Telomerase Reverse Transcriptase Promoter Methylation Research Articles

Risk stratification by combining common genetic mutations and TERT promoter methylation in papillary thyroid cancer.

Risk stratification based on somatic mutations in TERT promoter and BRAF/RAS has been well established for papillary thyroid cancer (PTC), and there is emerging evidence showed that TERT promoter methylation was frequently observed in thyroid cancer patients with adverse features. This study was aimed to comprehensive explore the prognostic value of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation in PTC. The relationships of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation with clinical characteristics and outcomes of PTC were analyzed in 382 patients with PTC. TERT promoter mutation and hypermethylation were collectively observed in 52 (13.6%) samples and associated with BRAF/RAS mutation, aggressive clinical characteristics, and poor clinical outcomes of PTC. Coexistence of BRAF/RAS and TERT alterations was found in 45 of 382 (11.8%) PTC patients and strongly associated with old patient age, extrathyroidal extension, advanced pathologic T stage and metastasis. Importantly, patients with both BRAF/RAS and TERT alterations had higher rates of tumor recurrence (13.6% vs 1.5%, P = 0.042) and disease progression (24.4% vs 3.3%, P < 0.001) than patients without any alterations, and cox regression analysis revealed that the coexistence of BRAF/RAS and TERT alterations, but not BRAF/RAS or TERT alterations alone, increased the risk of progression-free interval with an adjusted HR of 10.35 (95% CI: 1.79-59.81, P = 0.009). This study suggested that comprehensively analysis of BRAF/RAS mutations, TERT promoter mutation and methylation is an effective strategy to identify high-risk patients with PTC.

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.

TERT Promoter Methylation Is Oxygen-Sensitive and Regulates Telomerase Activity.

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O2 environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.

TERT promoter methylation is associated with high expression of TERT and poor prognosis in papillary thyroid cancer.

The telomerase reverse transcriptase (TERT) is overexpressed and associated with poor prognosis in papillary thyroid cancer (PTC), the most common subtype of thyroid cancer. The overexpression of TERT in PTC was partially attributed to transcriptional activation by two hotspot mutations in the core promoter region of this gene. As one of the major epigenetic mechanisms of gene expression regulation, DNA methylation has been proved to regulate several tumor-related genes in PTC. However, the association of TERT promoter DNA methylation with TERT expression and PTC progression is still unclear. By treating PTC cell lines with demethylating agent decitabine, we found that the TERT promoter methylation and the genes' expression were remarkably decreased. Consistently, PTC patients with TERT hypermethylation had significantly higher TERT expression than patients with TERT hypomethylation. Moreover, TERT hypermethylated patients showed significant higher rates of poor clinical outcomes than patients with TERT hypomethylation. Results from the cox regression analysis showed that the hazard ratios (HRs) of TERT hypermethylation for overall survival, disease-specific survival, disease-free interval (DFI) and progression-free interval (PFI) were 4.81 (95% CI, 1.61-14.41), 8.28 (95% CI, 2.14-32.13), 3.56 (95% CI, 1.24-10.17) and 3.32 (95% CI, 1.64-6.71), respectively. The HRs for DFI and PFI remained significant after adjustment for clinical risk factors. These data suggest that promoter DNA methylation upregulates TERT expression and associates with poor clinical outcomes of PTC, thus holds the potential to be a valuable prognostic marker for PTC risk stratification.

Prognostic value and immune landscapes of TERT promoter methylation in triple negative breast cancer.

