Enzyme Telomerase Research Articles

Cell Immortalization: In Vivo Molecular Bases and In Vitro Techniques for Obtention.

Somatic human cells can divide a finite number of times, a phenomenon known as the Hayflick limit. It is based on the progressive erosion of the telomeric ends each time the cell completes a replicative cycle. Given this problem, researchers need cell lines that do not enter the senescence phase after a certain number of divisions. In this way, more lasting studies can be carried out over time and avoid the tedious work involved in performing cell passes to fresh media. However, some cells have a high replicative potential, such as embryonic stem cells and cancer cells. To accomplish this, these cells express the enzyme telomerase or activate the mechanisms of alternative telomere elongation, which favors the maintenance of the length of their stable telomeres. Researchers have been able to develop cell immortalization technology by studying the cellular and molecular bases of both mechanisms and the genes involved in the control of the cell cycle. Through it, cells with infinite replicative capacity are obtained. To obtain them, viral oncogenes/oncoproteins, myc genes, ectopic expression of telomerase, and the manipulation of genes that regulate the cell cycle, such as p53 and Rb, have been used.

Cancer-associated SMARCAL1 loss-of-function mutations promote alternative lengthening of telomeres and tumorigenesis in telomerase-negative glioblastoma cells.

Telomere maintenance mechanisms are required to enable the replicative immortality of malignant cells. While most cancers activate the enzyme telomerase, a subset of cancers uses telomerase-independent mechanisms termed alternative lengthening of telomeres (ALT). ALT occurs via homology-directed-repair mechanisms and is frequently associated with ATRX mutations. We previously showed that a subset of adult glioblastoma (GBM) patients with ATRX-expressing ALT-positive tumors harbored loss-of-function mutations in the SMARCAL1 gene, which encodes an annealing helicase involved in replication fork remodeling and the resolution of replication stress. However, the causative relationship between SMARCAL1 deficiency, tumorigenesis, and de novo telomere synthesis is not understood. We used a patient-derived ALT-positive GBM cell line with native SMARCAL1 deficiency to investigate the role of SMARCAL1 in ALT-mediated de novo telomere synthesis, replication stress, and gliomagenesis in vivo. Inducible rescue of SMARCAL1 expression suppresses ALT indicators and inhibits de novo telomere synthesis in GBM and osteosarcoma cells, suggesting that SMARCAL1 deficiency plays a functional role in ALT induction in cancers that natively lack SMARCAL1 function. SMARCAL1-deficient ALT-positive cells can be serially propagated in vivo in the absence of detectable telomerase activity, demonstrating that the SMARCAL1-deficient ALT phenotype maintains telomeres in a manner that promotes tumorigenesis. SMARCAL1 deficiency is permissive to ALT and promotes gliomagenesis. Inducible rescue of SMARCAL1 in ALT-positive cell lines permits the dynamic modulation of ALT activity, which will be valuable for future studies aimed at understanding the mechanisms of ALT and identifying novel anticancer therapeutics that target the ALT phenotype.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

Epigenetic changes of Arabidopsis MET1 cytosine methyltransferase mutants under salt stress

In this study, we investigated the morphological and molecular responses of Arabidopsis met1-7 and met1-3 mutants under salinity stress. Global DNA methylation changes of mutants were compared and genes known to be involved in DNA methylation in plants were analyzed. Also, the expression of the telomerase reverse transcriptase (TERT) gene as a stress response indicator was analyzed in order to examine the effects of stress on the telomerase enzyme in the mutants. We found that mutants have a higher rate of hypomethylation than Col-0, under all conditions. According to the qPCR analysis, expression levels of Domains Rearranged Methylase 2 (DRM2), Polymerase IV (Pol IV) and Polymerase V (Pol V) genes were higher in mutants than Col-0 plants. Contrary to expectations for the TERT gene, there was an increase in expression in the stress-applied mutant plants. Taken together, results suggest that the RNA-directed DNA methylation (RdDM) pathway is enhanced in mutants in order to deal with the lack of DNA methylation in CG sites. In conclusion, we believe that the morphological and molecular data obtained regarding the effects of NaCl application to met1 mutants will help us to understand the epigenetic basis of stress mechanisms.

