Ataxia Telangiectasia Mutated Protein Research Articles

Multidisciplinary Management of Ataxia Telangiectasia: Current Perspectives.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia mutated (ATM) gene. In the absence of a family history, the diagnosis of A-T is usually not made until the child is older and symptomatic. Classic A-T is characterized by a constellation of clinical symptoms including progressive ataxia, oculocutaneous telangiectasias and sinopulmonary disease and is usually associated with absence of ATM protein. Other laboratory features associated with A-T include elevated serum levels of alpha-fetoprotein (AFP) and increased chromosomal breakage with in vitro exposure to ionizing radiation. Sinopulmonary symptoms can occur to varying degrees across the lifespan. Some children will also have hypogammaglobulinemia and impaired antibody responses requiring supplemental gamma globulin. People with hypomorphic ATM mutations are often considered to have mild A-T with onset of ataxia and neurological progression occurring later in life with less impairment of the immune system. The risk of malignancy, however, is significantly increased in people with either classic or mild A-T. While hematological malignancies are most common in the first two decades of life, solid organ malignancies become increasingly common during young adulthood. Deterioration of neurologic function with age is associated with dysphagia with aspiration, growth faltering, loss of ambulation and decline in pulmonary function, morbidities that contribute to shortened life expectancy and decreased quality of life. Premature death is often due to malignancies or chronic respiratory insufficiency. A-T is currently managed with supportive care and symptomatic treatment. Current clinical trials, however, represent progress and hope towards disease-modifying therapies for A-T.

The DNA repair protein ATM as a target in autism spectrum disorder.

Impairment of the GABAergic system has been reported in epilepsy, autism, attention deficit hyperactivity disorder, and schizophrenia. We recently demonstrated that ataxia telangiectasia mutated (ATM) directly shapes the development of the GABAergic system. Here, we show for the first time to our knowledge how the abnormal expression of ATM affects the pathological condition of autism. We exploited 2 different animal models of autism, the methyl CpG binding protein 2–null (Mecp2y/–) mouse model of Rett syndrome and mice prenatally exposed to valproic acid, and found increased ATM levels. Accordingly, treatment with the specific ATM kinase inhibitor KU55933 (KU) normalized molecular, functional, and behavioral defects in these mouse models, such as (a) delayed GABAergic development, (b) hippocampal hyperexcitability, (c) low cognitive performances, and (d) social impairments. Mechanistically, we demonstrate that KU administration to WT hippocampal neurons leads to (a) higher early growth response 4 activity on Kcc2b promoter, (b) increased expression of Mecp2, and (c) potentiated GABA transmission. These results provide evidence and molecular substrates for the pharmacological development of ATM inhibition in autism spectrum disorders.

First-in-Human Trial of the Oral Ataxia Telangiectasia and RAD3-Related (ATR) Inhibitor BAY 1895344 in Patients with Advanced Solid Tumors.

Targeting the ataxia telangiectasia and RAD3-related (ATR) enzyme represents a promising anticancer strategy for tumors with DNA damage response (DDR) defects and replication stress, including inactivation of ataxia telangiectasia mutated (ATM) signaling. We report the dose-escalation portion of the phase I first-in-human trial of oral ATR inhibitor BAY 1895344 intermittently dosed 5 to 80 mg twice daily in 21 patients with advanced solid tumors. The MTD was 40 mg twice daily 3 days on/4 days off. Most common adverse events were manageable and reversible hematologic toxicities. Partial responses were achieved in 4 patients and stable disease in 8 patients. Median duration of response was 315.5 days. Responders had ATM protein loss and/or deleterious ATM mutations and received doses ≥40 mg twice daily. Overall, BAY 1895344 is well tolerated, with antitumor activity against cancers with certain DDR defects, including ATM loss. An expansion phase continues in patients with DDR deficiency. SIGNIFICANCE: Oral BAY 1895344 was tolerable, with antitumor activity in heavily pretreated patients with various advanced solid tumors, particularly those with ATM deleterious mutations and/or loss of ATM protein; pharmacodynamic results supported a mechanism of action of increased DNA damage. Further study is warranted in this patient population.See related commentary by Italiano, p. 14.This article is highlighted in the In This Issue feature, p. 1.

Ataxia Telangiectasia Mutated Protein Kinase: A Potential Master Puppeteer of Oxidative Stress-Induced Metabolic Recycling.

Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.

