Inhibition Of Telomerase Reverse Transcriptase Research Articles

Endoplasmic Reticulum Stress Promotes Telomerase Reverse Transcriptase Expression Contributes to Development of Allergic Rhinitis.

The Th2 cell polarization is a crucial factor in the pathogenesis of allergic diseases. The underlying mechanism requires further investigation. Telomerase has an immune-regulating ability. The aim of this study is to elucidate the association between telomerase and Th2 cell polarization in patients with allergic rhinitis (AR). CD4+ T cells were isolated from blood samples collected from AR patients and healthy control subjects. RNA sequencing was employed to analyze RNA samples extracted from CD4+ T cells. An AR mouse model was established using the ovalbumin-alum protocol. High telomerase gene activity and high endoplasmic reticulum (ER) stress status were observed in CD4+ T-cells in patients with AR. Positive correlation between the telomerase reverse transcriptase (TERT) gene expression in CD4+ T cells and AR response in patients with AR. TERT facilitated the degradation of Foxp3 proteins in CD4+ T cells, resulting in the polarization of Th2 cells. Sensitization with the ovalbumin-alum protocol enhanced the Tert expression in CD4+ T cells by exacerbating ER stress. Conditional inhibition of the Tert or eukaryotic translation initiation factor 2-α (Eif2a) expression in CD4+ T cells effectively attenuated experimental AR in mice. Elevated amounts of telomerase in CD4+ T cells were found in CD4+ T cells of subjects with AR. Telomerase promoted Th2 cell polarization by inducing Foxp3 protein degradation and promotes GATA3 activation. Inhibition of TERT or eIF2a alleviated experimental AR.

Copy-number-gain of telomerase reverse transcriptase (hTERT) is associated with an unfavorable prognosis in esophageal adenocarcinoma

Esophageal adenocarcinoma exhibits one of the highest mortality rates among all cancer entities. Multimodal therapy strategies have improved patients’ survival significantly. However, patients in early stages are currently limited to receiving only local therapies, even though some patients within this group showcase short survival periods. Until now, there has been no widely established clinically used biomarker to detect these high-risk patients. Telomerase reverse transcriptase (TERT), a gene encoding a crucial subunit of the telomerase enzyme, plays a significant role in establishing cancer cell immortality and is under suspicion for its potential contribution to tumor progression. Therefore, we aimed to evaluate the clinical relevance of the TERT amplification status. We included 643 patients with esophageal adenocarcinoma, who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. The TERT amplification status was characterized using fluorescence in situ hybridization. Clinicopathological values and patients’ overall survival were compared between patients with and without TERT amplification. Further sub-cohort analyses were conducted for patients with pT1N0-3 tumor stage. Eighty-One patients (12.6%) exhibited TERT amplification. Patients with amplified TERT showed significantly worse overall survival (median OS: 22.6 vs. 36.8 months, p = 0.009). Interestingly, TERT amplification could be characterized as an independent risk factor for worse overall survival in multivariate analysis in patients with pT1N0-3 tumor stage (HR = 2.440, 95% CI 1.095–5.440, p = 0.029). In this study, we describe the TERT amplification status as an independent risk factor for worse survival in patients diagnosed with esophageal adenocarcinoma at pT1N0-3 tumor stage, encompassing cases involving tumor infiltration of the lamina propria, muscularis mucosae, and/or submucosa. Based on our findings, we put forth the proposition that evaluating the TERT amplification status may serve as a valuable tool in identifying a specific subgroup of patients, namely those with TERT amplification and pT1N0-3 tumor-stage esophageal adenocarcinoma. The patients of this subgroup could potentially benefit from enhanced follow-up protocols, more aggressive treatment approaches, or possible targeted TERT inhibition therapies, all aimed at improving their overall clinical outcomes.

Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach.

