Ataxia Telangiectasia Mutated Expression Research Articles

Hepatitis C Virus Mediated Inhibition of miR-181c Activates ATM Signaling and Promotes Hepatocyte Growth.

Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma. We previously observed that HCV infection of hepatocytes transcriptionally down-regulates miR-181c expression through CCAAT/enhancer binding protein β (C/EBP-β). Here, we examined the role of miR-181c in the regulation of cell cycle progression in relation to HCV infection. In silico analysis suggested that ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. Our results demonstrated that ATM expression is higher in HCV-infected hepatocytes and chronic HCV-infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3' untranslated region of ATM, and the presence of ATM in miR-181c-associated RNA-induced silencing complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Furthermore, introduction of miR-181c significantly inhibited phospho-cyclin-dependent kinase 2 (CDK2) and cyclin-A expression, arresting cell cycle progression, whereas overexpression of miR-181c promoted apoptosis of HCV-infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in the xenograft human hepatocellular carcinoma mouse model. Together, our results suggest that HCV infection suppresses miR-181c in hepatocytes, resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provide mechanistic insight into understanding the role of miR-181c in HCV-associated hepatocyte growth promotion, and may have the potential for therapeutic intervention.

ATM and ATR Expression Potentiates HBV Replication and Contributes to Reactivation of HBV Infection upon DNA Damage.

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

Hsa_circ_0001546 acts as a miRNA-421 sponge to inhibit the chemoresistance of gastric cancer cells via ATM/Chk2/p53-dependent pathway

Circular RNAs (circRNAs) are a new class of noncoding RNAs, play a crucial role in tumor initiation and development. Hsa_circ_0001546 is a novel circular RNA that was downregulated in gastric cancer (GC) tissues, however its function and mechanism in GC has not been studied. Our study verified that circ_0001546 was decreased in GC and correlated with the poor prognosis. Next, Pull-down assay and dual-luciferase reporter assay verified that miR-421 was a target of circ_0001546 while ATM (Ataxia telangiectasia mutated) was target by miR-421. Overexpression of circ_0001546 inhibited the proliferation and chemoresistance of HGC-27 cells, and increased the expression of ATM. In addition, circ_0001546 overexpression reversed the effect of miR-421 overexpression. What is more, circ_0001546 inhibits the chemoresistance of HGC-27 cells to L-OPH (Oxaliplatin) may through the activation of the ATM/checkpoint kinase 2 (Chk2)/p53-dependent signaling pathway. In summary, our study proved that circ_0001546 sponges miR-421 to upregulate the expression level of ATM and inhibit the proliferation and chemoresistance through the activation of the ATM/Chk2/p53-dependent pathway.

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.

Prognostic relevance of ATM protein in uveal melanoma and its association with clinicopathological factors.

Uveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Ataxia-telangiectasia mutated (ATM) protein is an activator of DNA damage response and its role in uveal melanoma (UM) is still unexplored. Therefore, the study aims to detect the expression and localization of ATM protein and its association with clinicopathological parameters METHODS: Expression of nuclear ATM (nATM) was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. Prognostic significance was determined by the Kaplan-Meier analysis and the multivariate analysis by Cox's hazard proportional method. Loss of nATM was observed in 65% of cases, which was statistically significant with the reduced disease-free survival (p = 0.042). This loss was more frequently found in cases with high-risk histopathological factors like epithelioid cell type, tumor infiltrating lymphocytes and high pigmentation which might help in the progression of melanoma. On multivariate analysis, extraocular spread and loss of nATM were found to be independent prognostic factors (p < 0.05). Our data suggest that loss of nATM protein might serve as a poor prognostic marker in the pathogenesis of uveal melanoma which may lead to increased risk of metastasis.

HPV infection associated DNA damage correlated with cervical precancerous lesions and cancer in the highest area of cervical cancer mortality, Longnan, China.