Treatment options for patients with triple-negative breast cancer (TNBC) remain limited to mainstay therapies owing to a lack of efficacious therapeutic targets. Accordingly, there is an urgent need to discover and identify novel molecular targets for the treatment and diagnosis of this disease. In this study, we analyzed the correlation of telomerase reverse transcriptase (TERT) methylation status with TERT expression, prognosis, and immune infiltration in TNBC and identified the role of TERT methylation in the regulation TNBC prognosis and immunotherapy. Data relating to the transcriptome, clinicopathological characteristics and methylation of TNBC patients were obtained from The Cancer Genome Atlas (TCGA) database. TERT expression levels and differential methylation sites (DMSs) were detected. The correlations between TERT expression and DMSs were calculated. Kaplan-Meier curves was plotted to analyze the relationship between the survival of TNBC patients and the DMSs. The correlations of DMSs and TERT expression with several immunological characteristics of immune microenvironment (immune cell infiltration, immunomodulators, immune-related biological pathways, and immune checkpoints) were assessed. The results were validated using 40 TNBC patients from Sun Yat-sen University Cancer Center (SYSUCC). Six DMSs were identified. Among them, four sites (cg11625005, cg07380026, cg17166338, and cg26006951) were within the TERT promoter, in which two sites (cg07380026 and cg26006951) were significantly related to the prognosis of patients with TNBC. Further validation using 40 TNBC samples from SYSUCC showed that the high methylation of the cg26006951 CpG site was associated with poor survival prognosis (P=0.0022). TERT expression was significantly correlated with pathological N stage and clinical stage, and cg07380026 were significantly associated with pathological T and N stages in the TCGA cohort. Moreover, the methylation site cg26006951, cg07380026 and TERT expression were significantly correlated with immune cell infiltration, common immunomodulators, and the level of the immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) in TNBC patients. TERT promotertypermethylation plays an important role in TERT expression regulation and tumor microenvironment in TNBC. It is associated with overall survival and LAG-3 expression. TERT promoter hypermethylation may be a potential molecular biomarker for predicting response to the TERT inhibitors and immune checkpoint inhibitors in TNBC.

TERT promoter mutations and methylation for telomerase activation in urothelial carcinomas: New mechanistic insights and clinical significance.

Telomerase, an RNA-dependent DNA polymerase synthesizing telomeric TTAGGG sequences, is primarily silent in normal human urothelial cells (NHUCs), but widely activated in urothelial cell-derived carcinomas or urothelial carcinomas (UCs) including UC of the bladder (UCB) and upper track UC (UTUC). Telomerase activation for telomere maintenance is required for the UC development and progression, and the key underlying mechanism is the transcriptional de-repression of the telomerase reverse transcriptase (TERT), a gene encoding the rate-limiting, telomerase catalytic component. Recent mechanistic explorations have revealed important roles for TERT promoter mutations and aberrant methylation in activation of TERT transcription and telomerase in UCs. Moreover, these TERT-featured genomic and epigenetic alterations have been evaluated for their usefulness in non-invasive UC diagnostics, recurrence monitoring, outcome prediction and response to treatments such as immunotherapy. Importantly, the detection of the mutated TERT promoter and TERT mRNA as urinary biomarkers holds great promise for urine-based UC liquid biopsy. In the present article, we review recent mechanistic insights into altered TERT promoter-mediated telomerase activation in UCs and discuss potential clinical implications. Specifically, we compare differences in senescence and transformation between NHUCs and other types of epithelial cells, address the interaction between TERT promoter mutations and other factors to affect UC progression and outcomes, evaluate the impact of TERT promoter mutations and TERT-mediated activation of human endogenous retrovirus genes on UC immunotherapy including Bacillus Calmette-Guérin therapy and immune checkpoint inhibitors. Finally, we suggest the standardization of a TERT assay and evaluation system for UC clinical practice.

Targeted Long-Read Bisulfite Sequencing Identifies Differences in the TERT Promoter Methylation Profiles between TERT Wild-Type and TERT Mutant Cancer Cells.

