IDH-Mutant Glioblastoma (Secondary GBM)
Frequency: ~10% of all GBM cases
Frequency: ~10% of all GBM cases
Isocitrate dehydrogenase (IDH) enzymes, of which there are three isoforms, are essential enzymes that participate in several major metabolic processes, such as the Krebs cycle, glutamine metabolism, lipogenesis and redox regulation.1,2,3
IDH1 is located in the cytoplasm and peroxisomes, whereas IDH2 and IDH3 are located in the mitochondrial matrix.4
The catalytic sites of IDH1 and IDH2 exhibit affinity for the substrate, isocitrate, together with nicotinamide adenine dinucleotide phosphate (NADP+) and a divalent metal cation, usually magnesium or manganese,5
resulting in the formation of α-ketoglutarate (α-KG).
IDH3, which also catalyses the transformation from isocitrate into α-KG, employs nicotinamide adenine dinucleotide (NAD+) as its cofactor. The catalytic activity of IDH requires homodimerisation along with an alteration in the enzyme conformation; isocitrate binding changes the structure of the enzyme from an open to a closed conformation.6
IDH1 is located in the cytoplasm and peroxisomes, whereas IDH2 and IDH3 are located in the mitochondrial matrix.4
The catalytic sites of IDH1 and IDH2 exhibit affinity for the substrate, isocitrate, together with nicotinamide adenine dinucleotide phosphate (NADP+) and a divalent metal cation, usually magnesium or manganese,5
resulting in the formation of α-ketoglutarate (α-KG).
IDH3, which also catalyses the transformation from isocitrate into α-KG, employs nicotinamide adenine dinucleotide (NAD+) as its cofactor. The catalytic activity of IDH requires homodimerisation along with an alteration in the enzyme conformation; isocitrate binding changes the structure of the enzyme from an open to a closed conformation.6
Substrate recognition depends on the amino acid residues in the active site, whereas the frequent mutated active site residue in cancer is arginine 132 (R132).5
Mutations in IDH are prevalent in human malignancies. In glioma, IDH mutations are recognised in >80% of WHO grade II/III cases.7 In WHO grade IV glioblastoma (GBM), IDH mutations are also found frequent in secondary GBM, which account for 73% of clinical cases, whereas they are less seen in primary GBM (3.7%).8
A follow-up investigation showed that the presence of IDH mutations predict a favourable disease outcome with prolonged median survival in GBM:
In addition, IDH-mutated glioma is more likely to develop a hypermutation phenotype, which is associated with worsened prognosis.9 In non-central nervous system (non-CNS) malignancies, IDH mutations are identified in acute myeloid leukaemia (AML; 16% among all clinical cases),10 intrahepatic cholangiocarcinoma (23% among all clinical cases)11 and central/periosteal chondrosarcoma (56% among all clinical cases).12 The investigation of these non-CNS tumours with similar IDH mutation provides valuable information for glioma research, whereas in the present review we tend to be focussed on IDH-mutated glioma.
Mutations in IDH are prevalent in human malignancies. In glioma, IDH mutations are recognised in >80% of WHO grade II/III cases.7 In WHO grade IV glioblastoma (GBM), IDH mutations are also found frequent in secondary GBM, which account for 73% of clinical cases, whereas they are less seen in primary GBM (3.7%).8
A follow-up investigation showed that the presence of IDH mutations predict a favourable disease outcome with prolonged median survival in GBM:
- IDH wild type: 15 months;
- IDH mutant: 31 months
- IDH wild type: 20 months;
- IDH mutant: 65 months.7
In addition, IDH-mutated glioma is more likely to develop a hypermutation phenotype, which is associated with worsened prognosis.9 In non-central nervous system (non-CNS) malignancies, IDH mutations are identified in acute myeloid leukaemia (AML; 16% among all clinical cases),10 intrahepatic cholangiocarcinoma (23% among all clinical cases)11 and central/periosteal chondrosarcoma (56% among all clinical cases).12 The investigation of these non-CNS tumours with similar IDH mutation provides valuable information for glioma research, whereas in the present review we tend to be focussed on IDH-mutated glioma.
8HB9 IDH1 R132H Mutant in Complex with NADPH
5LGE IDH1 mutant (R132H) in complex with NADP+ BAY 1436032
6ADG IDH1 R132H in complex with AG-881
8T7O R132H mutant of IDH1 bound to AG-120
8VH9 IDH1 R132Q in complex with NADPH
8VHA IDH1 R132Q in complex with NADPH and Alpha-Ketoglutarate
8VHD IDH1 R132Q in complex with NADPH and Isocitrate
8VHE IDH1 R132Q in Complex with NADPH-TCEP Adduct
8VHB IDH1 R132Q in complex with NADPH and Alpha-Ketoglutarate
5LGE IDH1 mutant (R132H) in complex with NADP+ BAY 1436032
6ADG IDH1 R132H in complex with AG-881
8T7O R132H mutant of IDH1 bound to AG-120
8VH9 IDH1 R132Q in complex with NADPH
8VHA IDH1 R132Q in complex with NADPH and Alpha-Ketoglutarate
8VHD IDH1 R132Q in complex with NADPH and Isocitrate
8VHE IDH1 R132Q in Complex with NADPH-TCEP Adduct
8VHB IDH1 R132Q in complex with NADPH and Alpha-Ketoglutarate
6VFZ (IDH2) R140Q Homodimer +m NADPH and AG-881 (Vorasidenib)
3BLV Yeast IDH1, IDH2 with Citrate Bound in the Regulatory Subunits
5I96 Mitochondrial (IDH2) R140Q Homodimer in Complex with AG-221 (Enasidenib)
4JA8 IDH2 R140Q with AGI-6780
6ADI IDH2 R140Q in complex with AG-881
5I95 Mitochondrial IDH2 R140Q Homodimer bound to NADPH and alpha-Ketoglutaric acid
5H3F mouse IDH2 complexed with isocitrate
3BLV Yeast IDH1, IDH2 with Citrate Bound in the Regulatory Subunits
5I96 Mitochondrial (IDH2) R140Q Homodimer in Complex with AG-221 (Enasidenib)
4JA8 IDH2 R140Q with AGI-6780
6ADI IDH2 R140Q in complex with AG-881
5I95 Mitochondrial IDH2 R140Q Homodimer bound to NADPH and alpha-Ketoglutaric acid
5H3F mouse IDH2 complexed with isocitrate
8GRG active mutant of the alpha gamma heterodimer of IDH3
8GRH active mutant of the alpha gamma heterodimer of IDH3 + CIT
8GRU active mutant of the alpha beta heterodimer of IDH3 in complex with ICT, NAD and Ca
7CE3 IDH3 holoenzyme in APO form.
