Enhanced DNA Repair Capacity Research Articles

SMAD4 Limits PARP1 dependent DNA Repair to Render Pancreatic Cancer Cells Sensitive to Radiotherapy

AbstractDysregulation of SMAD4 (i.e. somatic mutation) is strongly associated with poor pancreatic ductal adenocarcinoma (PDAC) prognosis, yet the molecular mechanisms remain underlying this relationship obscure. Previously, we discovered that SMAD4 mutation renders pancreatic cancer resistant to radiotherapy via promotion of autophagy. In the current work, we observed a downregulation of the protein level of SMAD4 in PDAC as compared with adjacent normal tissue, and that such SMAD4low PDAC failed to benefit from chemotherapy. Furthermore, we observed that SMAD4 depletion dramatically enhanced DNA repair capacity in response to irradiation (IR) or a radiomimetic chemical. Interestingly, we found the radiomimetic chemical having induced a robust translocation of SMAD4 into the nucleus, where a direct interaction was shown to occur between the MH1 domain of SMAD4 and the DBD domain of PARP1. Functionally, the SMAD4-PARP1 interaction was found to perturb the recruitment of PARP1 to DNA damage sites. Accordingly, the combination of olaparib and radiotherapy was indicated in vivo and in vitro to specifically reduce the growth of SMAD4-deficient PDAC by attenuating PARP1 activity. Collectively, our results revealed a novel molecular mechanism for the involvement of the SMAD4-PARP1 interaction in DNA repair with a vital role in radiotherapy response in PDAC. Based on our set of findings, our findings offer a new combined therapeutic strategy for SMAD4 deficient PDAC that can significantly reduce pancreatic cancer radiotherapy resistance.

Potential Strategies for Overcoming Drug Resistance Pathways Using Propolis and Its Polyphenolic/Flavonoid Compounds in Combination with Chemotherapy and Radiotherapy

Conventional cancer treatments include surgical resection, chemotherapy, hyperthermia, immunotherapy, hormone therapy, and locally targeted therapies such as radiation therapy. Standard cancer therapies often require the use of multiple agents, which can activate nuclear factor kappa B (NF-κB) in tumor cells, leading to reduced cell death and increased drug resistance. Moreover, the use of multiple agents also contributes to added toxicity, resulting in poor treatment outcomes. Cancer cells gradually develop resistance to almost all chemotherapeutics through various mechanisms, such as drug efflux, alterations in drug metabolism and transport, changes in signal transduction pathways, enhanced DNA repair capacity, evasion of apoptosis, increased mutations, reactivation of drug targets, interaction with the cancer microenvironment, cancer cell-stroma interactions, epithelial–mesenchymal transition (EMT)-mediated chemoresistance, epigenetic modifications, metabolic alterations, and the effect of cancer stem cells (CSCs). Developing new strategies to improve chemotherapy sensitivity while minimizing side effects is essential for achieving better therapeutic outcomes and enhancing patients’ quality of life. One promising approach involves combining conventional cancer treatments with propolis and its flavonoids. These natural compounds may enhance tumor response to treatment while reducing toxicity. Propolis and its components can sensitize cancer cells to chemotherapeutic agents, likely by inhibiting NF-κB activation, reprogramming tumor-associated macrophages (TAMs; an M2-like phenotype), and thereby reducing the release of matrix metalloproteinase (MMP)-9, cytokines, chemokines, and the vascular endothelial growth factor (VEGF). By reducing TAMs, propolis and its components may also overcome EMT-mediated chemoresistance, disrupt the crosstalk between macrophages and CSCs, inhibit the maintenance of stemness, and reverse acquired immunosuppression, thus promoting an antitumor response mediated by cytotoxic T-cells. This review highlights the potential of flavonoids to modulate the responsiveness of cancer to conventional treatment modalities. The evidence suggests that novel therapeutic strategies incorporating flavonoids could be developed to improve treatment outcomes. The positive effects of combining propolis with chemotherapeutics include reduced cytotoxicity to peripheral blood leukocytes, liver, and kidney cells. Therefore, polyphenolic/flavonoid components may hold potential for use in combination with chemotherapeutic agents in the clinical treatment of various types of cancers.

