DNA Repair-deficient Cells Research Articles

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations.

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be converted into mutations that promote carcinogenesis or the development of genetically inherited diseases. As a result, researchers and clinicians require tools that can detect DNA damage and mutations with exceptional sensitivity. In this study, we describe a massively parallel sequencing tool termed Mutation And DNA Damage Detection-seq (MADDD-seq) that is capable of detecting O6-methyl guanine lesions and mutations simultaneously, with a single assay. To illustrate the dual capabilities of MADDD-seq, we treated WT and DNA repair deficient yeast cells with the DNA-damaging agent MNNG and tracked DNA lesions and mutations over a 24-htime period. This approach allowed us to identify thousands of DNA adducts and mutations in a single sequencing run and gain deep insight into the kinetics of DNA repair and mutagenesis.

Exploring DNA repair deficient CHO cell response to low dose rate radiation

IntroductionDNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. Materials and methodsChinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. ResultsNHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DiscussionThese findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.

Multi-scale characterisation of homologous recombination deficiency in breast cancer

BackgroundHomologous recombination is a robust, broadly error-free mechanism of double-strand break repair, and deficiencies lead to PARP inhibitor sensitivity. Patients displaying homologous recombination deficiency can be identified using ‘mutational signatures’. However, these patterns are difficult to reliably infer from exome sequencing. Additionally, as mutational signatures are a historical record of mutagenic processes, this limits their utility in describing the current status of a tumour.MethodsWe apply two methods for characterising homologous recombination deficiency in breast cancer to explore the features and heterogeneity associated with this phenotype. We develop a likelihood-based method which leverages small insertions and deletions for high-confidence classification of homologous recombination deficiency for exome-sequenced breast cancers. We then use multinomial elastic net regression modelling to develop a transcriptional signature of heterogeneous homologous recombination deficiency. This signature is then applied to single-cell RNA-sequenced breast cancer cohorts enabling analysis of homologous recombination deficiency heterogeneity and differential patterns of tumour microenvironment interactivity.ResultsWe demonstrate that the inclusion of indel events, even at low levels, improves homologous recombination deficiency classification. Whilst BRCA-positive homologous recombination deficient samples display strong similarities to those harbouring BRCA1/2 defects, they appear to deviate in microenvironmental features such as hypoxic signalling. We then present a 228-gene transcriptional signature which simultaneously characterises homologous recombination deficiency and BRCA1/2-defect status, and is associated with PARP inhibitor response. Finally, we show that this signature is applicable to single-cell transcriptomics data and predict that these cells present a distinct milieu of interactions with their microenvironment compared to their homologous recombination proficient counterparts, typified by a decreased cancer cell response to TNFα signalling.ConclusionsWe apply multi-scale approaches to characterise homologous recombination deficiency in breast cancer through the development of mutational and transcriptional signatures. We demonstrate how indels can improve homologous recombination deficiency classification in exome-sequenced breast cancers. Additionally, we demonstrate the heterogeneity of homologous recombination deficiency, especially in relation to BRCA1/2-defect status, and show that indications of this feature can be captured at a single-cell level, enabling further investigations into interactions between DNA repair deficient cells and their tumour microenvironment.

ATR Inhibitor Synergizes PARP Inhibitor Cytotoxicity in Homologous Recombination Repair Deficiency TK6 Cell Lines.

The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors (olaparib, talazoparib, or veliparib) and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations.

Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper.

We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening

CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells.

Exploring the molecular and functional cellular response to hydrazine via transcriptomics and DNA repair mutant cell lines.

Hydrazine is a rodent carcinogen and is classified as a probable human carcinogen by IARC. Though hydrazine is positive in both in vitro and in vivo DNA strand break (comet) assays, hydrazine was reported to be negative in an in vitro mutation Muta Mouse lung epithelial cell (FE1) test, as well as in a regulatory-compliant, in vivo Big Blue mouse mutation test. In this article, mechanistic studies explored the cellular response to hydrazine. When tested in a regulatory-compliant mouse lymphoma assay, hydrazine yielded unusual, weakly positive results. This prompted an investigation into the transcriptional response to hydrazine in FE1 cells via RNA sequencing. Amongst the changes identified was a dose-dependent increase in G2/M DNA damage checkpoint activation associated genes. Flow cytometric experiments in FE1 cells revealed that hydrazine exposure led to S-phase cell cycle arrest. Clonogenic assays in a variety of cell lines harboring key DNA repair protein deficiencies indicated that hydrazine could sensitize cells lacking homology dependent repair proteins (Brca2 and Fancg). Lastly, hprt assays with hydrazine were conducted to determine whether a lack of DNA repair could lead to mutagenicity. However, no robust, dose-dependent induction of mutations was noted. The transcriptional and cell cycle response to hydrazine, coupled with functional investigations of DNA repair-deficient cell lines support the inconsistencies noted in the genetic toxicology regulatory battery. In summary, while hydrazine may be genotoxic, transcriptional and functional processes involved in cell cycle regulation and DNA repair appear to play a nuanced role in mediating the mutagenic potential.