Telomerase reverse transcriptase (TERT), the catalytic component of telomerase, may also contribute to carcinogenesis via telomere-length independent mechanisms. Our previous in vitro and in vivo studies demonstrated that short-term telomerase inhibition by BIBR1532 impairs cell proliferation without affecting telomere length. Here, we show that the impaired cell cycle progression following short-term TERT inhibition by BIBR1532 in in vitro models of B-cell lymphoproliferative disorders, i.e., Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), and B-cell malignancies, i.e., Burkitt's lymphoma (BL) cell lines, is characterized by a significant reduction in NF-κB p65 nuclear levels leading to the downregulation of its target gene MYC. MYC downregulation was associated with increased expression and nuclear localization of P21, thus promoting its cell cycle inhibitory function. Consistently, treatment with BIBR1532 in wild-type zebrafish embryos significantly decreased Myc and increased p21 expression. The combination of BIBR1532 with antineoplastic drugs (cyclophosphamide or fludarabine) significantly reduced xenografted cells' proliferation rate compared to monotherapy in the zebrafish xenograft model. Overall, these findings indicate that short-term inhibition of TERT impairs cell growth through the downregulation of MYC via NF-κB signalling and supports the use of TERT inhibitors in combination with antineoplastic drugs as an efficient anticancer strategy.

QSAR, pharmacophore modeling and molecular docking studies to identify structural alerts for some nitrogen heterocycles as dual inhibitor of telomerase reverse transcriptase and human telomeric G-quadruplex DNA

BackgroundTelomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA are amongst the favorable target for researchers to discover novel and more effective anticancer agents. To understand and elucidate structure activity relationship and mechanism of inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA, a QSAR modeling and molecular docking were conducted.ResultsTwo robust QSAR model were obtained which consist of full set QSAR model (R2: 0.8174, CCCtr: 0.8995, Q2loo: 0.7881, Q2LMO: 0.7814) and divided set QSAR model (R2: 0.8217, CCCtr: 0.9021, Q2loo: 0.7886, Q2LMO: 0.7783, Q2-F1: 0.7078, Q2-F2: 0.6865, Q2-F3: 0.7346) for envisaging the inhibitory activity of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The analysis reveals that carbon atom exactly at 3 bonds from aromatic carbon atom, nitrogen atom exactly at six bonds from planer nitrogen atom, aromatic carbon atom within 2 A0 from the center of mass of molecule and occurrence of element hydrogen within 2 A0 from donar atom are the key pharmacophoric features important for dual inhibition of TERT and human telomeric G-quadruplex DNA. To validate this analysis, pharmacophore modeling and the molecular docking is performed. Molecular docking analysis support QSAR analysis and revealed that, dual inhibition of TERT and human telomeric DNA is mainly contributed from hydrophobic and hydrogen bonding interactions.ConclusionThe findings of molecular docking, pharmacophore modelling, and QSAR are all consistent and in strong agreement. The validated QSAR analyses can detect structural alerts, pharmacophore modelling can classify a molecule's consensus pharmacophore involving hydrophobic and acceptor regions, whereas docking analysis can reveal the mechanism of dual inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The combination of QSAR, pharmacophore modeling and molecular docking may be useful for the future drug design of dual inhibitors to combat the devastating issue of resistance.Graphical abstract

PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1.

Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS. These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Abstract P154: Pgc1a Regulates The Endothelial Response To Fluid Shear Stress Via Telomerase Reverse Transcriptase Control Of Heme Oxygenase-1

Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow that is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. Here we have identified peroxisome proliferator gamma coactivator-1α (PGC-1α) as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo . Compared to oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) increased PGC-1α expression and its transcriptional co-activation. PGC-1α was required for laminar FSS-induced expression of telomerase reverse transcriptase (TERT) in vitro and in vivo via its association with ERRα and KLF4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo . Among the flow-responsive genes sensitive to TERT status was heme oxygenase-1 (HMOX1), a gene required for endothelial alignment to laminar FSS. Thus, these data suggest an important role for a PGC-1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Metabolic imaging detects elevated glucose flux through the pentose phosphate pathway associated with TERT expression in low-grade gliomas.

