Advancements in research on the role of the key glycolytic enzyme hexokinase 2 in the regulation of tumor immune evasion (Review)

Introduction

Metabolic reprogramming is a fundamental characteristic of cancer development and progression (1), and it is primarily marked by a notable increase in aerobic glycolysis (2). This atypical activation of aerobic glycolysis not only meets the energy and biosynthetic demands of rapidly proliferating malignant tumor cells but also contributes to the formation of a hypoxic, acidic and nutrient-deprived tumor microenvironment (TME). Such an environment facilitates tumor immune evasion and presents a notable challenge to the antitumor immune response (3,4). Hexokinase 2 (HK2), the initial and critical rate-limiting enzyme in glycolysis, is markedly upregulated in various solid tumors [such as colorectal cancer (5), triple-negative breast cancer (6), hepatocellular carcinoma (7), pancreatic cancer (8), esophageal cancer (9), etc.], where it serves a pivotal role in catalyzing glycolytic processes and influencing the malignant behavior of tumors; it is also closely related to poor patient prognosis (10–18). The regulatory function of HK2 in immune evasion has increasingly attracted attention for research (3,19–21). Previous research indicates that the functions of HK2 encompass multiple immune escape mechanisms (3,20,22). The expression and post-translational modification of HK2 are modulated by a variety of signal transduction pathways and oncogenic factors, which further facilitate immune evasion through both glycolysis-dependent and independent mechanisms. These mechanisms impact immune cell distribution (23), modulate the expression of immune checkpoint molecules (20) and contribute to the establishment of an immunosuppressive microenvironment (3). Furthermore, HK2-mediated hyperactive glycolysis in tumor cells competes with immune cells for glucose, induces metabolic reprogramming in immune cells, suppresses the activation and function of immune cells, and releases large quantities of metabolites to establish an immunosuppressive niche, thereby impairing immune molecule expression and further weakening the antitumor immune response (24,25). Although small-molecule inhibitors [such as 3-bromopyruvic acid (26) and lonidamine (27)] and gene-editing tools that target HK2 can reverse metabolism-dependent immunosuppression, their clinical application is constrained by off-target toxicity, compensatory metabolism and delivery efficiency (21,28–30). Emerging strategies focusing on subcellular localization and non-catalytic intervention may offer novel directions to overcome these clinical translation bottlenecks (3).

At present, an expanding body of research has substantiated the multifaceted role of HK2 in bridging tumor metabolic reprogramming and immune evasion; however, comprehensive analyses remain limited. In light of this, the present review aims to summarize the research advancements regarding the regulation of immune escape in malignant tumors by HK2 and the role of HK2 in metabolic-immune interactions, with the objective of providing references and theoretical underpinnings for the development of novel immunotherapeutic strategies.

HK2 and its role in tumor glycolysis

HK serves as the rate-limiting enzyme that initiates the glycolytic pathway by catalyzing the phosphorylation of glucose to glucose-6-phosphate (G-6-P), thereby facilitating energy production and biosynthesis (31). In mammals, five isoforms of HK have been identified, HK1-4 and HK domain-containing 1, with HK2 being markedly upregulated in malignancies, and strongly associated with tumor aerobic glycolysis and malignant progression (14,32). Unlike other isomers, HK2 has unique structural features: Although its secondary structure is very similar to that of HK1 and HK3, both composed of two symmetrical α-helical regions linked to glucokinase-catalyzed equivalent domains, HK2 is the only isomer that maintains catalytic activity in both domains (2), which allows for more efficient glycolysis (3). The N-terminal hydrophobic domain of HK2 directly anchors to the voltage-dependent anion channel (VDAC) located on the outer mitochondrial membrane. This crucial interaction results in the formation of a transmembrane-coupled complex with the adenine nucleotide translocator on the inner membrane. Such a configuration effectively prevents inhibition by G-6-P and establishes a ‘metabolic compartmentalization’ effect, thereby facilitating efficient coordination between glycolysis and mitochondrial energy metabolism within localized cellular regions (33). As a result, ATP produced by the mitochondria is directly utilized by HK2 to initiate glycolysis, creating a metabolic relay. This mechanism rapidly supplies the energy, metabolic intermediates and precursors necessary for tumor cell proliferation within a short period (34), thereby serving a pivotal role in cancer cell proliferation, drug resistance and immune evasion (35). Concurrently, HK2 localized to the mitochondrial outer membrane serves a critical role in inhibiting the opening of the mitochondrial permeability transition pore, stabilizing the mitochondrial membrane potential, suppressing the release of cytochrome c and blocking apoptotic signaling pathways, thereby facilitating tumor cell survival (36,37). Furthermore, HK2 is subject to O-GlcNAcylation, a process that enhances cell proliferation, migration and invasion in non-small cell lung cancer, and mitigates mitochondrial damage, and thus, facilitates tumor progression (38).

