Ataxia Telangiectasia Mutated Function Research Articles

ATAXIA TELANGIECTASIA MUTATED PROTECTS AGAINST LIPOPOLYSACCARIDE-INDUCED BLOOD-BRAIN BARRIER DISRUPTION BY REGULATING ATK/DRP1-MEDIATED MITOCHONDRIAL HOMEOSTASIS.

Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of ATM in lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) disruption remains unknown. This study aimed to investigate the role and underlying mechanism of ATM in the regulation of the BBB function in sepsis. Methods: We used LPS to induce BBB disruption in vivo and to establish an in vitro model of cerebrovascular endothelial cells. Blood-brain barrier disruption was assessed by measuring Evans blue leakage and expression of vascular permeability regulators. To investigate the role of ATM, its inhibitor AZD1390 and clinically approved doxorubicin, an anthracycline that can activate ATM, were administered as scheduled. To explore the underlying mechanism, protein kinase B (AKT) inhibitor MK-2206 was administered to block the AKT/dynamin-related protein 1 (DRP1) pathway. Results: Lipopolysaccharide challenge induced significant BBB disruption, ATM activation, and mitochondrial translocation. Inhibiting ATM with AZD1390 aggravated BBB permeability as well as the following neuroinflammation and neuronal injury, while activation of ATM by doxorubicin abrogated these defects. Further results obtained in brain microvascular endothelial cells showed that ATM inhibition reduced the phosphorylation of DRP1 at serine (S) 637, promoted excessive mitochondrial fission, and resulted in mitochondrial malfunction. By activating ATM, doxorubicin increased the protein binding between ATM and AKT and promoted the phosphorylated activation of AKT at S473, which could directly phosphorylate DRP1 at S637 to repress excessive mitochondrial fission. Consistently, the protective role of ATM was abolished by the AKT inhibitor MK-2206. Conclusions: Ataxia telangiectasia mutated protects against LPS-induced BBB disruption by regulating mitochondrial homeostasis, at least in part, through the AKT/DRP1 pathway.

Selective inhibition of ATM-dependent double-strand break repair and checkpoint control synergistically enhances the efficacy of ATR inhibitors.

Ataxia Telangiectasia and Rad3-Related Protein (ATR) kinase regulates a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell cycle checkpoints controlled by Ataxia Telangiectasia Mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1 phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell cycle checkpoints and DSB repair, lowered the p53 protective barrier and extended the life of ATR inhibitor induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Further, a combination study in 26 patient-derived xenograft models of triple negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared to single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.

Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation.

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions. Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

Ataxia Telangiectasia Mutated and MSH2 Control Blunt DNA End Joining in Ig Class Switch Recombination.

Class-switch recombination (CSR) produces secondary Ig isotypes and requires activation-induced cytidine deaminase (AID)-dependent DNA deamination of intronic switch regions within the IgH (Igh) gene locus. Noncanonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal switch regions. Ataxia telangiectasia mutated (ATM)-dependent phosphorylation of AID at serine 38 (pS38-AID) promotes its interaction with apurinic/apyrimidinic endonuclease 1 (APE1), a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene mutS homolog 2 (Msh2). Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele (Atmf) and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared with AtmD/D and Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from AtmD/DMsh2-/- B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared with wild-type, AtmD/D, or Msh2-/- B cells. These data indicate that the absence of both ATM and MSH2 blocks nonhomologous end joining, leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end joining during CSR through pS38-AID.

Characteristics of Rare Germline ATM Variants in Chronic Lymphocytic Leukemia (CLL)