8GRD IDH3 in complex with ADP and Mg
8GRB active mutant of the alpha beta heterodimer of human IDH3
6KDY alpha bata heterodimer of human IDH3 in complex with NAD
6L59 IDH3 in complex with CIT, Mg and ATP binding at allosteric site and Mg, ATP binding at active site.
8GRH active mutant of the alpha gamma heterodimer of IDH3 + CIT
8GRU active mutant of the alpha beta heterodimer of IDH3 in complex with ICT, NAD and Ca
7CE3 IDH3 holoenzyme in APO form.
8GRD IDH3 in complex with ADP and Mg
8GRB active mutant of the alpha beta heterodimer of human IDH3
6KDY alpha bata heterodimer of human IDH3 in complex with NAD
6L59 IDH3 in complex with CIT, Mg and ATP binding at allosteric site and Mg, ATP binding at active site.
IDH mutations that are associated with cancer tend to localise to the arginine residue that is crucial for the recognition of isocitrate (R132 for IDH1, R140 or R172 for IDH2).7 Missense mutations in the IDH1 gene result in the replacement of a strong, positively charged arginine residue at position 132 with lower-polarity amino acids such as histidine (H), lysine (K) or cysteine (C), which impedes the formation of hydrogen bonds with the α-carboxyl and β-carboxyl sites of isocitrate.13,14
The mutant IDH enzyme therefore exhibits decreased affinity for isocitrate, along with an elevated preference for NADPH. However, only one copy of the IDH gene is mutated in tumours and, in tumour cells harbouring heterozygous IDH mutations, the main forms of IDH dimers are presumed to be heterodimers that contain a version of wild-type IDH1 and a version with the R132H mutation. As a result, in IDH-mutant cells, the IDH1 wild-type component of the dimer converts isocitrate into α-KG to produce NADPH, whereas the mutant part of the dimer exhibits neomorphic activity, converting α-KG into D-2-hydroxyglutarate (D-2-HG) in an NADPH-dependent manner
The mutant IDH enzyme therefore exhibits decreased affinity for isocitrate, along with an elevated preference for NADPH. However, only one copy of the IDH gene is mutated in tumours and, in tumour cells harbouring heterozygous IDH mutations, the main forms of IDH dimers are presumed to be heterodimers that contain a version of wild-type IDH1 and a version with the R132H mutation. As a result, in IDH-mutant cells, the IDH1 wild-type component of the dimer converts isocitrate into α-KG to produce NADPH, whereas the mutant part of the dimer exhibits neomorphic activity, converting α-KG into D-2-hydroxyglutarate (D-2-HG) in an NADPH-dependent manner
Metabolic Reprogramming
IDH-mutant enzymes cause the accumulation of D-2-HG at concentrations as high as 5–30 mM15 in the cytoplasm, thereby draining carbohydrates from the Krebs cycle.18 The Krebs cycle is adjusted to compensate for fluctuations in the metabolic pathways.19 A 13C metabolic flux analysis suggested that IDH1-mutated cells exhibit increased oxidative metabolism in the Krebs cycle, whereas reductive glutamine metabolism is suppressed.20
With the depletion of cellular metabolism, several non-Krebs-cycle sources of carbohydrates are recruited to compensate for the loss of α-KG.21,22
Waitkus et al.23 demonstrated that glutamate dehydrogenase 2, an enzyme that catalyses the conversion of glutamate into α-KG and that is expressed at high levels in the brain, is important for relieving the metabolic liabilities in the context of IDH mutants. This finding is confirmed by the observation that IDH-mutated glioma cells are more sensitive to the inhibition of glutaminase,24 suggesting that glutaminolysis serves as a key compensatory pathway to maintain metabolic homoeostasis.
With the depletion of cellular metabolism, several non-Krebs-cycle sources of carbohydrates are recruited to compensate for the loss of α-KG.21,22
Waitkus et al.23 demonstrated that glutamate dehydrogenase 2, an enzyme that catalyses the conversion of glutamate into α-KG and that is expressed at high levels in the brain, is important for relieving the metabolic liabilities in the context of IDH mutants. This finding is confirmed by the observation that IDH-mutated glioma cells are more sensitive to the inhibition of glutaminase,24 suggesting that glutaminolysis serves as a key compensatory pathway to maintain metabolic homoeostasis.
