hTERT Promoter Mutations Research Articles

Advancements in Telomerase-Targeted Therapies for Glioblastoma: A Systematic Review.

Glioblastoma (GBM) is a primary CNS tumor that is highly lethal in adults and has limited treatment options. Despite advancements in understanding the GBM biology, the standard treatment for GBM has remained unchanged for more than a decade. Only 6.8% of patients survive beyond five years. Telomerase, particularly the hTERT promoter mutations present in up to 80% of GBM cases, represents a promising therapeutic target due to its role in sustaining telomere length and cancer cell proliferation. This review examines the biology of telomerase in GBM and explores potential telomerase-targeted therapies. We conducted a systematic review following the PRISMA-P guidelines in the MEDLINE/PubMed and Scopus databases, from January 1995 to April 2024. We searched for suitable articles by utilizing the terms "GBM", "high-grade gliomas", "hTERT" and "telomerase". We incorporated studies addressing telomerase-targeted therapies into GBM studies, excluding non-English articles, reviews, and meta-analyses. We evaluated a total of 777 records and 46 full texts, including 36 studies in the final review. Several compounds aimed at inhibiting hTERT transcription demonstrated promising preclinical outcomes; however, they were unsuccessful in clinical trials owing to intricate regulatory pathways and inadequate pharmacokinetics. Direct hTERT inhibitors encountered numerous obstacles, including a prolonged latency for telomere shortening and the activation of the alternative lengthening of telomeres (ALT). The G-quadruplex DNA stabilizers appeared to be potential indirect inhibitors, but further clinical studies are required. Imetelstat, the only telomerase inhibitor that has undergone clinical trials, has demonstrated efficacy in various cancers, but its efficacy in GBM has been limited. Telomerase-targeted therapies in GBM is challenging due to complex hTERT regulation and inadequate inhibitor pharmacokinetics. Our study demonstrates that, despite promising preclinical results, no Telomerase inhibitors have been approved for GBM, and clinical trials have been largely unsuccessful. Future strategies may include Telomerase-based vaccines and multi-target inhibitors, which may provide more effective treatments when combined with a better understanding of telomere dynamics and tumor biology. These treatments have the potential to be integrated with existing ones and to improve the outcomes for patients with GBM.

Pathology of Hepatocellular Carcinoma and Tumor-Bearing Liver Tissue in Association with hTERT Promoter Mutation.

The hTERT promoter mutation represents a common and early event in hepatocarcinogenesis, but its linkage to the morphological status of the underlying liver tissue is poorly understood. We analyzed the connection between the histopathological changes in tumor-bearing liver tissue and the occurrence of the hTERT promoter mutation in hepatocellular carcinoma (HCC), correlated with clinical data. The study cohort comprised 160 histologically confirmed HCC in patients with or without cirrhosis that were investigated for the hTERT promoter mutation. We evaluated the frequency of the hTERT promoter mutation in patients with HCC with or without cirrhosis and correlated it with potential clinical and histopathological drivers. In particular, we examined tumor-bearing noncirrhotic liver tissue regarding inflammation; the modified histological activity index (mHAI), fibrosis, and steatosis; and its correlation with the frequency of the hTERT promoter mutation in HCC. We evaluated overall survival with multivariate Cox regression. Furthermore, we compared hTERT antibody immunohistochemistry and molecular hTERT promoter mutation analysis of both HCC and background liver tissue. The hTERT promoter mutation was especially related to HCC in cirrhotic compared with noncirrhotic liver (p < 0.001) and independently of cirrhosis in patients ≥ 60 years (p = 0.005). Furthermore, the hTERT promoter mutation was associated with cirrhosis caused by alcohol toxicity and hepatitis C virus infection. In noncirrhotic liver tissue, the frequency of hTERT-promoter-mutated HCC increased with the degree of inflammation and fibrosis. Nevertheless, 25% of the hTERT-promoter-mutated HCC developed in normal liver tissue without HCC risk factors. Multivariate Cox regression analysis did not reveal an influence of the hTERT promoter mutation in HCC on overall survival at 3, 5, and 16 years. Immunohistochemical analysis with the hTERT antibodies LS-B95 and 2D8 in hTERT-promoter-mutated HCC and hTERT-wildtype HCC showed a mildly stronger immunoreaction compared with the tumor-bearing liver tissue (LS-B95: p < 0.01, 2D8: p < 0.01). Our study reveals a connection between pathological changes in tumor-bearing liver tissue and the hTERT promoter mutation in most HCC, even in noncirrhotic liver tissue. Immunohistochemical hTERT antibodies do not discriminate between hTERT-promoter-mutated and wildtype HCC.