Background The use of next-generation sequencing in the clinic has increasingly uncovered new variants of unknown significance (VUS), which are often ignored clinically. Ataxia telangiectasia mutated (ATM) is a tumor-suppressor gene that has a critical role in DNA double-strand break repair and has been implicated in the pathogenesis of CLL. In this study, we assessed rare germline ATM variants including VUS using a novel variant calling algorithm, evaluated their impact on the clinical characteristics of CLL and functionally assessed their effect on ATM kinase activity. Methods We evaluated the entire ATM coding region via germline whole exome sequencing from 885 CLL patients representing three different previously reported cohorts (327 DFCI; 276 German CLL Study Group, and 282 ICGC), along with a cohort of ancestry matched controls. We utilized DeepVariant (Poplin et al Nature Biotech 2018) to detect germline variants, followed by a standardized quality control workflow (AlDubayan et al JAMA 2020). All individuals harbored ATM germline variants. Variants were annotated using dbSNP, Clinvar and Varsome, and variants with GnomAD population allele frequency (AF) < 1% were defined as rare. For clinical characterization of CLL patients with rare germline ATM missense variants, we focused on the DFCI cohort. For functional analysis, we evaluated ATM kinase activity by ATM and KAP1 phosphorylation in response to 1 Gy gamma-irradiation using flow cytometry in primary CLL samples with/without rare germline ATM missense variants. We also generated CRISPR/Cas9 engineered cell lines to evaluate the function of ATM p.L2307F which was frequent in our CLL cohort and which we previously demonstrated as enriched in CLL, yet generally annotated as a VUS. Results We identified a total of 130 ATM germline variants among the CLL cases, including 87 missense, and 9 splice site alterations. 80 of 87 missense variants were rare and 59 were annotated as VUS. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In 63 DFCI patients with rare missense variants, the median age at diagnosis was 55 (31-82), 21.7% had a familial history of CLL and 29% had del(11q). Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to gamma-irradiation was reduced in 12 patients assessed (8 rare and 4 non-rare) with germline ATM variants (ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P<0.01). Similar results were seen when the analysis was restricted to the 8 CLL patients with rare ATM variants. Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del 11q + ATM variant group) compared to 13 of those with del(11q) alone (del 11q group) (P<0.01) (Figure 1). Altogether, these results suggest that germline ATM variants have a functional impact on DNA damage response in primary CLL cells. We previously identified the VUS variant ATM p.L2307F as particularly enriched among CLL patients (Tiao G et al., Leukemia. 2017). We therefore generated ATM L2307F knock-in NALM-6 cell lines via CRISPR/Cas9. We generated 7 cell lines including ATM wild type (+/+), ATM monoallelic mutated (+/L2307F), ATM monoallelic knocked out (+/-), ATM monoallelic mutated with knock out of the other allele (-/L2307F), and ATM biallelic knockout (-/-). The induction of both pATM and pKAP1 levels by gamma irradiation was reduced in proportion to ATM mutational status as shown in Figure 2. Conclusions We demonstrate that rare ATM germline variants are common in CLL and affect the DNA damage response in primary CLL cells when controlling for somatic 11q deletion. We further characterize the p.L2307F variant using CRISPR and demonstrate that this allele represents a functional hypomorph in cell-based assays. Further studies are needed to assess the impact of each individual rare germline variant on the function of ATM, CLL pathogenesis and the inherited risk of CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of atm in Zebrafish as a Model of Ataxia-Telangiectasia Syndrome.

Ataxia–telangiectasia mutated (ATM) is a key DNA damage signaling kinase that is mutated in humans with ataxia–telangiectasia (A-T) syndrome. This syndrome is characterized by neurodegeneration, immune abnormality, cancer predisposition, and premature aging. To better understand the function of ATM in vivo, we engineered a viable zebrafish model with a mutated atm gene. Zebrafish atm loss-of-function mutants show characteristic features of A-T-like motor disturbance, including coordination disorders, immunodeficiency, and tumorigenesis. The immunological disorder of atm homozygote fish is linked to the developmental blockade of hematopoiesis, which occurs at the adulthood stage and results in a decrease in infection defense but, with little effect on wound healing. Malignant neoplasms found in atm mutant fish were mainly nerve sheath tumors and myeloid leukemia, which rarely occur in A-T patients or Atm−/− mice. These results underscore the importance of atm during immune cell development. This zebrafish A-T model opens up a pathway to an improved understanding of the molecular basis of tumorigenesis in A-T and the cellular role of atm.