STEM-08. OVERCOMING GLIOBLASTOMA STEM CELL RESISTANCE TO RADIATION THROUGH TARGETING THIOREDOXIN REDUCTASE 1

Abstract Glioblastoma, the most aggressive primary tumor of the central nervous system, presents challenges due to its resistance to standard treatment, including radiation therapy (RT). Glioblastoma stem cells (GSCs), known for their chemo-radioresistance, employ adaptive mechanisms to counteract therapy-induced reactive oxygen species (ROS) levels. This includes modulating the activity of thioredoxin (Trx), a key player in the redox system. Furthermore, GSCs exhibit enhanced DNA repair capacity by upregulating DNA repair proteins, such as RAD51 to survive following ROS-induced DNA damage in response to radiation. Our TCGA analysis revealed elevated expression of thioredoxin reductase 1 (TrxR1) in patients diagnosed with GBM and a significant positive correlation between TrxR1 and RAD51. We hypothesized that GSCs might enhance their antioxidant capacity following RT to suppress RT-induced ROS elevation. Hence, combining RT with TrxR1 inhibitors might overcome GSCs radioresistance while reducing RAD51 expression. Auranofin (Au), a drug FDA-approved for rheumatoid arthritis inhibits TrxR1 activity with subsequent ROS increase. We assessed Au cytotoxicity on GSCs with and without ionizing radiation using Alamar Blue and neutrosphere formation assays and ROS assay for quantitative analysis of ROS levels. We evaluated the molecular mechanisms associated with Au and IR-induced cell death in GSCs using western blotting analysis. Our findings suggest that resistance of GSCs to IR was accompanied with increased expression of TrxR1 and RAD51. On the other hand, Au alone significantly decreased GSC viability and neurosphere formation and sensitized GSCs to IR within the nanomolar range (significantly lower than the corresponding IC50s). Au alone and combined with IR increased ROS, while expression of both RAD51 and TrxR1 was decreased. Our study provides insights into repurposing the TrxR1 inhibitor, Au and its combination with RT as a novel therapeutic strategy to overcome GSC radioresistance and ultimately improve the dismal outcome of patients diagnosed with GBM.

Abstract 3337: The impact of black raspberry as a whole food approach on DNA damage-induced by tobacco carcinogens in the oral cavity and urine of smokers

Abstract Introduction. We recently reported that black raspberries (BRB) powder inhibits dibenzo[def,p]chrysene (also known as dibenzo[a,l]pyrene; DBP) and its diolepoxides-induced DNA damage, mutagenesis and oral squamous cell carcinoma (OSCC) development in mice. We also showed that BRB enhanced DNA repair capacity of DNA damage-induced by DBP and altered genetic and epigenetic markers in a manner consistent with its cancer prevention activity. Polycyclic aromatic hydrocarbons (PAHs) are recognized as potential etiological agents in the development of oral cancer in smokers. In particular, benzo[a]pyrene (B[a]P) and DBP are detected in cigarette smoke and the environment; they can induce DNA damage, mutagenesis and carcinogenesis in the oral cavity of rodents. Consequently, their DNA adducts are regarded as the most direct markers of genotoxicity and can be used as biomarkers of cancer risk. Therefore, to translate our preclinical findings, we initiate a clinical study to examine the effects of BRB lozenges on DNA damage in smokers’ buccal cells. Experimental procedures. As an initial step for method development to examine the effects of BRB in humans, we conducted a study using LC-MS/MS analysis with isotope labelled internal standard to detect and quantify DNA adducts derived from B[a]P and DBP in buccal cells of cigarette smokers and non-smokers. Participants in this study include 21 smokers and 16 non-smokers. The urinary level of 8-oxodG (a marker of oxidative DNA damage) of smokers was also analyzed by LC-MS/MS. Results. Our results are the first to report that levels (mean ± S.D.) of BPDE-N2-dG were significantly higher in smokers (20.18 ± 8.40 adducts/108 dG) than in non-smokers (0.84 ± 1.02 adducts/108 dG). Likewise, levels of DBPDE-N6-dA in smokers (5.49 ± 3.41 adducts/108 dA) were significantly higher than non-smokers (2.76 ± 2.29 adducts/108 dA). Furthermore, we found that the baseline level of 8-oxodG in smokers’ urine before consumption of BRB lozenges is 18.92 ± 11.53 pmol/mL (n = 6). This level was significantly decreased by 16% (p = 0.006) during the consumption of BRB lozenges, and the level was decreased by 23% (p = 0.06) after cessation of BRB lozenges. Conclusion. Collectively, the results of this clinical study demonstrats that PAHs in tobacco smoke can contribute to the development of oral cancer in humans. Our results also suggest that BRB inhibited oxidative DNA damage in smokers’ urine. Currently, we are focusing on examining the effects of BRB on DNA damages induced by B[a]P and DBP in smokers. Supported by NIH grant #CA173465. Citation Format: Kun-Ming Chen, Yuan-Wan Sun, Nicolle M. Krebs, Lisa Reinhart, Dongxiao Sun, Elizabeth P. Bond, Jacek Krzeminski, Krishne Gowda, Shantu Amin, Susan Mallery, John P. Richie, Karam El-Bayoumy. The impact of black raspberry as a whole food approach on DNA damage-induced by tobacco carcinogens in the oral cavity and urine of smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3337.

