Catalytic Subunit Of Telomerase Research Articles

Telomerase Reverse Transcriptase Prevents Doxorubicin-induced Microvascular Endothelial Dysfunction

Introduction & Objective: Doxorubicin (Dox) is a frequently used chemotherapeutic known to induce cardiovascular (CV) complications by impairing microvascular- and cardiac function. We previously submitted an abstract showing that Dox-induced microvascular dysfunction preceded Dox-induced cardiac dysfunction and thus microvascular dysfunction might contribute to the damage of the heart itself.>Microvascular function specifically relies upon the adequate release of the vasodilator nitric oxide (NO). Our previous work showed that Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase, is necessary for physiological release of NO to mediate vasodilation. NO suppresses reactive oxygen species (ROS) levels. While Dox correlates with increased ROS production and suppressed TERT activity, attenuated TERT activity may explain the elevated levels of ROS after Dox exposure. The objective of this study was to determine whether TERT transcriptional activation is suffcient to protect against Dox-induced microvascular endothelial- and cardiac dysfunction. We utilized cycloastragenol (CAG) as transcriptional activator. Hypothesis: We hypothesized that TERT transcriptional activation is suffcient to prevent or attenuate Dox-induced endothelial dysfunction. Methods: WT or TERT loss-of-function rats on a Sprague Dawley background were assigned to untreated control-, CAG-, Dox and CAG+Dox groups. Animals were ~14 weeks of age to start the six-week protocol. Dox and CAG+Dox animals received one weekly IP Dox-injection (3x 4mg/kg*BW). CAG administer via diet with 50-60mg of CAG/day*BW. Microvascular function was assessed six-weeks after the first dose of Dox via videomicroscopy. Vasodilator responses to flow (flow-mediated dilation; FMD) and mediators of dilation (NO, H2O2) via fluorescent probes were evaluated. Cardiac function was evaluated using echocardiography. Human microvessels were treated either with 100nM Dox or 1μM CAG or a combination of both. Results: Dox treatment resulted in reduction in FMD in both WT and TERT mutant animals. Smooth-muscle dependent vasodilation was not impaired indicating endothelial impairment. CAG prevented Dox-induced endothelial dysfunction in WT but not TERT mutant rats. AGS499 another activator of TERT showed similar results suggestion a TERT mediated phenotype. Dox-treated WT animals exhibited an increase in H2O2 but not NO production. This phenotype was opposite in CAG+Dox WT animals. TERT mutant animals showed an increase in H2O2 but not NO production even without Dox. To assess which vasodilator mediates dilation to flow, we used L-NAME and PEG-Catalase to block NO- and H2O2, respectively. In CAG+Dox WT animals, L-NAME blocked the vasodilatory response to flow, while PEG-Cat blocked the vasodilatory response non-significantly. Similarly, we observed this protective effect of TERT in the human microcirculation. Human adipose microvessels treated over-night with Dox showed no vasodilatory response to flow while CAG+Dox treated vessels dilated normally similar to untreated control vessels. Smooth muscle function was not affected in neither group. Conclusion: We show evidence that TERT prevents Dox-induced endothelial dysfunction in an in vivo model and using human tissue. We highlight the specificity to TERT by a) using a genetic model lacking normal TERT activity and b) showing similar results using two different compounds which have been shown to activate TERT/promote TERT transcriptional activation. Ultimately, the present data shows that CAG is a potential therapeutic to counteract the detrimental effects of Dox treatment in the CV system via TERT signaling. RO1 HL133029-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB096: Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma

