Activation Of DNA Repair Pathways Research Articles

Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs increase the accessibility of human pericentromeric heterochromatin by disrupting HP1α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of de-compaction to be accessed. Our results support a model in which the 3D organization of heterochromatin dictates the spatial activation of DNA repair pathways and is key to preventing the activation of HR within clustered repeats and the onset of chromosomal translocations.

Curcumin sensitizes carboplatin treatment in triple negative breast cancer through reactive oxygen species induced DNA repair pathway.

As patients with triple-negative breast cancer (TNBC) have a very weak response to hormone inhibition or anti-HER2 therapy, traditional chemotherapy is commonly used in these patients. Recently, carboplatin has been approved for the clinical treatment of TNBC. However, several patients exhibit resistance to carboplatin treatment. Therefore, strategies to enhance the antitumor effect of carboplatin need to be explored. In our study, we investigated the function of curcumin in increasing the response to carboplatin. MTT and colony formation assays were used to evaluate cell viability after carboplatin and curcumin treatment. In addition, we conducted flow cytometric and Western blot analyses to examine cellular apoptosis. Subsequently, molecular and biochemical experiments were used to explore the mechanism by which curcumin sensitized TNBC to carboplatin treatment. We demonstrated that different TNBC cells responded differently to carboplatin. Low-dose carboplatin killed CAL-51 cells but barely influenced CAL-51-R and MDA-MB-231 cells. To improve the sensitivity of resistant TNBC cells to carboplatin, combined treatment with curcumin was applied and was found to inhibit proliferation and induce apoptosis. Mechanistically, curcumin exerted its anticancer effect by increasing reactive oxygen species (ROS) production, which downregulated the DNA repair protein RAD51, leading to upregulation of γH2AX. As expected, ROS scavenger NAC reversed the inhibitory effect on growth and DNA repair pathway activity mediated by curcumin. Collectively, our data demonstrate that curcumin sensitizes TNBC to the anticancer effect of carboplatin by increasing ROS-induced DNA damage, thus providing an effective combination treatment strategy for TNBC.

The Activation оf DNА Repair Pathways after Ultra-Short Pulsed Electron Beam Irradiation in Human Cells

The aim of this work was to estimate the differences in activation of DNA repair pathways of DNA double-strand and single-strand breaks induced by ultrashort pulse electron beam irradiation in human K-562 cells. The activation of HR, NHEJ and BER DNA repair pathways was studied at non-lethal, sub-lethal and lethal doses of irradiation. Our results indicate that the activation of the specific repair pathways and repair kinetics depend on the irradiation dose.

Pharmacologic Ascorbate Enhances the Therapeutic Index of ATM-Inhibitor Based Chemoradiation for Colorectal Cancer

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Host DNA repair response to oxidative damage is modulated by Trypanosoma cruzi in a strain-dependent manner

The conservation of genomic integrity and stability is essential for cell survival. DNA Damage Responses (DDRs) are considered of paramount importance for all living beings and involve mechanisms of cell cycle regulation and damage-specific DNA repair pathways. Hydrogen peroxide (H2O2) is a compound that, in supraphysiological concentrations, damages biomolecules including the DNA, causing base modifications and strand breaks. There is evidence that Trypanosoma cruzi, the protozoan that causes Chagas disease, interferes in the host cell's DNA metabolism. In order to investigate the influence of T. cruzi infection over the host cell capacity to withstand and repair DNA damage, we analyzed L6 cells infected with Berenice, and Colombiana T. cruzi strains according to their viability, proliferation, morphology, DNA degradation, expression of DNA repair, and cell cycle genes following H2O2 treatment. It was noted that T. cruzi infection might act as either a stressor or a protective element of host DNA, depending on the strain and H2O2 concentration. Cells infected with Berenice strain and treated with 0.8 mM H2O2 presented a reduced DNA damage response intensity (e.g., BER and HR). Infection with T. cruzi Colombiana prevented the activation of DNA repair pathways in response to 0.8mM and 1.6mM H2O2 (NER and MMR). Nevertheless, since cellular viability was not significantly compromised in Colombiana-infected cells following the oxidative insult, it is possible that the parasite directly influenced the host DNA repair machinery. Our results support the notion that T. cruzi is able to modulate the host cell DNA metabolism in a strain-dependent manner, an event which can be explored in future drug development strategies.

