ORCID Profile
0000-0002-0864-1254
Current Organisations
Children's Medical Research Institute
,
University of Sydney
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Cell Development, Proliferation and Death | Cell and Nuclear Division | Developmental Genetics (incl. Sex Determination) | Biochemistry and Cell Biology | Biochemistry and Cell Biology not elsewhere classified | Genetics
Publisher: American Association for Cancer Research (AACR)
Date: 15-02-2007
DOI: 10.1158/0008-5472.CAN-06-3672
Abstract: The maintenance of telomere length is essential for the indefinite proliferation of cancer cells. This is most often achieved by the activation of telomerase however, a substantial number of cancers lack detectable telomerase activity and are classified as using an alternative lengthening of telomeres (ALT) pathway. We showed recently that ALT cells have a high level of extrachromosomal telomeric circles (t circles) that may be a specific marker of the ALT phenotype. The mechanism underlying t circle production and the requirement of t circles in ALT remain unclear. Understanding the specific requirements of ALT is key to developing diagnostic tools and therapies that target this pathway and is critical for the treatment of cancers in which ALT is prevalent, including cancers of neuroepithelial and mesenchymal origin. In this study, we used short hairpin RNAs directed at either Xrcc3 or Nbs1, two proteins involved in the homologous recombination pathway, to determine the role of these proteins in t circle production and the requirement of t circles in maintaining the ALT pathway. We show that Xrcc3 and Nbs1 are indeed required for the production of t circles in human ALT. However, these cells continue to proliferate in the absence of t circles, suggesting that they are not required for the survival of ALT cells. [Cancer Res 2007 (4):1513–9]
Publisher: Springer Science and Business Media LLC
Date: 17-09-2019
DOI: 10.1038/S41467-019-12255-W
Abstract: Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.
Publisher: Springer Science and Business Media LLC
Date: 25-11-2020
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481334.V1
Abstract: Supplementary Tables S1-S5
Publisher: Cold Spring Harbor Laboratory
Date: 04-07-2020
DOI: 10.1101/2020.07.03.187385
Abstract: BRG1 (encoded by SMARCA4 ) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programs and cancer cell behaviour. Our investigation of SMARCA4 revealed that BRG1 is universally overexpressed in 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programs. Unexpectedly, depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation ( KLK2 , PCAT1 and VAV3 ). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the oncogenic transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. Our data have revealed that BRG1 has capacity to drive oncogenesis by coordinating oncogenic pathways dependent on BRG1 for proliferation, cell cycle progression and DNA replication.
Publisher: Springer Science and Business Media LLC
Date: 30-11-2020
DOI: 10.1038/S41556-020-00605-6
Abstract: Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.
Publisher: Cold Spring Harbor Laboratory
Date: 20-03-2020
DOI: 10.1101/2020.03.18.997817
Abstract: High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G 1 transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell ision. Y-box-binding protein-1, YB-1, is essential for cell ision, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of ision, specifically being required to initiate cytokinesis.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481337
Abstract: Supplementary Figures S1-S16
Publisher: Wiley
Date: 29-08-2014
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481334
Abstract: Supplementary Tables S1-S5
Publisher: Cold Spring Harbor Laboratory
Date: 09-03-2018
DOI: 10.1101/279877
Abstract: We used super-resolution microscopy to investigate the role of macromolecular telomere structure in chromosome end protection. In murine and human cells with reduced TRF2, we find that ATM-activation at chromosome ends occurs with a structural change from t-loops to linearized chromosome ends through t-loop unfolding. Comparably, we find Aurora B kinase regulates telomere linearity concurrent with ATM activation at telomeres during mitotic arrest. Using a separation of function allele, we find that the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that telomere linearity and ATM activation occur separately from telomere fusion via non-homologous end-joining (NHEJ). Further, we show that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1-arrest when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that regulate ATM activation at chromosome ends independent of mechanisms to suppress chromosome end fusion.
Publisher: Springer Science and Business Media LLC
Date: 10-05-2018
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.MAD.2007.11.006
Abstract: A substantial number of human tumors utilize a telomerase-independent telomere length maintenance mechanism referred to as alternative lengthening of telomeres (ALT). Although it is known that ALT is a telomere-specific, loss of function phenotype, which involves lengthening of telomeres by homologous recombination-mediated replication of telomeric DNA, many of the details of these processes require elucidation. Here we discuss the current literature on ALT and telomere capping, specifically focusing on how alterations in telomere capping functions may permit activation of ALT and explain the phenotypic characteristics of cells in which this occurs.
