DNA Damage Recognition Research Articles

Abstract 831: TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM

Abstract Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy. Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [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 831. doi:10.1158/1538-7445.AM2017-831

Abstract 983: Identification of potent, highly selective and orally available ATR inhibitor BAY 1895344 with favorable PK properties and promising efficacy in monotherapy and combination in preclinical tumor models

Abstract The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress (e.g. in oncogene-driven tumor cells). Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Herein we report the identification of the potent, highly selective and orally available ATR inhibitor BAY 1895344 by a collaborative effort involving medicinal chemistry, pharmacology, DMPK and computational chemistry. The chemical structures of lead compound BAY-937 and clinical candidate BAY 1895344 as well as the main SAR trends within this novel class of naphthyridine derivatives will be disclosed for the first time. The novel lead compound BAY-937 revealed promising inhibition of ATR (IC50 = 78 nM) and high kinase selectivity in vitro. In cellular mechanistic assays BAY-937 inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 380 nM) demonstrating the anticipated mode of action. Moreover, BAY-937 was shown to inhibit proliferation of a variety of tumor cell lines with low- to sub-micromolar IC50 values. In initial xenograft studies, BAY-937 revealed moderate activity in monotherapy and in combination with cis-platin. However, BAY-937 also revealed low aqueous solubility, low bioavailability (rat) and activity in the hERG patch clamp assay. Extensive lead optimization efforts led to the identification of the novel, orally available ATR inhibitor BAY 1895344. In vitro, BAY 1895344 was shown to be a very potent and highly selective ATR inhibitor (IC50 = 7 nM), which potently inhibits proliferation of a broad spectrum of human tumor cell lines (median IC50 = 78 nM). In cellular mechanistic assays BAY 1895344 potently inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 36 nM). Moreover, BAY 1895344 revealed significantly improved aqueous solubility, bioavailability across species and no activity in the hERG patch-clamp assay. BAY 1895344 also demonstrated very promising efficacy in monotherapy in DNA damage deficient tumor models as well as combination treatment with DNA damage inducing therapies. The start of clinical investigation of BAY 1895344 is planned for early 2017. Citation Format: Ulrich T. Luecking, Julien Lefranc, Antje Wengner, Lars Wortmann, Hans Schick, Hans Briem, Gerhard Siemeister, Philip Lienau, Christoph Schatz, Benjamin Bader, Gesa Deeg, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. Identification of potent, highly selective and orally available ATR inhibitor BAY 1895344 with favorable PK properties and promising efficacy in monotherapy and combination in preclinical tumor models [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 983. doi:10.1158/1538-7445.AM2017-983

Abstract 836: ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models

Abstract The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, induction of DNA repair, or cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first and only approved targeted alpha therapy so far. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It exhibits strong cytotoxic effects on adjacent cells via the induction of DNA DSB. Here, we disclose for the first time the structure and functional characterization of the novel ATR kinase inhibitor BAY 1895344. In vitro, BAY 1895344 is a selective low-nanomolar inhibitor of ATR kinase activity, potently inhibiting proliferation of a broad spectrum of human tumor cell lines (median IC50 of 78 nM). A clear separation between highly sensitive (IC50 <10 nM) and less sensitive cell lines was observed. The majority of the sensitive cell lines are characterized by mutations affecting the ATM (ataxia telangiectasia mutated) pathway. In cellular mechanistic assays BAY 1895344 inhibited hydroxyurea-induced H2AX phosphorylation demonstrating the anticipated mode of action. BAY 1895344 is an ATR inhibitor that exhibits strong in vivo anti-tumor efficacy in monotherapy in a variety of xenograft models of different indications that are characterized by DDR deficiencies, inducing stable disease in ovarian and colorectal cancer or even complete tumor remission in mantle cell lymphoma models. In addition, we could demonstrate that combination treatment with BAY 1895344 and Radium-223 exhibits clear synergistic anti-tumor activity in a bone metastases xenograft model of CRPC. Our findings validate the concept of synthetic lethality of genetically determined DNA repair deficiency and ATR blockade by demonstrating strong monotherapy efficacy of the highly potent ATR inhibitor BAY 1895344 in a variety of tumor indications. Furthermore, the mechanism-based combination potential of DNA damage induction by Radium-223 with BAY 1895344 creates a powerful new treatment option for CRPC patients with bone metastases. The start of clinical investigation of BAY 1895344 is planned early 2017. Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Philip Lienau, Gesa Deeg, Eleni Lagkadinou, Li Liu, Sven Golfier, Christoph Schatz, Arne Scholz, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models [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 836. doi:10.1158/1538-7445.AM2017-836