ATM Expression as a Prognostic Marker in Patients With Advanced Biliary Tract Cancer Treated With First-line Gemcitabine and Platinum Chemotherapy

Biliary tract cancer (BTC) has a poor prognosis due to its highly invasive and metastatic potential. Ataxia-telangiectasia mutated (ATM) is a key regulator of DNA damage response and an emerging therapeutic target; however, the association between the expression of ATM and the prognosis in advanced BTC is unknown. We aimed to identify the relationship between ATM expression, clinicopathological characteristics, and survival outcomes in patients with advanced BTC. We analyzed 113 patients with advanced BTC who received first-line gemcitabine and platinum. The tumor location was intrahepatic cholangiocarcinoma (IH-CCC) in 43 patients, extrahepatic cholangiocarcinoma (EH-CCC) in 49, and gallbladder (GB) cancer in 21 patients. Fifty-four patients (47.8%) exhibited loss of ATM protein expression. The overall response rate (ORR) of ATM loss and intact ATM was 13.3% and 19.6%, respectively. In a subgroup analysis, EH-CCC patients with ATM loss tended to have improved PFS after platinum-based chemotherapy compared to those with intact ATM (7.9 vs. 6.2 months, respectively; p=0.050). We demonstrated that ATM loss could be a prognostic marker after platinum-based chemotherapy in patients with advanced EH-CCC.

Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.

DDRE-05. DECREASED EXPRESSION OF DNA DAMAGE RESPONSE PATHWAYS CORRELATES WITH SYNERGISTIC ACTIVITY OF TEMOZOLOMIDE WITH ATR INHIBITORS IN GLIOBLASTOMA

Abstract The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most important gaps in enabling the development of effective targeted cancer therapies. Increasing evidence supports the notion that DNA damage response (DDR) signaling plays an important role in inducing radiation and temozolomide (TMZ) resistance in glioblastoma (GBM) and hence, it has emerged as a molecular target for therapeutic development. The cellular response to TMZ therapy is critically mediated by MGMT and DDR signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related protein (ATR). ATR is a primary sensor of DNA damage initiating subsequent repair leading to cell survival. Here, we show that three clinical grade ATR inhibitors (Bay1895344, AZD6738 and VE-822) had similar selective sensitivity pattern across eight glioma-like stem cells (GSC) lines tested with three (GSC28, GSC11, GSC274) being the most sensitive, and three (GSC300, GSC272, and GSC20) being the most resistant cell lines. Interestingly, all three ATR inhibitors showed a significant synergism with TMZ (Combination index and Bliss model) in GSC300, GSC272, and GSC20, and antagonism in GSC28, GSC11, and GSC274. Further analysis showed that the synergistic group was resistant to single agent ATR inhibitor, whereas antagonistic group was inherently sensitive to ATR inhibitor. The synergistic response was not associated with any major genetic alterations such as MGMT status, EGFR amplification, PTEN deletion/mutation, or p53 deletion/mutation. However, GSEA analysis of baseline RNA-seq data of the 6 cell lines showed an enhanced expression of six DNA damage/repair/response pathways in the antagonistic group. It suggests that a low level of DNA damage/repair/response pathways could be a marker of synergy for TMZ with ATR inhibitors in GBM. Further studies are underway to validate this observation in more GSC lines in vitro and in vivo.

Predictive and Prognostic Value of DNA Damage Response Associated Kinases in Solid Tumors.

Dysfunctional DNA repair with subsequent genome instability and high mutational burden represents a major hallmark of cancer. In established malignant tumors, increased DNA repair capacity mediates resistance to DNA-damaging therapeutics, including cytotoxic drugs, radiotherapy, and selected small molecules including inhibitors of poly (ADP-ribose) polymerase (PARP), Ataxia Telangiectasia Mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), and Wee1 kinase (Wee1). In addition, DNA repair deficiency is not only associated with sensitivity to selected anticancer drugs, but also with increased mutagenicity and increased neoantigen load on tumor cells, resulting in increased immunogenicity and improved response to CTLA4- or PD-(L)1 targeting monoclonal antibodies. DNA damage response (DDR) is composed of complex signalling pathways, including the sensing of the DNA damage, signal transduction, cellular response pathways to DNA damage, and activation of DNA repair. DNA double strand breaks (DSBs) are the most dangerous form of DNA damage. Tumor cells are characterised by frequent accumulation of DSBs caused by either endogenous replication stress or the impact of cancer treatment, most prominently chemotherapy and radiotherapy. Therefore, response of cancer cells to DSBs represents a crucial mechanism for how tumors respond to systemic treatment or radiotherapy, and how resistance develops. Ample clinical evidence supports the importance of DDR associated kinases as predictive and prognostic biomarkers in cancer patients. The ATM-CHK2 and ATR-CHK1-WEE1 pathways initiate DNA DSB repair. In the current review, we focus on major DDR associated kinases including ATM, ATR, CHK1, CHK2, and WEE1, and discuss their potential prognostic and predictive value in solid malignancies.