Telomerase reverse transcriptase (TERT) is essential for tumor proliferation, including in low-grade oligodendrogliomas (LGOGs). Since TERT is silenced in normal cells, it is also a therapeutic target. Therefore, noninvasive methods of imaging TERT are needed. Here, we examined the link between TERT expression and metabolism in LGOGs, with the goal of leveraging this information for noninvasive magnetic resonance spectroscopy (MRS)-based metabolic imaging of LGOGs. Immortalized normal human astrocytes with doxycycline-inducible TERT silencing, patient-derived LGOG cells, orthotopic tumors, and LGOG patient biopsies were studied to determine the mechanistic link between TERT expression and glucose metabolism. The ability of hyperpolarized [U-13C, U-2H]-glucose to noninvasively assess TERT expression was tested in live cells and orthotopic tumors. TERT expression was associated with elevated glucose flux through the pentose phosphate pathway (PPP), elevated NADPH, which is a major product of the PPP, and elevated glutathione, which is maintained in a reduced state by NADPH. Importantly, hyperpolarized [U-13C, U-2H]-glucose metabolism via the PPP noninvasively reported on TERT expression and response to TERT inhibition in patient-derived LGOG cells and orthotopic tumors. Mechanistically, TERT acted via the sirtuin SIRT2 to upregulate the glucose transporter GLUT1 and the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase. We have, for the first time, leveraged a mechanistic understanding of TERT-associated metabolic reprogramming for noninvasive imaging of LGOGs using hyperpolarized [U-13C, U-2H]-glucose. Our findings provide a novel way of imaging a hallmark of tumor immortality and have the potential to improve diagnosis and treatment response assessment for LGOG patients.

Abstract PO-020: Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence

Abstract Upon reactivation in cancer, telomerase adds TTAGGG DNA repeats to the ends of chromosomes to prevent telomere erosion and senescence despite deregulated cell proliferation. The telomerase catalytic core consists of telomerase reverse transcriptase (TERT) and the template RNA, telomerase RNA (TR). While TR is broadly expressed, TERT is silenced in somatic cells, leading to progressive telomere shortening with each cell division leading to replicative senescence or apoptosis. That most cancers can maintain telomere integrity despite continued cell division has made TERT an attractive target for cancer therapy. We recently reported NU-1, a novel covalent telomerase inhibitor derived from the natural product chrolactomycin, that irreversibly blocks the telomerase activity of TERT. In examining mechanisms of action, our RNA-seq analysis found that NU-1 affects not only telomere maintenance but also DNA damage response (DDR) pathways. TERT has previously been implicated in multiple cancer-related pathways, including cell survival and repair of DNA double strand breaks (DSBs). To further explore the effects of TERT inhibition on the DDR, we used the human ER+ breast cancer cell line MCF7 to examine radiation response in the presence or absence of TERT inhibitors, including NU-1, chrolactomycin, BIBR1532 and MST-312. TERT inhibition was sufficient to delay both the resolution of γH2AX foci, a marker for DDR signaling, and DSB repair based on neutral comet assay. Surviving cells displayed increased cellular senescence after TERT inhibition. Importantly, only a minor fraction of the persistent γH2AX foci induced by TERT inhibitors colocalized with telomeres, based on binding of PNA probes. Examining mechanisms of DSB repair, NU-1 or BIBR1532 appeared to primarily inhibit the non-homologous end joining (NHEJ) DSB repair pathway versus less effect on homologous recombination (HR) repair, using Traffic Light Reporter assays. Similarly, NU-1 enhanced loss of cell proliferation after treatment with irinotecan, doxorubicin, etoposide, or paclitaxel in multiple TERT-expressing cancer cell lines. This effect was absent in the TERT negative Saos2 cell line. These results establish NU-1 as a chemical probe to explore TERT catalytic activity as a determinant of DNA damage tolerance and suggest telomerase inhibitors as tumor-specific radio/chemo-sensitizers. Citation Format: Yue Liu, Stephen J. Kron, Rick C. Betori, Karl A. Scheidt, Grant Frost, Ding Wu, Don J. Wolfgeher, Elena V. Efimova, Scott B. Cohen. Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-020.

