The dynamic equilibrium of DNA methylation is orchestrated by the antagonistic interplay between DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) dioxygenases. Emerging pan-cancer analyses have revealed divergent TET family expression patterns: TET1 is transcriptionally silenced in 63% of hepatocellular carcinomas, whereas TET2 is mutated in 17% of gliomas (TCGA data). NB-specific epigenetic studies (17–19) identified TET3 as a potential prognostic biomarker, with its expression inversely correlating with the mitotic–karyorrhexis index. Elevated TET3 expression is correlated with improved 5-year survival (42%), and the tumor-suppressive role of TET3 is mechanistically linked to the 5mC hydroxylation-mediated maintenance of open chromatin states at neurodifferentiation-associated genes (e.g., PHOX2B). Contrastingly, TET1 drives oncogenesis through β2-adrenergic receptor pathway activation, inducing cAMP–PKA signaling that stabilizes MYCN protein (3.2-fold extended half-life). This isoform additionally partners with the histone demethylase KDM6B to form a transcriptional activation complex promoting tumor progression (20). The development of isoform-selective TET1 inhibitors and TET3 agonists represents a promising frontier for epigenetic therapy in NB ( Figure 2A ).

Pioneering work by the Alaminos group (21) first elucidated the critical association among CpG island hypermethylation, MYCN amplification, and poor clinical outcomes in NB. Therapeutically, DNA methyltransferase inhibitors (e.g., decitabine) demonstrate dual efficacy, exhibiting standalone antiproliferative effects while synergistically enhancing conventional chemotherapeutics, a paradigm validated across multiple pediatric NB clinical trials. Notably, cisplatin-resistant models and high-risk NB subtypes exhibit marked upregulation of DNMT3A/B isoforms. Selective targeting of DNMT3B with nanaomycin A induces tumor-selective apoptosis through global methylation reduction (22), revealing a vulnerability in treatment-refractory disease. With the advancement of genome-wide methylation profiling technologies, high-resolution methylation mapping promises to resolve long-standing debates about the cellular origins of NB while informing precision epigenetic therapies (23).

Emerging evidence positions histone deacetylases (HDAC8/HDAC10) as central regulators of NB proliferation, differentiation, and chemosensitivity. Preclinical studies demonstrated that pan-HDAC inhibitors such as vorinostat suppress tumor growth through dual mechanisms: cell cycle arrest (G1 phase accumulation) and intrinsic apoptosis activation (caspase-3 cleavage) (24). These agents synergize with conventional chemotherapy or radiation, achieving enhanced tumor regression in xenograft models. Second-generation inhibitors (e.g., panobinostat, romidepsin) further exhibit blood–brain barrier penetration efficacy and display promise in central nervous system-metastasized NB subtypes (25), with six active HDAC-targeted clinical trials currently recruiting pediatric patients with NB. Paradoxically, although HDAC inhibition broadly suppresses oncogenic programs, context-dependent activation of differentiation pathways could underlie its therapeutic duality.

Beyond HDAC targeting, multilayered histone methylation networks involving WDR5 (H3K79me modulator), EZH2 (H3K27me3 writer), and PRTM5 (H3K4me3 eraser) orchestrate NB plasticity. WDR5–MYC complexes drive super-enhancer formation at oncogenic loci, whereas EZH2-mediated PRC2 activation silences tumor suppressors such as CLU and CADM1. PRMT5-centric arginine methylation sustains spliceosome integrity in MYCN-amplified cells. Pharmacological disruption of these nodes (e.g., EZH2 inhibitor tazemetostat, PRMT5 inhibitor GSK3326595) induces differentiation and reverses chemoresistance in preclinical models (26). These findings collectively map NB’s epigenetic vulnerabilities, facilitating the development of novel combinatorial strategies that simultaneously target histone-modifying enzymes and lineage-specific oncogenic drivers ( Figure 2B ).

The precise equilibrium between histone acetylation and deacetylation serves as a master epigenetic switch governing transcriptional plasticity, with its dysregulation representing a hallmark of tumorigenesis. In high-risk NB, HDAC8 and HDAC10 exhibit pathologic overexpression (27, 28), establishing them as actionable targets. Pharmacologic HDAC inhibition both suppresses tumor proliferation and chemosensitizes resistant cells to doxorubicin, potentially overcoming treatment barriers. Preclinical studies identified valproic acid as a broad-spectrum HDAC inhibitor capable of inducing a triad of effects: proliferation arrest, mitochondrial apoptosis, and neural differentiation.

Vorinostat, the first FDA-approved pan-HDAC inhibitor, demonstrates mechanistically distinct antitumor activity in NB. This drug triggers G2/M phase blockade through CDK1/cyclin B dysregulation and activates intrinsic apoptosis via Bim/PUMA transcriptional induction. Clinical translation has revealed striking efficacy. Combined with ¹³¹I-metaiodobenzylguanidine (MIBG), vorinostat elevates objective response rates in relapsed/refractory NB from 14% to 32% and extends median progression-free survival by 5.8 months (ASCO 2021 data) (29). Synergistic potential was further evidenced by its combined use with panobinostat, which extended survival in TH-MYCN transgenic mice by 62%, and with GD2-targeted immunotherapy, in which co-treatment enhanced antibody-dependent cellular cytotoxicity (30). These multimodal regimens exemplify the evolving paradigm of epigenetic-immune interplay in NB precision medicine.

