Knockdown Of Ataxia Telangiectasia Mutated Research Articles

Inhibition of ATM promotes PD-L1 expression by activating JNK/c-Jun/TNF-α signaling axis in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer. Anti-PD-1/PD-L1 treatment for advanced TNBC is still limited to PD-L1-positive patients. Ataxia telangiectasia mutated (ATM) is a switch molecule for homologous recombination and repair. In this study, we found a significant negative correlation between ATM and PD-L1 in 4 TNBC clinical specimens by single-cell RNA sequencing (scRNA-seq), which was confirmed by immunochemical staining in 86 TNBC specimens. We then established ATM knockdown TNBC stable cell lines to perform in vitro studies and animal experiments, proving the negative regulation of PD-L1 by ATM via suppression of tumor necrosis factor-alpha (TNF-α), which was confirmed by cytokine array analysis of TNBC cell line and analysis of clinical specimens. We further found that ATM inhibits TNF-α via inactivating JNK/c-Jun by scRNA-seq, Western blot and luciferase reporter assays. Finally, we identified a negative correlation between changes in phospho-ATMS1981 and PD-L1 levels in TNBC post- and pre-neoadjuvant therapy. This study reveals a novel mechanism by which ATM negatively regulates PD-L1 by downregulating JNK/c-Jun/TNF-α in TNBC, shedding light on the wide application of immune checkpoint blockade therapy for treating multi-line-resistant TNBC.

Reduced expression of phosphorylated ataxia-telangiectasia mutated gene is related to poor prognosis and gemcitabine chemoresistance in pancreatic cancer

BackgroundLoss of expression of the gene ataxia-telangiectasia mutated (ATM), occurring in patients with multiple primary malignancies, including pancreatic cancer, is associated with poor prognosis. In this study, we investigated the detailed molecular mechanism through which ATM expression affects the prognosis of patients with pancreatic cancer.MethodsThe levels of expression of ATM and phosphorylated ATM in patients with pancreatic cancer who had undergone surgical resection were analyzed using immunohistochemistry staining. RNA sequencing was performed on ATM-knockdown pancreatic-cancer cells to elucidate the mechanism underlying the invlovement of ATM in pancreatic cancer.ResultsImmunohistochemical analysis showed that 15.3% and 27.8% of clinical samples had low levels of ATM and phosphorylated ATM, respectively. Low expression of phosphorylated ATM substantially reduced overall and disease-free survival in patients with pancreatic cancer. In the pancreatic cancer cell lines with ATM low expression, resistance to gemcitabine was demonstrated. The RNA sequence demonstrated that ATM knockdown induced the expression of MET and NTN1. In ATM knockdown cells, it was also revealed that the protein expression levels of HIF-1α and antiapoptotic BCL-2/BAD were upregulated.ConclusionsThese findings demonstrate that loss of ATM expression increases tumor development, suppresses apoptosis, and reduces gemcitabine sensitivity. Additionally, loss of phosphorylated ATM is associated with a poor prognosis in patients with pancreatic cancer. Thus, phosphorylated ATM could be a possible target for pancreatic cancer treatment as well as a molecular marker to track patient prognosis.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation.

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

Macamide B suppresses lung cancer progression potentially via the ATM signaling pathway.

Macamides are a class of bioactive natural products obtained from Lepidium meyenii (maca), which have been reported to exert inhibitory activity in cancer. However, their role in lung cancer is currently unknown. In the present study, macamide B was shown to inhibit the proliferation and invasion of lung cancer cells, as determined by Cell Counting Kit-8 and Transwell assays, respectively. By contrast, macamide B induced cell apoptosis, as determined by Annexin V-FITC assay. Moreover, combined treatment with macamide B and olaparib, an inhibitor of poly (ADP-ribose) polymerase, further suppressed the proliferation of lung cancer cells. At the molecular level, the expression of ataxia-telangiectasia mutated (ATM), RAD51, p53 and cleaved caspase-3 were significantly increased by macamide B, as determined by western blotting, whereas the expression levels of Bcl-2 were decreased. By contrast, when ATM expression was knocked down by small interfering RNA technology in A549 cells treated with macamide B, the expression levels of ATM, RAD51, p53 and cleaved caspase-3 were reduced, whereas those of Bcl-2 were increased. Consistently, cell proliferation and invasive ability were partially rescued by ATM knockdown. In conclusion, macamide B inhibits lung cancer progression by inhibiting cell proliferation and invasion, and inducing apoptosis. Furthermore, macamide B may participate in regulating the ATM signaling pathway. The present study provides a potential new natural drug for treating patients with lung cancer.

