Ataxia Telangiectasia Mutated Pathway Research Articles

Harmine alleviated STZ-induced rat diabetic nephropathy: A potential role via regulating AMPK/Nrf2 pathway and deactivating ataxia-telangiectasia mutated (ATM) signaling

Diabetic nephropathy (DN) is a serious kidney disorder driven by diabetes and affects people all over the world. One of the mechanisms promoting NF-κB-induced renal inflammation and injury has been theorized to be ATM signaling. On the other hand, AMPK, which can be activated by the naturally occurring alkaloid harmine (HAR), has been proposed to stop that action. As a result, the goal of this study was to evaluate the therapeutic effectiveness of HAR against streptozotocin (STZ)-induced DN in rats through AMPK-mediated inactivation of ATM pathways. Twenty male Wistar rats were grouped into 4 groups, as follow: CONT, DN, HAR (10 mg/kg), DN + HAR, where HAR was daily administered I.P. once for 2 weeks. The renal AMPK and PGC-1α expressions, as well as Sirt1 levels, were assessed. To ascertain the oxidative reactions, renal Nrf2 expression, HO-1, MDA, and TAC concentrations were measured. As parts of ATM pathways, ATM and p53 expressions, in addition to GSK-3β levels were determined. Renal expression of NEMO, TNF-α, and IL-6 levels were also estimated. Moreover, histopathological and immunohistochemical detection of Bcl-2, Bax, and caspase 3 were reported. Results indicated that HAR intake notably alleviated STZ-induced kidney damage by triggering AMPK and Sirt1, which in turn boosted PGC-1α, improved NRf2/HO-1 axis, and lowered ROS production. As a consequence, HAR blocked the ATM-triggered renal inflammation and minimized caspase-3 expression by repressing the Bax/Bcl2 ratio. Because of its ability to activate AMPK/Nrf2 axis, HAR may represent an emerging avenue for future DN therapy by blocking ATM pathways.

PIM Kinase Inhibition Attenuates the Malignant Progression of Metastatic Hepatoblastoma.

Hepatoblastoma is the most common primary pediatric liver tumor. Children with pulmonary metastases at diagnosis experience survival rates as low as 25%. We have shown PIM kinases play a role in hepatoblastoma tumorigenesis. In this study, we assessed the role of PIM kinases in metastatic hepatoblastoma. We employed the metastatic hepatoblastoma cell line, HLM_2. PIM kinase inhibition was attained using PIM3 siRNA and the pan-PIM inhibitor, AZD1208. Effects of PIM inhibition on proliferation were evaluated via growth curve. Flow cytometry determined changes in cell cycle. AlamarBlue assay assessed effects of PIM kinase inhibition and cisplatin treatment on viability. The lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. PIM kinase inhibition resulted in decreased HLM_2 proliferation, likely through cell cycle arrest mediated by p21. Combination therapy with AZD1208 and cisplatin resulted in synergy, potentially through downregulation of the ataxia-telangiectasia mutated (ATM) kinase DNA damage response pathway. When assessing the combined effects of pharmacologic PIM kinase inhibition with cisplatin on HLM_2 cells, we found the agents to be synergistic, potentially through inhibition of the ATM pathway. These findings support further exploration of PIM kinase inhibition as a therapeutic strategy for metastatic hepatoblastoma.

Abstract 6212: Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors

Abstract Ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated (ATM) protein kinases play key roles in the DNA damage response (DDR) by responding to replication stress and double-strand DNA breaks respectively to pause the cell cycle and promote DNA repair. ATR kinase inhibition in cancer cells disrupts cell cycle control and causes unrepaired DNA lesions and cytotoxicity. Accordingly, several ATR inhibitors (ATRi) are in clinical development as monotherapies and in combination with DNA damaging chemotherapies and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that while treatment of cancer cells with an ATRi inhibits the ATR pathway, it simultaneously activates the ATM signaling pathway as shown by increased levels of p-ATM, p-CHK2, p-KAP1 and p-p53. In p53 wild-type cancer cells, ATR inhibition causes an ATM-mediated G1 cell cycle arrest which diminishes the DNA lesions and cytotoxic effects of ATR inhibition. Combination of an ATRi with a selective ATM inhibitor (ATMi) synergistically potentiated efficacy in cancer cells in vitro and increased efficacy in vivo at doses that did not show overt toxicities. In a panel of patient-derived xenograft (PDX) models of triple-negative breast cancer treated with an ATRi/ATMi combination, substantial improvement in efficacy was observed. Thus, activation of the ATM pathway by an ATRi acts as a compensatory resistance pathway for ATR inhibition. These results suggest a novel and efficacious combination approach for cancer therapy by dual inhibition of these two key DDR kinases. Citation Format: Audrey Turchick, Astrid Zimmermann, Li-Ya Chiu, Heike Dahmen, Brian Elenbaas, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6212.