Regulatory mechanisms of HK2-mediated tumor immune evasion via distinct signaling pathways

Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway

Activation of the PI3K/AKT signaling pathway facilitates cellular metabolic reprogramming (4) and is frequently associated with immunosuppressive effects (21). Research indicates that the AKT-induced nuclear translocation of HK2 enhances the transcription of programmed death-ligand 1 (PD-L1) through hypoxia-inducible factor 1-α (HIF-1α), which promotes immune evasion in gastric cancer cells (39). AKT further activates the downstream mechanistic target of rapamycin complex 1 (mTORC1), leading to the stabilization of HIF-1α, which subsequently binds to hypoxia-response elements (HREs) within the HK2 promoter. This interaction establishes a positive feedback loop that upregulates HK2 transcription, induces M2 macrophage polarization and suppresses the functions of T cells and natural killer (NK) cells (40). The inhibition of PI3K activity or the application of 2-deoxy-D-glucose (2-DG) can reverse these effects (41). A clinical study has corroborated a positive association between the expression of PI3K/AKT and HK2 in hepatocellular carcinoma (HCC), with a marked increase in PD-L1 positivity observed in groups exhibiting high co-expression levels (42). The programmed cell death protein 1 (PD-1)/PD-L1 pathway inhibits T cell glycolysis and promotes autophagy through negative regulation of HK2 activity (43). Inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit β, such as AZD8186, disrupt the AKT-HK2 signaling pathway, leading to decreased lactate production and the restoration of T cell functionality (44). In glioma models, the combination of HK2 inhibitors with PD-1 antibodies has been demonstrated to enhance the infiltration of CD8+ T cells (3,5). Future research should focus on developing precise stratification strategies based on the subcellular localization of HK2 and investigate the potential of PI3K isoform-specific inhibitors.

Mitogen-activated protein kinase (MAPK) pathway

The MAPK signaling pathway facilitates signal transduction through a hierarchical kinase cascade, predominantly involving the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to −3), p38 (α, β, γ and δ), and ERK5 families (45). HK2 activates ERK1/2 via the Raf/MEK pathway, leading to the induction of cyclin A1 expression, the downregulation of p27, the initiation of DNA replication stress, the release of damage-associated molecular patterns, the activation of chronic inflammatory responses and the recruitment of immunosuppressive cells (46,47). Furthermore, HK2 enhances the expression of pyruvate dehydrogenase kinase 1 through the JNK-c-Jun pathway, thereby inhibiting CD8+ T cell infiltration, or modulates the nucleocytoplasmic shuttling of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) via p38 MAPKβ (48). hnRNPA1 interacts with the 3′ untranslated region of PD-L1 mRNA to maintain its stability, consequently suppressing T cell activity (49) and fostering the development of an immunosuppressive microenvironment. In the context of colorectal cancer, fructose attenuates MAPK/signal transducer and activator of transcription (STAT)1 signaling through the HK2- inositol 1,4,5-trisphosphate receptor type 3-Ca2+ axis, thereby inhibiting the polarization of M1-type tumor-associated macrophages (TAMs) and further diminishing antitumor immune responses (50). In melanoma, the splicing of hnRNPA1 regulated by HK2 leads to the production of immunosuppressive human leukocyte antigen G mRNA isoforms, which subsequently diminish the cytotoxic efficacy of NK cells (51). Additionally, the pro-inflammatory mediator, macrophage migration inhibitory factor, enhances HK2 expression through the MAPK-ERK signaling pathway, establishing an immunosuppressive positive feedback loop that synergistically promotes immune evasion by the tumor (52).

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway

The NF-κB signaling pathway is activated by chronic inflammation, oncogenic mutations or microenvironmental stressors (53). HK2 interacts with the NF-κB pathway through mechanisms that are both dependent and independent of its metabolic functions. A seminal study conducted by Guo et al (3) demonstrated that HK2 possessed non-metabolic roles in the cytoplasm, functioning as a protein kinase to directly phosphorylate the threonine 291 residue of the NF-κB inhibitor nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα). This phosphorylation event facilitated the degradation of IκBα via the µ-calpain-proteasome pathway, thereby releasing the NF-κB dimer (RelA/p50) to translocate into the nucleus and initiate the transcription of immune checkpoint proteins, including PD-L1 (3). Concurrently, G-6-P, produced through HK2 catalysis, activates the mTORC1 signaling pathway, which promotes the phosphorylation of the IκB kinase complex and indirectly increases NF-κB activity (54). Simultaneously, NF-κB directly interacts with the HK2 promoter region, thereby augmenting its transcriptional activity and activating downstream glycolysis-related genes, which collectively constitute a self-reinforcing regulatory network known as the ‘NF-κB-HK2-Warburg effect’ (42).

In pancreatic ductal adenocarcinoma models, the HK2-IκBα-NF-κB axis markedly upregulates PD-L1 expression, diminishes CD8+ T cell infiltration and stabilizes β-catenin, thereby enhancing the self-renewal capacity of CD133+ tumor stem cells and further mediating immunosuppression (55). In sarcoma, the canonical NF-κB pathway upregulates HK2 expression, promoting lactate production and suppressing mitochondrial oxidative phosphorylation, which results in the acidification of the TME (56). Lactate contributes to the establishment of an immunosuppressive microenvironment by activating regulatory T cells (Tregs) and M2 macrophages, while inhibiting dendritic cell (DC) maturation (57,58). Preclinical studies have demonstrated that a combined therapeutic approach utilizing the HK2-specific inhibitor 2-DG and the NF-κB pathway inhibitor BAY11-7082 enhanced antitumor immune responses through dual mechanisms: The suppression of PD-L1 protein expression and the reversal of T cell exhaustion phenotypes (59,60). This synergistic therapeutic effect lays the theoretical groundwork for the clinical advancement of innovative immunometabolic combination therapies.

