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Review

The Biological and Clinical Role of the Telomerase Reverse Transcriptase Gene in Glioblastoma: A Potential Therapeutic Target?

by
Vincenzo Di Nunno
1,†,
Marta Aprile
2,†,
Stefania Bartolini
1,*,
Lidia Gatto
3,
Alicia Tosoni
1,
Lucia Ranieri
1,
Dario De Biase
4,5,
Sofia Asioli
6,7 and
Enrico Franceschi
1
1
Nervous System Medical Oncology Department, IRCCS Istituto delle Scienze Neurologiche di Bologna, 40139 Bologna, Italy
2
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138 Bologna, Italy
3
Department of Oncology, Azienda Unità Sanitaria Locale (AUSL) Bologna, 40139 Bologna, Italy
4
Solid Tumor Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna, Italy
5
Department of Pharmacy and Biotechnology (FaBit), University of Bologna, 40126 Bologna, Italy
6
IRCCS Istituto delle Scienze Neurologiche di Bologna, 40139 Bologna, Italy
7
Department of Biomedical and Neuromotor Sciences (DIBINEM), Surgical Pathology Section, Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italy
*
Author to whom correspondence should be addressed.
†
Co-first authors—These authors contributed equally to this work.
Cells 2024, 13(1), 44; https://doi.org/10.3390/cells13010044
Submission received: 25 October 2023 / Revised: 15 December 2023 / Accepted: 18 December 2023 / Published: 25 December 2023
(This article belongs to the Topic Novel Discoveries in Oncology)
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Abstract

:
Glioblastoma IDH-wildtype represents the most lethal and frequent primary tumor of the central nervous system. Thanks to important scientific efforts, we can now investigate its deep genomic assessment, elucidating mutated genes and altered biological mechanisms in addition to its clinical aggressiveness. The telomerase reverse transcriptase gene (TERT) is the most frequently altered gene in solid tumors, including brain tumors and GBM IDH-wildtype. In particular, it can be observed in approximately 80–90% of GBM IDH-wildtype cases. Its clonal distribution on almost all cancer cells makes this gene an optimal target. However, the research of effective TERT inhibitors is complicated by several biological and clinical obstacles which can be only partially surmounted. Very recently, novel immunological approaches leading to TERT inhibition have been investigated, offering the potential to develop an effective target for this altered protein. Here, we perform a narrative review investigating the biological role of TERT alterations on glioblastoma and the principal obstacles associated with TERT inhibitions in this population. Moreover, we discuss possible combination treatment strategies to overcome these limitations.

1. Introduction

Telomeres are repeated nucleotide sequences located at the chromosomal extremities. Telomere erosion due to multiple cell divisions finally triggers a DNA damage response, replicative senescence, and growth arrest in somatic cells. Not surprisingly, the preservation of telomere integrity is a critical hallmark that cancer cells must acquire to become “immortal”. Tumor cells have two different methods to maintain telomere integrity. These are represented by telomerase reverse transcriptase (TERT) alterations and a telomerase-independent mechanism called “alternative lengthening of telomeres” (ALT) that depends on ATRX/DAXX (a-thalassemia/mental retardation syndrome X-linked/Death-associated protein 6) complex. The “alternative lengthening of telomeres” (ALT), which depends on the ATRX/DAXX complex, is mutually exclusive with TERT in gliomas [1]. This last mechanism is a type of homologous recombination called break-induced telomere synthesis. Normally, homologous recombination is necessary to repair broken DNA strands by adding nucleotides that are complementary to the undamaged DNA segment. This same mechanism is used to extend telomeres [1].
TERT promoter mutations are the most frequent non-coding hotspot alteration in human cancers [2]. They have been described in several tumor entities, comprising hepatocellular carcinomas [3], urothelial carcinomas of the bladder [4], and melanomas, where they were described for the first time [5].
The fact that TERT alterations are shared by several different malignancies reflects the importance of this gene for cancer. Physiologically, TERT encodes for the catalytic subunit of a ribonucleoprotein complex responsible for telomere maintenance called “telomerase”. Conversely, TERT promoter (TERTp) variants or TERT overexpression are associated with enhanced telomerase activity and cell immortalization [6].
TERT promoter mutations are a very frequent event in central nervous system tumors (CNS) [7], especially in gliomas [8,9,10]. In particular, in patients with GBM IDH-wildtype, TERT alterations could reach 80–90% incidence [8]. Furthermore, TERT promoter mutations are an early genetic event in gliomagenesis, explaining the observed homogeneous distribution among cellular tumor subclones [11]. IDH-wildtype glioblastoma, according to the Central Nervous System World Health Organization Classification (CNS WHO), 5th edition [12], is a diffuse, astrocytic glioma with wild-type IDH and H3, exhibiting one or more of the following histological or genetic features: microvascular proliferation, necrosis, TERT promoter mutation, EGFR gene amplification, and +7/−10 chromosome copy-number changes (CNS WHO grade 4).
Considering the prevalence and incidence of this genomic alteration, the possibility of developing specific inhibitors for TERT-altered tumors is a very attractive opportunity for clinical oncology. Thanks to its ubiquitous distribution and elevated incidence, TERT appears as the perfect potential target in glioblastomas. However, several factors are limiting TERT inhibition, and, to date, no experimental compounds targeting TERT have been approved. Several strategies to inhibit TERT have been tested across multiple cancer types, comprising small molecule inhibitors and TERT-based immunotherapy, in particular, vaccines.
This review aims to provide an update of the evidence on the biology and physiopathology of TERT, exploring promising novel strategies and limitations to TERT inhibition in patients with glioblastomas.