The effects of metformın, acetylsalıcylıc acıd and ıbuprofen on telomerase enzyme actıvıty: ınhıbıtory effect of ıbuprofen

Telomerase enzyme is necessary for the elongation of telomeres while telomerase being critical for aging and cancer. Metformin, ibuprofen, and acetylsalicylic acid used in this research are drugs that millions of people already use and that many are likely to use in future. In this study, the effects of these drugs on telomerase activity of Mus musculus swiss albino mice in liver tissue were investigated and the telomerase activity was measured with a PCR-ELISA based kit. In the study a possible connection between telomerase enzyme activity and activities of antioxidant enzymes was also investigated by determining the activity of superoxide dismutase (SOD) and catalase enzymes. The data obtained show that metformin slightly decreased telomerase enzyme activity in low dose application; however, this change was not statistically significant. In ibuprofen application, there was a significant inhibitory effect when high doses were used; whereas, there was a slight inhibitory effect at low doses. In acetylsalicylic acid application, a slight activator effect was detected; it was not statistically significant, though. Metformin was observed to increase catalase and SOD activities in general while low and high doses of acetyl salicylic acid showed different effects. In addition, ibuprofen caused a statistically significant increase in liver SOD values. It is important to note that this study demonstrated a significant inhibitory effect of ibuprofen on telomerase enzyme activity in animal models..

Potential Anti-Cancer Effect of Helenalin as a Natural Bioactive Compound on the Growth and Telomerase Gene Expression in Breast Cancer Cell Line.

The telomerase gene is overexpressed in the majority of tumors and cancers compared to normal and healthy cells, and on the other hand, this enzymatic protein is overactive, therefore, the telomerase enzyme is considered a primary target for diagnostic and therapeutic purposes in most cancers. This has been hypothesized that Helenalin has anti-telomerase activity in a wide range of cancers and Tumor tissues. In this study, we investigated the inhibitory effect of helenalin extract on telomerase gene expression in the T47D breast cancer cell line. We used the MTT assay to evaluate the cytotoxic effect of different concentrations of helenalin on the T47D breast cancer cell line at 24, 48, and 72 hours. Besides, the expression of the hTERT gene in T47D cell lines treated with 1.0 and 5.0 µM helenalin after 24, 48, and 72 h incubation times was investigated through real-time PCR. According to the MTT assay, the inhibitory effect of helenalin on T47D cell proliferation is time and dose-dependent. Moreover, the results of Real-time PCR showed that exposure of T47D cell lines to helenalin led to a significant Decreasing in the expressional values of the hTERT gene as a time and dose-dependent procedure compared with the control group (P ≤ 0.05). These preliminary results demonstrated the cytotoxic potential of helenalin through inhibition of hTERT against T47D breast cancer cells.

Telomerase Inhibition by MST-312 Sensitizes Breast Cancer Cells to the Anti-cancer Properties of Plumbagin.

Breast cancer is the most common cause of malignancy and the second most common cause of death due to cancer in women. This heterogeneous disease is currently broadly classified as estrogen receptor (ER), progesterone receptor (PR) positive luminal tumors, human epidermal growth factor receptor 2 (HER2) amplified tumors and triple-negative breast cancers (TNBC). Phytochemicals are proven to be promising anti-cancer chemotherapeutics agents with minimal cytotoxic effects on normal cells. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a phytochemical derived from the roots of Plumbago zeylanica and it is known to possess anti-cancer properties similar to other compounds of naphthoquinones. In about 90% of cancer cells, the telomerase enzyme activity is revived to add telomeric repeats to evade apoptosis. In this study, a combinatorial approach of combining the anti-cancer compound plumbagin to induce genotoxicity and a potent telomerase inhibitor, MST-312 (synthetic derivative of tea catechins), was used to determine the combinational treatment-induced lethality in breast cancer cells such as MDA-MB-231 (TNBC) and MCF-7 (lumina) cells. MDA-MB-231 cells were responsive to combination treatment in both short-term (48 h) and long-term treatment (14 days) in a synergistic manner, whereas in MCF-7, the combination treatment was more effective in the long-term regimen. Furthermore, the cytotoxic effects of the plumbagin and MST-312 combination treatment were not recoverable after the short-term treatment. In conclusion, a combination treatment of MST-312 and plumbagin is proven to be more effective than a single plumbagin compound treatment in inducing DNA damage and telomere dysfunction leading to greater genome instability, cell cycle arrest and eventually cell death in cancer cells.

