Telomere Maintenance Mechanisms Research Articles

Pan-cancer analysis of telomere maintenance mechanisms.

Telomeres, protective caps at chromosome ends, maintain genomic stability and control cell lifespan. Dysregulated telomere maintenance mechanisms (TMM) are cancer hallmarks, enabling unchecked cell proliferation. We conducted a pan-cancer evaluation of TMM using RNA sequencing data from The Cancer Genome Atlas (TCGA) for 33 different cancer types and analyzed the activities of telomerase-dependent (TEL) and alternative lengthening of telomeres (ALT) TMM pathways in detail. To further characterize the TMM profiles, we categorized the tumors based on their ALT and TEL TMM pathway activities into five major phenotypes: ALT high TEL low, ALT low TEL low, ALT middle TEL middle, ALT high TEL high, and ALT low TEL high. These phenotypes refer to variations in telomere maintenance strategies, shedding light on the heterogeneous nature of telomere regulation in cancer. Moreover, we investigated the clinical implications of TMM phenotypes by examining their associations with clinical characteristics and patient outcomes. Specific TMM profiles were linked to specific survival patterns, emphasizing the potential of TMM profiling as a prognostic indicator and aiding in personalized cancer treatment strategies. Gene ontology analysis of the TMM phenotypes unveiled enriched biological processes associated with cell cycle regulation (both TEL and ALT), DNA replication (TEL), and chromosome dynamics (ALT) showing that telomere maintenance is tightly intertwined with cellular processes governing proliferation and genomic stability. Overall, our study provides an overview of the complexity of transcriptional regulation of telomere maintenance mechanisms in cancer.

Extrachromosomal telomere DNA derived from excessive strand displacements

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

Abstract LB185: High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing

Abstract Telomere length is an important biomarker of cellular replicative potential and aging, but existing measurement methods are limited in resolution and accuracy. During human carcinogenesis, telomeres shorten profoundly in the earliest stages that precede tumor invasion. Telomere shortening can either halt cancer progression by inducing senescence and cell death or can promote cancer development by destabilizing chromosomes and generating chromosomal rearrangements. Stabilization of telomeres, typically by the upregulation of telomerase, is required for progression of nearly all human cancers. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change during aging and following carcinogenesis. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human stem cells, in cells from the peripheral blood of healthy individuals and patients with genetic defects in telomere maintenance, cancer patients, and cancer cell lines. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres in white blood cells. In patients with inherited defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. Next, we investigated the telomere lengths of a cohort of twenty colorectal tumors with patient-matched healthy colonic epithelia and found that two-thirds of colorectal tumors have telomere length distributions significantly shorter than the surrounding normal tissue. Finally, we explore the utility of telomere length distributions in cancer cell lines as clinically actionable disease biomarkers. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease. Citation Format: Santiago Sanchez, Jessica Gu, Anudeep Golla, William Shomali, Annika Martin, Dirk Hockemeyer, Sharon Savage, Steven Artandi. High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing [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 LB185.

Telomere-lengthening germline variants predispose to a syndromic papillary thyroid cancer subtype

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. 10% to 15% of individuals show familial clustering with three or more affected members, but the factors underlying this risk are unknown. In a group of recently studied individuals with POT1 pathogenic variants and ultra-long telomere length, PTC was the second most common solid tumor. We tested whether variants in POT1 and four other telomere-maintenance genes associated with familial cancer underlie PTC susceptibility. Among 470 individuals, we identified pathogenic or likely pathogenic variants in three genes encoding telomere-binding proteins: POT1, TINF2, and ACD. They were found in 4.5% and 1.5% of familial and unselected cases, respectively. Individuals harboring these variants had ultra-long telomere length, and 15 of 18 (83%) developed other cancers, of which melanoma, lymphoma, and sarcoma were most common. Among individuals with PTC and melanoma, 22% carried a deleterious germline variant, suggesting that a long-telomere syndrome might be clinically recognizable. Successive generations had longer telomere length than their parents and, at times, developed more cancers at younger ages. Tumor sequencing identified a single oncogenic driver, BRAF p.Val600Glu, in 10 of 10 tumors studied, but no telomere-maintenance mechanism, including at the TERT promoter. These data identify a syndromic subset of PTCs with locusheterogeneity and telomere lengthening as a convergent mechanism. They suggest these germline variants lower the threshold to cancer by obviating the need for an acquired telomere-maintenance mechanism in addition to sustaining the longevity of oncogenic mutations.

