Ataxia Telangiectasia Mutated Activation Research Articles

Chlamydia trachomatis Inhibits Homologous Recombination Repair of DNA Breaks by Interfering with PP2A Signaling.

Cervical and ovarian cancers exhibit characteristic mutational signatures that are reminiscent of mutational processes, including defective homologous recombination (HR) repair. How these mutational processes are initiated during carcinogenesis is largely unclear. Chlamydia trachomatis infections are epidemiologically associated with cervical and ovarian cancers. Previously, we showed that C. trachomatis induces DNA double-strand breaks (DSBs) but suppresses Ataxia-telangiectasia mutated (ATM) activation and cell cycle checkpoints. The mechanisms by which ATM regulation is modulated and its consequences for the repair pathway in C. trachomatis-infected cells remain unknown. Here, we found that Chlamydia bacteria interfere with the usual response of PP2A to DSBs. As a result, PP2A activity remains high, as the level of inhibitory phosphorylation at Y307 remains unchanged following C. trachomatis-induced DSBs. Protein-protein interaction analysis revealed that C. trachomatis facilitates persistent interactions of PP2A with ATM, thus suppressing ATM activation. This correlated with a remarkable lack of homologous recombination (HR) repair in C. trachomatis-infected cells. Chemical inhibition of PP2A activity in infected cells released ATM from PP2A, resulting in ATM phosphorylation. Activated ATM was then recruited to DSBs and initiated downstream signaling, including phosphorylation of MRE11 and NBS1 and checkpoint kinase 2 (Chk2)-mediated activation of the G2/M cell cycle checkpoint in C. trachomatis-infected cells. Further, PP2A inhibition led to the restoration of C. trachomatis-suppressed HR DNA repair function. Taking the data together, this study revealed that C. trachomatis modulates PP2A signaling to suppress ATM activation to prevent cell cycle arrest, thus contributing to a deficient high-fidelity HR pathway and a conducive environment for mutagenesis.IMPORTANCEChlamydia trachomatis induces DNA double-strand breaks in host cells but simultaneously inhibits proper DNA damage response and repair mechanisms. This may render host cells prone to loss of genetic integrity and transformation. Here we show that C. trachomatis prevents activation of the key DNA damage response mediator ATM by preventing the release from PP2A, leading to a complete absence of homologous recombination repair in host cells.

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

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

Inhibitory effect of Korean Red Ginseng extract on DNA damage response and apoptosis in Helicobacter pylori–infected gastric epithelial cells

BackgroundHelicobacter pylori increases reactive oxygen species (ROS) and induces oxidative DNA damage and apoptosis in gastric epithelial cells. DNA damage activates DNA damage response (DDR) which includes ataxia-telangiectasia-mutated (ATM) activation. ATM increases alternative reading frame (ARF) but decreases mouse double minute 2 (Mdm2). Because p53 interacts with Mdm2, H. pylori–induced loss of Mdm2 stabilizes p53 and induces apoptosis. Previous study showed that Korean Red Ginseng extract (KRG) reduces ROS and prevents cell death in H. pylori–infected gastric epithelial cells. MethodsWe determined whether KRG inhibits apoptosis by suppressing DDRs and apoptotic indices in H. pylori–infected gastric epithelial AGS cells. The infected cells were treated with or without KRG or an ATM kinase inhibitor KU-55933. ROS levels, apoptotic indices (cell death, DNA fragmentation, Bax/Bcl-2 ratio, caspase-3 activity) and DDRs (activation and levels of ATM, checkpoint kinase 2, Mdm2, ARF, and p53) were determined. ResultsH. pylori induced apoptosis by increasing apoptotic indices and ROS levels. H. pylori activated DDRs (increased p-ATM, p-checkpoint kinase 2, ARF, p-p53, and p53, but decreased Mdm2) in gastric epithelial cells. KRG reduced ROS and inhibited increase in apoptotic indices and DDRs in H. pylori–infected gastric epithelial cells. KU-55933 suppressed DDRs and apoptosis in H. pylori–infected gastric epithelial cells, similar to KRG. ConclusionKRG suppressed ATM-mediated DDRs and apoptosis by reducing ROS in H. pylori–infected gastric epithelial cells. Supplementation with KRG may prevent the oxidative stress-mediated gastric impairment associated with H. pylori infection.

