Glioblastoma (GBM) represents the most prevalent and aggressive type of primary malignant brain tumor found in adults. It is distinguished by its rapid growth, resistance to standard treatment modalities, and poses a significant risk to both the survival and quality of life of affected individuals [1]. Presently utilized therapeutic strategies, such as surgical intervention, radiotherapy, chemotherapy, and tumor-treating fields, demonstrate restricted effectiveness owing to significant tumor heterogeneity and the presence of the blood-brain barrier, which obstructs the delivery of pharmacological agents, while research on the core molecular mechanisms driving GBM progression faces significant challenges [2, 3, 4]. In the past few years, there have been notable developments in the field of molecular pathology, especially regarding the identification of mutations in isocitrate dehydrogenase (IDH) and the associated oncometabolite D-2-hydroxyglutarate (D-2-HG), have brought hope for elucidating the core mechanisms underlying GBM pathogenesis and development [5, 6]. IDH plays a vital role as an enzyme in the tricarboxylic acid (TCA) cycle, where it is chiefly tasked with promoting the oxidative decarboxylation of isocitrate, resulting in the formation of alpha-ketoglutarate ($\alpha$-KG). Notably, mutations in IDH are observed in more than 80% of gliomas, specific point mutations occur in the IDH1 or IDH2 genes, such as IDH1 Arginine 132 (Arg132) or IDH2 Arginine 172 (Arg172) [7, 8]. The mutant IDH assumes a “neomorphic” role, facilitating the conversion of $\alpha$-KG into substantial quantities of D-2-HG, which abnormally accumulates within cellular environments, often achieving millimolar levels. The elevated concentrations of D-2-HG serve as competitive inhibitors for a range of $\alpha$-KG-dependent dioxygenases, with particular emphasis on histone demethylases such as jumonji domain containing 2A (JMJD2A) and jumonji domain containing 2C (JMJD2C), as well as members of the ten-eleven translocation (TET) family of DNA demethylases. This inhibition triggers a widespread hypermethylation of both histones and DNA across the genome, culminating in the emergence of a unique “glioma CpG island methylator phenotype” (G-CIMP). This epigenetic alteration silences tumor suppressor genes such as cyclin dependent kinase inhibitor 2A (CDKN2A), while aberrantly activating oncogenes like platelet derived growth factor receptor alpha (PDGFRA) through methylation-dependent regulation, ultimately causing cell differentiation arrest and uncontrolled proliferation, thereby promoting gliomagenesis [9, 10, 11]. Furthermore, D-2-HG can induce hypermethylation of promoter regions of interferon signaling-related genes [e.g., interferon regulatory factor 1 (IRF1), signal transducer and activator of transcription 1 (STAT1)], thereby suppressing anti-tumor immune responses.

The importance of the oncometabolite D-2-HG seems to be intricately nuanced and may exhibit dual characteristics depending on the context [12]. A notable clinical contradiction exists whereby gliomas harboring IDH mutations, which are marked by the accumulation of D-2-HG, exhibit a considerably improved prognosis. Specifically, the median overall survival for patients with these mutations is reported to be 3.8 years, in contrast to just 1.1 years for those with the wildtype variant. Furthermore, these IDH-mutant gliomas demonstrate heightened responsiveness to temozolomide (TMZ) treatment [13]. At the molecular level, studies suggest that D-2-HG can exert tumor-suppressive effects in certain contexts, for instance, by downregulating the integrin subunit beta 4 (ITGB4)/phosphatidylinositol 3-kinase (PI3K)/Akt serine/threonine kinase (AKT) signaling axis, thereby inhibiting proliferation and promoting apoptosis. This function contrasts sharply with its known oncogenic activity of inhibiting $\alpha$-KG-dependent dioxygenases and inducing epigenetic silencing, underscoring a dose-, time-, or tissue-dependent duality in its mechanism of action [14]. Research indicates that D-2-HG inhibits the RNA demethylase fat mass and obesity-associated protein (FTO), leading to elevated levels of N6-methyladenosine (m6A) modifications on mRNA transcripts, such as activating transcription factor 5 (ATF5) and MYC Proto-Oncogene, BHLH Transcription Factor (MYC). This consequently reduces the stability of these oncogenic transcripts, providing an explanation for the restricted growth observed in IDH-mutant tumors [15]. Despite considerable advancements in this field, current research efforts remain predominantly confined to single-omics approaches, emphasizing linear signaling pathways rather than systems-level network effects. To address this gap, our study focuses on reconstructing the molecular network linking D-2-HG to GBM pathogenesis through integrative multi-omics analysis. We utilize machine learning-based analyses of topological and functional enrichment to identify key hub molecules within the network. This is subsequently complemented by molecular docking simulations to assess target binding affinity and the thermodynamics of interactions. Through this approach, we aim to clarify the complex regulatory network governed by D-2-HG, this work aims to identify key drivers of GBM oncogenesis and progression, potentially revealing novel therapeutic targets to overcome current treatment limitations.

