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PDF file - 322K, Supplemental Figure 1. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancer whole cohort (A), Lymph node negative (B) and lymphnode positive (C). Supplemental Figure 2. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancerlow risk, no adjuvant therapy (A), high risk, received endocrine therapy (B) and high risk, received CMF chemotherapy (C). Supplemental Figure 3. Disease free survival (DFS) and distant metastasis free survival (DMFS) in breast cancerER negative tumours (A), Triple negative tumours (B). Supplemental Figure 4. Clonogenic survival assay (A), Annexin-V FITC assay (B).

PDF file - 104K, Supplemental Table S1: Clinicopathological characteristics of whole breast cancer cohort Supplemental Table S2: Clinicopathological characteristics of validation setSupplementary Table S3: Antigens, primary antibodies, clone, source, optimal dilution and scoring system used for each immunohistochemical marker Supplementary Table S4: siRNA constructs for XRCC1 knockdown

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n = 1,297) and validated in an independent cohort of estrogen receptor (ER)-α–negative breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia–mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G2–M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5); 1621–34. ©2012 AACR.

The poly(ADP-ribose) polymerase (PARP) family of proteins has been implicated in numerous cellular processes, including DNA repair, translation, transcription, telomere maintenance, and chromatin remodeling. Best characterized is PARP1, which plays a central role in the repair of single strand DNA damage, thus prompting the development of small molecule PARP inhibitors (PARPi) with the intent of potentiating the genotoxic effects of DNA damaging agents such as chemo- and radiotherapy. However, preclinical studies rapidly uncovered tumor-specific cytotoxicity of PARPi in a subset of cancers carrying mutations in the

Highlights • PARP1-dependent PARylation post-translationally modifies and regulates STAT5. • Catalytic PARP inhibition reduces STAT5 stability. • PARP1 loss results in reduced STAT5 signaling and activation of downstream targets. • STAT5-activated cancers are sensitive to PARP inhibition. • PARP inhibition overcomes TKI-resistance in FLT3-ITD AML., Signal transducer and activator of transcription 5 (STAT5) signaling plays a pathogenic role in both hematologic malignancies and solid tumors. In acute myeloid leukemia (AML), internal tandem duplications of fms-like tyrosine kinase 3 (FLT3-ITD) constitutively activate the FLT3 receptor, producing aberrant STAT5 signaling, driving cell survival and proliferation. Understanding STAT5 regulation may aid development of new treatment strategies in STAT5-activated cancers including FLT3-ITD AML. Poly ADP-ribose polymerase (PARP1), upregulated in FLT3-ITD AML, is primarily known as a DNA repair factor, but also regulates a diverse range of proteins through PARylation. Analysis of STAT5 protein sequence revealed putative PARylation sites and we demonstrate a novel PARP1 interaction and direct PARylation of STAT5 in FLT3-ITD AML. Moreover, PARP1 depletion and PARylation inhibition decreased STAT5 protein expression and activity via increased degradation, suggesting that PARP1 PARylation of STAT5 at least in part potentiates aberrant signaling by stabilizing STAT5 protein in FLT3-ITD AML. Importantly for translational significance, PARPis are cytotoxic in numerous STAT5-activated cancer cells and are synergistic with tyrosine kinase inhibitors (TKI) in both TKI-sensitive and TKI-resistant FLT3-ITD AML. Therefore, PARPi may have therapeutic benefit in STAT5-activated and therapy-resistant leukemias and solid tumors.

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.