Bone Marrow Transplantation as Therapy for Ataxia-Telangiectasia: A Systematic Review

Simple SummaryAtaxia-Telangiectasia is a rare neurodegenerative disease and patients die in their early forties mainly because of immunodeficiency, leukemia and lymphoma. In this work we describe the different routes of diagnosis, the recent treatments, and the emerging therapies for the disease. Bone marrow transplantation from siblings or an unrelated donor is becoming an option of therapy for selected Ataxia-Telangiectasia patients to deal with the immunodeficiency and to prevent leukemia and lymphoma. The patients require a non-myeloablative conditioning before bone marrow transplantation because they are radiosensitive to ionizing radiations and radiomimetics. The eligibility of patients for transplantation such as the range of age, spectrum of clinical features, and conditioning are still to be determined, transplant protocol guidelines must be defined and longer follow-ups are necessary to evaluate the risks and side effects associated with transplantation.Ataxia-Telangiectasia (A-T) is a rare autosomal recessive disorder, first reported in 1926, caused by a deficiency of ATM (Ataxia-Telangiectasia Mutated) protein. The disease is characterized by progressive cerebellar neurodegeneration, immunodeficiency, leukemia, and lymphoma cancer predisposition. Immunoglobulin replacement, antioxidants, neuroprotective factors, growth, and anti-inflammatory hormones are commonly used for A-T treatment, but, to date, there is no known cure. Bone marrow transplantation (BMT) is a successful therapy for several forms of diseases and it is a valid approach for tumors, hemoglobinopathies, autoimmune diseases, inherited disorders of metabolism, and other pathologies. Some case reports of A-T patients have shown that BMT is becoming a good option, as a correct engraftment of healthy cells can restore some aspects of immunologic capacity. However, due to a high risk of mortality as a result of a clinical and cellular hypersensitivity to ionizing radiation and radiomimetic drugs, a specific non-myeloablative conditioning is required before BMT. Although BMT might be considered as one promising therapy for the treatment of immunological defects and cancer prevention in selected A-T patients, the therapy is currently not recommended or recognized and the eligibility of A-T patients for BMT is a point to deepen and deliberate.

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp

Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22–70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (H2O2). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Activation of ATM kinase by ROS generated during ionophore-induced mitophagy in human T and B cell malignancies.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor, also possesses non-nuclear functions owing to its presence in extra-nuclear compartments, including peroxisomes, lysosomes, and mitochondria. ATM is frequently altered in several human cancers. Recently, we and others have shown that loss of ATM is associated with defective mitochondrial autophagy (mitophagy) in ataxia-telangiectasia (A-T) fibroblasts and B-cell lymphomas. Further, we reported that ATM protein but not ATM kinase activity is required for mitophagy. However, the mechanism of ATM kinase activation during ionophore-induced mitophagy is unknown. In the work reported here, using several ionophores in A-T and multiple T-cell and B-cell lymphoma cell lines, we show that ionophore-induced mitophagy triggers oxidative stress-induced ATMSer1981 phosphorylation through ROS activation, which is different from neocarzinostatin-induced activation of ATMSer1981, Smc1Ser966, and Kap1Ser824. We used A-T cells overexpressed with WT or S1981A (auto-phosphorylation dead) ATM plasmids and show that ATM is activated by ROS-induced oxidative stress emanating from ionophore-induced mitochondrial damage and mitophagy. The antioxidants N-acetylcysteine and glutathione significantly inhibited ROS production and ATMSer1981 phosphorylation but failed to inhibit mitophagy as determined by retroviral infection with mt-mKeima construct followed by lysosomal dual-excitation ratiometric pH measurements. Our data suggest that while ATM kinase does not participate in mitophagy, it is activated via elevated ROS.

Genomic and Clinicopathologic Characterization of ATM-deficient Prostate Cancer.

The ATM (ataxia telangiectasia mutated) gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, although ATM-deficient prostate cancers have not been well-characterized. We genetically validated a clinical grade IHC assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMAs). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes. ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for biallelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of biallelic inactivation, compared with zero of four of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in grade group 5 (17/181; 9%) compared with all other grades (8/650; 1%; P < 0.0001). Of those with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 50% (6/12) with heterogeneous ATM protein loss had detectable pathogenic ATM alterations. In surgically treated patients, ATM loss was not significantly associated with clinical outcomes in random-effects Cox models after adjusting for clinicopathologic variables. ATM loss is enriched among high-grade prostate cancers. Optimal evaluation of ATM status requires both genomic and IHC studies and will guide development of molecularly targeted therapies.