Abstract Impaired telomere length (TL) maintenance in ovarian tissue may play a pivotal role in epithelial ovarian carcinoma (OvC) onset. TL in either target or surrogate tissue (blood) is currently being investigated as a predictor in anti-OvC therapy or as a biomarker of the disease progression, respectively. There is presently an urgent need for an appropriate approach to chemotherapy response prediction. We performed a monochrome multiplex qPCR measurement of TL in peripheral blood and tumor tissues of 209 OvC patients. Methylation status and gene expression of shelterin complex and telomerase catalytic subunit (hTERT) were determined within tumor tissues by High-Throughput DNA methylation profiling and RNA sequencing (RNA-Seq) analysis, respectively. The patients sensitive to cancer treatment (n=46) had shorter telomeres in peripheral blood cells compared to those treatment-resistant (n=93; P=0.037). Cancer tissue TL lower than the median predisposed to better overall survival (P=0.002). Gene expression of TPP1, which recruits telomerase to telomeres, was positively associated with TL in tumor tissue (P=0.026). TL measured in peripheral blood cells could serve as a marker of platinum therapy response in OvC patients. Additionally, TL determined in tumor tissue provides information on OvC patients' overall survival. The study was financed by the project NPO (LX22NPO05102), the Czech Health Research Council (NU21-03-00145), and the Czech Science Foundation (21-27902S). Citation Format: Kristyna Tomasova, Karolina Seborova, Michal Kroupa, Josef Horak, Ludmila Vodickova, Lukas Rob, Martin Hruda, Marcela Mrhalova, Alena Bartakova, Jiri Bouda, Thomas Fleischer, Vessela Kristensen, Pavel Vodicka, Radka Vaclavikova. Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB096.

Telomere maintenance mechanisms in neuroblastoma: New insights and translational implications

The clinical behaviour of neuroblastoma ranges from spontaneous tumour regression or maturation into benign ganglioneuroma to fatal progression despite intensive treatment [1,2]. The mechanisms underlying the distinct phenotypes have remained uncertain yet. A number of recent studies noticed that activation of telomere maintenance mechanisms (TMM) in the malignant neuroblasts is strongly associated with high-risk disease and poor outcome, whereas TMM are absent in spontaneously regressing low-risk tumours, suggesting that telomere maintenance is essential to drive neuroblasts into the fully malignant state. Stabilization of the telomeres above a critical threshold is essential for cancer cells to gain replicative immortality and has thus been considered a hallmark of cancer [3]. In most high-risk neuroblastomas, TMM is conferred by induction of telomerase, either through genomic rearrangements of the TERT locus, encoding for the catalytic subunit of telomerase, or amplification of the MYCN proto-oncogene, whereas the alternative lengthening of telomeres (ALT) pathway is activated in approximately one-quarter of high-risk tumours [4–8]. We here review recent advances on our understanding of the mechanistic role of telomere maintenance in neuroblastoma pathogenesis, and discuss potential implications on neuroblastoma risk assessment and on the development of novel therapies directed against TMM.

MTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

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 mutations in atypical melanocytic lesions: A series of seven cases with adverse melanoma-specific outcome

The majority of melanocytic proliferations can be readily categorized as benign or malignant based on histologic assessment under the microscope by a trained dermatopathologist. However, a subset of lesions, termed Atypical Melanocytic Proliferations (AMPs), are histologically ambiguous, leading to possible diagnostic error and suboptimal treatment. Mutations in the promoter region of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), are commonly found in melanomas but are rare in melanocytic nevi. In this study, we aimed to determine the prevalence of hot spot TERT promoter (TERT-p) mutations in AMPs with adverse melanoma-specific outcome. Studies were approved by respective institutional review boards. Using a multi-center database, we identified seven cases of melanocytic proliferations with a clinical follow-up period of at least 4 years, which were initially diagnosed as AMPs, and which recurred either as melanoma at site of prior biopsy or as metastatic melanoma. Sequencing of the TERT-p region showed hotspot mutations in three cases (43 %), suggesting that TERT-p mutations are enriched and could aid in the identification of AMPs with adverse outcome. In comparison with existing ancillary techniques for prognostication of AMPs, TERT-p mutation analysis may have advantages in terms of cost effectiveness and turnaround time, and is a promising diagnostic parameter with potential widespread utility.

Characterization of novel mutations in the TEL-patch domain of the telomeric factor TPP1 associated with telomere biology disorders.