PARP1-modulated chromatin remodeling is a new target for cancer treatment.

Cancer progression requires certain tumorigenic mutations in genes encoding for different cellular and nuclear proteins. Altered expressions of these mutated genes are mediated by post-translational modifications and chromatin remodeling. Chromatin remodeling is mainly regulated by the chromatin remodeling enzyme complexes and histone modifications. Upon DNA damage, Poly-(ADP-ribose) Polymerase1 (PARP1) plays a very important role in the induction of chromatin modifications and activation of DNA repair pathways to repair the DNA lesion. It has been targeted to develop different anti-cancer therapeutic interventions and PARP inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for clinical use. But it has been found that the cancer cells often develop resistance to these PARP inhibitors and chromatin remodeling helps in enhancing this process. Hence, it may be beneficial to target PARP1-mediated chromatin remodeling, which may allow to reverse the drug resistance. In the current review, we have discussed the role of chromatin remodeling in DNA repair, how PARP1 regulates modifications of chromatin dynamics, and the role of chromatin modifications in cancer. It has also been discussed how the PARP1-mediated chromatin remodeling can be targeted by PARP inhibitors alone or in combination with other chemotherapeutic agents to establish novel anti-cancer therapeutics. We have also considered the use of PARG inhibitors that may enhance the action of PARP inhibitors to target different types of cancers.

Abstract LB113: Regulation of PARP-1 expression in mammary tumor cell lines after treatment with olaparib

Abstract Introduction: Breast cancer is the most common tumor type among women and has a high mortality rate, which is associated with late detection of the disease and treatment failure due to the acquisition of resistance to protocol treatments. It is known that a variety of antitumor drugs are capable of inducing chemoresistance by activating DNA repair pathways. Among these, the PARPs are involved in the recognition and repair of simple-strand breaks. As a consequence, new drugs such as PARP inhibitors (olaparib), are being investigated as potential therapeutic targets in cancer, as an alternative to evade the resistance of tumor cells to chemotherapeutic agents, such as those in the class of platinum salts. In this context, the aim of this study was to evaluate the PARP expression after olaparib tratament with in mammary tumor cell lines. Materials and Methods: Mammary tumor cell lines MDA-MB-468 and CF41 was cultured in DMEM high glucose culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with different concentrations of carboplatin. Once stablished the concentration of 10µM for carboplatin and 10µM of olaparib, the protein and gene expression of PARP-1 were detected by immunofluorescence and real time PCR, respectively. Results: There was a significantly decrease of cell viability after treatment with different concentrations of carboplatin in 24 hours. Both PARP-1 protein and gene expression significantly decreased after treatment with carboplatin and olaparib. In some cases, the treatment action was potentiated when performed in combination. Conclusion: Our results suggest the efficacy of olaparib in controlling the mechanism of DNA repair activated by PARPs in human and canine breast cancer cells. Citation Format: Marina Gobbe Moschetta-Pinheiro, Jucimara Colombo, Murilo de Souza Tuckumantel, Gabriela Karam Rebolho, Debora Aparecida Zuccari. Regulation of PARP-1 expression in mammary tumor cell lines after treatment with olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB113.