Fibroblasts from Retinoblastoma Patients Show Radiosensitivity Linked to Abnormal Localization of the ATM Protein

ABSTRACT Purpose/Aim of the study Retinoblastoma (Rb) is a rare form of pediatric cancer that develops from retina cells. Bilateral and some unilateral forms of Rb are associated with heterozygous germline mutations of the (retinoblastoma 1) RB1 gene. RB1 mutations are also associated with a significant risk of secondary malignancy like head and neck tumors. Hence, to date, even if Rb patients are less subjected to radiotherapy to treat their primary ocular tumors, their healthy tissues may be exposed to significant doses of ionizing radiation during the treatment against their secondary malignancies with a significant risk of adverse tissue reactions (radiosensitivity) and/or radiation-induced cancer (radiosusceptibility). However, the biological role of the Rb protein in response to radiation remains misunderstood. Since the ataxia telangiectasia mutated (ATM) protein is a key protein of radiation response and since untransformed skin fibroblasts are a current model to quantify cellular radiosensitivity, we investigated here for the first time the functionality of the ATM-dependent signaling and repair pathway of the radiation-induced DNA double-strand breaks (DSB) in irradiated skin fibroblasts derived from Rb patients Materials and methods The major biomarkers of the DSB repair and signaling, namely clonogenic cell survival, micronuclei, nuclear foci of the phosphorylated forms of the X variant of the H2A histone (γH2AX), the phosphorylated forms of the ATM protein (pATM) and the meiotic recombination 11 nuclease (MRE11) were assessed in untransformed skin fibroblasts derived from three Rb patients. Results Skin fibroblasts from Rb patients showed significant cellular radiosensitivity, incomplete DSB recognition, delay in the ATM nucleo-shuttling and exacerbated MRE11 nuclease activity. Treatment with statin and bisphosphonates led to significant complementation of these impairments. Conclusions Our findings strongly suggest the involvement of the ATM kinase in the radiosensitivity/radiosusceptibility phenotype observed in Rb cases.

ATM loss disrupts the autophagy-lysosomal pathway

ABSTRACT ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm−/− neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm−/− lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm−/− neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T. Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A1; CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB:cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide;FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate;Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2;MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol bindingprotein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM:standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 likekinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.

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.

Inhibition of ATM Increases the Radiosensitivity of Uveal Melanoma Cells to Photons and Protons

Treatment of uveal melanoma (UM) is generally successful, with local primary tumour control being at 90–95%. Localized radiotherapy in the form of plaque brachytherapy or proton beam radiotherapy is the most common treatment modality in the UK. However, the basic mechanisms of radiation response, DNA repair and tissue reactions in UM have not been well documented previously. We have investigated the comparative radiosensitivity of four UM cell lines in response to exogenous radiation sources (both X-rays and protons), and correlated this with DNA repair protein expression and repair efficiency. We observed a broad range of radiosensitivity of different UM cell lines to X-rays and protons, with increased radioresistance correlating with elevated protein expression of ataxia telangiectasia mutated (ATM), a protein kinase involved in the signaling and repair of DNA double strand breaks. The use of an ATM inhibitor in UM cell lines enhanced radiosensitivity following both X-ray and proton irradiation, particularly in cells that contained high levels of ATM protein which are otherwise comparatively radioresistant. In proton-irradiated compared with non-irradiated primary enucleated UM patient samples, there was no significant difference in ATM protein expression. Our study therefore suggests that ATM is a potential target for increasing the radiosensitivity of more resistant UM subgroups.

Role of DNA damage in the progress of chronic tubule‑interstitial injury

Tubulointerstitial fibrosis (TIF) is a common final endpoint of chronic allograft nephropathy. Over the years, several hypotheses have been developed to explain the progression of TIF, including mechanisms such as inflammation, epithelial‑mesenchymal transition, senescence, chronic hypoxia and reactive oxygen species. Furthermore, TIF is reportedly induced by the 'damage‑proliferation‑death' cycle. In the present study, an AA renal fibrosis model was established invitro to investigate whether the vicious proliferation‑death cycle is a pathophysiological process of TIF following chronic injury to the kidneys. Results from the present study revealed that cell death was associated with the entrance of cells into the cell cycle. Genetic knockdown of p21 was observed to increase cell cycle progression and the proliferative rate of cells, which overall promoted increased rates of cell death. In addition, the activation of the DNA damage response (DDR) signaling pathway was demonstrated to be crucial to the initiation of the vicious cycle of 'proliferation‑death'. Ataxia telangiectasia mutated (ATM) is an important molecule of the DDR and the genetic knockdown of ATM induced apoptosis, increased cell proliferation and promoted cell death. The increase in apoptosis was suggested to be due to the decreased expression levels of p21 observed following the genetic knockdown of ATM. In conclusion, the present study suggested that the crosstalk between the ATM and p21 protein may serve an important role in the regulation of the 'proliferation‑death' cycle in the progress of chronic tubulointerstitial injury.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Adefovir dipivoxil induces DNA replication stress and augments ATR inhibitor-related cytotoxicity.

Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.