2. TERT Role and Implications in Tumors

Telomerase is expressed in stem cells of proliferative tissues such as blood and skin [13]. When activating hot-spot mutations in the promoter region of TERT (pTERT) occur, these result in an upregulation of telomerase complex activity and thus constitute a relevant mechanism for the immortalization of tumor cells. This makes pTERT mutations one of the most common alterations shared in solid malignancies, including CNS primary tumors.
In tumors, re-activation of telomerase and telomere length maintenance constitute a key step in tumorigenesis. As previously reported, telomeres are guanine (G)-rich nucleotide repeats at the end of chromosomes. Without specific maintenance mechanisms, telomeres progressively shorten with mitotic cell divisions due to the intrinsic properties of DNA replication machinery. This is known as an “end replication problem”. The sequence loss at the end of chromosomes is consecutively responsible for cellular senescence or a process of cell death known as a “telomere crisis”.
Telomerase is recruited to the 3′ tail of telomeric DNA by the shelterin complex, which consists of six proteins that are involved in the activation of the whole enzymatic complex: telomeric repeat-binding factor 1 (TRF1), TRF2, Ras-related protein 1 (RAP1), TRF1-interacting nuclear factor 2 (TIN2), tripeptidyl peptidase 1 (TPP1), and protection of telomeres protein 1 (POT1) (Table 1 and Figure 1) [14]. Among the shelterins, TPP1 has a key role in recruiting telomerase to telomeres and activating telomere synthesis.
Indeed, TPP1 engages POT1. The POT1 binds the single-stranded telomeric DNA, and TIN2. The TIN2 interacts with the double-stranded telomeric DNA through TRF1 and TRF2 [14].
The role of TERT is to directly prevent this mechanism through the elongation of telomeres. In particular, TERT acts as a reverse transcriptase and employs its internal RNA molecule (hTR) as a template to add hexameric 5′-TTAGGG-3′ tandem repeats at chromosomal ends. Telomeric DNA consists of both a double-stranded DNA and a single-stranded DNA.
TPP1 engages POT1, which binds the single-stranded telomeric DNA, and TIN2. This last interacts with the double-stranded telomeric DNA through TRF1 and TRF2 [14]. Recently, a better definition of the cryo-electron microscopy structures of TERT and hTR allowed a clearer interpretation of the TERT–hTR interaction, and the TERT–TPP1 interaction, providing new potential drug targets [15].
Table 1. Principal alterations leading to telomerase reverse transcriptase (TERT) gain of function and a summary of TERT principal biological activities. ETS = E26 transformation-specific family transcription factors; POT1 = protection of telomeres protein 1; RAP1 = Ras-related protein 1; TIN2 = TRF1-interacting nuclear factor 2; TPP1 = Tripeptidyl peptidase 1; TRF 1 = telomeric repeat-binding factor 1; TRF2 = telomeric repeat-binding factor 2 [14].