Simple SummaryTERT promoter methylation is enriched in cancers lacking TERT genetic alterations (wild-type cancers), but its functional impact on TERT transcription remains elusive. We developed a long-read bisulfite-sequencing platform to characterize the TERT promoter methylation profile at a single-molecule level. In wild-type cancer cell lines, both epialleles were hypermethylated symmetrically on the TERT distal promoter. In the core and proximal promoter, by contrast, the transcribed epialleles were significantly more hypomethylated than the silent epialleles. Decitabine-therapy reduced the core and proximal (not the distal) promoter methylation and reactivated the silent allele. We showed that TERT allele-specific expression is amenable to in vitro epigenetic manipulation in wild-type cancers.Background: TERT promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to TERT activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of TERT promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged TERT genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (p < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the TERT distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between TERT wild-type and genetically altered cancer cell lines. The causal relation between TERT promoter methylation and gene expression remains to be established.

EPEN-23. Interaction of epigenetic regulation and telomerase re-activation in high-risk ependymoma

Ependymomas (EPN) account for 10% of pediatric CNS tumors. Among the ten subgroups characterized by DNA methylation profiling, tumors located in the supratentorial region that harbor ZFTA fusions (e.g. ZFTA-RELA), and tumors in the posterior fossa region group A (PF-A) represent the most aggressive entities. As currently therapy success relies on the extent of tumor resection and druggable targets are so far widely missing, new therapeutic approaches are urgently needed. Epigenetic dysfunction, resulting in aberrant histone modifications as well as altered DNA methylation patterns, majorly contributes to the aggressiveness of high-risk EPN. In earlier studies, we discovered that high-risk EPN is composed of a cellular hierarchy initiating from stem-cell like populations, frequently showing telomerase re-activation. Considering that epigenetic mechanisms regulate stemness maintenance and telomerase reverse transcriptase (TERT), we studied the impact of epigenetically active drugs on differentiation and telomerase re-activation in these tumors. Accordingly, we first investigated the basal expression levels of TERT and EZH2 in a panel of patient-derived high-risk EPN cell models of different subtypes (n=7). Interestingly, both, TERT and EZH2, were highly expressed predominantly in ZFTA-RELA cell models. Corroboratively, increased sensitivity of ZFTA-RELA cells towards the EZH2 inhibitor DZNep was observed in cell viability and clonogenic assays. While HDAC inhibitors were similarly active across high-risk EPN cell models, the BET inhibitor JQ1 more efficiently reduced survival of ZFTA-RELA cells. Treatment with DZNep resulted in a loss of H3K27me3 histone marks accompanied by decreased ubiquitination of H2AK119 in the investigated ZFTA-RELA cell models, and induced apoptosis indicated by PARP cleavage. Currently, impacts of direct or pharmacological EZH2 blockade on TERT promoter methylation, induction of senescence and differentiation are analyzed. Summarizing, we proof varying efficacy of epigenetically active drugs in high-risk EPN subgroups, in particular EZH2 inhibition in ZFTA-RELA cell models.

Associations of Maternal Polyunsaturated Fatty Acids With Telomere Length in the Cord Blood and Placenta in Chinese Population.