ObjectivesThis study was to assess whether human papillomavirus (HPV) resulting in genetic instability is one reason for the high incidence and mortality of cervical cancer in Longnan.MethodsBetween 2012 and 2016, a total of 346 samples from Longnan were collected and divided into four groups: cervicitis group (n=57), cervical intraepithelial neoplasia I group (CIN I, n=63), CIN II/III group (n=79) and invasive squamous cell carcinoma group (SCC, n=147). HPV E6/E7 mRNA was detected by Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA). The markers of DNA damage response (DDR) – ataxia telangiectasia mutated (ATM) pSer1981, H2AX pSer139 (γH2AX), Chk2 pThr68 and P53 – were analyzed by immunohistochemistry.ResultsThe activation of ATM, γH2AX, Chk2 and P53 was increased with increasing severity of cervical lesion. A significant difference of ATM expression in simple infection was also shown accompanied by the cervical lesion. The expression of γH2AX between HPV16+ and HPV16- specimens, γH2AX and P53 between HPV58+ and HPV58- groups had statistical significance. The expression and copy number of HPV E6/7 mRNA increases with the cervical lesion severity. A significant difference was shown for P53 expression between HPV E6/7 mRNA+ and mRNA- specimens. A close correlation with CHK2 expression for HPV E6/7 mRNA+ and HPV16 E6/7 mRNA+ specimens and γH2AX and CHK2 expression for SCC specimens was shown between low and high viral load groups.ConclusionsDDR, HPV genotypes and HPV E6/E7 oncogene expression correlated with the level of dysplasia of cervical lesions. HPV infection resulted in genetic instability may be one reason for the high incidence and mortality in Longnan.

Inhibition of ATM reverses EMT and decreases metastatic potential of cisplatin-resistant lung cancer cells through JAK/STAT3/PD-L1 pathway

BackgroundThe cisplatin-resistance is still a main course for chemotherapy failure of lung cancer patients. Cisplatin-resistant cancer cells own higher malignance and exhibited increased metastatic ability, but the mechanism is not clear. In this study, we investigated the effects of Ataxia Telangiectasia Mutated (ATM) on lung cancer metastasis.Materials and methodsCisplatin-resistant A549CisR and H157CisR cell line were generated by long-term treating parental A549 and H157 cells (A549P and H157P) with cisplatin. Cell growth, cell migration and cell invasion were determined. Gene expressions were determined by Western Blot and qPCR. Tumor metastasis was investigated using a xenograft mouse model.ResultsThe IC50 of the cisplatin-resistant cells (A549CisR and H157CisR cells) to cisplatin was 6–8 higher than parental cells. The A549CisR and H157CisR cells expressed lower level of E-cadherin and higher levels of N-cadherin, Vimentin and Snail compared to the parental A549P and H157P cells, and exhibited stronger capabilities of metastatic potential compared to the parental cells. The ATM expression was upregulated in A549CisR and H157CisR cells and cisplatin treatment also upregulated expression of ATM in parental cells, The inhibition of ATM by using specific ATM inhibitor CP466722 or knock-down ATM by siRNA suppressed Epithelial-to-Mesenchymal transition (EMT) and metastatic potential of A549CisR and H157CisR cells. These data suggest that ATM mediates the cisplatin-resistance in lung cancer cells. Expressions of JAK1,2,、 STAT3 、PD-L1 and ATM were increased in A549CisR and H157CisR cells and could by induced by cisplatin in parental lung cancer cells. Interestedly, ATM upregulated PD-L1 expression via JAK1,2/STAT3 pathway and inhibition of ATM decreased JAK/STAT3 signaling and decreased PD-L1 expression. The treatment of PD-L1 neutralizing Ab reduced EMT and cell invasion. Inhibition of JAK1,2/STAT3 signaling by specific inhibitors suppressed ATM-induced PD-L1 expression, EMT and cell invasion. Importantly, inhibition of ATM suppressed EMT and tumor metastasis in cisplatin-resistant lung cancer cells in an orthotopic xenograft mouse model.ConclusionsOur results show that ATM regulates PD-L1 expression through activation of JAK/STAT3 signaling in cisplatin-resistant cells. Overexpression of ATM contributes to cisplatin-resistance in lung cancer cells. Inhibition of ATM reversed EMT and inhibited cell invasion and tumor metastasis. Thus, ATM may be a potential target for the treatment of cisplatin-resistant lung cancer.