Table 1. Principal alterations leading to telomerase reverse transcriptase (TERT) gain of function and a summary of TERT principal biological activities. ETS = E26 transformation-specific family transcription factors; POT1 = protection of telomeres protein 1; RAP1 = Ras-related protein 1; TIN2 = TRF1-interacting nuclear factor 2; TPP1 = Tripeptidyl peptidase 1; TRF 1 = telomeric repeat-binding factor 1; TRF2 = telomeric repeat-binding factor 2 [14].
TERT AlterationsTERT Function
Hotspot mutations of the TERT promoter gene result in TERT hyperexpression or hyperactivation [14].Telomere elongation by telomerase activity. This function is mediated by six different proteins: (TRF1), TRF2, RAP1, TIN2, TPP1, and POT1 [14]. All these proteins constitute the shelterins complex.
Increased TERT m-RNA expression mediated by TERT promoter mutations and ETS interaction mediated by the GA-binding protein (GABP). Repression of growth inhibitory factors [16].
Resistance to apoptosis diminished the capacity for DNA repair [17,18].
Besides its central role in telomere length maintenance, we have increasing data about telomere length-independent functions of TERT. In particular, TERT enhances cell proliferation through repression of growth inhibitory factors [16], impairment of DNA damage responses, and resistance to apoptosis [17,18].
Overall, TERT expression is regulated by multiple factors on genetic and epigenetic levels, including promoter mutations, promoter methylations, chromosomal rearrangements, and amplifications [19].
On a transcriptional level, a TERTp mutation implies the creation of transcriptionally active mutant promoters, such as a novel binding site for E26 transformation-specific (ETS) family transcription factors. The E26 transcription factors facilitate TERT mRNA expression. The GA-binding protein (GABP) has been identified as the only ETS transcription factor able to bind the mutated ETS motif [20].
The GA-binding protein transcription factor subunit alpha (GABPα) is a multimer made of two kinds of subunits that can selectively bind to the mutant TERT promoter: GABPα (a DNA-binding subunit), and GABPβ (a transactivating subunit). GABPβ exists in two alternative paralogues, GABPβ1 and GABPβ2. GABPβ1L (GABPβ1 long), a potential druggable target, is one of the two isoforms of GABPβ1. The isoform GABPβ1S (GABPβ1 short) differs from GABPβ1L due to the different site of GABPα binding.
The β1S can dimerize with GABPα, and both β1L and β2 have a leucine-zipper domain that mediates the tetramerization of two GABPαβ heterodimers. Among ETS transcription factors, GABP is the only one able to bind neighboring native ETS motifs and mutant ETS motifs as a heterotetrametric complex [21]. The GABP tetramer-forming isoforms are critical in activating the mutant TERT promoter; for this reason, these are under evaluation as potential therapeutic targets (Figure 2).