Abstract 2995: The ectopic expression of miR-380-5p interferes with the aggressive traits of diffuse malignant peritoneal mesothelima cells

Abstract Telomerase activity (TA) and the alternative lengthening of telomere (ALT) pathway are two telomere maintenance mechanisms (TMM) yet identified in human cancers. Although the activation of either TMM seems to be equivalent in supporting tumor cell immortalization (a hallmark of cancer), the contribution of TA and ALT to tumor progression and aggressiveness may be different. Mounting evidence suggests that cancer cell aggressiveness could be associated with a TA↔ALT shift that would allow the acquisition of distinct aggressive traits. We previously showed that an ALT-like phenotype emerged in diffuse malignant peritoneal mesothelioma (DMPM) cells upon miR-380-5p-dependent inhibition of TA (Cimino-Reale G, et al. J Hematol Oncol. 2017). To analyze the transcriptomic changes associated with the emergence of such a phenotype, gene expression profiling was carried out. Results showed that 3331 genes were differentially expressed (FDR<0.05, adjusted for multiple hypothesis testing) in miR-380-5p- vs. preNeg-transfected DMPM cells, out of which 1673 genes were up-regulated and 1658 genes down-regulated. To get biological meaning about the molecular events occurring upon miR-380-5p reconstitution, Gene Set Enrichment Analysis was performed. Results showed a positive enrichment of 188 gene sets, restricted for an FDR < 0.05, out of which signatures related to DNA replication, DNA recombination and telomere maintenance via recombination were present. Conversely, only 1 gene set (GO: positive regulation of blood vessel endothelial cell migration) was negatively enriched at significant level. Notably, among the leading edge genes, PTGS2, which encodes for cyclooxygenase-2 (COX-2) and represents a predicted target of miR-380-5p (miRWalk), was the most significantly down-regulated gene (FDR< 0.000001; log(fold-change): -2.15). Notably, a complete abrogation of PTGS2 expression levels and COX-2 protein amounts was observed in miR-380-5p- compared to preNeg-transfected cells upon validation by qRT-PCR and western blotting, respectively. This evidence was paralleled by reduced amounts of phospho-FAK (focal adhesion kinase) and impaired migrating/invading capabilities of cells ectopically expressing miR-380-5p. Moreover, a reduction of Vimentin and a slight increase in P-Cadherin protein levels were also observed in cells ectopically expressing miR-380-5p, likely indicating the occurrence of a reverse-EMT program. Finally, a pronounced increase in the sensitivity to Cisplatin and Doxorubicin was also appreciable in miR-380-5p- vs. preNeg-transfected cells, suggesting that miRNA-mediated induction of a reverse-EMT may, at least in part, contribute to improve the response of DMPM cells to chemotherapeutic drugs. Citation Format: Stefano Percio, Lorenzo Di Pietro, Eisa Naghshineh, Marcello Deraco, Nadia Zaffaroni, Marco Folini. The ectopic expression of miR-380-5p interferes with the aggressive traits of diffuse malignant peritoneal mesothelima cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2995.

Abstract 418: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

Abstract The Bloom Syndrome helicase (BLM) is critical for Alternative Lengthening of Telomeres (ALT), a homology directed repair mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT positive cells to assemble a replication stress associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-lagging strand flap formation and ALT in a BLM dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, single-stranded nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM directed HDR mechanism that sustains telomere length in a subset of human cancers. Citation Format: Aravind M. Krishnan, Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Youngho Kwon, Patrick Sung, Roger A. Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 418.