DNA methyltransferase inhibitors (DNMTis) modulate genome-wide methylation patterns, previously linked by our group to downregulation of DNA double strand break repair (DSBR) that sensitizes non-small cell lung cancer (NSCLC) to poly(ADP-ribose) polymerase inhibitors (PARPis). DNMTi hypomethylation of endogenous retroviral (ERV) elements produces cytosolic aggregation of double-stranded RNA (dsRNA) that activates antiviral interferon (IFN) and inflammasome signaling. Accordingly, DNMTi treatment of NSCLC cell lines (A549, H460) increases expression of 1) multiple ERV transcripts; 2) dsRNA sensors RIG-I and MDA5; 3) downstream mitochondrial antiviral signaling protein (MAVS); 4) MAVS-activated antiviral kinases (TBK1, IKKα/β) and transcriptional regulators (IRF3/7, NFκB); and 5) IFNβ and IFN-stimulated genes (ISGs). A second mechanism of cellular antiviral signaling is STING (stimulator of interferon genes) activation triggered by cytosolic dsDNA. Following DNMTi treatment, we observe mitochondrial dysfunction including impairment of oxidative phosphorylation, reactive oxygen species generation, and reduced mitochondrial membrane potential. This dysfunction is associated with increased mitochondrial DNA (mtDNA) damage and accumulation of cytosolic mtDNA, leading to increased STING expression and IFN activation that can be abrogated by siSTING or IKK inhibition. Treatment with PARPi, which activates STING via cytosolic accumulation of damaged nuclear DNA, further enhances DNMTi-induced IFN signaling. We previously linked IFN activation to DSBR downregulation in breast and ovarian cancer, and now present TCGA analysis in NSCLC indicating a similar negative correlation, corroborated by reduced DSBR in NSCLC cell lines after IFNβ treatment. Computational interrogation indicates multiple DSBR promoters contain IFN-stimulated response elements (ISREs), including potential negative regulators, that may account for these findings. Notably, we find that precise patterns of immune activation may be TP53 dependent: NSCLC cell lines with missense mutations (H23, H441, H1115), as observed in ~30% patients, exhibit increased NFκB inflammasome signaling and decreased IFN activation compared to wildtype (A549, H460). TP53 mutation is associated with significantly reduced baseline STING expression, potentially affording a more robust DNMTi treatment response; accordingly, TP53-mutant NSCLC cells exhibit significantly greater sensitivity to DNMTi/PARPi. Our new data expands on our published work to further understanding of novel mechanisms of 1) inflammasome induction and 2) mitochondrial dysfunction through which DNMTis activate cellular antiviral signaling and downregulate DSBR to induce PARPi sensitivity in NSCLC. We also identify TP53 as a possible biomarker for future personalization of DNMTi therapy. Citation Format: Rachel Abbotts, Lora Stojanovic, Michael J. Topper, Brian M. Polster, Stephen B. Baylin, Feyruz V. Rassool. DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity [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 5593.

The genetic material of all organisms is susceptible to modification. In some instances, these changes are programmed, such as the formation of DNA double strand breaks during meiotic recombination to generate gamete variety or class switch recombination to create antibody diversity. However, in most cases, genomic damage is potentially harmful to the health of the organism, contributing to disease and aging by promoting deleterious cellular outcomes. A proportion of DNA modifications are caused by exogenous agents, both physical (namely ultraviolet sunlight and ionizing radiation) and chemical (such as benzopyrene, alkylating agents, platinum compounds and psoralens), which can produce numerous forms of DNA damage, including a range of "simple" and helix-distorting base lesions, abasic sites, crosslinks and various types of phosphodiester strand breaks. More significant in terms of frequency are endogenous mechanisms of modification, which include hydrolytic disintegration of DNA chemical bonds, attack by reactive oxygen species and other byproducts of normal cellular metabolism, or incomplete or necessary enzymatic reactions (such as topoisomerases or repair nucleases). Both exogenous and endogenous mechanisms are associated with a high risk of single strand breakage, either produced directly or generated as intermediates of DNA repair. This review will focus upon the creation, consequences and resolution of single strand breaks, with a particular focus on two major coordinating repair proteins: poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1).

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

The human genome is under constant stress from thousands of DNA damage lesions that are formed as indirect products of endogenous metabolic or enzymatic processes, or generated from exposure to exogenous environmental physical and chemical agents. A number of highly conserved DNA repair pathways have evolved to identify and resolve these lesions, preventing a collection of potentially deleterious consequences. At the molecular level, DNA damage can arrest RNA transcription or DNA replication processes, or can lead to mutagenic by-pass or gross chromosomal rearrangement events. At the cellular level, such outcomes can result in apoptosis, senescence, or transformation, consequences known to underlie disease and aging. Consistently, germline and somatic mutations at DNA repair gene loci, as well as epigenetic inactivation or loss of heterozygosity, have been linked to an array of cancer diagnoses, including familial cancer syndromes, polymorphism-associated predispositions, and sporadic malignancies, as well as to neurological disease and premature aging features. DNA damage also plays an important role in the cytotoxicity of numerous cancer treatments, namely those involving radiotherapy or commonly used chemotherapeutic drugs. More recently, molecular targets within DNA repair pathways have been the focus of drug development efforts, both as possible potentiators of existing anticancer agents, and as targeted monotherapies within a synthetic lethality paradigm.