ATM expression is attenuated by promoter hypermethylation in human ovarian endometriotic stromal cells.

A number of genes involved in the pathogenesis of endometriosis are silenced by the hypermethylation of their promoter regions. We assessed the effect and mechanism of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) (10μM) on the cell cycle in human endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) by flow cytometry. The DNA methylation status of G2/M checkpoint regulators were investigated by methylation-specific PCR. The expression of ataxia telangiectasia mutated (ATM) and the effect of 5-aza-dC on its expression were also evaluated by quantitative RT-PCR and western blotting analysis. 5-aza-dC treatment resulted in the cell cycle arrest of ECSCs at the G2/M phase. In contrast, 5-aza-dC did not affect the cell cycle of NESCs. The promoter region of the ATM gene was hypermethylated in ECSCs, but not in NESCs. ATM mRNA expression was attenuated in ECSCs compared to that in NESCs. Further, 5-aza-dC was found to restore ATM expression in ECSCs by its promoter demethylation. Our findings indicate that ATM promoter hypermethylation occurs in endometriosis, and that ATM silencing is involved in tumorigenesis during this disease; moreover, selective DNA demethylating agents and molecular target drugs against ATM silencing are promising for the treatment of endometriosis.

Arecoline-regulated ataxia telangiectasia mutated expression level in oral cancer progression.

Ataxia telangiectasia mutated (ATM) regulates DNA repair and cell cycle. The present study analyzed arecoline-induced ATM expression during oral cancer progression. In vitro studies were performed using oral squamous cell carcinoma (OSCC) cell lines treated with arecoline to analyze cell response and ATM regulation. in vivo studies were performed using immunohistochemistry to detect ATM expression in normal, oral potentially malignant disorder (OPMD), and OSCC tissues. Low-dose arecoline induced cell proliferation, ATM promoter activity, and DNA repair. High-dose arecoline induced cell cycle arrest, apoptosis, and DNA damage. ATM was overexpressed in OPMD tissues but was downregulated in OSCC tissues. ATM expression level was associated with the risk of developing dysplasia, buccal-OSCC, and with OSCC survival rate. High ATM expression helps DNA repair mechanisms to maintain the cells in the OPMD stage, but low ATM expression causes DNA damage accumulation to increase cell malignancy.

Clinicopathological significance of ataxia telangiectasia-mutated (ATM) kinase and ataxia telangiectasia-mutated and Rad3-related (ATR) kinase in MYC overexpressed breast cancers

PurposeMYC transcription factor has critical roles in cell growth, proliferation, metabolism, differentiation, transformation and angiogenesis. MYC overexpression is seen in about 15% of breast cancers and linked to aggressive phenotypes. MYC overexpression also induces oxidative stress and replication stress in cells. ATM signalling and ATR-mediated signalling are critical for MYC-induced DNA damage response. Whether ATM and ATR expressions influence clinical outcomes in MYC overexpressed breast cancers is unknown.MethodsWe investigated ATM, ATR and MYC at the transcriptional level [Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1950)] and at the protein level in the Nottingham series comprising 1650 breast tumours. We correlated ATM, ATR and MYC expressions to clinicopathological features and survival outcomes.ResultsIn MYC over expressed tumours, high ATR or low ATM levels were associated with aggressive breast cancer features such as higher tumour grade, de-differentiation, pleomorphism, high mitotic index, high-risk Nottingham Prognostic Index, triple negative and basal-like breast cancers (all adjusted p values < 0.05). Tumours with low ATM or high ATR levels in conjunction with MYC overexpression also have worse overall breast cancer-specific survival (BCSS) (p value < 0.05).ConclusionsWe conclude that ATR/ATM-directed stratification and personalisation of therapy may be feasible in MYC overexpressed breast cancer.

