Publications by authors named "Roger R Reddel"

105 Publications

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Sci Rep 2021 Aug 23;11(1):17052. Epub 2021 Aug 23.

AIST-INDIA DAILAB, DBT-AIST International Center for Translational & Environmental Research (DAICENTER), National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, 305-8565, Japan.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21-mediated signaling to limit proliferation in TEP cells.
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http://dx.doi.org/10.1038/s41598-021-96096-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382839PMC
August 2021

Alternative lengthening of telomeres is not synonymous with mutations in ATRX/DAXX.

Nat Commun 2021 03 10;12(1):1552. Epub 2021 Mar 10.

Cancer Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW, Australia.

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http://dx.doi.org/10.1038/s41467-021-21794-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946928PMC
March 2021

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

Clin Cancer Res 2021 Mar 11;27(5):1438-1451. Epub 2020 Dec 11.

Department of Pathology, School of Medical Sciences, UNSW Sydney, Sydney, New South Wales, Australia.

Purpose: gene rearrangement with transcriptional superenhancers leads to overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with -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 -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 -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 -rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with -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 -rearranged neuroblastoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3044DOI Listing
March 2021

Role of POT1 in Human Cancer.

Cancers (Basel) 2020 Sep 24;12(10). Epub 2020 Sep 24.

Cancer Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead NSW 2145, Australia.

Telomere abnormalities facilitate cancer development by contributing to genomic instability and cellular immortalization. The Protection of Telomeres 1 (POT1) protein is an essential subunit of the shelterin telomere binding complex. It directly binds to single-stranded telomeric DNA, protecting chromosomal ends from an inappropriate DNA damage response, and plays a role in telomere length regulation. Alterations of have been detected in a range of cancers. Here, we review the biological functions of POT1, the prevalence of germline and somatic mutations across cancer predisposition syndromes and tumor types, and the dysregulation of POT1 expression in cancers. We propose a framework for understanding how POT1 abnormalities may contribute to oncogenesis in different cell types. Finally, we summarize the clinical implications of POT1 alterations in the germline and in cancer, and possible approaches for the development of targeted cancer therapies.
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http://dx.doi.org/10.3390/cancers12102739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598640PMC
September 2020

Strategies to enable large-scale proteomics for reproducible research.

Nat Commun 2020 07 30;11(1):3793. Epub 2020 Jul 30.

ProCan®, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.
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http://dx.doi.org/10.1038/s41467-020-17641-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393074PMC
July 2020

End Products of Telomere Research.

Cell Stem Cell 2020 06;26(6):804-805

Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia.

Most rare inherited telomere biology disorders and some common aging-related diseases are associated with shortened telomeres. In this issue of Cell Stem Cell, insights into one of the rarest genetic causes of these disorders led to the discovery (Nagpal et al., 2020) of small molecules that lengthen telomeres.
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http://dx.doi.org/10.1016/j.stem.2020.05.006DOI Listing
June 2020

Author Correction: The FANCM-BLM-TOP3A-RMI complex suppresses alternative lengthening of telomeres (ALT).

Nat Commun 2019 Nov 20;10(1):5345. Epub 2019 Nov 20.

Telomere Length Regulation Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, 2145 NSW, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-13097-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863874PMC
November 2019

Addressing the Challenges of High-Throughput Cancer Tissue Proteomics for Clinical Application: ProCan.

Proteomics 2019 11 24;19(21-22):e1900109. Epub 2019 Sep 24.

ProCan, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, 2145, Australia.

The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in the development of mass spectrometry (MS)-based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big-data analytics that are refined in other fields of 'omics research. ProCan (ProCan is a registered trademark) is a program aiming to generate high-quality tissue proteomic data across a broad spectrum of cancer types. It is based on data-independent acquisition-MS proteomic analysis of annotated tissue samples sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high-throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, sample preparation, raw data acquisition, and data analysis. The ultimate goal is to establish a large proteomics knowledge-base that, in combination with other cancer 'omics data, will accelerate cancer research.
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http://dx.doi.org/10.1002/pmic.201900109DOI Listing
November 2019

The FANCM-BLM-TOP3A-RMI complex suppresses alternative lengthening of telomeres (ALT).

Nat Commun 2019 05 28;10(1):2252. Epub 2019 May 28.

Telomere Length Regulation Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, 2145, NSW, Australia.

The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.
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http://dx.doi.org/10.1038/s41467-019-10180-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538672PMC
May 2019

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

J Cell Sci 2019 03 14;132(5). Epub 2019 Mar 14.