Telomeres are nucleoprotein structures that protect the chromosome ends from degradation and fusion. Telomerase is a ribonucleoprotein complex essential to maintain the length of telomeres. Germline defects that lead to short and/or dysfunctional telomeres cause telomere biology disorders (TBDs), a group of rare and heterogeneous Mendelian diseases including pulmonary fibrosis, dyskeratosis congenita, and Høyeraal-Hreidarsson syndrome. TPP1, a telomeric factor encoded by the gene ACD, recruits telomerase at telomere and stimulates its activity via its TEL-patch domain that directly interacts with TERT, the catalytic subunit of telomerase. TBDs due to TPP1 deficiency have been reported only in 11 individuals. We here report four unrelated individuals with a wide spectrum of TBD manifestations carrying either heterozygous or homozygous ACD variants consisting in the recurrent and previously described in-frame deletion of K170 (K170∆) and three novel missense mutations G179D, L184R, and E215V. Structural and functional analyses demonstrated that the four variants affect the TEL-patch domain of TPP1 and impair telomerase activity. In addition, we identified in the ACD gene several motifs associated with small deletion hotspots that could explain the recurrence of the K170∆ mutation. Finally, we detected in a subset of blood cells from one patient, a somatic TERT promoter-activating mutation that likely provides a selective advantage over non-modified cells, a phenomenon known as indirect somatic genetic rescue. Together, our results broaden the genetic and clinical spectrum of TPP1 deficiency and specify new residues in the TEL-patch domain that are crucial for length maintenance and stability of human telomeres in vivo.

Dynamics of TERT regulation via alternative splicing in stem cells and cancer cells.

Part of the regulation of telomerase activity includes the alternative splicing (AS) of the catalytic subunit telomerase reverse transcriptase (TERT). Although a therapeutic window for telomerase/TERT inhibition exists between cancer cells and somatic cells, stem cells express TERT and rely on telomerase activity for physiological replacement of cells. Therefore, identifying differences in TERT regulation between stem cells and cancer cells is essential for developing telomerase inhibition-based cancer therapies that reduce damage to stem cells. In this study, we measured TERT splice variant expression and telomerase activity in induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and non-small cell lung cancer cells (NSCLC, Calu-6 cells). We observed that a NOVA1-PTBP1-PTBP2 axis regulates TERT alternative splicing (AS) in iPSCs and their differentiation into NPCs. We also found that splice-switching of TERT, which regulates telomerase activity, is induced by different cell densities in stem cells but not cancer cells. Lastly, we identified cell type-specific splicing factors that regulate TERT AS. Overall, our findings represent an important step forward in understanding the regulation of TERT AS in stem cells and cancer cells.

Electroacupuncture pretreatment preserves telomerase reverse transcriptase function and alleviates postoperative cognitive dysfunction by suppressing oxidative stress and neuroinflammation in aged mice.

Elderly patients often exhibit postoperative cognitive dysfunction (POCD), a postsurgical decline in memory and executive function. Oxidative stress and neuroinflammation, both pathological characteristics of the aged brain, contribute to this decline. This study posits that electroacupuncture (EA) stimulation, an effective antioxidant and anti-inflammatory modality, may enhance telomerase reverse transcriptase (TERT) function, the catalytic subunit of telomerase known for its protective properties against cellular senescence and oxidative damage, to alleviate POCD in aged mice. The animal POCD model was created by subjecting aged mice to abdominal surgery, followed by EA pretreatment at the Baihui acupoint (GV20). Postoperative cognitive function was gauged using the Morris water maze (MWM) test. Hippocampal TERT mRNA levels and telomerase activity were determined through qPCR and a Telomerase PCR ELISA kit, respectively. Oxidative stress was assessed through superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) levels. Iba-1 immunostaining determined the quantity of hippocampal microglia. Additionally, western blotting assessed TERT, autophagy markers, and proinflammatory cytokines at the protein level. Abdominal surgery in aged mice significantly decreased telomerase activity and TERT mRNA and protein levels, but increased oxidative stress and neuroinflammation and decreased autophagy in the hippocampus. EA-pretreated mice demonstrated improved postoperative cognitive performance, enhanced telomerase activity, increased TERT protein expression, improved TERT mitochondrial localization, and reduced oxidative damage, autophagy dysfunction, and neuroinflammation. The neuroprotective benefits of EA pretreatment were diminished following TERT knockdown. Our findings underscore the significance of TERT function preservation in alleviating surgery-induced oxidative stress and neuroinflammation in aged mice. Anovel neuroprotective mechanism of EA stimulation is highlighted, whereby modulation of TERT and telomerase activity reduces oxidative damage and neuroinflammation. Consequently, maintaining TERT function via EA treatment could serve as an effective strategy for managing POCD in elderly patients.