Few studies have investigated the correlation between maternal polyunsaturated fatty acids (PUFAs) and telomeres in offspring, and the underlying influential mechanisms. In this study, we assessed the associations of maternal PUFAs with telomere length (TL) and DNA methylation of the telomerase reverse transcriptase (TERT) promoter in the cord blood and the placenta. A total of 274 pregnant women and their newborn babies were enrolled in this study. Maternal blood before delivery, the cord blood, and the placenta at birth were collected. Fatty acids in maternal erythrocytes and cord blood cells were measured by gas chromatography (GC). TL in the cord blood and the placenta was determined using real-time quantitative PCR (qPCR) by calculating the product ratio of telomeric DNA to the single-copy gene β-globin. The TERT promoter methylation was analyzed by DNA bisulfite sequencing. The associations of maternal fatty acids with TL were analyzed by univariate and multivariate regression. We found that low concentrations of docosapentaenoci acid (DPA, C22: 5n-3) and total n-3 PUFAs, adrenic acid (ADA, C22: 4n-6), and osbond acid (OA, C22: 5n-6) and high concentrations of linoleic acid (LA, C18: 2n-6) in maternal erythrocytes were associated with the shortened TL in cord blood cells (estimated difference in univariate analysis −0.36 to −0.46 for extreme quintile compared with middle quintile), and that low concentrations of cord blood docosahexaenoic acid (DHA, C22: 6n-3) were related to the shortened TL in cord blood cells. Differently, high concentrations of α-linolenic acid (LNA, C18: 3n-3), eicosatrienoic acid (EA, C20: 3n-3), DHA, and γ-linoleic acid (GLA, C18:3n-6) in maternal erythrocytes were associated with the shortened TL in the placenta (estimated difference in univariate analysis −0.36 to −0.45 for higher quintiles compared with the middle quintile). Further examination demonstrated that the concentrations of DHA and total n-3 PUFAs in maternal erythrocytes had positive associations with DNA methylation of the TERT promoter in the cord blood instead of the placenta. These data suggest that maternal PUFAs are closely correlated to infant TL and the TERT promoter methylation, which are differently affected by maternal n-3 PUFAs between the cord blood and the placenta. Therefore, keeping higher levels of maternal n-3 PUFAs during pregnancy may help to maintain TL in the offspring, which is beneficial to long-term health.

Alternative Lengthening of Telomeres (ALT) and Telomerase Reverse Transcriptase Promoter Methylation in Recurrent Adult and Primary Pediatric Pituitary Neuroendocrine Tumors.

Neoplastic cells acquire the ability to proliferate endlessly by maintaining telomeres via telomerase, or alternative lengthening of telomeres (ALT). The role of telomere maintenance in pituitary neuroendocrine tumors (PitNETs) has yet to be thoroughly investigated. We analyzed surgical samples of 24 adult recurrent PitNETs (including onset and relapses for 14 of them) and 12 pediatric primary PitNETs. The presence of ALT was assessed using telomere-specific fluorescence in situ hybridization, methylation of telomerase reverse transcriptase promoter (TERTp) by methylation-specific PCR, and ATRX expression by immunohistochemistry. Among the adult recurrent PitNETs, we identified 3/24 (12.5%) ALT-positive cases. ALT was present from the onset and maintained in subsequent relapses, suggesting that this mechanism occurs early in tumorigenesis and is stable during progression. ATRX loss was only seen in one ALT-positive case. Noteworthy, ALT was observed in 3 out of 5 aggressive PitNETs, including two aggressive corticotroph tumors, eventually leading to patient's death. ALT-negative tumors (87.5%) were classified according to their low (29.2%), medium (50%), and high (8.3%) telomere fluorescence intensity, with no significant differences emerging in their molecular, clinical, or pathological characteristics. TERTp methylation was found in 6/24 cases (25%), with a total concordance in methylation status between onset and recurrences, suggesting that this mechanism remains stable throughout disease progression. TERTp methylation did not influence telomere length. In the pediatric cohort of PitNETs, TERTp methylation was also observed in 4/12 cases (33.3%), but no case of ALT activation was observed. In conclusion, ALT is triggered at onset and maintained during tumor progression in a subset of adult PitNETs, suggesting that it could be used for clinical purposes, as a potential predictor of aggressive behavior.

Evaluation of Molecular Determinants Related to Outcomes in Recurrent High Grade Glioma Treated With Hypofractionated Stereotactic Radiotherapy (HFSRT) and Immune Checkpoint Blockade