Transforming growth factor β (TGF-β) pathway

The TGF-β pathway facilitates the differentiation of immunosuppressive cells and diminishes immune cell cytotoxicity through mechanisms that are both SMAD-dependent and independent, thereby promoting an immune-tolerant phenotype (61). In models characterized by energy deficiency induced by oligomycin A-mediated inhibition of oxidative phosphorylation, highly metastatic HCT116 cells stimulated by TGF-β contribute to the formation of an immunosuppressive microenvironment through the upregulation of HK2 expression (62). Furthermore, TGF-β modulates HK2 expression by activating the SMAD3 pathway, while HK2 directly interacts with TGF-β receptor I (TβRI) via its structural domain, stabilizing the TβRI-TβRII complex and enhancing the phosphorylation efficiency of SMAD2/3 (31,63). This receptor-kinase interaction creates a positive feedback loop that intensifies TGF-β-induced epithelial-mesenchymal transition and stromal fibrosis, thereby physically obstructing T cell infiltration into tumor beds (64–66). Furthermore, the TGF-β/SMAD signaling pathway directly inhibits the transcription of major histocompatibility complex class I (MHC-I) heavy chains and antigen processing-associated transporters (67). Concurrently, HK2 activates histone deacetylases through metabolic byproducts, leading to further silencing of the epigenetic regulatory regions of MHC-I genes and impairment of the recognition and binding of tumor-specific antigens by immune cells (68).

Janus kinase (JAK)/STAT pathway

The JAK/STAT pathway, a pivotal axis for cytokine signal transduction, facilitates tumor immunosuppression through aberrant activation of STAT3/STAT5 and alters the TME via metabolic reprogramming (69). In colorectal cancer, upregulation of HK2 enhances STAT3 phosphorylation, reduces the infiltration of immune cells such as CD8+ T cells and NK cells, and markedly increases PD-L1 levels, and these effects are reversed by JAK/STAT3 inhibitors (5). Mechanistically, phosphorylated STAT3 directly binds to the PD-L1 promoter, and HK2 indirectly promotes PD-L1 transcription by augmenting STAT3 activity (70).

SUMOylation has been identified as a crucial factor in the cellular response to mitochondrial stress, acting as a sensor and mediator of stress signals to maintain mitochondrial homeostasis (65). This regulatory mechanism is particularly relevant in the context of HK2, suggesting that deSUMOylation likely enhances the interaction between HK2 and VDAC1, thereby activating JAK2 kinase activity and establishing an HK2-JAK2/STAT3-immunosuppressive positive feedback loop (71,72). Epigenetic modifications serve a notable role in this process. Specifically, methyltransferase-like 3 stabilizes HK2 mRNA through N6-methyladenosine modification, and its upregulation enhances HK2 translation via insulin-like growth factor 2 mRNA-binding protein 2, while concurrently activating STAT3-dependent PD-L1 transcription (73). Furthermore, the accumulation of lactate mediated by HK2 inhibits glycolysis in T cells, diminishes interferon γ (IFN-γ) secretion and activates the JAK-STAT pathway through paracrine signaling, thereby further impairing antitumor immunity (74) (Table I).

Table I. Regulatory mechanisms of HK2-mediated tumor immune evasion via distinct signaling pathways.

Table I.

Regulatory mechanisms of HK2-mediated tumor immune evasion via distinct signaling pathways.

First author/s, year	Signaling pathway	HK2 interaction mechanism	Impact on immune cells	Key outcomes	(Refs.)
Huang et al, 2023;	PI3K/AKT	AKT induces nuclear translocation of HK2,	Suppresses T cell and NK cell	Promotes immune checkpoint	(39,40,43)
Ranjbar et al, 2023;		upregulating PD-L1 transcription via HIF-1α.	functions. Induces M2 macrophage	expression and inhibits antitumor
Kawasaki et al, 2021		mTORC1 stabilizes HIF-1α, forming a HK2-PD-L1 positive feedback loop.	polarization.	immunity.
Kawasaki et al, 2021;	MAPK	HK2 activates ERK1/2 via Raf/MEK, inhibiting	Impairs T cell activation. Recruits	Regulates immune checkpoint	(43,50,51)
Yan et al, 2024;		CD8+ T cell infiltration. p38 MAPKβ modulates	immunosuppressive cells (such as	stability via RNA-binding proteins,
Dunnett et al, 2025		hnRNPA1, stabilizing PD-L1 mRNA.	Tregs).	fostering a chronic inflammatory microenvironment.
Chen et al, 2022;	NF-κB	HK2 directly phosphorylates IκBα, releasing	Reduces CD8+ T cell infiltration.	Establishes a ‘NF-κB-HK2-	(42,54–56)
Jiang et al, 2024;		NF-κB to drive PD-L1 transcription. G-6-P	Enhances self-renewal of tumor	Warburg effect’ positive feedback
Tong et al, 2024;		activates mTORC1, indirectly enhancing NF-κB	stem cells.	loop, suppressing antigen
Londhe et al, 2018		activity.		presentation.
Zhong et al, 2025;	TGF-β	TGF-β upregulates HK2 via SMAD3. HK2	Suppresses MHC-I expression.	Epigenetically silences MHC-I	(62,68)
Santarpia et al, 2015		stabilizes the TβRI-TβRII complex, enhancing SMAD2/3 phosphorylation.	Promotes EMT and stromal fibrosis, blocking T cell infiltration.	genes via histone deacetylase activation by metabolic byproducts.
Ren et al, 2020;	JAK/STAT	HK2 enhances STAT3 phosphorylation, promoting	Reduces CD8+ T and NK cell	Forms an ‘HK2-JAK2/STAT3’	(70,71,73)
Shangguan et al, 2021;		PD-L1 transcription. Lactate activates JAK-STAT	infiltration. Induces T cell	positive feedback loop,
Wang et al, 2020		via paracrine signaling, impairing T cell function.	exhaustion.	synergistically suppressing antitumor immunity.