Abstract 2072: DNA repair pathway score is associated with cancer aggressiveness and worse survival in hepatocellular carcinoma (HCC)

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. A prognostic biomarker based on cancer biology can tailor treatments according to patient prognosis. DNA repair mechanisms activated following DNA damage are essential to suppress carcinogenesis. Hence, deficiencies in DNA damage repair pathways promote carcinogenesis. The role of DNA repair genes in HCC is actively being investigated, but contradictory reports on some DNA repair-related genes have complicated our overall understanding of DNA repair in HCC progression. Though it is important to study the function of each individual DNA repair-related gene, it is more important to understand the role of the entire DNA repair pathway in HCC progression. We hypothesize that the enhancement of the DNA repair pathway is associated with worse survival in HCC patients. Method: A total of 749 HCC patients from 5 cohorts (TCGA, GSE6764, GSE89377, GSE56545, GSE76427) were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC utilizing a DNA repair pathway score, which was calculated by Gene Set Variation Analysis (GSVA) with Hallmark DNA repair gene set. Results: We found that DNA repair pathway was enhanced by stepwise carcinogenic process of HCC and significantly enhanced in higher grade HCC compared with low grade tumor in two cohorts (both p < 0.001). DNA repair high HCC was associated with worse survival (disease-free survival: p < 0.001, disease-specific survival: p = 0.002, and overall survival: p < 0.001), elevated intratumor heterogeneity (p = 0.036) and mutation load (p < 0.001), but not with fraction of immune cell infiltration nor immune response in the TCGA cohort. HCC tumors with DNA repair high score enriched proliferation-related (E2F targets, G2M checkpoint, mitotic spindle, MYC target v1 and v2) and other cancer aggressiveness-related (unfolded protein response, PI3K/AKT/MTOR signaling, MTORC1, notch signaling, and WNT-β catenin signaling) gene sets. Interestingly, these features were more pronounced in early HCC compared to advanced HCC. DNA repair high HCC was associated with elevated mutation load, enriched proliferation- and other cancer aggressiveness-related gene sets and was associated with worse survival in early (grade 1 and grade 2) but not in advanced (grade 3) HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. Conclusion: The DNA repair score may be used to better understand and predict prognosis in HCC. Citation Format: Masanori Oshi, Tae Hee Kim, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Leonid Cherkassky, Kazuaki Takabe. DNA repair pathway score is associated with cancer aggressiveness and worse survival in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2072.

Pharmacologic ascorbate enhances the therapeutic index of ATM-inhibitor based chemoradiation for colorectal cancer.

3609 Background: Ataxia telangectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury. Methods: Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57blj/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors. Results: Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated P-ATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT. Conclusions: P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

NSMF promotes the replication stress-induced DNA damage response for genome maintenance.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.

DNA repair pathway activation features in follicular and papillary thyroid tumors, interrogated using 95 experimental RNA sequencing profiles

DNA repair can prevent mutations and cancer development, but it can also restore damaged tumor cells after chemo and radiation therapy. We performed RNA sequencing on 95 human pathological thyroid biosamples including 17 follicular adenomas, 23 follicular cancers, 3 medullar cancers, 51 papillary cancers and 1 poorly differentiated cancer. The gene expression profiles are annotated here with the clinical and histological diagnoses and, for papillary cancers, with BRAF gene V600E mutation status. DNA repair molecular pathway analysis showed strongly upregulated pathway activation levels for most of the differential pathways in the papillary cancer and moderately upregulated pattern in the follicular cancer, when compared to the follicular adenomas. This was observed for the BRCA1, ATM, p53, excision repair, and mismatch repair pathways. This finding was validated using independent thyroid tumor expression dataset PRJEB11591. We also analyzed gene expression patterns linked with the radioiodine resistant thyroid tumors (n = 13) and identified 871 differential genes that according to Gene Ontology analysis formed two functional groups: (i) response to topologically incorrect protein and (ii) aldo-keto reductase (NADP) activity. We also found RNA sequencing reads for two hybrid transcripts: one in-frame fusion for well-known NCOA4-RET translocation, and another frameshift fusion of ALK oncogene with a new partner ARHGAP12. The latter could probably support increased expression of truncated ALK downstream from 4th exon out of 28. Both fusions were found in papillary thyroid cancers of follicular histologic subtype with node metastases, one of them (NCOA4-RET) for the radioactive iodine resistant tumor. The differences in DNA repair activation patterns may help to improve therapy of different thyroid cancer types under investigation and the data communicated may serve for finding additional markers of radioiodine resistance.

