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Background In selected patients with early-stage malignant pleural mesothelioma (MPM), a multimodal therapy that includes surgical cytoreduction, chemotherapy, and/or radiotherapy is recommended. Several clinical trials have demonstrated the beneficial effects of immune checkpoint inhibitors in pretreated MPM patients with advanced disease. Recent clinical data have suggested that the combination of chemotherapy and checkpoint inhibition might improve efficacy. Trial Design The NICITA (nivolumab with chemotherapy in pleural mesothelioma after surgery) trial is a prospective, 1:1 randomized, open-label, multicenter phase II clinical trial ( ClinicalTrials.gov identifier, NCT04177953 ). Ninety-two patients with MPM epithelioid subtype, who had undergone extended pleurectomy and decortication with or without hyperthermic intrathoracic chemoperfusion, will be included to receive adjuvant treatment. All patients will receive ≤ 4 cycles of platinum-based chemotherapy with pemetrexed (arms A and B). Patients in arm B will additionally receive nivolumab, together with the adjuvant chemotherapy, and subsequently for ≤ 12 cycles as maintenance therapy. The primary endpoint of this study is the time-to-next-treatment. The secondary endpoints include progression-free survival, overall survival, proportion of patients with treatment beyond progression, duration of treatment beyond progression in this population, and quality of life. Conclusion This prospective trial will contribute data to assess the efficacy of standard chemotherapy combined with nivolumab in the context of multimodal management of early-stage MPM. The study is currently enrolling patients.

The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT, transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT, we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length.

The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of

Background Availability of potent anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) has pushed the median survival of ALK+ non-smallcell lung cancer (NSCLC) patients to over five years. In particular, second-generation ALK TKI have demonstrated superiority compared to the first-generation compound crizotinib and are meanwhile standard first-line treatment. However, clinical courses of individual patients vary widely, with secondary development of drug resistance and intracranial progression remaining important problems. While these limitations highlight the need for better disease monitoring and additional therapeutic tools, molecular tumor features are increasingly recognized as crucial determinants of clinical outcome. This trial aims to optimize management of ALK+ NSCLC by analyzing the efficacy of second-generation ALK inhibitors in conjunction with deep longitudinal phenotyping across two treatment lines. Methods/design In this exploratory prospective phase II clinical trial, newly diagnosed ALK+ NSCLC patients will be randomized into two treatment arms, stratified by presence of brain metastases and ECOG performance status: brigatinib (experimental arm) vs. any other approved second-generation ALK TKI. Tumor tissue and blood samples will be collected for biomarker analysis at the beginning and throughout the study period to investigate baseline molecular tumor properties and analyze the development of acquired drug resistance. In addition, participating investigators and patients will have the possibility of fast-track molecular tumor and ctDNA profiling at the time of disease progression using state-of-the-art next-generation sequencing (NGS), in order to support decisions regarding next-line therapy. Discussion Besides supporting therapeutic decisions for enrolled patients, the ABP trial primarily aims to deepen the understanding of the underlying biology and facilitate development of a framework for individualized management of ALK+ NSCLC according to molecular features. Patients with low molecular risk and the perspective of a “chronic disease” will be distinguished from “high-risk” cases, molecular properties of which will be utilized to elaborate improved methods of non-invasive monitoring and novel preclinical models in order to advance therapeutic strategies. Trial registration Clinicaltrials.gov, NCT04318938. Registered March 182,020, https://www.clinicaltrials.gov/ct2/show/NCT04318938 Eudra-CT, 2019–001828-36. Registered September 302,019, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2019-001828-36

Cell segmentation in microscopy images is a common and challenging task. In recent years, deep neural networks achieved remarkable improvements in the field of computer vision. The dominant paradigm in segmentation is using convolutional neural networks, less common are recurrent neural networks. In this work, we propose a new deep learning method for cell segmentation, which integrates convolutional neural networks and gated recurrent neural networks over multiple image scales to exploit the strength of both types of networks. To increase the robustness of the training and improve segmentation, we introduce a novel focal loss function. We also present a distributed scheme for optimized training of the integrated neural network. We applied our proposed method to challenging data of glioblastoma cell nuclei and performed a quantitative comparison with state-of-the-art methods. Insights on how our extensions affect training and inference are also provided. Moreover, we benchmarked our method using a wide spectrum of all 22 real microscopy datasets of the Cell Tracking Challenge.

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.

The ends of linear chromosomes, the telomeres, comprise repetitive DNA sequences that are protected by the shelterin protein complex. Cancer cells need to extend these telomere repeats for their unlimited proliferation, either 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. To provide a reference 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 2,000 human and over 1,100 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 on a statistics page. With these features TelNet can be integrated into the annotation of genes identified from bioinformatics analysis pipelines to determine possible connections with TM networks as illustrated by an exemplary application. We anticipate that TelNet will be a helpful resource for researchers that study TM processes.