Kadmiyum toksisitesi oluşturulan ratlarda arı sütünün farklı dozlarının oksidatif stres ve telomeraz enzimi üzerine etkileri

Purpose: Cadmium (Cd) is a toxic metal that seriously threatens human health due to environmental pollution, is widely used in industry and agriculture, and causes oxidative stress and tissue damage. This study aims to examine the effect of royal jelly (RJ) on oxidative status and telomerase enzyme activity in tissue damage induced by Cd.
 Materials and Methods: The experimental design was made with 6 rats in each group. A total of 6 groups were created: control group, Cd group, 250 mg/kg RJ group, Cd + 250 mg/kg RJ group, 400 mg/kg RJ group, Cd + 400 mg/kg RJ group. In the study, total oxidant status and total antioxidant status in blood serum were investigated by colorimetric method, and telomerase enzyme activity in ovarian tissue was investigated by ELISA method.
 Results: Cd caused an increase in oxidative capacity (23.80 ± 2.4) and a significant decrease was determined after RJ applications compared to the control group. After RJ application, the best total antioxidant response was observed in the 250 mg/kg RJ and Cd + 250 mg/kg RJ groups. Cd significantly reduced telomerase enzyme activity (0.90 ± 0.13). RJ administered for treatment after Cd application increased telomerase levels up to the control level (1.40 ± 0.05). The best treatment response was observed in the Cd + 250 mg/kg RJ group (1.42 ± 0.05).
 Conclusion: Cd causes oxidative stress and that RJ may have curative effects by increasing the antioxidant capacity and telomerase enzyme activity RJ is a promising natural product and can contribute to recovery.

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS).

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. Many ALT features are conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA TERRA, and its ability to form RNA-DNA hybrids, has been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. We observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to 'normal' senescence, we report that RNA-DNA hybrids slow the rate of SAS, likely through the elongation of critically short telomeres, however decreasing the rate of telomere shortening may contribute to this effect. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence before and after survivor formation.

Telomere, Telomerase and The Aging Heart

BackgroundAging is a physiological process in all populations with time and finally leads to cellular senescence and organ dysfunction. Telomere's primary function is the protection of DNA with the help of the telomerase enzyme. Telomerase activity declines with time, and with each cell division, telomere length decreases. ObjectiveTo review the association between short telomere and aging-related heart diseases. MethodsThe previous literature was narratively reviewed. Results and ConclusionsOverview of telomere length and cardiovascular diseases. Unique variations of telomere length are affected by genetic and non‐genetic factors. Genetic and environmental factors cause individual variation in telomere length (TL). A healthy lifestyle as a healthy diet and physical activity maintains TL, while hypertension, diabetes, atherosclerosis, Hepatitis C virus, and human immune deficiency virus are cardiovascular risk factors that increase oxidative stress, induce endothelial inflammation, and accelerate telomere shortening. Telomere shorting plays an essential role in the pathogenesis of aging-associated heart diseases. Short telomeres cause cellular senescence and dysfunction, contributing to atherogenesis and decreasing repair and regenerative capacity in the cardiovascular system. Telomere and telomerase are associated with heart aging. However, Telomerase and telomere-associated proteins, which regulate TL, may participate in the therapeutic strategies for ischemic heart diseases and heart failure.

Virtual screening and drug repositioning of FDA-approved drugs from the ZINC database to identify the potential hTERT inhibitors.

The length of the telomeres is maintained with the help of the enzyme telomerase constituting of two components, namely, a core reverse transcriptase protein (hTERT) and RNA (hTR). It serves as a significant and universal cancer target. In silico approaches play a crucial role in accelerating drug development processes, especially cancer drug repurposing is an attractive approach. The current study is aimed at the repurposing of FDA-approved drugs for their potential role as hTERT inhibitors. Accordingly, a library of 2,915 sets of FDA-approved drugs was generated from the ZINC database in order to screen for novel hTERT inhibitors; later on, these were subjected to molecular docking analysis. The top two hits, ZINC03784182 and ZINC01530694, were shortlisted for molecular dynamic simulation studies at 100 ns based on their binding scores. The RMSD, RMSF, Rg, SASA, and interaction energies were calculated for a 100-ns simulation period. The hit compounds were also analyzed for antitumor activity, and the results revealed promising cytotoxic activities of these compounds. The study has revealed the potential application of these drugs as antitumor agents that can be useful in treating cancer and can serve as lead compounds for further in vivo, in vitro, and clinical studies.