McBrayer et al.25 further highlighted the dependency of IDH1-mutated cells on glutaminolysis, as D-2-HG functions as an inhibitor of the branched-chain amino acid transaminase (BCAT1/2), thereby decreasing the levels of glutamate. Furthermore, the consumption of NADPH by IDH mutants compromises de novo lipogenesis, resulting in an increased dependence on exogenous lipid sources for cellular growth.2 This is accompanied by the stimulation, by D-2-HG, of glutamine-derived lipogenesis under hypoxic condition to meet the needs for lipid productivity.26
Lactate dehydrogenase A (LDHA) catalyses the transformation of pyruvate formed by glycolysis into L-lactate,27 and the expression of LDHA is thus considered to be a hallmark of Warburg phenotype, allowing rapid glycolytic flux to meet the demands for cellular proliferation.28 Although LDHA is highly expressed in a variety of cancer cells, it is silenced in glioma tissue specimens and patient-derived glioma cells with IDH mutants.29,30
Silencing of LDHA (and of several other glycolysis genes including CA9 and VEGFA) has been found to be associated with hypermethylation in the promoter region of these genes in response to D-2-HG. The overall epigenetic silencing of the glycolytic pathway might explain the slow-growing nature of IDH-mutated glioma as compared with their IDH wild-type counterparts.30,31 In support of this hypothesis, in a recent study, the acquisition of the Warburg phenotype was associated with more aggressive gliomas and was found to occur at the CpG island methylator phenotype (G-CIMP) in gliomas described below, which is specific for astrocytoma.32
In addition, IDH mutations lead to the neomorphic enzyme activity, which redirects the Krebs cycle for D-2-HG production. The resultant decrease in α-KG levels might affect the level of hypoxia-inducible factor subunit HIF-1α,33 as α-KG is normally needed for prolyl hydroxylases (PHD) to hydroxylate and promote the degradation of HIF. However, the detailed molecular mechanism on how HIF is regulated in the context of IDH mutation is currently unclear. Other lines of evidence showed that D-2-HG, but not L-2-HG, stimulates the activity of the prolyl hydroxylase PHD2, which results in the reduced expression of HIF-1/2α.34 More effort is encouraged to elucidate the relationship between D-2-HG and the hypoxia-sensing pathway in glioma and other IDH1-mutated malignancies.
Overall, the acquisition of mutant IDH results in substantial reprogramming of cellular metabolism. Glutamine and/or glutamate serve as key substrates to compensate for the metabolic impact by strengthening synthetic pathways for lipids and glutathione. Interestingly, IDH-mutated glioma shows a distinctive metabolic pattern compared with other solid tumours—most notably, the remarkably reduced glycolysis, the metabolic hallmark of fast proliferating malignancies. The unique metabolic pathways in IDH-mutated glioma not only explain the slow-growing nature of this disease but also suggest that developing targeted strategies for IDH-mutant-specific metabolic patterns could be a valuable approach for future glioma therapeutics.
Lactate dehydrogenase A (LDHA) catalyses the transformation of pyruvate formed by glycolysis into L-lactate,27 and the expression of LDHA is thus considered to be a hallmark of Warburg phenotype, allowing rapid glycolytic flux to meet the demands for cellular proliferation.28 Although LDHA is highly expressed in a variety of cancer cells, it is silenced in glioma tissue specimens and patient-derived glioma cells with IDH mutants.29,30
Silencing of LDHA (and of several other glycolysis genes including CA9 and VEGFA) has been found to be associated with hypermethylation in the promoter region of these genes in response to D-2-HG. The overall epigenetic silencing of the glycolytic pathway might explain the slow-growing nature of IDH-mutated glioma as compared with their IDH wild-type counterparts.30,31 In support of this hypothesis, in a recent study, the acquisition of the Warburg phenotype was associated with more aggressive gliomas and was found to occur at the CpG island methylator phenotype (G-CIMP) in gliomas described below, which is specific for astrocytoma.32
In addition, IDH mutations lead to the neomorphic enzyme activity, which redirects the Krebs cycle for D-2-HG production. The resultant decrease in α-KG levels might affect the level of hypoxia-inducible factor subunit HIF-1α,33 as α-KG is normally needed for prolyl hydroxylases (PHD) to hydroxylate and promote the degradation of HIF. However, the detailed molecular mechanism on how HIF is regulated in the context of IDH mutation is currently unclear. Other lines of evidence showed that D-2-HG, but not L-2-HG, stimulates the activity of the prolyl hydroxylase PHD2, which results in the reduced expression of HIF-1/2α.34 More effort is encouraged to elucidate the relationship between D-2-HG and the hypoxia-sensing pathway in glioma and other IDH1-mutated malignancies.
Overall, the acquisition of mutant IDH results in substantial reprogramming of cellular metabolism. Glutamine and/or glutamate serve as key substrates to compensate for the metabolic impact by strengthening synthetic pathways for lipids and glutathione. Interestingly, IDH-mutated glioma shows a distinctive metabolic pattern compared with other solid tumours—most notably, the remarkably reduced glycolysis, the metabolic hallmark of fast proliferating malignancies. The unique metabolic pathways in IDH-mutated glioma not only explain the slow-growing nature of this disease but also suggest that developing targeted strategies for IDH-mutant-specific metabolic patterns could be a valuable approach for future glioma therapeutics.