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., The molecular genetic landscape of leiomyosarcoma (LMS) is largely unknown. Here, the authors identify frequent DNA copy number alterations, whole-genome duplication, TP53 and RB1 inactivation, alternative telomere lengthening, and genomic imprints of defective DNA repair via homologous recombination as a potential therapeutic target in LMS patients.

Pediatric glioblastoma (pedGBM) represent a highly malignant primary brain tumor with recurrent mutations in the chromatin remodeler ATRX and the histone variant H3.3 that is typically associated with a fatal outcome. ATRX acts as suppressor of the alternative lengthening of telomeres (ALT) pathway, which is frequently activated in pedGBM. However, telomere features of pedGBMs have not been studied in detail, and ALT-positive model cell lines are lacking. Here, we systematically characterized a panel of pedGBM models that carry a representative set of recurrent genomic mutations for a variety of telomere features. These included the presence of ALT-associated promyelocytic leukemia nuclear bodies and C-circles, a specific type of extrachromosomal telomeric repeats, the telomere repeat content, and phosphorylation of histone H3.3 at serine 31. From an integrated analysis of seven pedGBM cell lines and 57 primary tumor samples we identified cell lines and tumors that represent the different telomere maintenance mechanisms and conclude the following: (i) A positive signal in the C-circle assay is a reliable ALT marker. (ii) ALT features occur heterogeneously and one pedGBM subgroup uses a ‘non-canonical’ ALT mechanism in the presence of wild-type ATRX. (iii) The spreading of H3.3S31 phosphorylation during mitosis is associated with loss of ATRX but not with ALT per se. (iv) In contrast to a previous study in glioma stem cells, we did not find a hypersensitivity of ALT cells towards the ATR inhibitor VE-821. (v) ALT-positive pedGBMs can be reliably identified from a classification scheme developed here that evaluates various combinations of cytogenetic and/or genomic data. Thus, our findings elucidate further details of the ALT pathway in pedGBMs, provide valuable models for evaluating ALT targeted therapies in a preclinical setting, and introduce an ALT classification scheme for primary tumor samples.

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 provides 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 sub-populations 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.Abbreviations3D-TIM3D targeted imagingALTalternative lengthening of telomeresAPBALT-associated PML-NBCLSMconfocal laser scanning fluorescence microscopyECTRextrachromosomal telomeric repeatFFPEformalin-fixed, paraffin-embeddedFISHfluorescence in situ hybridizationIFImmunofluorescencepedGBMpediatric glioblastomaPMLpromyelocytic leukemiaPML-NBPML nuclear bodyPNApeptide nucleic acidROIregion of interestTMAtissue microarrayTMMtelomere maintenance mechanismSMLMsingle molecule localization microscopy

Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.

Leiomyosarcomas (LMS) are malignant tumors of smooth-muscle origin that occur across age groups. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown, and few therapeutic options exist for LMS patients. To detect somatic mutations, copy number alterations, and structural rearrangements, we performed whole-exome and transcriptome sequencing of 49 and 37 LMS tumors, respectively, and performed integrative analysis. Recurrence analysis identified TP53, RB1, and ATRX as significantly mutated genes and various other cancer-associated genes mutated at low frequency, indicating substantial mutational heterogeneity. Copy number analysis revealed widespread chromosomal gains and losses and highly rearranged genomes in all tumors. Additionally, chromothripsis and whole-genome duplication were detected in 35% and 51% of cases, respectively. Principle component analysis and unsupervised hierarchical clustering of transcriptome data revealed three distinct subgroups of patients. Furthermore, we detected multiple non-recurrent fusion transcripts resulting from chromosomal rearrangements, many of which were predicted to result in loss of TP53 and RB1 function. In-depth analysis of these loci revealed protein-damaging microdeletions, intragenic or distal inversions, and exon skipping events as additional, previously unrecognized mechanisms of TP53 and RB1 disruption. Integration of whole-exome and transcriptome data demonstrated biallelic disruption of TP53 and RB1 in 92% and 94% of cases, respectively, and tumors with wildtype RB1 displayed loss of CDKN2A expression, overexpression of CCND1, or mutation of MAX resulting in CDK4 and CCND2 overexpression as alternative mechanisms of RB1 suppression. We also detected alternative lengthening of telomeres (ALT) in 78% of cases, and identified recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing novel insight into the genetic basis of this mechanism. Finally, most tumors displayed hallmarks of “BRCAness”, including alterations in homologous recombination DNA repair genes and enrichment of specific mutational signatures, and cultured LMS cells were sensitive towards olaparib and cisplatin. This comprehensive genomic and transcriptomic analysis has unveiled that LMS is characterized by mutational heterogeneity, genomic instability, near-universal inactivation of TP53 and RB1, and frequent whole-genome duplication. Furthermore, we have established that most LMS tumors rely on ALT to escape replicative senescence, and identified recurrent alterations in a broad spectrum of telomere maintenance genes. Finally, our findings uncover “BRCAness” as potentially actionable feature of LMS tumors, and provide a rich resource for guiding future investigations into the mechanisms underlying LMS development and the design of novel therapeutic strategies. Citation Format: Priya Chudasama, Sadaf Mughal, Mathijs Sanders, Daniel Hübschmann, Inn Chung, Aurélie Ernst, Bernd Kasper, Hans-Georg Kopp, Sebastian Bauer, Karsten Rippe, Benedikt Brors, Marcus Renner, Peter Hohenberger, Claudia Scholl, Stefan Fröhling. Integrative genomic and transcriptomic analysis of leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4336.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