Investigation of DNA repair-related SNPs underlying susceptibility to papillary thyroid carcinoma reveals MGMT as a novel candidate gene in Belarusian children exposed to radiation

BackgroundGenetic factors may influence an individual’s sensitivity to ionising radiation and therefore modify his/her risk of developing papillary thyroid carcinoma (PTC). Previously, we reported that common single nucleotide polymorphisms (SNPs) within the DNA damage recognition gene ATM contribute to PTC risk in Belarusian children exposed to fallout from the Chernobyl power plant accident. Here we explored in the same population the contribution of a panel of DNA repair-related SNPs in genes acting downstream of ATM.MethodsThe association of 141 SNPs located in 43 DNA repair genes was examined in 75 PTC cases and 254 controls from the Gomel region in Belarus. All subjects were younger than 15 years at the time of the Chernobyl accident. Conditional logistic regressions accounting for radiation dose were performed with PLINK using the additive allelic inheritance model, and a linkage disequilibrium (LD)-based Bonferroni correction was used for correction for multiple testing.ResultsThe intronic SNP rs2296675 in MGMT was associated with an increased PTC risk [per minor allele odds ratio (OR) 2.54 95% CI 1.50, 4.30, Pper allele = 0.0006, Pcorr.=0.05], and gene-wide association testing highlighted a possible role for ERCC5 (PGene = 0.01) and PCNA (PGene = 0.05) in addition to MGMT (PGene = 0.008).ConclusionsThese findings indicate that several genes acting in distinct DNA repair mechanisms contribute to PTC risk. Further investigation is needed to decipher the functional properties of the methyltransferase encoded by MGMT and to understand how alteration of such functions may lead to the development of the most common type of thyroid cancer.

MiRNA regulation is important for DNA damage repair and recognition in malignant pleural mesothelioma.

Platin-containing regimes are currently considered as state-of-the-art therapies in malignant pleural mesotheliomas (MPM) but show dissatisfying response rates ranging from 6 to 16% only. Still, the reasons for the rather poor efficacy remain largely unknown. A clear stratification of patients based on new biomarkers seems to be a promising approach to enhance clinical management, which would be a long-needed improvement for MPM patients but does not seem likely soon unless new biomarkers can be validated. Twenty-four formalin-fixed, paraffin-embedded (FFPE) tumour specimens were subjected to a miRNA expression screening of 800 important miRNAs using digital quantification via the nCounter technique (NanoString). We defined a small subset of miRNAs regulating the key enzymes involved in the repair of platin-associated DNA damage. Particularly, the TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main miRNA targets within this context. The TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main players for risk stratification in patients suffering from this severe disease. Taking the specific molecular profile of the tumour into account can help to enhance the clinical management prospectively and to smooth the way to better response prediction.

Effects of Alcohol on CHK-1 and Phospho-CHK-1 in Neuronal Stem Cell losses in FASD.

Fetal Alcohol Spectrum Disorders (FASD) are associated with changes in proliferation and induced cell losses of neuronal stem cells during development. Alcohol induced oxidative stress has been noted in previous studies to cause chronic genomic instability and DNA damage. This damage results in the halting of S-Phase cell cycle progress and, in many cases, either induces DNA repair or apoptosis of the cell. However, alcohol-induced apoptosis has been show to work in a p53 independent fashion. Typically, proteins CDC25a and c as well as CHK-1 and 2 monitors DNA damage and help to determine cellular fate in p53 independent apoptosis. A study utilizing alcohol treated neuronal stem cells resulted in approximately 40% of the population maintaining DNA damage without inducing p53 dependent apoptotic mechanisms. The goal of this study was to elucidate the role, expression and localization of checkpoint kinase-1 (CHK-1) during the G1-S phase transition and to determine any major difference in phosphorylated, CHK-1 between control and alcohol treated neuronal stem cells. A Western Blot was run to determine quantitative presence of Phosphorylated CHK-1 and Immuno-fluorescent staining was used to quantitatively measure the abundance of Phosphorylated CHK-1 in groups. Image analysis determined that there was a significant difference in Phosphorylated Chk-1 in alcohol-treated cells. The increased level of Phospho-CHK-1 indicates recognition of DNA damage, initiation of signaling G1/S phase transition and possible induction of apoptosis. Understanding the mechanistic changes in alcohol induced proliferation and cell losses may help in defining treatment paradigms effective in treating FASD.