Abstract 2075: Systemic small molecule TREX1 inhibitors to selectively activate STING in the TME of metastatic disease

Abstract Genetic evidence from human disease and mouse genetic knock-out studies identify the Stimulator of Interferon Genes (STING) pathway as a critical innate immune sensor for the development of immunity. Tumor cells can evolve to avoid immune recognition through inactivating the STING pathway by diverse mechanisms, indicating that it is important to generating tumor-specific immunity. However, the clinical activity of STING agonists given by intratumoral (IT) administration has not compared well to preclinical studies. The scientific hypothesis supporting these first clinical studies is that localized CD8+ T cell priming would have activity against distal non-injected tumors, but findings that tumors in advanced malignancies have unique antigenic repertoires suggests that this approach may have limited activity against distal tumors. Global innate activation in metastases may therefore be necessary to prime a broadly active CD8+ T cell population targeting diverse antigens, in addition having the benefit of reversing the immune suppressive tumor microenvironment (TME). However, ubiquitous expression of STING presents a significant challenge to achieving a therapeutic index with systemic delivery of direct STING agonists. Selective activation of the STING pathway may be achieved through targeted inhibition of TREX1, a cytosolic DNA exonuclease that modulates cGAS/STING signaling. Expression of TREX1, in contrast to STING, is increased in tumor cells due to elevated levels of cytosolic DNA resulting from genetic instability, DNA repair mutation, inflammatory mediators or DNA-modifying anti-cancer therapies. These observations provide the principal scientific rationale to selectively activate the STING pathway in metastatic disease through targeted inhibition of TREX1. Utilizing published TREX1 X-ray crystal structures to guide medicinal chemistry, we discovered small molecule inhibitors of TREX1 and transformed these molecules from >100 µM leads into a series with drug-like physicochemical properties and picomolar potency against both human and mouse TREX1. We evaluated the activity of lead molecules in cell-based assays, in which TREX1 inhibition enhanced cGAS/STING signaling, and advanced molecules with desired pharmacokinetic profiles to mouse tumor studies. We observed significant anti-tumor activity in mice with CT26 tumors given a combined therapy of low dose doxorubicin to induce dsDNA breaks and increase TME TREX1 expression along with lead series TREX1 molecule inhibitors. Recognizing that TREX1 is a DNA repair enzyme, we also showed that TREX1 inhibitors were cytotoxic in DNA repair deficient human tumor cell lines, informing advancement of this new class of STING therapeutics as a clinical approach to both activate the cGAS/STING pathway to initiate immune recognition, as well as to inhibit DNA repair orthogonal to existing tumor-driver DNA repair mutations. Citation Format: Brian Francica, Dara Burdette, Ryan Clark, Jamie Cope, David Freund, Anja Holtz, Peppi Prasit, Chan Whiting, Thomas W. Dubensky. Systemic small molecule TREX1 inhibitors to selectively activate STING in the TME of metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2075.

Cytotoxicity and genotoxicity of blue LED light and protective effects of AA2G in mammalian cells and associated DNA repair deficient cell lines

Light emitting diode (LED) devices emit narrow bands of the blue, green, and red light spectrum rather than the continuous spectrum emitted from sunlight and fluorescent light bulbs. LED devices have become considerably common in society, and the fluence of blue light from LED devices is more intense than other light sources. Previous studies presented that the blue light spectrum may harness potentially inimical genotoxicity. Therefore, the aim of this study was to investigate this potential cytotoxicity and genotoxicity, as well as identify the mechanism of the cellular effects induced by blue LED light exposure in mammalian cell lines with their DNA repair deficient mutants. Our results demonstrated that blue LED light induced both oxidative stress to cells and cytotoxic and genotoxic effects including reduction of clonogenicity, cell cycle arrest, induction of sister chromatid exchanges, endoreduplicated chromosomes, and increased frequency of HPRT locus mutations. In DNA repair deficient cells, particularly those involving double strand break repair deficiency, cells presented hypersensitivity to blue LED light exposure. Blue LED light also induced chromosome aberrations more in DNA repair deficient cells than wild type cells. The cytotoxicity of blue LED light was reduced by an effective antioxidant, ascorbic acid 2-glucoside, which can suppress blue LED light induced oxidative stress. These results indicated that prolonged, high intensity exposure to blue LED light induces genotoxic stress to cells, and oxidative stress induced by blue LED light is targeting DNA to induce these biological effects.