Abstract P054: RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key mediator of cellular DNA damage repair (DDR) and is activated in response to DNA replication stress. ATR is attractive as a drug target in tumors with loss-of-function alterations in complimentary DDR pathways, including ataxia-telangiectasia mutated (ATM) and BRCA. RESULTS: RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor. RP-3500 is highly potent with ATR kinase IC50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. It is highly selective with &amp;gt;30-fold selectivity over mTOR and &amp;gt;2000-fold selectivity over ATM, DNA-PK and PI3Kα kinases. Preclinical tumor xenograft models harboring synthetic lethal (SNIPRx) gene mutations whose loss of function sensitizes to RP-3500 were selected for in vivo studies. RP-3500 treatment resulted in potent single-agent in vivo efficacy and/or tumor regression in multiple models at minimum effective doses (MED) of 5–7 mg/kg once daily. Pharmacodynamic assessments validated target engagement, with a proportional relationship between tumor pCHK1(Ser345) inhibition and circulating RP-3500 plasma levels (IC80 = 18.6 nM). Circulating free plasma levels of RP-3500 at the MED indicate that exposure above the in vivo tumor pCHK1(Ser345) IC80 for 10–12 hours is sufficient for efficacy and dose proportional phosphorylation of DNA damage markers γ-H2AX, pDNA-PKcs and pKAP1. In vitro, tumor cells with ATM loss exhibited increased susceptibility to RP-3500 and a 3-day compound exposure was sufficient to generate a sustained DNA damage response compared to cells with functional ATM expression. In ATM-deficient mouse models, short-duration intermittent (weekly 3 days on/4 days off or 5 days on/2 days off) dosing schedules maximized tumor growth inhibition while minimizing the impact on hematology parameters, including red blood cell depletion. These results emphasize the reversible nature of erythroid toxicity with RP-3500 and the advantage of intermittent dosing schedules to alleviate anemia. The 3 days on/4 days off intermittent treatment schedule also substantially improved the efficacy and tolerability of RP-3500 and PARP inhibitor combinations compared to continuous treatment schedules. Intermittent treatments of RP-3500 given concomitantly with reduced doses of olaparib or niraparib demonstrated synergistic efficacy in Granta-519 (ATM mutn) and SUM149PT (BRCA1mutn) models with minimal hematological adverse effects and superior efficacy to sequential treatment. CONCLUSIONS: These results provide a strong preclinical rationale to support clinical investigation of the novel ATR inhibitor RP-3500 on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a means of maximizing clinical benefit. RP-3500 is currently evaluated in the ongoing phase 1 TRESR (Treatment Enabled by SNIPRx) study (NCT04497116). Citation Format: Anne Roulston, Michal Zimmerman, Robert Papp, Alex Skeldon, Charles Pellerin, Émilie Dumas-Bérube, Valerie Dumais, Stephane Dorich, Sara Fournier, Li Li, Marie-Ève Leclaire, Shou Yun Yin, Amandine Chefson, Hunain Alam, William Yang, Chloe Fugère-Desjardins, Sabrina Hammond, Kathryn Skorey, Amina Mulani, Victoria Rimkunas, Artur Veloso, Martine Hamel, Rino Stocco, Yael Mamane, Zuomei Li, Jordan Young, Mike Zinda, Cameron Black. RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P054.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

ATM inhibition enhances Auranofin-induced oxidative stress and cell death in lung cell lines.

Ataxia-Telangiectasia (A-T), a pleiotropic chromosomal breakage syndrome, is caused by the loss of the kinase Ataxia-telangiectasia mutated (ATM). ATM is not only involved in the response to DNA damage, but also in sensing and counteracting oxidative stress. Since a disturbed redox balance has been implicated in the pathophysiology of A-T lung disease, we aimed to further explore the interplay between ATM and oxidative stress in lung cells. Using a kinetic trapping approach, we could demonstrate an interaction between the trapping mutant TRX1-CS and ATM upon oxidative stress. We could further show that combined inhibition of thioredoxin reductase (TrxR) and ATM kinase activity, using Auranofin and KU55933 respectively, induced an increase in cellular reactive oxygen species (ROS) levels and protein oxidation in lung cells. Furthermore, ATM inhibition sensitized lung cells to Auranofin-induced cell death that could be rescued by ROS scavengers. As a consequence, targeted reduction of ATM by TRX1 could serve as a regulator of oxidative ATM activation and contribute to the maintenance of the cellular redox homeostasis. These results highlight the importance of the redox-active function of ATM in preventing ROS accumulation and cell death in lung cells.

SATMF Suppresses the Premature Senescence Phenotype of the ATM Loss-of-Function Mutant and Improves Its Fertility in Arabidopsis.