[i] AKT, protein kinase B; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-regulated kinase; G-6-P, glucose-6-phosphate; HIF-1α, hypoxia-inducible factor 1α; HK2, hexokinase 2; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; IκBα, inhibitor of nuclear factor kappa Bα; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MHC-I, major histocompatibility complex class I; mTORC1, mechanistic target of rapamycin complex 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; NK cell, natural killer cell; PD-L1, programmed death-ligand 1; PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activator of transcription; TβRI/II, transforming growth factor β receptor I/II; TGF-β, transforming growth factor β; Treg, regulatory T cell.

HK2 facilitates the establishment of an immunosuppressive microenvironment

HK2 regulates the function of immune cells

The phenomenon of tumor immune escape is intricately linked to the immune subtypes present within the TME (75). However, the specific role of HK2 within these subtypes remains poorly understood. A comprehensive analysis utilizing data from The Cancer Genome Atlas and the International Union of Immunological Societies Immunology Databases revealed that HK2 expression is markedly elevated in immunosuppressive tumors, including pancreatic and liver cancers. This elevated expression is associated with the accumulation of Tregs and M2-type macrophages (76). Such an immunosuppressive milieu may further facilitate tumor cell immune escape through metabolic modulation mediated by HK2 (38). Conversely, in immune-active tumors, such as melanoma, a negative association between HK2 expression and T helper type 1 (Th1) cytokines (such as IFN-γ and C-X-C motif chemokine ligand 9) suggests that HK2 may alter the composition and functionality of immune cells by modulating the expression of chemokines and antigen-presenting molecules (77). Furthermore, research has demonstrated that knockout of HK2 can markedly increase the expression of MHC-II and its antigen-presenting capabilities in DCs, while also modulating the Th17/Treg balance in CD4+ T cells. This modulation subsequently influences the specificity and intensity of the immune response (78,79). These observations underscore the pivotal role of HK2 in determining immune cell subtypes. Consequently, these findings imply that HK2 is not only critical within tumor cells but also potentially modulates immune responses by affecting the functionality of immune cells within the tumor immune microenvironment.

Acidic TME

Lactate, a central metabolic product of HK2-driven glycolysis, has transitioned from being considered merely a ‘metabolic waste product’ (80) to a multifunctional mediator, acting as a signaling molecule, energy substrate and immunoregulatory agent that governs tumor immune evasion (81,82). Tumor cells continuously produce lactate through HK2 hyperactivation, which increases extracellular acidification rates, contributes to the formation of an acidic TME and establishes an immunosuppressive niche through multiple pathways (such as inhibiting the function of effector immune cells, promoting the expansion of inhibitory immune cells, disrupting antigen presentation and upregulating checkpoints) (30). As shown in Fig. 1, the elevated lactate levels mediated by HK2 result in a reduction in the calcium influx in T cells, inhibition of IFN-γ secretion and prevention of the nuclear translocation of activated T cell nuclear factors, as well as the expression of the autophagy factor focal adhesion kinase family interacting protein of 200 kDa (57,83,84). These processes contribute to T cell exhaustion, apoptosis and a diminished potential for memory T cell differentiation (85,86). Application of the HK2 inhibitor 2-DG counteracts these effects, thereby restoring the antitumor immune function of T cells (87). HK2-driven glycolysis creates intracellular and extracellular pH gradients, which directly induce the expansion of Tregs and upregulate PD-1 expression. Furthermore, pH gradients downregulate major MHC-II/CD80 expression through HIF-1α, thereby impairing the antigen-processing capacity of DCs and the polarization of helper T cells (88–91). Lactate also upregulates interleukin (IL)-10 and downregulates acidification-dependent natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1) by suppressing the activation of NF-κB (92), leading to suppressed cytotoxicity in NK cells (93,94). Additionally, lactate reduces the expression of inducible nitric oxide synthase, C-C motif chemokine ligand (CCL)2 and IL-6 in M1 macrophages by inhibiting the activity of the NF-κB signaling pathway, while the STAT3/HIF-1α axis promotes macrophage M2 polarization, facilitating immune escape (95,96). Furthermore, the elevation of lactate mediated by HK2 upregulates inhibitory molecules such as PD-L1, cytotoxic T-lymphocyte-associated protein 4 and TGF-β (97–99), induces histone lysine lactylation, and establishes a ‘glycolysis-lactylation-immunosuppression’ positive feedback loop (100,101).

Figure 1. HK2-driven lactate metabolism shapes an immunosuppressive tumor microenvironment. Tumor cells utilize HK2-driven glycolysis to convert glucose to lactate, resulting in extracellular acidification (low pH) and the formation of an immunosuppressive niche. Elevated lactate levels dysregulate immune cell functions through multiple mechanisms. HK2, hexokinase 2; 2-DG, 2-deoxy-D-glucose; IFN-γ, interferon γ; NFAT, nuclear factor of activated T-cells; FIP200, FAK family-interacting protein of 200 kDa; MHC-II, major histocompatibility complex class II; NKG2D, natural killer group 2D; DNAM-1, DNAX accessory molecule-1; iNOS, inducible nitric oxide synthase; CXCL2, C-X-C motif chemokine ligand 2; IL-6/10, interleukin 6/10; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; MCT, monocarboxylate transporter; DC, dendritic cell; NK cell, natural killer cell; Treg, regulatory T cell; PD-L1, programmed death-ligand 1; HIF-1α, hypoxia-inducible factor 1α.