Recurrent high-grade glioma (rHGG) continues to cause increased morbidity and mortality. To date, salvage therapy for rHGG remains ineffective, even with novel immune checkpoint blockade approaches. Here we evaluate molecular factors that may influence outcomes in rHGG patients treated with HFSRT and immunotherapy.This retrospective analysis identified 76 continuous rHGG patients treated with HFSRT and PD-1 inhibitors on or off trial protocol with > 3 months of follow-up from 2010 to 2020. Clinical characteristics, treatment, outcomes, and available molecular alterations in IDH1 (n = 74), PTEN (n = 54), EGFRvIII (n = 64) or amplification (n = 56), CDKN2A (n = 42), TERT (n = 42) and MGMT promoter methylation (n = 69) were collected. Time to recurrence (TTR) and overall survival (OS) were estimated from the end of HFSRT with univariable (UVA) Kaplan-Meier methods compared via log-rank test as well as Cox regression analyses.The median age at HFSRT was 55 years (range: 22-83) with a KPS of 80 (range: 50-100). The majority (63%) of patients were male, 35% had > 2 recurrences of HGG before HFSRT and 55% underwent salvage surgery. A total of 11 patients (15%) received bevacizumab (Bev) prior to HFSRT. The median HFSRT dose was 35 Gy (range: 30-40 Gy) given in 5-10 fractions. Sixty-six (87%) patients were treated with concurrent HFSRT and Bev followed by immunotherapy. At a median follow-up from HFSRT of 13.8 months (range: 8.7-18.9), 55 (72%) patients relapsed with a median TTR of 6.94 months (5.0-8.87) and OS of 10.1 months (7.4-12.9); this included Bev-naïve and -resistant patients. Bev vs no Bev with HFSRT improved TTR (13.7 vs 6.3 months, P < 0.001), though Bev with HFSRT was not associated with improved OS. Of interest, no Bev prior to HFSRT improved OS from HFSRT (12.0 vs 8.4 months, P = 0.01). No molecular factors were associated with TTR after HFSRT, though IDH1-mut (P = 0.001) and wild-type PTEN (P = 0.02) and EGFR (P = 0.02) were related with prolonged OS. Similarly, mutations in TERT were associated with inferior OS (26.7 vs 60.7 months, P < 0.001). MGMT methylation status was not associated with TTR of OS.Salvage treatments for rHGG remain a challenge. This study provides further insight into prognostic molecular determinants in the setting of HFSRT, PD-1 blockade and Bev.E. Guvenli: None. D. Grass: None. D.E. Oliver: None. A.B. Etame: None. N. Tran: None. R.J. Macaulay: None. P. Forsyth: Research Grant; NIH/NCI, CDMRP, Department of Defense, Pfizer, State of Florida - Bankhead Coley, Moffitt Center of Excellence Celgene project, NIH/NCI. Honoraria; BTG, NIH, Ziopharm, Tocagen, NCRI. Consultant; AbbVie, Inc, Ziopharm, Novellus, NCI Neuro-Oncology Branch Peer Review, Physical Sciences Oncology Network, Tocagen. Advisory Boar. S. Sahebjam: Research Grant; Merck, Bristol Myers Squibb, Brooklyn Immunotherapeutics. Advisory Board; Merck, Boehringer Ingelheim. Travel Expenses; Eli Lilly. H.M. Yu: Honoraria; UpToDate. Speaker's Bureau; BrainLab. Advisory Board; Novocure, AbbVie.

Association of telomere length and telomerase methylation with n-3 fatty acids in preschool children with obesity

BackgroundTelomeres play a crucial role in cellular survival and its length is a predictor for onset of chronic non-communicable diseases. Studies on association between telomeres and obesity in children have brought discrepant results and the underlying mechanisms and influential factors are to be elucidated. This study aimed to investigate changes in telomere length and telomerase reverse transcriptase (TERT) DNA methylation, and further to determine their correlation with n-3 polyunsaturated fatty acids (PUFAs) in preschool children with obesity.MethodsForty-six preschool children with obesity aged 3 to 4 years were included in the study, with equal numbers of age- and gender-matched children with normal weight as control. Leukocyte telomere length was determined by the ratio of telomeric product and single copy gene obtained using real-time qPCR. DNA methylation of TERT promoter was analyzed by bisulfite sequencing. Fatty acids in erythrocytes were measured by gas chromatography with a total of 15 fatty acids analyzed. The total saturated fatty acids (SFAs), total n-6 PUFAs, total n-3 PUFAs, and the ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) were calculated. Then the correlation between leukocyte telomere length, TERT promoter methylation and fatty acids was determined.ResultsIn preschool children with obesity, leukocyte telomeres were shortened and had a negative association with the body mass index. The methylated fractions in 13 of 25 CpG sites in the TERT promoter were increased by approximately 3 to 35% in the children with obesity compared to the normal weight children. Erythrocyte lauric acid and total SFAs, lenoleic acid and total n-6 PUFAs were higher, and DHA was lower in the children with obesity than those in the children with normal weight. Correlative analysis showed that leukocyte telomere length had a positive association with total SFAs and DHA, and a negative association with the AA/DHA ratio. However, no association between erythrocyte DHA and the TERT promoter methylation was found.ConclusionThese data indicate that the reduced body DHA content and increased AA/DHA ratio may be associated with shortened leukocyte telomeres in child obesity, which is probably not involved in the TERT promoter methylation.

