ATM-mediated DNA Damage Response Research Articles

SIRT1 regulates osteoblast senescence through SOD2 acetylation and mitochondrial dysfunction in the progression of Osteoporosis caused by Cadmium exposure

Environmental Cadmium (Cd) is a toxicant with widespread exposure, documented adverse effects on bone homeostasis, and makes the onset of osteoporosis (OP), one of the age-related chronic diseases an enormous burden to modern societies worldwide. Aging is the largest risk factor for a multitude of age-related diseases and osteoblasts senescence reduces bone formation and is a key factor for osteoporosis. Despite anti-aging molecules the nuclear silent information regulator of transcription 1 (SIRT1) actions in chondrocytes and bone cells are critical for normal skeletal development and homeostasis, much less is known about the role of SIRT1 in osteoporosis. Here, we aim to demonstrate that SIRT1 mediates osteoblasts' senescence response to OP caused by Cd. The senescent osteoblasts accumulation and their viability were analyzed after Cd exposure. To explore the effects and mechanism of SIRT1 in Cd-induced osteoblastic senescence, we generated SIRT1-overexpressed osteoblast and SIRT1 conditional overexpression in the rat femur. Meanwhile, the OP rat model was established by removing bilateral ovaries. We found decreased SIRT1 expression and senescent osteoblasts accumulation after Cd exposure. Meanwhile, Cd exposure increased P53, P16INK4a, and P21CIPI proteins level, triggered DNA damage response (DDR) through the phosphorylation of ATM and H2AX, and caused mitochondrial dysfunction by the increased acetylation of SOD2 and excessive mitophagy. SIRT1 overexpression attenuated DDR and mitochondrial dysfunction and downregulated the increase of hall makers senescence caused by Cd in osteoblasts. We found overexpression of osteoblastic SIRT1 protects against Cd-induced senescence, which is likely driven by ATM-mediated DDR and SOD2ace-mediated mitochondrial dysfunction. Our study demonstrates the mechanism of SIRT1 in mediating bone homeostasis via senescence. Further mechanistic studies using specific SIRT1 mutations elucidating how SIRT1 modulates bone cell senescence, will provide new therapeutic strategies for human osteoporosis.

ATM mediated DNA double‐strand breaks accumulate in LRRK2 G2019S Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over one million people in the US. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited and idiopathic PD. We were the first to show that mitochondrial DNA (mtDNA) damage is caused by the most common mutation in LRRK2 (G2019S) and inhibition of LRRK2 kinase activity restores mtDNA integrity in PD models. However, whether aberrant LRRK2 kinase activity due to PD-linked mutations has broad impact on nuclear genome integrity is unknown. Using LRRK2G2019S/G2019S knock-in (KI) human embryonic kidney 293 (HEK293) cells obtained by CRISPR/Cas9 gene editing, our preliminary results indicate nuclear DNA damage is increased, including DNA double-strand breaks (DSBs) as assayed by a neutral comet assay. Consistent with DSB accumulation, we observed significantly increased γ-H2AX and 53BP1 foci. ATM is activated by DSBs and phosphorylates several key proteins that initiate the DNA damage response, cell cycle arrest, DNA repair or apoptosis. We found that basal levels of ATM pS1981 are increased in this in vitro LRRK2 G2019S model, which is an autophosphorylation site that correlates with DNA damage-mediated activation. Additionally, downstream substrates CHK2 (pT68), and P53 (pS15) are similarly increased in LRRK2G2019S/G2019S KI cells compared to isogenic wild-type control cells, substantiating that the ATM-mediated DNA damage response pathway has been up-regulated with the LRRK2 G2019S mutation. Blocking either LRRK2 or ATM kinase activity pharmacologically, significantly reversed LRRK2 G2019S-induced γ-H2AX foci. Overall these results suggest DSBs accumulate in LRRK2 PD, which in turn activate a sustained ATM mediated DNA damage response, which may lead to cell cycle arrest, aberrant DNA repair, and/or cell death. Further understanding of the functional relationship between LRRK2 and ATM offers new molecular insights into PD pathogenesis.

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.

Usnic Acid Inhibits Proliferation and Migration through ATM Mediated DNA Damage Response in RKO Colorectal Cancer Cell.