Abstract 1639: Telomere dynamics in aging and cancer by nanopore long-read sequencing

Abstract Telomeres are the protective, nucleoprotein structure at the ends of linear eukaryotic chromosomes. The accurate measurement of both telomeric length and composition of individual telomeres in mammalian cells has been challenged by the length and repetitive nature of telomeres. With the advent of third generation sequencing technologies, it is now technically possible to sequence entire telomeres and map them to individual chromosome arms. Here, we report a reliable method to enrich, sequence and analyze human telomeres using Oxford Nanopore Technologies long-read sequencing. To enrich for telomeric sequences we combine the ligation of adapters complementary to the telomeric G-overhang with restriction enzyme digest to sequence the telomeric C-strand and part of the adjacent subtelomere. The subtelomeric information is harvested to map individual telomeric reads to specific chromosome arms and even alleles. We have measured bulk, chromosome-arm-specific telomere length dynamics during cellular aging of cultured primary cells and in a patient-derived aging cohort. To address the impact of the telomere maintenance mechanism on telomere length and composition, we have sequenced matched pairs of fibroblasts and induced pluripotent stem cells, as well as five well-established telomerase- and ALT-positive cancer cell lines. Our results suggest that based on nanopore telomere long-read sequencing ALT-positive cells can be easily discriminated from normal and telomerase-positive cancer cells. Further, telomere sequencing allows to evaluate the methylation status of the subtelomeric CpG islands adjacent to telomeres. In summary, nanopore telomere long-read sequencing allows to measure the length and composition of individual telomeres and their mapping to specific chromosome arms. Telomere long read sequencing methods will be valuable tools to study telomere biology during aging and cancer. Citation Format: Tobias T. Schmidt, Carly Tyer, Preeyesh Rughani, Candy Haggblom, Jeff Jones, Xiaoguang Dai, Kelly A. Frazer, Fred H. Gage, Sissel Juul, Scott Hickey, Jan Karlseder. Telomere dynamics in aging and cancer by nanopore long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1639.

RNA analysis of the longest living vertebrate Greenland shark revealed an abundance of LINE-like elements in its transcriptome

The Greenland shark (Somniosus microcephalus) is an enigmatic species known for its exceptional longevity and extraordinary adaptations to the cold environment. This animal lives in the arctic and subarctic regions of the North Atlantic Ocean. Surprisingly, even though it is a vertebrate with the longest known lifespan, its transcriptome has not been studied yet. Therefore, we isolated and analyzed RNA in the Greenland shark samples. Our findings reveal some important information about the possible genetic mechanisms that could contribute to its longevity. We identified a highly expressed long interspersed nuclear element-like transcript (LINE-like) that is supposed to be associated with extended lifespan and resilience to age-related diseases, possibly through an improved telomere maintenance mechanism. This research not only contributes to our understanding of the biology and evolution of the Greenland shark but could also have implications for human longevity research.

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.

Mechanisms of telomere maintenance and associated therapeutic vulnerabilities in malignant gliomas.

A majority of cancers (~85%) activate the enzyme telomerase to maintain telomere length over multiple rounds of cellular division. Telomerase-negative cancers activate a distinct, telomerase-independent mechanism of telomere maintenance termed Alternative lengthening of telomeres (ALT). ALT uses homologous recombination to maintain telomere length and exhibits features of break-induced DNA replication. In malignant gliomas, the activation of either telomerase or ALT is nearly ubiquitous in pediatric and adult tumors, and the frequency with which these distinct telomere maintenance mechanisms is activated varies according to genetically-defined glioma subtypes. In this review, we summarize the current state of the field of telomere maintenance mechanisms (TMMs) and their relevance to glioma biology and therapy. We review the genetic alterations and molecular mechanisms leading to telomerase activation or ALT induction in pediatric and adult gliomas. With this background, we review emerging evidence on strategies for targeting TMMs for glioma therapy. Finally, we comment on critical gaps and issues for moving the field forward to translate our improved understanding of glioma telomere maintenance into better therapeutic strategies for patients.