Orchestrating nucleic acid-protein interactions at chromosome ends: telomerase mechanisms come into focus.

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA-DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein-nucleic acid interactions that potentiate telomerase's unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase 'anchor site' proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein-protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.

Zeolitic Imidazolate Framework-8 (ZIF-8) as a Drug Delivery Vehicle for the Transport and Release of Telomerase Inhibitor BIBR 1532.

Telomerase is constitutively overexpressed in the majority of human cancers and telomerase inhibition provides a promising broad-spectrum anticancer therapeutic strategy. BIBR 1532 is a well-known synthetic telomerase inhibitor that blocks the enzymatic activity of hTERT, the catalytic subunit of telomerase. However, water insolubility of BIBR 1532 leads to low cellular uptake and inadequate delivery and thus, limits its anti-tumor effects. Zeolitic imidazolate framework-8 (ZIF-8) is considered as an attractive drug delivery vehicle for improved transport, release and anti-tumor effects of BIBR 1532. Herein, ZIF-8 and BIBR 1532@ZIF-8 were synthesized, respectively, and the physicochemical characterizations confirmed the successful encapsulation of BIBR 1532 in ZIF-8 coupled with an improved stability of BIBR 1532. ZIF-8 could alter the permeability of lysosomal membrane probably by the imidazole ring-dependent protonation. Moreover, ZIF-8 encapsulation facilitated the cellular uptake and release of BIBR 1532 with more accumulation in the nucleus. BIBR 1532 encapsulation with ZIF-8 triggered a more obvious growth inhibition of cancer cells as compared with free BIBR 1532. A more potent inhibition on hTERT mRNA expression, aggravated G0/G1 arrest accompanied with an increased cellular senescence were detected in BIBR 1532@ZIF-8-treated cancer cells. Our work has provided preliminary information on improving the transport, release and efficacy of water-insoluble small molecule drugs by using ZIF-8 as a delivery vehicle.

Telomerase deficiency and dysfunctional telomeres in the lung tumor microenvironment impair tumor progression in NSCLC mouse models and patient-derived xenografts

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death. Tumor progression depends on interactions of cancer cells with the tumor microenvironment. Here, we find increased copy number and mRNA expression of the catalytic subunit of telomerase, TERT, in tumors from NSCLC patients, contributing to a lower survival. Moreover, TERT expression in NSCLC patients from the TCGA cohort is mainly associated to the reduced infiltration of CD8+ T lymphocytes, as well as to increased infiltration of myeloid-derived suppressor cells (MDSCs). We also show that TERT deficiency and dysfunctional telomeres induced by 6-thio-dG treatment in mice reduced lung tumor implantation and vascularization, increased DNA damage response, cell cycle arrest and apoptosis, as well as reduced proliferation, inflammation, lung tumor immunosupression and invasion upon induction of a Lewis lung carcinoma (LLC). Furthermore, 6-thio-dG-treated human NSCLC xenografts exhibited increased telomere damage, cell cycle arrest and apoptosis, as well as reduced proliferation, resulting in a reduced tumor growth. Our results show that targeting telomeres might be an effective therapeutic strategy in NSCLC.

Telomerase RNA in Hymenoptera (Insecta) switched to plant/ciliate-like biogenesis.

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.

TERT silencing alters the expression of ARG1, GLUL, VIM, NES genes and hsa-miR-29b-3p in the T98G cell line

The central function of telomerase is maintaining the telomere length. However, several extra-telomeric roles have been identified for this protein complex. In this study, we evaluated the effect of the silencing of the catalytic subunit of telomerase (TERT) on the expression of candidate microRNAs, cell activation markers and glial-related genes in a glioblastoma cell line (T98G). The silencing was performed by a siRNA and the qPCR method was used to analyze the expression of TERT and downstream genes. Flow cytometry was used to quantify the TERT protein, and bioinformatics analysis was carried out to analyze the functions of microRNA target genes. Here, it was observed that after a 50% reduction of the TERT gene, the expression of ARG1 (Arginase 1) was upregulated, whereas NES (Nestin), GLUL (Glutamate-Ammonia Ligase), VIM (Vimentin) and the hsa-miR-29b-3p microRNA were downregulated (P-value <0.05). A bioinformatic analysis showed that target genes of hsa-miR-29b are associated with focal adhesion, the PI3K-Akt signaling pathway, among others. These results are important because they contribute to the knowledge of extratelomeric functions by providing relevant evidence about novel genes modulated by TERT.

