New somatic TERT promoter variants enhance the Telomerase activity in Glioblastoma

The catalytic activity of human Telomerase Reverse Transcriptase (TERT) compensates for the loss of telomere length, eroded during each cell cycle, to ensure a correct division of stem and germinal cells. In human tumors, ectopic TERT reactivation, most frequently due to hotspot mutations in the promoter region (TERTp), i.e. c.1-124 C > T, c.1-146 C > T, confers a proliferative advantage to neoplastic cells. In gliomas, TERTp mutations (TERTpmut) mainly occur in oligodendroglioma and glioblastoma. We screened, for TERTp hotspot mutations, 301 adult patients with gliomas and identified heterozygous mutations in 239 cases: 94% of oligodendroglioma, 85% of glioblastoma, and 37.5% of diffuse/anaplastic astrocytoma. Besides the recurrent c.1-124 C > T and c.1-146 C > T, two cases of glioblastoma harbored novel somatic TERTp variants, which consisted of a tandem duplications of 22 nucleotides, i.e. a TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, both located downstream c.1-124 C > T and c.1-146 C > T. In silico analysis predicted the formation of 119 and 108 new transcription factor’s recognition sites for TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, respectively. TERTp duplications (TERTpdup) mainly affected the binding capacity of two transcription factors’ families, i.e. the members of the E-twenty-six and the Specificity Protein/Krüppel-Like Factor groups. In fact, these new TERTpdup significantly enhanced the E-twenty-six transcription factors’ binding capacity, which is also typically increased by the two c.1-124 C > T/c.1-146 C > T hotspot TERTpmut. On the other hand, they were distinguished by enhanced affinity for the Krüppel proteins. The luciferase assay confirmed that TERTpdup behaved as gain-of-function mutations causing a 2,3-2,5 fold increase of TERT transcription. The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors, with the identification of new recurrent somatic gain-of-function mutations, occurring in 0.8% of glioblastoma IDH-wildtype. Electronic supplementary material The online version of this article (10.1186/s40478-020-01022-4) contains supplementary material, which is available to authorized users.


Introduction
The abnormal reactivation of human Telomerase Reverse Transcriptase (TERT) is a common hallmark of human solid tumors. Although it may be caused by an identical 11 bp 'CCC CTT CCGGG' sequence, resulting in the creation of de novo consensus binding motifs for E-twenty-six (ETS) transcription family members. These new binding sites recruit a larger number of ETS factors, enhancing the transcription of TERT [3].
TERT promoter mutations (TERTp mut ) typically occur in tumors that arise from low self-renewal tissue, such as melanomas, thyroid, hepatobiliary carcinoma, and central nervous system (CNS) tumors, with a variable frequency, that range from 15 to 90% of cases, in diverse histological subtypes [10,14,28]. In CNS tumors, TERTp mut are typically associated with glioblastoma (GBM) (70-80%) and oligodendroglioma (ODG) (60-70%), whereas their frequency decreases in other glioma subtypes, such as diffuse/ anaplastic astrocytoma (DA/AA) (30-40%), medulloblastoma (20-30%), and meningioma (about 7%) [10,25,27]. Although the clinical value of TERTp mut , in refining the diagnostic classification of gliomas, is widely accepted [6], its role as prognostic/predictive biomarker is still largely debated. TERTp mut have been associated with a poor disease outcome in GBM IDH-wildtype (GBM IDH wt ), but there is no full agreement on its impact on DA/AA [6,15,16,22,24,29]. It is worth noting, however, that DA/AA IDH-wildtype (DA/AA IDH wt ) harboring genomic abnormalities typically associated with GBM, i.e. TERTp mutations, or EGFR amplification, or gain of whole chromosome 7 in combination with monosomy of chromosome 10, have a clinical outcome similar to, or only slightly longer, than GBM [4]. Thus, the cIMPACT NOW (Update 3) recommended to use one of these molecular criteria to classify this subgroup of astrocytomas as "diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV" and to revise the classification of DA/AA IDH wt , accordingly [4].
Herein, we report two new TERTp mutations that were identified in two patients with GBM IDH wt . Both these new variants originated from the duplication of a stretch of 22 nucleotides at TERTp (TERTp dup ) and, although slightly different, shared an overlapping sequence of 12 nucleotides. We demonstrated the somatic nature of one of these TERTp dup and that, enhancing the binding affinity for ETS transcription factors (TFs), they both elicit the TERT transcription, thus widening the spectrum of recurrent gain-of-function mutations of TERTp in GBM.