Upon genome damage, large-scale protein sumoylation occurs from yeast to humans to promote DNA repair. Currently, the underlying mechanism is largely unknown. Here we show that, upon DNA break induction, the budding yeast SUMO ligase Siz2 collaborates with the ssDNA-binding complex RPA (replication protein A) to induce the sumoylation of recombination factors and confer damage resistance. Both RPA and nuclease-generated ssDNA promote Siz2-mediated sumoylation. Mechanistically, the conserved Siz2 interaction with RPA enables Siz2 localization to damage sites. These findings provide a molecular basis for recruiting SUMO ligases to the vicinity of their substrates to induce sumoylation upon DNA damage.

Protection of chromosomal ends from erroneous recognition as double‐strand breaks (DSBs) is critical for maintaining genome integrity. Unprotected ends can activate DNA repair pathways leading to catastrophic fusions or rearrangements of chromosomes. A study in The EMBO Journal identifies Uls1 as a new player in this end‐protection process, utilizing SUMO and ubiquitin modifications to prevent chromosomal fusions by clearing a polysumoylated form of Rap1. Telomeres, the ends of linear chromosomes, pose a tremendous challenge for eukaryotic cells, because their resemblance to DNA DSBs puts them at constant risk of being targeted by repair pathways. Thus, these ends must be kept off the radar of the two major DSB repair pathways, non‐homologous end joining (NHEJ) and homologous recombination (HR). A number of studies in the model system budding yeast previously identified a central role for the Rap1 protein in preventing NHEJ at telomeres. Rap1 is an evolutionarily conserved protein that binds to the double‐stranded DNA region of telomeres. It inhibits NHEJ via at least three different pathways (Marcand et al , 2008). Two of these are mediated by its binding partners Rif2 and Sir4, respectively, with a less well‐characterized third pathway independent of either interactor. How these pathways enable NHEJ evasion is not entirely clear, but recent findings of posttranslational modifications of telomere‐binding proteins, particularly by SUMO, hinted at a possible role for dynamic protein modifications in regulating telomeric functions. Attachment of the SUMO protein to a lysine residue in target proteins can change both their interaction profiles, by allowing other proteins to bind or impairing interactions, and their biochemical functions. Additionally, since the SUMO polypeptide itself contains lysines, poly‐SUMO chains can be generated, which …

The Ku70-Ku80 ring complex encloses DNA ends to facilitate telomere maintenance and DNA break repair. Many studies focus on the ring-forming regions of subunits Ku70 and Ku80. Less is known about the Ku70 C-terminal tail, which lies outside the ring. Our results suggest that this region is responsible for dynamic sumoylation of Yku70 upon DNA association in budding yeast. Mutating a cluster of five lysines in this region largely eliminates Yku70 sumoylation. Chromatin immunoprecipitation analyses show that yku70 mutants with these lysines replaced by arginines exhibit reduced Ku-DNA association at both telomeres and internal DNA breaks. Consistent with this physical evidence, Yku70 sumoylation deficiency is associated with impaired ability to block DNA end resection and suppression of multiple defects caused by inefficient resection. Correlating with these, yku70 mutants with reduced sumoylation levels exhibit shorter telomeres, increased G overhang levels, and altered levels of non-homologous end joining. We also show that diminution of sumoylation does not affect Yku70 protein levels or its interactions with protein and RNA partners. These results suggest a model whereby Yku70 sumoylation upon DNA association strengthens Ku-DNA interaction to promote multiple functions of Ku.

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.

Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed ‘yeast Endonuclease G (EndoG)’, is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5′–3′ exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog ‘EXOG’, a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5′–3′ exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.