the DNMT3A R882 mutation. Genes near these hypomethylated enhancers were enriched for hematopoietic development pathways, enriched strongly for the HOXB gene cluster (132). Additional evidence highlighting the significance of enhancer regulatory regions in AML pathogenesis arises from a condition known as enhancer hijacking, characterized by recurrent translocations involving enhancer elements within the myeloid compartment. Patients with AML exhibiting inv (3) or t (3, 3) demonstrate the relocation of the GATA-binding protein 2 (GATA2) enhancer to the EVI1 locus (133, 134). This results in a dual effect of improper EVI1 overexpression alongside GATA2 downregulation, established contributors to AML formation (133, 134).

Of note, a direct correlation between the abnormal suppression of super-enhancers and leukemogenesis has been established in only a limited number of studies (38). Tatsumi et al. (38) revealed that the sustained suppression of GFI1 super-enhancer by LSD1 was crucial for the maintenance of erythroleukemia cells. Moreover, as this repression may be readily reversed by the LSD1 inhibitors NCD38 and NCD25, LSD1-mediated suppression of super-enhancers presents a prospective target for erythroleukemia. While the DNA methylation state in the CA remained mostly unchanged among these cells, it has been shown that the combination of LSD1 inhibitors and the DNA hypomethylating drug, 5-azacytidine, resulted in enhanced efficacy against AML (135). The postponed reduction of CD235a and slight growth inhibition in UT7-EPO may be attributed to the activation of alternative super-enhancers, such as ERG-SE, since Tatsumi et al. (38) previously established that ERG super-enhancer activation by NCD38 facilitated the reduction of CD235a.

Emerging research on cancer has also demonstrated the role of heritable epigenetic modifications in cancer evolution (109). Pastore et al. (109) found a comprehensive examination of the CLL epigenetic landscape with respect to the intraleukemic variety of epigenetic and transcriptional factors. At the H3K27ac regulatory areas, they found substantial chromatin rewiring that was mediated by certain families of transcription factors, namely the NFAT and TCF/LEF families. Pastore et al. (109) demonstrated through the targeted bisulfite sequencing capture test that these regulatory regions exhibited the most significant DNA methylation alterations. In addition, the DNA methylation-RNA mutual information is significantly lower in CLL samples compared to normal B cells, which show coordinated epigenetic transcriptional control leading to higher pairwise mutual information. Pastore et al. (109) analyzed matched DNA methylation and mRNA single-cell data, revealing a greater increase in single-cell mutual information in CLL compared to normal B cells, facilitating direct exploration of this phenomenon. This finding lends credence to the idea that intra-leukemic epigenetic diversity is partially to blame for the modest role of promoter DNA methylation in explaining transcriptional variability in bulk cancer studies (136).

Pastore et al. (109) simulated the combinatorial patterns of histone modifications, DNA methylation, and gene expression to broaden the evaluation of epigenetic coordination beyond epigenetic levels. In particular, genes tagged with H3K27me3^hi/H3K4me3^low/H3K27ac^low tend to have a consistent transcriptional output in normal B cells, whereas in CLL, the expression levels of these genes are highly unpredictable (137). The therapeutic potential of targeting enhancer methylation is highlighted by emerging strategies combining DNA hypomethylating agents and chromatin modifiers, such as LSD1 inhibitors, to disrupt leukemogenic transcriptional networks. These findings emphasize the critical role of super-enhancer regulation in hematological malignancies and its potential as a therapeutic avenue.