The oncogenic effects of D-2-HG in GBM primarily stem from gain-of-function mutations in the IDH gene, particularly the IDH1 R132H mutation [23, 24]. This mutation confers a neomorphic activity upon IDH1, enabling it to excessively reduce the normal TCA cycle metabolite $\alpha$-KG into D-2-HG, leading to its abnormal accumulation within tumor cells, with concentrations reaching millimolar levels [25]. D-2-HG competitively inhibits a range of $\alpha$-KG-dependent dioxygenases, thereby inducing widespread epigenetic dysregulation [26]. The core oncogenic mechanism involves the potent inhibition of TET family DNA demethylases and histone demethylases [e.g., lysine demethylase (KDM) family] by D-2-HG. This silences numerous genes associated with cell differentiation and tumor suppression are silenced, trapping glioma cells in an undifferentiated, proliferative state [10]. Furthermore, D-2-HG can inhibit prolyl hydroxylases (PHDs), stabilizing hypoxia-inducible factor HIF-1$\alpha$, which promotes tumor angiogenesis, glycolysis, and adaptation to the hypoxic microenvironment [27, 28]. Notably, the oncogenic action of D-2-HG involves significant synergistic enhancing factors and is closely linked to the activity states of specific epigenetic regulators [29]. For instance, in IDH-wildtype GBM, arginine methyltransferase protein arginine methyltransferase 1 (PRMT1) is recruited to 5hmC-enriched regions to catalyze activating histone marks. However, in the IDH-mutant context, D-2-HG accumulation may inhibit protein arginine methyltransferase 5 (PRMT5) function, leading to a relative dominance of PRMT1 activity, which can drive alternative oncogenic pathways, illustrating the plasticity of tumor epigenetic regulation [30]. Beyond direct epigenetic regulation, D-2-HG promotes GBM progression through other pathways, with its role in shaping the tumor immune microenvironment being particularly important [31, 32]. D-2-HG secreted by tumor cells can be taken up by CD8+ T cells in the microenvironment. It directly inhibits the activity of lactate dehydrogenase (LDH), disrupting the glycolytic process in CD8+ T cells [33, 34]. This metabolic disruption impairs T cell proliferation and cytokine production [e.g., interferon gamma (IFN-$\gamma$)], ultimately facilitating tumor immune escape [35].

A comprehensive analysis of multi-omics data, utilizing sophisticated machine learning techniques alongside bioinformatics methodologies, has systematically revealed potential molecular targets that connect D-2-HG to the development of GBM. Through a rigorous screening process, six core genes—EIF4EBP1, FABP3, KCNQ2, EPCAM, S1PR5, and GRM3—were found to be particularly prominent. Differential expression analysis revealed that EIF4EBP1 was upregulated in GBM, while FABP3, KCNQ2, EPCAM, S1PR5, and GRM3 were downregulated. The application of machine learning models has substantiated the significant diagnostic potential of the identified gene signature. Interpretative analysis utilizing SHAP values revealed that S1PR5 (SHAP value = 0.0622) and KCNQ2 (SHAP value = 0.0438) emerged as the most pivotal predictors. Furthermore, molecular docking simulations provided additional evidence for the biological implications of these results, demonstrating a robust binding affinity between D-2-HG and the products of the core genes, facilitated by specific interactions among amino acids.

