Telomeric Foci Research Articles

Alternative lengthening of telomeres (ALT) in pediatric high-grade gliomas can occur without ATRX mutation and is enriched in patients with pathogenic germline mismatch repair (MMR) variants.

To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. We performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n=40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade gliomas (pHGGs) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. ALT is common in pHGGs (n=24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ pHGGs and in 30% of ALT- pHGGs. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. We demonstrate that ATRX is mutated in only a subset of ALT+ pHGGs, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with the development of ALT in patients with pHGG.

Genetic and Epigenetic Inheritance at Telomeres.

Transgenerational inheritance can occur at telomeres in distinct contexts. Deficiency for telomerase or telomere-binding proteins in germ cells can result in shortened or lengthened chromosome termini that are transmitted to progeny. In human families, altered telomere lengths can result in stem cell dysfunction or tumor development. Genetic inheritance of altered telomeres as well as mutations that alter telomeres can result in progressive telomere length changes over multiple generations. Telomeres of yeast can modulate the epigenetic state of subtelomeric genes in a manner that is mitotically heritable, and the effects of telomeres on subtelomeric gene expression may be relevant to senescence or other human adult-onset disorders. Recently, two novel epigenetic states were shown to occur at C. elegans telomeres, where very low or high levels of telomeric protein foci can be inherited for multiple generations through a process that is regulated by histone methylation.Together, these observations illustrate that information relevant to telomere biology can be inherited via genetic and epigenetic mechanisms, although the broad impact of epigenetic inheritance to human biology remains unclear.

Gametes deficient for Pot1 telomere binding proteins alter levels of telomeric foci for multiple generations

Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.

ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner.

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer.

Modulation of telomere protection by the PI3K/AKT pathway

Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.

Abstract 3465: Context-dependent effects of ATRX loss on telomere biology in glioma cells

Abstract Telomeres progressively shorten with each cell division, leading to critically short telomeres during tumorigenesis. While most cancers activate telomerase to maintain their telomeres, a subset of cancers utilize a recombination-based process known as alternative lengthening of telomeres, or ALT. ALT has been widely characterized mostly in the context of immortalized cells, and characteristic features include ultrabright telomeric foci, colocalization of telomere foci with PML (ALT-associated PML bodies, or APBs), extrachromosomal telomeric DNA (e.g. c-circles), and overall telomere length heterogeneity. While the overall rate of ALT in cancer is low (roughly 5%, overall), the prevalence of ALT is concentrated in certain cancer subtypes, including gliomas. Gliomas employing ALT have a common genomic signature: inactivating mutation of ATRX, a chromatin-modifying protein that functions at the telomeres. In order to study the role of ATRX loss in the biology of ALT in cancer, we have characterized a panel of six human cell lines derived from pediatric high grade glioma, two of which are ALT(+) and lack functional ATRX. Furthermore, in four pediatric and three adult high grade glioma cell lines that endogenously express functional ATRX, we knocked out ATRX using CRISPR/Cas9n and knocked down ATRX expression using a stable shRNA-mediated approach. Knockout or knockdown of ATRX was successful in these seven glioma cell lines. Overall, knockout and/or knockdown of ATRX induced the formation of ultrabright telomeric foci, as assessed by telomere FISH, in two out of seven glioma cell lines. However, the effect of ATRX loss on formation of c-circles and APBs was more variable: the presence of ultrabright telomeric foci was not predictive of the presence of c-circles or ALT-associated PML bodies. These results indicate that ATRX loss, alone, is not necessarily sufficient to activate the ALT pathway in gliomas. Further examination of ALT-competent and ALT-resistant cell lines from our cohort will yield clues as to additional ALT-associated gene expression changes. In addition, our results suggest that previously identified hallmarks of ALT (notably c-circles and APBs) may be more variable in cancer cells compared to immortalized normal cell lines. The ALT pathway is an intriguing target for synthetic lethality in the cancer subtypes that employ it; our hope is to better understand the ALT phenotype in cancer so that we may effectively translate ALT-specific therapeutics to the clinic. Citation Format: Jacqueline A. Brosnan-Cashman, Ming Yuan, Anthony J. Rizzo, Kaylar M. Myers, Walter T. Barry, Christine Davis, David M. Esopi, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Context-dependent effects of ATRX loss on telomere biology in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2017-3465