Cancer Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia

Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.
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http://dx.doi.org/10.1242/jcs.222349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432714PMC
March 2019

Regulation of the interferon-gamma (IFN-γ) pathway by p63 and Δ133p53 isoform in different breast cancer subtypes.

Oncotarget 2018 Jun 26;9(49):29146-29161. Epub 2018 Jun 26.

Pathology Department, University of Otago, Dunedin, New Zealand.

The family consists of three sets of transcription factor genes, , and , each of which expresses multiple RNA variants and protein isoforms. Of these, is mutated in 25-30% of breast cancers. How mutations affect the interaction of family members and their isoforms in breast cancer is unknown. To investigate this, 3 independent breast cancer cohorts were stratified into 4 groups based on oestrogen receptor (ER) and mutation status. Using bioinformatic methodologies, principal signalling pathways associated with the expression of family members were identified. Results show an enrichment of IFN-γ signalling associated with RNA in wild type (wt), ER negative (ER-) tumours and with RNA in mutant (m) ER positive (ER+) tumours. Moreover, tumours with low IFN-γ signalling were associated with significantly poorer patient outcome. The predicted changes in expression of a subset of RNAs involved in IFN-γ signalling were confirmed . Our data show that different members of the family can drive transcription of genes involved in IFN-γ signalling in different breast cancer subgroups.
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http://dx.doi.org/10.18632/oncotarget.25635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044385PMC
June 2018

Telomere sequence content can be used to determine ALT activity in tumours.

Nucleic Acids Res 2018 06;46(10):4903-4918

Telomere Length Regulation Unit, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.
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http://dx.doi.org/10.1093/nar/gky297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007693PMC
June 2018

Functional dissection of breast cancer risk-associated promoter variants.

Oncotarget 2017 Sep 26;8(40):67203-67217. Epub 2017 May 26.

Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

The multi-cancer susceptibility locus at 5p15.33 includes , encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the promoter. Collectively, our functional studies of the - breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which promoter variants may affect breast cancer risk.
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http://dx.doi.org/10.18632/oncotarget.18226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620167PMC
September 2017

BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.

EMBO J 2017 10 6;36(19):2907-2919. Epub 2017 Sep 6.

Telomere Length Regulation Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia

Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM-TOP3A-RMI (BTR) dissolvase complex is required for ALT-mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3-dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA. This process is counteracted by the SLX4-SLX1-ERCC4 complex, which promotes resolution of the recombination intermediate, resulting in telomere exchange in the absence of telomere extension. Our data are consistent with ALT being a conservative DNA replication process, analogous to break-induced replication, which is dependent on BTR and counteracted by SLX4 complex-mediated resolution events.
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http://dx.doi.org/10.15252/embj.201796889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623873PMC
October 2017

MatCol: a tool to measure fluorescence signal colocalisation in biological systems.

Sci Rep 2017 08 21;7(1):8879. Epub 2017 Aug 21.

Bioinformatics Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia.

Protein colocalisation is often studied using pixel intensity-based coefficients such as Pearson, Manders, Li or Costes. However, these methods cannot be used to study object-based colocalisations in biological systems. Therefore, a novel method is required to automatically identify regions of fluorescent signal in two channels, identify the co-located parts of these regions, and calculate the statistical significance of the colocalisation. We have developed MatCol to address these needs. MatCol can be used to visualise protein and/or DNA colocalisations and fine tune user-defined parameters for the colocalisation analysis, including the application of median or Wiener filtering to improve the signal to noise ratio. Command-line execution allows batch processing of multiple images. Users can also calculate the statistical significance of the observed object colocalisations compared to overlap by random chance using Student's t-test. We validated MatCol in a biological setting. The colocalisations of telomeric DNA and TRF2 protein or TRF2 and PML proteins in >350 nuclei derived from three different cell lines revealed a highly significant correlation between manual and MatCol identification of colocalisations (linear regression R = 0.81, P < 0.0001). MatCol has the ability to replace manual colocalisation counting, and the potential to be applied to a wide range of biological areas.
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http://dx.doi.org/10.1038/s41598-017-08786-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566543PMC
August 2017

Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres.

Cell Rep 2017 06;19(12):2544-2556

Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.
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http://dx.doi.org/10.1016/j.celrep.2017.05.087DOI Listing
June 2017

Withaferin-A kills cancer cells with and without telomerase: chemical, computational and experimental evidences.

Cell Death Dis 2017 04 20;8(4):e2755. Epub 2017 Apr 20.

