TelomereHunter - in silico estimation of telomere content and composition from cancer genomes.

BACKGROUND: Establishment of telomere maintenance mechanisms is a universal step in tumor development to achieve replicative immortality. These processes leave molecular footprints in cancer genomes in the form of altered telomere content and aberrations in telomere composition. To retrieve these telomere characteristics from high-throughput sequencing data the available computational approaches need to be extended and optimized to fully exploit the information provided by large scale cancer genome data sets. RESULTS: We here present TelomereHunter, a software for the detailed characterization of telomere maintenance mechanism footprints in the genome. The tool is implemented for the analysis of large cancer genome cohorts and provides a variety of diagnostic diagrams as well as machine-readable output for subsequent analysis. A novel key feature is the extraction of singleton telomere variant repeats, which improves the identification and subclassification of the alternative lengthening of telomeres phenotype. We find that whole genome sequencing-derived telomere content estimates strongly correlate with telomere qPCR measurements (r = 0.94). For the first time, we determine the correlation of in silico telomere content quantification from whole genome sequencing and whole genome bisulfite sequencing data derived from the same tumor sample (r = 0.78). An analogous comparison of whole exome sequencing data and whole genome sequencing data measured slightly lower correlation (r = 0.79). However, this is considerably improved by normalization with matched controls (r = 0.91). CONCLUSIONS: TelomereHunter provides new functionality for the analysis of the footprints of telomere maintenance mechanisms in cancer genomes. Besides whole genome sequencing, whole exome sequencing and whole genome bisulfite sequencing are suited for in silico telomere content quantification, especially if matched control samples are available. The software runs under a GPL license and is available at https://www.dkfz.de/en/applied-bioinformatics/telomerehunter/telomerehunter.html .

TelNet - a database for human and yeast genes involved in telomere maintenance.

BACKGROUND: The ends of linear chromosomes, the telomeres, comprise repetitive DNA sequences in complex with proteins that protects them from being processed by the DNA repair machinery. Cancer cells need to counteract the shortening of telomere repeats during replication for their unlimited proliferation by reactivating the reverse transcriptase telomerase or by using the alternative lengthening of telomeres (ALT) pathway. The different telomere maintenance (TM) mechanisms appear to involve hundreds of proteins but their telomere repeat length related activities are only partly understood. Currently, a database that integrates information on TM relevant genes is missing. DESCRIPTION: To provide a resource for studies that dissect TM features, we here introduce the TelNet database at http://www.cancertelsys.org/telnet/ . It offers a comprehensive compilation of more than 2000 human and 1100 yeast genes linked to telomere maintenance. These genes were annotated in terms of TM mechanism, associated specific functions and orthologous genes, a TM significance score and information from peer-reviewed literature. This TM information can be retrieved via different search and view modes and evaluated for a set of genes as demonstrated for an exemplary application. CONCLUSION: TelNet supports the annotation of genes identified from bioinformatics analysis pipelines to reveal possible connections with TM networks. We anticipate that TelNet will be a helpful resource for researchers that study telomeres.

Quantification of telomere features in tumor tissue sections by an automated 3D imaging-based workflow.

The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular subpopulations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.

PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening.

The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.

Telomere dysfunction and chromothripsis.

Chromothripsis is a recently discovered form of genomic instability, characterized by tens to hundreds of clustered DNA rearrangements resulting from a single dramatic event. Telomere dysfunction has been suggested to play a role in the initiation of this phenomenon, which occurs in a large number of tumor entities. Here, we show that telomere attrition can indeed lead to catastrophic genomic events, and that telomere patterns differ between cells analyzed before and after such genomic catastrophes. Telomere length and telomere stabilization mechanisms diverge between samples with and without chromothripsis in a given tumor subtype. Longitudinal analyses of the evolution of chromothriptic patterns identify either stable patterns between matched primary and relapsed tumors, or loss of the chromothriptic clone in the relapsed specimen. The absence of additional chromothriptic events occurring between the initial tumor and the relapsed tumor sample points to telomere stabilization after the initial chromothriptic event which prevents further shattering of the genome.

Integrative genomic and transcriptomic analysis of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1, widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of "BRCAness", including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies.

Cancer Cells with Alternative Lengthening of Telomeres Do Not Display a General Hypersensitivity to ATR Inhibition.

Telomere maintenance is a hallmark of cancer as it provides cancer cells with cellular immortality. A significant fraction of tumors uses the alternative lengthening of telomeres (ALT) pathway to elongate their telomeres and to gain an unlimited proliferation potential. Since the ALT pathway is unique to cancer cells, it represents a potentially valuable, currently unexploited target for anti-cancer therapies. Recently, it was proposed that ALT renders cells hypersensitive to ataxia telangiectasia- and RAD3-related (ATR) protein inhibitors (Flynn et al., Science 347, 273). Here, we measured the response of various ALT- or telomerase-positive cell lines to the ATR inhibitor VE-821. In addition, we compared the effect of the inhibitor on cell viability in isogenic cell lines, in which ALT was active or suppressed. In these experiments, a general ATR inhibitor sensitivity of cells with ALT could not be confirmed. We rather propose that the observed variations in sensitivity reflect differences between cell lines that are unrelated to ALT.

PML body meets telomere: the beginning of an ALTernate ending?

The unlimited proliferation potential of cancer cells requires the maintenance of their telomeres. This is frequently accomplished by reactivation of telomerase. However, in a significant fraction of tumors an alternative lengthening of telomeres (ALT) mechanism is active. The molecular mechanism of the ALT pathway remains elusive. In particular, the role of characteristic complexes of promyelocytic leukemia nuclear bodies (PML-NBs) with telomeres, the ALT-associated PML-NBs (APBs), is currently under investigation. Here, we review recent findings on the assembly, structure and functions of APBs. It is discussed how genomic aberrations in ALT-positive cancer cells could result in the formation of APBs and in ALT activity. We conclude that they are important functional intermediates in what is considered the canonical ALT pathway and discuss deregulations of cellular pathways that contribute to the emergence of the ALT phenotype.