Type 1 diabetes upregulates metastasis-associated protein 1- phosphorylated histone 2AX signaling in the testis

Under the sustained hyperglycemic state, oxidative stress induces irreparable DNA double-strand breaks resulting in germ cell death and testicular atrophy. Although molecular mechanisms underlying DNA damage repair in testicular cells are gradually getting unraveled, the effects on DNA double-strand breaks sensing are not precisely known. In this study, using streptozotocin-induced type 1 diabetic rats, we report that hyperglycemic state for one month or three months does not increase the levels of ataxia telangiectasia mutated (ATM) protein- an upstream kinase responsible for the phosphorylation of histone 2AX (Ɣ-H2AX)- after the formation of DNA double-strand breaks. The ATM expression is seminiferous epithelial stage-dependent in spermatogonia and primary spermatocytes, and the pattern of stage-dependent expression varies in diabetic rats, especially after three-month-long diabetes. However, the levels of metastasis-associated protein-1 (MTA1), an essential protein for ATM function, increase although not in a time-dependent manner. The amount of DNA double-strand breaks increases in a time- and stage-dependent manner as indicated by increased Ɣ-H2AX levels, especially in spermatogonia and primary spermatocytes, and in late spermatids in some tubular stages. Although ATM levels do not increase in diabetic rats, protein is expressed more or less in same testicular cells in which Ɣ-H2AX is expressed indicating that ATM might play a vital role in the phosphorylation of the histone. We conclude that diabetes upregulates MTA1-Ɣ-H2AX signaling in diabetic rat testis as a response to time-dependent increases in DNA double-strand breaks.

Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response.

STUDY QUESTIONDoes ovarian follicle activation by phosphatase homologue of chromosome-10 (PTEN) inhibition affect DNA damage and repair in bovine oocytes and granulosa cells?SUMMARY ANSWERPTEN inhibition promotes bovine non-growing follicle activation but results in increased DNA damage and impaired DNA repair capacity in ovarian follicles in vitro.WHAT IS KNOWN ALREADYInhibition of PTEN is known to activate primordial follicles but may compromise further developmental potential. In breast cancer cells, PTEN inhibition represses nuclear translocation of breast cancer susceptibility 1 (BRCA1) and Rad51; this impairs DNA repair resulting in an accumulation of damaged DNA, which contributes to cell senescence.STUDY DESIGN, SIZE, DURATIONBovine ovarian tissue fragments were exposed to control medium alone or containing either 1 or 10 μM bpv(HOpic), a pharmacological inhibitor of PTEN, in vitro for 24 h. A sub-group of tissue fragments were collected for Western blot analysis after bpv(HOpic) exposure. The remainder were incubated in control medium for a further 5 days and then analysed histologically and by immunohistochemistry to detect DNA damage and repair pathways.PARTICIPANTS/MATERIALS, SETTING, METHODSBovine ovaries were obtained from abattoir-slaughtered heifers. Tissue fragments were exposed to either control medium alone or medium containing either 1 μM or 10 μM bpv(HOpic) for 24 h. Tissue fragments collected after 24 h were subjected to Akt quantification by Western blotting (six to nine fragments per group per experiment). Follicle stage and morphology were classified in remaining fragments. Immunohistochemical analysis included nuclear exclusion of FOXO3 as a marker of follicle activation, γH2AX as a marker of DNA damage, meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM), Rad51, breast cancer susceptibility 1 (BRCA1) and breast cancer susceptibility 2 (BRCA2) as DNA repair factors. A total of 29 550 follicles from three independent experiments were analysed.MAIN RESULTS AND THE ROLE OF CHANCETissue fragments exposed to bpv(HOpic) had increased Akt phosphorylation at serine 473 (pAkt/Akt ratio, 2.25- and 6.23-fold higher in 1 and 10 μM bpv(HOpic) respectively compared to control, P < 0.05). These tissue fragments contained a significantly higher proportion of growing follicles compared to control (78.6% in 1 μM and 88.7% in 10 μM versus 70.5% in control; P < 0.001). The proportion of morphologically healthy follicles did not differ significantly between 1 μM bpv(HOpic) and control (P < 0.001) but follicle health was lower in 10 μM compared to 1 μM and control in all follicle types (P < 0.05). DNA damage in oocytes, indicated by expression of γH2AX, increased following exposure to 1 μM bpv(HOpic) (non-growing, 83%; primary follicles, 76%) and 10 μM (non-growing, 77%; primary, 84%) compared to control (non-growing, 30% and primary, 59%) (P < 0.05 for all groups). A significant reduction in expression of DNA repair proteins MRE11, ATM and Rad51 was observed in oocytes of non-growing and primary follicles of treatment groups (primary follicles in controls versus 10 μM bpv(HOpic): MRE, 68% versus 47%; ATM, 47% versus 18%; Rad51, 48% versus 24%), P < 0.05 for all groups. Higher dose bpv(HOpic) also resulted in lower expression of BRCA1 compared to control and 1 μM bpv(HOpic) (P < 0.001) in non-growing and primary follicles. BRCA2 expression was increased in oocytes of primary follicles in 1 μM bpv(HOpic) (36%) compared to control (20%, P = 0.010) with a marked decrease in 10 μM (1%, P ≤ 0.001). Granulosa cells of primary and secondary follicles in bpv(HOpic) groups showed more DNA damage compared to control (P < 0.05). However, bpv(HOpic) did not impact granulosa cell DNA repair capacity in secondary follicles, but BRCA1 declined significantly in higher dose bpv(HOpic).LARGE-SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis study focuses on non-growing follicle activation after 6 days culture and may not reflect DNA damage and repair capacity in later stages of oocyte and follicle growth.WIDER IMPLICATIONS OF THE FINDINGS In vitro activation of follicle growth may compromise the bidirectional signalling between oocyte and granulosa cells necessary for optimal oocyte and follicle health. This large animal model may be useful in optimising follicle activation protocols with a view to transfer for clinical application.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by Indonesia endowment fund for education. No competing interest.TRIAL REGISTRATION NUMBERNot applicable.

