In this entry, we discuss the use of the alkylating agent temozolomide (TMZ) in the treatment of IDH-mutant gliomas. We describe the challenges associated with TMZ in clinical (drug resistance and tumor recurrence) and preclinical settings (variabilities associated with in vitro models) in treating IDH-mutant glioma.
1. Introduction
Gliomas are the most common primary malignant tumors in the central nervous system. Grade 2 and 3 gliomas are referred to as lower grade gliomas (LGG) and harbor mutations in the isocitrate dehydrogenase (IDH) gene
[1]. IDH-mutant gliomas have a slower growth rate and longer survival than IDH wild type (IDH-wt) tumors
[1][2][1,2]. IDH-mutant gliomas are classified into two subgroups based on the presence (astrocytoma) or absence (oligodendroglioma) of chromosome arms 1p/19q
[3] and histological criteria
[4]. Recently, the European Association of Neuro-Oncology (EANO) stratified IDH-mutant gliomas into three WHO grades: oligodendroglioma, WHO grade 2 or 3; astrocytoma, WHO grade 2 or 3; astrocytoma, WHO grade 4
[5]. Although slower growing (at a rate of ~4–5 mm per year
[6]), the majority of IDH-mutant LGGs eventually undergo malignant progression due to activation of the PI3K/mTOR pathway as a result of PTEN loss
[7][8][7,8] or enhanced PDGF signaling
[9]. Detailed molecular diagnostic markers, and other common molecular and pathway alterations in IDH-mutant gliomas are summarized in .
Table 1. Molecular diagnostic markers and common genetic alterations in IDH-mutant glioma.
Category |
Alterations |
Oligodendroglioma WHO Grade 2 |
Oligodendroglioma WHO Grade 3 |
Astrocytoma WHO Grade 2/3 |
Astrocytoma WHO Grade 4 |
Diagnostic markers |
IDH1 or IDH2 mutation |
Present |
Present |
Present |
Present |
G-CIMP |
Present |
Present |
Present |
Present |
ATRX |
|
|
Inactivated |
Inactivated |
1p (FUBP1) / 19q (CIC) codeletion |
Present |
Present |
|
|
TERT |
Activated |
Activated |
|
|
9p21 (CDKN2A/B) |
|
|
|
Inactivated |
|
Necrosis and/or microvascular proliferation |
|
|
|
|
Present |
Other genomic alterations |
TP53 |
|
|
Inactivated |
Inactivated |
Myc |
|
Activated |
|
|
TCF12 |
|
Inactivated |
|
|
10q (PTEN/MGMT) |
|
|
|
Inactivated |
Signaling pathways |
Activation of PI3K signaling through loss of PTEN and gain of mTOR |
Activation of cell cycle signaling through gain of CDK4, CDK6 and cyclin E2 |
The IDH gene encodes the enzyme isocitrate dehydrogenase, which converts isocitrate to α-ketoglutarate (α-KG). α-KG an intermediate of the tricarboxylic acid (TCA) cycle that contributes to the production of NADPH. NADPH is necessary to reduce oxidized glutathione to glutathione, which directly neutralizes free radicals and reactive oxygen species (ROS). Overall, 65% of total NADPH in glioblastoma (GBM) is driven by the enzymatic activity of IDH, which is reduced to 38% when IDH is mutated
[10]. There are three IDH isoforms, IDH1, IDH2, and IDH3, which are encoded by different genes. IDH1 is localized in the cytosol and peroxisomes, while IDH2 and IDH3 are located in the mitochondria. Among them, IDH1 is most frequently mutated in gliomas and harbors a monoallelic missense mutation of arginine to histidine at position 132 (IDH1
R132H) at the catalytic site of the enzyme. IDH mutation produces a neomorphic enzyme that converts α-KG to D-(R)-2-hydroxyglutarate (2-HG), leading to the accumulation of 2-HG in the tumor
[11]. The oncometabolite 2-HG is a competitive inhibitor of α-KG-dependent enzymes, including DNA demethylases (family of TET enzymes) and histone demethylases (family of Jumonji enzymes)
[12][13][12,13]. This inhibition modifies the epigenetic status of histones and DNA, resulting in a plethora of cellular changes, including DNA hypermethylation
[14] and altered histone methylation
[15] ().
Figure 1. Epigenetic alterations induced by IDH mutations and potential drug targets for TMZ combination therapy. (a) Cellular epigenetic regulation without IDH mutation; (b) cellular epigenetic alterations in IDH-mutant gliomas. AZA, 5-azacitidine; DAC, Decitabine; DNMT1, DNA methyltransferase 1; HDAC, Histone deacetylase; 2-HG, D-(R)-2-hydroxyglutarate; HMT, Histone methyltransferase; KDM, Histone demethylase; TET, Ten-eleven translocation methylcytosine dioxygenases; VPA, Valproic acid; 5mC, 5-Mehylcytosine. Me: Methyl group; Ac, Acetyl group.