Selected Articles from This Issue

Ataxia telangiectasia mutated (ATM) kinase controls key cell cycle checkpoints and the repair of DNA double strand breaks and has been considered an attractive intervention point in DNA damage response for cancer therapy. Here, Zimmermann et al. introduce two novel ATM inhibitors. These highly potent and selective small molecules effectively suppress the ATM pathway, enhance antitumor effect of radiation and topoisomerase I inhibitors and synergize with PARP inhibitors in cancer cells and animal models of human cancer. They represent new molecular tools with potential for combination with key cancer therapies.ERα antagonists and degraders are highly effective in the treatment of the majority of ERα+ breast cancers but resistance mechanisms, including ERα mutations that facilitate ligand-independent activation, blunt the effectiveness of these therapies. Here, Furman et al. report on the development H3B-6545, a novel covalent inhibitor of ERa which demonstrates superiority over standard-of-care fulvestrant in both ERWT and ERαMUT breast tumor models. Consistent with these preclinical findings, ongoing clinical trials of H3B-6545 are demonstrating preliminary activity in ER+, HER2− metastatic breast cancer patients with both ERαWT and ERαY537S mutations.Pancreatic cancer is a devastating malignancy with a 5-year survival rate of only 9%. Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in pancreatic cancer. Here, Ho and colleagues have constructed a GPC1-targeted immunotoxin derived from a bacterial toxin. Small molecule screening has been performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. The GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment for patients with pancreatic tumors.Despite the approval of immune checkpoints in small cell lung cancer (SCLC), it remains a rapidly progressing and fatal form of cancer. Recently, antibody drug conjugates (ADCs) have gained clinical traction in breast, bladder, and hematologic cancers. Here, Wiedemeyer and colleagues develop ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC that contains a non-cleavable linker which eliminates toxic metabolites associated with first generation calicheamicin ADCs. In mice, ABBV-011 potently regresses patient-derived xenografts of SCLC. ABBV-011 is being tested in a Phase 1 study and has potential as a first-in-class ADC therapy for SCLC.

Inhibitory effect of tanshinone IIA, resveratrol and silibinin on enterovirus 68 production through inhibiting ATM and DNA-PK pathway

BackgroundHuman enterovirus 68 (EV68) is a primary etiological agent for respiratory illnesses, while no effective drug has yet used in clinics largely because the pathogenesis of EV68 is not clear. DNA damage response (DDR) responds to cellular DNA breaks and is also involved in viral replication. Three DDR pathways includes ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). Natural products proved to be an excellent source for the discovery and isolation of novel antivirals. Among them, tanshinone IIA, resveratrol, silibinin, rutin and quercetin are reported to target DDR, therefore their roles in anti-EV68 are investigated in this study. PurposeThis study investigated the anti-EV68 ability of various natural compounds related to DDR. Study design and methodsThe methods include cell counting, flow cytometry, western blot, Immunofluorescence staining, comet assays, quantitative real-time RT PCR and short interfering RNAs (siRNAs) for analysis of cell number, cell cycle, protein expression, protein location, DNA damage, mRNA level and knock down target gene, respectively. ResultsEV68 infection induced DDR. Down-regulation or inhibition of ATM or DNA-PK lowered DDR in EV68-infected cells and mitigated viral protein expression, however, down-regulation or inhibition of ATR unexpectedly up-regulated DDR, and promoted viral protein expression. Meanwhile tanshinone IIA, resveratrol, and silibinin inhibited ATM and/or DNA-PK activation and decreased viral proliferation, while rutin and quercetin inhibited ATR activation and promoted viral production. The role of them in ATM, DNA-PK and ATR activation was consistent with previous reports. ConclusionTanshinone IIA, resveratrol and silibinin inhibited EV68 proliferation through inhibiting ATM and/or DNA-PK activation, and they were effective anti-EV68 candidates.