The Molecular Interplay between Human Oncoviruses and Telomerase in Cancer Development.

Human oncoviruses are able to subvert telomerase function in cancer cells through multiple strategies. The activity of the catalytic subunit of telomerase (TERT) is universally enhanced in virus-related cancers. Viral oncoproteins, such as high-risk human papillomavirus (HPV) E6, Epstein-Barr virus (EBV) LMP1, Kaposi's sarcoma-associated herpesvirus (HHV-8) LANA, hepatitis B virus (HBV) HBVx, hepatitis C virus (HCV) core protein and human T-cell leukemia virus-1 (HTLV-1) Tax protein, interact with regulatory elements in the infected cells and contribute to the transcriptional activation of TERT gene. Specifically, viral oncoproteins have been shown to bind TERT promoter, to induce post-transcriptional alterations of TERT mRNA and to cause epigenetic modifications, which have important effects on the regulation of telomeric and extra-telomeric functions of the telomerase. Other viruses, such as herpesviruses, operate by integrating their genomes within the telomeres or by inducing alternative lengthening of telomeres (ALT) in non-ALT cells. In this review, we recapitulate on recent findings on virus-telomerase/telomeres interplay and the importance of TERT-related oncogenic pathways activated by cancer-causing viruses.

Successive Detection of Telomerase by IHC and Assessment of Telomere Length by Q-FISH in Paucicellular Cumulus Samples from Cumulus-Oocyte Complexes Obtained in Assisted Reproduction Programs

This paper suggests an approach for the use of a single paucicellular histological sample to investigate two characteristics indicative of the cell’s functional potential: 1) the content of telomerase reverse transcriptase (TERT) and 2) the telomere length. An algorithm has been suggested for the successive detection of the catalytic telomerase subunit with immunohistochemical staining and assessment of telomere length with Q-FISH on the same set of cells. The described approach uses three incontestable advantages of the FISH technique, namely, the possibility of analyzing target sequences in individual cells and paucicellular samples, the performance of several successive hybridization rounds with different FISH probes on the same sample, and the examination of genomic regions consisting of repeats. This approach has been applied to assess the TERT content and telomere length in cumulus cells from human cumulus-oocyte complexes obtained from assisted reproduction programs. This approach provides an opportunity to examine the correlation between the TERT content and telomere length in cumulus cells surrounding the oocyte and the oocyte's capacity for fertilization, as well as the subsequent pre- and post-implantation development of the resulting embryo.

Establishment and Characterization of hTERT Immortalized Hutchinson-Gilford Progeria Fibroblast Cell Lines.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients’ fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50–100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes.

Noncanonical Role of Telomerase in Regulation of Microvascular Redox Environment With Implications for Coronary Artery Disease.

Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and β-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.

Abstract PL03-02: Somatic mutations, germline risk and ancestry in lung adenocarcinoma