The intricate patterns of super-enhancer DNA methylation in breast cancer underscore their pivotal role in tumor biology, influencing both oncogene activation and tumor suppressor gene silencing. Aberrant DNA methylation at super-enhancers, characterized by hypomethylation in enhancer regions and hypermethylation in regulatory regions such as CpG islands (CGIs), is a hallmark of breast cancer progression. Jin et al. (117) indicated that most DNA methylation changes in breast cancer are located in the gene body region rather than the promoter region, with the 3’ UTR exhibiting hypomethylation in breast tumor tissue. Hypermethylation in TSS1500, 5’ UTR, and first exon regions diminishes gene expression in breast tumor tissue (117). In breast cancer, upregulated genes are predominantly CGI hypermethylation-independent, while upregulated genes are predominantly associated with enhancer hypomethylation, implicating divergent gene silencing and activation mechanisms (117). Indeed, CGI hypermethylation represses FBLN2, CEBPA, and FAT4 expression, while enhancer hypomethylation enhances ERBB2 and ESR1 expression (117). Besides, Heyn et al. (13) found that 14% of the super-enhancers examined experienced alterations in CpG methylation in their corresponding human tumors, such as normal breast tissue compared to breast cancer. The predominant DNA methylation alteration, a reduction in CpG methylation, was identified in 75% of cases, whereas 25% of super-enhancers exhibited an increase in DNA methylation in cancer samples (13). The hypomethylation events were notably unspecific, correlating with the general loss of DNA methylation typically seen in cancer samples, with the exception of CRC, where they were highly specific to super-enhancer loci (13). Consequently, to identify functional epigenetic modifications, Heyn et al. (13) opted to concentrate on the hypermethylated events, which were prevalent in genes linked to transcriptional and metabolic processes as well as angiogenesis. Significantly, hypermethylation was corroborated using DNA methylation microarray analysis in a distinct cohort of 714 primary cancer cases, wherein 58.1% of the examined DNA hypermethylation at super-enhancers was validated. The findings indicate that the hypermethylation identified in the cancer cells reflects modified DNA methylation patterns at super-enhancer regions in primary tumors (13). Heyn et al. (13) determined that copy number variations (CNVs) did not bias the primary cancer samples, as they identified substantial differences in DNA methylation levels between normal and CNV samples in only a negligible proportion of the super-enhancers. Notably, through oxidative bisulfite treatment combined with DNA methylation analyses, they eliminated the possibility that the increase in DNA methylation found in cancer was attributable to an elevation of 5-hydroxymethylation, a particular cytosine modification that interferes with 5-methylation in bisulfite-based analyses and was found to be enriched in conventional enhancer regions (13). To clarify the functional implications of the observed cancer-specific super-enhancer DNA methylation alterations, they examined the effect of tumor-associated increases in super-enhancer DNA methylation on gene expression. Similar to the proximal regulatory gene regions, which are known to exhibit a general repressive effect due to DNA methylation, Heyn et al. (13) identified a correlation between increased DNA methylation in breast super-enhancer regions and the repression of associated genes in MDA-MB-468PT cell lines. They confirmed the association between super-enhancer hypermethylation and the transcriptional silencing of the related genes across several breast tumor types. Heyn et al. (13) demonstrated that super-enhancers were influenced by their CpG methylation in normal cells and specific aberrant DNA methylation events in cancer target them, potentially affecting the expression of downstream genes. They proposed that localized alterations in transcription factor binding influence super-enhancer DNA methylation patterns, therefore affecting target gene expression. Consequently, the levels of DNA methylation at super-enhancers signify regulatory activity and additionally identify associated transcription factors. In cancer, the altered expression of critical transcription factors creates new super-enhancers that promote oncogene expression. This phenomenon was partially elucidated by identifying FOXQ1 as a potential factor influencing the differential DNA methylation at CRC super-enhancers and the overexpression of significant oncogenes, including RNF43 and MYC. Heyn et al. (13) underscore that the creation of comprehensive databases of DNA methylomes at base resolution might enhance the comprehension of the regulatory roles of DNA methylation beyond the extensively examined proximal promoter gene areas.