Publisher: The Company of Biologists
Date: 2019
DOI: 10.1242/JCS.222349
Abstract: 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 PML nuclear bodies (PML NBs) which 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 killing ATRX-deficient cells. Infection of co-cultured primary and ATRX-null cancer cells revealed that mutant HSV-1 selectively killed ATRX-null 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.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481325.V1
Abstract: HT10806TG_Panobinostat treatment
Publisher: Springer Science and Business Media LLC
Date: 06-2015
DOI: 10.1038/NATURE14513
Publisher: Elsevier BV
Date: 12-2012
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481325
Abstract: HT10806TG_Panobinostat treatment
Publisher: Springer Science and Business Media LLC
Date: 11-03-2012
DOI: 10.1038/NSMB.2245
Publisher: Elsevier BV
Date: 12-2012
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481328
Abstract: HT10806TG_CBL0137 treatment
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481322
Abstract: HT10806TG_Combination treatment
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481331.V1
Abstract: HT10806TG_Control
Publisher: Elsevier BV
Date: 02-2020
Publisher: Wiley
Date: 23-05-2014
DOI: 10.1111/ACEL.12234
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.C.6530664.V1
Abstract: AbstractPurpose: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. Experimental Design: The effects of the drug combination on cancer growth were examined i in vitro /i and in animal models of i MYCN /i - lified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. Results: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137 anobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. Conclusions: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies. /
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.C.6530664
Abstract: AbstractPurpose: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. Experimental Design: The effects of the drug combination on cancer growth were examined i in vitro /i and in animal models of i MYCN /i - lified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. Results: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137 anobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. Conclusions: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies. /
Publisher: Cold Spring Harbor Laboratory
Date: 13-05-2022
DOI: 10.1101/2022.05.12.491744
Abstract: Replication stress is an endemic threat to genome stability. For reasons unknown, replication stress response factors become essential during peri-implantation development. This coincides with a stem cell potency switch from the naïve to the primed state. Using genetically matched, chimera-derived mouse naïve embryonic (mESC) and primed epiblast stem cells (mEpiSC) we found that replication stress management differs between potency states. Primed mEpiSCs rely on Atr activity to prevent replication catastrophe, minimize genomic damage, avoid apoptosis, and re-enter the cell cycle. Conversely, under replications stress, mESCs readily activate Atm regardless of Atr activity, undergo replication catastrophe, and induce apoptosis. Primed pluripotent cells therefore engage Atr to counteract replication difficulties and maintain viability, whereas cells in the naïve state are more readily cleared under the same conditions. We anticipate these ergent strategies enable pluripotent cells of different potency states to meet associated proliferative or developmental demands during early development.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 04-10-2019
Abstract: The telomerase protein hTERT has a noncanonical role in promoting telomere protection by heat shock protein 70.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481331
Abstract: HT10806TG_Control
Publisher: Oxford University Press (OUP)
Date: 24-10-2009
DOI: 10.1093/NAR/GKP814
Publisher: Wiley
Date: 12-02-2009
Publisher: Cold Spring Harbor Laboratory
Date: 24-10-2018
DOI: 10.1101/451708
Abstract: “Replication stress” describes phenomena that alter DNA replication rates 1–3 . Multiple architectural challenges within the confined nuclear volume must be navigated during replication to prevent or repair replication stress. Cellular mechanisms potentiating changes in nuclear architecture that facilitate DNA replication remain unclear. Here we show that the ATR, IPMK and mTOR kinases regulate actin polymerisation in human cells to alter nuclear architecture and promote replication fork repair. We demonstrate that replication stress activates mTOR, in an ATR and IPMK-dependent manner, to induce polymerisation of nuclear filamentous actin (F-actin). mTOR and ATR then counteract replication stress-induced nuclear envelope deformation and increase nuclear volume through their regulation of actin dynamics. Additionally, we reveal that FANCD2 labelled replication forks colocalise with actin filaments in late S-phase. mTOR and ATR then regulate the mobility, speed and directionality of stalled replication foci within the three-dimensional nuclear architecture. Importantly, we find nuclear F-actin also acts as a substrate for the directed migration of stalled replication foci to the nuclear periphery. Suppressing mTOR and ATR-dependent actin forces prevents replication fork restart and promotes chromosome segregation errors in primary and cancer cell lines. Together, these data reveal that ATR and mTOR regulate actin dynamics in the replication stress response to alter nuclear architecture and maintain genome stability.
Publisher: Elsevier BV
Date: 07-2013
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481328.V1
Abstract: HT10806TG_CBL0137 treatment
Publisher: MDPI AG
Date: 09-2020
Abstract: High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell ision.
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.MOLCEL.2018.06.025
Abstract: Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.
