Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway.

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

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.

Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution.

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.

Imetelstat-mediated alterations in fatty acid metabolism to induce ferroptosis as a therapeutic strategy for acute myeloid leukemia.

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing substantial disease control in AML.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Heritable defects in telomere and mitotic function selectively predispose to sarcomas.

Cancer genetics has to date focused on epithelial malignancies, identifying multiple histotype-specific pathways underlying cancer susceptibility. Sarcomas are rare malignancies predominantly derived from embryonic mesoderm. To identify pathways specific to mesenchymal cancers, we performed whole-genome germline sequencing on 1644 sporadic cases and 3205 matched healthy elderly controls. Using an extreme phenotype design, a combined rare-variant burden and ontologic analysis identified two sarcoma-specific pathways involved in mitotic and telomere functions. Variants in centrosome genes are linked to malignant peripheral nerve sheath and gastrointestinal stromal tumors, whereas heritable defects in the shelterin complex link susceptibility to sarcoma, melanoma, and thyroid cancers. These studies indicate a specific role for heritable defects in mitotic and telomere biology in risk of sarcomas.

TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT.

In some types of cancer, telomere length is maintained by the alternative lengthening of telomeres (ALT) mechanism. In many ALT cancers, the α-thalassemia/mental retardation syndrome X-linked (ATRX) gene is mutated leading to the conclusion that the ATRX complex represses ALT. Here, we report that most high-grade pediatric osteosarcomas maintain their telomeres by ALT, and that the majority of these ALT tumors are ATRX wild-type (wt) and instead carry an amplified 17p11.2 chromosomal region containing TOP3A. We found that TOP3A was overexpressed in the ALT-positive ATRX-wt tumors consistent with its amplification. We demonstrated the functional significance of these results by showing that TOP3A overexpression in ALT cancer cells countered ATRX-mediated ALT inhibition and that TOP3A knockdown disrupted the ALT phenotype in ATRX-wt cells. Moreover, we report that TOP3A is required for proper BLM localization and promotes ALT DNA synthesis in ALT cell lines. Collectively, our results identify TOP3A as a major ALT player and potential therapeutic target.

Targeting telomeres: advances in telomere maintenance mechanism-specific cancer therapies.

Cancer cells establish replicative immortality by activating a telomere-maintenance mechanism (TMM), be it telomerase or the alternative lengthening of telomeres (ALT) pathway. Targeting telomere maintenance represents an intriguing opportunity to treat the vast majority of all cancer types. Whilst telomerase inhibitors have historically been heralded as promising anticancer agents, the reality has been more challenging, and there are currently no therapeutic options for cancer types that use ALT despite their aggressive nature and poor prognosis. In this Review, we discuss the mechanistic differences between telomere maintenance by telomerase and ALT, the current methods used to detect each mechanism, the utility of these tests for clinical diagnosis, and recent developments in the therapeutic strategies being employed to target both telomerase and ALT. We present notable developments in repurposing established therapeutic agents and new avenues that are emerging to target cancer types according to which TMM they employ. These opportunities extend beyond inhibition of telomere maintenance, by finding and exploiting inherent weaknesses in the telomeres themselves to trigger rapid cellular effects that lead to cell death.

Functional interaction between compound heterozygous TERT mutations causes severe telomere biology disorder.

Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.

Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers.

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomeric DNA comprises terminal tracts of G-rich tandem repeats, which are inherently difficult for the replication machinery to navigate. Structural aberrations that promote activation of the alternative lengthening of telomeres (ALT) pathway of telomere maintenance exacerbate replication stress at ALT telomeres, driving fork stalling and fork collapse. This form of telomeric DNA damage perpetuates recombination-mediated repair pathways and break-induced telomere synthesis. The relationship between replication stress and DNA repair is tightly coordinated for the purpose of regulating telomere length in ALT cells, but has been shown to be experimentally manipulatable. This raises the intriguing possibility that induction of replication stress can be used as a means to cause toxic levels of DNA damage at ALT telomeres, thereby selectively disrupting the viability of ALT cancers.

A DNA-fiber protocol for single molecule analysis of telomere (SMAT) length and extension events in cancer cells.

Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism. It is active in approximately 10-15% of cancers. We present a DNA-fiber protocol, combining YOYO-1 staining of genomic DNA, telomere fluorescence in situ hybridization (FISH), and EdU labeling of nascent DNA, to measure telomere extension events in ALT cancer cells. The protocol can be used to delineate ALT-mediated telomere extension. For complete details on the use and execution of this protocol, please refer to Barroso-Gonzalez et al. (2021).

qmotif: determination of telomere content from whole-genome sequence data.

Motivation: Changes in telomere length have been observed in cancer and can be indicative of mechanisms involved in carcinogenesis. Most methods used to estimate telomere length require laboratory analysis of DNA samples. Here, we present qmotif, a fast and easy tool that determines telomeric repeat sequences content as an estimate of telomere length directly from whole-genome sequencing. Results: qmotif shows similar results to quantitative PCR, the standard method for high-throughput clinical telomere length quantification. qmotif output correlates strongly with the output of other tools for determining telomere sequence content, TelSeq and TelomereHunter, but can run in a fraction of the time-usually under a minute. Availability and implementation: qmotif is implemented in Java and source code is available at https://github.com/AdamaJava/adamajava, with instructions on how to build and use the application available from https://adamajava.readthedocs.io/en/latest/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Anti-recombination function of MutSα restricts telomere extension by ALT-associated homology-directed repair.

Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ∼15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT.

TELO-SCOPE study: a randomised, double-blind, placebo-controlled, phase 2 trial of danazol for short telomere related pulmonary fibrosis.

INTRODUCTION: Recent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening. METHODS AND ANALYSIS: A multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population. ETHICS AND DISSEMINATION: Ethics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences. TRIAL REGISTRATION NUMBERS: NCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools.

Chromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging.

A novel cause of DKC1-related bone marrow failure: Partial deletion of the 3' untranslated region.

Telomere biology disorders (TBDs), including dyskeratosis congenita (DC), are a group of rare inherited diseases characterized by very short telomeres. Mutations in the components of the enzyme telomerase can lead to insufficient telomere maintenance in hematopoietic stem cells, resulting in the bone marrow failure that is characteristic of these disorders. While an increasing number of genes are being linked to TBDs, the causative mutation remains unidentified in 30-40% of patients with DC. There is therefore a need for whole genome sequencing (WGS) in these families to identify novel genes, or mutations in regulatory regions of known disease-causing genes. Here we describe a family in which a partial deletion of the 3' untranslated region (3' UTR) of DKC1, encoding the protein dyskerin, was identified by WGS, despite being missed by whole exome sequencing. The deletion segregated with disease across the family and resulted in reduced levels of DKC1 mRNA in the proband. We demonstrate that the DKC1 3' UTR contains two polyadenylation signals, both of which were removed by this deletion, likely causing mRNA instability. Consistent with the major function of dyskerin in stabilization of the RNA subunit of telomerase, hTR, the level of hTR was also reduced in the proband, providing a molecular basis for his very short telomeres. This study demonstrates that the terminal region of the 3' UTR of the DKC1 gene is essential for gene function and illustrates the importance of analyzing regulatory regions of the genome for molecular diagnosis of inherited disease.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.