Cohort
The study was carried out on a cohort of 301 patients, affected by primary CNS tumours, and referred to our laboratory during the last 10 years (Table 1). There were 175 males and 126 females (ratio 1.4:1) with a median age of 64 (range age: 20-86). According to the WHO 2016, the diagnosis was: grade II DA IDH wt (6 cases) and DA IDH-mutant (DA IDH mut ) (10 cases); grade III AA IDH wt (6 cases) and AA IDH mut (= 10); grade IV GBM IDH wt (= 241) and GBM IDH mut (= 10); grade II/III ODG (= 15). Three patients had a diagnosis of uncommon glioma ( Table 1). The study was approved by Institutional Bioethics Committee (University of Perugia and Santa Maria della Misericordia Hospital of Perugia-Italy, Protocol no.2843/16); all patients gave informed consent for sample collection and molecular analyses, in agreement with the Declaration of Helsinki.

Index cases
A 71-year-old male (UPN#131) had a left frontal lesion of 24 mm diameter, partially infiltrating the corpus callosum; the second case (UPN#171), a male of 78 years, presented with a right frontal lesion. Histopathology and immunohistochemistry were consistent with a diagnosis of GBM IDH wt , in both patients. In case UPN#131, neoplastic cells showed marked cytoplasmic and nuclear pleomorphism; there was a discrete number of atypical mitotic figures, widespread necrosis, a diffuse GFAP positivity (100%), and few neoplastic elements (20%) with strong nuclear TP53 stain. Case UPN#171, was characterized by striking atypia of neoplastic cells, diffuse necrosis, vascular proliferation, strong and diffuse positivity for GFAP and nuclear TP53 (> 70%) (Fig. 1). No IDH1/IDH2 hotspot mutations were detected, while both cases showed MGMT promoter methylation. Monosomy of chromosome 10 co-occurred with EGFR amplification (UPN#131) or with gain of the whole chromosome 7 (UPN#171).

In silico TERTp mut functional analysis: JASPAR tool
This bioinformatic tool estimates the binding affinity and the number of TFs binding sites for the input sequence provided in FASTA format. A relative threshold score of 80% and Δ relative score ≥ 0.05 (mutant's relative scorewildtype's relative score) were chosen to define the statistically significant changes induced by TERTp mut , as previously reported [1]. The JASPAR CORE predicted the effects of the four different TERTp mut that we detected in our patients, i.e. the two new TERTp dup , the TERTp -

In vitro TERTp mut functional study: luciferase assay
To study the effect of TERTp mut on the expression of TERT, a luciferase assay was done for the TERTp dup detected in case UPN#171, the TERTp -146 (UPN#205), and the TERTp -124 (UPN#216). The TERT dup of case UPN#131 could not be studied due to lack of material. A TERTp wildtype (TERTp wt ) construct, already available in the laboratory, was also used as reference (Additional file 2: Table S2) [21]. TERT core promoter (310 bp) was amplified with specific primers reported in Table S3 (Additional file 3: Table S3), introducing cleavage sites for BglII (forward) and HindIII (reverse) restriction enzymes. Then, TERTp mut constructs were inserted in pGEM-T easy plasmid (Promega, Madison WI, USA) and cloned in Electromax DH10BT1 cells (Invitrogen, Milan, Italy) to increase the amount of mutant DNA. Finally, the inserts were subcloned in pGL4.10[luc2] vectors (Promega, Madison WI, USA) upstream of LUC2 gene, encoding for luciferase enzyme of Photinus Pyralis and resequenced. An empty pGL4. 10[luc2] vector was also used as negative control. Luciferase assay was performed using the GBM U87-MG cell line, maintained in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, Monza, Italy) with 10% fetal bovine serum, and 0.5% streptomycin/ penicillin at 37 °C/5% CO 2 . U87-MG cells were seeded in a 6-multiwell plate (3 × 10 5 cells/ml), co-trasfected with 3 µg of modified pGL4.

In vitro analysis confirms the increasing of TERT transcriptional activity induced by its promoter mutations
In vitro luciferase assay was carried out to evaluate whether the new TERTp -110-89 variant induced an increase of TERT transcriptional activity, enhancing its expression, similarly to TERTp -124 and TERTp -146 [12,21]. In Table S12 (Additional file 12: Table S12) we reported raw data referred to the fluorescence emission values, expressed in Relative Luciferase Activity (RLA), of both Photinus Pyralis and Renilla Reniformis luciferase enzymes, for all samples. Our experiments demonstrated that all three variants caused a significant increase of TERT transcription by 2.3-2.5 fold than wildtype (TERTp -110-89 vs TERTp wt : P < 0,0001; TERTp -124 vs TERTp wt : P < 0,0315; TERTp -146 vs TERTp wt : P < 0,0001; Mann-Whitney U test) (Fig. 4). On the other hand, no differences on the levels of TERT expression were present between the diverse TERTp variants, indicating they may all behave as gain-of-function mutations, likely exerting the same consequences on TERT transcription.