Novel nanomedicines to overcome cancer multidrug resistance.

Chemotherapy remains a powerful tool to eliminate malignant cells. However, the efficacy of chemotherapy is compromised by the frequent emergence of intrinsic and acquired multidrug resistance (MDR). These chemoresistance modalities are based on a multiplicity of molecular mechanisms of drug resistance, including : 1) Impaired drug uptake into cancer cells; 2) Increased expression of ATP-binding cassette efflux transporters; 3) Loss of function of pro-apoptotic factors; 4) Enhanced DNA repair capacity; 5) Qualitative or quantitative alterations of specific cellular targets; 6) Alterations that allow cancer cells to tolerate adverse or stressful conditions; 7) Increased biotransformation or metabolism of anticancer drugs to less active or completely inactive metabolites; and 8) Intracellular and intercellular drug sequestration in well-defined organelles away from the cellular target. Hence, one of the major aims of cancer research is to develop novel strategies to overcome cancer drug resistance. Over the last decades, nanomedicine, which focuses on targeted delivery of therapeutic drugs into tumor tissues using nano-sized formulations, has emerged as a promising tool for cancer treatment. Therefore, nanomedicine has been introduced as a reliable approach to improve treatment efficacy and minimize detrimental adverse effects as well as overcome cancer drug resistance. With rationally designed strategies including passively targeted delivery, actively targeted delivery, delivery of multidrug combinations, as well as multimodal combination therapy, nanomedicine paves the way towards efficacious cancer treatment and hold great promise in overcoming cancer drug resistance. Herein, we review the recent progress of nanomaterials used in medicine, including liposomal nanoparticles, polymeric nanoparticles, inorganic nanoparticles and hybrid nanoparticles, to surmount cancer multidrug resistance. Finally, the future perspectives of the application of nanomedicine to reverse cancer drug resistance will be addressed.

Abstract A51: The role of ATF6-mediate AP-1 signaling in promoting PARP inhibitor-resistant ovarian cancer