Treatment of LGGs includes surgery, radiation, and chemotherapy with either procarbazine/lomustine/vincristine (PCV) or temozolomide (TMZ). Here, we focus on the use of TMZ in IDH-mutant LGGs. First, we will present the effect of IDH mutation on cellular metabolism, epigenetic modifications, and the targeted therapies associated with these alterations. Second, we will discuss the use of TMZ in the treatment of IDH-mutant gliomas, including its toxicity, TMZ-associated molecular signature in tumor recurrence, and drug resistance, and discuss the synthetic lethality opportunities that emerge with TMZ treatment of IDH-mutant gliomas. Third, we will discuss the challenges of using TMZ to treat IDH-mutant gliomas in the preclinical setting, including non-consensus TMZ dosage and regimen, variable methods in measuring cell viability, and difficulties in culturing IDH-mutant glioma cell lines.
2. Temozolomide Treatment in IDH-Mutant Gliomas
Standard-of-care treatment for gliomas includes maximal surgical resection, possibly followed by radiotherapy (RT) and chemotherapy with PCV or TMZ. Several randomized clinical trials
[16][39] investigating dosing (EORTC 22844
[17][40]) and timing (EORTC 22845
[18][41]) for RT in LGGs show that RT alone provides no significant benefit for overall survival. Similarly, TMZ alone showed no significant difference in progression-free survival in patients with LGGs compared with the efficacy of RT (EORTC 22033-26033
[19][42]). However, RT combined with TMZ or PCV resulted in an overall survival benefit in patients
[20][43]. IDH-mutant oligodendrogliomas benefit from the addition of PCV to RT (RTOG 9802
[21][22][44,45], RTOG 9402
[23][46], EORTC 26951
[24][47], and NOA-04
[25][48]), while RT plus TMZ treatment shows more benefit in astrocytomas in clinical (EORTC 26053-22054 (CATNON)
[26][49], RTOG 0424
[27][50]), and retrospective
[28][51] studies. The interim analysis of the CATNON trial indicate a trend toward benefit with concurrent TMZ in IDH-mutant tumors, but not in IDH-wt gliomas. Thus, EANO recommends RT + TMZ for the treatment of newly diagnosed astrocytomas. For oligodendrogliomas, EANO recommends RT + PCV for initial treatment, and TMZ is only recommended for recurrent tumors not being pre-treated with TMZ
[5].
Currently, there are no mature data comparing TMZ and PCV or their combination with radiation for LGGs. The ongoing clinical trial ALLIANCE-N0577-CODEL comparing RT + TMZ with RT + PCV for anaplastic oligodendrogliomas with 1p/19q co-deletion could potentially provide a more definitive comparison between the two regimens
[29][52]. Both PCV and TMZ have been associated with grade 3 and 4 hematologic toxicities. Clinicians largely suggest TMZ to patients instead of PCV (>85%)
[30][53], considering the relative difficulty of administering intravenous vincristine and the greater toxicity of PCV
[31][54], whereas TMZ is easy to administer and generally well tolerated
[20][32][43,55]. The ongoing phase III EORTC-1635-BTG (Wait or Treat?) is a randomized phase III trial comparing early adjuvant treatment with radiotherapy and adjuvant temozolomide to active surveillance in patients with resected IDH-mutant astrocytoma. Here, we summarize the current challenges related to TMZ in gliomas with a particular focus on IDH-mutant tumors.
2.1. Mechanisms of TMZ Toxicity
TMZ is administered orally in capsules at a dose of 150–200 mg/m
2 for 5 out of 28 days for 6–12 cycles
[5]. TMZ is a lipophilic DNA alkylating prodrug, and the cytotoxicity of TMZ is mediated by the addition of methyl groups to DNA. TMZ is an imidazotetrazine derivative of dacarbazine. Under neutral pH and aqueous conditions, it spontaneously decarboxylates to generate 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC), which is further degraded to 4-amino-5-imidazole-carboxamide (AIC), and a highly reactive methyldiazonium ion that acts as a DNA methylating species
[33][56] (a). About 60–80% of methyl groups are added at DNA guanine residues (N
7-MeG), 10–20% of the methyl groups are added at adenine (N
3-MeA), and 10% of methyl groups at guanine (O
6-MeG)
[34][57] (b). Single damaged bases, N
7-MeG and N
3-MeA, are readily removed by the rapid and efficient base excision repair (BER) system before replication. Therefore, the key toxic insult of TMZ is attributed to the O
6-meG lesions
[35][36][58,59].
Figure 23. TMZ molecular structure, metabolism, toxicity, and resistance. (a) TMZ structure and metabolism, (b) DNA methylation upon TMZ, (c) DNA base mispair upon DNA methylation, (d) mechanism of TMZ toxicity with intact MMR, BER, and HR, (e) mechanism of TMZ resistance with functional MGMT and non-functional MMR. AIC, 4-Amino-5-imidazolecarboxamide; BER, Base-excision repair; HR, Homologous repair; MGMT, O6-methylguanine-DNA-methyltransferase; MMR, Mismatch repair; MLH, MutL homologue; MSH, MutS homologue; PMS, Post-meiotic segregation; TMZ, Temozolomide.