The identified core genes exert their functions through a cross-regulatory axis involving metabolism, epigenetics, and signaling pathways, and may exhibit significant synergistic or antagonistic interactions with the oncogenic metabolite D-2-HG produced by IDH mutation. D-2-HG, which accumulates due to IDH mutations, can competitively inhibit $\alpha$-KG-dependent dioxygenases, leading to epigenetic dysregulation such as DNA hypermethylation, which may interact with the functions of these core genes [14]. EIF4EBP1 (Eukaryotic Translation Initiation Factor 4E-Binding Protein 1) serves as a pivotal effector within the mTOR signaling pathway. In the context of glioma, its function extends beyond mere translational repression, exhibiting a tight coupling with the metabolic microenvironment. Functioning primarily as a negative regulator of mRNA translation, EIF4EBP1 acts as a direct substrate of mTOR and inhibits the assembly of the translation initiation complex by sequestering eIF4E [36]. Investigations reveal a dual regulatory mechanism for EIF4EBP1 in IDH-mutant gliomas. On one hand, D-2-HG accumulation precipitates metabolic stress (“energy crisis”), activating adenosine monophosphate-activated protein kinase (AMPK). Activated AMPK suppresses mechanistic target of rapamycin complex 1 (mTORC1), keeping EIF4EBP1 hypophosphorylated. This drives “translational reprogramming” to prioritize stress-response proteins, conferring a survival advantage in harsh environments. On the other hand, EIF4EBP1 overexpression correlates with tumor aggressiveness and is frequently observed in GBM [37]. This upregulation is driven by oncogenic transcription factors like MYB proto-oncogene like 2 (MYBL2) and Transcription factor ETS Proto-Oncogene 1 (ETS1) rather than genomic alterations [36]. The prognostic value of EIF4EBP1 extends to other cancers. In hepatocellular carcinoma (HCC), it predicts poor survival [38]. In lung adenocarcinoma (LUAD), compounds like Sauchinone exert anticancer effects by downregulating EIF4EBP1 [39]. Similarly, in renal cell carcinoma, inhibitors like PLX51107 block progression by suppressing EIF4EBP1. These findings support targeting EIF4EBP1 as a viable strategy [40]. Separately, FABP3 has been identified in GBM as an interaction partner for the tumor-homing peptide CooP, mediating the peptide’s targeted binding to tumor cells [41]. The MF of FABP3 primarily involves the regulation of lipid metabolism and transport. In invasive glioma cells, FABP3 maintains lysosomal membrane stability by preserving its lipid composition, specifically through the transport of polyunsaturated fatty acids (PUFAs). The loss of FABP3 function leads to lysosomal membrane permeabilization (LMP) and subsequent cell death [42]. KCNQ2 encodes a critical subunit of voltage-gated potassium channels, which are essential for regulating neuronal excitability, primarily by mediating the M-current. Loss-of-function mutations in KCNQ2, often in conjunction with KCNQ3 forming heterotetrameric channels, are a well-established cause of epilepsy and a spectrum of neurodevelopmental disorders [43]. The epithelial cell adhesion molecule (EpCAM), also designated as CD326, is a type I transmembrane glycoprotein predominantly expressed in epithelial tissues and epithelial-derived malignancies. It facilitates homotypic cell-cell adhesion and participates in critical cellular processes, including intracellular signaling, migration, proliferation, and differentiation. Its significant overexpression in a wide spectrum of carcinomas underscores its importance as a diagnostic biomarker and a promising therapeutic target in oncology [44]. In gliomagenesis, EPCAM exerts a significant influence on intercellular connectivity by regulating the stability and turnover of key tight junction proteins, including claudin-7 and claudin-1. This perturbation of junctional integrity compromises cell-cell adhesion and epithelial barrier function, a pathological hallmark that becomes increasingly pronounced in high-grade gliomas [45]. S1PR5, a member of the sphingosine-1-phosphate (S1P) receptor family, may regulate the progression of brain tumors through interactions with other G protein-coupled receptors (GPCRs). Research has identified a specific interaction between S1PR5 and the type 2 cannabinoid receptor (CB2). Furthermore, activation of S1PR5 was found to negatively modulate the CB2 receptor-mediated pro-tumorigenic effects in the human GBM cell line U-87 MG [46]. The findings suggest that S1PR5 may act as a negative regulatory factor for CB2 signaling, potentially exerting a protective function within the pathological process of glioma by counteracting CB2-mediated tumor-promoting effects. KCNQ2 encodes a voltage-gated potassium channel, and its functional loss leads to membrane depolarization, subsequently activating calcium-dependent signaling pathways [e.g., nuclear factor of activated T-cells (NFAT)], which ultimately promote tumor cell proliferation and inhibit differentiation. GRM3 (metabotropic glutamate receptor 3) functions primarily by modulating glutamate signaling pathways to influence tumor biological behavior [47, 48]. In the specific context of IDH-mutant gliomas, the mechanistic role of GRM3 exhibits a distinct metabolic dependency. Given the high structural homology between D-2-HG and glutamate, D-2-HG acts as a “metabolic mimetic” ligand, directly binding to the extracellular domain of GRM3. Specifically, activation of GRM3—whether by endogenous glutamate or high concentrations of D-2-HG—recruits Gi/o proteins, which subsequently inhibit adenylyl cyclase and significantly suppress intracellular cAMP levels. This sustained low-cAMP environment relieves protein kinase A (PKA)-mediated inhibition of downstream signaling and aberrantly regulates the extracellular signal-regulated kinase (ERK) pathway, thereby sustaining tumor cell survival and proliferation [48]. Additionally, GRM3 plays a regulatory role in the self-renewal and differentiation processes of glioma stem cells (GSCs). Its expression is positively correlated with key GSC markers, including the transcription factor SRY-box transcription factor 2 (SOX2), suggesting its contribution to the maintenance of stemness in this critical cell population [49].