Abstract 3412: Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by short telomeres. The majority of cancers overcome this critical replication barrier by upregulating the telomerase enzyme, a telomere-specific reverse transcriptase. However, a subset of cancers lack telomerase, and telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is dependent on homologous recombination. Across a variety of tumor types, our laboratory and others have reported a robust correlation between ALT and recurrent cancer-associated somatic inactivating mutations in the ATRX-DAXX chromatin remodeling complex. In a previous comprehensive cancer survey of ALT, we reported that ALT was highly prevalent in some tumor types (e.g. astrocytomas, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive primary prostate cancer (out of 1,176 analyzed). However, we subsequently found a subset of metastatic prostate cancers that harbor ALT, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting ALT, with the explicit purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic targets for men with ALT-positive lethal metastatic prostate cancer. Inactivating mutations in either ATRX or DAXX using the CRISPR/Cas9n system were introduced into the genetically well-characterized, telomerase-positive (ALT-negative) prostate cancer cell lines, LAPC-4 and CWR22Rv1. Resulting mutant subclones were compared to their parental (or empty vector) counterparts. In these new models, abolishing ATRX expression was sufficient to induce the ALT phenotype in both LAPC-4 and CWR22Rv1, as assessed by multiple biomarkers of ALT, including the presence of bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and extrachromosomal telomere c-circles. Interestingly, abolishing DAXX expression induced ALT in only a subset (3/5) of LAPC-4 DAXX KO clones, and a subset (4/19) of CWR22Rv1 DAXX KO clones. We have successfully activated the ALT telomere maintenance phenotype in two prostate cancer cell lines through CRISPR-mediated targeted gene deletion. We are currently utilizing these isogenic cell lines to further characterize and elucidate the underlying biology of cancers harboring ALT, with the goal of pharmacologically targeting the molecular features unique to the ALT phenotype (e.g. ATR inhibition). The identification of ALT-specific drugs may pave the way for the development of new targeted treatments for the subset of men with ALT-positive lethal metastatic prostate cancer, and more broadly, other ALT-positive cancers. ALT is easily detected in clinical tissue samples, and thus would serve as a predictive biomarker for personalized medicine. Citation Format: Mindy K. Graham. Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3412. doi:10.1158/1538-7445.AM2017-3412

Abstract 3464: CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines

Abstract Cancer cells must find a way to subvert replicative senescence in order to achieve cellular immortality. While most malignancies (&amp;gt; 90 %) overcome this critical barrier by reactivating the telomerase enzyme, a telomere-specific reverse transcriptase; other cancers (5 - 10 %) utilize a telomerase-independent pathway of telomere maintenance, referred to as Alternative Lengthening of Telomeres (ALT). ALT is thought to utilize homologous recombination and DNA damage repair (DDR) machinery to maintain the chromosome ends. Importantly, this mechanism appears to be cancer-specific and dependent on alterations in chromatin dynamics at the telomeres, almost invariably facilitated by inactivating mutations in the genes, ATRX and DAXX. The protein products of these genes are responsible for the deposition of histone variant H3.3 at repetitive regions of DNA (e.g. telomeres) and when altered, the affected cells tend to exhibit the ALT phenotype. Pancreatic neuroendocrine tumors (PanNETs) are an example of a tumor type that often employs the ALT pathway with coincident somatic mutations in either the ATRX or DAXX genes. PanNETs are the second most common pancreatic malignancy, with only 40% of affected patients surviving past 10 years. Recently, we and others have demonstrated that in primary PanNETs, ALT is an independent prognostic marker, associated with aggressive clinicopathologic behavior and reduced recurrence-free survival. In addition, current data suggest that ALT-positive cancer cells are sensitive to inhibition of the DNA-damage mediator, ATR. In light of these findings, there seems to be an opportunity to develop ALT specific therapeutic modalities that would have considerable clinical utility. Models of ALT-positive PanNET pathology are needed to further progress in this direction, unfortunately, there are currently no ALT-positive PanNET cell lines. BON1 and QGP1, the two well characterized PanNET cell lines that are available, are both ALT-negative and retain expression of telomerase, ATRX, and DAXX. In an attempt recapitulate the ALT phenotype in a PanNET cell line, we derived a panel of isogenic cell lines from both BON1 and QGP1 by utilizing the CRISPR-Cas9 nickase system to target ATRX and DAXX. In both cell lines, we have successfully established 5 independent subclones with inactivating mutations in either ATRX or DAXX, validated by Sanger sequencing. We are currently characterizing these novel subclones for the hallmarks of ALT, including the presence of ultrabright telomeric foci, dramatic telomere length heterogeneity, and the presence of extrachromosomal telomeric DNA (e.g. c-circles). Once validated, we plan to investigate whether these ATRX or DAXX-deficient subclones are more sensitive to DDR pathway inhibition (i.e. ATR, ATM, CHK1 and CHK2) pharmacologically or with RNA interference in order to identify new therapeutic strategies for PanNETs. Citation Format: Anthony J. Rizzo, Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Alan K. Meeker, Christopher M. Heaphy. CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3464. doi:10.1158/1538-7445.AM2017-3464