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3.

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC invitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.

Abstract P6-12-08: Phase I study of low dose oral cyclophosphamide (C) plus the poly-ADP-ribose- polymerase (PARP) inhibitor veliparib (V) in women with HER2/neu-negative inoperable locally advanced/metastatic breast cancer (MBC): NCI P8853

Abstract BACKGROUND: PARP, an essential nuclear enzyme, is involved in the recognition of DNA damage and facilitation of DNA base-excision repair (BER). PARP inhibition sensitizes tumor cells to cytotoxic agents which induce DNA damage, including C. Metronomic dosing of C may optimize potential for synergy with PARP inhibitors, and also inhibits angiogenesis (Kerbel et al, Nat Rev Cancer, 4:423-36, 2004) and may enhance anti-tumor immunity (Ghiringhelli et al. Cancer Immunol Immunother 56:641–648, 2007) V is an oral small molecule inhibitor of PARP which potentiates the antineoplastic activity of DNA damaging agents such as C in MX-1 breast xenograft model (Donawho et al Clin Cancer Res 13:2728-37, 2007). We performed a phase I trial of metronomic dose oral C plus V in patients with MBC. METHODS: The primary objective was to determine the safety and identify the recommended phase II dose (RPTD) of the combination of low-dose oral C once daily in combination with V (100, 200, 300 mg) administered BID for 21 days using a standard 3+3 design. Eligibility included HER2/neu negative MBC, ECOG PS 0-1, and at least 1 prior chemotherapy regimen for MBC. Dose limiting toxicity (DLT) was defined as any Grade 3 non-hematological toxicity or Grade 4 thrombocytopenia/neutropenia occurring during cycle 1. After the RPTD of V was shown to be 200 mg BID with C 50 mg daily, the trial was amended to increase the C dose to 75, 100 and then 125 mg daily until hematologic toxicity was dose-limiting. RESULTS: 31 patients were enrolled, 19 treated with 50 mg of C and 12 treated at higher doses (75-125 mg), with V doses ranging from 50 mg-300 mg BID (see table);5 patients with not evaluable due to rapid disease progression (N=2), non-compliance (N=2), or tumor pain that was not a DLT (N=1). Median age was 52 years (28-72 years), 14 (45 %) had triple negative disease, all had at least 1 prior chemotherapy regimen for metastasis (median 2, range 1-8), and, 7 had germline BRCA mutations, (3 BRCA1 and 4 BRCA2). When combined with 50 mg C daily, RPTD of V was 200 mg PO BID, with nausea being DLT at 300 mg BID. DLT was not observed in any of the 9 additional patients. The median number of cycles given was 3 (range 1-14). Clinical benefit (response or stable disease for at least 24 weeks) occurred in 3/7 (43%), 1/3 (33%) and 1/16(6%) for BRCA mutated, BRCA negative and BRCA unknown, respectively. Median progression-free survival was 4.3 months (1.2-10.9 months) for BRCA mutated patients and 2 months (0.7-10 months) for non-mutated. CONCLUSIONS: The combination of oral continuous dosing of V (200 mg PO BID) with metronomic C (50, 75, 100 and 125 mg daily) is well tolerated and shows antitumor activity in patients with BRCA mutation associated MBC. The RPTD is C 125 mg daily plus V 200 mg BID, although further escalation of the C dose may be feasible since DLT was not seen at this dose level. Dose LevelsDose Level# Patients/Evaluable# DLTType of DLTDL 1 :V 50mg , C 50mg3/30 DL 2 :V 100 mg, C 50mg4/30 DL 3 :V 200 mg, C 50mg6/61HeadacheDL 4 :V 300 mg, C 50mg6/52Nausea (N=2)DL 3A :V 200 mg; C 75mg3/30 DL 3B :V 200 mg, C 100mg6/30 DL 3C :V 200 mg, C 125mg3/30 Citation Format: Anampa JD, Patel M, Pellegrino C, Fehn K, Makower D, Oh S-y, Noah K, Chen A, Sparano JA, Andreopoulou E. Phase I study of low dose oral cyclophosphamide (C) plus the poly-ADP-ribose- polymerase (PARP) inhibitor veliparib (V) in women with HER2/neu-negative inoperable locally advanced/metastatic breast cancer (MBC): NCI P8853 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-08.