Probing the Efficiency of 13-Pyridylalkyl Berberine Derivatives to Human Telomeric G-Quadruplexes Binding: Spectroscopic, Solid State and In Silico Analysis.

The interaction between the series of berberine derivatives 1-5 (NAX071, NAX120, NAX075, NAX077 and NAX079) and human telomeric G-quadruplexes (G4), which are able to inhibit the Telomerase enzyme's activity in malignant cells, was investigated. The derivatives bear a pyridine moiety connected by a hydrocarbon linker of varying length (n = 1-5, with n number of aliphatic carbon atoms) to the C13 position of the parent berberine. As for the G4s, both bimolecular 5'-TAGGGTTAGGGT-3' (Tel12) and monomolecular 5'-TAGGGTTAGGGTTAGGGTTAGGG-3' (Tel23) DNA oligonucleotides were considered. Spectrophotometric titrations, melting tests, X-ray diffraction solid state analysis and in silico molecular dynamics (MD) simulations were used to describe the different systems. The results were compared in search of structure-activity relationships. The analysis pointed out the formation of 1:1 complexes between Tel12 and all ligands, whereas both 1:1 and 2:1 ligand/G4 stoichiometries were found for the adduct formed by NAX071 (n = 1). Tel12, with tetrads free from the hindrance by the loop, showed a higher affinity. The details of the different binding geometries were discussed, highlighting the importance of H-bonds given by the berberine benzodioxole group and a correlation between the strength of binding and the hydrocarbon linker length. Theoretical (MD) and experimental (X-ray) structural studies evidence the possibility for the berberine core to interact with one or both G4 strands, depending on the constraints given by the linker length, thus affecting the G4 stabilization effect.

Identification of the Telomere elongation Mutation in Drosophila.

Telomeres in Drosophila melanogaster, which have inspired a large part of Sergio Pimpinelli work, are similar to those of other eukaryotes in terms of their function. Yet, their length maintenance relies on the transposition of the specialized retrotransposons Het-A, TART, and TAHRE, rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Our previous work has led to the isolation of a dominant mutation, Tel1, that caused a several-fold elongation of telomeres. In this study, we molecularly identified the Tel1 mutation by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel1 to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel1 to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel1 telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.

The Role of Telomerase in Breast Cancer's Response to Therapy.

Currently, breast cancer appears to be the most widespread cancer in the world and the most common cause of cancer deaths. This specific type of cancer affects women in both developed and developing countries. Prevention and early diagnosis are very important factors for good prognosis. A characteristic feature of cancer cells is the ability of unlimited cell division, which makes them immortal. Telomeres, which are shortened with each cell division in normal cells, are rebuilt in cancer cells by the enzyme telomerase, which is expressed in more than 85% of cancers (up to 100% of adenocarcinomas, including breast cancer). Telomerase may have different functions that are related to telomeres or unrelated. It has been shown that high activity of the enzyme in cancer cells is associated with poor cell sensitivity to therapies. Therefore, telomerase has become a potential target for cancer therapies. The low efficacy of therapies has resulted in the search for new combined and more effective therapeutic methods, including the involvement of telomerase inhibitors and telomerase-targeted immunotherapy.

Anticancer activity, hTERT expression and telomere length analysis in SH-SY5Y neuroblastoma cell lines applied to docetaxel