Abstract The purpose of this study is to define the role of AP-1 mediated DNA repair in PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSOC). AP-1 is a dimeric transcription factor comprising Jun, Fos, and/or ATF proteins and its signaling is known to regulate multiple DNA repair genes, including PARP1. Wnt activation can promote AP-1 signaling and upregulates expression of AP-1 subunits. We previously published that hyperactive Wnt signaling in PARPi-resistant HGSOC cells promotes DNA repair and attenuates PARPi response. We therefore hypothesized that Wnt signaling drives PARPi resistance thorough increased AP1-signaling and subsequent enhanced DNA repair. We performed transcription factor analysis of RNA sequencing data from TP53/BRCA2-mutated olaparib-sensitive (PEO1) and -resistant (PEO1-OR) HGSOC cells. We observed increased AP-1 target genes in resistant cells and confirmed elevated AP-1 activity with a reporter assay. Quantitative PCR (qPCR) demonstrated that a majority of AP-1 subunits were upregulated in olaparib-resistant cells, namely JUN, ATF5, and ATF6. An shRNA screen was performed to determine which AP-1 subunits are necessary to convey olaparib resistance. Four out of five shRNAs targeting ATF6 effectively resensitized PARPi-resistant cells to olaparib. We confirmed that ATF6 protein was elevated in PEO1-OR cells. To further explore the role of ATF6 in AP-1 mediated PARPi resistance, ATF6 was stably knocked down in PEO1-OR cells. Colony formation assays confirmed that loss of ATF6 resensitized cells to olaparib. Knockdown of ATF6 also resulted in decreased transcription of PARP1, as measured by qPCR, and higher baseline levels of DNA damage, as measured by phosphorylated histone gamma-H2AX. Future studies will evaluate ATF6-dependent DNA repair in in vitro and in vivo models of PARPi-resistant HGSOC. In summary, these data indicate activation of AP-1 signaling through increased expression of ATF6 is promoting enhanced DNA repair capacity, which could be contributing to PARPi resistance. Thus, pharmacologic inhibition of AP-1 or ATF6 could provide a targetable approach to overcome resistance and improve patient outcomes. Citation Format: Alexandra N. McMellen, Benjamin G. Bitler. The role of ATF6-mediate AP-1 signaling in promoting PARP inhibitor-resistant ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A51.

Glioblastomas located in proximity to the subventricular zone (SVZ) exhibited enrichment of gene expression profiles associated with the cancer stem cell state.

Conflicting results have been reported in the association between glioblastoma proximity to the subventricular zone (SVZ) and enrichment of cancer stem cell properties. Here, we examined this hypothesis using magnetic resonance (MR) images derived from 217 The Cancer Imaging Archive (TCIA) glioblastoma subjects. Pre-operative MR images were segmented automatically into contrast enhancing (CE) tumor volumes using Iterative Probabilistic Voxel Labeling (IPVL). Distances were calculated from the centroid of CE tumor volumes to the SVZ and correlated with gene expression profiles of the corresponding glioblastomas. Correlative analyses were performed between SVZ distance, gene expression patterns, and clinical survival. Glioblastoma located in proximity to the SVZ showed increased mRNA expression patterns associated with the cancer stem-cell state, including CD133 (P = 0.006). Consistent with the previous observations suggesting that glioblastoma stem cells exhibit increased DNA repair capacity, glioblastomas in proximity to the SVZ also showed increased expression of DNA repair genes, including MGMT (P = 0.018). Reflecting this enhanced DNA repair capacity, the genomes of glioblastomas in SVZ proximity harbored fewer single nucleotide polymorphisms relative to those located distant to the SVZ (P = 0.003). Concordant with the notion that glioblastoma stem cells are more aggressive and refractory to therapy, patients with glioblastoma in proximity to SVZ exhibited poorer progression free and overall survival (P < 0.01). An unbiased analysis of TCIA suggests that glioblastomas located in proximity to the SVZ exhibited mRNA expression profiles associated with stem cell properties, increased DNA repair capacity, and is associated with poor clinical survival.

Arsenic Trioxide and (-)-Gossypol Synergistically Target Glioma Stem-Like Cells via Inhibition of Hedgehog and Notch Signaling.

Glioblastoma is one of the deadliest malignancies and is virtually incurable. Accumulating evidence indicates that a small population of cells with a stem-like phenotype is the major culprit of tumor recurrence. Enhanced DNA repair capacity and expression of stemness marker genes are the main characteristics of these cells. Elimination of this population might delay or prevent tumor recurrence following radiochemotherapy. The aim of this study was to analyze whether interference with the Hedgehog signaling (Hh) pathway or combined Hh/Notch blockade using small-molecule inhibitors can efficiently target these cancer stem cells and sensitize them to therapy. Using tumor sphere lines and primary patient-derived glioma cultures we demonstrate that the Hh pathway inhibitor GANT61 (GANT) and the arsenic trioxide (ATO)-mediated Hh/Notch inhibition are capable to synergistically induce cell death in combination with the natural anticancer agent (−)-Gossypol (Gos). Only ATO in combination with Gos also strongly decreased stemness marker expression and prevented sphere formation and recovery. These synergistic effects were associated with distinct proteomic changes indicating diminished DNA repair and markedly reduced stemness. Finally, using an organotypic brain slice transplantation model, we show that combined ATO/Gos treatment elicits strong growth inhibition or even complete elimination of tumors. Collectively, our data show for the first time that ATO and Gos, two drugs that can be used in the clinic, represent a promising targeted therapy approach for the synergistic elimination of glioma stem-like cells.