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication.

Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2β. Upon DNA cleavage, a covalent intermediate DNA-TOP2β (TOP2βcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2βcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2β proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2β and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2β with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2β also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2β. This study identifies TOP2β as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2β levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2β reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2β through DNA break formation.

Cellular response to endogenous DNA damage: DNA base modifications in gene expression regulation

The integrity of the genetic information is continuously challenged by numerous genotoxic insults, most frequently in the form of oxidation, alkylation or deamination of the bases that result in DNA damage. These damages compromise the fidelity of the replication, and interfere with the progression and function of the transcription machineries. The DNA damage response (DDR) comprises a series of strategies to deal with DNA damage, including transient transcriptional inhibition, activation of DNA repair pathways and chromatin remodeling. Coordinated control of transcription and DNA repair is required to safeguardi cellular functions and identities. Here, we address the cellular responses to endogenous DNA damage, with a particular focus on the role of DNA glycosylases and the Base Excision Repair (BER) pathway, in conjunction with the DDR factors, in responding to DNA damage during the transcription process. We will also discuss functions of newly identified epigenetic and regulatory marks, such as 5-hydroxymethylcytosine and its oxidative products and 8-oxoguanine, that were previously considered only as DNA damages. In light of these resultsthe classical perception of DNA damage as detrimental for cellular processes are changing. and a picture emerges whereDNA glycosylases act as dynamic regulators of transcription, placing them at the intersection of DNA repair and gene expression modulation.

Enhanced DNA Repair Pathway is Associated with Cell Proliferation and Worse Survival in Hepatocellular Carcinoma (HCC).

Simple SummaryWe studied the relationship between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in 749 hepatocellular carcinoma (HCC) patients from 5 cohorts using a DNA repair pathway score. We show that the DNA repair pathway was enhanced by the stepwise carcinogenic process of HCC, notably in grade 3 compared to grade 1 or 2 HCC. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor cytolytic activity. The expression of proliferation- and other cancer aggressiveness-related gene sets was also increased. Interestingly, these features were more pronounced in low-grade compared to high-grade HCC. In conclusion, the DNA repair score may be used to understand the role of DNA repair pathways in patient prognosis and treatment sensitivity and be used to improve patient outcome. To our knowledge, this is the first study using DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC.Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. In this study, a total of 749 HCC patients from 5 cohorts were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC, utilizing a DNA repair pathway score. Our findings suggest that the DNA repair pathway was not only enhanced by the stepwise carcinogenic process of HCC, but also significantly enhanced in grade 3 HCC compared with grade 1 and 2 tumors. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor immune response. HCC tumors with a DNA repair high score enriched the cell proliferation- and other cancer aggressiveness-related gene sets. Interestingly, these features were more pronounced in grade 1 and 2 HCC compared to grade 3 HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. The DNA repair score may be used to better understand and predict prognosis in HCC.

A Combined Epithelial Mesenchymal Transformation and DNA Repair Gene Panel in Colorectal Cancer With Prognostic and Therapeutic Implication.

BackgroundEpithelial mesenchymal transformation (EMT) and DNA repair status represent intrinsic features of colorectal cancer (CRC) and are associated with patient prognosis and treatment responsiveness. We sought to develop a combined EMT and DNA repair gene panel with potential application in patient classification and precise treatment.MethodsWe comprehensively evaluated the EMT and DNA repair patterns of 1,652 CRC patients from four datasets. Unsupervised clustering was used for classification. The clinical features, genetic mutation, tumor mutation load, and chemotherapy as well as immunotherapy sensitivity among different clusters were systematically compared. The least absolute shrinkage and selection operator regression method was used to develop the risk model.ResultsThree distinct CRC clusters were determined. Clustet1 was characterized by down-regulated DNA repair pathways but active epithelial markers and metabolism pathway and had intermediate prognosis. Clustet2 was characterized by down-regulated both epithelial markers and DNA repair pathways and had poor outcome. Clustet3 presented with activation of DNA repair pathway and epithelial markers had favorable prognosis. Clustet1 might benefit form chemotherapy and Clustet3 had a higher response rate to immunotherapy. An EMT and DNA repair risk model related to prognosis and treatment response was developed.ConclusionsThis work developed and validated a combined EMT and DNA repair gene panel for CRC classification, which may be an effective tool for survival prediction and treatment guidance in CRC patients.