Abstract 3466: ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition

Abstract Overcoming the end-replication problem is a major hurdle for cancer cells due to progressive telomere shortening that results from excessive cell division during tumorigenesis. While most cancers activate telomerase to maintain their telomere length, about five percent of cancers use a telomerase-independent telomere maintenance mechanism, termed alternative lengthening of telomeres (ALT). Despite its low overall frequency in cancer, ALT is enriched in several cancer subtypes, including pediatric high grade glioma (HGG). Previous work in our laboratory suggests that nearly half of pediatric HGG utilize the ALT mechanism. To date, therapeutic options are largely ineffective for children with HGG, reflected in the dismal five-year survival rate, which is less than 33 percent. Our goal is to better harness ALT as a clinical marker in pediatric HGG, specifically by identifying drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pediatric HGG cell lines. Two of the six cell lines in this panel display hallmarks of the ALT pathway. To assess the potential of ALT as a therapeutic biomarker in pediatric HGG, we measured cell viability in the presence of inhibitors, stratifying our analysis by ALT status. ALT is predicted to occur via a homologous-recombination-based mechanism. As such, we focused our attention on inhibitors of the DNA damage response, as well as agents that induce DNA damage. Treatment with inhibitors of DNA-PK, RAD51, and MRE11 did not result in significant differences in cell viability when stratified by ALT status, nor did treatment with temozolomide or hydroxyurea. However, inhibitors of ATR (VE-821, VE-822/VX-970, and AZD-6738) and CHK1 (MK-8776) led to significantly greater reductions in cell viability in ALT-positive cells. In order to further our understanding of the mechanism through which ATR inhibition preferentially targets ALT-positive cell lines, we examined the effect of these inhibitors on ALT-specific properties, such as the presence of ultrabright telomeric foci and extrachromosomal telomeric DNA (c-circles). Preliminary results indicate that ATR inhibition does not diminish these ALT-associated properties and, therefore, the differential sensitivity to these agents is unlikely to be due to directly blocking the ALT telomere maintenance mechanism. Continued study of the effect of ATR/CHK1 inhibition on ALT-positive cells will yield further insight into the mechanism of ALT-specific toxicity. Overall, we have identified ATR/CHK1 as a promising pathway to target for ALT-positive pediatric HGG. Our goal is to better understand the relationship between ATR/CHK1 signaling and ALT in order to effectively translate this observation to the clinic, both for pediatric HGG and for other ALT-positive cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Anthony J. Rizzo, Ezgi Goger, Kaylar M. Myers, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2017-3466

Detection of Dysfunctional Telomeres in Oncogene-Induced Senescence.

Expressing oncogenes in normal somatic human cells leads to cellular senescence after just a few cell division cycles. In cells that are more resistant to culture stresses, such as human dermal fibroblasts, this oncogene-induced senescence (OIS) is a result of a DNA damage response (DDR) that is activated due to the formation of DNA lesions at both non-telomeric and telomeric DNA sequences. DNA lesions can be visualized as DDR foci by immunofluorescence microscopy using antibodies against a number of DDR factors, including ϒ-H2AX and 53BP1. Over time and as cells remain arrested in OIS, non-telomeric DDR foci progressively become resolved, while telomeric DDR foci, also called dysfunctional telomeres, persist. Here we describe a protocol to detect dysfunctional telomeres in cultured human cells, to monitor a temporal enrichment of dysfunctional telomeres in cells that had undergone OIS, and to detect dysfunctional telomeres in paraffin-embedded and formalin-fixed human tissue.

Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.