Based on the current literature review, we have identified that these key genes are implicated in tumorigenesis and cancer progression, with most having been investigated in the context of GBM. Notably, no existing studies have reported direct or indirect interactions between these key genes and D-2-HG. However, through integrated machine learning and molecular docking approaches, our study reveals a close association between these genes and D-2-HG. Given that D-2-HG has been established as a driver in the formation of IDH-mutant glioma [50], these findings highlight substantial and valuable research avenues that remain unexplored, underscoring the significance of our current investigation. For instance, EIF4EBP1 acts as a key downstream effector of the mTORC1 complex. Upon phosphorylation by mTORC1, EIF4EBP1 releases the translation initiation factor eIF4E, thereby initiating cap-dependent translation and facilitating the synthesis of pro-oncogenic proteins (e.g., cyclins, growth factors). This process ultimately drives the proliferation, survival, and metabolic reprogramming of glioma cells [51]. By affecting the adenosine monophosphate (AMP)/ATP ratio, D-2-HG could indirectly modulate AMPK activity. This would engage the AMPK-mTORC1-EIF4EBP1 signaling axis, potentially establishing a negative feedback mechanism that limits uncontrolled proliferation and restricts tumor overgrowth [52, 53]. This finding corroborates the clinical observation that IDH-mutant gliomas typically exhibit a slower growth rate and a more favorable prognosis.

This research encompasses several fundamental limitations. While the sample size is adequate for initial analysis, it could restrict the wider applicability and generalizability of the findings to larger populations. Additionally, while factors such as age, genetic predispositions, and lifestyle choices were somewhat accounted for, there may still be residual influences that could affect the findings. Furthermore, the outcomes predicted through computational methods necessitate further experimental verification to establish their true biological significance and precision, a crucial aim for our forthcoming investigations. Consequently, subsequent investigations ought to focus on augmenting both the magnitude and heterogeneity of the participant group, in addition to utilizing various methodologies, such as in vitro and in vivo studies, to substantiate and corroborate the significant findings. This approach will enhance the reliability and applicability of the research outcomes. Notwithstanding these limitations, the comprehensive examination of the interconnected molecular pathways between D-2-HG and GBM presented in this research carries considerable implications for public health. Future research should focus on the interactions between these key genes and D-2-HG to explore potential targeted therapeutics for improving the survival prognosis of glioma patients.

The present research indicates that D-2-HG could play a role in the development of GBM through the modulation of particular genes. Molecular docking analyses demonstrated notable binding affinity between D-2-HG and its corresponding target proteins. These results establish a basis for additional exploration into the pathways through which D-2-HG may facilitate the onset and advancement of GBM. Future research should focus on elucidating the quantitative relationship between D-2-HG levels and GBM formation and prognosis, as well as exploring potential therapeutic interventions using targeted drugs or natural products for GBM treatment.

No supplementary files are uploaded with this submission. The original data supporting this work are available from the corresponding author upon reasonable request.

JZ and QBW formulated the overall research objectives and purposes of this paper; YFZ is responsible for data retrieval,data processing and analysis in this study; XFY has done important work in molecular docking and image integration; QLL contributed to drafting the manuscript and interpreting the data for the work.; QLL and JZ performed valuable work in revising the paper’s content and reviewing the data processing. All authors contributed to critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

This research is to mine the data in the public database, mainly the geo database. This database is open source and does not need additional ethical approval.

We would like to express our gratitude to all those who helped us during the writing of this manuscript. Thanks to all the peer reviewers for their opinions and suggestions.

This research received no external funding.

The authors declare no conflict of interest.

During the preparation of this work, the authors used ChatGPT-3.5 in order to check spelling and grammar. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.