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n = 1,297) and validated in an independent cohort of estrogen receptor (ER)-α–negative breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia–mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G2–M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5); 1621–34. ©2012 AACR.

Advanced cancer is a leading cause of death in the developed world.[1][1] Chemotherapy and ionising radiation are the two main treatment modalities currently available to improve outcomes in patients with disseminated malignancy. The cytotoxicity of many of these agents is directly related to their

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant (V-C8(Rev1)). b) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested synthetic lethality (SL) in CH ovary cells expressing a dominant–negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. Synthetic lethality was also demonstrated in CH cells expressing a dominant–negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising synthetic lethality target in cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the US. Poly (ADP-ribose) polymerase inhibitors (PARPi) show efficacy as radiosensitizers in NSCLC, but arguably have gained greater importance in cancer therapeutics by exhibiting synthetic lethality (SL) in tumors with defects in the homologous recombination (HR) pathway of double strand break repair (DSBR). Mechanistically, PARPi cytotoxicity is partially mediated through trapping of PARP1 at sites of DNA damage, with next generation PARPis such as talazoparib (Tal) being potent PARP trappers. Our group has previously demonstrated that combining Tal with a DNA methyltransferase inhibitor (DNMTi) enhances PARPi cytotoxicity in acute myeloid leukaemia and breast cancer models. DNMTis are cytosine analogs that become incorporated into replicating DNA, where they covalently bind their target enzyme DNMT1. When combined with PARPi, DNMTis enhance PARP trapping at damage sites, which collapse into cytotoxic double strand breaks (DSB) when encountered by a replication fork. In the current study, we demonstrate synergistic cytotoxicity of Tal in combination with the DNMTi 5-azacytidine (Aza) in NSCLC cell lines by combination index analyses and colony forming assays. Further potentiation of Tal+Aza cytotoxicity is observed following induction of DNA damage via IR. These results were substantiated in an A549 xenograft model (n=8 per group) where Tal+Aza+IR significantly reduced tumor volume and increased survival compared to vehicle (p Citation Format: Rachel Abbotts, Michael Topper, Daniel Fontaine, Christopher Biondi, Eun Yong Choi, Rena Lapidus, Stephen Baylin, Feyruz Rassool. DNA methyltransferase inhibitors sensitize NSCLC cells to PARP inhibitors by induction of a double strand break repair defect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2823.

FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p = 4.89 × 10(-57)), high mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)), PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)), integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5 (p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p = 9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps < 0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps < 0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps < 0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps < 0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer.

Chronic inflammation is a driving force for gastric carcinogenesis. Reactive oxygen species (ROS) generated during the inflammatory process generates DNA damage that is processed through the DNA repair pathways. In this study, we profiled key DNA repair proteins (single-strand-selective monofunctional uracil-DNA glycosylase 1 [SMUG1], Flap endonuclease 1 [FEN1], X-ray repair cross-complementing gene 1 [XRCC1], and Ataxia telangiectasia mutated [ATM]) involved in ROS-induced oxidative DNA damage repair in gastric cancer and correlated to clinicopathological outcomes. High expression of SMUG1, FEN1, and XRCC1 correlated to high T-stage (T3/T4) (p-values: 0.001, 0.005, and 0.02, respectively). High expression of XRCC1 and FEN1 also correlated to lymph node-positive disease (p-values: 0.009 and 0.02, respectively). High expression of XRCC1, FEN1, and SMUG1 correlated with poor disease-specific survival (DSS) (p-values: 0.001, 0.006, and 0.05, respectively) and poor disease-free survival (DFS) (p-values: 0.001, 0.001, and 0.02, respectively). Low expression of ATM correlated to lymph node positivity (p=0.03), vascular invasion (p=0.05), and perineural invasion (p=0.005) and poor DFS (p=0.001) and poor DSS (p=0.003). In the multivariate Cox model, high XRCC1 and low ATM were independently associated with poor survival (p=0.008 and 0.011, respectively). Our observation supports the hypothesis that DNA repair factors are promising biomarkers for personalized therapy in gastric cancer. Antioxid. Redox Signal. 18, 2392–2398.