Hypoxic TME

The interaction of HK2 with mitochondrial VDAC1 leads to localized microenvironmental hypoxia, which accelerates lactate accumulation and abnormal angiogenesis, thereby further exacerbating hypoxia (100,102). As shown in Fig. 2, intratumoral hypoxia predominantly inhibits the prolyl hydroxylase-mediated stabilization of HIF-1α (103). Activation of HIF-1α also enhances HK2 expression (104). The hypoxia-HIF-1α axis directly interacts with HREs in the HK2 promoter, amplifying the hypoxia process in the TME and forming an immunosuppressive feedforward loop (105,106). Clinical investigations have demonstrated that the HK2-mediated upregulation of HIF-1α in breast and lung cancer is positively associated with PD-L1 expression, and patients with high HK2 expression exhibit reduced response rates to immune checkpoint inhibitors (107,108). Mechanistically, the activation of HIF-1α by HK2 facilitates its binding to HREs within the PD-L1 promoter, leading to upregulation of PD-L1 transcription (109). Additionally, this activation promotes the post-translational modifications of PD-L1 through the activation of the PI3K/AKT/mTOR signaling pathway (110). Furthermore, HK2-mediated activation of HIF-1α results in the upregulation of microRNA-224, which suppresses the natural cytotoxicity triggering receptor 1/NKp46 pathway and thereby protects tumors from NK cell-mediated attack (111). HIF-1α also induces the secretion of CCL28 and TGF-β by tumor cells, facilitating the recruitment of Tregs to tumor sites (112,113), and sustains immunosuppressive function of CCL28 and TGF-β by enhancing the expression of forkhead box P3 (Foxp3) (114). Concurrently, HIF-1α promotes the polarization of TAMs towards the M2 phenotype through the upregulation of vascular endothelial growth factor (VEGF) and IL-10. These M2 TAMs subsequently secrete arginase 1, IL-10 and other factors (such as TGF-β, PD-L1) that inhibit the activity of CD8+ T cells (115). Furthermore, HIF-1α activates the STAT3 pathway, leading to the expansion of myeloid-derived suppressor cells, which further suppress the antitumor effects of NK and T cells (116). HK2-activated HIF-1α directly interacts with HREs in the MHC-I gene promoter or recruits histone deacetylases to induce chromatin compaction, thereby inhibiting MHC-I transcription (30). HIF-1α inhibits the IFN-γ-mediated upregulation of MHC-I by suppressing the activity of IFN regulatory factors (117). Additionally, HIF-1α impairs DC differentiation and antigen-presenting capabilities through the induction of VEGF and IL-6, resulting in inadequate T cell priming (118). HK2 further enhances the expression of HIF-1α and monocarboxylate transporters, facilitating HK2-mediated lactate efflux and exacerbating the immunosuppressive TME (119). HIF-1α also upregulates CD73 expression, which catalyzes the conversion of ATP to adenosine, subsequently inhibiting T cell activation and NK cell cytotoxicity via adenosine A2A receptor interaction (120–122). Furthermore, HIF-1α induces the expression of indoleamine 2,3-dioxygenase, leading to tryptophan depletion and kynurenine acid production in the microenvironment, thereby suppressing T cell proliferation and promoting Treg differentiation (123,124).

Figure 2. HK2-HIF-1α hypoxic feedforward loop orchestrates multimodal immunosuppression in the tumor microenvironment. This schematic depicts the self-reinforcing hypoxic feedforward loop initiated by HK2-VDAC1 mitochondrial interaction, which induces localized hypoxia and stabilizes HIF-1α. The activated HIF-1α axis drives immunosuppression through: i) Transcriptional upregulation of PD-L1 via HRE binding and PI3K/AKT/mTOR-mediated post-translational modification; ii) recruitment of Tregs via CCL28/TGF-β secretion and Foxp3 stabilization; iii) polarization of TAMs to M2 phenotype through VEGF/IL-10, leading to Arg-1/IL-10 secretion that suppresses CD8+ T cells; iv) STAT3-dependent expansion of MDSCs; v) inhibition of MHC-I and DC antigen presentation; vi) IDO-induced tryptophan depletion promoting Treg differentiation. Concomitant abnormal angiogenesis (via VEGF) and lactate accumulation further exacerbate hypoxia, completing the immunosuppressive circuit; vii) The upregulation of microRNA-224 caused by HIF-1α activation inhibits the natural cytotoxicity trigger receptor 1/NKp46 pathway of NK cells. HK2, hexokinase 2; HIF-1α, hypoxia-inducible factor 1α; VDAC1, voltage-dependent anion channel 1; PD-L1, programmed death-ligand 1; HRE, hypoxia-response element; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; Treg, regulatory T cell; CCL28, C-C motif chemokine ligand 28; TGF-β, transforming growth factor β; Foxp3, forkhead box P3; TAM, tumor-associated macrophage; VEGF, vascular endothelial growth factor; IL, interleukin; Arg-1, arginase 1; STAT3, signal transducer and activator of transcription 3; MDSC, myeloid-derived suppressor cell; MHC-I, major histocompatibility complex class I; DC, dendritic cell; IDO, indoleamine 2,3-dioxygenase; MCT, monocarboxylate transporter; Trp, tryptophan; PHD, prolyl hydroxylase domain; IRF, interferon regulatory factors.