Regulation of host factor γ-H2AX level and location by enterovirus A71 for viral replication

ABSTRACT Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent in vitro and in vivo data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.

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

DNA damage has been linked to neuropathology. Diverse DNA damage response (DDR) pathways help preserve DNA integrity in the nervous system during both the developmental and mature stages. Mutations of factors in various signaling pathways responsive to different types of DNA damage have been associated with developmental syndromes with neurological symptoms (McKinnon, 2009; Araújo and Kuraoka, 2019; Khokhlova et al., 2020). For example, mutations in a single gene encoding ataxia-telangiectasia mutated (ATM), which is activated by DNA double-strand breaks for checkpoint initiation, can lead to ataxia telangiectasia (McKinnon, 2004). It is believed that neuropathology can result from cellular DNA damage, or even DNA damage within the mitochondria, as has been shown to correlate with aging and Parkinson’s disease (Bender et al., 2006). However, attributing all the mechanisms underlying neurological diseases caused by DDR gene deficiencies to the accumulation of genetic alterations is an oversimplification. In particular, non-canonical DDRs have exhibited examples of uncoupling of upstream triggers (DNA damage) and downstream events. Other than single/double-strand breaks of DNA, for instance, telomere maintenance and oxidative stress in cells can also activate DDR (Burgess and Misteli, 2015). The consequences of DDR have been typically characterized as cell-cycle arrest, expression of repair genes, apoptosis, etc. However, the specific effects of DDR pathway activation in various types of cells, especially the terminally differentiated cells, warrant further exploration. Whether the DDR machinery can function beyond its traditional role remains an open question. A recent study identified a novel role of the DDR pathway in neurons. Using an axonal injury and repair paradigm in Drosophila melanogaster, this study uncovered the ATM and rad3-related (ATR) pathway as a key regulator in axon regeneration (Li et al., 2021). As a serine/threonine kinase, ATR responds to DNA single-strand breaks and replication stress. ATR, together with ATM, mediates the two major checkpoints to protect the integrity of the genome. The ATM pathway is dispensable for cell survival and mutations of its components are frequently observed in tumor cells. The ATR pathway appears more critical for viability, as mice embryos bearing ATR loss-of-function mutations die at an early developmental stage, confounding investigations into ATR’s role out of the cell cycle (Yazinski and Zou, 2016). In this study, gene candidates were specifically manipulated in subtypes of sensory neurons, revealing a new function of the ATR pathway. After knocking down the fly homolog of ATR, meiotic 41, in class III dendritic arborization sensory neurons, the authors noticed increased capacity of these neurons to regrow axons after injury, and the same effect was observed in ATR null flies (Li et al., 2021). This finding suggests that, in non-dividing cells, DDR pathways have developed non-canonical functions to help cells adapt to their environment and cope with their unique perturbations. It is intriguing to ask what the upstream signal is that activates the ATR pathway in the scenario of axon injury, whether it is still DNA damage, and whether other components in the same pathway are also involved. A large range of genomic aberrations can activate the ATR pathway. In addition, it has been reported that mechanical stress on the nuclear envelope can relocalize ATR to the nuclear membrane to protect the perinuclear chromatin, suggesting that ATR directly or indirectly senses mechanical stimuli to prevent cells from mechanical insults (Kumar et al., 2014). In the context of axon regeneration, it has been shown that a mechanosensitive ion channel, Piezo, inhibits axon regrowth after injury by sensing the mechanical force in the environment around the axon tip (Song et al., 2019). While possible mechanisms for Piezo activation to generate a regenerative halt have been proposed, it is exciting to find that ATR is implicated in the Piezo-induced inhibition of axon regeneration. This study demonstrated that, instead of DNA damage, ATR responds to mechanical stimuli from the environment. γH2AX (phosphorylation of the Ser-139 residue of the histone variant H2AX) staining, which detects DNA-damage events in situ, showed no difference between injured and uninjured fly sensory neurons. Moreover, targeting the replication protein A complex, the machinery that monitors single-stranded DNAs and recruits ATR, did not result in a similar axon regeneration phenotype as ATR ablation. These results thus suggested that DNA damage is not directly induced by axon injury and that a signal transduction pathway is activated downstream of ATR, but not by DNA damage per se. In the following experiment using a cultured cell model of osmotic stress to induce ATR clustering in the nucleus, PIEZO1 was demonstrated to be required and act upstream, as ATR clustering was significantly reduced in the PIEZO1 knock-out cell line. By genetic interaction