ALT neuroblastoma chemoresistance due to telomere dysfunction-induced ATM activation is reversible with ATM inhibitor AZD0156.

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

Abstract 1442: Targeting ATM kinase and mTOR signaling reverses bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy

Abstract Internal tandem duplication (ITD) mutations in FMS-like tyrosine kinase 3 (FLT3) are among the most common mutations in acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD causes constitutive activation of FLT3, and the strong evidence that activated FLT3 drives leukemogenesis has led to the development of several FLT3-targeted inhibitors. However, clinical studies of FLT3-targeted inhibitors have demonstrated much more effective clearing of leukemic burden in the periphery than in the bone marrow, implicating the bone marrow microenvironment as a potential contributor to drug resistance. As previously reported, the conditioned media of human bone marrow stromal cells (CM-BMSC) protects human FLT3-ITD AML cells from the killing effect of FLT3-targeted therapy despite complete inhibition of FLT3. We further show that genetic knockdown of Ataxia Telangiectasia Mutated (ATM) in combination with FLT3 inhibition substantially reverses the protection from cell death mediated by CM-BMSC. While autophosphorylation activity of ATM is downregulated upon FLT3 inhibition in regular media, CM-BMSC prevented the loss of ATM activity. Interestingly, genetic knockdown of ATM in human FLT3-ITD AML cells impaired the activity of mTOR Complex 1, and unbiased analyses of gene expression and signaling pathways by RNA-seq analysis and Reverse Phase Protein Arrays (RPPA) showed that mTOR signaling is downregulated with FLT3 inhibition in regular media, coinciding with marked inhibition of the protein translation machinery. Both mTOR signaling and protein translation were maintained during FLT3 inhibition in the presence of CM-BMSC. Furthermore, human FLT3-ITD AML cells treated with mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib reversed the protection mediated by CM-BMSC, and combination therapies are currently being tested in mouse models. These data suggest that ATM kinase and mTOR signaling play a key role in bone marrow stoma-mediated protection against FLT3 targeted therapy. Findings from this research provide new insights into the mechanism of bone marrow stroma-mediated protection of FLT3-ITD AML from FLT3-targeted therapy, identifying additional candidates for combinatorial therapies designed to overcome the protective effects of bone marrow stromal cells and improve patient outcomes. Citation Format: Hae J. Park, Mark A. Gregory, Vadym Zaberezhnyy, James DeGregori. Targeting ATM kinase and mTOR signaling reverses bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1442.

Abstract PO-017: Radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo

Abstract While radiotherapy (RT) is fundamental for the treatment of brain tumors, irradiation of the brain frequently causes devastating effects on cognitive function and quality of life. DNA damage within neural stem cells (NSC) is a key factor in the pathogenesis of radiation-induced cognitive dysfunction. The ataxia telangiectasia mutated (ATM) kinase is a central protein in the DNA damage response and a critical determinant of tumor cell survival after radiation. ATM inhibition potently radiosensitizes preclinical models of GBM in vitro and in vivo. A novel, brain penetrant ATM inhibitor AZD1390, which is predicted to achieve brain tumor concentrations in the range of 1-5nM, is currently in early phase clinical evaluation in combination with RT. In marked contrast to observations in tumor models, genetic knockdown of ATM has radioprotective effects on NSC in vitro; the proposed mechanism is via suppression of p53 mediated apoptosis. The purpose of this study was to investigate the impact of AZD1390 on survival responses and mode of death in NSCs exposed to RT in vitro and in vivo. NSCs were derived from the telencephalon of E13 mouse embryos. Cells were treated with AZD1390 (0.1-10nM) 1 hour prior to ionizing radiation (IR; 0-5 Gy). Mode and timing of cell death was interrogated using IncuCyte live cell analysis to measure proliferation, cytotoxicity and apoptosis up to 72 hours post-IR. Cell viability and neurosphere formation assays were also used to measure radiation sensitivity in vitro. C57BL/6 mice received 20Gy hemibrain irradiation +/- 7-day treatment with AZD1390 (10mg/kg). Immunohistochemistry for Ki67 and Sox2 was used to assess effects on NSC in the subventricular zone (SVZ) 50 days post-irradiation. In vitro AZD1390 (1-10nM) inhibited ATM kinase function within 1 hour, evidenced by abrogation of KAP1 and p53 phosphorylation. NSCs primarily undergo apoptosis in response to IR. AZD1390 at 1 and 3nM significantly reduced apoptosis in irradiated NSCs (ratios of annexin V area under the curve 1.95 and 2 respectively); 10 nM had no effect on this parameter. Proliferation rates and cell viability after radiation were preserved at all drug concentrations. AZD1390 at 1nM did not modulate radiation effects on neurosphere formation whereas at 10nM a radiosensitizing effect was observed (ratio of SF[3Gy]=0.25). In vivo, IR decreased the number of Ki67 positive proliferating cells (92% reduction) and Sox2-positive cells (24% reduction) in the SVZ after 50 days; these effects were not exacerbated by addition of AZD1390. Acute effects (24 hours post-IR) are under investigation. We demonstrate in vitro that AZD1390 has radioprotective effects on NSCs at clinically achievable concentrations. In vivo, treatment with AZD1390 did not enhance the effects of radiation on NSCs in the SVZ. In the context of its profound radiosensitizing effects on GBM models, the absence of radiosensitization of NSCs both in vitro and in vivo strengthens the rationale for evaluating AZD1390 in combination with RT in GBM patients. Citation Format: Rodrigo Guttierez-Quintana, David J. Walker, Mark R. Jackson, Natividad Gomez-Roman, Sandeep Chahal, Stephen T. Durant, Anthony J. Chalmers. Radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-017.