Abstract ND05: Discovery and first structural disclosure of AZD5305: A next generation, highly selective PARP1 inhibitor and trapper

Abstract Poly ADP-ribose polymerase (PARP) inhibitors have demonstrated clinical effectiveness in the treatment of homologous recombination deficient tumors; however, hematological and other toxicities have limited their use as a class of agents, both as monotherapy and as combination with already poorly tolerated chemotherapies. Recent literature suggests that inhibition of PARP2, a feature shared by all the current clinical PARP inhibitors, is not required for efficacy and may in fact be associated with hematological effects. These observations led to our hypothesis that a highly selective and potent PARP1 inhibitor and trapper would retain efficacy while improving therapeutic index, thus expanding clinical combinations options. A high throughput screen against PARP1 and PARP2 identified a compound series with selectivity for binding to PARP1 over PARP2. This set of compounds was refined further by screening for the ability to trap PARP1 to DNA and selectively inhibit growth of BRCA mutant cell lines. Further lead optimization efforts utilizing PARP1 enzyme structural information, lead to the discovery of to AZD5305. AZD5305 is a highly potent inhibitor of PARP1, with significant PARP1-DNA trapping activity, no PARP2-activity, nor binding activity to any other members of the PARP family. AZD5305 has excellent secondary pharmacology and physicochemical properties as well as high oral bioavailability in preclinical species. AZD5305 has shown unique properties in preclinical toxicology models. When dosed daily for 14 days in rats at clinically equivalent doses, olaparib (a PARP1/2 inhibitor), causes up to 50% reduction in hemoglobin, in line with clinically observed anemia. Under the same experimental conditions, AZD5305 had no effects on any hematological parameters measured, when dosed at predicted clinically efficacious doses. In vitro, AZD5305 inhibits growth selectively in cell lines with deficiencies in DNA repair pathways, with IC50s in the single-digit nM range, while having no or minimal growth inhibitory effects in other cells (IC50s >10μM). The difference observed between the effects on DNA repair deficient and proficient cell lines is unprecedented with PARP inhibitors and indicates the great potential for AZD5305 to demonstrate an improved therapeutic index in the clinic. Treatments of xenografts and PDX models with AZD5305 at doses ≥ 0.1 mg/kg QD, compared to olaparib 100 mg/kg QD, resulted in greater depth of tumor regressions, as well as significantly longer duration of regressions following cessation of dosing. Overall, AZD5305 is a next generation PARP inhibitor and trapper, with enhanced anticancer efficacy and an improved safety profile in preclinical models compared to other PARP inhibitors. These features make AZD5305 an exciting and unique clinical candidate, with multiple clinical options as monotherapy and in combination. AZD5305 is in Ph1 clinical trials. Citation Format: Elisabetta Leo, Jeffrey Johannes. Discovery and first structural disclosure of AZD5305: A next generation, highly selective PARP1 inhibitor and trapper [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 ND05.

Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2'-Deoxyuridine and 5-Bromo-2'-Deoxyurdine to Mammalian Cells.

BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU‘s effects have not been extensively studied yet. Therefore, we investigated EdU’s potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were observed in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concentrations (similar to manufacturers suggested concentration; >5–10 μM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with additional precautions.

