Ataxia Telangiectasia-mutated And Rad3-related Pathway Research Articles

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake.

Effects of Sepantronium Bromide (YM-155) on the Whole Transcriptome of MDA-MB-231 Cells: Highlight on Impaired ATR/ATM Fanconi Anemia DNA Damage Response.

Sepantronium bromide (YM-155) is believed to elicit apoptosis and mitotic arrest in tumor cells by reducing (BIRC5, survivin) mRNA. In this study, we monitored changes in survivin mRNA and protein after treating MDA-MB-231 cells with YM-155 concurrent with evaluation of whole transcriptomic (WT) mRNA and long intergenic non-coding RNA at 2 time points: 8 h sub-lethal (83 ng/mL) and 20 h at the LC50 (14.6 ng/mL). The data show a tight association between cell death and the precipitating loss of survivin protein and mRNA (-2.67 fold-change (FC), p<0.001) at 20 h, questioning if the decline in survivin is attributed to cell death or drug impact. The meager loss of survivin mRNA was overshadowed by enormous differential change to the WT in both magnitude and significance for over 2000 differentially up/down-regulated transcripts: (+22 FC to -12 FC, p<0.001). The data show YM-155 to up-regulate transcripts in control of circadian rhythm (NOCT, PER, BHLHe40, NFIL3), tumor suppression (SIK1, FOSB), histone methylation (KDM6B) and negative feedback of NF-kappa B signaling (TNFAIP3). Down-regulated transcripts by YM-155 include glucuronidase (GUSBP3), numerous micro-RNAs, DNA damage repair elements (CENPI, POLQ, RAD54B) and the most affected system was the ataxia-telangiectasia mutated (ATM)/Fanconi anemia E3 monoubiquitin ligase core complexes (FANC transcripts - A/B/E/F/G/M), FANC2, FANCI, BRCA1, BRCA2, RAD51, PALB2 gene and ATR (ATM- and Rad3-Related) pathway. In conclusion, these findings suggest that a primary target of YM-155 is the loss of replicative DNA repair systems.

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.

Abstract 3632: Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound that was used for treating arthritis. Its primary mechanism of action is the inhibition of thioredoxin reductase (TRR) activity. It consequently induces oxidative/ER stress and also inhibits Heat shock protein (Hsp) 90 chaperone function. Ataxia-telangiectasia mutated and Rad3-related (ATR), Brca1, Chk1 and DNA-PK are Hsp90 client proteins that participate in DNA repair pathways. Based on our observation that AF induces DNA damage, we evaluated whether treatment with AF also impairs Hsp90-dependent DNA damage repair pathways in Mantle Cell Lymphoma (MCL) cells. It has been reported that ATM mutations are found in about 50% cases of MCL. Earlier studies have shown that ATM mutant MCL cells are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors in vitro and in vivo. We hypothesized that by inhibiting Hsp90-dependent DNA repair pathways (such as the ATR pathway), AF would sensitize ATM-proficient MCL cells to PARP inhibition by ABT-888. We demonstrate that treatment of ATM-proficient MCL cell lines, JeKo-1, MO2058 and Z138C with AF induced reactive oxygen species (ROS) levels and resulted in a concomitant decrease in TRR activity. Microarray analysis of AF-treated primary MCL cells revealed that AF significantly altered heat shock response genes, ER and oxidative stress-inducible genes and a subset of genes that were regulated by ATM or Brca1. Treatment with AF also induced Hsp90 hyperacetylation and resulted in the depletion of Hsp90 client proteins including ATR, AKT and Cyclin D1 in MCL cells. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (&amp;lt; 15% apoptosis) (p &amp;lt; 0.01). Co-treatment of MCL cells with AF and the PARP inhibitor ABT-888 resulted in synergistic increase in apoptosis of ATM-proficient MCL cells, with an increase in the induction of γH2AX foci and depletion of p-Chk1 (a downstream target of ATR signaling). This was accompanied with a concomitant increase in the levels of the ER stress-induced transcription factor CHOP (C/EBP homologous protein), cleaved Caspase 3 and PARP. Collectively, our data suggests the synthetic lethal effects observed by the simultaneous inhibition of DNA repair pathway(s) and PARP activity can be extended to ATM-proficient MCL cells. These pre-clinical studies create a strong rationale to determine the in vivo activity of the combination ABT-888 with AF in MCL. Citation Format: Siddhartha Ganguly, Trisha Home, Sumedha Gunewardena, Scott Weir, Joseph McGuirk, Rekha M. Rao. Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2015-3632

David Gardner-Medwin

Sepantronium bromide (YM-155) is believed to elicit apoptosis and mitotic arrest in tumor cells by reducing (BIRC5, survivin) mRNA. In this study, we monitored changes in survivin mRNA and protein...

Human Parvovirus B19 DNA Replication Induces a DNA Damage Response That Is Dispensable for Cell Cycle Arrest at Phase G 2 /M

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

Targeted Mutations in the ATR Pathway Define Agent-Specific Requirements for Cancer Cell Growth and Survival

Many anticancer agents induce DNA strand breaks or cause the accumulation of DNA replication intermediates. The protein encoded by ataxia-telangiectasia mutated and Rad 3-related (ATR) generates signals in response to these altered DNA structures and activates cellular survival responses. Accordingly, ATR has drawn increased attention as a potential target for novel therapeutic strategies designed to potentiate the effects of existing drugs. In this study, we use a unique panel of genetically modified human cancer cells to unambiguously test the roles of upstream and downstream components of the ATR pathway in the responses to common therapeutic agents. Upstream, the S-phase-specific cyclin-dependent kinase (Cdk) 2 was required for robust activation of ATR in response to diverse chemotherapeutic agents. While Cdk2-mediated ATR activation promoted cell survival after treatment with many drugs, signaling from ATR directly to the checkpoint kinase Chk1 was required for survival responses to only a subset of the drugs tested. These results show that specifically inhibiting the Cdk2/ATR/Chk1 pathway via distinct regulators can differentially sensitize cancer cells to a wide range of therapeutic agents.