Nuclear and Cytoplasmic hTERT, Tumor-Infiltrating Lymphocytes, and Telomere Elongation Leukocytes Are Independent Factors in the Response to Neoadjuvant Treatment in HER2-Enriched Breast Cancer

HER2-enriched tumors are responsible for 20% of breast tumors and have high rates of immune infiltrates in the tumor stroma that respond favorably to neoadjuvant chemotherapy. In the context of tumors, telomeres control cell death and prevent tumor cells from replicating discontinuously, leading to their immortalization. This study aimed to evaluate the presence of tumor-infiltrating lymphocytes, hTERT expression, hTERT promoter mutation, and leukocyte telomere length in HER2-enriched breast tumors. A total of 103 cases were evaluated, 19 with pathologic complete response. The TILs percentage was above ≥10 in 44 cases (43%) and significantly present in patients ≥50 years of age. hTERT staining positivity was mostly nuclear, significantly present in the non-pCR group, and associated with a lower survival rate. Leukocyte telomeres were elongated for HER2-enriched tumors, and in multivariate analysis, shortening was associated with an increased risk of death. Overall, our results show that the nuclear and cytoplasmic presence of hTERT may indicate a worse prognosis and that leukocyte telomere elongation is a protective factor.

Association of human telomerase reverse transcriptase promoter mutation with unfavorable prognosis in glioma: A systematic review and meta-analysis.

Glioma is one of the most malignant and aggressive tumors, with an extremely poor prognosis. Human telomerase reverse transcriptase (hTERT) promoter mutation is regarded as a risk factor in tumor growth. Although the prevalence of hTERT promoter (pTERT) mutation in gliomas has been investigated, the results are inconsistent. This meta-analysis aims to investigate the prognostic value of hTERT in glioma patients and its interaction with other biomarkers. We searched 244 citations from four databases: PubMed (2000-2021), Web of Science (2000-2021), Embase (2010-2021), and Cochrane Library (2000-2021) with 28 articles included. We calculated hazard ratios (HRs) using the random effect model and the pooled result suggested that TERT promoter mutation predicted poorer overall survival (HR: 1.53, 95% confidence interval [CI]: 1.34-1.75, P < 0.001, I2: 49.9%, pheterogeneity:0.002) and progression-free survival (HR: 1.55, 95% CI: 1.27-1.88, P < 0.001, I2: 0.0%, pheterogeneity: 0.473). For subgroup analysis, we analyzed multiple factors including iso-citrate dehydrogenase (IDH) genotype, age, diagnosis, pTERT region, so as to locate the sources of heterogeneity. Interestingly, in IDH mutant subgroup, pTERT mutation became a beneficial prognostic factor (HR: 0.73, 95% CI: 0.57-0.93, I2: 22.3%, pheterogeneity: 0.277), which is contrary to the results in pooled analysis. In general, pTERT mutation may result in shorter survival time in glioma patients, but longer survival time when glioma patients are combined with IDH mutation.

Evaluation of possible role of the hTERT gene rs2853669 polymorphism in the development of colorectal cancer as a genetic risk factor

Colorectal cancer (CRC) is the second deadliest malignancy. Human telomerase reverse transcriptase (hTERT) gene has been identified as one of the potential cancer susceptibility genes. We evaluated the relationship between the risk of CRC and CRC’s clinicopathological features of the hTERT rs2853669 (A > G/T > C, by the chain direction) polymorphism in Turkish population. The rs2853669 polymorphism was investigated with the LightCycler 96 device in 100 CRC patients and 327 controls. We found that the rs2853669 polymorphism AG/GG genotypes in genetic models reduced the risk of CRC. However, there was no significant relationship between rs2853669 polymorphism and clinicopathological features of CRC in studied population. The results of this study showed that the risk of colorectal cancer is significantly reduced in the individuals having the G (C) allele. Our recommendation is to analyze the hTERT gene expression by studying the hTERT promoter mutations with this polymorphism in colorectal cancer.