Heterochromatic Repeat Clustering Imposes a Physical Barrier on Homologous Recombination to Prevent Chromosomal Translocations

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters containing homologous sequences from different chromosomes. These clusters are protected from DNA damage and are refractory to DNA repair through homologous recombination (HR). Moreover, double stranded DNA breaks (DSBs) occurring within chromocenters relocate away from the core domain to be repaired. The mechanisms by which pericentric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon DSBs induced specifically in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs in human pericentromeric heterochromatin increase the accessibility of the domain by disrupting HP1 α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of decompaction to be accessed. Our results are consistent with a model in which the 3D organization of the different heterochromatic regions dictates the spatial activation of DNA repair pathways and is key to prevent the activation of HR within clustered repeats and the onset of chromosomal translocations.

Abstract 6269: Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway

Abstract Background: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. Peptide Receptor Radionuclide Therapy (PRRT) is a novel therapy approved for treating NETs with a treatment response rate of 20-30%. To improve therapeutic response of PRRT, 3-AP (Triapine), which is a ribonucleotide reductase (RR) inhibitor with antineoplastic and radiation sensitizing properties, can be used in combination with PRRT. The purpose of our study is to examine effects of Triapine treatment on cellular proliferation, apoptosis and activation of DNA repair pathway in NET cells in vitro. Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) To determine effective dose range of Triapine (500 - 2500nM), proliferation was measured at 48, 72, and 96 h with sulforhodamine B (SRB) assay. (ii) To evaluate the effects of radiosensitivity, cultures were treated with increasing doses (250 - 5,000nM), received 2 Gy radiation (XRT) 1h later, exposed to Triapine for 24 hours, rinsed, and fed Triapine-free media; colonies were counted and quantified with SRB assays 14 days later and survival curves were generated. (iii) To further characterize cellular responses, cells were treated with Triapine (250 - 5,000 nM), irradiated 1h later, and harvested 24 h later to examine expression levels of Cyclin D1, cleaved PARP, p-DNA-PK (S2056), p-ATM (S1981) and p-ATR (S428) by Western blot. Results: (i) SRB proliferation assay demonstrated the effective dose range, determined by half maximal inhibitory concentration (IC-50), was 1,3 µM in BON cells and 1.73 µM in QGP-1 cells at 96 h. (ii) Exposure to Triapine before irradiation resulted in a significant increase in the radiosensitivity of each of the tumor cell lines with dose enhancement factors at a surviving fraction of 0.10 of 1.8 and 1.6 for BON and QGP-1, respectively. (iii) Triapine treatment alone activated DNA repair pathway in a dose-dependent fashion as determined by pDNA-PK (S2056) expression. Increasing p-ATM (S1981) expression was observed in a dose-dependent fashion in QGP-1 cells, but not in BON cells. In addition, p-ATR (S428) expression was not significantly different in either cell line. (iv) Triapine treatment alone or in combination with 2 and 4 Gy XRT resulted in dose-dependent decrease in cyclin D1 expression in both cell lines, however, no significant increase in cleaved PARP expression at 24 and 48h. Conclusion: Our results demonstrated rapid and dose dependent decrease in cyclin D1 expression after Triapine treatment, dose-dependent activation of DNA-PK, the key component of the non-homologous end joining pathway and significant increase in the radiosensitivity of each of the tumor cell lines. In conclusion, our findings provide a rationale for inclusion of Triapine as a radiosensitizer in combination with PRRT in treating NETs. Citation Format: Zeta Chow, Aman Chauhan, Lowell Anthony, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6269.