Abstract Like most cancer types, lung cancer is caused predominantly by somatic genome alterations. In lung adenocarcinoma, the most common histological type of lung cancer, these alterations are known to occur in multiple pathways including the p53 pathway, the receptor tyrosine kinase (RTK)/Ras/Raf/MAP kinase (MAPK) pathway and PI3 kinase pathway, the Rb1/cell cycle pathway, the Swi/Snf pathway, the Myc pathway, and the telomerase pathway, among others. Genome-wide association studies comparing polymorphisms in germline DNA from patients with lung cancer and from unaffected control subjects have led to the identification of multiple germline risk alleles for lung adenocarcinoma, including the HLA locus, the telomerase catalytic subunit gene locus, and, notably, the nicotinic acetylcholine receptor locus, where polymorphisms appear to modulate individual propensity for smoking. The components of inherited lung cancer risk, particularly in nonsmokers, are not fully understood, but there appear to be variations in lung adenocarcinoma mutations in distinct ancestry groups, which may be related to inherited factors. In particular, the frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity. Somatic EGFR mutation rates are higher in lung cancers from patients with East Asian ancestry and lower in lung cancers from patients with European or African ancestry. Somatic KRAS and STK11 mutation rates show the opposite pattern, higher in lung cancers from patients with European or African ancestry and lower in lung cancers from patients with East Asian ancestry. In patients from Latin America, somatic lung cancer EGFR mutation frequencies vary by country, with higher frequencies in Peru, intermediate frequencies in Mexico, Colombia, Costa Rica and Brazil, and lower frequencies in Argentina. To explore the relationship between the somatic genome in lung cancer and ethnicity related germline risk, we performed cancer gene panel sequencing of DNA from over 1,000 lung cancers from Mexico and Colombia, countries with significant population admixture. We developed methods to infer ancestry from the tumor DNA sequence based on coverage of single nucleotide polymorphisms. This study revealed associations between ancestry and somatic lung cancer genomic alterations, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local ancestry score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics, rather than environmental exposure, could underlie these disparities. Our study suggests that the variation in EGFR and KRAS somatic mutation frequency in lung cancer is associated with genetic ancestry in patients from Latin America, and suggests further studies to identify germline alleles that underpin this association. If we find a germline locus or loci that could impact the development of lung cancers with EGFR and/or KRAS mutation, this might help in improving lung cancer prevention and screening for populations of Latin American origin and others. Furthermore, multiple studies now highlight the importance of EGFR mutation screening and EGFR-directed targeted therapy for lung cancer patients in Latin America and with origins in Latin America. Citation Format: Matthew L. Meyerson. Somatic mutations, germline risk and ancestry in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr PL03-02.

Landscape analysis of urothelial carcinoma (UC) by telomerase reverse transcriptase (TERT) alterations.

4524 Background: TERT is a catalytic subunit of telomerase, the unique enzyme that confers immortality to cells and is expressed in &gt;90% of cancer cells. Mutations in the TERT promoter region (pTERTmut) are the most prevalent noncoding mutations in cancer. Recent data suggest pTERTmut are associated with improved outcomes in patients with UC treated with immune checkpoint inhibitors. We evaluated the molecular and immune landscape of UC with and without pTERTmut. Methods: UC tissue samples were analyzed for DNA alterations (NextSeq, 592 Genes; NovaSeq, WES) and mRNA expression (NovaSeq, WTS). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). PD-L1 expression was assessed by immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by fragment analysis, IHC and NGS. TMB-H was based on a cut-off of &gt; 10 mut/MB. We compared alterations between samples with and without detected pathogenic pTERTmut. Significance was determined by Mann–Whitney U, X2, and Fischer-Exact and p adjusted for multiple comparisons (q) was &lt; 0.05 using Benjamini-Hochberg. Results: Overall, 1686 UC samples were analyzed, 1166 from primary lesions and 499 from a lymph node or metastatic site. pTERTmut was present in 68% of primary and 61% of metastatic tumors, and correlated with modest increase of TERT expression (1.18 fold, p=0.015). pTERTmut was associated with less frequent alterations in TP53, KMT2D, CCND1, MYC, KEAP1 and less MSI/dMMR. By contrast, pTERTmut was associated with more frequent alterations in ARID1A, TSC1, PIK3CA and TMB-H (all q&lt;0.05). Over 41% of pTERTmut were TMB-H. TERTp mutations were not associated with FGFR alterations. The frequency of co-occurring mutations was similar by specimen site. In evaluating the immune landscape (Table), pTERTmut was associated with higher expression of PD-L1 (IHC, mRNA), PD-L2 (mRNA) and TIM3 (mRNA) in tumors from primary sites (all p and q&lt;0.05), but not in metastatic sites. Investigation of tumor-associated immune cells demonstrated that pTERTmut correlated with higher percentage of M1-macrophages and CD8+ T cells in primary tumors, and was inversely-correlated with NK cells in metastatic sites (all q&lt;0.05). Conclusions: This is the largest analysis looking at the molecular and immune landscape of pTERTmut UC tumors. We observed differential patterns of DNA alterations and tumor immune microenvironment based on pTERTmut status. Further work is needed to understand differences in these molecular cohorts and the association of these data with clinical outcomes. [Table: see text]