ATM and P53 differentially regulate pancreatic beta cell survival in Ins1E cells.

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.

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

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

Ataxia-telangiectasia mutated interacts with Parkin and induces mitophagy independent of kinase activity. Evidence from mantle cell lymphoma.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently altered in human cancers including mantle cell lymphoma. Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy in both murine thymocytes and in ataxia-telangiectasia cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in mantle cell lymphoma and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 mantle cell lymphoma cells possess single copy kinase-dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATMproficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial reactive oxygen species. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132, suggesting that the ATMParkin interaction is important for Parkin stability. Neither loss of ATM kinase activity in primary B-cell lymphomas nor inhibition of ATM kinase in mantle cell lymphoma, ataxia-telangiectasia and HeLa cell lines mitigated FCCP- or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-UbSer65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of the role of ATM in mitophagy in mantle cell lymphoma and other B-cell lymphomas.

MiR‐26a attenuates cardiac apoptosis and fibrosis by targeting ataxia–telangiectasia mutated in myocardial infarction

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia-telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.

ATM Expression Is Elevated in Established Radiation-Resistant Breast Cancer Cells and Improves DNA Repair Efficiency.

Repair of damaged DNA induced by radiation plays an important role in the development of radioresistance, which greatly restricts patients' benefit from radiotherapy. However, the relation between radioresistance development and DNA double-strand break repair pathways (mainly non-homologous end joining and homologous recombination) and how these pathways contribute to radioresistance are unclear. Here, we established a radioresistant breast cancer cell line by repeated ionizing radiation and studied the alteration in DNA repair capacity. Compared with parental sham-treated cells, radioresistant breast cancer cells present elevated radioresistance, enhanced malignancy, increased expression of Ataxia-telangiectasia mutated (ATM), and increased DNA damage repair efficiency, as reflected by accelerated γ-H2AX kinetic. These defects can be reversed by ATM inhibition or ATM knockdown, indicating a potential link between ATM, DNA repair pathway and radiosensitivity. We propose that cancer cells develop elevated radioresistance through enhanced DNA damage repair efficiency mediated by increased ATM expression. Our work might provide a new evidence supporting the potential of ATM as a potential target of cancer therapy.

ATM Deficiency Accelerates DNA Damage, Telomere Erosion, and Premature T Cell Aging in HIV-Infected Individuals on Antiretroviral Therapy.

HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends—telomeres, leading to accelerated telomere erosion—a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro, recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection.

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.