Usnic Acid (UA), also known as lichenol, has been reported to have inhibitory effects on a variety of cancer cells, but its specific mechanism remained to be elucidated. Tumor chemotherapy drugs, especially DNA damage chemotherapeutic drugs, target Chromosomal DNA, but their spontaneous and acquired drug resistance are also an urgent problem to be solved. Therefore, drug combination research has become the focus of researchers. Here, we evaluated the tumor-suppressing molecular mechanism of UA in colorectal cancer cells RKO from the perspective of the ATM-mediated DNA damage signaling pathway through H2O2 simulating DNA damage chemotherapeutic drugs. CCK8 cell proliferation assay was used to determine the inhibition of RKO cells by hydrogen peroxide and UA alone or in combination, and wound healing assay was applied to determine the effect of the drug on cell migration. Transfected cells with miRNA18a-5p mimics and inhibitors, MDC and DCFH-DA staining for the measurement of autophagy and ROS, cell cycle and apoptosis were detected by flow cytometry, expressions of microRNA and mRNA were determined by fluorescence quantitative PCR, and protein by Western blot. We found that UA can upregulate ATM via miR-18a to activate the DNA damage signaling pathway and inhibit the proliferation and migration of RKO cells in a concentration-dependent manner. At the same time, DNA damage responses, including cell cycle, autophagy, apoptosis and ROS levels, are also regulated by UA. Therefore, UA combined with DNA damage chemotherapeutic drugs may be an effective treatment for cancer.

MiR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

BackgroundThe Epstein-Barr virus (EBV) is closely associated with several types of malignancies. EBV is normally present in the latent state in the peripheral blood B cell compartment. The EBV latent-to-lytic switch is required for virus spread and virus-induced carinogenesis. Immunosuppression or DNA damage can induce the reactivation of EBV replication. EBV alone is rarely sufficient to cause cancer. In this study, we investigated the roles of host microRNAs and environmental factors, such as DNA-damage agents, in EBV reactivation and its association with lymphomagenesis.MethodsWe first analyzed the publicly available microRNA array data containing 45 diffuse large B-cell lymphoma patients and 10 control lymph nodes or B cells with or without EBV infection. In situ hybridization for miR-18a and immunohistochemitry were performed to evaluate the correlation between the expression of miR-18a and nuclear EBV protein EBNA1 in lymphoid neoplasm. The proliferative effects of miR-18a were investigated in EBV-positive or –negative lymphoid neoplasm cell lines. EBV viral load was measured by a quantitative real-time EBV PCR and FISH assay. The genomic instability was evaluated by CGH-array.ResultsIn this study, we analyzed the publicly available microRNA array data and observed that the expression of the miR-17-92 cluster was associated with EBV status. In situ hybridization for miR-18a, which is a member of the miR-17-92 cluster, showed a significant upregulation in lymphoma samples. miR-18a, which shares the homolog sequence with EBV-encoded BART-5, promoted the proliferation of lymphoma cells in an EBV status-dependent manner. The DNA-damaging agent UV or hypoxia stress induced EBV activation, and miR-18a contributed to DNA damaging-induced EBV reactivation. In contrast to the promoting effect of ATM on the lytic EBV reactivation in normoxia, ATM inhibited lytic EBV gene expression and decreased the EBV viral load in the prescence of hypoxia-induced DNA damage. miR-18a reactivated EBV through inhibiting the ATM-mediated DNA damage response (DDR) and caused genomic instability.ConclusionsTaken together, these results indicate that DNA-damaging agents and host microRNAs play roles in EBV reactivation. Our study supported the interplay between host cell DDR, environmental genotoxic stress and EBV.

Abstract A178: Targeting the ATM-mediated DNA damage response pathway for cancer treatment

Abstract Epigenetic modulators have emerged as potent therapeutic agents for treatment of patients with cancers. We have developed dual-function small molecules targeting activation of ATM, the DNA damage response transducer, ataxia telangiectasia mutated gene product, and inhibition of histone deacetylases (HDACi). The goal is to develop a single-molecule therapeutic that optimizes the protective effects of ATM activation in normal tissue while targeting HDAC inhibition to sensitize cancerous tissue to radiation. The candidate compound SP-1-161 is a hydroxamic acid that was rationally designed by optimizing ATM activation in the cap domain. In vitro assays against a panel of Class I and Class II HDAC enzymes identified SP-1-161 as a pan-HDAC inhibitor with nanomolar potency (IC50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and activates ATM (p-ATM/S1987) in MCF7 cells. The cytotoxicity values (IC50) were in low micro-molar ranges in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines. Radiation clonogenic survival assays demonstrated that the compound protected nonmalignant 184A1 cells (from D0 = 1 Gy to D0 =1.4 Gy) while increasing sensitivity of malignant MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Furthermore, in vivo plasma pharmacokinetics following oral administration revealed the area under the curve of 415.79 ng/ml × h with the half-life 1.94 h. Together, our results demonstrate that SP-1-161 is an unprecedented radiation chemotherapeutic agent for treating cancers and protecting normal cells, potentially by targeting the DDR signaling pathways. Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Targeting the ATM-mediated DNA damage response pathway for cancer treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A178.