Glucose-deprived TME

The synergistic effects of the HK2-mediated glycolytic enhancement and impaired vascular exchange result in limited glucose availability within the TME, causing intratumoral hypoglycemia (125). Under these glucose-deprivation conditions, the TME selectively impairs glucose-dependent immune cells (126). As shown in Fig. 3, the hypoglycemic TME driven by HK2 activates AMP-activated protein kinase (AMPK), inhibits the signal of MTORC1-activated cell metabolic synthesis, downregulates HIF-1α expression, reduces IL-12 secretion and fails to effectively activate naïve T cells (127–129). The secretion of effector cytokines such as IFN-γ and tumor necrosis factor-α is also diminished (130). Under conditions of HK2-induced glucose deprivation, T cells are compelled to shift towards fatty acid oxidation and mitochondrial respiration. However, this metabolic shift is inefficient due to the suppression of carnitine palmitoyltransferase 1A translation mediated by glucose deprivation, ultimately leading to T cell exhaustion (131). Hypoglycemia induces DCs to adopt a tolerogenic phenotype characterized by elevated PD-L1 and IL-10 expression, further inhibiting T cell responses (128). By contrast, Tregs within the hypoglycemic TME have enhanced oxidative phosphorylation through Foxp3-mediated metabolic reprogramming, relying on fatty acid and lactate metabolism to maintain their immunosuppressive functions. This metabolic adaptation provides Tregs with a competitive advantage in nutrient competition, thereby exacerbating antitumor immune suppression (132). Furthermore, glucose deprivation triggers the activation of the AMPK pathway in tumor cells. AMPK phosphorylates Hu antigen R (HuR) at Ser318, facilitating its translocation from the nucleus to the cytoplasm (133). In melanoma cells, the cytoplasmic localization of HuR increases from 20 to 60% under hypoglycemic conditions, which enhances its binding affinity to PD-L1 mRNA, and thus, stabilizes PD-L1 mRNA (134).

Figure 3. HK2-mediated glycolysis drives glucose starvation in the tumor immune microenvironment, suppressing antitumor immunity. This schematic depicts how HK2-driven glycolytic flux creates a hypoglycemic TME that selectively impairs immunocompetent cells while favoring immunosuppressive populations. Cancer cells exacerbate glucose scarcity through GLUT1-mediated glucose uptake and HK2-dependent pyruvate production, triggering AMPK activation. Phosphorylated AMPK induces nuclear-to-cytoplasmic translocation of HuR via Ser318 phosphorylation, enhancing HuR binding to PD-L1 mRNA to stabilize PD-L1 transcripts. Concurrently, hypoglycemia forces effector T cells toward inefficient fatty acid oxidation due to suppressed CPT1A translation, culminating in T cell exhaustion and diminished IFN-γ/TNF-α secretion. DCs adopt a tolerogenic phenotype with elevated PD-L1/IL-10 expression, impairing T cell priming. Conversely, Tregs thrive through Foxp3-mediated metabolic reprogramming, utilizing lactate and fatty acids to enhance OXPHOS and maintain immunosuppressive function, thereby outcompeting effector cells for nutrients. This metabolic imbalance establishes a self-perpetuating immunosuppressive circuit. HK2, hexokinase 2; GLUT1, glucose transporter type 1; AMPK, AMP-activated protein kinase; HuR, Hu antigen R; PD-L1, programmed death-ligand 1; CPT1A, carnitine palmitoyltransferase 1A; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; DC, dendritic cell; IL, interleukin; Treg, regulatory T cell; Foxp3, forkhead box P3; OXPHOS, oxidative phosphorylation; TME, tumor microenvironment.

HK2 and therapeutic strategies for combating tumor immune evasion

Advancements in drug research, development and clinical trials targeting HK2

Advancements in the development of novel HK2 inhibitors have reached the clinical research phase, in which they have shown notable antitumor activity (Table II). Small molecule inhibitors, such as benserazide and benitrobenrazide (BNBZ), have been shown to decrease glucose uptake, lactic acid production and intracellular ATP levels by specifically binding to HK2 and inhibiting its enzymatic function (135,136). This mechanism induces apoptosis in tumor cells and suppresses tumor cell proliferation. BNBZ has exhibited notable antitumor effects in xenograft mouse models using SW1990 and SW480 cells, highlighting its potential as a promising HK2 inhibitor (136). Similarly, benserazide has been shown to effectively reduce tumor growth in a SW480 cell xenograft mouse model without significant toxic effects (135). The nanoparticle formulation of benserazide further enhances its antitumor efficacy and specificity in targeting tumors (135).

Table II. Summary of HK2 inhibitors in development.

Table II.

Summary of HK2 inhibitors in development.

First author/s, year	Inhibitor	Preclinical/Clinical efficacy and mechanism	Key limitations/challenges	(Refs.)
Zheng et al, 2021	BNBZ	Suppresses T cell and NK cell functions. Induces M2 macrophage polarization.	Promotes immune checkpoint expression and inhibits antitumor immunity.	(136)
Li et al, 2017	Benserazide	Impairs T cell activation. Recruits immunosuppressive cells (such as Tregs).	Regulates immune checkpoint stability via RNA-binding proteins, fostering a chronic inflammatory microenvironment.	(135)
Lozzi et al, 2019	VDA-1102 (Almavid™)	First-in-class; disrupts HK2-VDAC1 interaction to restore apoptosis. ORR >60% in phase II for CTCL/AK.	As a newer mechanism, long-term safety profile is still being established.	(138)
		Stable plasma concentration and linear PK in pediatric patients with brain tumors.
Zheng et al, 2023	Ketoconazole	Disrupts HK2-AIMP2 interaction, restoring apoptosis.	Antifungal dose may differ from anticancer dose.	(139)
		Synergizes with radiotherapy to suppress HCC growth and overcome radioresistance.	Potential for drug interactions and liver toxicity.
Agnihotri et al, 2019	Posaconazole (Azoles)	Targets HK2-expressing glioblastoma cells.	Similar to ketoconazole; efficacy as a monotherapy in cancer is unproven.	(150)
Jiang et al, 2025	Ikarugamycin	Inhibits glycolytic flux in pancreatic cancer cells by targeting HK2. Antitumor activity in murine models without significant cytotoxicity.	Natural product; often face challenges in synthesis, scalability and bioavailability.	(141)
Bao et al, 2020;	Ganoderma sinense	High binding affinity to HK2.	Very early-stage discovery; mechanism and	(142,143)
Wang et al, 2023	steroid		efficacy need extensive validation.
Lin et al, 2016	2,6-disubstituted glucosamines	Potent and selective for HK2 over HK1. Inhibits glycolysis in cancer cell lines.	New chemical class; no in vivo or toxicity data reported yet.	(140)
Zhong et al, 2022	3-BP	Induces apoptosis and ER stress. Inhibits survival/proliferation of colon cancer cells.	Non-selective and highly reactive, leading to significant toxicity issues.	(149)
Xu et al, 2020;	HK2-silencing	Inhibits tumor cell proliferation and metastasis.	Lack of a safe, efficient and specific in vivo delivery	(144–148)
Ye et al, 2020;	RNA		system; Potential off-target effects, immunogenicity
Zheng et al, 2023;			and instantaneity and other issues.
Zhu et al, 2017;
Guo et al, 2020