DAILAB, National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba 305 8565, Japan.

Maintenance of telomere length is the most consistent attribute of cancer cells. Tightly connected to their capacity to overcome replicative mortality, it is achieved either by activation of telomerase or an Alternative mechanism of Lengthening of Telomeres (ALT). Disruption of either of these mechanisms has been shown to induce DNA damage signalling leading to senescence or apoptosis. Telomerase inhibitors are considered as potential anticancer drugs but are ineffective for ALT cancers (~15% of all cancers). Withaferin-A (Wi-A), a major constituent of the medicinal plant, Withania somnifera (Ashwagandha), has been shown to exert anti-tumour activity. However, its effect on either telomerase or ALT mechanisms has not been investigated. Here, by using isogenic cancer cells with/without telomerase, we found that Wi-A caused stronger cytotoxicity to ALT cells. It was associated with inhibition of ALT-associated promyelocytic leukemia nuclear bodies, an established marker of ALT. Comparative analyses of telomerase positive and ALT cells revealed that Wi-A caused stronger telomere dysfunction and upregulation of DNA damage response in ALT cells. Molecular computational and experimental analyses revealed that Wi-A led to Myc-Mad mediated transcriptional suppression of NBS-1, an MRN complex protein that is an essential component of the ALT mechanism. The results suggest that Wi-A could be a new candidate drug for ALT cancers.
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http://dx.doi.org/10.1038/cddis.2017.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477593PMC
April 2017

Limiting Thymic Precursor Supply Increases the Risk of Lymphoid Malignancy in Murine X-Linked Severe Combined Immunodeficiency.

Mol Ther Nucleic Acids 2017 Mar 10;6:1-14. Epub 2016 Dec 10.

Gene Therapy Research Unit, Children's Medical Research Institute, The University of Sydney and The Sydney Children's Hospitals Network, Westmead, NSW 2145, Australia; Discipline of Child and Adolescent Health, The University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

In early gene therapy trials for SCID-X1, using γ-retroviral vectors, T cell leukemias developed in a subset of patients secondary to insertional proto-oncogene activation. In contrast, we have reported development of T cell leukemias in SCID-X1 mice following lentivirus-mediated gene therapy independent of insertional mutagenesis. A distinguishing feature in our study was that only a proportion of transplanted γc-deficient progenitors were transduced and therefore competent for reconstitution. We hypothesized that reconstitution of SCID-X1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy. To test this hypothesis, in the absence of transduction, SCID-X1 mice were reconstituted with serially fewer wild-type hematopoietic progenitors. A robust inverse correlation between hematopoietic progenitor cell dose and T-lymphoid malignancy was observed, with earlier disease onset at lower cell doses. Malignancies were of donor origin and carried activating Notch1 mutations. These findings align with emerging evidence that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Although insertional proto-oncogene activation is required for the development of malignancy in humans, failure of γc-deficient thymocytes to effectively compete with this at-risk cell population may have also contributed to oncogenesis observed in early SCID-X1 trials.
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http://dx.doi.org/10.1016/j.omtn.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363493PMC
March 2017

Whole-genome landscape of pancreatic neuroendocrine tumours.

Nature 2017 03 15;543(7643):65-71. Epub 2017 Feb 15.

QIMR Berghofer Medical Research Institute, Herston Road, Brisbane 4006, Australia.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
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http://dx.doi.org/10.1038/nature21063DOI Listing
March 2017

DNA methylation mediated up-regulation of TERRA non-coding RNA is coincident with elongated telomeres in the human placenta.

Mol Hum Reprod 2016 11 7;22(11):791-799. Epub 2016 Sep 7.

Murdoch Childrens Research Institute-Cancer and Disease Epigenetics, Royal Children's Hospital Flemington Road, Parkville, Melbourne, Victoria 3052, Australia

Study Question: What factors regulate elongated telomere length in the human placenta?

Summary Answer: Hypomethylation of TERRA promoters in the human placenta is associated with high TERRA expression, however, no clear mechanistic link between these phenomena and elongated telomere length in the human placenta was found.

What Is Known Already: Human placenta tissue and trophoblasts show longer telomere lengths compared to gestational age-matched somatic cells. However, telomerase (hTERT) expression and activity in the placenta is low, suggesting a role for an alternative lengthening of telomeres (ALT). While ALT is observed in 10-15% of human cancers and in some mouse stem cells, ALT has never been reported in non-cancerous human tissues.