ATR signalling: more than meeting at the fork

Preservation of genome integrity via the DNA-damage response is critical to prevent disease. ATR (ataxia telangiectasia mutated- and Rad3-related) is essential for life and functions as a master regulator of the DNA-damage response, especially during DNA replication. ATR controls and co-ordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints and DNA repair. Since its identification 15 years ago, a model of ATR activation and signalling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. In the present article, we review the ATR signalling process, focusing on mechanistic findings garnered from the identification of new ATR-interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation and point out the significant gaps in our understanding of this essential genome-maintenance pathway.

Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

The DNA damage signaling pathways mediated by the ataxia-telangiectasia mutated (ATM) and the ATM and Rad3-related (ATR) kinases play crucial roles in the maintenance of genomic integrity and may function as an anti-cancer barrier during early tumorigenesis. Although the ATM and ATR pathways share some of their downstream functions, the DNA damage that evoke these two pathways are distinct. While ATM plays a primary role in the response to double-stranded DNA breaks (DSBs), ATR controls the response to a much broader spectrum of DNA damage, including many that interfere with DNA replication. And, unlike ATM, ATR is crucial for maintaining genomic integrity during S phase of the cell cycle, and is indispensable for cell survival. Clearly, revealing the DNA structure that elicits the ATR pathway would be a critical step toward understanding the essential function of ATR and the genomic instability that it counters. The versatility of the ATR pathway in DNA damage response suggests that this pathway is likely able to sense a common signal generated by different types of DNA damage and genomic instability. Two simple structures commonly generated at sites of DNA repair and stressed DNA replication forks are single-stranded DNA coated with replication protein A (RPA-ssDNA) and junctions of singleand doublestranded DNA. Both of these structures have been implicated in the activation of ATR checkpoint by a number of studies using different model organisms. In this issue of Genes & Development, Cimprich and colleagues (MacDougall et al. 2007) report that circular singlestranded DNA (ssDNA) annealed with primers specifically triggers the ATR-mediated checkpoint responses in Xenopus egg extracts, revealing the first defined DNA structure sufficient to activate the ATR checkpoint pathway.

Herpes simplex virus type I disrupts the ATR-dependent DNA-damage response during lytic infection

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATR-signaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, gammaH2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.

Menin Localizes to Chromatin Through an ATR-CHK1 Mediated Pathway After UV-Induced DNA Damage

Menin is the tumor suppressor protein product of the gene identified in MEN1 syndrome. Evidence suggests menin binds DNA and interacts with proteins implicated in DNA damage pathways. The canonical cellular response to UV-induced DNA damage involves activation of the ataxia-telangiectasia-mutated and Rad3-related (ATR) kinase pathway. HEK293 cells were irradiated in a UV chamber. Menin's cellular location before and after UV irradiation was investigated by extracting four separate cellular components--a soluble, two chromatin and a nuclear matrix. To block the ATR pathway, we treated with 5 microM of caffeine for 1 h before irradiation. The ATR pathway was further investigated by transiently transfecting HEK293 cells with two mammalian CHK1 expression constructs--full length CHK1 and truncated active CHK1. A 24-h post UV-irradiation time course was studied and demonstrated menin concentration in the chromatin peaked at 4 h. At 4 h post-irradiation, menin concentration in the chromatin increased in a dose dependent manner and demonstrated a 2.8-fold maximal increase. HEK293 cells were pretreated with caffeine, an inhibitor of the ATR. Caffeine decreased menin localization to the chromatin after UV. Constitutively active CHK1 (1-365) transfection increased chromatin-bound menin, mimicking UV irradiation. Menin localizes to the chromatin after UV irradiation. Caffeine blocks menin localization to the chromatin after UV-irradiation. Over expressing active CHK1 (1-365) increased chromatin-bound menin, similar to UV. The data suggest menin localization to chromatin after UV irradiation is the result of an ATR-CHK1 dependent pathway.

Involvement of the ATR- and ATM-Dependent Checkpoint Responses in Cell Cycle Arrest Evoked by Pierisin-1

Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N(2) of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G(2)-M-phase delay. In the colony survival assays, Rad17(-/-) DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM(-/-) DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling.

Caffeine Inhibits Human Immunodeficiency Virus Type 1 Transduction of Nondividing Cells

Caffeine is an efficient inhibitor of DNA repair and DNA damage-activated checkpoints. We have shown recently that caffeine inhibits retroviral transduction of dividing cells, most likely by blocking postintegration repair. This effect may be mediated at least in part by a cellular target of caffeine, the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase. In this study, we present evidence that caffeine also inhibits efficient transduction of nondividing cells. We observed reduced transduction in caffeine-treated growth-arrested cells as well as caffeine-treated terminally differentiated human neurons and macrophages. Furthermore, this deficiency was observed with a human immunodeficiency virus type 1 (HIV-1) vector lacking Vpr, indicating that the effect is independent of the presence of this viral protein in the infecting virion. Finally, we show that HIV-1 transduction of nocodazole-arrested cells is reduced in cells that express an ATR dominant-negative protein (kinase-dead ATR [ATRkd]) and that the residual transduction of ATRkd-expressing cells is relatively resistant to caffeine. Taken together, these data suggest that the effect(s) of caffeine on HIV-1 transduction is mediated at least partly by the inhibition of the ATR pathway but is not dependent on the caffeine-mediated inhibition of cell cycle checkpoints.