O
6-meG is considered the most genotoxic base modification due to the subsequent nucleotide mispairing with thymine (T) instead of cytosine (C) during DNA replication (c). During replication, DNA polymerase inserts T opposite O
6-meG. The mismatch repair (MMR) system can detect and repair these mismatches through the MutS and MutL complexes. The MutS recognition complex, including MutSα (an MSH2/MSH6 heterodimer) and MutSb (MSH2/ MSH3 heterodimer), identifies base–base mismatches and binds the O
6-meG: T mismatch. Upon binding to the mismatch, the MutS complex recruits MutL (MLH1/PMS2 dimer) to the site of DNA damage. Together, these proteins excise a stretch of single-stranded DNA (ssDNA) containing the mispaired T, creating a gap in the DNA, while leaving the O
6-meG adduct on the template strand intact
[37][60]. DNA polymerase fills the gap by reinserting T opposite O
6-meG, triggering another round of MMR which leads to repeated attempts to repair the same base T. This futile MMR cycling and accumulation of ssDNA gaps lead to successively longer DNA reinsertion and excision, which generates double strand breaks (DSBs) in subsequent rounds of replication and induce cell cycle arrest in G2/M phase, apoptosis and autophagy
[38][61]. Thus, it needs two cell divisions for the emergence of TMZ cytotoxicity
[39][62] (d).
However, O
6-meG lesions can be directly removed by O
6-methylguanine DNA methyltransferase (MGMT) through covalent transfer, a process that effectively repairs the alteration prior to replication (e). MGMT promoter methylation is a predictive biomarker of TMZ response in GBM. In general, the repair of O
6-meG depends on the number of MGMT molecules per cell and the rate of MGMT regeneration
[40][63]. In summary, the cytotoxicity from TMZ depends on low MGMT levels
[41][64] and an intact MMR pathway
[42][65].
2.2. Maintenance of TMZ Sensitivity
Efforts have focused on maintaining TMZ sensitivity by reducing MGMT levels or attenuating the activity of the BER and HR pathways for the duration of TMZ treatment to prevent resistance.
MGMT. O
6–benzylguanine (O
6–BG) is a potent inhibitor of the repair protein O
6–alkylguanine–DNA alkyltransferase (AGT) that effectively inhibits MGMT activity by suicide inactivation. O
6-BG binds and inactivates AGT, and until new AGT protein is synthesized, the cells have increased sensitivity to TMZ
[43][44][45][66,67,68], leading to several clinical trials combining O
6-BG and TMZ
[44][46][47][48][49][50][67,69,70,71,72,73]. A phase II study showed that one-day dosing of O
6-BG plus TMZ restored TMZ sensitivity in patients with TMZ-resistant anaplastic IDH-mutant gliomas
[50][73] ().
Table 2. Preclinical and clinical studies of combination therapy of TMZ with DNA damage repair pathway inhibitors.
Targeting DNA Damage Repair |
Synergistic with TMZ |
Preclinical Model |
Clinical Trial |
Arms |
Tumor Type |
Phase |
Year |
MGMTi |
O6BG + TMZ |
GBM PDX [45]; astrocytoma or GBM patient [44] | GBM PDX [68]; astrocytoma or GBM patient [67] |
NCT00006474 |
O6-BG + TMZ |
Astrocytoma |
I |
2001–2004 |
NCT00389090 |
O6-BG + TMZ |
Gliomas |
II |
(2006–2009)Terminated |
NCT00613093 |
O6-BG + TMZ |
GBM |
II |
2002–2008 |
NCT00275002 |
O6-BG + TMZ |
Pediatrichigh-grade gliomas |
II |
2006–2010 |
PARPi |
Olaparib + TMZ |
U87-IDH mutant, U251-IDH mutant cell lines [51] | U87-IDH mutant, U251-IDH mutant cell lines [81] |
NCT03212742 |
Olaparib + TMZ + IMRT |
GBM |
I/IIa |
2017–2022 |
NCT04394858 |
Olaparib + TMZ |
Pheochromocytoma and paraganglioma |
II |
2020–2023 |
Veliparib (ABT-888)+TMZ |
GBM BTICs and xenografts [52] | GBM BTICs and xenografts [80] |
NCT01026493 |
ABT-888 + TMZ |
Recurrent GBM |
I/II |
2010–2016 |
NCT01514201 |
RT+ ABT-888 + TMZ |
Children with newly diagnosed DIPG |
I/II |
2012–2018 |
NCT02152982 |
veliparib + TMZ vs. placebo + TMZ |
GBM |
II/III |
2014–2020 |
NCT03581292 |
RT + TMZ + veliparib |
GBM |
II |
2018–2024 |
Pamiparib (BGB-290) + TMZ |
GBM, GL261 murine glioma cells xenografts [53] | GBM, GL261 murine glioma cells xenografts [82] |
NCT03150862 |
BGB-290 + RT vs. BGB-290 + TMZ |
GBM |
1b/2 |
2017–2021 |
NCT03914742 |
BGB-290 + TMZ |
Recurrent IDH mutant glioma |
I/II |
2020–2023 |
NCT03914742 |
BGB-290 + TMZ |
IDH mutant glioma |
I |
2019–2027 |