Gilbert et al.53 showed that IDH1-mutated glioma cells exhibit strong oxidative stress, as evidenced by an enhanced expression of manganese superoxide dismutase and protein carbonylation. This increased stress was confirmed by subsequent investigations showing that IDH-mutated cancers are more prone to oxidative damage.52,54 Also was confirmed elevated oxidative stress that is closely related to the acquisition of IDH mutants, leading to oxidative damage in biomolecules such as DNA and lipids.51 Owing to the substantially increased oxidative burden, inhibiting antioxidant pathways, such as the synthesis of glutathione, which is mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), could be a valuable strategy for targeting IDH1-mutated solid tumours.55 In addition, proline synthesis has been reported to maintain redox homoeostasis in mitochondria in IDH1-mutated cells. Enhanced activity of pyrroline 5-carboxylate reductase 1-mediated glutamate-to-proline transformation in IDH-mutated cells alongside the oxidation of NADH partially uncouples the electron transport chain from Krebs cycle activity, thus maintaining anabolism in cancer cells
Direct targeting of mutant IDH
Given that the neomorphic activity of IDH mutants correlates with malignant transformation, direct targeting of the mutant enzyme has been a heavily pursued strategy. Rohle et al.57 reported the first synthetic inhibitor of the IDH mutant, AGI-5198, which blocks the production of D-2-HG and impairs IDH1-mutated xenograft growth in vivo.
Second generation of IDH-mutant inhibitors, ivosidenib (AG-120) and vorasidenib (AG-881), are currently approved by the Food and Drug Administration as a therapeutic option for IDH-mutated AML.58 These IDH-mutant inhibitors exhibit an improved brain-to-plasma ratio, suggesting that they might be effective for IDH1-mutated glioma.60 Several other IDH-mutant inhibitors, such as BAY1436032, have shown tumour-suppressing effects as experimental therapeutics for the treatment of AML and astrocytoma in animal models.61,62
Given that the neomorphic activity of IDH mutants correlates with malignant transformation, direct targeting of the mutant enzyme has been a heavily pursued strategy. Rohle et al.57 reported the first synthetic inhibitor of the IDH mutant, AGI-5198, which blocks the production of D-2-HG and impairs IDH1-mutated xenograft growth in vivo.
Second generation of IDH-mutant inhibitors, ivosidenib (AG-120) and vorasidenib (AG-881), are currently approved by the Food and Drug Administration as a therapeutic option for IDH-mutated AML.58 These IDH-mutant inhibitors exhibit an improved brain-to-plasma ratio, suggesting that they might be effective for IDH1-mutated glioma.60 Several other IDH-mutant inhibitors, such as BAY1436032, have shown tumour-suppressing effects as experimental therapeutics for the treatment of AML and astrocytoma in animal models.61,62
Two clinical studies (NCT03127735 and NCT02746081) are currently ongoing to confirm these findings in patients with IDH1-mutated AML or advanced solid tumours, respectively.
Despite the promising success of the IDH-mutant inhibitors, several studies have indicated the potential limitations of their application. For example, Johannessen et al.63 discovered that, although the IDH-mutant inhibitor AGI-5198 successfully reduces neomorphic activity, it relieves hypermethylation phenotype but to a much less extent, as evidenced by elevated histone-3 methylation. In addition, Sulkowski et al.64 reported that
AGI-5198 relieves the burden of DNA damage in cancer cells, which might increase their resistance to genotoxic therapies, such as radiation and chemo agents. This phenomenon has been confirmed by another study showing that AGI-5198 confers radioprotective effects on IDH1-mutated cancer cells.65 Overall, targeting IDH-mutant neomorphic activity is a straightforward strategy and has shown efficacy against haematopoietic malignancies in humans and several experimental models for solid cancers. In addition to suppressing D-2-HG production, a combined approach with other agents, such as inhibitors of critical enzymes in metabolic or DNA repair pathways, might be helpful to improve the disease outcome (see the discussion below on synthetic lethality).
Despite the promising success of the IDH-mutant inhibitors, several studies have indicated the potential limitations of their application. For example, Johannessen et al.63 discovered that, although the IDH-mutant inhibitor AGI-5198 successfully reduces neomorphic activity, it relieves hypermethylation phenotype but to a much less extent, as evidenced by elevated histone-3 methylation. In addition, Sulkowski et al.64 reported that
AGI-5198 relieves the burden of DNA damage in cancer cells, which might increase their resistance to genotoxic therapies, such as radiation and chemo agents. This phenomenon has been confirmed by another study showing that AGI-5198 confers radioprotective effects on IDH1-mutated cancer cells.65 Overall, targeting IDH-mutant neomorphic activity is a straightforward strategy and has shown efficacy against haematopoietic malignancies in humans and several experimental models for solid cancers. In addition to suppressing D-2-HG production, a combined approach with other agents, such as inhibitors of critical enzymes in metabolic or DNA repair pathways, might be helpful to improve the disease outcome (see the discussion below on synthetic lethality).
RTK pathway.
RTK (receptor tyrosine kinase) signaling is the most frequently altered signaling pathway in GBM, especially in IDH-wildtype GBM tumors. RTK is a cell-surface receptor that binds growth factors, the family of which includes EGFR, PDGFR, TGFR, FGFR, MET, and VEGFR, and is an essential component of signal transduction pathways that mediate cell-to-cell communication.
In GBM, the activation of RTK signaling through the PI3K/AKT/mTOR pathway induces cell proliferation, migration, differentiation, and survival.
The most common targets of the RTK pathway are EGFR and PTEN, the former acting in an oncogenic role while the latter acting as a tumor suppressor.
In GBM cells, the activation of EGFR and the PI3K/AKT/mTOR signaling could be achieved either through amplification of the EGFR (resulting in overexpression of EGFR) and/or EGFR mutation. The negative regulator of the pathway, PTEN, could be inactivated through mutation or deletion, and thus facilitates the pathway activation and induces cell migration, invasion, and survival.