Targeting SWI/SNF complex dysregulation represents a therapeutic mechanism in NB. Approximately 25% of human cancers harbor mutations in genes encoding mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes (31). Core components include ATPase subunits (SMARCA4/BRG1 and SMARCA2/BRM) and structural subunits (e.g., SMARCC1/2, SMARCD1/2/3) (32). Mechanistically, SMARCB1 mutations induce the mislocalization of mSWI/SNF complexes at gene promoter regions accompanied by RNA polymerase II dysfunction and altered H3K27ac signatures. In NB, mutations in the ARID1A/ARID1B subunits of the SWI/SNF chromatin remodeling complex promote tumor progression and correlate with poor prognosis.

Emerging evidence has revealed the histone acetyltransferase EP300 as an epigenetic linchpin in NB pathogenesis, particularly through MYCN transcriptional regulation (33). Mechanistically, EP300 acetylates histones at MYCN super-enhancer regions, thereby sustaining oncogene addiction in MYCN-amplified tumors. Pioneering work by Durbin et al. (33) developed JQAD1, a PROTAC-based EP300 degrader that achieves tumor-selective depletion (90% reduction at 100 nM) through VHL E3 ligase recruitment. This agent induces rapid apoptosis (caspase-3 activation within 8 h) in MYCN-driven models while demonstrating exceptional safety margins (normal cell viability > 85%), establishing targeted protein degradation as a breakthrough paradigm.

Paralleling these advances, the histone methyltransferase EZH2 exhibits co-operative oncogenesis in MYCN-amplified NB. MYCN transcriptionally upregulates EZH2 (34) while physically interacting with its N-terminal domain to stabilize MYCN protein through PRC2-catalytic-independent mechanisms (35), creating a feed-forward malignancy loop. Pharmacologic disruption using catalytic inhibitors (GSK126, IC₅₀ = 6 nM; JQEZ5, IC₅₀ = 8 nM) induces tumor regression in orthotopic models (36), with current clinical efforts exploring combination regimens with BET inhibitors.

Despite these successes, structural biology limitations persist, as the domain structure has been resolved for fewer than 30% of epigenetic regulators, hampering the rational design of isoform-selective agents. Next-generation approaches currently prioritize covalent EZH2 inhibitors (e.g., MS1943) and dual EZH2/HDAC degraders to overcome compensatory resistance mechanisms, although tumor-selective delivery remains a critical translational barrier.

ncRNAs have emerged as master epigenetic regulators governing NB tumorigenesis, with distinct subclasses, namely miRNAs, lncRNAs, and circular RNAs (circRNAs), orchestrating malignant hallmarks through multilayered gene regulatory networks (37–39). Clinically, circulating miRNAs exhibit diagnostic potential through their exosome-mediated intercellular communication and stability in biofluids (40). For instance, miR-124 acts as a tumor suppressor by reversing therapy resistance in mesenchymal-type NB cells, and its targeted upregulation using PP121 (a tyrosine/PI3K kinase inhibitor) synergizes with BDNF-activated bufalin to induce neural differentiation and apoptosis (41, 42). This exemplifies the therapeutic potential of miRNA modulation in overcoming NB chemoresistance.

The lncRNA landscape reveals equally critical oncogenic drivers (37). DUXAP8, which is overexpressed in > 60% of high-risk NB tumors, accelerates tumor progression via dual mechanisms: sequestering miR-29 to derepress NOL4L-mediated cell cycle activation and enhancing metastatic potential through TWIST1 stabilization. CRISPR-mediated DUXAP8 silencing reduces xenograft tumor growth by 72%, validating its therapeutic candidacy.

The discovery of circRNAs has unveiled a new regulatory layer in NB pathogenesis, with MYCN amplification profoundly altering circRNA landscapes (43). Comparative deep sequencing analysis of five MYCN-amplified NB tumors versus matched normal tissues revealed 2242 significantly downregulated circRNAs, among which three tumor-suppressive circRNAs exhibited particularly promising therapeutic potential. Specifically, circTBC1D4 functions as a molecular sponge for oncogenic miR-21, thereby de-repressing PDCD4 expression and restoring apoptosis sensitivity in treatment-resistant cells. circNAALAD2 directly interacts with the PHLPP2 phosphatase to suppress AKT hyperphosphorylation, effectively inhibiting PI3K-driven survival pathways. circTGFBR3 structurally stabilizes the AXIN1–APC destruction complex, leading to β-catenin degradation and Wnt pathway suppression (44).

The primary mechanisms and functions of these epigenetic drugs in NB treatment are summarized in Table 1 . These agents demonstrate multitarget regulatory capabilities in NB disease progression by modulating key nodes including apoptosis, proliferation, and epigenetic modifications, offering multifaceted mechanisms and potential therapeutic targets for neuroblastoma therapy. However, few epigenetic drugs have advanced to clinical trial phases.

Although the number of active clinical trials for epigenetic modifiers in NB remains limited, a substantial pool of potential novel epigenetic targets awaits exploration. The epigenetic regulatory genes with therapeutic potential for NB identified in current preclinical studies ( Table 2 ) will facilitate the development of new compounds (epigenetic drugs).