[i] 3-BP, 3-bromopyruvate; AIMP2, aminoacyl transfer RNA synthetase complex-interacting multifunctional protein 2; AK, actinic keratosis; BNBZ, benitrobenrazide; CTCL, cutaneous T-cell lymphoma; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; HK1, hexokinase 1; HK2, hexokinase 2; mCRPC, metastatic castration-resistant prostate cancer; NK cell, natural killer cell; ORR, objective response rate; PK, pharmacokinetics; Treg, regulatory T cell; VDAC1, voltage-dependent anion channel 1; CR, complete response.

VDA-1102 (Almavid™), the first small molecule targeting the HK2-VDAC1 interaction, disrupts HK2 mitochondrial localization to restore cancer cell apoptosis. In a phase II trial for cutaneous T-cell lymphoma and actinic keratosis, VDA-1102 achieved an objective response rate (ORR) >60% with manageable toxicity (137). The subcutaneous formulation of VDA-1102 exhibited a stable plasma concentration (>24 h) and dose-linear pharmacokinetics in pediatric patients with brain tumors, indicating broad-spectrum potential in solid tumors (138).

Ketoconazole, a United States Food and Drug Administration-approved antifungal agent and HK2 inhibitor, disrupts the interaction between HK2 and aminoacyl transfer RNA synthetase complex-interacting multifunctional protein 2 (AIMP2). This reduces autophagic lysosome-dependent degradation of AIMP2, restoring AIMP2-mediated apoptosis and synergizing with radiotherapy to suppress HCC growth. This drug-repurposing strategy overcomes radioresistance with translational advantages in terms of safety and cost (139).

A novel series of 2,6-disubstituted glucosamines has been identified as potent and selective HK2 inhibitors. These compounds demonstrate significant selectivity over HK1 and have been shown to inhibit glycolysis in cancer cell lines, underscoring their potential as therapeutic agents (140). Ikarugamycin, a polycyclic tetramide macrolide compound isolated from Actinomycetes associated with mangroves, has been shown to inhibit the glycolytic flux in pancreatic cancer cells by targeting HK2 and exhibits antitumor activity in murine models without exhibiting significant cytotoxicity (141). Another promising development in the field is the identification of natural HK2 inhibitors from Ganoderma sinense. For example, a specific steroid compound, (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol, has been identified as having a high binding affinity for HK2, suggesting its potential as a drug candidate for cancer therapy (142). This discovery is complemented by a broader study that systematically reviews the characteristics of HK2 inhibitors, emphasizing the need for compounds with improved efficacy and selectivity due to the poor performance of existing inhibitors when used alone (143).

RNA interference-based therapy, which silences the expression of HK2, has been shown to inhibit tumor cell proliferation and metastasis, thereby demonstrating notable therapeutic potential (144–148). The clinical potential of HK2 inhibitors is further supported by studies on their role in inducing apoptosis and endoplasmic reticulum stress in cancer cells. For instance, the HK2 inhibitor 3-bromopyruvate has been shown to inhibit the survival and proliferation of colon cancer cells, suggesting its utility in combination therapies (149). Additionally, the use of azole antifungals, such as ketoconazole and posaconazole, has demonstrated efficacy in targeting HK2-expressing glioblastoma cells, providing a basis for repurposing these compounds in clinical trials (150). These findings collectively underscore the therapeutic promise of HK2 inhibitors and their potential to advance to clinical applications (Table II).

Potential of HK2 as a relevant predictive biomarker

As the role of HK2 in tumor immune evasion becomes clearer, its utility as a predictive biomarker for immunotherapy is gaining attention. In bladder cancer research, the immune expression of glycolysis-related proteins, including HK2, has been assessed. The findings indicated that high HK2 expression was closely associated with independent prognostic factors for progression-free survival and overall survival, suggesting its utility as a prognostic biomarker for patients with this disease (151). In colorectal cancer, HK2 expression is associated with tumor size, depth of invasion, liver metastasis and TNM stage, effectively predicting the recurrence risk and overall mortality. Thus, HK2 serves as an important prognostic biomarker (12). Additionally, in patients with lung cancer, HK2 expression serves as a biomarker for identifying a novel subset of circulating tumor cells associated with poor prognosis. This finding is instrumental for prognostic evaluation prior to treatment (152). Positron emission tomography utilizing 18F-fluorodeoxyglucose (18F-FDG) is a widely employed technique for assessing tumor glucose metabolism. HK2 expression is associated with the uptake of 18F-FDG by tumors (153). Consequently, HK2 detection can facilitate the early diagnosis, staging and evaluation of the treatment response in tumors (10). Furthermore, the development of a ternary scoring system incorporating HK2, PD-L1 and HIF-1α for predicting the response of patients with liver cancer is a promising approach that leverages the interplay between metabolic pathways, immune checkpoints and hypoxic conditions within the TME (154). This model is substantiated by several studies that highlight the critical roles of these biomarkers in HCC prognosis and treatment response (155–157).