Yang et al. (118) comprehensively examined the methylation landscape of super-enhancers in The Cancer Genome Atlas (TCGA) BRCA cohort and identified differential methylation sites (DMS) associated with super-enhancers, demonstrating significant efficacy for risk classification of BRCA patients. Alterations in the distribution of super-enhancer DNA methylation in cancer cells correlate with the aberrant activation or repression of transcription in the respective target genes (20, 96). The acquired DNA methylation indicates that transcription factors influence the localized activity of super-enhancers, and the modulation of the DNA methylation profile by trans-acting factors affects the transformation process in carcinogenesis (96). Consequently, Yang et al. (118) found that the DMS in the super-enhancer regions could serve as an indicator for patient prognosis. The CPM derived from various methylation sites in the super-enhancer regions can assess the prognosis of breast cancer. Abnormal DNA methylation in the promoter region of an oncogene or tumor suppressor gene is characteristic of tumors; these aberrant methylations contribute to the tumorigenesis and spread of breast cancer (138, 139). This suggests that alterations in methylation status within enhancer regions of BRCA facilitate tumorigenesis and progression, underscoring the significance of DNA methylation localization in the development of cancer biomarkers.

Super-enhancer DNA methylation represents a pivotal mechanism in the epigenetic regulation of lung cancer, influencing the expression of oncogenes and tumor suppressor genes through alterations in enhancer activity. Researchers investigated the epigenetics of SCLC by tracing the locations of lactotransferrin (LTF) binding sites, enhancers dependent on H3K27ac histone modification, and promoter DNA methylation (122). Using the SCLC cell line databases (https://discover.nci.nih.gov/SclcCellMinerCDB/), Pongor et al. (122) integrated whole-genome DNA methylation, enhancer H3K27ac ChIP-seq, and ChIP-seq for main LTFs driving SCLC pathogenesis (NEUROD1, ASCL1, and POU2F3) to expand the understanding of gene regulation in SCLC and discover the potential value of the MethylationEPIC 850k array. Algorithms were programmed to automatically measure the amounts of methylation in the promoter and gene bodies, copy number, and promoter acetylation for every gene (122). They also showed how each epigenetic marker predicts gene expression by analyzing their chromosomal distribution (122). Moreover, Pongor et al. (122) found that enhancer regions have lower DNA methylation levels and that genic regions encapsulated by super-enhancers are often absolutely hypomethylated. The increased promoter mark H3K4me3, which prevents the deposition of DNA methylation to broader genomic areas in super-enhancers, is likely associated with this (140, 141). The profile of genes covered by super-enhancers, like FOXA1/2, NEUROD1, and NKX2-1, was shown in this unexpectedly negative connection between expression and gene methylation (122). In line with the super-enhancer analysis, phylogenetic analysis of the H3K27ac findings for the enhancers with the highest signal intensity range across cell lines revealed that the cell lines clustered into three main groups, distinguishing the NEUROD1, ASCL1, and POU2F3 subtypes and validating the unique enhancer signatures of the SCLC subtypes (142). Based on the subtypes, four primary clusters were identified in the cluster heatmap of the H3K27ac signals (122). Clusters 1, 2, and 3 were shown to have an abundance of DNA sequence motifs from ASCL1, POU2F3, and NEUROD1, according to the HOMER sequence motif analysis (122). Recent efforts have also been made to apply this categorization to clinical samples (143, 144). These subtypes can be grouped according to their super-enhancer activity, according to recent basic investigations (122). Using variable enhancers, rather than only super-enhancers, also revealed the clumping. It was expected that there would be minimal overlap between the ASCL1, NEUROD1, and POU2F3 binding sites, given that each subtype has its own increased enhancer signal. Furthermore, ASCL1, NEUROD1, and POU2F3 binding sites had reduced DNA methylation levels (145). Lastly, Pongor et al. (122) showed that levels of promoter enhancer signals are powerful gene expression predictors, even more so than DNA methylation and copy number obtained from DNA methylation.