Punicalagin from pomegranate promotes human papillary thyroid carcinoma BCPAP cell death by triggering ATM-mediated DNA damage response

Punicalagin (PUN), a component derived from pomegranate, is well known for its anticancer activity. Our previous work revealed that PUN induces autophagic cell death in papillary thyroid carcinoma cells. We hypothesized that PUN triggers DNA damage associated with cell death because DNA damage was reported as an inducer of autophagy. Our results showed that PUN treatment caused DNA breaks as evidenced by the significant enhancement in the phosphorylation of H2A.X. However, reactive oxygen species and DNA conformational alteration, 2 common inducing factors in DNA damage, were not involved in PUN-induced DNA damage. The phosphorylation of ataxia-telangiectasia mutated gene-encoded protein (ATM) but not ataxia telangiectasia and Rad3-related protein (ATR) was up-regulated in a time- and dosage-dependent manner after PUN treatment. KU-55933, an inhibitor of ATM, inhibited the phosphorylation of ATM induced by PUN and reversed the decreased cell viability caused by PUN. Thus, we demonstrated that PUN induces cell death of papillary thyroid carcinoma cells by triggering ATM-mediated DNA damage response, which provided novel mechanisms and potential targets for the better understanding of the anticancer actions of PUN.

Abstract B32: CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, combined with DDR agents provides a novel approach to increasing therapeutic index

Abstract Topoisomerase I inhibitors are used as standard-of-care chemotherapy in many types of cancer but are associated with significant toxicities. There is potential to improve their efficacy further by combining with inhibitors of the DNA damage response, such as the poly ADP ribose polymerase (PARP) inhibitor olaparib. However, while preclinical data highlight the improved efficacy of this combination, subsequent clinical trials have struggled due to dose limiting myelotoxicity. CRLX101 is an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a potent topoisomerase I inhibitor. This agent is preferentially targeted to tumours and demonstrated a generally favorable toxicity profile in the clinic. Here, we explored the molecular mechanism and therapeutic potential of combining CRLX101 with either olaparib or the WEE1 G2 checkpoint kinase inhibitor AZD1775, by testing both efficacy and safety in preclinical models. In vitro studies using NCI-H417a small cell lung cancer (SCLC) cells demonstrated that combination with both olaparib and AZD1775 potentiated the efficacy of CRLX101 although by different mechanisms. Cellular analyses revealed that CRLX101 treatment alone predominantly activated ATM-mediated DNA damage response and resulted in late S/G2 cell cycle arrest. Combination with a PARP inhibitor further enhanced the CRLX101-induced DNA damage response and prolonged cell cycle arrest in late S/G2 phase. In contrast, WEE1 inhibition abrogated late S/G2 cell cycle arrest induced by CRLX101, resulting in aberrant mitotic entry and enhanced cell death. Our in vivo studies using wild type Wistar rat model showed that CRLX101, olaparib and AZD1775, are well tolerated as single agents. However, concurrent combination of CRLX101 with either olaparib or AZD1775 resulted in a dose-dependent decrease in hematological parameters. We investigated sequenced schedules and demonstrated that at a 24h delay between the CRLX101 and olaparib mitigates much of the combined bone marrow toxicity, while improving the efficacy above CRLX101 alone in xenograft tumors from NCI-H417a cells. Collectively, these preclinical data demonstrate increased anti-tumor efficacy of CRLX101 when combined with DDR inhibitors. The combination schedule for CRLX101 and olaparib identified in our preclinical models as providing an increased therapeutic index has been used to develop protocols to test this combination in a relapsed (2nd line) SCLC human clinical trial (in collaboration with NCI). Citation Format: Lenka Oplustil O'Connor, Anderson T. Wang, David Jones, Rajesh Odedra, Michael Spreadborough, Joanne Wilson, Aaron Smith, Peter Cotton, Jaimini Reens, Jen Barnes, Victoria Sheridan, Andres Tellez, Alan Lau, Claire Sadler, Mark J. O'Connor, Scott Eliasof. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, combined with DDR agents provides a novel approach to increasing therapeutic index [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B32.

Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures.

The ATM gene and its protein product, the ATM protein kinase, were identified as a result of attempts to understand the molecular basis of the genetic disorder, ataxia-telangiectasia (A-T). The cardinal symptom of A-T is neurodegeneration expressed primarily as progressive cerebellar atrophy. A major tool in the investigation of ATM functions in the cerebellum is cerebellar organotypic cultures, which allow cerebellar slices to live in culture for several weeks without losing their viability and organization. These cultures are amenable to various treatments and manipulations and provide a close look at Purkinje cells in their almost natural environment. We optimized the protocol for establishing and maintaining these cultures and provide here examples of readouts of the DNA damage response in cerebellar organotypic cultures treated with a DNA-damaging agent.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Abstract 3721: A camptothecin-containing nanoparticle-drug conjugate combination with DDR agents provides a novel approach to increasing therapeutic index

Abstract Topoisomerase I inhibitors are used as standard-of-care chemotherapy in many types of cancer but are associated with significant toxicities. There is potential to improve their efficacy further by combining with inhibitors of the DNA damage response, such as the PARP inhibitor olaparib. However, while preclinical data highlight the improved efficacy of this combination, subsequent clinical trials have struggled due to dose limiting myelotoxicity. CRLX101 is an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (the most potent topoisomerase I inhibitor known). This agent is preferentially targeted to tumours and demonstrated a favourable toxicity profile in the clinic. Here, we explored the molecular mechanism and therapeutic potential of combining CRLX101 with either olaparib or the WEE1 inhibitor AZD1775, by testing both efficacy and safety in preclinical models. In vitro studies using NCI-H417a SCLC cells demonstrated that combination with both olaparib and AZD1775 potentiated the efficacy of CRLX101 although by different mechanisms. Cellular analyses revealed that CRLX101 treatment alone predominantly activated ATM-mediated DNA damage response and resulted in late S/G2 cell cycle arrest. Combination with a PARP inhibitor further enhanced the CRLX101-induced DNA damage response and prolonged cell cycle arrest in late S/G2 phase. In contrast, WEE1 inhibition abrogated late S/G2 cell cycle arrest induced by CRLX101, resulting in aberrant mitotic entry and enhanced cell death. Our in vivo studies using wild type Wistar rat model showed that CRLX101, olaparib and AZD1775, are well tolerated as single agents. However, concurrent combination of CRLX101 with either olaparib or AZD1775 resulted in a dose-dependent decrease in haematological parameters. We investigated sequenced schedules and demonstrated that at a 24h delay between the CRLX101 and olaparib mitigates much of the combined bone marrow toxicity, while improving the efficacy above CRLX101 alone in xenograft tumours from NCI-H417a cells. Collectively, these preclinical data demonstrate increased anti-tumour efficacy of CRLX101 when combined with DDR inhibitors. The combination schedule for CRLX101 and olaparib identified in our preclinical models as providing an increased therapeutic index has been used to develop protocols to test this combination in a relapsed (2nd line) SCLC human clinical trial (in collaboration with NCI). Citation Format: Lenka Oplustil O’Connor, Anderson T. Wang, David R. Jones, Rajesh Odedra, Michael Spreadborough, Joanne Wilson, Aaron Smith, Peter Cotton, Jaimini Reens, Jen Barnes, Victoria Sheridan, Scott Eliasof, Andres Tellez, Alan Lau, Claire Sadler, Mark J. O’Connor. A camptothecin-containing nanoparticle-drug conjugate combination with DDR agents provides a novel approach to increasing therapeutic index. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3721.

Influence of ATM-Mediated DNA Damage Response on Genomic Variation in Human Induced Pluripotent Stem Cells.

Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage, cellular DNA damage response (DDR), and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs, we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM-deficient iPSCs relative to wild-type iPSCs. Specifically, the early replicating regions had increased CNV losses during retroviral (RV) reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DDR reveals unique vulnerability of early replicating open chromatin to RV vectors.