X-ray repair cross-complementing gene 1 (XRCC1) is essential for DNA base excision repair, single strand break repair and nucleotide excision repair. We investigated clinicopathological and functional significance of XRCC1 expression in ovarian cancers. XRCC1 protein expression was evaluated in 195 consecutive human ovarian cancers and correlated with clinicopathological variables and survival outcomes. Functional preclinical studies were conducted in a panel of XRCC1 deficient and proficient Chinese hamster and Human cancer cells for cisplatin chemosensitivity. Clonogenic assay, neutral COMET assay, γH2AX immunocytochemistry and flow cytometric analyses were performed in cells. In ovarian cancer, 48% of the tumors were positive for XRCC1 expression and significantly associated with higher stage (p = 0.006), serous type tumors (p = 0.008), suboptimal de-bulking (p = 0.004) and platinum resistance (p < 0.0001). Positive XRCC1 had twofold increase of risk of death (p = 0.007) and progression (p < 0.0001). In the multivariate Cox model, XRCC1 expression was independently associated with cancer specific [p = 0.038] and progression free survival [p = 0.003]. Preclinically, XRCC1 negative cells were sensitive to cisplatin compared to XRCC1 positive cells. Sensitivity to cisplatin in XRCC1 negative cells was associated with accumulation of DNA double strand breaks and G2/M cell cycle arrest. XRCC1 expression is associated with adverse clinicopathological and survival outcomes in patients. Preclinical data provides mechanistic functional evidence for cisplatin sensitivity in XRCC1 negative cells. XRCC1 is a promising predictive biomarker in ovarian cancer.

Genomic DNA possesses an inherent instability, at risk from damage by spontaneous base lesions, metabolic by-products, and exogenous sources such as ultraviolet light, ionising radiation and chemical agents. Unrepaired, this damage could result in non-canonical base pairing during replication, leading to the propagation of potentially mutagenic lesions. A number of DNA repair mechanisms have evolved to ensure genomic integrity can be preserved. So critical are these repair pathways that mutations within constituent genes are associated with several cancer predisposition syndromes such as hereditary non-polyposis carcinoma coli (HNPCC) or BRCA-deficient breast and ovarian cancer syndromes (Sweasy, Lang, and DiMaio 2006). Cancer therapies commonly rely upon the induction of DNA damage to exert their effects. Upregulation of DNA repair pathways in cancer is common and may impact upon response to therapy and contribute to development of treatment resistance. Inhibition of DNA repair offers exciting possibilities for the future treatment of cancer. DNA repair constituents may also be used as biomarkers to predict tumour response to treatment and improve outcome prognostication. Pharmacological inhibition of DNA repair might potentiate the effects of anticancer agents, improving response rates, overcoming resistance, and improving outcomes. Furthermore, there may be scope to specifically target tumour cells using DNA repair inhibitors by exploiting genetic differences with normal tissue. Base excision repair (BER) is critical for the repair of damage induced by alkylating chemotherapy agents such as temozolomide and dacarbazine. Targeting BER has shown considerable promise in the form of poly (ADP-ribose) polymerase (PARP) inhibitors, and a number of groups are now focusing on other BER targets. This chapter will provide an overview of the BER pathway, with specific consideration of the compelling evidence base for targeting the critical enzyme apurinic/apyrimidinic endonuclease I (APE1) for cancer therapy.