Leaf senescence is the final stage of leaf development. It is accompanied by the remobilization of nutrients from senescent leaves to developing organs. The occurrence of senescence is the consequence of integrating intrinsic and environmental signals. DNA damage triggered by stresses has been regarded as one of the reasons for senescence. To prevent DNA damage, cells have evolved elaborate DNA repair machinery. The ataxia telangiectasia mutated (ATM) functions as the chief transducer of the double-strand breaks (DSBs) signal. Our previous study suggests that ATM functions in lifespan regulation in Arabidopsis. However, ATM regulatory mechanism on plant longevity remains unclear. Here, we performed chemical mutagenesis to identify the components involved in ATM-mediated longevity and obtained three dominant mutants satmf1~3, suppressor of atm in fertility, displaying delayed senescence and restored fertility in comparison with atm mutant. Molecular cloning and functional analysis of SATMF (suppressor of atm in fertility) will help to understand the underlying regulatory mechanism of ATM in plants, and shed light on developing new treatments for the disease Ataxia-telangiectasia.

The Importance of ATM and ATR in Physcomitrella patens DNA Damage Repair, Development, and Gene Targeting.

Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana (A. thaliana) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrella patens (P. patens) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR, are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast (Saccharomyces cerevisiae). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens, suggesting that they represent adaptations to different demands for the maintenance of genome stability.

ATM Promotes RAD51-Mediated Meiotic DSB Repair by Inter-Sister-Chromatid Recombination in Arabidopsis.

Meiotic recombination ensures accurate homologous chromosome segregation during meiosis and generates novel allelic combinations among gametes. During meiosis, DNA double strand breaks (DSBs) are generated to facilitate recombination. To maintain genome integrity, meiotic DSBs must be repaired using appropriate DNA templates. Although the DNA damage response protein kinase Ataxia-telangiectasia mutated (ATM) has been shown to be involved in meiotic recombination in Arabidopsis, its mechanistic role is still unclear. In this study, we performed cytological analysis in Arabidopsis atm mutant, we show that there are fewer γH2AX foci, but more RAD51 and DMC1 foci on atm meiotic chromosomes. Furthermore, we observed an increase in meiotic Type I crossovers (COs) in atm. Our genetic analysis shows that the meiotic phenotype of atm rad51 double mutants is similar to the rad51 single mutant. Whereas, the atm dmc1 double mutant has a more severe chromosome fragmentation phenotype compared to both single mutants, suggesting that ATM functions in concert with RAD51, but in parallel to DMC1. Lastly, we show that atm asy1 double mutants also have more severe meiotic recombination defects. These data lead us to propose a model wherein ATM promotes RAD51-mediated meiotic DSB repair by inter-sister-chromatid (IS) recombination in Arabidopsis.

Inhibitory effect of alpha-lipoic acid on mitochondrial dysfunction and interleukin-8 expression in interleukin-1beta-stimulated ataxia teleangiectasia fibroblasts.

Ataxia telangiectasia (A-T) is an inherited neurodegenerative disease caused by mutation in the ataxia telangiectasia mutated (ATM) gene, leading to loss of function in the encoded protein ATM. Because ATM functions to reduce oxidative stress by up-regulating antioxidant enzymes, oxidative stress is a prevalent A-T phenotype and a mediator of the inflammation that drives A-T pathology. Reactive oxygen species (ROS) levels and the expression of pro-inflammatory cytokine interleukin-8 (IL-8) were higher in A-T cells than in normal cells. ROS are related to mitochondrial dysfunction and activation of nuclear factor kappa B (NF-κB) to induce IL-8 expression. Alpha-lipoic acid (α-LA), a naturally occurring thiol compound, shows an antioxidant effect in various cells. This study is aimed to determine if α-LA confers protection against NF-κB activation, IL-8 expression, and mitochondrial dysfunction in A-T cells which are exposed to the inflammatory cytokine IL-1β. A-T fibroblasts were treated with or without α-LA. The levels of intracellular and mitochondrial ROS, mRNA and protein levels of IL-8, mitochondrial membrane potential (MMP), ATP levels, and DNA binding activity of NF-κB were determined. As a result, IL-1β increased NF-κB activation, IL-8 expression, intracellular and mitochondrial ROS levels, but decreased MMP and ATP level in A-T cells. Pretreatment of A-T cells with α-LA inhibited IL-1β-induced activation of NF-κB, IL-8 expression, and mitochondrial dysfunction by reducing ROS levels. In conclusion, supplementation with α-LA may be beneficial for reducing the oxidative stress-induced mitochondrial dysfunction and IL-8 production associated with A-T.