3. TERTp Mutations in GBM IDH-Wildtype

In the last two decades, genome-wide sequencing technologies have allowed a deep molecular characterization of neoplasms. Glioblastoma (both IDH-wildtype and, now, reclassifying astrocytoma IDH-mutant grade 4 CNS WHO) was the first cancer studied within the Cancer Genome Atlas Program (TCGA), whose aim was to list and describe the major cancer-causing genome alterations [22]. Novel evidence reported TERTp mutations as the most frequent genetic event in GBM IDH-wildtype.
TERTp mutations are mainly transitions caused by the substitution of the pyrimidine nucleotide cytidine with thymidine (C>T). The C228T (c.–124C>T, g.1295228 on GRCh37) and C250T (c.–146C>T, g.1295250 on GRCh37) mutations occur upstream of the transcriptional start site and represent the most frequent hotspot TERTp mutations. Both transitions generate an identical 11bp sequence which constitutes a novel ETS binding motif.
A different mutational position in TERT promoters is linked to a different TERT mutated expression. Indeed, C228T and C250T mutations show a 14-fold and 7-fold increase in mRNA expression, respectively [5].
Apart from GBM IDH-wildtype, TERTp mutations have been described in almost 100% of oligodendrogliomas, 80–90% of molecular/non-molecular GBM IDH-wildtype, and 7% of IDH mutant astrocytomas [10,12,23]. Overall, the C228T mutation has been reported with a higher frequency than the C250T mutation.
Among TERTp-mutated gliomas, a strong association has been observed with older age [1,24]. TERTp mutations are more frequent in adults than in pediatric patients [25]. TERTp mutation and ALT (secondary to ATRX mutation) are complementary mechanisms for telomere lengthening in GBM IDH-wildtype and are mutually exclusive.
Several studies investigated the prognostic role for TERTp in GBM IDH-wildtype patients with conflicting results (Table 2) [23,26,27,28,29,30,31,32,33,34]. After the advent of the Central Nervous System World Health Organization Classification (CNS WHO), 5th edition, TERTp should be considered an essential factor for the molecular diagnosis of GBM IDH-wildtype without a validated prognostic role [35]. Moreover, it has been observed as a co-occurrence with the chromosome 7 gain and chromosome 10 loss (+7/−10) [1].
Chromosomal abnormalities involving genes that drive proliferation, such as EGFR and PDGFA on chromosome 7, probably happen earlier than TERTp mutations in GBM IDH-wildtype. Subsequently, TERTp mutations could be necessary later for the clonal expansion of cancer cells [36].
Further studies have focused on the relationship between TERT regulation and oncogenic pathways involved in cell proliferation. The B Raf proto-oncogene p.Val600Glu mutation (BRAF p.V600E) can induce TERT upregulation in tumors; however, this genetic event is extremely uncommon in GBM IDH-wildtype [28]. Focusing on the more frequent EGFR activation, reported in approximately 57% of GBM IDH-wildtype, McKinney et al. recently demonstrated that GABP receives signals from EGFR through AMP-activated protein kinase (AMPK). On the other hand, GABP binds TERT and activates telomerase. The authors hypothesized targeting EGFR may therefore decrease the activity of mutant TERTp, in particular, combination therapies targeting EGFR could downregulate proliferation and reduce TERT activity [37].
Finally, in diffuse astrocytic glioma adult type, IDH-wildtype, and H3-wildtype, the TERTp mutation seemed to be associated with the poorest prognosis. Ceccarelli et al. analyzed TCGA diffuse gliomas and found mutations of TERTp in 85% of cases. They confirmed significant TERT upregulation in TERTp mutant cases [1].
Berzero et al. [38] demonstrated that patients with strictly defined astrocytoma IDH-wt grade 2 with isolated pTERTmut do not have the same prognosis as those with glioblastoma IDH-wt. Giannini C and Giangaspero F highlighted that clinicians and pathologists should be aware of these conclusions [39]. A TERT mutation identification could not be sufficient to assume that the tumor will behave as glioblastoma, IDH-wildtype (WHO CNS grade 4) as proposed in the cIMPACT-NOW update 6 [40] and it may be too late for the results of this paper to be incorporated in the upcoming 2021 WHO classification for CNS Tumor. Indeed, a novel type of IDH-wildtype glioma is characterized by gliomatosis cerebri-like growth pattern, TERT promoter mutation, and distinct epigenetic profile, as recently described by Meuch A. et al. [41]. The patients’ outcome in this study was better compared to IDH-wildtype glioblastomas, with a median progression-free survival of 58 months and overall survival of 74 months (both p-value < 0.0001). Therefore, the identification of pTERT mutation is an important step in the diagnostic and prognostic predictive process. However, its effective role in this setting should be pondered considering other molecular alterations identified as well as other histopathological, clinical, and neuroradiological features of the disease.