Identification of h-TERT Promoter Mutations in Germline DNA from North Indian Lung Carcinoma Patients.

Lung cancer is a severe and the leading cause of cancer related deaths worldwide. The recurrent h-TERT promoter mutations have been implicated in various cancer types. Thus, the present study is extended to analyze h-TERT promoter mutations from the North Indian lung carcinoma patients. Total 20 histopathologically and clinically confirmed cases of lung cancer were enrolled in this study. The genomic DNA was extracted from venous blood and subjected to amplification using appropriate h-TERT promoter primers. Amplified PCR products were subjected for DNA Sanger sequencing for the identification of novel h-TERT mutations. Further, these identified h-TERT promoter mutations were analysed for the prediction of pathophysiological consequences using bioinformatics tools such as Tfsitescan and CIIDER. The average age of patients was 45 ± 8years which was categorized in early onset of lung cancer with predominance of male patients by 5.6 fold. Interestingly, h-TERT promoter mutations were observed highly frequent in lung cancer. Identified mutations include c. G272A, c. T122A, c. C150A, c. 123 del C, c. C123T, c. G105A, c. 107 Ins A, c. 276 del C corresponding to -168 G>A, -18T>A, -46 C>A, -19 del C, -19 C>T, -1 G>A, -3 Ins A, -172 del C respectively from the translation start site in the promoter of the telomerase reverse transcriptase gene which are the first time reported in germline genome from lung cancer. Strikingly, c. -18T>A [C.T122A] was found the most prevalent variant with 75% frequency. Notwithstanding, other mutations viz c. -G168A [c. G272A] and c. -1 G>A [c. G105A] were found to be at 35% and 15% frequency respectively whilst the rest of the mutations were present at 10% and 5% frequency. Additionally, bioinformatics analysis revealed that these mutations can lead to either loss or gain of various transcription factor binding sites in the h-TERT promoter region. Henceforth, these mutations may play a pivotal role in h-TERT gene expression. Taken together, these identified novel promoter mutations may alter the epigenetics and subsequently various transcription factor binding sites which are of great functional significance. Thereby, it is plausible that these germline mutations may involve either as predisposing factor or direct participation in the pathophysiology of lung cancer through entangled molecular mechanisms.

Evaluation of TERT promoter mutations in tumor biopsies and urine sediment of Moroccan bladder cancer patients

Background: Human telomerase reverse transcriptase (hTERT) promoter mutations are common genetic events in bladder cancer (BC) and have been recognized as potential biomarkers for BC diagnosis and prognosis. Detection of TERT promoter mutations as urine-based tests has previously been reported to detect primary and recurrent BC. This study was planned to evaluate hTERT promoter mutations in both biopsies and urines from BC patients to assess the interest and the usefulness of introducing these biomarkers for better management of BC in Morocco.

Usefulness of Cell-Free Human Telomerase Reverse Transcriptase Mutant DNA Quantification in Blood for Predicting Hepatocellular Carcinoma Treatment Efficacy.

Although the usefulness of liquid biopsy as a biomarker in the treatment of hepatocellular carcinoma (HCC) has been suggested, its usefulness in transcatheter arterial chemoembolization (TACE) or tyrosine kinase inhibitor (TKI) therapies has not been reported in detail. In this study, we investigated the clinical value of a cell‐free (cf)DNA quantification system targeting the human telomerase reverse transcriptase (hTERT) promoter mutation in advanced HCC treatment. Plasma from 67 patients with advanced HCC, treated with TACE and TKI, was used for extraction of cfDNA. We defined cfDNA with the hTERT promoter C228T mutation as circulating mutant DNA (mutant DNA) and without the mutation as circulating wild‐type DNA (wild‐type DNA). We analyzed the changes in mutant and wild‐type DNA levels during HCC treatment and examined the relationship between changes in the cfDNA level and the clinical course. Mutant DNA was detected in 73.1% (49/67) of the patients during HCC treatment. In univariate analysis, factors associated with detection of mutant DNA before treatment were the intrahepatic maximum tumor diameter (P = 0.015) and protein induced by vitamin K absence (PIVKAII) (P = 0.006). The degree of mutant DNA change after TACE was significantly correlated with tumor volume (P < 0.001), reflecting the treated tumor volume. Responders with peak cfDNA levels within 1 week of TKI initiation had significantly better progression‐free survival than nonresponders (P = 0.004). Conclusion: Changes in blood hTERT promoter mutant DNA levels during TACE or TKI treatment indirectly reflect the amount of HCCs and are useful for predicting long‐term treatment responses.