Publisher: Wiley
Date: 07-2015
DOI: 10.1002/0471142956.CY1240S73
Abstract: In cancer cells, telomere length maintenance occurs largely via the direct synthesis of TTAGGG repeats at chromosome ends by telomerase, or less frequently by the recombination‐dependent alternative lengthening of telomeres (ALT) pathway. The latter is characterized by the atypical clustering of telomeres within promyelocytic leukemia (PML) nuclear bodies, which harbor proteins that are linked with DNA repair and recombination activity. For this reason, it is speculated that these associated PML bodies represent the sites of the recombination that maintains telomere length. The protocols described here can be employed for the routine investigation of the structural integrity of telomeres and the association of proteins at telomeres in normal cells, challenged cells, and archived formalin‐fixed paraffin‐embedded clinical tissue specimens that may have activated the ALT pathway. © 2015 by John Wiley & Sons, Inc.
Publisher: Proceedings of the National Academy of Sciences
Date: 27-03-2019
Abstract: To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia–telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.
Publisher: Cold Spring Harbor Laboratory
Date: 24-06-2022
DOI: 10.1101/2022.06.24.497492
Abstract: Faithful DNA replication requires the orderly firing of replication origins across the genome. At present, we lack details around how origins are selected for activation and the subsequent impact of this on replication dynamics. Here, we have investigated how chromatin organisation contributes to replication initiation and dynamics by intersecting ChIP-seq, Hi-C, Repli-seq, and OK-seq data from primary and tumour cells lines. We found replication initiation is significantly enriched at TAD boundaries, co-localizing with CTCF and cohesin in early and mid S-phase. Strong replication fork directionality (RFD) from initiation zones in TAD boundaries could occur in a bi- or uni-directional manner, which highly correlated with replication timing. While TAD boundaries were largely invariant, a minority of initiation zones were shared across cell lines, indicative of cell type specific regulation. These data are consistent with chromatin structure organizing replication initiation and dynamics, ensuring orderly completion of replication from TAD boundaries into TAD internal regions.
Publisher: Informa UK Limited
Date: 06-2005
Publisher: Proceedings of the National Academy of Sciences
Date: 10-02-2003
Abstract: The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC cl loader and proliferating cell nuclear antigen polymerase cl , respectively. Here, we demonstrate functional similarities between the replication and checkpoint cl loader/DNA cl pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two in idually purified checkpoint complexes also form a supercomplex in vitro , which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is cl ed around the DNA.
Publisher: Cold Spring Harbor Laboratory
Date: 03-02-2023
DOI: 10.1101/2023.02.01.526591
Abstract: The shelterin component telomeric repeat-binding factor 2 (TRF2) is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2 TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, resulting in a telomeric DNA damage response. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2 TRFH domain. We identify APOD53 as our most promising compound. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2 TRFH domain and induces phenotypes consistent with TRF2 TRFH domain mutants. These include induction of a telomeric DNA damage response in the absence of fusions, increased telomeric replication stress, and impaired recruitment of regulator of telomere elongation helicase 1 (RTEL1) and structure-specific endonuclease subunit (SLX4) to telomeres. We demonstrate that APOD53 impairs cell growth in both a telomerase-positive and an ALT cell line, while sparing the viability of non-cancerous cells. Finally, we find that co-treatment with APOD53 and the G4 stabilizer RHPS4 further exacerbates telomere replication stress.
Publisher: Elsevier BV
Date: 10-2021
DOI: 10.1016/J.TCB.2021.06.002
Abstract: The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.
Publisher: EMBO
Date: 09-12-2011
Abstract: Replicative senescence is accompanied by a telomere‐specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early‐passage normal human cells and increase in number with increasing cumulative cell isions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end‐to‐end fusions and mostly result from strand‐independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1‐phase cells just before senescence and after bypassing senescence by inactivation of wild‐type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53‐dependent senescence.
Publisher: Elsevier BV
Date: 12-2006
Publisher: Elsevier BV
Date: 10-2007
Publisher: MDPI AG
Date: 29-01-2020
Abstract: Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location.
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.PBIOMOLBIO.2019.04.002
Abstract: DNA replication plays a central role in genome health. Deleterious alteration of replication dynamics, or "replication stress", is a key driver of genome instability and oncogenesis. The replication stress response is regulated by the ATR kinase, which functions to mitigate replication abnormalities through coordinated efforts that arrest the cell cycle and repair damaged replication forks. mTOR kinase regulates signaling networks that control cell growth and metabolism in response to environmental cues and cell stress. In this review, we discuss interconnectivity between the ATR and mTOR pathways, and provide putative mechanisms for mTOR engagement in DNA replication and the replication stress response. Finally, we describe how connectivity between mTOR and replication stress may be exploited for cancer therapy.