Abstract P3-11-18: Partial RALBP1 loss depletes DNA repair pathway genes

Abstract TP53 (p53) functions as a stress-responsive, genome protective, tumor suppressor whose functions are lost or altered a majority of breast cancers. TP53 homozygous null mice uniformly die of spontaneous malignancy. The human RALBP1 gene (18p11.22) encodes is a stress-protective, anti-apoptotic mercapturic acid pathway transporter protein (RLIP76 or Rlip) regulated by the Ral/Rac/Rho pathways. In stark contrast to TP53 null mice, RALBP1-null mice are highly cancer resistant, even to the most potent known chemical carcinogens benzo[a]pyrene or dimethylbenzanthracene. We recently reported that pharmacologically or chemically induced haploinsufficiency of Rlip switches off spontaneous carcinogenesis in p53-null mice (Proc. Natl. Acad Sci USA, 2018; 115:3918), indicating an existential role of Rlip in p53-deficient cancer cells and suggesting the possibility that DNA-repair deficient cells could be particularly susceptible to Rlip depletion. Results of present studies demonstrated that RALBP1 depletion by antisense inhibited the growth of the MDA-MB231 cell line in-vitro and in xenografts. Remarkably, Rlip depletion with antisense or upon treatment with an orange-derived flavanone (2-hydroxyflavanone) depleted the expression of PALB2 as well as BRCA1 and BRCA2 in triple negative breast cancer cell lines, and synergistically increased the efficacy of PARP inhibition. These findings suggest that drugs that inhibit or deplete Rlip could be useful in sensitizing DNA-repair sufficient triple negative breast cancer to PARP inhibitors. Citation Format: Sanjay Awasthi, Chhanda Bose, Catherine Jones, Rakhshanda Rahman, Sharad S. Singhal, Sharda P. Singh. Partial RALBP1 loss depletes DNA repair pathway genes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-18.

WIP1 Contributes to the Adaptation of Fanconi Anemia Cells to DNA Damage as Determined by the Regulatory Network of the Fanconi Anemia and Checkpoint Recovery Pathways.

DNA damage adaptation (DDA) allows the division of cells with unrepaired DNA damage. DNA repair deficient cells might take advantage of DDA to survive. The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs), and deficiencies in this pathway cause a fraction of breast and ovarian cancers as well as FA, a chromosome instability syndrome characterized by bone marrow failure and cancer predisposition. FA cells are hypersensitive to ICLs; however, DDA might promote their survival. We present the FA-CHKREC Boolean Network Model, which explores how FA cells might use DDA. The model integrates the FA pathway with the G2 checkpoint and the checkpoint recovery (CHKREC) processes. The G2 checkpoint mediates cell-cycle arrest (CCA) and the CHKREC activates cell-cycle progression (CCP) after resolution of DNA damage. Analysis of the FA-CHKREC network indicates that CHKREC drives DDA in FA cells, ignoring the presence of unrepaired DNA damage and allowing their division. Experimental inhibition of WIP1, a CHKREC component, in FA lymphoblast and cancer cell lines prevented division of FA cells, in agreement with the prediction of the model.

Editor's Note: Prevention of Ultraviolet Radiation-Induced Immunosuppression by (-)-Epigallocatechin-3-Gallate in Mice Is Mediated through Interleukin 12-Dependent DNA Repair.

Purpose: Solar UV radiation–induced immunosuppression is considered to be a risk factor for melanoma and nonmelanoma skin cancers. We previously have shown that topical application of (−)-epigallocatechin-3-gallate (EGCG) prevents UV-induced immunosuppression in mice. We studied whether prevention of UV-induced immunosuppression by EGCG is mediated through interleukin 12 (IL-12)–dependent DNA repair. Experimental Design: IL-12 knockout (KO) mice on C3H/HeN background and DNA repair–deficient cells from xeroderma pigmentosum complementation group A ( XPA ) patients were used in this study. The effect of EGCG was determined on UV-induced suppression of contact hypersensitivity and UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in mice and XPA -deficient cells using immunohistochemistry and dot-blot analysis. Results: Topical treatment with EGCG prevented UV-induced suppression of the contact hypersensitivity in wild-type (WT) mice but did not prevent it in IL-12 KO mice. Injection of anti-IL-12 monoclonal antibody to WT mice blocked the preventive effect of EGCG on UV-induced immunosuppression. EGCG reduced or repaired UV-induced DNA damage in skin faster in WT mice as shown by reduced number of CPDs + cells and reduced the migration of CPD + antigen-presenting cells from the skin to draining lymph nodes. In contrast, this effect of EGCG was not seen in IL-12 KO mice. Further, EGCG was able to repair UV-induced CPDs in XPA -proficient cells obtained from healthy person but did not repair in XPA -deficient cells, indicating that nucleotide excision repair mechanism is involved in DNA repair. Conclusions: These data identify a new mechanism by which EGCG prevents UV-induced immunosuppression, and this may contribute to the chemopreventive activity of EGCG in prevention of photocarcinogenesis.

