Alternative Lengthening Of Telomeres Research Articles

The C250T Mutation of TERTp Might Grant a Better Prognosis to Glioblastoma by Exerting Less Biological Effect on Telomeres and Chromosomes Than the C228T Mutation.

TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients. Overall, there were no differences in outcomes between patients with mutated TERTp-wt or TERTp. However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp-wt (p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively (p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes. In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp-wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

Unzipped genome assemblies of polyploid root-knot nematodes reveal unusual and clade-specific telomeric repeats

Using long-read sequencing, we assembled and unzipped the polyploid genomes of Meloidogyne incognita, M. javanica and M. arenaria, three of the most devastating plant-parasitic nematodes. We found the canonical nematode telomeric repeat to be missing in these and other Meloidogyne genomes. In addition, we find no evidence for the enzyme telomerase or for orthologs of C. elegans telomere-associated proteins, suggesting alternative lengthening of telomeres. Instead, analyzing our assembled genomes, we identify species-specific composite repeats enriched mostly at one extremity of contigs. These repeats are G-rich, oriented, and transcribed, similarly to canonical telomeric repeats. We confirm them as telomeric using fluorescent in situ hybridization. These repeats are mostly found at one single end of chromosomes in these species. The discovery of unusual and specific complex telomeric repeats opens a plethora of perspectives and highlights the evolutionary diversity of telomeres despite their central roles in senescence, aging, and chromosome integrity.

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 (TMMs) is activated varies according to genetically defined glioma subtypes. In this review, we summarize the current state of the field of 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 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.

Abstract B023: SMARCAL1 is a novel candidate therapeutic target for ALT-positive osteosarcoma

Abstract The DNA translocase SMARCAL1 has been characterized by our laboratory and others as a DNA replication-associated factor that promotes the remodeling of stalled replication forks to facilitate the repair of DNA damage and the restart of DNA synthesis. Recent genome-wide analyses of CRISPR-Cas9 loss-of-function screens in pediatric cancer cell lines have suggested an essential function for SMARCAL1 in a subset of pediatric tumors (Dharia NV et al., Nature Genetics, 2021). In agreement with these observations, pan-cancer analysis of dependency scores in over 1,000 cell lines from the DepMap project revealed an important role for SMARCAL1 in tumors of mesenchymal origin and especially in osteosarcoma. We found that SMARCAL1 dependency in osteosarcoma is selectively associated with lack of telomerase expression. In the absence of telomerase, cancer cells mostly rely on a recombination-based mechanism, known as ALT (Alternative Lengthening of Telomeres), for the maintenance of their telomere length. Surprisingly, by comparing genome-wide CRISPR screening data of known ALT-positive vs ALT-negative osteosarcoma cell lines, we found that SMARCAL1 is among the genes that are most selectively essential in ALT-positive tumors. We validated in multiple cellular models a strict correlation and functional relationship between ALT status and SMARCAL1 dependency. In line with previous findings (Cox KE et al., Cell Reports, 2016), we observed that SMARCAL1 localizes to a subset of telomeres in ALT-positive cells and loss of SMARCAL1 induces accumulation of telomere damage. SMARCAL1 enzymatic activity and its RPA-binding function contribute to suppress DNA damage in ALT-positive cells. Furthermore, our studies suggest that SMARCAL1 protects telomere stability in ALT-positive cancer cells by limiting PRIMPOL-mediated restart of stalled replication forks. In addition, SMARCAL1 deficiency enhances cell-intrinsic immune response and anti-tumor immunity. Collectively, our studies have uncovered SMARCAL1 as a novel candidate therapeutic target for the treatment of ALT-positive cancer patients. In these patients, SMARCAL1 inhibition may have a dual beneficial effect by promoting selective cancer cell toxicity combined with stimulation of anti-tumor immunity and enhancement of immunotherapy response. Citation Format: Angelo Taglialatela, Xiao Chen, Zahra Fatema Khan, Filemon Dela Cruz, Dua Shivi, Dillon Oswald, Azeroglu Benura, Eros Lazzerini Denchi, Min Jaewon, Andrew L. Kung, Alberto Ciccia. SMARCAL1 is a novel candidate therapeutic target for ALT-positive osteosarcoma [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 B023.

METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma.

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+NB.

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.