analyses in flies, Piezo and ATR were demonstrated to function in the same genetic pathway during axon regeneration. Furthermore, nitric oxide, produced by nitric oxide synthases, was suggested to act as the signal from Piezo to ATR (Li et al., 2021). Previous work has established that calcium elevation immediately after axotomy is critical for the initiation of growth cone reformation and that calcium, as a second messenger, is derived from extracellular and internal sources and spreads rapidly to the whole axon and cell body (Mahar and Cavalli, 2018). However, the identity of a possible signal communicating between the axon and nucleus when regenerating axons elongate remains unknown. This study presented evidence that nitric oxide is a strong candidate, as it is produced in the axon tip and propagates to the nucleus, enabling neurons to respond to the mechanical force from the extracellular matrix (Li et al., 2021) (Figure 1).Figure 1: The ataxia-telangiectasia mutated and rad3-related (ATR) pathway responds to mechanical force to inhibit axon regeneration.Following Piezo activation, nitric oxide synthase (NOS) produces nitric oxide (NO), which propagates along the axon to the nucleus. Then, ATR, under the regulation of ATR interacting protein (ATRIP), TOPBP1, and the 9-1-1 complex, phosphorylates CHEK1 and inhibits CDC25. CDC25 dephosphorylates CDK1 to promote axon regeneration. Through the axis of ATR-CHEK1-CDC25-CDK1, axon regeneration is inhibited.Although ATR relocalization at the nuclear envelope induced by osmotic stress is independent of replication protein A-single-stranded DNA nucleofilaments, phosphorylated CHEK1 and ATR interacting protein were found at the nuclear envelope with ATR (Kumar et al., 2014). During axon regeneration, fly homologs of CHEK1/grapes and ATR interacting protein/mus304 were also found to participate in the ATR-activation-induced regeneration inhibition. In the fly sensory neurons, knockdown of CHEK1/grapes or ATR interacting protein/mus304 recapitulated the axon regeneration enhancement caused by ATR deficiency. Moreover, overexpression of CHEK1/grapes sufficiently inhibited axon regeneration (Li et al., 2021). These results indicate that other components of the ATR pathway, mainly in the execution steps, cooperate to carry out this non-canonical function. In DNA repair, after ATR recruitment to replication protein A-single-stranded DNA, its activity is regulated by TOPBP1 (DNA Topoisomerase II Binding Protein 1), which is mediated by the 9-1-1 complex composed of RAD9-HUS1-RAD1 and RAD17. After CHEK1 is phosphorylated by ATR, it can in turn phosphorylate and inhibit CDC25, which is a phosphatase for CDK1. Although RAD17 and TOPBP1 were not found to respond to the osmotic-stress-induced ATR relocalization (Kumar et al., 2014), they are indispensable for axon regeneration inhibition. Knockdown of fly homologs of RAD17, TOPBP1, and RAD1 in the sensory neurons all significantly increased axon regeneration, while loss of function of fly homologs of CDC25 and CDK1 reduced axon regeneration (Li et al., 2021). It is thus demonstrated that ATR-CHEK1-CDC25-CDK1 is an axis regulating axon regeneration in response to the mechanical force from the extracellular matrix (Figure 1). CDK1 phosphorylation at the end of this axis typically leads to cell cycle arrest. However, this study provided evidence that, in terminally differentiated cells, such as neurons, the ATR-activation machinery, together with its downstream effectors, modulates cellular regeneration when encountering axonal injury. Therefore, cells are capable of utilizing or repurposing seemingly “redundant/obsolete” regulatory pathways to adapt to unusual scenarios, such as cellular stress. It remains a fascinating question when/how such a functional switch was made, from an evolutionary standpoint. Discovering novel neuronal intrinsic regulators for axon regeneration is one of the key areas for the development of new therapeutics for nervous system trauma (Mahar and Cavalli, 2018). The ATR pathway identified in Drosophila thus provides potential targets for treatments. This study further tested the conservation of the ATR pathway in axon regeneration in mammalian injury models, confirming that manipulations of ATR and CHEK1 in mice neurons in vitro and in vivo also affect axon regeneration. Lastly, ATR and CHEK1 pharmacological inhibitors have been developed to treat various types of cancers, and they were found to promote axon regeneration in cultured mammalian neurons and flies. Other DNA repair pathways, such as ATM, were proved not essential for regulating axon regeneration in the injured fly sensory neurons, demonstrating the specificity of the ATR pathway (Li et al., 2021). However, we cannot rule out the possibility that the ATM-CHEK2 pathway may be involved under other injury paradigms in other cell types. To conclude, this study raises the possibility that pathways with conventionally known functions may exhibit non-canonical roles in unexpected situations, and suggests that utilizing the screening platform in Drosophila will be a fruitful strategy to identify more factors controlling axon regeneration. The present work was supported by the National Institutes of Health Grant, 1R01NS107392 (to YS). C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y.