Abstract IA04: Mutational spectra of environmental carcinogens as biomarkers for early detection of cancer

Abstract In a multicellular organism, somatic mutations represent a permanent record of the biochemical perturbations experienced by a cell in its local microenvironment. Akin to a perpetual recording device, with every replication, genomic DNA accumulates mutations in patterns that reflect: i) the sequence context-dependent formation of DNA damage, due to environmental or endogenous reactive species, including spontaneous processes; ii) the activity of DNA repair pathways, which, depending on the type of lesion, can erase, ignore, or exacerbate the mutagenic consequences of that DNA damage; and iii) the choice of replication machinery that synthesizes the nascent genomic copy. These three factors result in a richly contoured sequence context-dependent mutational spectra, each characteristic of each type of DNA damage. Such mutagenic legacy, if appropriately decoded, can reveal the local history of genome-altering events such as chemical or pathogen exposures, metabolic stress, and inflammation, which in turn can provide an indication of the underlying causes and mechanisms of genetic disease. Modern tools have positioned us to develop a deep mechanistic understanding of the cellular factors and pathways that modulate a mutational process and, in turn, provide opportunities for better diagnostic and prognostic biomarkers, better exposure risk assessment, and even actionable therapeutic targets. One such tool is duplex consensus sequencing (DS), which is a type of highly accurate sequencing that allows the recording of mutational spectra of carcinogens from heterogenous collections of cells. Using DS, we identified the characteristic high-resolution mutational spectrum of the liver carcinogen aflatoxin B1 in a mouse model, 6-9 months before aflatoxin-induced tumors are detectable. In human terms, this timeline corresponds to the detection of a carcinogen exposure, through their mutational spectrum, 1-2 decades before clinical disease. Our DS spectrum is highly similar to the mutational spectra of aflatoxin recorded in cell culture, mouse tumors, and the computationally extracted mutational signature of aflatoxin (signature 24) from human tumors, suggesting that its features reflect fundamental sequence-context dependent biochemical processes of lesion formation, repair avoidance, and misreplication. Importantly, while the exposure to carcinogen, as quantified through its mutational imprint, increases the odds of cancer development, it does not provide direct information on the rate of tumor formation. Gender-specific differences and the presence of tumor-promoting factors are strong modulators of the rate of cancer development under the same carcinogen exposure conditions. Nevertheless, mutational spectra as biomarkers of carcinogen exposure present a significant clinical promise. The identification of at-risk individuals, based on their exposure mutational spectra, could lead to accelerated screening programs and early detection of malignancies at a surgically resectable stage. Citation Format: Bogdan I. Fedeles, Supawadee Chawanthayatham, Amanda L. Armijo, Robert G. Croy, John M Essigmann. Mutational spectra of environmental carcinogens as biomarkers for early detection of cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA04.

Insights into the Polyhexamethylene Biguanide (PHMB) Mechanism of Action on Bacterial Membrane and DNA: A Molecular Dynamics Study.

Polyhexamethylene biguanide (PHMB) is a cationic polymer with antimicrobial and antiviral properties. It has been commonly accepted that the antimicrobial activity is due to the ability of PHMB to perforate the bacterial phospholipid membrane leading ultimately to its death. In this study, we show by the means of atomistic molecular dynamics (MD) simulations that, while the PHMB molecules attach to the surface of the phospholipid bilayer and partially penetrate it, they do not cause any pore formation at least within the microsecond simulation times. The polymers initially adsorb onto the membrane surface via the favorable electrostatic interactions between the phospholipid headgroups and the biguanide groups and then partially penetrate the membrane slightly disrupting its structure. This, however, does not lead to the formation of any pores. The microsecond-scale simulations reveal that it is unlikely for PHMB to spontaneously pass through the phospholipid membrane. Our findings suggest that PHMB translocation across the bilayer may take place through binding to the phospholipids. Once inside the cell, the polymer can effectively "bind" to DNA through extensive interactions with DNA phosphate backbone, which can potentially block the DNA replication process or activate DNA repair pathways.