Given the significant impact of enhancer methylation on carcinogenesis in LUSC, Cho et al. (85) postulated that super-enhancers play crucial roles in the epigenetic regulation of cancer progression. Super-enhancer DMRs (seDMRs) were established based on the overlap between annotated super-enhancers and DMRs. Cho et al. (85) identified around 1000 seDMRs with an equal number of hypomethylated and hypermethylated areas and approximately 1500 target genes. They found <200 functional seDMRs (F-seDMRs) and Reactome pathways for “keratinization” and “surfactant metabolism” showed substantial enrichment for target genes of hypomethylated and hypermethylated F-seDMRs, respectively. Interestingly, all five enhanced Reactome pathways among hypermethylated F-seDMR targets were linked to surfactant metabolism. Cho et al. (85) discovered that two hypermethylated F-seDMRs target genes encoding surfactant protein A2, surfactant-associated protein A3, and surfactant protein D. These findings indicate that hypermethylation of super-enhancers may be a significant epigenetic regulatory mechanism for driving tumor growth in lung tissue. Overall, Cho et al. (85) conducted a functional enrichment analysis and found that enhancer methylation, rather than promoter methylation, plays a significant role in carcinogenesis and immune infiltration. This finding implies that enhancer methylation plays a significant role in canonical cancer pathways and neoplastic features unique to LUSC as compared to other forms of cancer. For example, disturbance of the keratinization and pulmonary surfactant pathways has been shown to alter clinical outcomes, mostly for LUSC31,35-37, and these parameters were shown to be related to eDMR targets in the current investigation. These results suggest that disruption of these pathways may be mediated by abnormal methylation of enhancers in LUSC. Taken together, studies on SCLC and LUSC have revealed that super-enhancers are often hypomethylated in genic regions, allowing for subtype-specific enhancer activity that drives oncogenic pathways. Conversely, hypermethylation of super-enhancers, such as those regulating surfactant protein genes, underscores their role in tumor progression and immune evasion.

The methylation state of super-enhancers is a key regulator of gene expression and cell fate, particularly in gastrointestinal (GI) cancer contexts such as CRC (146, 147). Super-enhancers are critical in driving the expression of oncogenes such as HSF1 (148). Ren et al. (148) found that HSF1 mutations may be partially responsible for HSF1 overexpression in CRC patients. Overexpression of HSF1 was associated with several driver genes in CRC, notably TP53, which showed the strongest correlation (148). This aligns with the findings of Isermann et al. (149), who reported that mutp53 can enhance HSF1 activity by disrupting the inhibitory WTp53-HSF1 interaction. Ren et al. (148) then investigated the influence of epigenetic changes (including DNA methylation) on HSF1 expression. Unexpectedly, they discovered a super-enhancer in the TSS of HSF1 mRNA. To further research the reasons for HSF1’s elevated expression in CRC, Ren et al. (148) examined the driver genes, which are important nodes of regulatory networks and signaling pathways. Using the TCGA portal database, they discovered that HSF1 expression was associated with several driver genes, such as TP53, APC, KRAS, and PIK3CA (148). In line with this finding, HSF1 expression was substantially related to mutant TP53 in the UALCAN database (148). Aside from genetic changes, tumors are also associated with epigenetic alterations, including histone modifications, non-coding RNAs, and DNA methylation. Using the UCSC database, Ren et al. (148) identified a strong H3K27ac signal at the transcription start site of HSF1. Furthermore, the expression of HSF1 correlates favorably with that of BRD4, the master reader that binds to acetylated histones and regulates gene transcription. These findings collectively suggested a possible role for super-enhancers in regulating HSF1 expression. Ren et al. (148) also investigated the link between HSF1 and DNA methylation in CRC, and they discovered that HSF1 had decreased levels of DNA methylation in CRC compared to normal tissues. In addition, the cBioPortal database demonstrated a clear negative connection between HSF1 expression and DNA methylation levels in CRC (148).