RTK (receptor tyrosine kinase) signaling is the most frequently altered signaling pathway in GBM, especially in IDH-wildtype GBM tumors. RTK is a cell-surface receptor that binds growth factors, the family of which includes EGFR, PDGFR, TGFR, FGFR, MET, and VEGFR, and is an essential component of signal transduction pathways that mediate cell-to-cell communication.
In GBM, the activation of RTK signaling through the PI3K/AKT/mTOR pathway induces cell proliferation, migration, differentiation, and survival.
The most common targets of the RTK pathway are EGFR and PTEN, the former acting in an oncogenic role while the latter acting as a tumor suppressor.
In GBM cells, the activation of EGFR and the PI3K/AKT/mTOR signaling could be achieved either through amplification of the EGFR (resulting in overexpression of EGFR) and/or EGFR mutation. The negative regulator of the pathway, PTEN, could be inactivated through mutation or deletion, and thus facilitates the pathway activation and induces cell migration, invasion, and survival.
Another commonly altered RTK pathway in GBM is the Ras pathway (Ras/BRAF/MEK) [67,68]. Active Ras (Ras-GTP) promotes cell cycle progression, cell survival, and migration through a cascade of downstream effectors. RTK has been suggested as a druggable target in GBM and is extensively investigated in clinical trials.
The retinoblastoma protein (RB) pathway is also found to be frequently altered in GBM and plays a crucial role in regulating tumorigenesis in GBM. The phosphorylation of RB protein, which is accomplished by the CDK4/Cyclin D1 complex, can inhibit the cell cycle progress from the G1 to S phase by binding with the E2F transcription factor. RB pathway could be joined with the TP53 pathway through CDKN2A, which encodes Ink4a and Arf proteins and plays an important role in activating RB and TP53, respectively.
The growth inhibition function of the RB pathway is often disrupted in GBM, most commonly due to inactivation of CDKN2A/CDKN2B and RB1 and amplification of CDK4 and CDK6. Methylation of the RB1 promoter, which is frequent in secondary GBM (IDH-mutant ones), can also result in decreased RB1 expression and cell-cycle checkpoint function and finally leads to dysregulated cell cycle and uncontrolled cell proliferation. CDK4 and CDK6 inhibitors have shown promising antitumor efficacy in GBM and are being studied in clinical trials
The growth inhibition function of the RB pathway is often disrupted in GBM, most commonly due to inactivation of CDKN2A/CDKN2B and RB1 and amplification of CDK4 and CDK6. Methylation of the RB1 promoter, which is frequent in secondary GBM (IDH-mutant ones), can also result in decreased RB1 expression and cell-cycle checkpoint function and finally leads to dysregulated cell cycle and uncontrolled cell proliferation. CDK4 and CDK6 inhibitors have shown promising antitumor efficacy in GBM and are being studied in clinical trials
7PE9 DEPTOR bound to human mTOR complex 2
7TZO apo structure of human mTORC2 complex
6ZWO mTOR complex 2, focused on one half
7PE8 DEPTOR bound to human mTOR complex 2, focussed on one protomer
7TZO apo structure of human mTORC2 complex
6ZWO mTOR complex 2, focused on one half
7PE8 DEPTOR bound to human mTOR complex 2, focussed on one protomer
5FLC human mTOR Complex 1
7PEA DEPTOR bound to human mTOR complex 1
7PEB DEPTOR bound to human mTOR complex 1, focussed on one protomer
6SB2 mTORC1 bound to active RagA/C GTPases
6SB0 mTORC1 bound to PRAS40-fused active RagA/C GTPases
9ED7 mTOR on membrane
9F45 human LST2 bound to human mTOR complex 1
7PEA DEPTOR bound to human mTOR complex 1
7PEB DEPTOR bound to human mTOR complex 1, focussed on one protomer
6SB2 mTORC1 bound to active RagA/C GTPases
6SB0 mTORC1 bound to PRAS40-fused active RagA/C GTPases
9ED7 mTOR on membrane
9F45 human LST2 bound to human mTOR complex 1
EGFR — EGFRvIII (Δ2–7)
EGFR — EGFR (wild‑type; amp/overexpr)
PIK3CA — PI3K catalytic subunit (p110α)
PTEN — PTEN phosphatase (loss‑of‑function)
AKT1/2/3 — AKT kinases (example: AKT1 E17K)
KRAS/NRAS — RAS GTPases (oncogenic)
BRAF — BRAF kinase
TP53 — p53 tumor suppressor
IDH1 — IDH1 (R132H)
IDH2 — IDH2 (R172K)
CDKN2A/B — p16INK4a / p14ARF (structure shown: p16)
RB1 — Retinoblastoma protein (pRb “pocket”)
MGMT — O6‑methylguanine‑DNA methyltransferase
TERT — Telomerase reverse transcriptase (human telomerase)
CD274 — PD‑L1
PDCD1 — PD‑1 (CD279)
CTLA4 — CTLA‑4 (CD152)
LAG3 — LAG‑3 (CD223)
HAVCR2 — TIM‑3
CD276 — B7‑H3 (closest experimental: mouse ectodomain)
IDO1 — Indoleamine 2,3‑dioxygenase
HLA‑A/B/C — HLA Class I (example: HLA‑A*02:01)
HLA‑DR/DQ/DP — HLA Class II (example: HLA‑DR1)
B2M — β2‑microglobulin
TAP1/TAP2 — Peptide transporter (TAP)
EGFR (vIII) — EGFRvIII heterogeneity / mosaic expression
PROM1 — CD133 (Prominin‑1)
SOX2 — SOX2 transcription factor (HMG box)
CD44 — CD44 hyaluronan‑binding domain
MET — c‑MET receptor (kinase domain)
PDGFRA — PDGFR‑alpha (kinase domain)
FGFR1/2/3 — Fibroblast growth factor receptors (kinase domains)
NF1 — Neurofibromin (GAP‑related domain)
VEGFA — VEGF
TGFB1 — TGF‑β1
IL10 — Interleukin‑10
IL6 — Interleukin‑6
ARG1 — Arginase‑1
| Gene | Protein | Mutation/Alteration | Resistance Mechanism | Clinical Impact |