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress.

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

Caffeine disrupts ataxia telangiectasia mutated gene-related pathways and exacerbates acetaminophen toxicity in human fetal immortalized hepatocytes

Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.

LARP7 Protects Against Heart Failure by Enhancing Mitochondrial Biogenesis.

Heart failure (HF) is among the leading causes of morbidity and mortality, and its prevalence continues to rise. LARP7 (La ribonucleoprotein domain family member 7) is a master regulator that governs the DNA damage response and RNAPII (RNA polymerase II) pausing pathway, but its role in HF pathogenesis is incompletely understood. We assessed LARP7 expression in human HF and in nonhuman primate and mouse HF models. To study the function of LARP7 in heart, we generated global and cardiac-specific LARP7 knockout mice. We acutely abolished LARP7 in mature cardiomyocytes by Cas9-mediated LARP7 somatic knockout. We overexpressed LARP7 in cardiomyocytes using adeno-associated virus serotype 9 and ATM (ataxia telangiectasia mutated protein) inhibitor. The therapeutic potential of LARP7-regulated pathways in HF was tested in a mouse myocardial infarction model. LARP7 was profoundly downregulated in failing human hearts and in nonhuman primate and murine hearts after myocardial infarction. Low LARP7 levels in failing hearts were linked to elevated reactive oxygen species, which activated the ATM-mediated DNA damage response pathway and promoted LARP7 ubiquitination and degradation. Constitutive LARP7 knockout in mouse resulted in impaired mitochondrial biogenesis, myocardial hypoplasia, and midgestational lethality. Cardiac-specific inactivation resulted in defective mitochondrial biogenesis, impaired oxidative phosphorylation, elevated oxidative stress, and HF by 4 months of age. These abnormalities were accompanied by reduced SIRT1 (silent mating type information regulation 2 homolog 1) stability and deacetylase activity that impaired SIRT1-mediated transcription of genes for oxidative phosphorylation and energy metabolism and dampened cardiac function. Restoring LARP7 expression after myocardial infarction by either adeno-associated virus-mediated LARP7 expression or small molecule ATM inhibitor substantially improved the function of injured heart. LARP7 is essential for mitochondrial biogenesis, energy production, and cardiac function by modulating SIRT1 homeostasis and activity. Reduction of LARP7 in diseased hearts owing to activation of the ATM pathway contributes to HF pathogenesis and restoring LARP7 in the injured heart confers myocardial protection. These results identify the ATM-LARP7-SIRT1 pathway as a target for therapeutic intervention in HF.

Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos.

Cohesin is an evolutionarily conserved chromosome-associated protein complex essential for chromosome segregation, gene expression, and repair of DNA damage. Mutations that affect this complex cause the human developmental disorder Cornelia de Lange syndrome (CdLS), thought to arise from defective embryonic transcription. We establish a significant role for placental defects in the development of CdLS mouse embryos (Nipbl and Hdac8). Placenta is a naturally senescent tissue; we demonstrate that persistent DNA damage potentiates senescence and activates cytokine signaling. Mutant embryo developmental outcomes are significantly improved in the context of a wild-type placenta or by genetically restricting cytokine signaling. Our study highlights that cohesin is required for maintaining ploidy and the repair of spontaneous DNA damage in placental cells, suggesting that genotoxic stress and ensuing placental senescence and cytokine production could represent a broad theme in embryo health and viability.