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

Mitochondria at the crossroads of ATM-mediated stress signaling and regulation of reactive oxygen species

The Ataxia-telangiectasia mutated (ATM) kinase responds to DNA double-strand breaks and other forms of cellular stress, including reactive oxygen species (ROS). Recent work in the field has uncovered links between mitochondrial ROS and ATM activation, suggesting that ATM acts as a sensor for mitochondrial derived ROS and regulates ROS accumulation in cells through this pathway. In addition, characterization of cells from Ataxia-telangiectasia patients as well as ATM-deficient mice and cell models suggest a role for ATM in modulating mitochondrial gene expression and function. Here we review ROS responses related to ATM function, recent evidence for ATM roles in mitochondrial maintenance and turnover, and the relationship between ATM and regulation of protein homeostasis.

Rational Design of 5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4-((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (VX-970, M6620): Optimization of Intra- and Intermolecular Polar Interactions of a New Ataxia Telangiectasia Mutated and Rad3-Related (ATR) Kinase Inhibitor.

The DNA damage response (DDR) is a DNA damage surveillance and repair mechanism that can limit the effectiveness of radiotherapy and DNA-damaging chemotherapy, commonly used treatment modalities in cancer. Two related kinases, ataxia telangiectasia mutated (ATM) and ATM and Rad3-related kinase (ATR), work together as apical proteins in the DDR to maintain genome stability and cell survival in the face of potentially lethal forms of DNA damage. However, compromised ATM signaling is a common characteristic of tumor cells, which places greater reliance on ATR to mediate the DDR. In such circumstances, ATR inhibition has been shown to enhance the toxicity of DNA damaging chemotherapy to many cancer cells in multiple preclinical studies, while healthy tissue with functional ATM can tolerate ATR inhibition. ATR therefore represents a very attractive anticancer target. Herein we describe the discovery of VX-970/M6620, the first ATR inhibitor to enter clinical studies, which is based on a 2-aminopyrazine core first reported by Charrier ( J. Med. Chem. 2011 , 54 , 2320 - 2330 , DOI: 10.1021/jm101488z ).

Type 1 diabetes upregulates metastasis-associated protein 1- phosphorylated histone 2AX signaling in the testis

Under the sustained hyperglycemic state, oxidative stress induces irreparable DNA double-strand breaks resulting in germ cell death and testicular atrophy. Although molecular mechanisms underlying DNA damage repair in testicular cells are gradually getting unraveled, the effects on DNA double-strand breaks sensing are not precisely known. In this study, using streptozotocin-induced type 1 diabetic rats, we report that hyperglycemic state for one month or three months does not increase the levels of ataxia telangiectasia mutated (ATM) protein- an upstream kinase responsible for the phosphorylation of histone 2AX (Ɣ-H2AX)- after the formation of DNA double-strand breaks. The ATM expression is seminiferous epithelial stage-dependent in spermatogonia and primary spermatocytes, and the pattern of stage-dependent expression varies in diabetic rats, especially after three-month-long diabetes. However, the levels of metastasis-associated protein-1 (MTA1), an essential protein for ATM function, increase although not in a time-dependent manner. The amount of DNA double-strand breaks increases in a time- and stage-dependent manner as indicated by increased Ɣ-H2AX levels, especially in spermatogonia and primary spermatocytes, and in late spermatids in some tubular stages. Although ATM levels do not increase in diabetic rats, protein is expressed more or less in same testicular cells in which Ɣ-H2AX is expressed indicating that ATM might play a vital role in the phosphorylation of the histone. We conclude that diabetes upregulates MTA1-Ɣ-H2AX signaling in diabetic rat testis as a response to time-dependent increases in DNA double-strand breaks.

Formation of 53BP1 foci and ATM activation under oxidative stress is facilitated by RNA:DNA hybrids and loss of ATM-53BP1 expression promotes photoreceptor cell survival in mice