Study Design, Samples/materials, Methods: Human term placental tissue and matched cord blood mononuclear cells (CBMCs) were collected as part of the Peri/Postnatal Epigenetic Twins study (PETS). In addition, first trimester placental villi, purified cytotrophoblasts, choriocarcinoma cell lines and a panel of ALT-positive cancer cell lines were tested. Telomere length was determined using the Terminal Restriction Fragment (TRF) assay and a relative quantitative PCR method. DNA methylation levels at several CpG rich subtelomeric TERRA promoters were determined using bisulfite conversion and the SEQUENOM EpiTYPER platform. Expression of TERRA and hTERT was determined using quantitative RT-PCR. ALT was assessed using the C-circle assay (CCA).

Main Results And The Role Of Chance: The human placenta tissue and purified first trimester trophoblasts showed low subtelomeric (TERRA) DNA methylation compared to matched CBMCs and other somatic cells. Interestingly placental TERRA methylation was lower than ALT-cancer cell lines, previously reported to be hypomethylated at these loci. Low TERRA methylation was associated with higher expression of TERRA RNA in placenta compared to matched CBMCs. Detectable levels of C-circles were observed in first trimester placental villi, but not term placenta, suggesting that the ALT mechanism may be active in specific placental cells in early gestation. C-circle analysis of purified first trimester trophoblasts and ALT-associated PML bodies (APB) staining of first trimester villi cross-sections failed to identify this specific cell type population.

Limitations, Reasons For Caution: While first trimester villi showed detectable levels of C-circles, these levels were very low compared with those observed in ALT-positive tumours and cell lines. This is consistent with a small sub-population of ALT-positive cells but this requires further investigation. Finally, no mechanistic link was established between TERRA DNA methylation, the presence of C-circles and longer telomere length.

Wider Implications Of The Findings: Given the previously described role of TERRA ncRNA as a negative regulator of telomerase, the finding of elevated TERRA and long telomeres is counterintutive. ALT as a mechanism for telomere length maintenance has only been reported in certain human cancers, and recently in mouse embryonic stem cells and embryos. As with many aspects of cancer, it appears that ALT activity in tumours may be the inappropriate activation of a pathway found in very specific cell types in human development. Our data are the first supportive evidence for ALT in a non-cancerous human tissue, a result that requires further investigation and replication. The level of TERRA methylation in the human placenta is significantly lower than found in ALT cancer cell lines and somatic cells, raising the possibility of a novel mechanism in maintaining low methylation at subtelomeric regions.

Large Scale Data: Not applicable.

Study Funding And Competing Interests: This study was supported by NHMRC early career fellowship (B.N.), NHMRC Senior Research Fellowship (R.S.) and the Victoria Government Infrastructure Grant. R.R. holds a patent for the C-circle assay. No other conflicts declared.
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http://dx.doi.org/10.1093/molehr/gaw053DOI Listing
November 2016

The C-Circle Assay for alternative-lengthening-of-telomeres activity.

Methods 2017 02 3;114:74-84. Epub 2016 Sep 3.

Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia.

The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.
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http://dx.doi.org/10.1016/j.ymeth.2016.08.016DOI Listing
February 2017

Comparative analysis of whole genome sequencing-based telomere length measurement techniques.

Methods 2017 02 24;114:4-15. Epub 2016 Aug 24.

Telomere Length Regulation Group, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia. Electronic address:

Telomeres are regions of repetitive DNA at the ends of human chromosomes that function to maintain the integrity of the genome. Telomere attrition is associated with cellular ageing, whilst telomere maintenance is a prerequisite for malignant transformation. Whole genome sequencing (WGS) captures sequence information from the entire genome, including the telomeres, and is increasingly being applied in research and in the clinic. Several bioinformatics tools have been designed to determine telomere content and length from WGS data, and include Motif_counter, TelSeq, Computel, qMotif, and Telomerecat. These tools utilise different approaches to identify, quantify and normalise telomeric reads; however, it is not known how they compare to one another. Here we describe the details and utility of each tool, and directly compare WGS telomere length output with laboratory-based telomere length measurements. In addition, we evaluate the accessibility, practicality, speed, and additional features of each tool. Each tool was tested using a range of telomere read extraction criteria, to determine the optimal parameters for the specific WGS read length. The aim of this article is to improve the accessibility of WGS telomere length measurement tools, which have the potential to be applied to WGS cohorts for clinical as well as research benefit.
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http://dx.doi.org/10.1016/j.ymeth.2016.08.008DOI Listing
February 2017

Molecular mechanisms of activity and derepression of alternative lengthening of telomeres.