IMPLICATIONS AND POTENTIAL ROLE OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE (HTERT) IN BLADDER CARCINOGENESIS

Bladder cancer is a heterogeneous disease and ranks as 10th most common cancer worldwide. Urothelial carcinoma (UC) is the most common histologic type of BC and majority constitute of papillary tumors that are well-differentiated (low-grade). Several genetic changes may occur in bladder cancer, but hTERT promoter mutations and its expression has been detected in most cases of transitional cell carcinoma. Numerous researches have led to the findings which suggest that the hTERT promoter mutations in conjunction with the common polymorphism and hTERT expression have potential of being used as clinical biomarkers in bladder cancer. Further studies need to explore the potential use of hTERT gene in bladder cancer detection, diagnosis and prognosis. This review focuses on the role of hTERT in bladder tumors in the backdrop of various studies published.

Human telomerase is directly regulated by non-telomeric TRF2-G-quadruplex interaction.

SummaryHuman telomerase reverse transcriptase (hTERT) remains suppressed in most normal somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% of cancers. However, any direct causal link between telomeres and telomerase regulation remains unclear. Here, we show that the telomere-repeat-binding-factor 2 (TRF2) binds hTERT promoter G-quadruplexes and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. Two highly recurrent hTERT promoter mutations found in many cancers, including ~83% glioblastoma multiforme, that are known to destabilize hTERT promoter G-quadruplexes, showed loss of TRF2 binding in patient-derived primary glioblastoma multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation, and hTERT re-suppression. These results uncover a mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links important in neoplastic transformation, aging, and regenerative therapy.

Human Telomerase (hTERT) is Directly Regulated by the Non-Telomeric Telomere-Binding Factor TRF2

Human telomerase reverse transcriptase hTERT remains suppressed in most normal adult somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% human cancers. However any direct causal link between telomeres and telomerase regulation remains unclear. Here we show that the telomere-repeat-binding-factor TRF2 binds to the hTERT promoter and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. TRF2 interacts with hTERT promoter G-quadruplexes. Two highly recurrent hTERT promoter mutations found in many cancers, including ~83% glioblastoma-multiforme, that are known to destabilize hTERT promoter G-quadruplexes, obliterated TRF2 binding in patient-derived primary glioblastoma-multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation and hTERT re-suppression. These results uncover a previously unknown mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links that might be important in neoplastic transformation, ageing and regenerative therapy.

Therapeutic Targets in Telomerase and Telomere Biology of Cancers.

Telomeres play an important role to conserve genomic integrity by protecting the ends of chromosomes in normal cells. Since, their progressive shortening during successive cell division which lead to chromosomal instability. Notably, telomere length is perpetuated by telomerase in large majority of cancers, thereby ensure indefinite cell proliferation-a hallmark of cancer-and this unique feature has provided telomerase as the preferred target for drug development in cancer therapeutics. Cancer cells have acquired the potential to have telomere length maintenance by telomerase activation- up-regulation of hTERT gene expression in tumor cells is synchronized by multiple genetic and epigenetic modification mechanisms viz hTERT structural variants, hTERT promoter mutation and epigenetic modifications through hTERT promoter methylation which have been implicated in various cancers initiation and progression. In view of these facts, strategies have been made to target the underlining molecular mechanisms involved in telomerase reactivation as well as of telomere structure with special reference to distortion of sheltrin proteins. This review is focussed on extensive understanding of telomere and telomerase biology. which will provide indispensable informations for enhancing the efficiency of rational anticancer drug design. However, there is also an urgent need for better understanding of cell signalling pathways for alternative lengthening of telomere which is present in telomerase negative cancer for therapeutic targets.