MiR-192-5p reverses cisplatin resistance by targeting ERCC3 and ERCC4 in SGC7901/DDP cells.

Cisplatin chemoresistance is a clinical obstacle in the treatment of gastric cancer (GC). Enhanced DNA repair capacity may lead to cisplatin resistance. However, the detailed molecular mechanism of GC cisplatin resistance specifically involving nucleotide excision repair (NER) is not clear. However, the mechanism through which the NER pathway contributes to cisplatin resistance in GC is still unclear. In light of the crucial role of microRNAs (miRNAs) in regulating protein expression and biological behavior, we aimed to analyze the expression and function of miR-192-5p in the NER pathway and its role in cisplatin resistance in GC. Comet assays were performed to measure the amount of DNA damage and repair in the SGC7901 and SGC7901/DDP GC cell lines by observing the tail length. MiRNA expression levels in SGC7901/DDP and SGC7901 cells were detected by microarray. Quantitative real-time PCR (qRT-PCR) was carried out to confirm the expression level of miR-192-5p. Lentiviral vector transfection modifies miR-192-5p levels in SGC7901/DDP and SGC7901 cells. The IC50 values of cisplatin-treated cells were assessed by MTT assays. The protein level was determined by Western blot and immunohistochemistry. With enhanced DNA repair, the expression levels of ERCC3 and ERCC4 in SGC 7901DDP cells increased, while miR-192-5p was significantly downregulated in SGC7901/DDP compared with SGC7901 cells. ERCC3 and ERCC4 were identified as the main targets of miR-192-5p. Forced expression of miR-192-5p in SGC7901/DDP cells significantly inhibited the expression of ERCC3 and ERCC4, making GC cells more sensitive to cisplatin in vitro and in vivo. In contrast, knockdown of miR-192-5p expression in SGC7901 cells increased the expression of ERCC3 and ERCC4, resulting in cisplatin resistance in vitro and in vivo. MiR-192-5p partially reversed GC cisplatin resistance by targeting ERCC3 and ERCC4, which participate in the NER pathway, suggesting that miR-192-5p may be a potential biomarker and therapeutic target for GC cisplatin resistance.

Radiation Therapy Modulates DNA Repair Efficiency in Peripheral Blood Mononuclear Cells of Patients With Non-Small Cell Lung Cancer

There is growing interest in developing individually tailored cancer radiation therapy (RT), wherein patients with high intrinsic radiosensitivity are identified before commencing treatment, to minimize severe adverse reactions. In a previous retrospective study of severely radiosensitive RT patients, we established a functional assay with a high predictive capability. The assay involves exvivo irradiation of peripheral blood mononuclear cells and analysis of DNA repair using the γ-H2AX assay. It is unknown whether RS is a fixed phenomenon or is modulated under different conditions. We now report the impact of RT on the apparent radiosensitivity, as reflected by the assay. Peripheral blood mononuclear cells of 11 patients with non-small cell lung cancer were collected before, during, and after RT. Quantitative parameters derived from the nonlinear regression analysis of γ-H2AX foci were applied to examine the cellular radiation response. Although the repair rate and foci yield remained constant during and after RT, the "unrepairable" component of γ-H2AX foci decreased over the course of treatment in 7 patients, signifying a generally enhanced DNA repair capacity. Interestingly, enhanced repair capacity tended to be associated with a poorer response to RT. Although generalization of these results into normal and tumor tissues warrants further investigation, the findings of this study have important implications in future strategies for identifying radiosensitive individuals before exposure to RT. We can anticipate that the threshold values that will discriminate radiosensitive patients in a future prospective trial will differ from those established in the retrospective study.