Table 2. Studies assessing the prognostic role of telomerase reverse transcriptase (TERT) gene mutations in GBM IDH-wildtype. To date, TERT should not be considered a prognostic factor in glioblastoma.
Table 2. Studies assessing the prognostic role of telomerase reverse transcriptase (TERT) gene mutations in GBM IDH-wildtype. To date, TERT should not be considered a prognostic factor in glioblastoma.
Study ConnotationPopulation of Study (n)Frequency of TERTp MutationImpact of TERTp Mutations on Survival Outcome in GBM IDH-WildtypeAdditional Results
Nonoguchi
(2013) [33]		358 GBM (n = 322 primary GBM IDH-wildtype; n = 36 secondary GBM)55% (58% in primary GBM IDH-wildtype and 28% in secondary GBM)Shorter survival in both univariate and multivariate analysis after adjustment for age and gender. However, no difference in survival in multivariate analyses after adjusting for other genetic alterations, or when primary and secondary GBM were separately analyzed Positive correlation between TERTp and EGFR amplification, inverse correlations with IDH1 mutations and TP53 mutations.
Labussière M (2014) [27]395 GBM IDH-wildtype76%Shorter PFS and OSThe absence of both TERTp mutation and EGFR amplification is associated with longer survival in patients with GBM.
Mosrati M (2015) [29]92 GBM IDH-wildtype 86% Shorter OSTERT SNPs rs2736100 and rs10069690 correlate with an increased risk of GBM.
Spiegl-Kreinecker S (2015) [30]126 GBM (n= 120 GBM IDH-wildtype; n = 6 IDH1 mutated) 73% Shorter OSTERT SNP rs2853669 improves survival in wtTERTp GBM IDH-wildtype. The shortest OS was detected in TERTp-mutated GBM IDH-wildtype with homozygous rs2853669 alleles.
Simon M
(2015) [24]		192 GBM (n = 178 primary GBMGBM IDH-wildtype; n = 14 secondary GBM)77% (80% in primary GBM; 28% in secondary GBM)Shorter OS in all primary GBMPoorer survival in patients with primary GBM IDH-wildtype and TERTp mutations who did not carry the variant G-allele for the rs2853669 polymorphism
Nguyen NH (2017) [32]30375%No impact on OS MGMT methylated patients showed improved survival only in the presence of TERTp mutation (analogous result in the cohort from TCGA).
Shu C (2018) [34]304 GBM (273 GBM IDH-wildtype)66%No impact on OS The subgroup with both unmethylated MGMT promoter and TERTp mutation had the worst prognosis. The main factors affecting survival in this group were age and Ki-67 positivity.
Brito C
(2019) [31]		256 GBM (n = 245 GBM IDH-wildtype; n = 11 IDH mut.)88% in GBM IDH-wildtype; 25% in IDH mut.No impact on OS in GBM IDH-wildtypePTEN favorable prognostic factor in GBM IDH-wildtype and unfavorable for astrocytoma IDH-wildtype.
Kikuchi Z (2020) [26]147 GBM IDH-wildtype62%Shorter PFS and OSTERTp mutant GBM IDH-wildtype is associated with multifocal/distant lesions.
Berzero G (2021) [38]47 diffuse astrocytomas IDH wildtype51%Patients meeting criteria for molecular GBM had a shorter OS compared to those with gliomas not meeting molecular GBM criteria (42 vs. 57 months)Patients with isolated TERT promoter mutation (16/26) had a more favorable outcome (median OS 88 months).
Muench A. (2023) [41] 16 patients with diffuse glioma IDH-wildtype TERT mutated.TERT mutation in 12/15 cases.Patients gliomatosis cerebri-like growth pattern.Median progression-free survival of 58 months and overall survival of 74 months.
GBM IDH-wildtype without TERTp mutations (10–20%) showed ATRX-mutation and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1 (SMARCAL1) as mechanisms of ALT [19].
Some studies have investigated the potential predictive response of TERT mutations. In a metanalysis by Vuong HG et al., MGMT promoter methylation was related to a survival benefit in patients with TERT-mutated GBM but not in TERT-wt GBM IDH-wildtype receiving temozolomide [42]. However, it should be explained that this analysis also involved studies enrolling patients with IDH-mutated tumors previously defined as secondary GBM. Thus, the presence of a TERT mutation should not be considered a prognostic factor. Finally, some studies have focused on the investigation of specific TERT single nucleotide polymorphisms (SNPs). These studies have suggested that some SNPs could be associated with increased clinical aggressiveness (rs2736100) [29]. On the other hand, the polymorphism rs2853669 has been largely detected in lower-grade gliomas and is associated with a decreased TERT expression since it leads to the disruption of the ETS2 binding sequence [27,43,44]. Again, the majority of these restricted the analysis on patients with WHO-CNS 5th-defined IDH-wildtype glioblastoma. Giunco S et al. confirmed the possible predictive role of the re2853669 in a cohort of patients with IDH-wildtype glioblastoma [44].
In conclusion, to date, the assessment of TERT in GBM IDH-wildtype has important diagnostic implications without a clear contribution to prognosis and treatment response prediction.