|---|---|---|---|---|
| EGFR | EGFRvIII (variant III) | In-frame deletion Δ2-7 (exons 2-7) | PRIMARY TARGET - Loss of EGFRvIII expression after vaccination leads to immune e... | High anti-EGFRvIII titers (≥1:12,800) predict survival (HR=0.17, p<0.0001); 80% ... |
| EGFR | EGFR wild-type | Amplification/overexpression | Provides alternative growth signaling; may sustain tumor despite EGFRvIII loss | May enable bypass signaling when EGFRvIII is lost; maintains EGFR pathway activa... |
| PIK3CA | PI3K catalytic subunit | H1047R, E545K activating mutations | Constitutive PI3K/AKT/mTOR activation bypasses need for upstream receptor signal... | Enables growth independent of EGFRvIII; may reduce dependence on vaccine target |
| PTEN | PTEN phosphatase | Loss of function (deletion, mutation) | Loss leads to constitutive PI3K/AKT activation; enhances immunosuppression | PTEN loss associated with reduced T-cell infiltration and immune evasion |
| AKT1/2/3 | AKT kinases | Amplification, E17K activating mutations | Direct AKT activation bypasses upstream signals | May reduce vaccine efficacy through pathway bypass |
| KRAS/NRAS | RAS GTPases | G12, G13, Q61 activating mutations | Constitutive MAPK pathway activation independent of EGFR | Enables EGFR-independent proliferation |
| BRAF | BRAF kinase | V600E and other mutations | Direct MAPK activation bypassing upstream receptors | May confer resistance to EGFR-targeted approaches |
| TP53 | p53 tumor suppressor | R175H, R248W, R273H missense mutations | Impaired apoptosis; altered tumor immunogenicity; affects DNA damage response | May affect tumor antigenicity and immune recognition |
| IDH1 | Isocitrate dehydrogenase 1 | R132H (>90% of IDH1 mutations) | Creates 2-hydroxyglutarate; alters epigenome and immune landscape | IDH-mutant GBM has distinct biology; limited data on vaccine response in this su... |
| IDH2 | Isocitrate dehydrogenase 2 | R172K, R172M | Similar to IDH1; produces 2-HG oncometabolite | Limited data on vaccine efficacy |
| CDKN2A/B | p16INK4a, p14ARF | Homozygous deletion | Cell cycle dysregulation; may affect tumor proliferation and immune surveillance | Contributes to aggressive phenotype |
| RB1 | Retinoblastoma protein | Loss of function, deletion | Cell cycle checkpoint loss; affects proliferation | Part of cell cycle dysregulation network |
| MGMT | O6-methylguanine-DNA methyltransferase | Promoter methylation (epigenetic) | Affects temozolomide sensitivity; methylated status associated with better progn... | Not directly validated as predictor of rindopepimut response |
| TERT | Telomerase reverse transcriptase | Promoter mutations (C228T, C250T) | Maintains telomere length; enables replicative immortality | No direct link to vaccine response established |
| CD274 | PD-L1 | Overexpression | Binds PD-1 on T cells; inhibits T-cell activation and cytotoxic function | High PD-L1 may suppress vaccine-induced T-cell responses |
| PDCD1 | PD-1 (CD279) | Expression on TILs | Inhibitory receptor; dampens T-cell responses upon ligand binding | May limit effector function of vaccine-induced T cells |
| CTLA4 | CTLA-4 (CD152) | Expression on T cells and Tregs | Competes with CD28 for B7; reduces T-cell priming and enhances Treg function | May blunt vaccine-induced T-cell priming |
| LAG3 | LAG-3 (CD223) | Expression on exhausted T cells | Binds MHC-II; inhibits T-cell activation | May contribute to T-cell exhaustion after vaccination |
| HAVCR2 | TIM-3 | Co-expression with PD-1 | Multiple ligands; promotes T-cell exhaustion | May limit durability of vaccine responses |
| CD276 | B7-H3 | Overexpression | Inhibits T-cell activation; promotes tumor immune evasion | May contribute to vaccine resistance |
| IDO1 | Indoleamine 2,3-dioxygenase | Expression in tumor/myeloid cells | Catabolizes tryptophan; creates immunosuppressive metabolites | Metabolic suppression of vaccine-induced T cells |
| HLA-A/B/C | HLA Class I | Downregulation, LOH | Prevents CD8+ T-cell recognition of tumor antigens | Reduces T-cell component of vaccine response |
| HLA-DR/DQ/DP | HLA Class II | Low expression | Reduces CD4+ T-cell help and antigen presentation | May limit helper T-cell responses to vaccine |
| B2M | β2-microglobulin | Mutations, deletions | Loss disrupts HLA class I surface expression | Complete resistance to CD8+ T-cell recognition |
| TAP1/TAP2 | Peptide transporters | Downregulation, mutations | Impairs peptide transport to ER for MHC loading | Reduces antigen presentation efficiency |
| EGFR (vIII) | EGFRvIII heterogeneity | Mosaic expression | Antigen-negative clones escape vaccine pressure; documented antigen loss at prog... | MAJOR RESISTANCE MECHANISM - Antigen loss documented after vaccination |
| PROM1 | CD133 (Prominin-1) | Cancer stem cell marker | Stem-like cells may be less immunogenic; maintain tumor-initiating capacity | May sustain tumor despite immune responses |
| SOX2 | SOX2 transcription factor | Variable expression | Maintains stemness; associated with therapy resistance | Stem cell populations may evade vaccine-induced immunity |