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

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

Abstract A15: DNA damage response-targeted molecules for breast cancer therapy

Abstract Breast cancer genes 1 and 2 (BRACA1 and BRACA2) are known to be linked to breast cancer risk. BRCA1 is an ATM substrate and plays critical roles in the DNA damage response (DDR) and maintenance of genomic stability. Furthermore, recent studies have shown that the aberrant expression of DNA damage repair transducer, ataxia-telangiectasia mutated (ATM), is associated with breast cancers. To develop target-specific drugs, we rationally designed a dual-function small molecule, SP-1-303, by conferring ATM activation and HDAC inhibition activity. The compound was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor at an IC50 of 120 nM. Western analysis revealed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and ATM activation (p-ATM/S1987). Testing two ER-positive BC cell lines (MCF7 and T-47D), two triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effect of SP-1-303 on cell growth was examined by cytotoxicity assays. SP-1-303 demonstrated cytotoxicity (IC50) of ER-positive BC cells in a range of 0.2-0.3 μM, while the values for TNBC cells are in a range of 1-3 μM and 12 μM for normal breast epithelial cells, respectively. Furthermore, proteomic analysis of MCF7 cells following SP-1-303 treatment identified differential effects on expression of various DDR molecules and chromatin regulators, including ATM, BRCA1, TP53BP1, NBN, histones, and chromatin remodelers. Taken together, SP-1-303 shows specificity for ER-positive breast cancers, potentially by modulating the DDR signaling pathway. Citation Format: Alfredo Valenea, Scott Grindrod, Mira Jung. DNA damage response-targeted molecules for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A15.

The histone chaperone ASF1 regulates the activation of ATM and DNA-PKcs in response to DNA double-strand breaks

ABSTRACTThe Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.

The identification of the ATR inhibitor VE-822 as a therapeutic strategy for enhancing cisplatin chemosensitivity in esophageal squamous cell carcinoma

Inducing DNA damage is known to be one of the mechanisms of cytotoxic chemotherapy agents for cancer such as cisplatin. The endogenous DNA damage response confers chemoresistance to these agents by repairing DNA damage. The initiation and transduction of the DNA damage response (DDR) signaling pathway, which is dependent on the activation of ATM (ataxia-telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related), is essential for DNA damage repair, the maintenance of genomic stability and cell survival. Therefore, ATM or ATR inhibition is considered as a promising strategy for sensitizing cancer cells to chemotherapy. This study is aimed to explore the effect of ATR inhibitor on sensitizing ESCC (esophageal squamous cell carcinoma) cells to cisplatin, and whether ATM deficiency could impact the sensitization. We found that 21.5% of ESCC cases had ATM deficiency and that patients with ATR activation after neoadjuvant chemotherapy had worse chemotherapy response and poorer overall survival than that without ATR activation (32 mons vs. >140mons). Then, it was shown that VE-822 inhibited ATR-CHK1 pathway activation, leading to the accumulation of cisplatin-modified DNA. And it inhibited cell proliferation, induced cell cycle arrest in G1 phase and enhanced cell apoptosis. Moreover, VE-822 significantly sensitized ESCC cells to cisplatin, and these two drugs had synergistic effects, especially in ATM-deficient cells, in vitro and in vivo. Our results suggest that ATR inhibition combining with cisplatin is a new strategy for managing patients with ESCC, especially those with ATM-deficiency. However, this is an idea that requires further validation.

Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

Linker histone H1 is a master regulator of higher order chromatin structure, but its involvement in the DNA damage response and repair is unclear. Here, we report that linker histone H1.2 is an essential regulator of ataxia telangiectasia mutated (ATM) activation. We show that H1.2 protects chromatin from aberrant ATM activation through direct interaction with the ATM HEAT repeat domain and inhibition of MRE11-RAD50-NBS1 (MRN) complex-dependent ATM recruitment. Upon DNA damage, H1.2 undergoes rapid PARP1-dependent chromatin dissociation through poly-ADP-ribosylation (PARylation) of its C terminus and further proteasomal degradation. Inhibition of H1.2 displacement by PARP1 depletion or an H1.2 PARylation-dead mutation compromises ATM activation and DNA damage repair, thus leading to impaired cell survival. Taken together, our findings suggest that linker histone H1.2 functions as a physiological barrier for ATM to target the chromatin, and PARylation-mediated active H1.2 turnover is required for robust ATM activation and DNA damage repair.

Camptothecin induces G2/M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species.