Abstract 271: The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells

Abstract Therapeutic targeting of the DNA damage response (DDR) has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication stress, which activates Ataxia-telangiectasia and Rad3-related kinase (ATR). We have demonstrated previously, efficacy of the ATR inhibitor (AZD6738) in PDAC models, particularly when combined with gemcitabine (gem) (Wallez et al, MCT, 2018). This combination strongly induces activation of Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), indicating the induction of double-strand breaks. It has been suggested that ATM-deficiency can sensitize cancer cells to ATR inhibitors. This study sought to assess the relevance of this finding to PDAC and to interrogate the distinct roles of ATM and DNA-PK in response to ATRi/gem in PDAC cell lines. The ATM inhibitor, AZD0156, sensitized human pancreatic cancer cell lines (MIA PaCa-2, HPAF-II, AsPC-1) to AZD6738, as determined by SRB assays (e.g. MIA PaCa-2; AZD6738 GI50 = 3.8 µM, versus 1.3 µM in the presence of 30 nM AZD0156) and in longer term colony forming assays (surviving fraction (SF) (1 µM AZD6738); 78 +/- 0.9%, SF (1 µM AZD6738 + 30nM AZD0156); 11 +/- 4.2%). However, ATM knockdown with siRNA did not sensitize to AZD6738, nor to the combination of AZD6738/gem. This suggests that the presence of kinase-inhibited ATM at sites of DNA damage is more deleterious to PDAC cells than deficiency of ATM protein expression. We established that AZD6738/gem could induce phosphorylation of the canonical targets of ATM (Chk2, KAP1) in MIA PaCa-2 when ATM was knocked down or inhibited. DNA-PK was also activated in response to exposure to ATRi/gem and we postulated that it was responsible for the Chk2/KAP1 activation. We then demonstrated that Chk2/KAP1 activation could be abrogated by DNA-PK inhibition with NU7441. Furthermore, in an ATM-null pancreatic cancer cell line, AZD6738/gem-induced KAP1 phosphorylation was also abrogated by inhibition of DNA-PK. Thus, DNA-PK appears to be responsible for the downstream activation of Chk2 and KAP1, induced by AZD6738/gem in PDAC cells. Therefore, as well as revealing that inhibition of ATM using the novel inhibitor AZD0156 is highly deleterious to ATR inhibited PDAC cells, we have identified a compensatory mechanism via DNA-PK, which may be relevant for maximizing the therapeutic potential of DDR inhibitors in PDAC. Since DNA-PK clearly plays a dominant role in the DDR signaling pathways, we are now assessing whether DNA-PK inhibition synergizes with AZD6738+/- gem. Citation Format: Charles R. Dunlop, Yann Wallez, Sandra Bernaldo de Quirós Fernández, Saadia A. Karim, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 271.

Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos.

DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA-dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulation of the HR pathway in mammalian embryos.-Bohrer, R. C., Dicks, N., Gutierrez, K., Duggavathi, R., Bordignon, V. Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos.

P3.02-077 Platin Sensitivity and ATM-Deficiency in Non-Small Cell Lung Cancer

Platinum based antineoplastic therapies (platins) are a first line treatment for non-small cell lung cancer (NSCLC) that generate DNA breaks and stimulate DNA damage response pathways. An inability to repair damage generated by these agents leads to cytotoxicity and cell death. A key mediator of the DNA damage response is ataxia telangiectasia mutated (ATM), an activator of downstream targets involved in DNA repair, cell cycle arrest, and apoptosis. Our lab has demonstrated that cell lines lacking ATM show increased sensitivity to platins. We hypothesize that platin exposure will activate ATM and that cells deficient in ATM will be innately sensitive to platins. Here we assess the molecular action of ATM in response to platins to determine if ATM-deficiency is predictive of platin sensitivity. ATM status was determined in five NSCLC cell lines using western blotting and RT-qPCR. Cell lines were treated with varying concentrations cisplatin, carboplatin and oxaliplatin for 18-hours and assessed for ATM phosphorylation by western blot. Additionally, downstream targets of ATM (KAP-1, p53, and gamma-H2AX) were investigated to determine ATM pathway activation. Knockdown cell lines were generated using shRNA to ATM before testing for IR and cisplatin sensitivity using clonogenic assay. ATR and ATM inhibitors were tested on knockdown cell lines to investigate pathway response differences after cisplatin and IR. NSCLC cell lines NCI-H226, NCI-H460, and NCI-H522 were found to be ATM-proficient whereas cell lines NCI-H23 and NCI-H1373 were found to be ATM-deficient. ATM-proficient cell lines demonstrated an increased level of phosphorylated-ATM in response to treatments with cisplatin, carboplatin, and oxaliplatin. ATM knockdown cell lines were found to have increased sensitivity to IR, however analysis of cisplatin sensitivity was inconclusive with only 1 out of 4 showing increased sensitivity. ATR inhibition in combination with cisplatin caused a large increase in DNA damage response from ATM and DNA-PKcs suggesting an avenue for synthetic lethality. It is clear that platin exposure induced an ATM mediated signaling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA repair response. However, the predictive capabilities of ATM loss for platin sensitivity is still unclear. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low dose therapies that exploit this deficiency.