Neuroblastoma is the most common extracranial solid tumor of infancy in a broad range of clinical courses, ranging from spontaneous regression to fatal progression. Telomere maintenance plays an important role in genome stability and cell proliferation. Telomerase reverse transcriptase (hTERT in humans) is a catalytic subunit of the enzyme telomerase. In this study, it was aimed to determine the anticancer activity of the docetaxel chemotherapeutic agent in neuroblastoma cell line (SH-SY5Y) and to investigate its effect on hTERT gene expression level and telomere length. Molecular docking studies were performed on docetaxel with the crystal structure of telomerase. The electronic properties of docetaxel were calculated using the density functional theory (DFT) method. SH-SY5Y cells were treated for 24, 48 and 72 h with specific concentrations of docetaxel drug ranging from 1 to 100 µg/ml. IC50 doses of docetaxel were determined and administered to SH-SY5Y cells, followed by RNA isolation. hTERT and MYC gene expression levels and telomere length were measured in the docetaxel-treated sample using the RT-PCR method. In addition, theoretical analyzes were made. The IC50 values of docetaxel after 24, 48 and 72 h were 8.32±1.45 μg/ml, 7.67±2.56 μg/ml and 5.51±1.24 μg/ml, respectively. According to the results obtained, docetaxel was found to have the highest activity in 72 h of incubation. It was determined that the docetaxel drug decreased the expression level of the hTERT gene in SH-SY5Y cells. Telomere lengths were significantly reduced in the docetaxel treated SH-SY5Y cell line compared to the control group (p < 0.05). Molecular docking analysis results were in agreement with the experiments. Analysis results indicated a good interaction between docetaxel and the active site of telomerase. The results of this study, reinforced by molecular docking analyzes, might be proved valuable for the development of potent telomerase inhibitors.

Telomerase enzyme activity in patients with major depressive disorder: A pre and post-treatment study

BackgroundTelomerase is a cellular enzyme that prevents telomere shortening and promoting viability. The literature has reported shortened telomere length in patients with major depressive disorder (MDD). Methods35 patients with diagnosis of major depressive disorder (MDD) fulfilling DMS-5 criteria in the age range of 18–60 years, treatment-naïve after assessing the severity on HAM-D and HAM-A and 35 age and sex matched healthy controls were included in the study. Baseline peripheral blood monocytes (PBMC) telomerase enzyme was assessed in cases and controls and repeated in cases of MDD at 8th week after intervention with escitalopram for 8 weeks. ResultsPretreatment telomerase activity (TA) was elevated in cases as compared to controls and it was also significantly correlated to the severity of depression (p = 0.00). There was a significant positive difference in telomerase activity between non-responders (higher TA) and responders at baseline (p = 0.001) and 8th week (p = 0.012). The TA did not vary significantly amidst pretreatment and post-treatment, although it was slightly lower in the post-treatment group. LimitationsThe study has few limitations in the form of small sample size, shorter duration of follow-up, and leucocyte telomeres length (LTL) was not assessed. ConclusionThe index study concludes that TA is higher in drug naïve patients with MDD than age and sex matched healthy control. The non-responders had significantly higher TA as compared to responders at baseline and post-treatment which indicates TA as a potential biomarker in the underlying biological mechanism of MDD and in response to antidepressant pharmacotherapy.

The effect of some trace elements on the expression of telomerase gene in lung cancer

Lung cancer is a disorder that begins due to genetic and epigenetic changes. These changes cause the activation of oncogenes and the inactivation of tumor suppressor genes. Various factors influence the expression of these genes. In this research, we investigated the relationship between the number of trace elements zinc and copper and the ratio of these two in serum with the expression of the telomerase enzyme gene in lung cancer. For this purpose, we included 50 people with lung cancer in the study as the case group and 20 patients with non-tumor lung diseases as the control group. The TRAP assay method measured the telomerase activity in biopsy samples of lung tumor tissue. Also, serum copper and zinc were measured by atomic absorption spectrometry. The results showed that the mean serum concentration of copper and the ratio of copper to zinc in patients were significantly higher than in the controls (120.8 ± 5.7 versus 107.2 ± 6.5μg/dL)(P<0.05). However, there is no significant difference in the mean serum concentration of zinc between the two groups (p > 0.05). The patients' mean ratio of copper to zinc is significantly higher than the control group (1.6 ± 0.4 versus 1.1 ± 0.2)(p < 0.05). The average level of telomerase enzyme activity of patients showed a significant difference from the control group 32.8 ± 16.1 vs. zero percent) (p < 0.01). There is a direct and significant correlation between the serum level of copper and the level of telomerase enzyme activity in patients (r = 0.36 and p < 0.05). A positive correlation was observed between total serum copper concentration and the increasing age of patients (r = 0.39 and p < 0.01). The correlation between the ratio of copper to zinc and the serum copper concentration of the patients was positive and significant (r = 0.36 and p < 0.05), but the correlation between the amount of serum zinc and the ratio of copper to zinc was negative and significant (r = -0/ 72 and p < 0.01). The average copper serum concentration of people with small cell carcinoma (123.7 ± 2.8 μg/dL) compared to non-small cell carcinoma (117.6 ± 4.8 μg/dL) is high and significant (P< 0.05). Patients with small cell carcinoma had a high mean telomerase concentration (112 ± 0.57%) and significantly compared to non-small cell carcinomas (6.4 ± 2.5%)(P < 0.05). This situation regarding the zinc element and the ratio of copper to zinc in non-small cell carcinomas compared to small cell carcinomas were evaluated as non-significant (P>0.05). Based on the obtained results, it can be assumed that determining the amount of zinc and copper and the telomerase enzyme activity in lung cancer can have a biological role in the initiation and progression of tumor tissue, which requires more studies.