Abstract B049: DNA damage echoes are key to prostate cancer susceptibility to supraphysiologic androgen levels

Abstract Purpose: Prostate cancer cells are dependent on AR for growth and survival, but recent reports have shown a paradoxical role of supraphysiologic androgen in growth inhibition, known as hormone interference. Androgen stimulation in AR-overexpressing prostate cancer cells leads to formation of double-strand breaks (DSBs). Here, we investigate the potential mechanism of high androgen induced DSBs and growth inhibition and its effect on survival following androgen stimulation in combination with DNA damage repair inhibitors or defects. Experimental Design: AR-dependent LNCaP, AR overexpressing LNCaP and PC3, AR-independent PC3, and APIPC were used for this study. High levels of androgen (R1881)/ DHT and olaparib-induced DNA damage were evaluated by gH2AX and 53BP1 foci formation after 6 or 24h. For cell death assays the drug was administered continuously for 3 days following the plating day. DNA PKcs activity was determined by Ser2056foci formation and dissociation from chromatin was measured by Thr2609 foci. AR activity or DNA binding was evaluated by Ser81 AR phosphorylation. An ex vivo PDX model (LuCaP 96CR) was utilized to mimic the BRCA2 defect condition. Results: We demonstrated that supraphysiologic androgen treatment on androgen-dependent and AR-overexpressing prostate cancer cell lines resulted in a dose-dependent induction of widespread DSBs, which was augmented after PARP inhibition (olaparib). AR expression correlated with persistent DNA damage as AR-independent cells lacked DSBs. High androgen treatment significantly induced caspase activity and reduced survival in AR-overexpressing cells. Hyperactivated DNA PKcs was retained on chromatin following high androgen exposure in these cells, leading to impaired dissociation of DNA PKcs from chromatin and inducing DNA damage. AR phosphorylation at Ser81 was also in concordance with hyperphosphorylated DNA PKcs, particularly in AR-overexpressing cells following high androgen and olaparib treatment. HR-deficient, BRCA2-null cells induced more DSBs after high androgen treatment with or without olaparib and were susceptible to DNA PKcs inhibition following androgen treatment. In contrast to this, AR-overexpressing cells did not exhibit any DNA damage following DNA PKcs inhibition after high androgen exposure. Ex vivo PDX models demonstrated a synergistic interaction between olaparib and high levels of androgen-induced DSB formation as well as hyperactive DNA PKcs. Conclusions: These results suggest a potential mechanism of supraphysiologic androgen stimulated AR-induced DNA damage and proliferation inhibition. AR binds to DNA PKcs following high androgen and until AR levels become saturated DNA PKcs and AR stay on the chromatin, which leads to inefficient DNA damage repair. HR-deficient cells were particularly susceptible to high androgen. Our data provide a mechanistic rationale for the response of AR upregulated cells as well as DNA repair deficient cells to supraphysiologic androgen therapy in combination with PARP inhibition. Citation Format: Payel Chatterjee, Jared M. Lucas, Michael D. Nyquist, Eva Corey, Peter S. Nelson. DNA damage echoes are key to prostate cancer susceptibility to supraphysiologic androgen levels [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B049.

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1−/− mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1−/− cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1−/− cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

25IN - The relationship between DNA damage and immune checkpoint activation in cancer

Background: In cancer the presence of T cell immune infiltration has been recognised as a prognostic factor, however the mechanisms underpinning this response are not clearly defined. Our group and others have identified a relationship between type I Interferon immune signalling and loss of DNA repair, particularly the Fanconi Anemia pathway, in several different types of cancer. We therefore investigated the mechanism activating this immune response in the context of abnormal DNA repair. Methods: Preclinical isogenic cell line systems and human tumours were used to identify the relationship between loss of function of DNA repair genes and activation of immune signalling. A panel of Chemotherapeutic agents were studied for their effect on immune activation. Results: IHC analysis demonstrated that both intra-tumoral and stromal CD8+ and CD4+ T cell infiltration were associated with DNA repair deficient breast tumors. The CXCL10 and CCL5 cytokines as well as the immune checkpoint target PD-L1 were shown to be significantly up-regulated in DNA repair deficient tumours and in tissue culture models when compared to DNA proficent tumours. Furthermore, conditioned media from DNA repair deficient cell lines stimulated inward migration of peripheral blood mononuclear cells, when compared to media from proficient cells, indicating the presence of active cytokines. We identified constitutive activation of the innate immune pathway STING/TBK1/IRF3 specifically in DNA repair deficient tumour cells when compared to proficient cells and found that binding of the DNA sensor cGAS to cytosolic DNA fragments was required for this immune response. In addition, we identified several chemotherapeutic agents that were able to activate the immune response in DNA repair proficient cell lines through DNA damage, the development of cytoplasmic DNA fragments and the consequent activation of the STING/TBK1/IRF3 immune pathway. Conclusions: We have identified that the STING/TBK1/IRF3 immune pathway is constitutively activated by cytoplasmic DNA in DNA repair deficient cancers and may explain lymphocytic infiltration and the response to immune checkpoint based therapy. Some conventional chemotherapy agents such as doxorubicin are able to active this pathway in DNA repair proficient cancers and may represent a logical combination with immune checkpoint targeted drugs in clinical trials. Legal entity responsible for the study: Queen's University of Belfast Funding: McClay Foundation Disclosure: R. Kennedy: I receive payment as the Global VP and Medical Director for Almac Diagnostics.