Effects of p53 and ATRX inhibition on telomeric recombination in aging fibroblasts

In order to avoid replicative senescence, tumor cells must acquire a telomere maintenance mechanism. Beside telomerase activation, a minority of tumors employs a recombinational mechanism called Alternative Lengthening of Telomeres (ALT). Several studies have investigated the potential ALT stimulation by inactivation of ATRX in tumor cells, obtaining contrasting results. Differently, since ALT can be viewed as a mechanism to overcome telomere shortening-mediated replicative senescence, we have investigated the effects of the inhibition of ATRX and p53 in aging primary fibroblasts. We observed that senescence leads to a phenotype that seems permissive for ALT activity, i.e. high levels of ALT-associated PML bodies (APB), telomeric damage and telomeric cohesion. On the other hand, RAD51 is highly repressed and thus telomeric recombination, upon which the ALT machinery relies, is almost absent. Silencing of ATRX greatly increases telomeric recombination in young cells, but is not able to overcome senescence-induced repression of homologous recombination. Conversely, inhibition of both p53 and ATRX leads to a phenotype reminiscent of some aspects of ALT activity, with a further increase of APB, a decrease of telomere shortening (and increased proliferation) and, above all, an increase of telomeric recombination.

Abstract B019: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

Abstract Alternative Lengthening of Telomeres (ALT) is a homology-directed repair (HDR) mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. ALT cells exhibit more endogenous replication stress and DNA damage at the telomere compared to ALT-negative cells. The Bloom Syndrome helicase (BLM) is critical for ALT and promotes replication stress and DNA damage specifically at telomeres in ALT cells. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication stress-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-flap formation in a BLM-dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers (Jiang et al. submitted). These findings will be discussed and how BLM activity is a key determinant of the specific vulnerability of ALT cells to FANCM inhibition, which is currently being developed to target ALT-dependent cancers. Citation Format: Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Aravind Krishnan, Youngho Kwon, Patrick Sung, Roger Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B019.

Abstract B025: Exceptional response to the ataxia telangiectasia and Rad3-related inhibitor (ATRi), camonsertib, in a patient with alternative lengthening of telomeres (ALT)-positive metastatic melanoma

Abstract One hallmark of cancer tumorigenesis is the maintenance of telomere length which canonically occurs through the reactivation of telomerase. Alternative lengthening of telomeres (ALT) is a minor telomere maintenance mechanism that uses homologous recombination (HR) to maintain telomere length and is associated with replication stress and defects in genome maintenance. In preclinical models, ALT positivity (ALT+) has been shown to sensitize tumor cells to ataxia telangiectasia and Rad3-related inhibitors (ATRi). Here we describe a confirmed clinical and molecular response to the ATRi, camonsertib, in a patient with ALT+ metastatic melanoma. To our knowledge, this is the first report of clinical experience with ATRi in ALT+ tumors. A 63-year-old male was diagnosed in July 2019 with stage IV (cTX, cNX, pM1c[1]) metastatic melanoma and treated with nivolumab (nivo) + ipilumumab (ipi) with initial partial response (PR) documented after 3 months which was confirmed on subsequent nivo maintenance. Ipi was resumed following suspected pseudoprogression in March 2020, and continued until confirmed progression in June 2020. Genomic profiling of a liver biopsy collected at the time of progression identified somatic alterations in TP53 (p.S127F, p.R65fs), NF1 (p.K1263*), BRCA2 (p.R2482C) and ATRX (p.R418*), a germline CHEK2 mutation (p.T376fs), and high tumor mutational burden. The patient received additional targeted regimens with olaparib (ola): nivo ± ola and later ola ± trametinib; no response was documented, and progression was noted in April 2021. In June 2021, the patient received camonsertib monotherapy at an initial dose of 120 mg QD 3 days on/4 days off in the phase I TRESR trial (NCT04497116). The patient tolerated treatment well and continued at the same dose until dose was reduced via a change in schedule (to 2 weeks on/1 week off) at 9 months due to anemia. The patient had an initial RECIST PR (-36%) at 3 months with circulating tumor DNA (ctDNA) clearance. The target lesions continued to decrease with a best decline of -74% after about 1 year of treatment. The cancer progressed at 19 months (+105% from nadir, -47% from baseline) though due to ongoing clinical benefit treatment was continued for a total duration of 22 months. Interrogation of ctDNA tumor fraction in longitudinal plasma samples is ongoing and will be reported. To understand the long and deep response to camonsertib treatment in this patient, we further characterized the screening biopsy by a custom targeted sequencing panel, SyNthetic lethal Interactions for Precision Diagnostics (SNiPDx), and whole genome sequencing (WGS). Both the BRCA2 and gCHEK2 mutations were monoallelic and genomic signature analysis defined the tumor as HR-proficient, consistent with the lack of benefit from ola. WGS confirmed a biallelic loss of ATRX with a genomic signature consistent with an ALT+ phenotype. The clinical experience of ATRi therapy in patients with ALT+ tumors is limited but further investigation of ATRi such as camonsertib is warranted in this patient population. Citation Format: Timothy A. Yap, Ian M. Silverman, Adrienne Johnson, Chenfeng Meng, Joseph D. Schonhoft, Michal Zimmermann, Danielle Ulanet, Victoria Rimkunas, Maria Koehler. Exceptional response to the ataxia telangiectasia and Rad3-related inhibitor (ATRi), camonsertib, in a patient with alternative lengthening of telomeres (ALT)-positive metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B025.