Charlet et al. (12) examined the role of DNA methylation in super-enhancers through a comparative analysis with gene promoters in colon cancer by the highly methylated colon cancer cell line HCT116. The recently released 850K MethylationEPIC BeadChip Infinium array incorporates CpG sites in enhancer regions from the Fantom5 and ENCODE projects (150). However, Charlet et al. (12) utilized whole-genome bisulfite sequencing data from their NOMe-seq samples, which demonstrated that the active H3K27ac mark coexists with the typically repressive DNA methylation mark in standard enhancers and across extensive chromatin regions in super-enhancers. These sites likely represent functional elements that regulate gene expression; they serve as binding sites for transcription factors and exhibit the anticipated inverse relationships among DNA methylation, H3K27ac, and accessibility. The findings demonstrated that bivalent loci, characterized by a positive correlation between DNA methylation and H3K27ac, were predominantly located outside transcription factor-binding sites, where chromatin accessibility is reduced (12). The removal of cytosine methylation in DKO1 cells significantly impacts bivalent enhancer structures and results in a marked reduction of the H3K27ac mark. This effect was further validated through DNA demethylation induced by transient 5-Aza-CdR treatment. Charlet et al. (12) indicated that DNA methylation adversely affects H3K27ac levels at enhancers. Currently, it has been indicated that super-enhancers may completely collapse upon the removal of a single H3K27ac-enriched constituent or the absence of a transcription factor (33, 41). Charlet et al. (12) demonstrated that DNA methylation significantly influences both regular and super-enhancer regions upon genetic or transient removal.

Additionally, to clarify the roles of m3Es in CRC, Lin et al. (151) analyzed their distribution by integrating H3K4me3 and H3K27ac ChIP-Seq data. Their findings indicated that m3Es were extensively distributed throughout the human genome and played a crucial role in regulating inflammatory gene expression in CRC. Lin et al. (151) employed a native ChIP approach to eliminate interference from promoter H3K4me3 signals. These findings indicate that m3Es account for approximately 10% of total enhancers, suggesting their widespread distribution in the genome and significant roles in cellular functions. The AP-1/JUN transcription factor is implicated in the regulation of m3E activity and is closely linked to immune pathways and functions as an oncogene in various cancers. Recent epigenomic investigations and motif analyses focused on enhancer profiling have identified and validated the AP-1 family as a crucial group of oncogenic transcription factors (152, 153). Lin et al. (151) have established potential links among AP-1 members, tumor-specific Vm3Es, inflammation, and cancer, highlighting their significant roles in tumorigenesis and metastasis, particularly in the contexts of cancer, immunity, and stress response. In summary, in GI cancers, such as CRC, the interplay between super-enhancers and DNA methylation can emerge as a pivotal area of research, shedding light on tumorigenic mechanisms and potential therapeutic interventions.