Human Telomerase Reverse Transcriptase Gene Promoter Mutation in Serum of Patients with Hepatocellular Carcinoma

Background: Mutations in the human telomerase reverse transcriptase (hTERT) gene promoter have been reported in hepatocellular carcinoma (HCC); however, analyses of these mutations in liquid biopsies have been technically difficult because of the high GC content of the regions of interest within this promoter. We evaluated the feasibility and prognostic value of hTERT promoter mutations identified in circulating cell-free DNA (cfDNA) from the serum of patients with HCC. Objective: A cohort of HCC patients (n = 36) who were curatively treated by surgical resection between June 2003 and September 2014 were enrolled in this study. Methods: The presence of hTERT promoter mutations in cfDNA from the patients’ serum was analyzed via modified droplet digital polymerase chain reaction, and associations were sought between specific promoter mutations and patients’ disease-free survival (DFS). Results: The G>A hTERT mutation at –124 bp was detected in the serum of 25 patients (69%). Although no marked differences were observed between the characteristics of the serum mutation-positive and serum mutation-negative patient groups, the DFS of patients with the mutation was significantly shorter than that of the serum mutation-negative patients (p = 0.02). Among 18 clinicopathologic and background liver factors examined, the presence of the –124 bp G>A mutation was an independent and significant predictor of patients’ DFS (hazard ratio = 3.01, 95% confidence interval 1.11–10.5, p = 0.03) in multivariate analyses. Conclusions: The –124 bp G>A hTERT promoter mutation was observed in the serum of 69% of HCC patients who underwent surgical resection and was an independent predictor of disease progression in HCC.

ACTR-60. A PHASE 2, HISTORICALLY CONTROLLED STUDY TESTING THE EFFICACY OF TTFIELDS WITH ADJUVANT TEMOZOLOMIDE IN HIGH-RISK WHO GRADE II AND III ASTROCYTOMAS (FORWARD)

Abstract BACKGROUND A long-standing observation in neuro-oncology concerns a group of patients with WHO Grade II and III gliomas expressing isolated hTERT promoter mutations, which exhibit indolent histological features but are highly aggressive and resistant to standard chemoradiation therapy. These patients show an overall survival rate similar to glioblastoma WHO Grade IV, necessitating new treatment options. This represents the first study of its kind testing novel treatment for this rare subgroup of lower grade gliomas with high-risk cytogenetics. METHODS This Phase 2, single arm, multicenter open label study [NCT03906448] will enroll 100 adult patients with newly diagnosed WHO grade II and III astrocytoma without IDH mutations and 1p/19q codeletions and with isolated hTERT promoter mutations who have completed standard radiotherapy with concurrent temozolomide, followed by adjuvant temozolomide for 12 cycles in combination with 24 months of TTFields therapy. The primary endpoint is overall survival compared to historical controls. Secondary endpoints are safety, quality of life, and progression free survival. Correlative studies will examine the effect of TTFields on intratumoral immune microenvironment and the role of electric field distribution on disease control and outcomes. Sample size was based on improvement in median OS versus historical controls (N=59; median survival 22 months). Improvements in median overall survival was based on a one-sided Wald test of the Weibull regression coefficient (H0: beta=0, alpha=0.05). A consortium of 15 institutions across the US will participate, with the first enrollment May 2019. Total accrual of 100 is planned over 36 months. The survival results and the correlative studies are designed to provide new insights for future therapeutic development for these patients.

Prognostic significance of human telomerase reverse transcriptase promoter region mutations C228T and C250T for overall survival in spinal chordomas.