HIF-1α-Mediated Telomerase Reverse Transcriptase Activation Inducing Autophagy Through Mammalian Target of Rapamycin Promotes Papillary Thyroid Carcinoma Progression During Hypoxia Stress.

Background: It is important to properly understand the molecular mechanisms of aggressive tumors among papillary thyroid carcinomas (PTCs) that are often the most indolent. Hypoxia inducible factor-1α (HIF-1α), induced by hypoxia, plays pivotal roles in the development and metastasis of the many tumors, including PTCs. Upregulation of telomerase reverse transcriptase (TERT) activity is found in highly invasive PTCs. Further, previous studies have reported that autophagy serves as a protective mechanism to facilitate PTC cell survival. We, therefore, hypothesized that there was a link between HIF-1α, TERT, and autophagy in promoting PTC progression. Methods: Immunohistochemistry staining was conducted to evaluate the expressions of HIF-1α, TERT, and autophagy marker, LC3-II, in matched PTC tumors and corresponding nontumor tissues. Two PTC cell lines (TPC-1 and BCPAP) were used in subsequent cytological function studies. Cell viability, proliferation, apoptosis, migration, and invasion were assessed during hypoxia, genetic enhancement and inhibition of TERT, and chemical and genetic inhibition of autophagy. The protein expression levels of the corresponding biomarkers were determined by Western blotting, and autophagy flow was detected. We characterized the molecular mechanism of PTC cell progression. Results: The protein expression levels of HIF-1α, TERT, and LC3-II were upregulated in PTCs and were significantly correlated with high tumor-node-metastasis stage. Further, an in vitro study indicated that HIF-1α induced by hypoxia functioned as a transcriptional activator by binding with sequences potentially located in the TERT promoter and was positively correlated with the malignant behavior of PTC cell lines. Overexpression of TERT inhibited the kinase activity of mammalian target of rapamycin (mTOR), resulting in the activation of autophagy. Functionally, TERT-induced autophagy provided a survival advantage to PTC cells during hypoxia stress. Conclusions: We identified a novel molecular mechanism involving the HIF-1α/TERT axis, which promoted PTC progression by inducing autophagy through mTOR during hypoxia stress. These findings may provide a basis for the new treatment of aggressive PTCs.

Abstract 3792: Imetelstat inhibits RNA-editing mediated myeloproliferative neoplasm stem cell self-renewal