Publisher: Informa UK Limited
Date: 2008
DOI: 10.1128/MCB.01122-07
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481337.V1
Abstract: Supplementary Figures S1-S16
Publisher: Wiley
Date: 17-09-2003
DOI: 10.1046/J.1365-313X.2003.01882.X
Abstract: Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5'-TTTAGGG-3'/3'-AAATCCC-5' (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481343
Abstract: Supplementary Figure Legends S1-S16
Publisher: Springer Science and Business Media LLC
Date: 30-03-2010
DOI: 10.1038/NRG2763
Abstract: Unlimited cellular proliferation depends on counteracting the telomere attrition that accompanies DNA replication. In human cancers this usually occurs through upregulation of telomerase activity, but in 10-15% of cancers - including some with particularly poor outcome - it is achieved through a mechanism known as alternative lengthening of telomeres (ALT). ALT, which is dependent on homologous recombination, is therefore an important target for cancer therapy. Although dissection of the mechanism or mechanisms of ALT has been challenging, recent advances have led to the identification of several genes that are required for ALT and the elucidation of the biological significance of some phenotypic markers of ALT. This has enabled development of a rapid assay of ALT activity levels and the construction of molecular models of ALT.
Publisher: Springer Science and Business Media LLC
Date: 22-11-2009
DOI: 10.1038/NSMB.1725
Abstract: Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.
Publisher: Cold Spring Harbor Laboratory
Date: 24-09-2018
DOI: 10.1101/425264
Abstract: Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death promotes genome instability in replication stressed cells through erse mechanisms depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and genome instability.
Publisher: Elsevier BV
Date: 08-2002
DOI: 10.1086/341750
Publisher: Informa UK Limited
Date: 11-2004
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481343.V1
Abstract: Supplementary Figure Legends S1-S16
Publisher: American Association for Cancer Research (AACR)
Date: 16-05-2021
DOI: 10.1158/1078-0432.CCR-20-2357
Abstract: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN- lified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137 anobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.
Publisher: Cold Spring Harbor Laboratory
Date: 11-10-2022
DOI: 10.1101/2022.10.10.511510
Abstract: The Eyes Absent family of 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 molecular targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as a direct interactor and phosphatase substrate of both EYA4 and EYA1, with pY445 on PLK1 being the primary target site. EYA-mediated dephosphorylation of PLK1 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 the PLK1-activating proteins BORA and CEP192. 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 novel phosphotyrosine signalling network governing PLK1 and mitosis. This work provides a mechanism of cell killing for EYA phosphatase inhibitors with important therapeutic implications.
Publisher: Cold Spring Harbor Laboratory
Date: 26-03-2022
DOI: 10.1101/2022.03.25.485759
Abstract: Telomere shortening places a key limitation on cell proliferation 1 . In all vertebrates explored to date, this limitation is overcome by telomerase-dependent telomere extension. Failure to maintain telomere length results in premature ageing and functional impairments in highly replicative cell populations as telomeres erode 2 . Alternative lengthening of telomeres (ALT), a telomerase-independent mechanism, compensates for telomere loss in a subset of human cancer cell lines 2 . Here, we demonstrate that the highly regenerative newt Pleurodeles waltl lacks telomerase activity, contains telomeres distinct from all known vertebrates in both sequence and structure, and deploys ALT for physiological telomere maintenance. This constitutes the first report of telomerase-independent resolution of the end-replication problem at the whole-organism level within Chordata . P. waltl telomere biology is distinct amongst vertebrates and uses ALT at the whole-organism level.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22481322.V1
Abstract: HT10806TG_Combination treatment
Location: United States of America
Location: United States of America
Start Date: 2015
End Date: 2019
Funder: University of Sydney
View Funded ActivityStart Date: 2015
End Date: 2017
Funder: Cancer Council NSW
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2019
Funder: Cancer Institute NSW
View Funded ActivityStart Date: 2014
End Date: 2018
Funder: Australian Cancer Research Foundation
View Funded ActivityStart Date: 2009
End Date: 2012
Funder: National Cancer Institute
View Funded ActivityStart Date: 2013
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2018
Funder: Cancer Institute NSW
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2006
End Date: 2008
Funder: National Science Foundation
View Funded ActivityStart Date: 2021
End Date: 12-2023
Amount: $555,892.00
Funder: Australian Research Council
View Funded ActivityStart Date: 04-2022
End Date: 03-2026
Amount: $1,082,000.00
Funder: Australian Research Council
View Funded Activity