Long Noncoding RNA FAM201A Mediates the Radiosensitivity of Esophageal Squamous Cell Cancer by Regulating ATM and mTOR Expression via miR-101.

Background: The aim of the present study was to identify the potential long non-coding (lnc.)-RNA and its associated molecular mechanisms involved in the regulation of the radiosensitivity of esophageal squamous cell cancer (ESCC) in order to assess whether it could be a biomarker for the prediction of the response to radiotherapy and prognosis in patients with ESCC.Methods: Microarrays and bioinformatics analysis were utilized to screen the potential lncRNAs associated with radiosensitivity in radiosensitive (n = 3) and radioresistant (n = 3) ESCC tumor tissues. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed in 35 ESCC tumor tissues (20 radiosensitive and 15 radioresistant tissues, respectively) to validate the lncRNA that contributed the most to the radiosensitivity of ESCC (named the candidate lncRNA). MTT, flow cytometry, and western blot assays were conducted to assess the effect of the candidate lncRNA on radiosensitivity in vitro in ECA109/ECA109R ESCC cells. A mouse xenograft model was established to confirm the function of the candidate lncRNA in the radiosensitivity of ESCC in vivo. The putative downstream target genes regulated by the candidate lncRNA were predicted using Starbase 2.0 software and the TargetScan database. The interactions between the candidate lncRNA and the putative downstream target genes were examined by Luciferase reporter assay, and were confirmed by PCR.Results: A total of 113 aberrantly expressed lncRNAs were identified by microarray analysis, of which family with sequence similarity 201-member A (FAM201A) was identified as the lncRNA that contributed the most to the radiosensitivity of ESCC. FAM201A was upregulated in radioresistant ESCC tumor tissues and had a poorer short-term response to radiotherapy resulting in inferior overall survival. FAM201A knockdown enhanced the radiosensitivity of ECA109/ECA109R cells by upregulating ataxia telangiectasia mutated (ATM) and mammalian target of rapamycin (mTOR) expression via the negative regulation of miR-101 expression. The mouse xenograft model demonstrated that FAM201A knockdown improved the radiosensitivity of ESCC.Conclusion: The lncRNA FAM201A, which mediated the radiosensitivity of ESCC by regulating ATM and mTOR expression via miR-101 in the present study, may be a potential biomarker for predicting radiosensitivity and patient prognosis, and may be a therapeutic target for enhancing cancer radiosensitivity in ESCC.