BackgroundSpinal chordomas, a subtype of primary spinal column malignancies (PSCM), are rare tumors with poor prognosis, and we have limited understanding of the molecular drivers of neoplasia.MethodsStudy design was a retrospective review of prospectively collected data with cross-sectional survival. Archived paraffin embedded pathologic specimens were collected for 133 patients from 6 centers within Europe and North America between 1987 and 2012. Tumor DNA was extracted and the human telomerase reverse transcriptase (hTERT) promoter was sequenced. The hTERT mutational status was correlated with overall survival (OS) and time to first local recurrence.ResultsNinety-two chordomas, 26 chondrosarcomas, 7 osteosarcomas, 3 Ewing’s sarcomas, and 5 other malignant spinal tumors were analyzed. Median OS following surgery was 5.8 years (95% CI: 4.6 to 6.9) and median time to first local recurrence was 3.9 years (95% CI: 2.5 to 6.7). Eight chordomas, 2 chondrosarcomas, 1 Ewing’s sarcoma, and 1 other malignant spinal tumor harbored either a C228T or C250T mutation in the hTERT promoter. In the overall cohort, all patients with hTERT mutation were alive at 10 years postoperative with a median OS of 5.1 years (95% CI: 4.5 to 6.6) (P = 0.03). hTERT promoter mutation was observed in 8.7% of spinal chordomas, and 100% of chordoma patients harboring the mutation were alive at 10 years postoperative compared with 67% patients without the mutation (P = 0.05).ConclusionsWe report for the first time that hTERT promoter mutations C228T and C250T are present in approximately 8.7% of spinal chordomas. The presence of hTERT mutations conferred a survival benefit and could potentially be a valuable positive prognostic molecular marker in spinal chordomas.

Telomerase activity, TERT expression, hTERT promoter alterations, and alternative lengthening of the telomeres (ALT) in meningiomas - a systematic review.

Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.

Human telomerase reverse transcriptase in papillary thyroid cancer: gene expression, effects of silencing and regulation by BET inhibitors in thyroid cancer cells.

Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and β-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

HTERT promoter mutations in chondrosarcomas associate with progression and disease-related mortality

Chondrosarcomas are malignant skeletal tumors with chondroid differentiation. Prognosis is largely dependent on histological grading, which suffer from significant interobserver variability. Telomerase activity and abundant telomerase reverse transcriptase (hTERT) expression has previously been associated with chondrosarcoma grade and metastasis. We therefore analyzed the hTERT promoter in clinicopathologically well-characterized chondrosarcomas (grade 1–3) from 87 patients. Using Sanger sequencing we identified an activating −124 C > T mutation in 23 cases (26%). Promoter mutations were significantly associated with increased histological grade (8% of grade 1, 32% of grade 2 and 46% of grade 3, P = 0.002), suggesting a role in tumor progression. In four chondrosarcomas where the histopathological grade was heterogenous, the hTERT mutation was only identified in the higher-grade areas. Additionally, hTERT promoter mutations were significantly associated with worse metastasis-free survival (P = 0.018), chondrosarcoma-specific survival (P = 0.022) and older patient age (P = 0.003). These data suggest that hTERT promoter mutations are common in high grade conventional chondrosarcomas. Granted that additional studies can confirm these findings; hTERT promoter analysis could potentially serve as an adjuvant prognostic marker in routine chondrosarcoma grading. This study reinforces the rationale of telomerase targeted therapy in a subset of chondrosarcomas.

Abstract 4798: Targeting hTERT for treatment of glioblastoma (GBM)

Abstract Approximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis. Citation Format: Saumya Reddy Bollam, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Targeting hTERT for treatment of glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4798.

Long-Range Chromatin Interaction Regulates hTERT Expression Through Epigenetic Modifications

hTERT gene is heavily under control of epigenetic mechanisms which regulates its transcription. This transcriptional regulation is in most cases the limiting component for telomerase activity. However, telomerase is re-activated in 85% of cancers and this re-activation of hTERT has a complex mechanism in cancers. Recently, highly frequent substitution mutations have been observed in the proximal promoter of the hTERT gene. These correlated with increased hTERT expression and telomerase activity in cancers. These mutations create de novo ETS binding motifs, which alter the chromatin organization by mediating formation of long-range chromatin interaction between hTERT promoter and distal regions of chromatin with multiple ETS binding motifs. This specific chromatin interaction alters the epigenetic features of the hTERT promoter and explains how hTERT expression is increased in cancer cells with hTERT promoter mutations.