Abstract PR010: Understanding extrachromosomal telomere generation in ALT cancers

Abstract Introduction: Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of Cytosine-rich (CCCTAA) sequences. These C-circles are unique biomarkers of ALT cells and may be generated from extrachromosomal telomeric single-stranded DNAs (ssDNAs), also unique to ALT cells, but the generation process remains undefined. Methods and results: In order to investigate C-rich ssDNA, we developed a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both telomeric ssDNAs and C-circles are abundant in cytoplasm and nucleoplasm fractions, which supports the hypothesis that telomeric ssDNA accumulation may indeed precede C-circle formation. We also found that telomeric ssDNAs originate during Okazaki fragment processing during C-rich lagging strand DNA synthesis at telomere ends during telomere elongation in ALT cancers. During lagging strand priming and synthesis, the CST-PP complex (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) primes the telomeric C-rich strand; then, Polymerase delta and BLM helicase facilitate excessive strand displacement, leading to the accumulation of telomeric ssDNAs in the nucleoplasm and cytoplasm which is potential precursor material for C-circles in ALT cancers. Conclusion: Here, we introduce a highly sensitive 4SET method for studying telomeric ssDNA, which we used to propose a new model for the generation of telomeric C-rich ssDNAs and C-circles during ALT-mediated telomere elongation. Our work contributes to a growing body of knowledge pertaining to the idiosyncrasies of the ALT mechanism based on investigation of its unique biomarkers. Citation Format: Junyeop Lee, Jina Lee, Eric J. Sohn, Angelo Taglialatela, Roderick J. O’Sullivan, Alberto Ciccia, Jaewon Min. Understanding extrachromosomal telomere generation in ALT cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR010.

The ALT pathway generates telomere fusions that can be detected in the blood of cancer patients

Telomere fusions (TFs) can trigger the accumulation of oncogenic alterations leading to malignant transformation and drug resistance. Despite their relevance in tumour evolution, our understanding of the patterns and consequences of TFs in human cancers remains limited. Here, we characterize the rates and spectrum of somatic TFs across >30 cancer types using whole-genome sequencing data. TFs are pervasive in human tumours with rates varying markedly across and within cancer types. In addition to end-to-end fusions, we find patterns of TFs that we mechanistically link to the activity of the alternative lengthening of telomeres (ALT) pathway. We show that TFs can be detected in the blood of cancer patients, which enables cancer detection with high specificity and sensitivity even for early-stage tumours and cancers of high unmet clinical need. Overall, we report a genomic footprint that enables characterization of the telomere maintenance mechanism of tumours and liquid biopsy analysis.

Vorinostat, temozolomide or bevacizumab with irradiation and maintenance BEV/TMZ in pediatric high-grade glioma: A Children's Oncology Group Study.