In the present study, we report that camptothecin (CPT) caused irreversible cell cycle arrest at the G2/M phase, and was associated with decreased levels of cell division cycle 25C (Cdc25C) and increased levels of cyclin B1, p21, and phospho-H3. Interestingly, the reactive oxygen species (ROS) inhibitor, glutathione, decreased CPT-induced G2/M phase arrest and moderately induced S phase arrest, indicating that the ROS is required for the regulation of CPT-induced G2/M phase arrest. Furthermore, transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2), in the presence of CPT, increased the ROS’ level and further shifted the cell cycle from early S phase to the G2/M phase, indicating that Nrf2 delayed the S phase in response to CPT. We also found that CPT-induced G2/M phase arrest increased, along with the ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2)-Cdc25C axis. Additionally, the proteasome inhibitor, MG132, restored the decrease in Cdc25C levels in response to CPT, and significantly downregulated CPT-induced G2/M phase arrest, suggesting that CPT enhances G2/M phase arrest through proteasome-mediated Cdc25C degradation. Our data also indicated that inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibited CPT-induced p21 and cyclin B1 levels; however, inhibition of ERK blocked CPT-induced G2/M phase arrest, and inhibition of JNK enhanced apoptosis in response to CPT. Finally, we found that CPT-induced G2/M phase arrest circumvented apoptosis by activating autophagy through ATM activation. These findings suggest that CPT-induced G2/M phase arrest through the ROS-ATM-Chk2-Cdc25C axis is accompanied by the activation of autophagy.

Insufficiency of DNA repair enzyme ATM promotes naive CD4 T-cell loss in chronic hepatitis C virus infection

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci—hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.

Dysregulation of the Tumor Suppressor Tip60 and ATM Signaling Paradigm by the HIV Transactivator of Transcription

Despite the longevity afforded by modern anti‐retroviral therapy, long‐term HIV infection increases the risk for pulmonary co‐morbidities associated with cell cycle dysregulation including lung cancer and pulmonary arterial hypertension. As such, we postulate that select viral factors, in particular the HIV transactivator of transcription (Tat), interfere with cellular processes involving tumor suppressors and other cell cycle regulators during chronic infection. Tat is well known to inhibit the activity of the tumor suppressor Tat Interacting Protein 60 kDa (Tip60), an acetyltransferase critical in the progression to cell cycle arrest and/or apoptosis. In response to stressors such as DNA damage and oxidative stress, Tip60 activates the cell cycle master regulator kinase Ataxia Telangiectasia Mutated (ATM) via acetylation, initiating a signaling cascade that culminates in the activation of a DNA damage response resulting in both short and long term cellular arrest. Thus, we hypothesize that inhibition of Tip60 by Tat interferes with cellular arrest in responses to these stressors and results in abnormal proliferation. We addressed the hypothesis using HeLa cells stably transfected to express Tat (HeLa‐TatIII). Utilizing an ATP based luminescent cell viability assay, we observed accelerated cell proliferation in HeLa‐TatIII compared to its HeLa wild‐type counterpart. Furthermore, immunoprecipitation experiments in HeLa‐WT cells confirmed a physical interaction between Tat and Tip60. Acetylation by Tip60 results in autophosphorylation of ATM at ser1981, and this phosphorylation event is a canonical marker for ATM activation. As such, we determined the phosphorylation state of ATM ser1981 after treatment with doxorubicin in HeLa‐WT and HeLa‐TatIII cells. We found that, following treatment with doxorubicin, ATM phosphorylation at ser1981 was severely attenuated in HeLa‐TatIII cells compared to HeLa‐WTs, suggesting a role for Tat in the inhibition of ATM activation. Taken together, these data support the role of the HIV Tat protein in dysregulation of cell cycle arrest via ATM signaling pathways. Whether HIV promotes cellular proliferation in co‐morbidities such as lung cancer and pulmonary arterial hypertension via these pathways remains to be addressed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H2O2 (600 μM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H2O2, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H2O2. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H2O2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H2O2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H2O2-induced cytotoxicity. The relevance of this kinase in vivo merits further study.

The Landscape of Somatic Genetic Alterations in Breast Cancers From ATM Germline Mutation Carriers.