Abstract 4767: Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by telomere shortening. The majority of cancers overcome this critical barrier by up-regulating the enzyme telomerase, a telomere-specific reverse transcriptase. However, for a subset of cancers that lack telomerase, telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is thought to be dependent on homologous recombination. In a variety of tumor types, our laboratory and others have reported a strong correlation between the ALT phenotype and recurrent cancer-associated somatic inactivating mutations ATRX or DAXX, genes encoding chromatin remodeling proteins. In a previous comprehensive survey of the ALT phenotype in cancer, we reported that the ALT phenotype is highly prevalent in some tumor types (e.g. astrocytoma, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive cases in over 1,000 cases of primary prostate cancer. Notably, however, we found a subset of metastatic prostate cancer harbors the ALT phenotype, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting the ALT phenotype, with the purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic approaches for men with lethal metastatic prostate cancer harboring this phenotype. These novel cell lines were generated by inactivating ATRX using the CRISPR cas9 nickase system in a genetically well characterized prostate cancer cell line that normally expresses telomerase, as well as wild-type ATRX and DAXX. In this new model, abolishing ATRX expression is sufficient to induce the ALT phenotype, as assessed by multiple biomarkers of ALT, including bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and c-circles. Interestingly, compared to the parental line, cells with compromised ATRX function have significantly lower telomerase activity and a five-fold decreased expression of telomerase reverse transcriptase (TERT). Ultimately, we will use these isogenic cell lines to further characterize and elucidate the basic biology of cancers harboring ALT, and pharmacologically target the molecular features unique to the ALT phenotype. The identification of ALT-specific drugs will pave the way for the development of new targeted treatments for a subset of men with lethal metastatic prostate cancer that harbor this unique molecular phenotype, and more broadly, other cancers that share the ALT phenotype in common. Citation Format: Mindy K. Graham, Jacqueline Brosnan-Cashman, Anthony Rizzo, Michael Haffner, Alan Meeker, Christopher Heaphy. Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4767.

Abstract 3783: High resolution telomere FISH analysis of testicular germ cell tumors reveals telomere anomalies specific to cancer subtypes and demonstrates telomere shortening as an early event in TGCT carcinogenesis

Abstract Testicular germ cell tumor (TGCT) is the most common malignancy of young men between 15 and 40 years of age. TGCT is exceptional, as most men can be completely cured. We evaluated 76 TGCT cases using high resolution (single-cell) telomere-specific fluorescence in situ hybridization (FISH) combined with Oct4 immunofluorescence staining to delineate telomere length patterns in different TGCT subtypes, as well as in the precursor lesion, intratubular germ cell neoplasia unclassified type (ITGCNU). Telomere lengths were qualitatively scored by comparing the intensity of the telomere signals from cancer cells to benign reference cell types and then grouped (short, medium, and long). For isolated tumors and for those part of a mixed germ cell tumor (containing 2 or more TGCT types), we determined the overall trends in telomere length in comparison to reference cell populations. This comparison demonstrated medium telomeres for normal germ cells, short telomeres for ITGCNU and seminomas (p = 0.006 and p = 0.0005, respectively), and a wide range of average telomere lengths in the non-seminomas. Among the non-seminomas, embryonal carcinomas displayed the longest telomeres, on average, and were the only subtype (27% of cases) to display the alternative lengthening of telomeres (ALT) phenotype, as evident from abnormally large telomeric foci and telomere length heterogeneity. ATRX and DAXX protein expression was not absent in a subset of embryonal carcinoma cases. Compared to somatic tissues, germ cells have a high basal telomerase activity and longer telomeres. Hence, short telomeres in ITGCNU are surprising. Telomere attrition does not occur in normal germ cells; therefore, telomere shortening in ITGCNU may play a role in TGCT tumorigenesis. Embryonal carcinoma, the TGCT subtype that is most aggressive and has the worst prognosis, showed very long telomeres. Seminomas, with short telomeres, are exquisitely sensitive to standard platinum based chemotherapy. A recent study implicates telomere damage as a consequence of platinum based chemotherapeutic agents in a male germ cell line. Thus, the knowledge of telomere length differences and that of chemotherapeutic agents’ damage to telomeres together may help shed light on the differential sensitivities of TGCT subtypes to certain chemotherapeutic agents. In summary, TGCT can demonstrate varied telomere lengths depending on lesion subtype and whether the lesion occurs in the context of a mixed tumor. The presence of short telomeres in ITGCNU suggests a major role of telomere shortening in the pathogenesis of TGCT. In the future, telomere length information may also inform point-of-care diagnostic and treatment decisions for clinicians caring for TGCT patients. Citation Format: Mohammed T. Shekhani, John Barber, Christopher M. Heaphy, Leonardo Reis, Gunes Guner, Corinne Joshu, George J. Netto, Alan K. Meeker. High resolution telomere FISH analysis of testicular germ cell tumors reveals telomere anomalies specific to cancer subtypes and demonstrates telomere shortening as an early event in TGCT carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2015-3783