Aims: Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. Methods: An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. Results: Several specific APE1 inhibitors were isolated by this approach. The IC50 for APE1 inhibition ranged between 30 n and 50 μ. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. Conclusions: Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

The cytotoxicity of both chemotherapy and radiotherapy is to a large extent directly related to their ability to induce DNA damage. The ability of cancer cells to recognise and repair this damage contributes to therapeutic resistance. Sub-optimal DNA repair in normal tissue may impair normal tissue tolerance. Inter-individual differences in DNA repair pathways may also influence the natural history and progression of cancer and hence prognosis. The base excision repair (BER) pathway has evolved to repair base damage induced by endogenous and exogenous base targeting agents. Polymorphic variants of genes, mRNA expression and alterations in protein expression within BER, may alter DNA repair capacity and influence both cancer progression and clinical responses to chemotherapy and radiotherapy. We discuss the role of BER genes as potential predictive and prognostic markers in human cancer and review the current state of play within this field.

Background: Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. Methods: Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression of APE1 was analysed by IHC and correlated to clinicopathological variables. Results: In ovarian cancer, nuclear APE1 expression was seen in 71.9% (97 out of 135) of tumours and correlated with tumour type (P=0.006), optimal debulking (P=0.009), and overall survival (P=0.05). In gastro-oesophageal cancers previously exposed to neoadjuvant chemotherapy, 34.8% (16 out of 46) of tumours were positive in the nucleus and this correlated with shorter overall survival (P=0.005), whereas cytoplasmic localisation correlated with tumour dedifferentiation (P=0.034). In pancreatico-biliary cancer, nuclear staining was seen in 44% (32 out of 72) of tumours. Absence of cytoplasmic staining was associated with perineural invasion (P=0.007), vascular invasion (P=0.05), and poorly differentiated tumours (P=0.068). A trend was noticed with advanced stage (P=0.077). Conclusions: Positive clinicopathological correlations of APE1 expression suggest that APE1 is a potential drug target in ovarian, gastro-oesophageal, and pancreatico-biliary cancers.

Background: The role of DNA repair in estrogen receptor (ER) positive breast cancer is largely unknown. We hypothesized that DNA repair dysregulation could lead to evolution of aggressive phenotypes with increasing resistance to endocrine therapy. Methods: We immuno-profiled 1120 early stage ER positive breast tumours for Pol b, FEN1, APE1, XRCC1, SMUG1, PARP1, ATR, ATM, DNA-PK, BRCA1, CHK1, CHK2, TOPO2, and p53. Multivariate analysis was used to calculate a DNA repair prognostic index after controlling for other validated prognostic factors, confounders and adjuvant therapy. For additional validation, genomic expression as well as array CGH evaluation of key DNA repair genes was performed [discovery cohort (n = 128), external validation in Uppsala (n = 249) and METABRIC cohort (n = 1950)]. Neural network analysis was conducted in 249 tumours to map genetic interactions. Results: DNA repair immuno-profiling stratified ER positive breast tumours into four distinct sub-groups with increasing aggressiveness and endocrine resistance (p = 1.6 × 10 −27). Subgroup 1 had the best breast cancer specific survival, subgroups 2 & 3 have intermediate breast cancer specific survival and sub-group 4 had the worst breast cancer specific survival (Table). Survival analysis HR (95% CI)P ValuesSub-group 2 versus. Sub-group 11.6 (1.1-2.1)0.006Sub-group 3 versus. Sub-group 13.1 (2.3-4.3)1.5 × 10-12Sub-group 4 versus. Sub-group 15.7 (4.0-8.1)9.0 × 10−21 These distinct subgroups manifest progressively increasing tumour size, level of proliferation, de-differentiation, lymphovascular invasion, metastasis, altered apoptosis and increasing level of genomic instability (ps Conclusion: This is the first and largest study with 20 years long term follow-up data to provide compelling evidence that DNA repair dysregulation impacts ER positive breast cancer pathogenesis and drives evolution of aggressive phenotypes. Our data could transform stratification and the personalization landscape for ER positive tumours. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-01.

DNA base excision repair (BER) is critically involved in the processing of DNA base damage induced by alkylating agents. Pharmacological inhibition of BER (using PARP inhibitors), either alone or in combination with chemotherapy has recently shown promise in clinical trials. Human apurinic/apyrimidinic endonuclease 1(APE1) is an essential BER protein that is involved in the processing of potentially cytotoxic abasic sites that are obligatory intermediates in BER. Here we provide a summary of the basic mechanistic role of APE1 in DNA repair and redox regulation and highlight preclinical and clinical data that confirm APE1 as a valid anticancer drug target. Development of small molecule inhibitors of APE1 is an area of intense research and current evidence using APE1 inhibitors has demonstrated potentiation of cytotoxicity of alkylating agents in preclinical models implying translational applications in cancer patients.