Craniopharyngiomas, including Recurrent Cases, Lack TERT Promoter Hotspot Mutations.

Adamantinomatous craniopharyngiomas (ACP) are characterized by alterations in the CTNNB1 gene while almost all papillary craniopharyngiomas (PCP) harbor a canonical V600E mutation in the BRAF gene. Although other recurrent driver genes have not been described to date in craniopharyngiomas, the heterogeneous clinical course of these tumors might be associated with the acquisition of further genomic alterations. It is well known that telomerase reverse transcriptase (TERT) promoter (TERTp) alterations, including mutations or methylation, upregulate the expression of TERT and increase telomerase activity, promoting tumorigenesis. We investigated whether TERTp mutations or methylation are associated with tumor relapse in a subset of craniopharyngiomas. Samples from 42 patients with histologically confirmed craniopharyngioma were retrieved. We determined TERTp, BRAF, and CTNNB1 hotspot mutations in all samples using targeted sequencing and the TERTp methylation status by methylation-specific polymerase chain reaction (PCR) in 30 samples. While BRAF V600E mutations and CTNNB1 mutations were detected in 12 (28.6%) and 21 patients (50%) in the initial tumors and subsequent recurrences, respectively, none of the patients in our cohort, including those with multiple relapses, harbored a TERTp mutation. Furthermore, TERTp methylation was detected in 14 out of 24 cases (58.3%) with available primary samples; however, no correlation between TERTp methylation with the pathological subtype, genotype, or tumor aggressiveness was detected. These data suggest that elevated telomerase activity via acquisition of TERTp mutations is an infrequent pathway in the tumorigenesis of craniopharyngiomas, regardless of their clinical course.

Exploring the epigenetic regulation of telomerase reverse transcriptase (TERT) in human cancer cell lines.

Telomerase regulation, including TERT promoter methylation, has been of long-standing interest to cancer biologists. Rowland et al. have now vastly expanded their ongoing characterization of TERT promoter methylation in cancer cells, analyzing the methylation patterns of 833 cell lines from 23 human cancers. They document a highly conserved pattern of hypomethylation around the proximal promoter, as well as a more heterogeneous region of hypermethylation further upstream, both associated with active TERT expression in cancer cells. They further describe the interplay between activating TERT promoter mutations and allelic methylation and transcription patterns. This valuable dataset represents the most extensive characterization of TERT promoter methylation in cancer cells to date and will help guide the future study of transcriptional regulation of telomerase. Comment on: https://doi.org/10.1002/1878-0261.12786.

Allele-specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene (TERT) associates with TERT expression in multiple cancers.