DNA methylation, an epigenetic mechanism widely implicated in gene regulation, has emerged as a critical factor in modulating super-enhancer activity in various cancers, including HCC (22, 154). In HCC, aberrant DNA methylation patterns, particularly hypomethylation, have been identified at enhancer regions, leading to oncogene activation and global transcriptional reprogramming (96, 155). Xiong et al. (9) developed a method combining epigenomic and transcriptomic data to infer enhancer-target interactions, enabling the identification of genes regulated by differentially methylated enhancers in HCC. Their epigenomic study identified a hypomethylated CCAAT/enhancer-binding protein-beta (C/EBPβ) enhancer that promotes HCC tumorigenicity via global transcriptional reprogramming. Xiong et al. (9) discovered widespread hypomethylation of transcriptional enhancers in HCCs by analyzing DNA methylation in primary tumors, nontumor, and normal liver tissues. Their research identified an aberrantly methylated enhancer of prognostic importance, which forms a positive circuitry with its target gene to confer HCC characteristics such as angiogenesis, proliferation, and invasion (9). Xiong et al. (9) advanced the understanding of the HCC methylome. Although promoter hypermethylation and hypomethylation have been documented in HCC development, their whole-genome bisulfite sequencing (WGBS) analysis revealed a notable decrease in methylation across most differentially methylated elements (DMEs). Through nanoscale chromatin profiling of HCC tissues, they precisely mapped enhancers and confirmed enhancer hypomethylation-associated C/EBPβ overexpression (9). This was validated using bisulfite pyrosequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) on paired tumor and non-tumor HCC samples. Other HCC-related genes have been found, including SRC tyrosine kinase and ATG7, an autophagy-related pro-survival gene. This investigation discovered new enhancer-hypomethylated and over-expressed targets, including IFNGR2 and SLC45A4, which can play essential roles in HCC due to their involvement in controlling hepatitis B virus (HBV) viraemia and redox homeostasis (156). The HBx oncoprotein, which has been demonstrated to cause demethylation of distal regulatory areas, could be a contributing component to HCC tumorigenesis (9). The HBx TG HCC model recapitulates the C/ebpβ enhancer regulatory network, showing the role of HCC risk factors in enhancer dysregulation during carcinogenesis. Xiong et al. (9) indicated that C/EBPβ enhancer hypomethylation reactivates eRNA, resulting in C/EBPβ transcription. This transcription then binds to and activates its enhancer, creating a self-reinforcing cycle. They found a 3-kb eRNA that was unidirectionally transcribed for C/EBPβ gene regulation. Knocking down this eRNA reduces C/EBPβ gene reactivation by DNA demethylation, as well as inhibiting HCC cell proliferation and invasion. The findings of Xiong et al. (9) support the functional significance of other tumor-specific eRNAs, including androgen- and estrogen-dependent eRNAs in prostate and breast cancers, which play crucial roles in gene transcription. This epigenomic analysis highlights the tumorigenic role of the C/EBPβ enhancer in HCC. Deleting this enhancer significantly reduced HCC tumorigenicity, leading to genome-wide co-depletion of C/EBPβ and BRD4 occupancy, as well as severe dysregulation of gene expression. C/EBPβ functions at both promoters and enhancers, but the findings suggest that C/EBPβ/BRD4 complexes primarily target enhancers to regulate gene activation in HCC cells. Absence of the C/EBPβ enhancer leads to the loss of C/EBPβ/BRD4 from thousands of enhancers, resulting in decreased H3K27ac levels and gene expression. In summary, the function of super-enhancer DNA methylation in HCC highlights the intricate interplay between epigenetic modifications and oncogene activation. The hypomethylation of super-enhancers, as evidenced by the C/EBPβ enhancer, serves as a pivotal driver of HCC tumorigenicity through the reactivation of oncogenic transcriptional programs and enhancer remodeling.