Abstract B21: The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma

Abstract Glioblastoma (GBM) represents one of the most aggressive cancer types with the vast majority of patients succumbing to disease within the first five years. This dire prognosis reflects the limited efficacy of our frontline therapies which include radiation therapy and temozolomide (TMZ) chemotherapy. The cellular response to these therapies is critically mediated by DNA damage response signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia And Rad3-Related Protein (ATR). Preliminary studies from our laboratory suggest the ATR inhibitor VX-970 has single agent efficacy in both established and primary GBM cell lines. Moreover, combined treatment with TMZ demonstrated therapeutic responses that, following regression analysis, were deemed synergistic (p&amp;lt;0.001). Validation studies using siRNA confirmed ATR as a central mediator of TMZ sensitivity as knockdown of ATM elicited no added benefit when tested in combination. VX-970 also enhanced radiation effects in neurosphere formation assays to suggest it may also be used as a radiosensitizer. While the specific mechanism responsible for these observations has yet to be determined, VX-970 treatment was found to exert anti-proliferative effects in a dose-dependent manner. This collection of work suggests that ATR inhibition may represent a worthwhile treatment strategy in GBM. Citation Format: Danielle Burgenske, Ann Mladek, Jann Sarkaria. The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma [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 B21.

ATM mediates DAB2IP-deficient bladder cancer cell resistance to ionizing radiation through the p38MAPK and NF-κB signaling pathway.

Although surgery remains the standard therapy for the treatment of bladder cancer (BCa), the data from previous clinical studies suggest that there is an increase in the number of patients with a preference for bladder preservation strategies, including radiotherapy, to improve their life quality. Our preliminary results showed that disabled homolog 2 interactive protein (DAB2IP), a putative tumor suppressor gene, is often downregulated in BCa with a radioresistant phenotype. Subsequent investigations revealed that elevated expression of ataxia-telangiectasia mutated (ATM) induced by DAB2IP-knockdown may be the key event in BCa cell resistance to ionizing radiation (IR). However, how ATM is involved in the survival of DAB2IP-deficient cells exposed to IR remains to be fully elucidated. The present study knocked down the expression of ATM in DAB2IP-deficient BCa cells using RNA interference technology. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways were detected by western blot analysis and immunofluorescence assay, respectively. It was demonstrated that knockdown of ATM enhanced the response of DAB2IP-deficient BCa cells to IR, which may have resulted from delayed DNA double-strand break repair kinetics, compromised nuclear factor-κB translocation, inhibited phosphorylation of p38 and the induced activation of c-Jun N-terminal kinase. Taken together, these findings suggested that ATM may be an effective target in the radiotherapy of patients with DAB2IP-deficient BCa.

Ataxia-telangiectasia mutated (ATM) participates in the regulation of ionizing radiation-induced cell death via MAPK14 in lung cancer H1299 cells.

The role of Ataxia-telangiectasia mutated (ATM) in response to DNA damage has previously been studied, but its underlying mechanisms specific to ionizing radiation (IR) have remained to be elucidated. In this study, function of ATM on radiation-induced cell death in lung cancer H1299 cells was analysed. Human lung cancer cells, H1299, were used, and cell models with ATM(-/-) and MAPK14(-/-) were established by genetic engineering. Radiosensitivity was analysed using colony formation assays. Western blotting and co-immunoprecipitation were implemented to detect protein expression and interaction. MDC staining and GFP-LC3 relocalization were used to detect autophagy. Autophagy as well as phosphorylation of ATM was activated by ionizing radiation. Both the inhibitor of ATM, KU55933 and ATM silencing reduced phosphorylation of ATM and MAPKAPK2 expression. Both ATM(-/-) and MAPK14(-/-) cells displayed hypersensitivity. IR increased autophagy level by more than 129% in DMSO-treated cells, while only by 47% and 27% in KU55933-treated and ATM(-/-) cells respectively. MAPK14 knock-down alone gave rise to the basal autophagy level, but decreased notably after IR. KU55933 and ATM knock-down inhibited IR-induced autophagy by activating mTOR pathways. Both Beclin1-PI3KIII and Beclin1-MAPKAPK2 interactions as were remarkably affected by silencing either ATM or MAPK14. ATM promoted IR-induced autophagy via the MAPK14 pathway, mTOR pathway and Beclin1/PI3KIII complexes. MAPK14 contributed to radiosensitization of H1299 cells.