Galiellalactone induces cell cycle arrest and apoptosis through the ATM/ATR pathway in prostate cancer cells.

Galiellalactone (GL) is a fungal metabolite that presents antitumor activities on prostate cancer in vitro and in vivo. In this study we show that GL induced cell cycle arrest in G2/M phase, caspase-dependent apoptosis and also affected the microtubule organization and migration ability in DU145 cells. GL did not induce double strand DNA break but activated the ATR and ATM-mediated DNA damage response (DDR) inducing CHK1, H2AX phosphorylation (fH2AX) and CDC25C downregulation. Inhibition of the ATM/ATR activation with caffeine reverted GL-induced G2/M cell cycle arrest, apoptosis and DNA damage measured by fH2AX. In contrast, UCN-01, a CHK1 inhibitor, prevented GL-induced cell cycle arrest but enhanced apoptosis in DU145 cells. Furthermore, we found that GL did not increase the levels of intracellular ROS, but the antioxidant N-acetylcysteine (NAC) completely prevented the effects of GL on fH2AX, G2/M cell cycle arrest and apoptosis. In contrast to NAC, other antioxidants such as ambroxol and EGCG did not interfere with the activity of GL on cell cycle. GL significantly suppressed DU145 xenograft growth in vivo and induced the expression of fH2AX in the tumors. These findings identify for the first time that GL activates DDR in prostate cancer.

9: The ATM-mediated DNA damage response: Implications for cancer development and treatment

9: The ATM-mediated DNA damage response: Implications for cancer development and treatment

Methylated TRF2 associates with the nuclear matrix and serves as a potential biomarker for cellular senescence.

Methylation of N-terminal arginines of the shelterin component TRF2 is important for cellular proliferation. While TRF2 is found at telomeres, where it plays an essential role in maintaining telomere integrity, little is known about the cellular localization of methylated TRF2. Here we report that the majority of methylated TRF2 is resistant to extraction by high salt buffer and DNase I treatment, indicating that methylated TRF2 is tightly associated with the nuclear matrix. We show that methylated TRF2 drastically alters its nuclear staining as normal human primary fibroblast cells approach and enter replicative senescence. This altered nuclear staining, which is found to be overwhelmingly associated with misshapen nuclei and abnormal nuclear matrix folds, can be suppressed by hTERT and it is barely detectable in transformed and cancer cell lines. We find that dysfunctional telomeres and DNA damage, both of which are potent inducers of cellular senescence, promote the altered nuclear staining of methylated TRF2, which is dependent upon the ATM-mediated DNA damage response. Collectively, these results suggest that the altered nuclear staining of methylated TRF2 may represent ATM-mediated nuclear structural alteration associated with cellular senescence. Our data further imply that methylated TRF2 can serve as a potential biomarker for cellular senescence.

Pleiotropic Effects of the Trichloroethylene-Associated P81S VHL Mutation on Metabolism, Apoptosis, and ATM-Mediated DNA Damage Response

BackgroundThe risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations.MethodsVHL deficient (Vhl -/-) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided.ResultsThe P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation–induced apoptosis.ConclusionsThe TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response.

DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT

SummaryThe consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

Studying the cerebellar DNA damage response in the tissue culture dish

The cerebellum is exquisitely sensitive to deficiencies in the cellular response to specific DNA lesions. Genetic disorders caused by such deficiencies involve relentless, progressive cerebellar atrophy with striking loss of Purkinje and granule neurons. The reason for the extreme sensitivity of these cells to defective response to certain DNA lesions is unclear. This is particularly true for ataxia-telangiectasia (A-T) – a genomic instability syndrome whose major symptom is cerebellar atrophy. It is important to understand whether the DNA damage response in the cerebellum, particularly in Purkinje neurons, has special characteristics that stem from the unique features of these cells. Murine cerebellar organotypic cultures provide a valuable experimental system for this purpose since they retain the tissue organization for several weeks in culture and appear to provide the delicate Purkinje neurons with a physiological environment close to that in vivo. We have optimized this system and are using it to examine the Atm-mediated DNA damage response (DDR) in the cerebellum, with special emphasis on Purkinje cells. Our results to date, which indicate special chromatin organization in Purkinje cells that affects certain pathways of the DDR, demonstrate the usefulness of cerebellar organotypic cultures for addressing the above questions.