Dietary Nutrient Intake, Ethnicity, and Epigenetic Silencing of Lung Cancer Genes Detected in Sputum in New Mexican Smokers.

Lung cancer gene methylation detected in sputum assesses field cancerization and predicts lung cancer incidence. Hispanic smokers have higher lung cancer susceptibility compared with non-Hispanic whites (NHW). We aimed to identify novel dietary nutrients affecting lung cancer gene methylation and determine the degree of ethnic disparity in methylation explained by diet. Dietary intakes of 139 nutrients were assessed using a validated Harvard food frequency questionnaire in 327 Hispanics and 1,502 NHWs from the Lovelace Smokers Cohort. Promoter methylation of 12 lung cancer genes was assessed in sputum DNA. A global association was identified between dietary intake and gene methylation (Ppermutation = 0.003). Seventeen nutrient measurements were identified with magnitude of association with methylation greater than that seen for folate. A stepwise approach identified B12, manganese, sodium, and saturated fat as the minimally correlated set of nutrients whose optimal intakes could reduce the methylation by 36% (Ppermutation < 0.001). Six protective nutrients included vitamin D, B12, manganese, magnesium, niacin, and folate. Approximately 42% of ethnic disparity in methylation was explained by insufficient intake of protective nutrients in Hispanics compared with NHWs. Functional validation of protective nutrients showed an enhanced DNA repair capacity toward double-strand DNA breaks, a mechanistic biomarker strongly linked to acquisition of lung cancer gene methylation in smokers. Dietary intake is a major modifiable factor for preventing promoter methylation of lung cancer genes in smokers' lungs. Complex dietary supplements could be developed on the basis of these protective nutrients for lung cancer chemoprevention in smokers. Hispanic smokers may benefit the most from this complex for reducing their lung cancer susceptibility. Cancer Prev Res; 11(2); 93-102. ©2017 AACR.

Resistance to DNA damage and enhanced DNA repair capacity in the hypoxia-tolerant blind mole rat Spalax carmeli.

Blind mole rats of the genus Spalax are the only mammalian species to date for which spontaneous cancer has never been reported and resistance to carcinogen-induced cancers has been demonstrated. However, the underlying mechanisms are still poorly understood. The fact that Spalax spp. are also hypoxia-tolerant and long-lived species implies the presence of molecular adaptations to prevent genomic instability, which underlies both cancer and aging. We previously demonstrated the upregulation of transcripts related to DNA replication and repair pathways in Spalax Yet, to date, no direct experimental evidence for improved genomic maintenance has been demonstrated for this genus. Here, we show that compared with skin fibroblasts of the above-ground rat, Spalax carmeli skin fibroblasts in culture resist several types of genotoxic insult, accumulate fewer genotoxic lesions and exhibit an enhanced DNA repair capacity. Our results strongly support that this species has evolved efficient mechanisms to maintain DNA integrity as an adaptation to the stressful conditions in the subterranean habitat.

Alternative NHEJ pathway proteins as components of MYCN oncogenic activity in human neural crest stem cell differentiation: implications for neuroblastoma initiation

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.

Radiotherapy and Glioma Stem Cells: Searching for Chinks in Cellular Armor.

Radiation became a pillar of oncologic treatment in the last century and provided a powerful and effective locoregional treatment of solid malignancies. After achieving some of the first cures in lymphomas and skin cancers, it assumed a key role in curative treatment of epithelioid malignancies. Despite success across a variety of histologic types, glioblastoma (GBM), the most common primary brain tumor afflicting adults, remains ultimately resistant to current radiation strategies. While GBMs demonstrate an initial response, recurrence is essentially universal and fatal, and typically reoccur in the areas that received the most intense radiation. Glioma stem cells (GSCs), a subpopulation of tumor cells with expression profiles similar to neural stem cells and marked self-renewal capacities, have been shown to drive tumor recurrence and preclude curative radiotherapy. Recent research has shown that these cells have enhanced DNA repair capacity, elevated resistance to cytotoxic ion fluxes and escape multi-modality therapies. We will analyze the current understanding of GSCs and radiation by highlighting key discoveries probing their ability to withstand radiotherapy. We then speculate on novel mechanisms by which GSC can be made sensitive to or specifically targeted by radiation therapy.