Low ATM expression and progression-free and overall survival in advanced gastric cancer patients treated with first-line XELOX chemotherapy.

Gastric cancer (GC) is a leading cause of cancer-specific mortality with limited biologically informed treatments. The ataxia telangiectasia mutated (ATM) gene is critically involved in the repair of double-stranded DNA breaks and a component of DNA damage repair (DDR) pathways. Platinum salts are hypothesized to have increased efficacy in tumors deficient in DDR pathways. We sought to investigate an association between ATM status and response to XELOX in a homogenous first line GC patient cohort. A clinically annotated cohort of 137 Korean patients with advanced GC treated with first-line XELOX was retrospectively examined for ATM status by immunohistochemistry. Correlation between ATM expression and clinicopathologic variables was performed by two-tailed, unpaired t-tests and Fisher's exact tests. Kaplan-Meier survival analysis curves and Cox proportional hazards models were used to evaluate for independent predictors of disease-free survival (DFS) and overall survival (OS). Low ATM expression was observed in 19.0% (26/137) of patients and was not associated with clinicopathologic features or response rate to XELOX. Univariate, but not multivariable, logistic regression and Cox analysis identified ATM as an independent risk factor influencing OS and DFS. A higher ECOG score independently predicted worse survival [hazard ratio (HR) 2.96, P=0.016] and complete surgical resection independently protected against progression of disease (HR 0.69, P=0.007). Low ATM expression was not associated with increased response rates to XELOX in a single-institution cohort of advanced GC patients. Similarly, ATM status did not predict DFS or OS after platinum-based chemotherapy.

Molecular features unique to glioblastoma radiation resistant residual cells may affect patient outcome - a short report.

Previously we have shown, using a primary glioblastoma (GBM) cell model, that a subpopulation of innately radiation resistant (RR) GBM cells survive radiotherapy and form multinucleated and giant cells (MNGCs) by homotypic fusions. We also showed that MNGCs may cause relapse. Here, we set out to explore whether molecular characteristics of RR cells captured from patient-derived primary GBM cultures bear clinical relevance. Primary cultures were derived from 19 naive GBM tumor samples. RR cells generated from these cultures were characterized using various cell biological assays. We also collected clinicopathological data of the 19 patients and assessed associations with RR variables using Spearman's correlation test and with patient survival using Kaplan-Meier analysis. Significance was determined using a log-rank test. We found that SF2 (surviving fraction 2) values (p = 0.029), days of RR cell formation (p = 0.019) and percentage of giant cells (p = 0.034) in the RR population independently correlated with a poor patient survival. We also found that low ATM (Ataxia-telangiectasia mutated) expression levels in RR cells showed a significant (p = 0.002) negative correlation with SF2 values. A low ATM expression level in RR cells along with a high tumor volume was also found to negatively correlate with patient survival (p = 0.011). Finally, we found that the ATM expression levels in RR cells independently correlated with a poor patient survival (p = 0.014). Our data indicate that molecular features of innately radiation resistant GBM cells independently correlate with clinical outcome. Our study also highlights the relevance of using patient-derived primary GBM cultures for the characterization of RR cells that are otherwise inaccessible for analysis.

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.