Multi-omics analysis of pathological changes in the amygdala of rats subjected to chronic restraint stress

ObjectiveOverwhelming stress potentially results in the occurrence of many mental diseases. The amygdala is one region in the brain targeted by stress. Recent studies have shown that changes in the amygdala of subjects under stress are related to depression, anxiety and post-traumatic stress disorder (PTSD). However, researchers have not clearly elucidated the changes in the amygdala in response to stress and the underlying mechanism. We conducted several experiments to understand this mechanism. MethodsIn this study, we first established a rat model of chronic restraint stress (CRS) and observed the changes in behavior and neurons in the amygdala. Second, an integrated metabolomics and proteomics experiment was conducted to identify potential stress-related biomarkers. Finally, we validated two molecules of interest and detected four apoptosis-related proteins using Western blotting to further determine the related mechanisms. ResultsOur study revealed the presence of anxiety-like behaviors and pathological changes in amygdalar neurons in the rat model. In the multi-omics analysis, 19 potential molecules were identified. Western blotting confirmed consistent changes in the levels of Cry1 and Brcc36 obtained in previous results. The levels of proteins in the ataxia telangiectasia mutated (ATM) pathway were increased in the CRS group. ConclusionsCRS causes anxiety-like behaviors that are potentially related to decreased levels of GABA in the amygdala. Moreover, CRS potentially alters the levels of Cry1 and Brcc36 and results in circadian rhythm disorder and impairments in DNA repair and apoptosis in the amygdala through a mechanism mediated by the ATM pathway.

High Levels of ROS Impair Lysosomal Acidity and Autophagy Flux in Glucose-Deprived Fibroblasts by Activating ATM and Erk Pathways.

Under glucose deprivation, cells heavily mobilize oxidative phosphorylation to maintain energy homeostasis. This leads to the generation of high levels of ATP, as well as reactive oxygen species (ROS), from mitochondria. In nutrient starvation, autophagy is activated, likely to facilitate resource recycling, but recent studies suggest that autophagy flux is inhibited in cells undergoing glucose deprivation. In this study, we analyzed the status of autophagic flux in glucose-deprived human fibroblasts. Although lysosomes increased in quantity due in part to an increase of biogenesis, a large population of them suffered low acidity in the glucose-deprived cells. Autophagosomes also accumulated due to poor autolysis in these cells. A treatment of antioxidants not only restored lysosomal acidity but also released the flux blockade. The inhibition of ataxia telangiectasia mutated (ATM) serine/threonine kinase, which is activated by ROS, also attenuated the impairment of lysosomal acidity and autophagic flux, suggesting an effect of ROS that might be mediated through ATM activation. In addition, the activity of extracellular signal-regulated kinase (Erk) increased upon glucose deprivation, but this was also compromised by a treatment of antioxidants. Furthermore, the Erk inhibitor treatment also alleviated the failure in lysosomal acidity and autophagic flux. These together indicate that, upon glucose deprivation, cells undergo a failure of autophagy flux through an impairment of lysosomal acidity and that a high-level ROS-induced activation of Erk and ATM is involved in this impairment.

Abnormal expression of p-ATM/CHK2 in nasal extranodal NK/T cell lymphoma, nasal type, is correlated with poor prognosis

AimsThe aim of this study is to investigate the expression profiles of cell cycle related proteins in nasal extranodal NK/T cell lymphoma, nasal type (ENKTCL).MethodsThe expression profiles of cell cycle...

Ataxia telangiectasia mutated pathway disruption affects hepatic DNA and tissue damage in nonalcoholic fatty liver disease

To overcome the rising burdens of nonalcoholic fatty liver disease, mechanistic linkages in mitochondrial dysfunction, inflammation and hepatic injury are critical. As ataxia telangiectasia mutated (ATM) gene oversees DNA integrity and mitochondrial homeostasis, we analyzed mRNAs and total proteins or phosphoproteins related to ATM gene by arrays in subjects with healthy liver, fatty liver or nonalcoholic steatohepatitis. Functional genomics approaches were used to query DNA damage or cell growth events. The effects of fatty acid-induced toxicity in mitochondrial health, DNA integrity and cell proliferation were validated in HuH-7 cells, including by inhibiting ATM kinase activity or knckdown of its mRNA. In fatty livers, DNA damage and ATM pathway activation was observed. During induced steatosis in HuH-7 cells, lowering of ATM activity produced mitochondrial dysregulation, DNA damage and cell growth inhibition. In livers undergoing steatohepatitis, ATM was depleted with increased hepatic DNA damage and growth-arrest due to cell cycle checkpoint activations. Moreover, molecular signatures of oncogenesis were associated with upstream mechanistic networks directing cell metabolism, inflammation or growth that were either activated (in fatty liver) or inactivated (in steatohepatitis). To compensate for hepatic growth arrest, preoncogenic oval cell populations expressing connexin-43 and/or albumin emerged. These oval cells avoided DNA damage and proliferated actively. We concluded that ATM is a major contributor to the onset and progression of nonalcoholic fatty liver disease. Therefore, specific markers for ATM pathway dysregulation will allow prospective segregation of cohorts for disease susceptibility and progression from steatosis to steatohepatitis. This will offer superior design and evaluation parameters for clinical trials. Restoration of ATM activity with targeted therapies should be appropriate for nonalcoholic fatty liver disease.