Investigation of the relative biological effectiveness and uniform isobiological killing effects of irradiation with a clinical carbon SOBP beam on DNA repair deficient CHO cells.

Spread-out Bragg peak (SOBP) C ions have been used clinically in charged particle radiation therapy for years. An SOBP beam consists of various monoenergetic Bragg peaks; however, the biological effect of irradiation with an SOBP beam track has not been well-studied. In order to determine the clinically prospective molecular targets, radiosensitivity to the beam track in DNA repair deficient cell lines was investigated. A total of four distinct Chinese hamster ovary (CHO) cell lines, including CHO10B2 (wild-type), V3 (protein kinase DNA-activated catalytic polypeptide deficient), 51D1 (RAD51 paralog D deficient) and PADR9 [poly(ADP-ribose) polymerase (PARP) deficient], were irradiated with gamma-rays, C ions (290 MeV/n) and Fe ions (500 MeV/n), in order to compare cellular lethality. An OptiCell™ culture system was used to evaluate the lethality at distinct depths of SOBP C ions. Relative biological effectiveness (RBE) values of C ions (linear energy transfer (LET), 13 and 70 keV/µm) and Fe ions (LET, 200 keV/µm) were calculated from cell survival using colony formation assay with standard cell dishes. All cell lines exhibited similar sensitivity to 70 keV/µm C ions and 200 keV/µm Fe ions. Furthermore, V3 cells did not exhibit increased sensitivity to high LET C ions and Fe ions, compared with low LET gamma-rays and C ions, and 51D1 cells irradiated with 13 keV/µm C ions exhibited relatively high RBE values among the tested cell lines. Conversely, PADR9 cells exhibited low RBE values for 13 keV/µm C ions and high RBE values for 70 keV/µm C ions. Obtained using the OptiCell system, the survival fractions in the SOBP region were uniform for wild-type and PADR9 cells. Conversely, V3 and 51D1 cells exhibited decreased cell death in the distal region of the SOBP. These results indicated that PARP is a more effective target for clinical beam therapy, compared with the non-homologous end joining repair and homologous recombination repair pathways. PARP deficiency may be an optimal target for C ion therapy and the results of the present study may contribute to the development of a more effective heavy ion radiation therapy.

Multiple repair pathways mediate cellular tolerance to resveratrol-induced DNA damage

Resveratrol (RSV) has been reported to exert health benefits for the prevention and treatment of many diseases, including cancer. The anticancer mechanisms of RSV seem to be complex and may be associated with genotoxic potential. To better understand the genotoxic mechanisms, we used wild-type (WT) and a panel of isogenic DNA-repair deficient DT40 cell lines to identify the DNA damage effects and molecular mechanisms of cellular tolerance to RSV. Our results showed that RSV induced significant formation of γ-H2AX foci and chromosome aberrations (CAs) in WT cells, suggesting direct DNA damage effects. Comparing the survival of WT with isogenic DNA-repair deficient DT40 cell lines demonstrated that single strand break repair (SSBR) deficient cell lines of Parp1−/−, base excision repair (BER) deficient cell lines of Polβ−/−, homologous recombination (HR) mutants of Brca1−/− and Brca2−/− and translesion DNA synthesis (TLS) mutants of Rev3−/− and Rad18−/− were more sensitive to RSV. The sensitivities of cells were associated with enhanced DNA damage comparing the accumulation of γ-H2AX foci and number of CAs of isogenic DNA-repair deficient DT40 cell lines with WT cells. These results clearly demonstrated that RSV-induced DNA damage in DT40 cells, and multiple repair pathways including BER, SSBR, HR and TLS, play critical roles in response to RSV- induced genotoxicity.