Oxidative guanine base damage plays a dual role in regulating productive ALT-associated homology-directed repair

Cancer cells maintain telomeres by upregulating telomerase or alternative lengthening of telomeres (ALT) via homology-directed repair at telomeric DNA breaks. 8-Oxoguanine (8oxoG) is a highly prevalent endogenous DNA lesion in telomeric sequences, altering telomere structure and telomerase activity, but its impact on ALT is unclear. Here, we demonstrate that targeted 8oxoG formation at telomeres stimulates ALT activity and homologous recombination specifically in ALT cancer cells. Mechanistically, an acute 8oxoG induction increases replication stress, as evidenced by increased telomere fragility and ATR kinase activation at ALT telomeres. Furthermore, ALT cells are more sensitive to chronic telomeric 8oxoG damage than telomerase-positive cancer cells, consistent with increased 8oxoG-induced replication stress. However, telomeric 8oxoG production in G2 phase, when ALT telomere elongation occurs, impairs telomeric DNA synthesis. Our study demonstrates that a common oxidative base lesion has a dual role in regulating ALT depending on when the damage arises in the cell cycle.

Alternative lengthening of telomeres is seen in a proportion of oligodendrogliomas and is associated with a worse prognosis.

Summary Oligodendrogliomas are known to be mutated for TERTp, and in this report, we evaluated 112 IDH-mutant, 1p19q codeleted oligodendrogliomas for ALT by FISH, and FISH for copy number changes of CDKN2A, MYC, PDGFRA, EGFR, chromosomes +7/10 and TERT-rearrangement. Enigmatically, 35.7% of cases were ALT-positive in spite of the vast majority of them being TERTp-mutant. ALT was associated with a shorter PFS (p=0.009) and remained an independent prognosticator in multivariate analysis. ALT was also associated with MYC amplification. ALT-positive cases were further examined with targeted sequencing. No genes were found to be of prognostic significance in this group.

Fluorescence polarization assay for screening FANCM-RMI inhibitors to target the alternative lengthening of telomeres.

Alternative Lengthening of Telomeres (ALT) is a mechanism used by 10-15% of all cancers to achieve replicative immortality, bypassing the DNA damage checkpoint associated with short telomeres that leads to cellular senescence or apoptosis. ALT does not occur in non-cancerous cells, presenting a potential therapeutic window for cancers where this mechanism is active. Disrupting the FANCM-RMI interaction has emerged as a promising therapeutic strategy that induces synthetic ALT lethality in genetic studies on cancer cell lines. There are currently no chemical inhibitors reported in the literature, in part due to the lack of reliable biophysical or biochemical assays to screen for FANCM-RMI disruption. Here we describe the development of a robust competitive fluorescence polarization (FP) assay that quantifies target binding at the FANCM-RMI interface. The assay employs a labeled peptide tracer TMR-RaMM2 derived from the native MM2 binding motif, which binds to recombinant RMI1-RMI2 and can be displaced by competitive inhibitors. We report the methods for recombinant production of RMI1-RMI2, design and evaluation of the tracer TMR-RaMM2, along with unlabeled peptide inhibitor controls to enable ALT-targeted drug discovery.

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.

Native FISH: A low- and high-throughput assay to analyze the alternative lengthening of telomere (ALT) pathway.

Alternative lengthening of telomeres (ALT) is a telomerase-independent and recombination-based mechanism used by approximately 15% of human cancers to maintain telomere length and to sustain proliferation. ALT-positive cells display unique features that could be exploited for tailored cancer therapies. A key limitation for the development of ALT-specific treatments is the lack of an assay to detect ALT-positive cells that is easy to perform and that can be scaled up. One of the most broadly used assays for ALT detection, CCA (C-circle assay), does not provide single-cell information and it is not amenable to High-Throughput Screening (HTS). To overcome these limitations, we developed Native-FISH (N-FISH) as an alternative method to visualize ALT-specific single-stranded telomeric DNA. N-FISH produces single-cell data, can be applied to fixed tissues, does not require DNA isolation or amplification steps, and it can be miniaturized in a 384-well format. This protocol details the steps to perform N-FISH protocol both in a low- and high-throughput format to analyze ALT. While low-throughput N-FISH is useful to assay the ALT state of cell lines, we expect that the miniaturized N-FISH assay coupled with high-throughput imaging will be useful in functional genomics and chemical screens to identify novel cellular factors that regulate ALT and potential ALT therapeutic targets for cancer therapies directed against ALT-positive tumors, respectively.

Prevalence of alternative lengthening of telomeres in pediatric sarcomas determined by the telomeric DNA C-circle assay.