TGF-β controls stromal telomere length through epigenetic modifications.

The online version contains supplementary material available at 10.1007/s13205-022-03346-5.

Abstract A09: Telomerase activity as a biomarker to predict outcomes of CAR T-cell therapy

Abstract Introduction: Chimeric antigen receptor T-cells (CAR T-cells) and T-cells in general, remain in a quiescent state when unstimulated, showing no proliferative activity. In contrast, upon specific antigenic stimulation, CAR T-cells rapidly divide and kill their target cancer cells. However, one of the immunologic changes with aging is the progressive impairment of T-cell responses. This physiologic immunosenescence is associated with the shortening of telomeres. The telomerase enzyme is transiently upregulated in stimulated CAR T-cells preventing dramatic telomere shortening during rapid CAR T-cell division. Our previous study showed that during T-cell expansion, the period during which telomerase is active and the magnitude of its activity correlates with the donor’s age and overall health status (“healthy aging”) [1]. The aim of this study is to investigate whether the magnitude of telomerase activity in stimulated CAR T-cells is a predictive biomarker of outcomes in patients receiving CAR T-cell therapy.Methods: The study population comprises of older adult patients (age range: 50-84 years) receiving autologous anti-CD19 CAR T-cell therapy for relapsed or refractory diffuse large B-cell lymphoma, mantle cell lymphoma or follicular lymphoma. Peripheral blood samples are collected from patients pre-lymphodepletion and T-cells isolated from PBMCs (peripheral blood mononuclear cells). CAR T-cells are then generated from patient T-cells in the lab and telomerase activity analyzed over 2 weeks in culture. For each patient’s CAR T-cells, absolute values of telomerase activity are plotted to calculate the area under the curve (AUC). We are investigating the potential correlation of AUC of telomerase activity to disease response at 30- and 90-days post CAR T-cell infusion. Results: Preliminary data from eight patients is showing a clear correlation between telomerase activity and patient outcomes. Robust telomerase activity shows a positive correlation with complete responses on day 30 PET-CT scans and sustained clinical remission, while poor telomerase activity correlates with no response or progressive disease. We are expanding our study sample to 30-50 patients and updated data will be presented at the meeting. Conclusions: CAR T-cell therapy has been a game-changer for hematological malignancies; however therapeutic resistance and disease relapse remains a major concern. Understanding the mechanisms of resistance to CAR T-cell therapy is critical for advancing the field. Telomerase activity levels could be a potential predictive biomarker for CAR T-cell therapy outcomes and aid in personalizing treatment choices. Furthermore, future research on increasing telomerase activity may enhance CAR T-cell therapy function and ultimately patient outcomes. [1] Tedone et.al, Telomere length and telomerase activity in T cells are biomarkers of high-performing centenarians, Aging Cell, 2019 Citation Format: Mohammed E Sayed, Enzo Tedone, Emily Zhang, Helen Stephens, Farrukh T Awan, Jerry Shay, Praveen Ramakrishnan Geethakumari. Telomerase activity as a biomarker to predict outcomes of CAR T-cell therapy [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A09.

P02.01.B The telomere maintenance mechanism spectrum and its dynamics in gliomas

Abstract The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation indicates alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Here, we show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined in the dichotomy of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation and ATRX mutation. Moreover, we observed that a considerable proportion of gliomas lack both telomerase activity and ALT (Neither group). And this Neither group exhibited evidence of slow growth potential. From a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed but changes with glioma progression. Collectively, these results suggest that the TMM is a dynamic entity and that reflects the plasticity of the oncogenic biological status of tumor cells and that the TMM should be defined by the direct measurement of telomerase enzyme activity and evidence of ALT.