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer.

1014 Background: XRCC1 is essential for DNA base excision repair, single strand break repair and nucleotide excision repair. XRCC1 deficiency promotes genomic instability and may increase cancer risk. Methods: We evaluated XRCC1 immunohistochemically in early stage breast (n=2046), ovarian (n=157), gastric (n=140), colorectal (n=250) and pancreaticobiliary cancers (n=240). Pre-clinically, we evaluated a panel of XRCC1 deficient and proficient Chinese hamster ovary and human cancer cell lines. Double strand break repair (DSB) inhibitors targeting ATM (KU55933), DNA-PKcs (NU7441) and ATR (NU6027) were evaluated for synthetic lethality and cisplatin alone or in combination with DSB inhibitors for chemopotentiation. Results: In breast cancer,XRCC1 loss (16%) was associated with higher grade (p

Background Mutation of the tumour suppressor PTEN is well-characterised in many advanced malignancies, including 30-50% of metastatic melanomas. Emerging evidence suggests a new role for PTEN in DNA repair, through transcriptional downregulation of the critical HR protein RAD51. Tumour cells deficient in HR (such as BRCA-deficient breast and ovarian cancers) have been therapeutically targeted in a synthetic lethality (SL) strategy using PARP inhibitors that block single strand break repair (SSBR), a pathway related to base excision repair (BER). We have previously identified novel inhibitors of the critical BER enzyme APE1, and demonstrated these can induce SL in BRCA-deficient cell lines. In the current study, we have evaluated SL relationships in PTEN-proficient and -deficient melanoma systems for a potential novel treatment strategy. Method We screened a panel of melanoma cell lines (MeWo, MM418, SkMel28, SkMel30, HT144, UACC62) for PTEN, APE1 and RAD51 protein expression. We tested for SL using clonogenic survival assays in PTEN- and RAD51-deficient cell lines using our recently developed APE1 DNA repair domain inhibitors, methoxyamine (indirect APE1 inhibitor) and NU1025 (PARP inhibitor), either alone or in combination. Aldehyde reactive probe assay, neutral comet assay, γH2AX immunofluorescence and FACS analyses were performed to confirm biological activity in cells. Results Of the screened cell lines, SkMel28, HT144 and UACC62 were deficient in PTEN and RAD51 protein expression. APE1 inhibitors were more toxic to PTEN-deficient cells compared to PTEN-proficient cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-263. doi:1538-7445.AM2012-LB-263

Phosphatase and tensin homolog (PTEN) loss is associated with genomic instability. APE1 is a key player in DNA base excision repair (BER) and an emerging drug target in cancer. We have developed small molecule inhibitors against APE1 repair nuclease activity. In the current study we explored a synthetic lethal relationship between PTEN and APE1 in melanoma. Clinicopathological significance of PTEN mRNA and APE1 mRNA expression was investigated in 191 human melanomas. Preclinically, PTEN-deficient BRAF-mutated (UACC62, HT144, and SKMel28), PTEN-proficient BRAF-wildtype (MeWo), and doxycycline-inducible PTEN-knockout BRAF-wildtype MeWo melanoma cells were DNA repair expression profiled and investigated for synthetic lethality using a panel of four prototypical APE1 inhibitors. In human tumours, low PTEN mRNA and high APE1 mRNA was significantly associated with reduced relapse free and overall survival. Pre-clinically, compared to PTEN-proficient cells, PTEN-deficient cells displayed impaired expression of genes involved in DNA double strand break (DSB) repair. Synthetic lethality in PTEN-deficient cells was evidenced by increased sensitivity, accumulation of DSBs and induction of apoptosis following treatment with APE1 inhibitors. We conclude that PTEN deficiency is not only a promising biomarker in melanoma, but can also be targeted by a synthetic lethality strategy using inhibitors of BER, such as those targeting APE1.