The integration of single-cell transcriptomics and spatial transcriptomics has revolutionized the current understanding of cell dynamics and tissue structure, providing unprecedented insights into the study of gene expression patterns and cell interactions in natural spatial environments (158,159). This two-pronged research strategy is particularly important when monitoring the changes in HK2; it can achieve single-cell resolution analysis of HK2 expression profiles while maintaining spatial information, thereby deeply analyzing the expression distribution of HK2 in complex tissue environments and its interaction with other factors. This strategy can also provide in-depth insights into the metabolic heterogeneity within tumors (160,161). Based on single-cell transcriptome and spatial transcriptome techniques, the HK2-Glycolytic Activity Index shows good stability in various tumor types and is associated with the characteristics of immune infiltration in the tumor microenvironment, having the potential to guide the selection of CAR-T cell therapy (20). Furthermore, advanced computational tools, such as the Significant Process Inference Algorithm, enhance the detection of significant expression patterns and biological processes, even the subtle changes in HK2 expression across different cell populations (162). Furthermore, the application of deep learning models, such as KanCell, enhances the analysis of cellular heterogeneity by integrating single-cell RNA-sequencing and spatial transcriptomics data (163). These models can effectively capture non-linear relationships and optimize computational efficiency, providing a more accurate depiction of HK2 expression patterns and their implications in disease microenvironments (164). By leveraging these advanced technologies, researchers can gain a deeper understanding of the molecular mechanisms underlying HK2 regulation, and the impact on cellular metabolism and disease progression.

Integrated HK2-targeted therapy and immunotherapy strategies

Current research on tumor immune escape is focused on several advanced fields. A comprehensive investigation into the interactions among diverse cellular and molecular mechanisms within the TME, particularly the metabolic interactions between immune cells and tumor cells, will provide a theoretical foundation for the development of more effective therapeutic strategies. Studies have identified that TAMs facilitate tumor immune escape through multiple signaling pathways, such as IL-4/STAT6, MAPK/ERK and TLR-NF-κB (165–167). Therapeutic strategies aimed at targeting TAMs, including colony stimulating factor 1 receptor inhibitors and CD40 agonists, have progressed to clinical trials (nos. NCT04169672 and NCT04059588), illustrating their potential to mitigate immunosuppression and augment the effectiveness of immunotherapy (78,168,169). Conversely, the utilization of advanced technologies, such as single-cell sequencing and high-dimensional flow cytometry, offers a deeper understanding of the heterogeneity inherent in tumor immune evasion, thereby providing a more precise foundation for personalized treatment approaches (170).

Conclusions

The multifaceted role of HK2 in linking tumor metabolic reprogramming and immune escape is becoming increasingly evident. HK2, with its distinctive dual catalytic active domain and N-terminal mitochondrial anchoring capability, facilitates the ‘metabolic compartmentalization’ effect and ‘metabolic relay’, thereby mediating efficient glycolysis. HK2 has a bidirectional regulatory network with critical oncogenic pathways, including the PI3K/AKT, MAPK, NF-κB, TGF-β and JAK/STAT pathways, thereby enhancing the glycolysis-dependent immune evasion of tumors. The metabolic competition induced by the Warburg effect facilitates the influence of HK2 on the function of immune cells in the TME, leading to the formation of hypoxic and hypoglycemic niches. This process is characterized by abundant lactic acid excretion, which collectively influences immune cell distribution, alters immune cell function and modulates the expression of immune checkpoints, thereby further facilitating tumor immune escape. The protein kinase function, subcellular localization regulation of HK2 and its involvement in different immune subtypes have gained notable attention, offering a novel perspective for a comprehensive analysis of its non-glycolytic functions. Emerging HK2 inhibitors, along with multi-dimensional predictive models and their integration with immunotherapy, have demonstrated promising potential for clinical translation. Future research should focus on elucidating immune subtype-specific mechanisms, developing highly selective targeted therapeutics and conducting comprehensive analyses of HK2-related molecular characteristics. These efforts should aim to advance the clinical implementation of HK2-targeted intervention strategies in precision immunotherapy.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82505450), the Top Talent Support Program for Young and Middle-Aged People of Wuxi Health Committee (grant no. HB2023067), the Natural Science Foundation of Jiangsu Province (grant no. BK20240309), and the Natural Science Foundation of Nanjing University of Chinese Medicine (grant no. XZR2023095).

Availability of data and materials

Not applicable.

Authors' contributions

YQ participated in conceptualization, data curation and writing of the original draft. XZ participated in conceptualization, acquisition of funding, and reviewing and editing of the manuscript. DN and QT participated in reviewing and editing the manuscript. CJ participated in reviewing and editing the manuscript, and acquiring resources. Data authentication is not applicable. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

During the preparation of this work, artificial intelligence tools (ChatGPT) were used to improve the readability and language of the manuscript, and subsequently, the authors revised and edited the content produced by the artificial intelligence tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

References