Abstract Introduction: Myeloproliferative neoplasms (MPN) including blast crisis (BC) CML and myelofibrosis (MF) are characterized by the clonal proliferation of hematopoietic cell lineages, mostly in the marrow. BC CML gives rise to TKI resistant myeloid progenitors that activate Wnt/β-catenin signaling pathway. However, BCR-ABL TKI resistant BC CML exhibits a robust telomerase activity and presents at very low or undetectable level in normal cells, and telomerase plays a pivotal role in cancer cell growth and may serve as an ideal target for anticancer therapeutics. In addition, β-catenin had been reported to transcriptionally regulate human telomerase reverse transcriptase (TERT). Telomerase is composed of the enzymatic reverse transcriptase protein TERT and the RNA template TERC. Thus, we investigated the capacity of imetelstat, a telomerase inhibitor, which binds to the TERC subunit with high affinity to prevent self-renewal of malignant progenitors. Recent clinical trials showed early signs of efficacy in MF. But, it's role in selectively inhibiting leukemia stem cell (LSC) self-renewing in CML has not been elucidated. Methods and Results: Progenitors from BC CML were compared with chronic phase (CP) CML and primary normal samples, RNA-seq results revealed upregulation of TERT suggesting a role for TERT activation in BC CML progenitor transformation. Human MPN progenitor co-culture experiments revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 or 5 uM significantly inhibited (p < 0.001, ANOVA) in vitro replating of BC CML compared with aged bone marrow progenitors. Also, imetelstat (5 mM) significantly reduced replating of MF compared with normal progenitor samples. Treatment of humanized MPN mouse models, established with 5 different BC CML and 4 different MF samples, at 30 mg/kg of imetelstat, three times a week for 4 weeks resulted in a significant reduction of malignant progenitors and human CD45+ cells (p < 0.001, t test) in BC CML, and a significant reduction of human CD45+ cells (p<0.01, t test) in MF in both marrow and spleen, when compared with vehicle controls. FACS analysis revealed a significant reduction of activated β-catenin protein in BC CML engrafted human progenitors after imetelstat treatment (p < 0.01, t test) compared with vehicle control. In addition, a significant inhibition of TERT (p = 0.03) and TERC (p = 0.02) was observed in BC CML LSC isolated from imetelstat treated mouse marrow when compared with vehicle control. Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Recent RNA-sequencing analysis showed an increase in ADAR1 expression during MPN progression, and our RNA-seq analysis demonstrated a decreased ADAR1 gene expression (p < 0.005) as well as overall adenosine to inosine editing rates as measured by edits per million reads (p <0.005) following imetelstat treatment of BC CML engrafted mice when compared to mismatch control. Conclusions: Niche responsive interactions between the telomerase complex and the Wnt/β-catenin self-renewal pathway sensitize β-catenin activated MPN progenitors to imetelstat treatment in both the in vitro co-culture and in vivo humanized MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat. Citation Format: Wenxue Ma, Larisa Balaian, Cayla Mason, Raymond Diep, Jessica Pham, Qingfei Jiang, Jeremy Lee, Sheldon Morris, Phoebe Mondala, Ping Chen, Thomas Whisenant, Mary Donohoe, Benjamin Heyman, Edward Ball, Fei Huang, Catriona Jamieson. Imetelstat inhibits RNA-editing mediated myeloproliferative neoplasm stem cell self-renewal [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 3792.

Anti-Proliferative and Pro-Apoptotic Effects of Short-Term Inhibition of Telomerase In Vivo and in Human Malignant B Cells Xenografted in Zebrafish.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres’ length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430; tert−/−) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response; all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert−/− embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

Induction of Telomerase Catalytic Subunit Alternative Splicing by Apoptotic Endonuclease G in Mouse and Rat Lymphocytes

It is known that apoptotic endonuclease G (EndoG) induces alternative splicing (AS) of telomerase catalytic subunit TERT (telomerase reverse transcriptase) mRNA and inhibits telomerase activity in tumor cells and activated human T cells. The aim of this study was to investigate the possibility of TERT mRNA AS induction and inhibition of telomerase activity by EndoG in activated mouse and rat lymphocytes. To induce EndoG expression, mouse and rat CD4+, CD8+ T cells, B cells, and NK cells were transfected with the pEndoG-GFP plasmid or incubated with the DNA-damaging agent cisplatin in vitro. The increase in the EndoG expression resulted in decreased expression of full-length active TERT variant, enhanced synthesis of the truncated splicing variant, and decreased telomerase activity. An increase in the EndoG expression, a change in the mRNA pool of TERT splicing variants, and inhibition of telomerase activity were observed in mouse and rat lymphocytes after cisplatin administration in vivo. Thus, EndoG is capable of inducing TERT mRNA AS and regulating telomerase activity in mouse and rat lymphocytes.