Nat Struct Mol Biol 2015 Nov 4;22(11):875-80. Epub 2015 Nov 4.

Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.

Alternative lengthening of telomeres (ALT) involves homology-directed telomere synthesis. This multistep process is facilitated by loss of the ATRX or DAXX chromatin-remodeling factors and by abnormalities of the telomere nucleoprotein architecture, including altered DNA sequence and decreased TRF2 saturation. Induction of telomere-specific DNA damage triggers homology-directed searches, and NuRD-ZNF827 protein-protein interactions provide a platform for the telomeric recruitment of homologous recombination (HR) proteins. Telomere lengthening proceeds by strand exchange and template-driven DNA synthesis, which culminates in dissolution of HR intermediates.
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http://dx.doi.org/10.1038/nsmb.3106DOI Listing
November 2015

Is cell culture a risky business? Risk analysis based on scientist survey data.

Int J Cancer 2016 Feb 14;138(3):664-70. Epub 2015 Sep 14.

CellBank Australia, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia.

Cell culture is a technique that requires vigilance from the researcher. Common cell culture problems, including contamination with microorganisms or cells from other cultures, can place the reliability and reproducibility of cell culture work at risk. Here we use survey data, contributed by research scientists based in Australia and New Zealand, to assess common cell culture risks and how these risks are managed in practice. Respondents show that sharing of cell lines between laboratories continues to be widespread. Arrangements for mycoplasma and authentication testing are increasingly in place, although scientists are often uncertain how to perform authentication testing. Additional risks are identified for preparation of frozen stocks, storage and shipping.
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http://dx.doi.org/10.1002/ijc.29817DOI Listing
February 2016

A Common Cancer Risk-Associated Allele in the hTERT Locus Encodes a Dominant Negative Inhibitor of Telomerase.

PLoS Genet 2015 Jun 8;11(6):e1005286. Epub 2015 Jun 8.

Telomere Length Regulation Group, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.

The TERT-CLPTM1L region of chromosome 5p15.33 is a multi-cancer susceptibility locus that encodes the reverse transcriptase subunit, hTERT, of the telomerase enzyme. Numerous cancer-associated single-nucleotide polymorphisms (SNPs), including rs10069690, have been identified within the hTERT gene. The minor allele (A) at rs10069690 creates an additional splice donor site in intron 4 of hTERT, and is associated with an elevated risk of multiple cancers including breast and ovarian carcinomas. We previously demonstrated that the presence of this allele resulted in co-production of full length (FL)-hTERT and an alternatively spliced, INS1b, transcript. INS1b does not encode the reverse transcriptase domain required for telomerase enzyme activity, but we show here that INS1b protein retains its ability to bind to the telomerase RNA subunit, hTR. We also show that INS1b expression results in decreased telomerase activity, telomere shortening, and an increased telomere-specific DNA damage response (DDR). We employed antisense oligonucleotides to manipulate endogenous transcript expression in favor of INS1b, which resulted in a decrease in telomerase activity. These data provide the first detailed mechanistic insights into a cancer risk-associated SNP in the hTERT locus, which causes cell type-specific expression of INS1b transcript from the presence of an additional alternative splice site created in intron 4 by the risk allele. We predict that INS1b expression levels cause subtle inadequacies in telomerase-mediated telomere maintenance, resulting in an increased risk of genetic instability and therefore of tumorigenesis.
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http://dx.doi.org/10.1371/journal.pgen.1005286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459975PMC
June 2015

ATRX represses alternative lengthening of telomeres.

Oncotarget 2015 Jun;6(18):16543-58

Cancer Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia.

The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599288PMC
http://dx.doi.org/10.18632/oncotarget.3846DOI Listing
June 2015

Coordinated epigenetic remodelling of transcriptional networks occurs during early breast carcinogenesis.

Clin Epigenetics 2015 1;7:52. Epub 2015 May 1.

Epigenetic Research Laboratory, Genomics and Epigenetic Division, The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010 Australia ; St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Level 5 deLacy Building, St Vincent's Hospital, Victoria Street, Darlinghurst, NSW 2010 Australia.

Background: Dysregulation of the epigenome is a common event in malignancy; however, deciphering the earliest cancer-associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of human mammary epithelial cells (HMEC) and a matched variant cell population (vHMEC) that have spontaneously escaped senescence and undergone partial carcinogenic transformation. Using this model of basal-like breast carcinogenesis, we provide striking new insights into the very first epigenetic changes that occur during the initial stages of malignancy.