Outcomes for children with high-grade gliomas (HGG) remain poor. This multicenter phase II trial evaluated whether concurrent use of vorinostat or bevacizumab with focal radiotherapy (RT) improved 1-year event-free survival (EFS) compared to temozolomide in children with newly diagnosed HGG who received maintenance temozolomide and bevacizumab. Patients ≥ 3 and < 22 years with localized, non-brainstem HGG were randomized to receive RT (dose 54-59.4Gy) with vorinostat, temozolomide, or bevacizumab followed by 12 cycles of bevacizumab and temozolomide maintenance therapy. Among 90 patients randomized, the 1-year EFS for concurrent bevacizumab, vorinostat, or temozolomide with RT was 43.8% (±8.8%), 41.4% (±9.2%), and 59.3% (±9.5%), respectively, with no significant difference among treatment arms. Three- and five-year EFS for the entire cohort was 14.8% and 13.4%, respectively, with no significant EFS difference among the chemoradiotherapy arms. IDH mutations were associated with more favorable EFS (P = .03), whereas H3.3 K27M mutations (P = .0045) and alterations in PIK3CA or PTEN (P = .025) were associated with worse outcomes. Patients with telomerase- and alternative lengthening of telomeres (ALT)-negative tumors (n = 4) had an EFS of 100%, significantly greater than those with ALT or telomerase, or both (P = .002). While there was no difference in outcomes based on TERT expression, high TERC expression was associated with inferior survival independent of the telomere maintenance mechanism (P = .0012). Chemoradiotherapy with vorinostat or bevacizumab is not superior to temozolomide in children with newly diagnosed HGG. Patients with telomerase- and ALT-negative tumors had higher EFS suggesting that, if reproduced, mechanism of telomere maintenance should be considered in molecular-risk stratification in future studies.

Is serum-derived exosomal hTERT transcript a marker of oncogenic activity in primary brain tumors? An exploratory study.

In order to proliferate indefinitely, all tumors require a telomere maintenance mechanism. The expression of human telomerase reverse transcriptase (hTERT) enables telomere maintenance and provides cancer cells with limitless replicative potential. As such, it may serve as an attractive biomarker for oncogenic activity. This study explored whether a liquid biopsy that analyses blood derived exosomal hTERT transcript (e-hTERT-trans) may serve as such a biomarker in gliomas and meningiomas when compared to healthy controls. Exosomes were isolated from the pre-operative sera of patients' samples stored in the biobank of both Rabin and Sheba Medical Centers. The levels of e-hTERT-trans were measured in 81 healthy controls, 117 meningiomas, 17 low-grade gliomas, and 61 glioblastomas. Clinical parameters of the patients were collected retrospectively and compared to the levels of the e-hTERT-trans. The upper normal limit of controls e-hTERT-trans was 1.85 relative quantitation (RQ). The rate of detection increased with rising tumor grade and correlated with tumor recurrence in meningiomas: mean RQ without recurrence (2.17 ± 11.7) versus with recurrence (3.59 ± 4.42; p = 0.002). In glioblastomas, preoperative measurements correlated with tumor volume and with the disease course on serial sampling. We demonstrated for the first time that the expression of e-hTERT-trans transcript can be measured in the serum of primary brain tumors. This exosomal marker carries the potential to serve as a biomarker once used in conjunction with other clinical and radiological parameters. Future studies are required to investigate whether the sensitivity could be augmented and whether it can be implemented into routine patients care.

Telomere dynamics in human pluripotent stem cells

ABSTRACT Pluripotent stem cells (PSCs) are a promising source of stem cells for regenerative therapies. Stem cell function depends on telomere maintenance mechanisms that provide them with the proliferative capacity and genome stability necessary to multiply and regenerate tissues. We show here that established human embryonic stem cells (hESCs) have stable telomere length that is dependent on telomerase but not on alternative mechanisms based on homologous recombination pathways. Here, we show that human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar to ESCs. hiPSCs also acquire telomeric chromatin marks of ESCs including decreased abundance of tri-methylated histone H3K9 and H4K20 and HP1 heterochromatic marks, as well as of the shelterin component TRF2. These chromatin features are accompanied with increased abundance of telomere transcripts or TERRAs. We also found that telomeres of both hESCs and hiPSCs are well protected from DNA damage during telomere elongation and once full telomere length is achieved, and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hPSCs).

Telomere Maintenance Mechanisms in a Cohort of High-Risk Neuroblastoma Tumors and Its Relation to Genomic Variants in the TERT and ATRX Genes.

Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.