Background: Photoreceptors, light-sensing neurons in retina, are central to vision. Photoreceptor cell death (PCD) is observed in most inherited and acquired retinal dystrophies. But the underlying molecular mechanism of PCD is unclear. Photoreceptors are sturdy neurons that survive high oxidative and phototoxic stress, which are known threats to genome stability. Unexpectedly, DNA damage response in mice photoreceptors is compromised; mainly due to loss of crucial DNA repair proteins, ATM and 53BP1. We tried to understand the molecular function of ATM and 53BP1 in response to oxidative stress and how suppression of DNA repair response in mice retina affect photoreceptor cell survival. Methods: We use the state of art cell biology methods and structure-function analysis of mice retina. RNA:DNA hybrids (S9.6 antibody and Hybrid-binding domain of RNaseH1) and DNA repair foci (gH2AX and 53BP1) are quantified by confocal microscopy, in retinal sections and cultured cell lines. Oxidative stress, DNA double strand break, RNaseH1 expression and small-molecule kinase-inhibitors were used to understand the role of ATM and RNA:DNA hybrids in DNA repair. Lastly, retinal structure and function of ATM deficient mice, in Retinal degeneration 1 (Pde6brd1) background, is studied using Immunohistochemistry and Electroretinography. Results: Our work has three novel findings: firstly, both human and mice photoreceptor cells specifically accumulate RNA:DNA hybrids, a structure formed by re-hybridization of nascent RNA with template DNA during transcription. Secondly, RNA:DNA-hybrids promote ataxia-telangiectasia mutated (ATM) activation during oxidative stress and 53BP1-foci formation during downstream DNA repair process. Thirdly, loss of ATM -in murine photoreceptors- protract DNA repair but also promote their survival. Conclusions: We propose that due to high oxidative stress and accumulation of RNA:DNA-hybrids in photoreceptors, expression of ATM is tightly regulated to prevent PCD. Inefficient regulation of ATM expression could be central to PCD and inhibition of ATM-activation could suppress PCD in retinal dystrophy patients.

Formation of 53BP1 foci and ATM activation under oxidative stress is facilitated by RNA:DNA hybrids and loss of ATM-53BP1 expression promotes photoreceptor cell survival in mice.

Background: Photoreceptors, light-sensing neurons in retina, are central to vision. Photoreceptor cell death (PCD) is observed in most inherited and acquired retinal dystrophies. But the underlying molecular mechanism of PCD is unclear. Photoreceptors are sturdy neurons that survive high oxidative and phototoxic stress, which are known threats to genome stability. Unexpectedly, DNA damage response in mice photoreceptors is compromised; mainly due to loss of crucial DNA repair proteins, ATM and 53BP1. We tried to understand the molecular function of ATM and 53BP1 in response to oxidative stress and how suppression of DNA repair response in mice retina affect photoreceptor cell survival. Methods: We use the state of art cell biology methods and structure-function analysis of mice retina. RNA:DNA hybrids (S9.6 antibody and Hybrid-binding domain of RNaseH1) and DNA repair foci (gH2AX and 53BP1) are quantified by confocal microscopy, in retinal sections and cultured cell lines. Oxidative stress, DNA double strand break, RNaseH1 expression and small-molecule kinase-inhibitors were used to understand the role of ATM and RNA:DNA hybrids in DNA repair. Lastly, retinal structure and function of ATM deficient mice, in Retinal degeneration 1 (Pde6brd1) background, is studied using Immunohistochemistry and Electroretinography. Results: Our work has three novel findings: firstly, both human and mice photoreceptor cells specifically accumulate RNA:DNA hybrids, a structure formed by re-hybridization of nascent RNA with template DNA during transcription. Secondly, RNA:DNA-hybrids promote ataxia-telangiectasia mutated (ATM) activation during oxidative stress and 53BP1-foci formation during downstream DNA repair process. Thirdly, loss of ATM -in murine photoreceptors- protract DNA repair but also promote their survival. Conclusions: We propose that due to high oxidative stress and accumulation of RNA:DNA-hybrids in photoreceptors, expression of ATM is tightly regulated to prevent PCD. Inefficient regulation of ATM expression could be central to PCD and inhibition of ATM-activation could suppress PCD in retinal dystrophy patients.

Inflammation, a significant player of Ataxia-Telangiectasia pathogenesis?

Ataxia-Telangiectasia (A-T) syndrome is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, chromosome instability, radiosensitivity, and predisposition to malignancy. There is growing evidence that A-T patients suffer from pathologic inflammation that is responsible for many symptoms of this syndrome, including neurodegeneration, autoimmunity, cardiovascular disease, accelerated aging, and insulin resistance. In addition, epidemiological studies have shown A-T heterozygotes, somewhat like deficient patients, are susceptible to ionizing irradiation and have a higher risk of cancers and metabolic disorders. This review summarizes clinical and molecular findings of inflammation in A-T syndrome. Ataxia-Telangiectasia Mutated (ATM), a master regulator of the DNA damage response is the protein known to be associated with A-T and has a complex nuclear and cytoplasmic role. Loss of ATM function may induce immune deregulation and systemic inflammation.