Pathogenic germline variants in ataxia-telangiectasia mutated (ATM), a gene that plays a role in DNA damage response and cell cycle checkpoints, confer an increased breast cancer (BC) risk. Here, we investigated the phenotypic characteristics and landscape of somatic genetic alterations in 24 BCs from ATM germline mutation carriers by whole-exome and targeted sequencing. ATM-associated BCs were consistently hormone receptor positive and largely displayed minimal immune infiltrate. Although 79.2% of these tumors exhibited loss of heterozygosity of the ATM wild-type allele, none displayed high activity of mutational signature 3 associated with defective homologous recombination DNA (HRD) repair. No TP53 mutations were found in the ATM-associated BCs. Analysis of an independent data set confirmed that germline ATM variants and TP53 somatic mutations are mutually exclusive. Our findings indicate that ATM-associated BCs often harbor bi-allelic inactivation of ATM, are phenotypically distinct from BRCA1/2-associated BCs, lack HRD-related mutational signatures, and that TP53 and ATM genetic alterations are likely epistatic.

Anacardic acid inhibits pancreatic cancer cell growth, and potentiates chemotherapeutic effect by Chmp1A - ATM - p53 signaling pathway

BackgroundPancreatic cancer is one of the leading causes of cancer related death and its incidence has risen steadily. Although anticancer drugs have been developed based on the new molecular findings, the drugs have produced unsatisfactory results due to toxicity and resistance. Thus, a complementary therapeutic intervention is urgently needed for pancreatic cancer patients.MethodsThe aim of this study was to assess the potential therapeutic effect of Anacardic acid on pancreatic cancer in vitro and elucidate its underlying mechanisms. Human pancreatic cancer cells were treated with Anacardic acid and assessed for the cytotoxic effect using MTT and spheroid formation assays. Using the same methods, the synergy between Anacardic acid and 5-Fluorouracil or Gemcitabine was determined. To elucidate the underlying molecular mechanisms, Western blot analysis and immunocytochemistry were performed on cancer cells treated with Anacardic acid alone or in combination with 5-Fluorouracil or Gemcitabine. Chromatin Modifying Protein 1A (Chmp1A), Ataxia Telangiectasia Mutated (ATM), and p53 were the primary signaling molecules examined. In addition, Chmp1A was silenced with shRNA to examine the necessity of Chmp1A for the anticancer effect of Anacardic acid, 5-Fluorouracil, or Gemcitabine.ResultsAnacardic acid induced an anticancer effect in pancreatic cancer cell lines in a dose dependent manner, and increased the cytotoxicity of 5-Fluorouracil or Gemcitabine in MTT cell viability assays. In spheroid formation assays, spheroids formed were smaller in size and in number upon Anacardic acid treatment compared to control. Mechanistically, Anacardic acid exerted its anticancer activity via the activation of Chmp1A, ATM, and p53. Interestingly, 5-Fluorouracil and Gemcitabine also induced an increase in Chmp1A protein level, suggesting that Chmp1A might mediate the cytotoxic action of chemotherapeutics. Silencing experiments indicate that Chmp1A is required for the action of Anacardic acid, but not for 5-Fluorouracil or Gemcitabine.ConclusionsOur data suggests that Anacardic Acid might be a promising complementary supplement to slow the initiation or progression of pancreatic cancer.

EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein–Barr virus (EBV) infection. In NPC, miR‐BARTs, the EBV‐encoded miRNAs derived from BamH1‐A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV‐encoded miRNAs in a panel of NPC patient‐derived xenografts and an EBV‐positive NPC cell line by small RNA sequencing. Among the 40 miR‐BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV‐miRNAs, BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p could negatively regulate the expression of a key DNA double‐strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'‐UTR. Notably, the expression of these four miR‐BARTs represented more than 10% of all EBV‐encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT‐PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR‐BARTs in EBV‐positive NPC cells, we further demonstrated the novel function of miR‐BARTs in inhibiting Zta‐induced lytic reactivation. These findings imply that the four viral miRNAs work co‐operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract PD1-15: The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers

Abstract Introduction:Pathogenic and/or founder germline variants in the ataxia-telangiectasia mutated (ATM) gene confer an increased breast cancer (BC) risk. The protein kinase ATM plays a central role inDNA double-strand break-repair and in the activation of downstream targets such as p53 and BRCA1. We sought to define the repertoire of somatic genetic alterations of BCs from patients with pathogenic germline ATM mutations and whether somatic loss of heterozygosity (LOH) of ATM would be present in these cancers. Methods: 21 BCs from ATM germline mutation carriers were microdissected. Tumor and normal DNA samples were subjected to whole-exome sequencing (WES, n=12) or massively parallel sequencing targeting all coding regions and selected intronic and regulatory regions of 410 key cancer genes (n=9). Somatic mutations, copy number alterations, cancer cell fractions, large-scale state transitions (LSTs) and mutational signatures were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to assess LOH of the wild-type allele of ATM. Results: Of the patients included in this study, 71%, 24% and 5% of cases harbored ATM missense (all but one p.V2424G), frame-shift and nonsense germline mutations, respectively. All tumors were ER-positive and four (19%) were HER2-positive. The median age of the patients was 46 years (32–79 years). Our analyses revealed biallelic inactivation of ATM through LOH of the wild-type allele in 16 of 21 cases (76%), and second somatic ATM mutations were not found. The median number of non-synonymous somatic mutations was 38 (range 15-113) and 2 (range 0-8)in tumors subjected to WES and targeted sequencing, respectively. The repertoire of somatic genetic alterations of ATM-associated BCs was found to be heterogeneous, including clonal PIK3CA mutations (24%), GATA3 mutations (19%), FANCI amplifications (19%) and CCND1 amplifications (14%). Importantly, however, no somatic mutations affecting TP53 were found. Analysis of the WES data revealed that 5 (42%) ATM-associated BCs displayed high LST scores, all of which harbored bi-allelic ATM inactivation. In contrast to BRCA1- and BRCA2-associated BCs, which frequently display the mutational signature 3 associated with defective homologous recombination DNA repair, the ATM-associated BCs studied displayed the ageing mutational signature (i.e. signature 1). Comparison of the mutational profiles of the ATM--associated BCs subjected to WES (n=12) with those of BRCA1- (n=11) and BRCA2-associated (n=10) BCs from The Cancer Genome Atlas revealed that TP53 was more frequently mutated in BCs from BRCA1 germline mutation carriers (0% vs 72%, P<0.001), while no differences with BRCA2-associated BCs were found. Conclusion: ATM-associated BCs frequently display bi-allelic ATM inactivation through LOH of the wild-type allele and a subset of these cases displayed high levels of LSTs. These findings suggest that at least in a subset of ATM-associated BCs, biallelic inactivation of ATM rather than a dominant negative effect of the germline mutation may be the mechanism of inactivation of this tumor suppressor gene. The repertoire of somatic genetic alterations of ATM-associated BCs is heterogeneous, with a noticeable lack of TP53 somatic mutations. Citation Format: Weigelt B, Bi R, Kumar R, James PA, Thorne H, Couch FJ, Eccles DM, Blows F, Geyer FC, Li A, Selenica P, Lim RS, Blecua P, Shen R, Wen H, Robson ME, Reis-Filho JS, Chenevix-Trench G. The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-15.

Pine needle oil induces G2/M arrest of HepG2 cells by activating the ATM pathway.

Over the last two decades, inducing DNA damage of cancer cells by natural medicines has become a research hotspot in the field of cancer treatment. Although various natural medicines have anticancer effects, very few studies have been conducted to explore the anti-cancer effect of pine needle oil. In the present study, the role of pine needle oil in inducing G2/M arrest in HepG2 cells was investigated. The data revealed that pine needle oil could induce DNA damage in a dose-dependent manner. In the pine needle oil-treated HepG2 cells, the protein levels of phosphorylated (p)-ataxia-telangiectasia mutated (ATM), γ-H2A histone family, member X, p-p53, p-checkpoint kinase 2 and p-cell division cycle 25C were evidently increased, indicating that pine needle oil facilitated G2/M arrest in HepG2 cells through the ATM pathway. In response to the treatment with pine needle oil, ATM was activated in HepG2 cells, which subsequently phosphorylated downstream targets and induced G2/M arrest. In summary, the data of the present study indicated that pine needle oil induces G2/M arrest in HepG2 cells by facilitating ATM activation.

ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T

BackgroundAtaxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1.ResultsIn the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new “ATMdexa1 variants” originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients’ blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment.ConclusionsFor the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration.