| CD44 | CD44 glycoprotein | High in mesenchymal GBM | Stem cell marker; promotes invasion and therapy resistance | May contribute to treatment resistance |
| MET | c-MET receptor | Amplification, overexpression | Alternative RTK; activates PI3K and MAPK pathways independent of EGFR | Enables growth when EGFRvIII is lost or targeted |
| PDGFRA | PDGFR-alpha | Amplification, activating mutations | Alternative growth factor receptor; bypass EGFR dependence | Maintains proliferation despite EGFRvIII targeting |
| FGFR1/2/3 | Fibroblast growth factor receptors | Amplification, fusions (rare) | Alternative RTK signaling | Potential bypass mechanism |
| NF1 | Neurofibromin | Loss of function, deletion | Loss leads to RAS hyperactivation; MAPK pathway activation | Enables RAS/MAPK signaling independent of EGFR |
| VEGFA | VEGF | Overexpression | Inhibits dendritic cell maturation; promotes Treg recruitment; impairs T-cell tr... | Suppresses vaccine-induced immunity; BLOCKED by bevacizumab in ReACT |
| TGFB1 | TGF-β | Overexpression | Potent immunosuppressor; inhibits T-cell activation and NK function; promotes Tr... | Strongly suppresses vaccine efficacy |
| IL10 | IL-10 | Overexpression | Anti-inflammatory cytokine; inhibits T-cell and DC function | Dampens vaccine-induced responses |
| IL6 | IL-6 | Overexpression | Pro-tumorigenic; promotes MDSC expansion | May impair vaccine responses |
| ARG1 | Arginase-1 | Expression in MDSCs/M2 macrophages | Depletes L-arginine; inhibits T-cell proliferation | Metabolically suppresses T cells |
The above results demonstrate that more neutrophils were recruited to generate neutrophil extracellular traps (NETs) in the recurrent GBM after surgery.
To reveal the recruitment of neutrophils, we evaluated the expression of neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL15 at the surgical site by the multiplex immunofluorescence analysis.
The results reveal that neurons, astrocytes, and microglia are all capable of secreting these neutrophil chemokines. By quantifying the number of CXCL1, CXCL2, and CXCL15-positive cells, we observed that the ratio of these cells secreting CXCL1 and CXCL15 followed the same order, i.e., neurons > microglia > astrocytes > vascular endothelial cells, but the ratio of cells secreting CXCL2 followed the order of microglia > astrocytes > neurons > vascular endothelial cells.
The secretion of CXCL1, CXCL2, and CXCL15 leads to the accumulation of neutrophils at the surgery site. These results not only demonstrate that surgery promotes the generation of NETs in vivo, but also suggest that NETs are associated with the malignant progression and recurrence of GBM.
To reveal the recruitment of neutrophils, we evaluated the expression of neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL15 at the surgical site by the multiplex immunofluorescence analysis.
The results reveal that neurons, astrocytes, and microglia are all capable of secreting these neutrophil chemokines. By quantifying the number of CXCL1, CXCL2, and CXCL15-positive cells, we observed that the ratio of these cells secreting CXCL1 and CXCL15 followed the same order, i.e., neurons > microglia > astrocytes > vascular endothelial cells, but the ratio of cells secreting CXCL2 followed the order of microglia > astrocytes > neurons > vascular endothelial cells.
The secretion of CXCL1, CXCL2, and CXCL15 leads to the accumulation of neutrophils at the surgery site. These results not only demonstrate that surgery promotes the generation of NETs in vivo, but also suggest that NETs are associated with the malignant progression and recurrence of GBM.
Lipopolysaccharide (LPS) is a classical agent that increases the release of NETs by neutrophils and thus increases the level of NETs in the medium. Because the LPS source can affect the formation of NETs, it was investigate the effect of LPS obtained from three different sources, i.e., LPS-O55, LPS-O128 and LPS-PA.
Compared with LPS-O55 and LPS-O128, LPS-PA induced neutrophils to generate more NETs. A transwell assay was performed to evaluate the effects of NETs on the migration of GL261 cells in vitro. The GL261 cells were treated with medium containing 1% fetal bovine serum (FBS, control), neutrophils medium (NE CM), medium collected from neutrophils treated with LPS (LPS CM) or medium collected from neutrophils treated with LPS and DNase 1 (DNase 1 CM).
Compared with LPS-O55 and LPS-O128, LPS-PA induced neutrophils to generate more NETs. A transwell assay was performed to evaluate the effects of NETs on the migration of GL261 cells in vitro. The GL261 cells were treated with medium containing 1% fetal bovine serum (FBS, control), neutrophils medium (NE CM), medium collected from neutrophils treated with LPS (LPS CM) or medium collected from neutrophils treated with LPS and DNase 1 (DNase 1 CM).
After the increase in NETs in the medium following the incubation of neutrophils with LPS, the migration rate of GL261 cells increased five folds. In contrast to LPS, DNase 1 was used to eliminate NETs by degrading their DNA structure. As expected, the migration rate of GL261 cells was reduced in the DNase 1 CM group, further demonstrating that NETs can promote the migration of GL261 cells.