The formation and sequestration of heterochromatin during development

Chromatin is not randomly positioned in the nucleus, but is distributed in subdomains based on its degree of compaction and transcriptional status. Recent studies have shed light on the logic of chromatin distribution, showing that tissue-specific promoters drive distinct patterns of gene positioning during cell-type differentiation. In addition, the sequestration of heterochromatin at the nuclear envelope has been found to depend on lamin and lamin-associated proteins. On the chromatin side, H3K9 monomethylation, dimethylation and trimethylation were shown to be the critical signals for perinuclear anchoring in worm embryonic nuclei. Downregulation of an equivalent histone methyltransferase, G9a, in human cells has a similar effect. In worms, the sequestration of the terminal methyltransferase by repressed chromatin may facilitate the propagation of a heterochromatin compartment, much as the sequestration of the silent information regulatory complex does at telomeric foci in budding yeast. These results argue for conserved logic in eukaryotic nuclear organization.

Characterization of telomeric repeats in metaphase chromosomes and interphase nuclei of Syrian Hamster Fibroblasts

BackgroundRodents have been reported to contain large arrays of interstitial telomeric sequences (TTAGGG)n (ITS) located in pericentromeric heterochromatin. The relative sizes of telomeric sequences at the ends of chromosomes (TS) and ITS in Syrian hamster (Mesocricetus auratus) cells have not been evaluated yet, as well as their structural organization in interphase nuclei.ResultsFISH signal distribution analysis was performed on DAPI-banded metaphase chromosomes of Syrian hamster fibroblasts, and relative lengths of telomere signals were estimated. Besides well-distinguished FISH signals from ITS located on chromosomes ##2, 4, 14, 20 and X that we reported earlier, low-intensity FISH signals were visualized with different frequency of detection on all other metacentric chromosomes excluding chromosome #21. The analysis of 3D-distribution of TS in interphase nuclei demonstrated that some TS foci formed clearly distinguished associations (2–3 foci in a cluster) in the nuclei of cells subjected to FISH or transfected with the plasmid expressing telomeric protein TRF1 fused with GFP. In G0 and G1/early S-phase, the average total number of GFP-TRF1 foci per nucleus was less than that of PNA FISH foci in the corresponding cell cycle phases suggesting that TRF1 overexpression might contribute to the fusion of neighboring telomeres. The mean total number of GFP-TRF1 and FISH foci per nucleus was increased during the transition from G0 to G1/early S-phase that might be the consequence of duplication of some TS.ConclusionsThe relative lengths of TS in Syrian hamster cells were found to be moderately variable. All but one metacentric chromosomes contain ITS in pericentromeric heterochromatin indicating that significant rearrangements of ancestral genome occurred in evolution. Visualization of GFP-TRF1 fibrils that formed bridges between distinct telomeric foci allowed suggesting that telomere associations observed in interphase cells are reversible. The data obtained in the study provide the further insight in the structure and dynamics of telomeric sequences in somatic mammalian cells.

Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast

A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2-Sir3-Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3's ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2-Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.

N-Cyclic Bay-Substituted Perylene G-Quadruplex Ligands Have Selective Antiproliferative Effects on Cancer Cells and Induce Telomere Damage

A series of bay-substituted perylene derivatives is reported as a new class of G-quadruplex ligands. The synthesized compounds have differing N-cyclic substituents on the bay area and differing side chains on the perylene major axis. ESI-MS and FRET measurements highlighted the strongest quadruplex binders in this series and those showing the highest quadruplex/duplex selectivity. Several biological assays were performed on these compounds, which showed that compound 5 (PPL3C) triggered a DNA damage response in transformed cells with the formation of telomeric foci containing phosphorylated γ-H2AX and 53BP1. This effect mainly occurred in replicating cells and was consistent with Pot1 dissociation. Compound 5 does not induce telomere damage in normal cells, which are unaffected by treatment with the compound, suggesting that this agent preferentially kills cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs.