Abstract 4257: Dietary nutrient intake, ethnicity, and epigenetic silencing of lung cancer genes detected in sputum in New Mexican smokers

Abstract Promoter hypermethylation of lung cancer (LC) genes detected in sputum assesses field cancerization and predicts LC risk. Hispanic smokers have greater risk for methylation than non-Hispanic Whites (NHW). We aimed to identify novel dietary nutrients affecting methylation of LC genes in sputum and determine the degree of ethnic disparity explained by diet. A cross-sectional population-based study was conducted with dietary intake assessed using a validated Harvard food frequency questionnaire in 327 Hispanics and 1502 NHWs from the Lovelace Smokers cohort (LSC). Twelve LC genes with diverse cellular functions whose concomitant methylation strongly predicted LC risk were studied. A global association was identified between dietary intake and gene methylation (Ppermutation=0.003). Seventeen nutrient measurements were associated with methylation with magnitude greater than seen for folate and these nutrients together accounted for 36% of the variance in methylation. Six protective nutrients were identified and their insufficient intake in Hispanics explained approximately 42% of ethnic disparity in methylation. Functional validation of protective nutrients that also explained ethnic disparity was provided by showing an enhanced DNA repair capacity towards double-strand DNA breaks, a mechanistic biomarker strongly linked to acquisition of LC gene methylation in smokers. Dietary intake is a major modifiable factor for preventing promoter methylation of LC genes in smokers’ lungs. Altering the diet in Hispanics may alleviate ethnic disparity for methylation. Complex dietary supplements could be developed based on these protective nutrients for LC chemoprevention in smokers. This study was funded by NIH R01 CA097356, NIH/NCI P30 CA118100, and the State of New Mexico as a direct appropriation from the Tobacco Settlement Fund. Citation Format: Shuguang Leng, Maria Picchi, Piotr Filipczak, Frank Gilliland, Steven A. Belinsky. Dietary nutrient intake, ethnicity, and epigenetic silencing of lung cancer genes detected in sputum in New Mexican smokers [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 4257. doi:10.1158/1538-7445.AM2017-4257

Protective effect of Alstonia scholaris Linn. R. Br. against Bleomycin induced chromosomal damage in cultured human lymphocytes, in vitro

It is both interesting and necessary to identify and develop nontoxic radioprotective compounds. Bleomycin (BLM), a known radiomimetic drug was used as a clastogen in the present study. The possible protective effects against BLM (15 μg/ml) induced clastogenicity by aqueous and methanolic extracts from Alstonia scholaris bark, stem and leaves were compared. The treatment of bark extracts significantly (p < 0.01) reduced total chromosomal aberrations. Such a reduction was not seen in case of stem and leaf treatments. The dose of 50 μg/ml was fixed for all extracts throughout the study. To understand the mechanism involved with the protective property of bark extracts, sensitive G2 assay was performed. Lymphocyte cultures from 12 healthy volunteers were exposed to aqueous (50 μg/ml) and methanolic (50 μg/ml) extracts of A. scholaris bark alone as well as in combination with Bleomycin under two different growth phases, G0 and G2. There was a statistically significant reduction (p < 0.05) in the total chromatid breaks in all cultures which were exposed at G2 phase as compared to respective cultures exposed at G0 phase. The highest level (p < 0.0001) of reduction in total chromatid breaks was observed in cultures treated with aqueous bark extracts at G2 phase than those at G0 phase. This indicated that there could be certain compound(s) present in aqueous bark extracts which enhance DNA repair capacity. Therefore, the bark of A. scholaris could be further utilized to identify and bring out front line radio protective agents in the market with effective formulations.

Chemotherapeutic Drugs: DNA Damage and Repair in Glioblastoma.