Results: The first phase of malignancy is defined by coordinated changes in the epigenome. At the chromatin level, this is embodied in long-range epigenetic deregulation, which involves the concomitant but atypical acquisition or loss of active and repressive histone modifications across large regional blocks. Changes in DNA methylation also occurs in a highly coordinated manner. We identified differentially methylated regions (DMRs) in the very earliest passages of vHMECs. Notably, we find that differential methylation targets loci regulated by key transcription factors including p53, AHR and E2F family members suggesting that epigenetic deregulation of transcription factor binding is a key event in breast carcinogenesis. Interestingly, DMRs identified in vHMEC are extensively methylated in breast cancer, with hypermethylation frequently encroaching into neighbouring regions. A subset of vHMEC DMRs exhibited a strong basal-like cancer specific hypermethylation.

Conclusions: Here, we generated epigenome-wide maps of the earliest phase of breast malignancy and show long-range epigenetic deregulation and coordinated DNA hypermethylation targets loci regulated by key transcription factors. These findings support a model where induction of breast cancer occurs through epigenetic disruption of transcription factor binding leading to deregulation of cancer-associated transcriptional networks. With their stability and very early occurrence, vHMECs hypermethylated loci could serve as excellent biomarkers for the initial detection of basal breast cancer.
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http://dx.doi.org/10.1186/s13148-015-0086-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424562PMC
May 2015

STC1 expression is associated with tumor growth and metastasis in breast cancer.

Clin Exp Metastasis 2015 Jan 13;32(1):15-27. Epub 2014 Nov 13.

Cancer Research Unit, Children's Medical Research Institute, Westmead, NSW, Australia.

Stanniocalcin-1 (STC1) is a secreted glycoprotein implicated in several pathologies including retinal degeneration, cerebral ischemia, angiogenesis and inflammation. Aberrant STC1 expression has been reported in breast cancer but the significance of this is not clear. High levels of STC1 expression were found in the aggressive 4T1 murine mammary tumor cells and in the MDA-MB-231 human breast cancer line. To investigate its significance, stable clones with STC1 down-regulation using shRNA were generated in both tumor models. The consequences of STC1 down-regulation on cell proliferation, chemotactic invasion, tumor growth and metastasis were assessed. Down-regulation of STC1 in the 4T1 murine mammary tumor cells had a major impact on mammary tumor growth. This observation was replicated in a second tumor model with the MDA-MB-231 human breast cancer line, with a significant reduction in primary tumor formation and a major inhibition of metastasis as well. Interestingly, in both models, proliferation in vitro was not affected. Subsequent microarray gene expression profiling identified 30 genes to be significantly altered by STC1 down-regulation, the majority of which are associated with known hallmarks of carcinogenesis. Furthermore, bioinformatic analysis of breast cancer datasets revealed that high expression of STC1 is associated with poor survival. This is the first study to show definitively that STC1 plays an oncogenic role in breast cancer, and indicates that STC1 could be a potential therapeutic target for treatment of breast cancer patients.
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http://dx.doi.org/10.1007/s10585-014-9687-9DOI Listing
January 2015

Inherited bone marrow failure associated with germline mutation of ACD, the gene encoding telomere protein TPP1.

Blood 2014 Oct 9;124(18):2767-74. Epub 2014 Sep 9.

Children's Medical Research Institute, Westmead, NSW, Australia; University of Sydney, Sydney, NSW, Australia;

Telomerase is a ribonucleoprotein enzyme that is necessary for overcoming telomere shortening in human germ and stem cells. Mutations in telomerase or other telomere-maintenance proteins can lead to diseases characterized by depletion of hematopoietic stem cells and bone marrow failure (BMF). Telomerase localization to telomeres requires an interaction with a region on the surface of the telomere-binding protein TPP1 known as the TEL patch. Here, we identify a family with aplastic anemia and other related hematopoietic disorders in which a 1-amino-acid deletion in the TEL patch of TPP1 (ΔK170) segregates with disease. All family members carrying this mutation, but not those with wild-type TPP1, have short telomeres. When introduced into 293T cells, TPP1 with the ΔK170 mutation is able to localize to telomeres but fails to recruit telomerase to telomeres, supporting a causal relationship between this TPP1 mutation and bone marrow disorders. ACD/TPP1 is thus a newly identified telomere-related gene in which mutations cause aplastic anemia and related BMF disorders.
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http://dx.doi.org/10.1182/blood-2014-08-596445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215308PMC
October 2014
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