Telomerase reverse transcriptase mutations are independent predictor of disease-free survival in Middle Eastern papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Tumor recurrence occurs in ∼20% of PTCs and some reach advanced stages. Promoter mutation in the telomerase reverse transcriptase (TERT) gene is identified to be a prognostic marker in PTC. However, the contribution of TERT promoter mutation to cancer progression in PTC patients is still not fully understood. In this study, we investigated the incidence of TERT promoter mutations and TERT protein expression and their association with clinicopathological outcomes in a large cohort of PTC samples using direct sequencing technology and immunohistochemistry. Furthermore, two PTC cell lines were utilized to investigate role of TERT mutations in mediating metastasis. Two promoter hotspot mutations C228T and C250T were identified in 18.0% (167/927) of our cohort and were significantly associated with poor 5 years disease-free survival and distant metastasis of PTC. TERT protein overexpression was noted in 20.1% of our PTC cohort and was significantly associated with poor prognostic markers such as older age, extrathyroidal extension and Stage IV tumors. A significant association was also found between TERT overexpression and epithelial-mesenchymal transition (EMT) markers. Functional analysis showed that TERT inhibition reduced cell growth, invasion, migration and angiogenesis in PTC via suppression of EMT in PTC cells. Our results suggest that TERT promoter mutation is an independent predictor of disease-free survival and might drive the metastasis, and downregulation of TERT could potentiate antitumor and antimetastatic activities in PTC.

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with invitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma.

Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein–Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.

TERT alleviates irradiation-induced late rectal injury by reducing hypoxia-induced ROS levels through the activation of NF-κB and autophagy.

The hypoxic microenvironment which is present following irradiation has been proven to promote radiation-induced injury to normal tissues. Previous studies have demonstrated that telomerase reverse transcriptase (TERT) is regulated by hypoxia, and that it plays a protective role in the process of wound repair. However, its effects on radiation-induced injury remain unclear. In this study, we examined the effects of human TERT on irradiation-induced late rectal injury in fibroblasts under hypoxic conditions. We also performed in vivo experiments. The rectums of 5-week-old female C57BL/6N mice were irradiated locally with a single dose of 25 Gy. We then examined the fibrotic changes using hematoxylin and eosin staining, and Masson's staining. The expression of hypoxia inducible factor-1α (HIF-1α) and TERT was analyzed by immunohistochemistry. In in vitro experiments, apoptosis, reactive oxygen species (ROS) production and the autophagy level induced by exposure to hypoxia were assayed in fibroblasts. The association between TERT, nuclear factor-κB (NF-κB) and the autophagy level was examined by western blot analysis. The antioxidant effects of TERT were examined on the basis of the ratio of glutathione to glutathione disulfide (GSH/GSSG) and mitochondrial membrane potential. Rectal fibrosis was induced significantly at 12 weeks following irradiation. The HIF-1α and TERT expression levels increased in the fibrotic region. The TERT-overexpressing fibroblasts (transfected with an hTERT-expressing lentiviral vector) exhibited reduced apoptosis, reduced ROS production, a higher autophagy level, a higher GSH/GSSG ratio and stable mitochondrial membrane potential compared with the fibroblasts in which TERT had been silenced by siRNA. NF-κB was activated by TERT, and the inhibition of TERT reduced the autophagy level in the fibroblasts. These results demonstrate that TERT decreases cellular ROS production, while maintaining mitochondrial function and protecting the cells from hypoxia-induced apoptosis, which may thus attenuate the effects of irradiation-induced hypoxia on rectal injury following irradiation.

The neuroprotective role and mechanisms of TERT in neurons with oxygen–glucose deprivation

Telomerase reverse transcriptase (TERT) is reported to protect neurons from apoptosis induced by various stresses including hypoxia–ischemia (HI). However, the mechanisms by which TERT exerts its anti-apoptotic role in neurons with HI injury remain unclear. In this study, we examined the protective role and explored the possible mechanisms of TERT in neurons with HI injury in vitro. Primary cultured neurons were exposed to oxygen and glucose deprivation (OGD) for 3h followed by reperfusion to mimic HI injury in vivo. Plasmids containing TERT antisense, sense nucleotides, or mock were transduced into neurons at 48h before OGD. Expression and distribution of TERT were measured by immunofluorescence labeling and western blot. The expression of cleaved caspase 3 (CC3), Bcl-2 and Bax were detected by western blot. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The mitochondrial reactive oxygen species (ROS) were measured by MitoSOX Red staining. Fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (ΔΨm). We found that TERT expression increased at 8h and peaked at 24h in neurons after OGD. CC3 expression and neuronal apoptosis were induced and peaked at 24h after OGD. TERT inhibition significantly increased CC3 expression and neuronal apoptosis after OGD treatment. Additionally, TERT inhibition decreased the expression ratio of Bcl-2/Bax, and enhanced ROS production and ΔΨm dissipation after OGD. These data suggest that TERT plays a neuroprotective role via anti-apoptosis in neurons after OGD. The underlying mechanisms may be associated with regulating Bcl-2/Bax expression ratio, attenuating ROS generation, and increasing mitochondrial membrane potential.