Telomerase reverse transcriptase (TERT) is pathologically expressed in the vast majority of human cancers, but the epigenetic regulation of its expression is only beginning to be understood. In particular, the active TERT gene in cancer cells has been characterized as having a hypermethylated CpG island, opposite to the general association of DNA methylation with gene repression. Here, we analyzed TERT promoter CpG methylation in 833 human cancer cell lines representing 23 different tissue types and found hypermethylation of the upstream portion of the CpG island and more conserved hypomethylation of a region including the proximal TERT promoter and exon 1. In cell lines with monoallelic expression of TERT, we found allelic methylation of the proximal TERT promoter. This included cell lines with the -124 or -146 activating promoter mutation as well as wild-type TERT cancer lines. In these cell line types, decreased proximal promoter methylation is associated with the active allele. Compared to cells with monoallelic expression of TERT, lines with biallelic expression of TERT had even lower methylation in the proximal TERT promoter. Thus, in cell lines from cancers of many different tissues, the TERT proximal promoter has canonical DNA methylation, with low methylation correlating with increased TERT expression.

Abstract 157: Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

Abstract In cancers, the epigenetic regulation of telomerase expression is not fully characterized, with multiple conflicting reports. In particular, the role of DNA methylation of cytosine residues in the control of TERT expression remains unclear. Here, we have carried out the most comprehensive characterization of TERT promoter methylation using ultra-deep bisulfite sequencing spanning more than 310 CpGs within and surrounding the core TERT promoter in 96 different cell lines, including primary, immortalized, and cancer cell types, as well as in control and reference samples. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent G228A and G250A TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. Notably, in cancers, the hypermethylated alleles were associated with repressed expression while remaining unmethylated alleles were enriched with the open chromatin marks, H3K4me3 and H3K27ac. Furthermore, the hypomethylated alleles of the TERT promoter were responsible for the observed TERT expression in cancer. This association of hypomethylated alleles with active expression and hypermethylated alleles with repressed expression is particularly evident in cancers harboring TERT promoter mutations, and cancers displaying allele-specific expression. Reporter assays confirmed that DNA methylation of the TERT promoter greatly suppressed reporter expression in heterologous TERT promoter-reporter constructs. Overall, our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. Citation Format: Mindy K. Graham, David Esopi, Jacqueline A. Brosnan-Cashman, Anuj Gupta, Angelo De Marzo, William G. Nelson, Christopher M. Heaphy, Alan K. Meeker, Sarah J. Wheelan, Srinivasan Yegnasubramanian. Pervasive promoter hypermethylation of silenced TERT alleles in human cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 157.

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

BackgroundIn cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood.MethodsHere, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples.ResultsIn general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells.ConclusionsOur findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines.

Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression.

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

TERT and TERT promoter in melanocytic neoplasms: Current concepts in pathogenesis, diagnosis, and prognosis.

Located on chromosome locus 5p15.33, telomerase reverse transcriptase (TERT or hTERT) encodes the catalytic subunit of telomerase which permits lengthening and preservation of telomeres following mitosis. Mutations in TERT promoter (TERT-p) upregulate expression of TERT, allowing survival of malignant cells and tumor progression in wide variety of malignancies including melanoma. The objective of this review is to examine the roles of TERT and TERT-p in the pathogenesis, diagnosis, and prognostication of cutaneous melanoma. All studies of TERT or TERT-p in cutaneous melanocytic neoplasms with the following inclusion criteria were reviewed: publication date between 2010 and 2019, English language, and series of ≥3 cases were reviewed for evidence supporting the role of TERT in pathogenesis, diagnosis, and prognosis. Studies with <3 cases or focused primarily on mucosal or uveal melanocytic tumors were excluded. TERT-p mutations are frequent in chronic and non-chronic sun damage melanoma and correlate with adverse prognosis, inform pathogenesis, and may provide diagnostic support. While TERT-p mutations are uncommon in acral melanoma, TERT copy number gains and gene amplification predict reduced survival. Among atypical spitzoid neoplasms, TERT-p mutations identify biologically aggressive tumors and support the diagnosis of spitzoid melanoma. TERT-p methylation may have prognostic value in pediatric conventional melanoma and drive tumorigenesis in melanoma arising within congenital nevi. Finally, TERT-p mutations may aid in the differentiation of recurrent nevi from recurrent melanoma.