ATM and ATR checkpoint kinase pathways: A concise review

The ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR) DNA damage repair pathways serve as the surveillance system which keeps a check on different types of DNA damages and lesions, which includes DNA single-strand breaks, DNA double-strand breaks and other aberrant structures such as arrested replication forks during replication. The ATM and ATR kinases belong to PIKK class of kinases which activate a large number of downstream mediator and effector molecules. The main classes of effector kinases activated by ATM and ATR are checkpoint kinase 2 and checkpoint kinase 1, respectively. ATR works primarily with the RAD9-RAD1-HUS1 (9-1-1) complex, whereas ATM works with the MRE11– RAD50–NBS1 complex. Together ATM and ATR kinase protects the cells' genomic integrity and prevents random mutations to be carried into their progeny.

Cellular cytokine receptor signaling and ATM pathway intersections affect hepatic DNA repair

Pathways involving ataxia telangiectasia mutated (ATM) gene and its downstream partners and effectors are critical for the DNA damage response. Cell survival, proliferation and tissue homeostasis are dependent upon preservation of DNA integrity but additional intracellular mechanisms contribute in these processes. As receptor-mediated signaling with beneficial intersections in ATM pathways could have therapeutic significance, we interrogated such intersections with assays using HuH-7 cells (hepatocytes). These cells were subjected to acetaminophen toxicity, which is a leading cause of hepatic injury and acute liver failure in people. The ATM pathway was examined in HuH-7-ATM-Prom-tdT cells containing fluorescent td-Tomato transgene reporter for ATM promoter activity. Titrated doses of specific growth factors were used as ligands for receptor-mediated signaling. The contribution of JAK/STAT3 signaling was defined by the loss-of-function approach with the JAK antagonist, ruxolitinib. In these assays, impairment in ATM-related DNA damage response following acetaminophen toxicity was ameliorated by selected growth factors, including fibroblast growth factors, granulocyte colony stimulating factor and vascular endothelial growth factor. The JAK/STAT3 signaling was exclusive to granulocyte colony stimulating factor but concerned additional pathways in cases of other growth factors. Antagonism of JAK/STAT3 by ruxolitinib abrogated benefits in ATM pathway-mediated DNA repair; and identification of the ruxolitinib-sensitive component of cytoprotection allowed separations of these pathway intersections. Therefore, this subtractive approach for ATM and other regulators in pathways will be informative for DNA damage response. These mechanisms will benefit therapeutic development for ATM-related tissue and organ injuries.

Wild-type p53-induced phosphatase 1 down-regulation promotes apoptosis by activating the DNA damage-response pathway in amyotrophic lateral sclerosis

Accumulation of DNA damage has been detected in the spinal cord of patients as well as in the G93A mouse model of amyotrophic lateral sclerosis (ALS). Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that terminates DNA-damage responses via dephosphorylation of DNA-damage response proteins, namely ataxia-telangiectasia mutated (ATM) kinase, checkpoint kinase 2, and p53, thus enhancing cell proliferation. However, the role of Wip1, DNA-damage responses, and their interaction in ALS development remains to be elucidated. Here, we showed that Wip1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro. The DNA-damage response was activated in superoxide dismutase 1 (SOD1) G93A-transfected cells. However, increased expression of Wip1 improved cell viability and inhibited the DNA-damage response in mutated SOD1G93A cells. Further studies demonstrated that decreased Wip1 expression reduced cell viability and further activated the DNA-damage response in chronic H2O2-treated NSC34 cells. In contrast, Wip1 promoted cell survival and suppressed DNA damage-induced apoptosis during persistent DNA damage conditions. Over-expression of Wip1 in the central nervous system (CNS) can delay the onset of disease symptoms, extended the survival, decreased MN loss improved motor function and inhibit the DNA-damage response in SOD1 G93A mice. Furthermore, homeodomain-interacting protein kinase 2 (HIPK2) promoted the degradation of Wip1 via the ubiquitin-proteasome system during chronic stress. These findings indicate that persistent accumulation of DNA damage and subsequent chronic activation of the downstream DNA damage-response ATM and p53 pro-apoptotic signaling pathways may trigger neuronal dysfunction and neuronal death in ALS. Wip1 may play a protective role by targeting the DNA-damage response in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS.