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 2822: ATR inhibitors are active as single agents and in combination with PARP1 and ATM inhibitors in molecularly distinct subsets of small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is an aggressive form of lung cancer, notable for rapid emergence of drug resistance following initial chemotherapy. Rates of five-year overall survival are only 7% across all stages and only one drug, topotecan, is approved by the FDA for recurrent SCLC. As a result, the National Cancer Institute has named identifying novel vulnerabilities in SCLC as an urgent area of need. Increased expression, relative to non-small cell lung cancer (NSCLC), of numerous components of the DNA damage response (DDR) pathway, including poly (ADP-Ribose) polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM), is observed in SCLC. Thus, targeting DDR has emerged as an attractive therapeutic strategy in SCLC, bolstered by recent data demonstrating activity of PARP1 inhibitors (PARPi) in SCLC patients. Interestingly, data suggest that PARPi resistant models from other tumors may rely on another DDR component, ataxia telangiectasia and Rad3 related protein (ATR), for survival. ATR/ATR is also highly expressed in SCLC compared to NSCLC and normal lung tissue. Preclinical data have shown that treatment with ATR inhibitors (ATRi) is especially effective in p53- and ATM-deficient tumor models, a notable fact given that SCLC is universally p53-mutant and that ATM-mutant and ATM-deficient SCLC is a small, but significant proportion of all SCLC. We treated 22 human-derived SCLC cell line models with two clinically relevant ATRi, VX-970 (formerly VE-822) and AZD-6738, and observed single agent activity of both ATR inhibitors in a significant number of cell lines, with half-maximal inhibitory concentrations (IC50s) as low as 30 nM and &amp;gt;100-fold difference in IC50s between the most and least sensitive cell lines. Utilizing extensive genomic, transcriptomic and proteomic characterization of these cell lines, we then identified predictive biomarkers of response to ATRi in SCLC, including low ATM expression. As low ATM was associated with ATRi sensitivity, we tested whether the addition of an ATM inhibitor (AZD-0156) may further sensitize SCLC models to ATRi. We treated 12 SCLC cell lines with AZD-6738 and AZD-0156 in combination and identified a subset of lines in which synergy is observed between the two agents. Similarly, as targeting ATR has been shown to overcome PARPi resistance in other cancer types, we treated 12 SCLC cell lines with the ATRi AZD-6738 and the PARPi olaparib in combination and again observed a subset of lines in which the two agents acted synergistically. Interestingly, the lines in which ATRi+ATMi and ATRi+PARPi synergy is observed are distinct and include lines that were the most resistant to single-agent AZD-6738. Together, these data support further investigation of ATRi in SCLC and suggest that via the use of ATRi alone or in combination with ATMi or PARPi, multiple molecularly distinct subsets of SCLC can be effectively targeted. Citation Format: Carl M. Gay, Pan Tong, Lerong Li, C. Allison Stewart, Triparna Sen, Bonnie S. Glisson, John V. Heymach, Jing Wang, Lauren Averett Byers. ATR inhibitors are active as single agents and in combination with PARP1 and ATM inhibitors in molecularly distinct subsets of small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2822.

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.

An Exploratory Study of Radiation Dermatitis in Breast Cancer Patients.

Radiation dermatitis is observed in 95% of breast cancer patients receiving radiotherapy. The aim of this study was to explore the correlation between protein expression in tumor cells and the risk of developing radiation dermatitis. Breast cancer patients receiving postoperative radiotherapy were included in this study. Tumor specimens from 122 patients were examined by immunohistochemistry for the expression of Ki67, ataxia telangiectasia mutated (ATM) kinase, hypoxia-inducible factor-1-alpha (HIF-1a), inducible nitric oxide synthase (iNOS), and a-glucosidase (aGluc). The findings were correlated with the occurrence and severity of radiation dermatitis (Radiation therapy oncology group-RTOG grading scale), taking into consideration body weight and skin type (Fitzpatrick system). Data were explored further via pathway and network analyses. Correlation of radiation dermatitis (RTOG scale) with the observed increased expression of Ki67, ATM, iNOS, HIF-1a and aGluc, failed to reach statistical significance when skin type and/or body weight were considered. Network interactions of proteins involved in tumor growth (Ki67, ATM) and/or affect the oxidation state of the cell (HIF-1a, iNOS, aGluc) were revealed, that may contribute to the risk of developing acute radiation dermatitis. Correlation of the increased expression of the studied proteins and the occurrence and severity of radiation dermatitis in women undergoing postoperative radiotherapy, failed to reach statistical significance. Pathway and network analyses predicted that vasodilation and angiogenesis may contribute to radiation-induced dermatitis via mechanisms that need to be further explored. Our strategy serves as a paradigm for coupling histopathological data to molecular findings and network analyses for risk assessment in the clinic.