The Werner Syndrome Protein Suppresses Telomeric Instability Caused by Chromium (VI) Induced DNA Replication Stress

Telomeres protect the chromosome ends and consist of guanine-rich repeats coated by specialized proteins. Critically short telomeres are associated with disease, aging and cancer. Defects in telomere replication can lead to telomere loss, which can be prevented by telomerase-mediated telomere elongation or activities of the Werner syndrome helicase/exonuclease protein (WRN). Both telomerase and WRN attenuate cytotoxicity induced by the environmental carcinogen hexavalent chromium (Cr(VI)), which promotes replication stress and DNA polymerase arrest. However, it is not known whether Cr(VI)-induced replication stress impacts telomere integrity. Here we report that Cr(VI) exposure of human fibroblasts induced telomeric damage as indicated by phosphorylated H2AX (γH2AX) at telomeric foci. The induced γH2AX foci occurred in S-phase cells, which is indicative of replication fork stalling or collapse. Telomere fluorescence in situ hybridization (FISH) of metaphase chromosomes revealed that Cr(VI) exposure induced an increase in telomere loss and sister chromatid fusions that were rescued by telomerase activity. Human cells depleted for WRN protein exhibited a delayed reduction in telomeric and non-telomeric damage, indicated by γH2AX foci, during recovery from Cr(VI) exposure, consistent with WRN roles in repairing damaged replication forks. Telomere FISH of chromosome spreads revealed that WRN protects against Cr(VI)-induced telomere loss and downstream chromosome fusions, but does not prevent chromosome fusions that retain telomere sequence at the fusion point. Our studies indicate that environmentally induced replication stress leads to telomere loss and aberrations that are suppressed by telomerase-mediated telomere elongation or WRN functions in replication fork restoration.

G-quadruplex ligand SYUIQ-5 induces autophagy by telomere damage and TRF2 delocalization in cancer cells

Agents stabilizing G-quadruplexes have the potential to destroy the functional structure of telomere and could therefore act as antitumor agents. We previously reported that SYUIQ-5 could stabilize G-quadruplex, induce senescence, and inhibit c-myc gene promoter activity. In this study, we showed that SYUIQ-5 inhibited proliferation of CNE2 and HeLa cancer cells, triggered a rapid and potent telomere DNA damage response characterized by the formation of telomeric foci gamma-H2AX, and obviously induced autophagy with the features of increased LC3-II and a punctuated pattern of YFP-LC3 fluorescence. These phenomena may primarily depend on the delocalization of TRF2 from telomere, which was further degraded by proteasomes. Furthermore, overexpression of TRF2 inhibited SYUIQ-5-induced gamma-H2AX expression. Also, ATM was activated following SYUIQ-5 treatment. The pretreatment with ATM inhibitor ku55933 and ATM siRNA effectively reduced the production of gamma-H2AX and LC3-II. ATM knockdown partially antagonized the anticancer effects of SYUIQ-5. Moreover, inhibition of autophagy by short hairpin RNA against the autophagy-related gene ATG5 attenuated the cytotoxicity of SYUIQ-5. These results indicated that SYUIQ-5 triggered potent telomere damage through TRF2 delocalization from telomeres, and eventually induced autophagic cell death in cancer cells. Our findings exhibit a novel mechanism that is responsible for the antitumor effects of SYUIQ-5.

Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice

The telomere repeat-binding factor 1 (TERF1, referred to hereafter as TRF1) is a component of mammalian telomeres whose role in telomere biology and disease has remained elusive. Here, we report on cells and mice conditionally deleted for TRF1. TRF1-deleted mouse embryonic fibroblasts (MEFs) show rapid induction of senescence, which is concomitant with abundant telomeric gamma-H2AX foci and activation of the ATM/ATR downstream checkpoint kinases CHK1 and CHK2. DNA damage foci are rescued by both ATM and ATM/ATR inhibitors, further indicating that both signaling pathways are activated upon TRF1 deletion. Abrogation of the p53 and RB pathways bypasses senescence but leads to chromosomal instability including sister chromatid fusions, chromosome concatenation, and occurrence of multitelomeric signals (MTS). MTS are also elevated in ATR-deficient MEFs or upon treatment with aphidicolin, two conditions known to induce breakage at fragile sites, suggesting that TRF1-depleted telomeres are prone to breakage. To address the impact of these molecular defects in the organism, we deleted TRF1 in stratified epithelia of TRF1(Delta/Delta)K5-Cre mice. These mice die perinatally and show skin hyperpigmentation and epithelial dysplasia, which are associated with induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. p53 deficiency rescues mouse survival but leads to development of squamous cell carcinomas, demonstrating that TRF1 suppresses tumorigenesis. Together, these results demonstrate that dysfunction of a telomere-binding protein is sufficient to produce severe telomeric damage in the absence of telomere shortening, resulting in premature tissue degeneration and development of neoplastic lesions.