4. Development of TERT Inhibitors and Perspectives in GBM IDH-Wildtype Treatment

The development of TERT inhibitors is a very attractive possibility due to the high frequency of TERTp mutations, and their clonal distribution across cancer cells [11,45,46,47].
Nevertheless, the development of TERT inhibitors has long been hampered by a lack of molecular tridimensional and structural data.
Furthermore, there is a strong limitation related to the pre-clinical assessment of these compounds mainly due to the absence of in vivo models allowing an adequate evaluation [48].
To date, no therapies targeting TERT have been approved in clinical practice and few molecules have been tested [49]. This is mainly because we recently elucidated mechanisms associated with TERT activities in normal and neoplastic tissues. Furthermore, the toxicities of agents targeting TERT represent another issue limiting their investigation.
Moreover, despite TERT inhibition finally leading to cell cycle arrest and cell death in in vitro and in vivo models, this biological consequence occurs after reiterated cell divisions, necessary to achieve critically short telomere length [50].
Despite these potential limitations, several approaches have been explored in the development of targeted therapies in this field (Table 3).
The oligonucleotide inhibiting TERT enzymatic function, imetelstat (or GRN163L) binds the RNA template of human telomerase and acts as a competitive inhibitor of telomerase activity. Preclinical and clinical studies demonstrated a clinical efficacy of imetelstat in hematological malignancies including myelofibrosis, essential thrombocythemia, myelodysplastic syndromes, and acute myeloid leukemia.
In vitro, tumor-initiating cells isolated from primary GBM IDH-wildtype tumors and expanded as neurospheres showed a reduction in proliferation with imetelstat after approximately 15 to 20 population doublings. Again, the benefit of targeting TERT is delayed [56]. In vitro, promising results were obtained from the treatment of GBM IDH-wildtype neurosphere cells with imetelstat in association with ionizing radiation or temozolomide [56]
As regards off-target toxicity, normal brain tissue appears less susceptible to telomerase inhibition than brain cancer cells as the average telomere GBM IDH-wildtype length of GBM cells is shorter compared with normal human brain cells. Moreover, after the removal of imetelstat, telomerase activity is reversible to normal levels [56].
Unfortunately, on moving to the clinical phase, imetelstat resulted in very modest clinical activity in solid tumors, furthermore exposing patients to significant hematologic and hepatotoxic dose-limiting side effects [51].
In a phase 2 trial conducted in children with recurrent CNS tumors (n = 40), among grade 3/4 toxicities thrombocytopenia was registered in 32.5 % of patients, lymphopenia in 17.5 %, and neutropenia in 12.5 %. The study was closed after two patients died of intratumoral hemorrhage secondary to thrombocytopenia [57]. Currently, one phase 2 clinical trial is investigating imetelstat in younger patients with recurrent or refractory brain tumors (NCT01836549).
Given the previous disappointing results, further studies focused on alternative telomerase-dependent therapeutic approaches. To date, several strategies employing TERTp inhibition are under investigation [48].
Among telomerase inhibitors, BIBR1532 targets a critical site in the interaction between the hTR and TERT. However, its anti-tumor effects are burdened in vivo by water insolubility and low cellular uptake. Recent efforts to improve the release and efficacy of this small molecule led to promising results through Zeolitic imidazolate framework-8 (ZIF-8) as a delivery vehicle.
Inhibition of hTERT mRNA expression, cell cycle arrest, and increased cellular senescence were observed in cancer cells treated with the combination molecules [52]. However, they have not yet been tested on brain cancer cells.
A class of small molecules identified as potential telomerase inhibitors is constituted by G-quadruplex stabilizers. Folding of the 3′- overhang of telomeric DNA into G-quadruplex structures, which consist of a four-stranded helical guanine-rich DNA secondary structure, hampers access of telomerase to telomere ends. Molecules able to stabilize the telomeric G-quadruplex can cause telomere erosion and act as anticancer agents. Among these molecules, BRACO-19 induced viability loss in glioma cell lines, showing selectivity for cancer cells [53].
The 6-thio-2-deoxyguanosine (6-thio-dG) represents an interesting agent that has been employed as a drug able to restore or modify the immune-microenvironment. This compound can be incorporated into telomeres by telomerase in place of normal guanosine. This leads to telomere dysfunction due to interference with telomere structure and with telomere-binding proteins (e.g., shelterins). This mechanism, also known as the “telomere poisoning approach”, is linked to the onset of DNA-damage signals. Cells treated with 6-thio-dG release DNA fragments that are taken up by dendritic cells and activate a pathway involved in immune response, called STING (stimulator of interferon genes). In mouse models of colon cancer, lung cancer, and hepatocellular carcinoma (HCC), the 6-thio-dG induces immunogenic cell death [54]. This mechanism could be crucial to switching an immunologically cold tumor microenvironment into a hot tumor microenvironment [58,59]. Recent studies demonstrated the promising efficacy of a sequential administration of 6-thio-dG and anti-programmed death ligand 1 (PDL1) plus an inhibitor of the vascular endothelial growth factor (VEGF) in HCC [54]. To date, no studies are assessing the 6-thio-dG within GBM IDH-wildtype.