Despite improvements in therapeutic strategies, glioblastoma (GB) remains one of the most lethal cancers. The presence of the blood–brain barrier, the infiltrative nature of the tumor and several resistance mechanisms account for the failure of current treatments. Distinct DNA repair pathways can neutralize the cytotoxicity of chemo- and radio-therapeutic agents, driving resistance and tumor relapse. It seems that a subpopulation of stem-like cells, indicated as glioma stem cells (GSCs), is responsible for tumor initiation, maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity. Recently, attention has been focused on the pivotal role of the DNA damage response (DDR) in tumorigenesis and in the modulation of therapeutic treatment effects. In this review, we try to summarize the knowledge concerning the main molecular mechanisms involved in the removal of genotoxic lesions caused by alkylating agents, emphasizing the role of GSCs. Beside their increased DNA repair capacity in comparison with non-stem tumor cells, GSCs show a constitutive checkpoint expression that enables them to survive to treatments in a quiescent, non-proliferative state. The targeted inhibition of checkpoint/repair factors of DDR can contribute to eradicate the GSC population and can have a great potential therapeutic impact aiming at sensitizing malignant gliomas to treatments, improving the overall survival of patients.

The importance of staying small

Long-lived and cancer-resistant adult dwarf rodents have enhanced DNA repair capacity due to decreased GH/IGF-1 signaling during peripubertal development.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

Lomeguatrib, a O6-Methylguanine-DNA-Methyltransferase (MGMT) Inhibitor, Induces DNA Damage Induced Apoptosis By Targeting Double-Strand DNA Repair in Multiple Myeloma

Background/Aim:Multiple Myeloma (MM), characterized by aberrant accumulation of terminally differentiated plasma cells in the bone marrow, is promoted by ongoing DNA damage and genomic instability. O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair protein, transfers the alkyl group from guanine to its cysteine residue and is implicated as an important chemoresistance factor particularly in chemotherapy-induced DNA alkylation. A growing body of evidence has shown that enhanced DNA repair capacity stimulates cancer cell survival and facilitates genomic instability. Thus, targeting DNA repair activities including MGMT seems to be a promising strategy for the treatment of MM.Materials and Methods: In our study, MGMT expression was evaluated in MGUS, SMM and MM patients compared to normal plasma cells using publically available gene expression data sets (Gene Expression Omnibus; GSE47552). Furthermore, MGMT mRNA and protein levels were determined in human MM cell lines (NCI H929, RPMI 8226 and U266), three samples of control PBMCs, matched CD138+ myeloma and CD138- non-tumorigenic cells obtained from bone marrow aspirates of same MM patients [newly diagnosed (ND, n=12) and relapsed / refractory (RR, n=8)] by RealTime Ready PCR Assay and Western blot, respectively. The experiments with human cells were approved by the Ethical Committee of Ankara University School of Medicine. Following treatment of MM cells with Lomeguatrib for 48 hours, cell viability, cell cycle progression and apoptosis were assessed by MTT and flow cytometry. DNA damage levels were examined by alkaline comet assay and immunoblotting of ΥH2AX phosphorylation.Results: Comparable MGMT expression was detected between MM cells and normal plasma cells, and between ND CD138+ and RR CD138+ myeloma cells. Inhibition of MGMT activity by Lomeguatrib reduced MM cell viability and induced apoptosis in MGMT proficient but not in MGMT deficient MM cells in a dose-dependent manner. In contrast, Lomeguatrib did not affect the cell viability of PBMCs from three healthy donors and CD138- non-tumorigenic cells from three MM patients. Furthermore, Lomeguatrib perturbed cell cycle and decreased phosphorylation of G1/S specific CyclinE1 in these cells and led to an increase in DNA damage. Western blot analysis revealed a reduction in protein levels of Fen1, Ku-70 and Rad51 which are involved in double-strand DNA break repair.Conclusion: These results revealed that MGMT promotes repair of DNA double-strand breaks in MGMT proficient MM cells and MGMT inhibition enhances DNA damage-induced apoptosis, suggesting that inhibition of MGMT by Lomeguatrib might be a useful strategy to overcome unfaithful DNA repair in MM. This research has been supported by The Scientific and Technological Research Council of Turkey (No:113Z383). DisclosuresBeksac:Celgene, Janssen Cilag Amgen, Novartis, Takeda: Honoraria, Speakers Bureau.