Discussion
Abnormal genomic events that alter telomere elongation are common in gliomas. Particularly, mutually exclusive mutations affect the TERT or the ATRX chromatin remodeler (ATRX) genes, a critical regulator of telomere homeostasis by chromatin remodeling [9].
Our studies, on a cohort of 301 patients, confirmed previous data on the incidence and distribution of TERTp mut in diverse subtypes of CNS tumors. As expected, we found that TERTp mut were highly recurrent in ODG and GBM, and less frequent in DA/ AA (Additional file 4: Table S4). TERTp mut were significantly enriched in GBM IDH wt cases (83%) (Chi square, P < 0.001) (Additional file 55: Table S5), where they mainly occurred together with EGFR amplification (Chi square, P = 0.001) and/or monosomy 10/PTEN deletions (Chi square, P < 0.0001). Similarly, in DA/ AA, TERTp mut were highly recurrent in IDH wt cases, thus allowing the reclassification of 83% of these subgroup of astrocytomas as "diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV" [4]. Besides the two known TERTp -124 and TERTp -146 variants, we uncovered two new TERTp variants in two cases of GBM IDH wt (UPN#131 and UPN#171). These novel TERTp mut consisted of a 22 nucleotide tandem duplication, sharing a duplicated region of 12 nucleotides, from 1-100 to 1-89, from the ATG start site. Hitherto, somatic TERTp dup has been reported in three human tumors. The first one, a duplication of 41 nucleotides in the TERT core promoter, was detected in a case of ODG [3]. Afterwards, TERTp dup were found in a case of myelodysplastic syndrome (MDS) (c.1-110_1-101dup) and in a case of papillary thyroid carcinoma (c.1-104_1-83dup) [21,23]. Published TERTp dup as well as our cases, are located in the same core promoter region, that span 1-110/1-79 bp from the ATG start site. Furthermore, they are all located downstream TERTp -124 and TERTp -146 , i.e. at 13-23 nucleotides from TERTp -124 and 35-45 nucleotides from TERTp -146 , in a region that contains the binding sites for the TFs modulating TERT transcription. Interestingly, in silico analysis predicted these new TERT dup affect the transcriptional regulation of the gene through the creation of new binding sites for TFs that mainly belong to the ETS family (Fig. 2c, Additional file 7: Table S7). Likewise, an increased number of binding sites or an enhanced affinity for the ETS TFs, has been previously reported in a thyroid cancer harbouring a TERTp c.1-104_1-83dup variant, and in cases bearing TERTp -124 or TERTp -146 mutations [3,10,23]. Bioinformatic analyses were consistent with the luciferase data showing a significant increase of TERT expression in cells transfected with the new TERTp -110-89 variant as well as with the two recurrent TERTp mut .
Then, we sought to assess the possible inter-relationship between the four diverse TERTp mutations using the Venn diagram (Fig. 3a). All four TERTp variants were predicted to share an increase binding capacity for 18 ETS members ( Fig. 3a; Additional file 8: Table S8), which included GABPA, a putative oncogene in GBM. Namely, in vitro studies on GBM cell lines have demonstrated that this transcription factor is needful in mediating the transcriptional reactivation of TERT dependent from TERTp -124 or TERTp -146 [3,10,19]. Besides ETS TFs, all TERTp variants affected the binding capacity for TEAD1, a protein that belongs to TEF-1-related factors family, and that has been demonstrated to act as a putative oncogene in GBM, favoring cell infiltration in vitro/in vivo models [26].
Although TERTp -124 and TERTp -146 , and the new TERTp -100-79 and TERTp -110-89 variants, shared the same effects on the binding capacity for ETS members, the latters were characterized by the exclusive involvement of 30 TFs, mainly belonging to Sp/KLF family (Fig. 3a, Additional files 7 and 8: Tables S7 and S8). Sp/KLF TFs are involved in a plethora of cellular processes ranging from proliferation and differentiation, pluripotency and apoptosis, in normal and tumoral tissues [17].