Movement of the β-hairpin in the third zinc-binding module of UvrA is required for DNA damage recognition

Nucleotide excision repair (NER) is distinguished from other DNA repair pathways by its ability to process various DNA lesions. In bacterial NER, UvrA is the key protein that detects damage and initiates the downstream NER cascade. Although it is known that UvrA preferentially binds to damaged DNA, the mechanism for damage recognition is unclear. A β-hairpin in the third Zn-binding module (Zn3hp) of UvrA has been suggested to undergo a conformational change upon DNA binding, and proposed to be important for damage sensing. Here, we investigate the contribution of the dynamics in the Zn3hp structural element to various activities of UvrA during the early steps of NER. By restricting the movement of the Zn3hp using disulfide crosslinking, we showed that the movement of the Zn3hp is required for damage-specific binding, UvrB loading and ATPase activities of UvrA. We individually inactivated each of the nucleotide binding sites in UvrA to investigate its role in the movement of the Zn3hp. Our results suggest that the conformational change of the Zn3hp is controlled by ATP hydrolysis at the distal nucleotide binding site. We propose a bi-phasic damage inspection model of UvrA in which movement of the Zn3hp plays a key role in damage recognition.

Repair shielding of platinum-DNA lesions in testicular germ cell tumors by high-mobility group box protein 4 imparts cisplatin hypersensitivity

Cisplatin is the most commonly used anticancer drug for the treatment of testicular germ cell tumors (TGCTs). The hypersensitivity of TGCTs to cisplatin is a subject of widespread interest. Here, we show that high-mobility group box protein 4 (HMGB4), a protein preferentially expressed in testes, uniquely blocks excision repair of cisplatin-DNA adducts, 1,2-intrastrand cross-links, to potentiate the sensitivity of TGCTs to cisplatin therapy. We used CRISPR/Cas9-mediated gene editing to knockout the HMGB4 gene in a testicular human embryonic carcinoma and examined cellular responses. We find that loss of HMGB4 elicits resistance to cisplatin as evidenced by cell proliferation and apoptosis assays. We demonstrate that HMGB4 specifically inhibits repair of the major cisplatin-DNA adducts in TGCT cells by using the human TGCT excision repair system. Our findings also reveal characteristic HMGB4-dependent differences in cell cycle progression following cisplatin treatment. Collectively, these data provide convincing evidence that HMGB4 plays a major role in sensitizing TGCTs to cisplatin, consistent with shielding of platinum-DNA adducts from excision repair.

Patient-Specific Screening Using High-Grade Glioma Explants to Determine Potential Radiosensitization by a TGF-β Small Molecule Inhibitor

High-grade glioma (HGG), a deadly primary brain malignancy, manifests radioresistance mediated by cell-intrinsic and microenvironmental mechanisms. High levels of the cytokine transforming growth factor-β (TGF-β) in HGG promote radioresistance by enforcing an effective DNA damage response and supporting glioma stem cell self-renewal. Our analysis of HGG TCGA data and immunohistochemical staining of phosphorylated Smad2, which is the main transducer of canonical TGF-β signaling, indicated variable levels of TGF-β pathway activation across HGG tumors. These data suggest that evaluating the putative benefit of inhibiting TGF-β during radiotherapy requires personalized screening. Thus, we used explant cultures of seven HGG specimens as a rapid, patient-specific ex vivo platform to test the hypothesis that LY364947, a small molecule inhibitor of the TGF-β type I receptor, acts as a radiosensitizer in HGG. Immunofluorescence detection and image analysis of γ-H2AX foci, a marker of cellular recognition of radiation-induced DNA damage, and Sox2, a stem cell marker that increases post-radiation, indicated that LY364947 blocked these radiation responses in five of seven specimens. Collectively, our findings suggest that TGF-β signaling increases radioresistance in most, but not all, HGGs. We propose that short-term culture of HGG explants provides a flexible and rapid platform for screening context-dependent efficacy of radiosensitizing agents in patient-specific fashion. This time- and cost-effective approach could be used to personalize treatment plans in HGG patients.

MicroRNA-346 facilitates cell growth and metastasis, and suppresses cell apoptosis in human non-small cell lung cancer by regulation of XPC/ERK/Snail/E-cadherin pathway.