Effect of ATM on inflammatory response and autophagy in renal tubular epithelial cells in LPS-induced septic AKI.

The aim of the present study was to explore the role of ataxia-telangiectasia mutated (ATM) in lipopolysaccharide (LPS)-induced in vitro model of septic acute kidney injury (AKI) and the association between ATM, tubular epithelial inflammatory response and autophagy. The renal tubular epithelial cell HK-2 cell line was cultured and passaged, with HK-2 cell injury induced by LPS. The effects of LPS on HK-2 cell morphology, viability, ATM expression and inflammation were observed. Lentiviral vectors encoding ATM shRNA were constructed to knock down ATM expression in HK-2 cells. The efficiency of ATM knockdown in HK-2 cells was detected by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR). HK-2 cells transfected with the ATM shRNA lentivirus were used for subsequent experiments. Following ATM knockdown, corresponding controls were set up, and the effects of ATM on inflammation and autophagy were detected in HK-2 cells using RT-qPCR, western blotting and ELISA. After LPS stimulation, the HK-2 cells were rounded into a slender or fusiform shape with poorly defined outlines. LPS treatment reduced cell viability in a partly dose-dependent manner. LPS increased the expression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6, with the levels reaching its highest value at 10 µg/ml. IL-6 and IL-1β expression increased with increasing LPS concentration. These findings suggest that LPS reduced HK-2 cell viability whilst increasing the expression of inflammatory factors. Following transfection with ATM shRNA, expression levels of key autophagy indicators microtubule associated protein 1 light chain 3α I/II ratio and beclin-1 in the two ATM shRNA groups were also significantly reduced compared with the NC shRNA group. In summary, downregulation of ATM expression in HK-2 cells reduced LPS-induced inflammation and autophagy in sepsis-induced AKI in vitro, suggesting that LPS may induce autophagy in HK-2 cells through the ATM pathway leading to the upregulation of inflammatory factors.

N-Acetyl Cysteine as a Novel Polymethyl Methacrylate Resin Component: Protection against Cell Apoptosis and Genotoxicity.

The present study investigated the antiapoptotic and antigenotoxic capabilities of N-acetyl cysteine- (NAC-) containing polymethyl methacrylate (PMMA) resin. An in vitro Transwell insert model was used to mimic the clinical provisional restorations placed on vital teeth. Various parameters associated with cell apoptosis and genotoxicity were investigated to obtain a deeper insight into the underlying mechanisms. The exposure of human dental pulp cell (hDPC) cultures to the PMMA resin (Unifast Trad™) resulted in a rapid increase in reactive oxygen species (ROS) level beginning at 1 h, which was followed by time-dependent cell detachment and overt death. The formation of γ-H2AX and cell cycle G1 phase arrest indicated that oxidative DNA damage occurred as a result of the interactions between DNA bases and ROS, beyond the capacities of cellular redox regulation. Such oxidative DNA damage triggers the activation of p53 via the ataxia telangiectasia mutated (ATM) signaling pathway and the induction of intrinsic mitochondrial apoptosis. Oxidative stress, cell apoptosis, and DNA damage induced by the PMMA resin were recovered to almost the level of untreated controls by the incorporation of NAC. The results indicate that the PMMA resin induced the intrinsic mitochondrial apoptosis as a consequence of p53 activation via the ATM pathway in response to oxidative DNA damage. More importantly, the incorporation of NAC as a novel component into the Unifast Trad™ PMMA resin offers protective effects against cell apoptosis and genotoxicity. This procedure represents a beneficial strategy for developing more biocompatible PMMA-based resin materials.