Another promising strategy under evaluation is the inhibition of transcription factors involved in TERT reactivation. In particular, the GABPβ1L subunit of the GABP transcription factor seems to be crucial to achieving the downregulation of telomerase in TERTp mutant cells. Disruption of GABPβ1L selectively inhibits TERT, and subsequent telomere loss favors cell death in TERTp mutant cancer cells. Moving from gene knockdown experiments, reduced GABPβ1L levels impaired tumor growth in vitro and extended survival in xenografted mice [60]. Similar preclinical models confirmed GABPβ1L as a potential therapeutic target through post-editing technologies, leading to cell death in vitro in 30 to 80 days [60,61]. In vivo, intracranial xenografts of GABPβ1L knockout cells exhibited reduced proliferation [61]. To overcome the time required to manifest the biological effect of telomerase inhibition, some authors hypothesized a synergistic effect between TERT inhibition and DNA-damaging agents, such as radiotherapy and cytotoxic chemotherapeutic agents. Amen A.M. et al. found knockout of GABP1L impaired the growth of TERT promoter mutant cells and reduced tumor growth rate in vivo, leaving normal cells unaffected. Furthermore, they demonstrated that loss of TERT activation sensitizes GBM IDH-wildtype to DNA damage. In particular, reduction in GABPB1L and administration of temozolomide had synergistic anti-tumor effects in vivo [62]. Inhibiting TERT makes cancer cells more sensitive to DNA breaks, due to the downregulation of DNA-damage repair mechanisms.
Aquilanti et al. provided further evidence about TERT reactivation and its role in tumor viability, beyond glioma initiation.
TERT promoter mutant-cells undergoing TERT knockdown exhibited features of telomere crisis and cell death, such as the formation of chromatin bridges and cell cycle arrest. Of note, cell death is achieved only after several cell divisions, necessary to cause telomere erosion and telomere crisis.
Considering the time required to see their biological effect, the optimal setting for a TERT inhibitor treatment could be after a gross total resection in an adjuvant setting more than in advanced disease in which a tumor response is generally required in a shorter time interval [50].
Some authors have suggested the possibility of targeting kinases upstream of the GABP-TERT axis. Since EGFR and TERTp are functionally connected, EGFR and AMPK could be tested as therapeutic targets in combination with other therapies to induce telomere reduction and tumor cell-killing [37]. No studies exploring this strategy are available. This also considers the negative results observed with agents targeting EGFR in GBM IDH-wildtype [63].
Over the past two decades, multiple studies have also evaluated telomerase-based immunotherapy and in particular vaccines, including peptide vaccines, dendritic cell vaccines, and DNA vaccines [64]. TERT is an intracellular protein that can be recognized by T cells after being presented on the external cell surface by major histocompatibility complex (MHC) molecules. In vitro, TERT protein has been demonstrated to be immunogenic for peripheral blood T lymphocytes: cancer cells present TERT peptides that can be recognized by either CD4+ or CD8+ T cells. In vivo, experiments demonstrated T-cell responses are in some cases associated with the inhibition of tumor growth.
Thus far, TERT-based vaccination has been studied in patients with different types of cancer in several phase 1 and 2 trials, and one phase 3 trial in pancreatic cancer patients. However, from a critical evaluation of these trials, it seems clear that therapeutic TERT-based vaccination induces temporary disease stabilization as the best response, with a poor effect on tumor size [65].
Regarding GBM IDH-wildtype, vaccination was tested in seven patients treated with a dendritic cell (DC)-based vaccine targeting GBM IDH-wildtype stem cells. An immune response was identified in all patients. No patients developed adverse autoimmune events or other side effects. Progression-free survival was 2.9 times longer in vaccinated patients compared to controls [66].
Recently, a DNA vaccine has been tested for safety and efficacy in a phase 1/2 trial in GBM IDH-wildtype patients. The trial enrolled 52 patients with newly diagnosed GBM IDH-wildtype, who were further divided into two cohorts (A: unmethylated MGMT and B: methylated MGMT). These patients received INO-5401 (synthetic DNA plasmid encoding hTERT, WT-1, PSMA) plus INO-9012 (synthetic DNA plasmid encoding IL-12), with cemiplimab (PD-1 inhibitor). Hypofractionated RT with temozolomide was administered to all patients, followed by maintenance therapy in Cohort B only. Most adverse events were ≤ grade 2, with no grade ≥ 4 events. Median OS in Cohorts A and B was 17.9 months and 32.5 months, respectively. The INO-5401 + INO-9012 has an acceptable risk/benefit profile and elicits robust immune responses that correlate with enhanced survival when administered with cemiplimab and RT/TMZ to newly diagnosed GBM IDH-wildtype patients [55].
Currently, a phase 2 study (NCT02818426) is evaluating UCPVax, a therapeutic anti-cancer vaccine based on the telomerase-derived helper peptides designed to induce strong TH1 CD4 T-cell responses in cancer patients. The study enrolls patients with GBM IDH-wildtype, pre-treated with standard radiochemotherapy.