Super-enhancers in nervous system cancers are often modified through mechanisms, including DNA methylation, histone modifications, or interactions with specific TFs (95). Dhar et al. (92) found that some super-enhancers repressed medulloblastoma and described a novel tumor-suppressive mechanism in which MLL4, an H3K4 methyltransferase, was required to maintain wide H3K4me3 and super-enhancers at tumor suppressor genes. Specifically, Xu et al. (157) employed ChIP-seq to analyze H3K27ac, a marker of active chromatin, in glioblastoma (GBM) tissues, oligoastrocytoma, normal brain samples, and cell lines. This comprehensive mapping of active regulatory regions in GBM uncovered tumor- and subtype-specific enhancer-gene interactions, transcription factor networks, and oncogenic dependencies (157). Investigating differentially regulated active regulatory elements (AREs), particularly super-enhancers, provided insights into oncogenic pathways, molecular classification, and epigenetic mechanisms underlying GBM subtypes. The findings of Xu et al. (157) can help researchers better understand the epigenetic pathways that underlie both normal brain functions and GBM. According to their objective study of AREs and gene expression in main tissues, there is a favorable association between total enhancer signals and target gene expression. Current molecular classification relies heavily on gene expression markers; however, these data show that there is extra heterogeneity in GBM at the ARE level (157). Research on super-enhancers that regulate gene expression in a context-dependent manner has revealed tumor- and subtype-specific regulation of target genes, such as RFX2 and TGIF1, long non-coding RNAs (lncRNAs) such as MIR99AHG and LINC01094, and druggable targets, such as BRD4, MKNK2, and WEE1 (157). Despite the fact that the exact cell type that gives rise to each GBM tumor is still a mystery, animal genetic studies have shown that neural stem cells, early progenitors, astrocytes, and neurons can undergo oncogenic transformation into malignant gliomas (158). GBM-specific super-enhancer targets have an overrepresentation of TFs, which indicates a thorough reorganization of the transcriptional network. Consistent with this, TF-centric super-enhancer signature genes isolated from GBM may reliably categorize glioma patients according to tumor grade, survival rate, and other molecular characteristics in patient cohorts. A study by Xu et al. (157) suggests that therapeutic drugs targeting BET bromodomain proteins could disrupt fundamental regulatory circuits, offering a potential therapeutic strategy. Additionally, they have documented that the BET protein degrader ZBC260 exhibited potent anti-GBM action. Furthermore, Xu et al. (157) identified a wide range of functional targets and pathways driven by super-enhancers that are associated with subtype specificity, highlighting promising avenues for future research on the dependencies of actionable tumors. The ARE dataset serves as an optimal resource for conducting in-depth biological investigations, particularly concerning lncRNAs, and for testing related hypotheses. The findings of Xu et al. (157) lay the groundwork for developing more precise scoring systems that can effectively map the involvement of super-enhancer domains and their targets to specific subtypes.

Furthermore, to examine the relationship between super-enhancers and high 5hmC loci, Azizgolshani et al. (125) utilized super-enhancer coordinates identified in various brain-derived cell lines. While 5-hydroxymethylation in pediatric brain tumors has been explored, existing studies have not achieved genome-scale analysis at locus resolution (125). An association between 5hmC and anaplasia was identified through immunohistochemistry analysis of 5hmC across all classifications of brain tumors (159). Wu et al. (160) recently demonstrated that elevated levels of 5hmC correlate with poorer survival outcomes in pediatric posterior fossa ependymomas. An analysis at the nucleotide level revealed enrichment of high 5hmC loci in genes essential for normal craniofacial and neurodevelopment, thereby reinforcing the association between these tumors and developmental neurobiology (161). These findings indicate that differentially hypohydroxymethylated CpGs are enriched in molecular pathways commonly associated with childhood brain tumors, especially those involving WNT signaling and β-catenin binding. Accumulation of 5hmC at 5′ splicing sites in the exon–intron boundary has been proposed as a connection between this epigenetic marker and alternative splicing (162). Azizgolshani et al. (125) indicated that the high 5hmC sites are localized to 5′ untranslated regions, consistent with prior research. Furthermore, these loci are enriched in genes associated with the posttranslational regulation of gene expression, including DICER1, AGO2, and EIF2C2. The relationship between 5hmC and CTCF, a methylation-sensitive transcription factor associated with alternative splicing and RNA polymerase II regulation, has been documented in embryonal cells. Elevated levels of 5hmC are associated with diminished nucleosome binding to DNA and decreased CTCF attachment (163). 5hmC oscillates at 150 nucleotides, corresponding to the length of nucleosome-wound DNA, and is proposed to function as a linker that binds CTCF to DNA (164). In summary, the current study presented the first genome-wide cytosine-specific analysis of 5hmC in three categories of nervous system cancers: embryonal, glioma, and ependymoma. Taken together, the findings show that super-enhancers are pivotal in shaping the transcriptional and epigenetic landscapes of nervous system neoplasms.