In silico evaluation of TERT inhibition by anticancer drugs

The activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant tumors suggests that telomerase inactivation may represent a potential chemotherapeutic target. In this work, existing anticancer drugs were docked against telomerase reverse transcriptase (TERT) using a Lamarckian genetic algorithm (LGA). Autodock's scoring function was applied to each of the molecules in order to identify the inhibitor with the strongest pharmacological action. The structural insights provided by this study regarding binding poses and possible interactions, free energies of binding, and drug scores aided in the identification of potential inhibitory compounds. The ranks of the various ligands investigated were based on the final docked energy values. Among nine selected compounds, vindesine, temsirolimus, and cyclosporine were found to be more potent TERT inhibitors than the standard inhibitor, curcumin.

Telomerase Reverse Transcriptase Upregulation Attenuates Astrocyte Proliferation and Promotes Neuronal Survival in the Hypoxic–Ischemic Rat Brain

Telomerase reverse transcriptase (TERT) is tightly related to the resistance of cells to stress and injury. However, little is known about the roles of TERT in the nervous system. We try to investigate the effects of TERT on the function of astrocytes in developing rat brains subjected to hypoxia-ischemia. TERT expression was detected in rat brains with hypoxia-ischemia. In in vitro study, the function of astrocytes with TERT overexpression was measured, and the effects of astrocyte on neuronal apoptosis were examined. Moreover, overexpression or inhibition of TERT was conducted in vivo by gene transduction. Astrocyte proliferation was examined through Ki67 staining. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining and brain infarct volume calculation were used to detect neuronal injury. Both TERT mRNA and protein were upregulated in neurons within 2 days but shifted to astrocytes at Day 3 after hypoxia-ischemia. Astrocyte proliferation was inhibited with TERT overexpression due to the upregulation of cell-cycle regulatory protein p15. Meanwhile, the apoptosis of neurons increased, whereas neurons were cocultured with conditioned media from astrocytes with TERT inhibition compared with TERT overexpression due to the decrease of neurotrophin-3 expression in astrocytes. Furthermore, Ki67-positive astrocytes and neuronal injury were found to be inhibited in TERT-overexpressing rat brains with hypoxia-ischemia. TERT attenuates astrocyte proliferation and promotes neuronal survival in the developing rat brain after hypoxia-ischemia, partly through its enhancement of p15 and neurotrophin-3 expression in astrocytes.

Correlation of dyskerin expression with active proliferation independent of telomerase

Dyskerin, which is an important component of the telomerase complex and is needed for normal telomerase activity, is frequently overexpressed in neoplasia. Dyskerin also plays an essential role in ribosome biogenesis. Because protein synthesis increases during tumorigenesis, this led us to hypothesize that dyskerin expression would be upregulated independently of the cell immortalization mechanism. Dyskerin and telomerase reverse transcriptase (TERT) expression were examined in oral squamous cell carcinomas (OSCC) and patient-matched controls, as well as in a panel of telomerase-positive and telomerase-negative cells. Antisense inhibition of TERT was used to test the effects of downregulation of telomerase on dyskerin expression. Dyskerin was frequently overexpressed in OSCC and in immortalized and transformed keratinocytes relative to primary cells, independently of TERT and telomerase activity. Instead, dyskerin expression strongly correlated with cell proliferation rates. The role of dyskerin in tumorigenesis does not correlate with its function within the telomerase complex.