Alternative lengthening of telomeres (ALT) occurs in sarcomas and ALT cancers share common mechanisms of therapy resistance or sensitivity. Telomeric DNA C-circles are self-primed circular telomeric repeats detected with a PCR assay that provide a sensitive and specific biomarker exclusive to ALT cancers. We have previously shown that 23% of high-risk neuroblastomas are of the ALT phenotype. Here, we investigate the frequency of ALT in Ewing's family sarcoma (EFS), rhabdomyosarcoma (RMS), and osteosarcoma (OS) by analyzing DNA from fresh frozen primary tumor samples utilizing the real-time PCR C-circle Assay (CCA). We reviewed prior publications on ALT detection in pediatric sarcomas. DNA was extracted from fresh frozen primary tumors, fluorometrically quantified, C-circles were selectively enriched by isothermal rolling cycle amplification and detected by real-time PCR. The sample cohort consisted of DNA from 95 EFS, 191 RMS, and 87 OS primary tumors. One EFS and 4 RMS samples were inevaluable. Using C-circle positive (CC+) cutoffs previously defined for high-risk neuroblastoma, we observed 0 of 94 EFS, 5 of 187 RMS, and 62 of 87 OS CC+ tumors. Utilizing the ALT-specific CCA we observed ALT in 0% of EFS, 2.7% of RMS, and 71% of OS. These data are comparable to prior studies in EFS and OS using less specific ALT markers. The CCA can provide a robust and sensitive means of identifying ALT in sarcomas and has potential as a companion diagnostic for ALT targeted therapeutics.

Distinct modes of telomere synthesis and extension contribute to Alternative Lengthening of Telomeres

Alternative lengthening of telomeres (ALT) is a homology-directed repair mechanism that becomes activated in a subset of cancers to maintain telomere length. One of the defining features of ALT cells is the prevalence of extrachromosomal telomeric repeat (ECTR) DNA. Here, we identify that ALT cells engage in two modes of telomere synthesis. Non-productive telomere synthesis occurs during the G2 phase of the cell cycle and is characterized by newly synthesized internal telomeric regions that are not retained in the subsequent G1, coinciding with an induction of ECTR DNA. Productive telomere synthesis occurs specifically during the transition from G2 to mitosis and is defined as the extension of the telomere termini. While many proteins associated with break-induced telomere synthesis function in both non-productive and productive telomere synthesis, POLH specifically promotes productive telomere lengthening and suppresses non-productive telomere synthesis. These findings delineate the mechanism and cell cycle regulation of ALT-mediated telomere synthesis and extension.

The Altered Functions of Shelterin Components in ALT Cells

Telomeres are nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes. Telomeric DNA is bound by shelterin protein complex to prevent telomeric chromosome ends from being recognized as damaged sites for abnormal repair. To overcome the end replication problem, cancer cells mostly preserve their telomeres by reactivating telomerase, but a minority (10–15%) of cancer cells use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). Recent studies have found that shelterin components play an important role in the ALT mechanism. The binding of TRF1, TRF2, and RAP1 to telomeres attenuates ALT activation, while the maintenance of ALT telomere requires TRF1 and TRF2. POT1 and TPP1 can also influence the occurrence of ALT. The elucidation of how shelterin regulates the initiation of ALT remains elusive. This review presents a comprehensive overview of the current findings on the regulation of ALT by shelterin components, aiming to enhance the insight into the altered functions of shelterin components in ALT cells and to identify potential targets for the treatment of ALT tumor cells.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader.

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. Dysregulated R-loops can cause stalled replication forks and telomere instability. However, how R-loops are recognized and regulated, particularly at telomeres, is not well understood. We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination. Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses. In addition, ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway. Using the proximity-dependent biotin identification (BioID) technology, we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases, including DHX9. Importantly, ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9. Our findings suggest that ILF3 may function as a reader of telomeric R-loops, helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

Alternative lengthening of telomeres (ALT) cells viability is dependent on C-rich telomeric RNAs

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10–15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.

Telomeric repeat evolution in the phylum Nematoda revealed by high-quality genome assemblies and subtelomere structures.

Telomeres are composed of tandem arrays of telomeric-repeat motifs (TRMs) and telomere-binding proteins (TBPs), which are responsible for ensuring end-protection and end-replication of chromosomes. TRMs are highly conserved owing to the sequence specificity of TBPs, although significant alterations in TRM have been observed in several taxa, except Nematoda. We used public whole-genome sequencing data sets to analyze putative TRMs of 100 nematode species and determined that three distinct branches included specific novel TRMs, suggesting that evolutionary alterations in TRMs occurred in Nematoda. We focused on one of the three branches, the Panagrolaimidae family, and performed a de novo assembly of four high-quality draft genomes of the canonical (TTAGGC) and novel TRM (TTAGAC) isolates; the latter genomes revealed densely clustered arrays of the novel TRM. We then comprehensively analyzed the subtelomeric regions of the genomes to infer how the novel TRM evolved. We identified DNA damage-repair signatures in subtelomeric sequences that were representative of consequences of telomere maintenance mechanisms by alternative lengthening of telomeres. We propose a hypothetical scenario in which TTAGAC-containing units are clustered in subtelomeric regions and pre-existing TBPs capable of binding both canonical and novel TRMs aided the evolution of the novel TRM in the Panagrolaimidae family.