5. Conclusions

TERT mutations assume a key role in driving development and progression in patients with GBM IDH wt. Due to its central biological role, a great deal of effort has been spent on the research of effective TERT inhibitors in these patients. Despite these aspects, the high-grade toxicities reported in clinical trials as well as the latency required by TERT inhibitors to achieve a biological effect are important limits to the development of effective drugs. However, an improved understanding of the TERT molecular structure and TERT interactions with other proteins have brought attention to the possible development of treatment strategies involving TERT selective inhibitors and other agents. In particular, the most promising early results come from combination therapies where TERT inhibition is combined with other approaches including immunotherapy. This last combination assumes a particular interest since inhibition of TERT could change the tumor-associated microenvironment toward an immune-active one. In conclusion, TERT inhibition is far from being a reality in clinical practice. Combination strategies employing these inhibitors are interesting opportunities despite still being in clinical trial investigation.

Funding

The publication of this article was supported by “Ricerca Corrente” funding from the Italian Ministry of Health.

Conflicts of Interest

The authors declare no conflicts of interest.

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Cells 13 00044 g001
Figure 1. Telomerase recruitment through interaction with shelterin complex. TPP1—tripeptidyl peptidase 1; TRF1 = telomeric repeat-binding factor 1; TRF2 = telomeric repeat binding factor 2; TIN2 = TRF1-interacting nuclear factor 2; POT1 = protection of telomeres protein 1; hTR = RNA template of TERT [14].
Figure 1. Telomerase recruitment through interaction with shelterin complex. TPP1—tripeptidyl peptidase 1; TRF1 = telomeric repeat-binding factor 1; TRF2 = telomeric repeat binding factor 2; TIN2 = TRF1-interacting nuclear factor 2; POT1 = protection of telomeres protein 1; hTR = RNA template of TERT [14].
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Cells 13 00044 g002
Figure 2. Regulation of TERT transcription through ETS transcription factor GABP. GABP tetramers, constituted by GABPα (DNA-binding subunit) and GABPβ1L (transactivating subunit) bind both the mutant E26 binding motifs and the normal motifs nearby. They are critical in activating the mutant TERT promoter.
Figure 2. Regulation of TERT transcription through ETS transcription factor GABP. GABP tetramers, constituted by GABPα (DNA-binding subunit) and GABPβ1L (transactivating subunit) bind both the mutant E26 binding motifs and the normal motifs nearby. They are critical in activating the mutant TERT promoter.
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Table 3. Principal TERT inhibitors were discussed.
Table 3. Principal TERT inhibitors were discussed.
Agents under InvestigationMechanisms of Action and Preliminary Results
Imetelstat [51]Oligonucleotide acts as a competitive inhibitor of telomerase activity.
No clinical benefit emerged in small clinical trials. Significant grade 3–4 thrombocytopenia. To date, a single trial investigating imetelstat in young patients with recurrent brain tumors (NCT01836549).
BIBR1532 [52]A small molecule targeting the interaction between hTR and TERT. Water insolubility makes this drug difficult to manage. A specific delivery vehicle has been proposed (zeolitic imidazolate framework-8) to overcome this limit. To date, no evidence of efficacy within patients with GBM IDH-wildtype.
BRACO-19 [53]Acts as G-quadruplex stabilizers modifying the DNA structure making difficult the binding of the telomerase. No studies on patients with GBM IDH-wildtype.
6-thio-2-deoxyguanosine [54]An agent that can be incorporated into telomerase in place of normal guanosine leads to telomere dysfunction (“telomere poisoning approach”). This drug can mediate the modification of the tumor microenvironment from cold to hot in preclinical models. No studies on patients with GBM IDH-wildtype.
INO-5401 [55]A synthetic DNA plasmid encoding hTERT. It has been administered with cemiplimab, and INO 9012 (synthetic DNA plasmid encoding IL-12). Administration of INO 5401 and INO 9012 resulted in an OS of 32.5 months in patients with methylated glioblastoma.
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Di Nunno, V.; Aprile, M.; Bartolini, S.; Gatto, L.; Tosoni, A.; Ranieri, L.; De Biase, D.; Asioli, S.; Franceschi, E. The Biological and Clinical Role of the Telomerase Reverse Transcriptase Gene in Glioblastoma: A Potential Therapeutic Target? Cells 2024, 13, 44. https://doi.org/10.3390/cells13010044

AMA Style

Di Nunno V, Aprile M, Bartolini S, Gatto L, Tosoni A, Ranieri L, De Biase D, Asioli S, Franceschi E. The Biological and Clinical Role of the Telomerase Reverse Transcriptase Gene in Glioblastoma: A Potential Therapeutic Target? Cells. 2024; 13(1):44. https://doi.org/10.3390/cells13010044

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Di Nunno, Vincenzo, Marta Aprile, Stefania Bartolini, Lidia Gatto, Alicia Tosoni, Lucia Ranieri, Dario De Biase, Sofia Asioli, and Enrico Franceschi. 2024. "The Biological and Clinical Role of the Telomerase Reverse Transcriptase Gene in Glioblastoma: A Potential Therapeutic Target?" Cells 13, no. 1: 44. https://doi.org/10.3390/cells13010044

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