Determinants of growth and metastasis in cancer remain of great interest to define. MicroRNAs (miRNAs) have frequently emerged as tumor metastatic regulator by acting on multiple signaling pathways. Here we report the definition of miR-346 as a novel oncogenic microRNA that facilitates non-small cell lung cancer (NSCLC) cell growth and metastasis. XPC, an important DNA damage recognition factor in nucleotide excision repair was defined as a target for down-regulation by miR-346, functioning through direct interaction with the 3′-UTR of XPC mRNA. Blocking miR-346 by an antagomiR was sufficient to inhibit NSCLC cell growth and metastasis, an effect that could be phenol-copied by RNAi-mediated silencing of XPC. In vivo studies established that miR-346 overexpression was sufficient to promote tumor growth by A549 cells in xenografts mice, relative to control cells. Overall, our results defined miR-346 as an oncogenic miRNA in NSCLC, the levels of which contributed to tumor growth and invasive aggressiveness.

Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced.

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells.

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6-4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair-deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair-proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.

DDB2 increases radioresistance of NSCLC cells by enhancing DNA damage responses.

Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2 in NSCLC could be used as a biomarker to predict radiosensitivity of the patients. Targeting Chk1 can be used to increase the efficacy of radiotherapy in patients of NSCLC possessing high levels of DDB2.

Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage.

Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity.

Abstract 3598: Laser micro-irradiations of large amounts of cells for high content applications

Abstract A new methodical approach for the evaluation of photo-manipulated samples is presented and tested in various biological setups. The method is based on an atypical setting of a standard laser-scanning microscope (LSM) and image-processing software solution. Newly, a whole population of several dozen of cells is simultaneously micro-irradiated by a laser beam in a precisely defined pattern of collinear rays. Such laser induced striation pattern is automatically analyzed by specialized software routine providing quantitative assessment of various photo-manipulations, such as DNA-damage induced protein translocations or modifications, in both live and fixed cells. Automation of the process reduces workload and opens new possibilities for various dynamic studies and/or high content experiments. Results obtained during the testing of the method are presented and offer an insight into several DNA damage recognition and response pathways. Citation Format: Martin Mistrik, Eva Vesela, Tomas Furst, Ivo Frydrych, Jan Gursky, Jiri Bartek. Laser micro-irradiations of large amounts of cells for high content applications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3598.

Biodosimetry for High-Dose Exposures Based on Dicentric Analysis in Lymphocytes Released from the G2-Block by Caffeine.

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridizationtechnique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.

Metal binding mediated conformational change of XPA protein:a potential cytotoxic mechanism of nickel in the nucleotide excision repair

Nucleotide excision repair (NER) is a pivotal life process for repairing DNA nucleotide mismatch caused by chemicals, metal ions, radiation, and other factors. As the initiation step of NER, the xeroderma pigmentosum complementation group A protein (XPA) recognizes damaged DNA molecules, and recruits the replication protein A (RPA), another important player in the NER process. The stability of the Zn(2+)-chelated Zn-finger domain of XPA center core portion (i.e., XPA98-210) is the foundation of its biological functionality, while the displacement of the Zn(2+) by toxic metal ions (such as Ni(2+), a known human carcinogen and allergen) may impair the effectiveness of NER and hence elevate the chance of carcinogenesis. In this study, we first calculated the force field parameters for the bonded model in the metal center of the XPA98-210 system, showing that the calculated results, including charges, bonds, angles etc., are congruent with previously reported results measured by spectrometry experiments and quantum chemistry computation. Then, comparative molecular dynamics simulations using these parameters revealed the changes in the conformation and motion mode of XPA98-210 Zn-finger after the substitution of Zn(2+) by Ni(2+). The results showed that Ni(2+) dramatically disrupted the relative positions of the four Cys residues in the Zn-finger structure, forcing them to collapse from a tetrahedron into an almost planar structure. Finally, we acquired the binding mode of XPA98-210 with its ligands RPA70N and DNA based on molecular docking and structural alignment. We found that XPA98-210's Zn-finger domain primarily binds to a V-shaped cleft in RPA70N, while the cationic band in its C-terminal subdomain participates in the recognition of damaged DNA. In addition, this article sheds light on the multi-component interaction pattern among XPA, DNA, and other NER-related proteins (i.e., RPA70N, RPA70A, RPA70B, RPA70C, RPA32, and RPA14) based on previously reported structural biology information. Thus, we derived a putative cytotoxic mechanism associated with the nickel ion, where the Ni(2+) disrupts the conformation of the XPA Zn-finger, directly weakening its interaction with RPA70N, and thus lowering the effectiveness of the NER process. In sum, this work not only provides a theoretical insight into the multi-protein interactions involved in the NER process and potential cytotoxic mechanism associated with Ni(2+) binding in XPA, but may also facilitate rational anti-cancer drug design based on the NER mechanism.