Abstract 4054: Development of dual function small molecules as therapeutic agents for cancer treatment

Abstract Inhibitors of histone deactylases (HDACs) are emerging as potent anti-cancer agents for treatment of patients with hematopoietic and solid tumors. However, most of these compounds exhibit limitations, including off-targets and toxicity. To improve the efficacy and target specificity of drugs, we developed the compound, SP-1-161 conferring HDAC inhibition activity and ataxia-telangiectasia mutated (ATM) activation. The purpose of this study is to develop a therapy that optimizes the protective effects of ATM activation in normal tissue with the sensitizing effects of HDAC inhibition in cancerous tissue into a single molecule. The compound SP-1-161 was rationally designed by optimizing ATM activation by the cap domain of a hydroximic acid, screened against a panel of Class I and Class II HDAC enzymes, and identified 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 ATM activation in MCF7 cells. Phosphorylated ATM was gradually increased within 1 - 4 hrs in a time-dependent manner. SP-1-161 was then tested in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines to determine its cytotoxicity and effect on radiation clonogenic survival in combination with graded radiation exposure. The data showed that the cytotoxicity values (IC50) were low micro-molar ranges. Furthermore, SP-1-161 protected 184A1 cells (from D0 = 1 Gy to D0 = 1.4 Gy) while increasing sensitivity of MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Together, our results demonstrate that SP-1-161 is an unprecedented radio-chemo therapeutic agent for treatment of cancers while protecting normal cells. Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Development of dual function small molecules as therapeutic agents for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2017-4054

Abstract 3317: Autophagy protects ovarian cancer cells from olaparib-induced toxicity

Abstract Ovarian cancer (OC) is the most lethal gynecologic malignancy. Persistence of dormant, drug resistant cancer cells is one of the major factors that contribute to poor outcomes. The Poly (ADP) ribose polymerase (PARP) inhibitor, olaparib, has shown promising anti-cancer activity and has been approved by the FDA for the treatment of ovarian cancer with BRCA1/2 mutations. Olaparib targets DNA repair mechanisms within cancer cells and induces apoptosis. Both preclinical and clinical data indicate that efficacy of olaparib can be decreased by acquired resistance. One mechanism of resistance involves autophagy. Autophagy can degrade organelles and long-lived proteins to provide energy for cancer cells under nutrient poor conditions or in the presence of stress. The energy provided from autophagy is used to fuel the DNA repair process and metabolic needs in cancer cells. Autophagy can protect cancer cells from chemotherapy or can enhance the response to certain drugs. In this study, we have asked whether autophagy protects OC cells against the cytotoxic effects of olaparib or augments damage. We first determined PARP activity using a modified PARP activity assay in different OC cell lines. Olaparib-induced autophagy was determined by punctate localization of GFP-LC3, conversion of LC3I to LC3II on protein blots and electron microscopy. To test whether autophagy contributes to the resistance of OC cells to olaparib, we determined the impact of autophagy inhibition on the sensitivity of OC cells to olaparib by either using the pharmacologic inhibitors chloroquine (CQ), LYS05 or short interfering RNA (siRNA) against ATG7. Upon investigation of a potential mechanism of olaparib-induced autophagy we evaluated the production of reactive oxygen species (ROS) and the expression of JNK, Ataxia-telangiectasia mutated (ATM), mTOR and p70S6K. Inhibition of PARP by olaparib treatment, decreased PARP activity in a dose- dependent manner, leading to DNA damage judged by phosphorylation of γ-H2AX. Olaparib treatment decreased cell growth, caused cell cycle arrest and significantly enhanced apoptosis in 5 of 5 OC cell lines. Olaparib induced autophagy in 8 of 8 OC cell lines. Both pharmacologic and genetic inhibition of autophagy increased OC cells sensitivity to olaparib and induced apoptosis in 5 of 6 OC cell lines. Furthermore, flow cytometry and western blot analysis demonstrated that olaparib treatment increased ROS production and increased the phosphorylation of JNK and ATM while decreasing the phosphorylation of mTOR and p70S6K. Taken together, our data described autophagy as a potential mechanism of olaparib resistance in OC that may depend upon the activation of ATM and JNK by ROS downregulating pmTOR and p70S6K. Combination of olaparib with CQ, LYS05 or other autophagy inhibitor may provide a novel strategy to increase the efficacy of olaparib in OC. Current studies are underway to test our hypothesis in vivo. Citation Format: Janice M. Santiago-O'Farrill, Safia Essien, Mary Figueroa, Lan Pang, Ravi Amaravadi, Zhen Lu, Robert C. Bast. Autophagy protects ovarian cancer cells from olaparib-induced toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2017-3317