In addition, NETs affected the proliferation of GL261 cells . The colony formation test findings showed that, compared with those in the control group, the colony formation ability of the LPS CM group was approximately 2.4 times greater.
However, the colony formation points of the DNase 1 CM group were nearly the same as those of the control group. These results demonstrate that NETs play a significant role in the migration and proliferation of tumor cells and thus promote the malignant progression of GBM.
In addition, NETs affected the proliferation of GL261 cells . The colony formation test findings showed that, compared with those in the control group, the colony formation ability of the LPS CM group was approximately 2.4 times greater.
However, the colony formation points of the DNase 1 CM group were nearly the same as those of the control group. These results demonstrate that NETs play a significant role in the migration and proliferation of tumor cells and thus promote the malignant progression of GBM.
Mechanism of CS nanozyme in alleviating inflammation and inhibiting the formation of NETs. The flow cytometric analysis showed that the percentage of ROS-positive cells in the control group was 33%, which increased to 82% after the addition of LPS to the neutrophils.
In contrast, after the addition of the CS nanozyme, the ratio gradually decreased from 82 to 46%, indicating that the ROS level in neutrophils was efficiently reduced by the CS nanozyme.
The CLSM images showed that normal neutrophils produced less ROS, and their ROS levels increased after LPS stimulation. However, with increasing concentrations of the CS nanozyme, the fluorescence intensity of DCF gradually decreased, which further demonstrates that the CS nanozyme can effectively eliminate ROS.
As neutrophils generate ROS mainly through NADPH oxidase
The results show that LPS increased the activity of NADPH oxidase approximately 1.7 times than that of untreated neutrophils, and the CS nanozyme significantly decreased its activity to approximately one-eighth of untreated neutrophils at a concentration of 6.25 μg/mL.
In contrast, after the addition of the CS nanozyme, the ratio gradually decreased from 82 to 46%, indicating that the ROS level in neutrophils was efficiently reduced by the CS nanozyme.
The CLSM images showed that normal neutrophils produced less ROS, and their ROS levels increased after LPS stimulation. However, with increasing concentrations of the CS nanozyme, the fluorescence intensity of DCF gradually decreased, which further demonstrates that the CS nanozyme can effectively eliminate ROS.
As neutrophils generate ROS mainly through NADPH oxidase
The results show that LPS increased the activity of NADPH oxidase approximately 1.7 times than that of untreated neutrophils, and the CS nanozyme significantly decreased its activity to approximately one-eighth of untreated neutrophils at a concentration of 6.25 μg/mL.
These results demonstrate that the CS nanozyme can act as an NADPH oxidase inhibitor strongly by scavenging ROS in a dose-dependent manner.
Next, we analysed the expression levels of AKT, ERK, and p38, which are ROS-related kinases that stimulate neutrophils to generate NETs. The phosphorylation of these proteins increased after the addition of LPS to neutrophils but decreased after co-culturing with the CS nanozyme. These results further confirm the excellent ROS scavenging ability of the CS nanozyme .
With the activation of ROS-related kinases, neutrophils can also release inflammatory factors, such as TNF-α, IL-6 and IL-1β. Therefore, the CS nanozyme can also decrease the release of inflammatory factors from neutrophils.
Treatment of neutrophils with LPS increased TNF-α expression nearly six folds compared with that in untreated neutrophils.
Next, we analysed the expression levels of AKT, ERK, and p38, which are ROS-related kinases that stimulate neutrophils to generate NETs. The phosphorylation of these proteins increased after the addition of LPS to neutrophils but decreased after co-culturing with the CS nanozyme. These results further confirm the excellent ROS scavenging ability of the CS nanozyme .
With the activation of ROS-related kinases, neutrophils can also release inflammatory factors, such as TNF-α, IL-6 and IL-1β. Therefore, the CS nanozyme can also decrease the release of inflammatory factors from neutrophils.
Treatment of neutrophils with LPS increased TNF-α expression nearly six folds compared with that in untreated neutrophils.
GSDMD, a crucial protein closely related to ROS levels during pyroptosis, has recently been demonstrated to play an important role in the formation of NETs. GSDMD can be cleaved to generate N-GSDMD, which can form pores in the cell membrane and release inflammatory cytokines, such as IL-1β and IL-18, as well as damage-associated molecular patterns, including IL-1α, lactate dehydrogenase (LDH) and HMGB1.
Caspase-1 as upstream of GSDMD, is crucial for the formation of NETs and the release of IL-1β. The change of cleaved caspase-1 was similar to GSDMD, indicating a decrease in NETs released from neutrophils. Notably, the inhibitor DPI had the same effect as the CS nanozyme on the inhibition of NETs. Therefore, the CS nanozyme can serve as an inhibitor of NADPH oxidase to reduce ROS levels and decrease the expression levels of cleaved caspase-1 and N-GSDMD to alleviate inflammation and inhibit the formation of NETs
Caspase-1 as upstream of GSDMD, is crucial for the formation of NETs and the release of IL-1β. The change of cleaved caspase-1 was similar to GSDMD, indicating a decrease in NETs released from neutrophils. Notably, the inhibitor DPI had the same effect as the CS nanozyme on the inhibition of NETs. Therefore, the CS nanozyme can serve as an inhibitor of NADPH oxidase to reduce ROS levels and decrease the expression levels of cleaved caspase-1 and N-GSDMD to alleviate inflammation and inhibit the formation of NETs
