Repair Of DNA Single-strand Breaks Research Articles

Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.

A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established ‘isogenic’ human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.

Expression of Poly [ADP-Ribose] Polymerase 1 (PARP-1) and Uridine-Cytidine Kinase (UCK) 1 and 2 in Myelodysplastic Syndrome

IntroductionPoly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA breaks, thus protecting the cell from genomic instability. Overexpression of PARP1 may lead a cell to apoptotic or necrotic death, defining the cell's fate. Genetic defects are important in the pathogenesis of myelodysplastic syndromes (MDS) and the role of PARP1 in the apoptotic pathways seems to be promising, since new agents targeting PARP1 are available. PARP1 expression has never been studied in MDS.Uridine-Cytidine Kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. The UCK family consists of two members, UCK1 and UCK2, both expressed in several tumor cell types. UCK2 in particular has been found to be expressed in pancreatic tumors, neuroblastoma and other malignant cells, but not in normal tissues, although it is normally expressed in human placenta. The selective expression of UCK2 in certain tumor cells enables selective malignant cell targeting with UCK2-dependent pyrimidine analogues, while sparing normal tissues.AimOur aim was to detect PARP1, UCK1 and UCK2 mRNA in bone marrow samples of patients with MDS and investigate its correlations to the hematologic and prognostic characteristics of the patients.Patients and MethodsBone marrow samples were collected from patients with MDS. Quantification of PARP1 mRNA was performed by a SYBR-green based Real-Time PCR, and the results were expressed in correlation to those of the housekeeping gene of beta actin. Quantification of UCK1 and UCK2 was performed by a Real-Time PCR using specific oligonucleotides and TaqMan probes (Applied Biosystems, CA, USA) and all experiments were normalized to the housekeeping gene of glyceraldehyde-2-phosphate dehydrogenase (GAPDH). All reactions were performed on BIORADCFX96 (BIORAD Laboratories, CA, USA).PARP1 protein was also detected by western-blot using both simple films and the Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, CA, USA).ResultsFifty seven (57) patients with MDS were included in the study. The basic demographic and hematologic characteristics of the patients are shown in Table 1. The vast majority of the patients (92.9%) were treatment naïve. PARP1 mRNA levels were measured in 53 patients, while UCK1 and UCK2 mRNA levels were measured in all patients.The median PARP1 mRNA levels were 0.0259 (range 0.0003 - 0.6410) and showed a statistically significant correlation to the type of MDS (according to the WHO classification) (p=0.016) and to the IPSS score (p=0.001) (detailed results in Table). The lowest levels were observed in patients with RA (0.0053 vs 0.1477 for non-RA patients, p= 0.016), as well as in patients with low and intermediate 1 IPSS score (0.0107 vs 0.1831, p=0.01) that had almost 20 times lower PARP1 mRNA levels than patients with intermediate 2 and high risk MDS.The protein could only be detected in samples tested with the Azure c300 Chemiluminescent Western Blot Imaging System. The median value of PARP1 expression was 0.298 (0 - 1.625) and no correlations with the WHO classification or the IPSS were detected. To our knowledge, PARP1 detection by western blotting has never been performed in the past in MDS.Although both UCK1 and UCK2 were detected in the majority of the patients (40/57, 70.2% for UCK1 and 15/57, 73.7% for UCK2), no correlation was found between their levels and the type of MDS or the prognostic factors according to the IPSS or WPSS. The median levels were for UCK1 0.0202 (0 - 2.7431) and for UCK2 0.0169 (0 - 7.270). Detailed results are presented in Table 2.ConclusionsThe correlation of higher levels of PARP1 mRNA with higher risk MDS has never been reported in the past. This correlation, if confirmed by larger studies, may render PARP1 a prognostic factor for patients with MDS. Moreover, this result can lay the basis for the design of clinical trials evaluating the use of PARP1 inhibitors in patients with higher risk MDS. Although we didn't detect any correlation of UCK1 and UCK2 with the prognostic characteristics of MDS, their mere detection in clinical samples of patients with MDS is significant, since these molecules are not detected in every malignancy, but just in a subset of malignant diseases. This finding could be of value since targeted therapies are already available. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Kinetic aspects of enzyme-mediated repair of DNA single-strand breaks

In cells and bacteria, DNA can be damaged in different ways. The efficient damage repair, mediated by various enzymes, is crucial for their survival. Most frequently, the damage is reduced to single-strand breaks. In human cells, according to the experiments, the repair of such breaks can mechanistically be divided into four steps including (i) the break detection, (ii) processing of damaged ends, (iii) gap filling, and (iv) ligation of unbound ends of the broken strand. The first and second steps run in parallel while the third and fourth steps are sequential. The author proposes a kinetic model describing these steps. It allows one to understand the likely dependence of the number of breaks in different states on enzyme concentrations. The dependence of these concentrations on the rate of the formation of breaks can be understood as well. In addition, the likely role of unzipping and zipping of the fragments of broken ends of the strand in the ligation step has been scrutinized taking the specifics of binding of DNA stands into account.

PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2).

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.

Abstract 1223: NMS-P293, a novel potent and selective PARP-1 inhibitor with high antitumor efficacy and tolerability

Abstract Poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology. Inhibition of PARP-1 is synthetically lethal with loss of function of the BRCA1 and BRCA2 tumor suppressor genes, as well with additional DNA repair defects. Tumor cells harboring defects in DNA repair pathways can thus be selectively targeted with PARP-1 inhibitors, and several such compounds are at different stages of clinical investigation in diverse tumor types, either as single agents or in combination regimens. However none of these agents selectively inhibits PARP-1 within the PARP family of enzymes: all drugs currently in clinical development, as well as the vast majority of preclinical compounds described to date potently cross-inhibit PARP-2, due to the high sequence homology between the two enzymes. Although PARP-2 is reported to be involved in DNA single-strand break repair, its contribution to total cellular levels of DNA damage-induced PARP activity is minimal (5-10%). Gene ablation studies show that loss of both PARP-1 and -2 function is incompatible with normal embryonic development, while PARP-2 single knockout mice show a variety of defects, including impaired erythropoiesis, thymopoiesis adipogenesis and spermatogenesis, increased neuronal loss after ischemic damage and higher risk of pancreatitis following chemical insult. We therefore reasoned that a potent and highly selective PARP-1 inhibitor might represent a significant advancement over currently available agents targeting both PARP-1 and -2, since sparing of PARP-2 inhibition potentially limits on target side-effects and offers greater opportunity for combination with other chemotherapeutic agents. Here we describe the preclinical characterization of NMS-P293, a novel highly potent PARP-1 inhibitor possessing >200-fold selectivity versus PARP-2. In cells, NMS-P293 inhibits hydrogen peroxide induced poly ADP-ribose (PAR) synthesis with an IC50 in the single digit nanomolar range, confirming expected mechanism of action in cells. NMS-P293 is selectively active on tumor cell lines defective in the HR repair pathway, such as pTEN and BRCA mutated lines, while sparing DNA repair proficient cells and normal myelocytes. NMS-P293 possesses favorable ADME properties including a low efflux ratio, high cross-species metabolic stability, low clearance and nearly complete oral bioavailability in rodents and non rodents. Oral administration to mice bearing BRCA mutated breast cancer xenografts resulted in complete tumor regressions and cures. The highly favorable preclinical characteristics of NMS-P293 make this compound a promising candidate for further development. Citation Format: Alessia Montagnoli, Sonia Rainoldi, Antonella Ciavolella, Dario Ballinari, Francesco Caprera, Lucio Ceriani, Rosita Lupi, Marina Ciomei, Eduard Felder, Antonella Isacchi, Daniele Donati, Arturo Galvani, Gianluca Papeo. NMS-P293, a novel potent and selective PARP-1 inhibitor with high antitumor efficacy and tolerability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1223.

Abstract 4: Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma

Abstract PARP1 is a key enzyme of the Base Excision Repair (BER) pathway, facilitating the repair of base damage and single-strand DNA breaks. Activated PARP1 synthesizes poly (ADP-ribose) (PAR), triggering chromatin de-condensation to facilitate recruitment of BER proteins to complete repair. PARP1 activation is attenuated upon successful repair of the DNA lesion. However, unrepaired DNA breaks lead to continuous PARP1 activation and cell death. The molecular mechanism underlying PARP1 activation induced cell death was recently revealed as independent from NAD+ depletion. We have shown that PARP1 activation and PAR synthesis affect glycolysis by directly inhibiting the glycolytic enzyme, hexokinase 1 (HK1). Following on these discoveries, we decided to investigate global metabolic changes triggered by hyperactivation of PARP1. For this study, we used gas chromatography mass spectrometry (GC-MS) to quantify over 150 cellular metabolites and Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry (MRM LC-MS) to measure NAD+ metabolites. As a model, we tested glioblastoma cells overexpressing methylpurine DNA glycosylase (MPG) to enhance the PARP1-activation response to DNA damage induced by the alkylating agent MNNG. Simultaneously, to monitor independence from the DNA damaging effect of NAD+ depletion, we utilized an inhibitor of NAD+ biosynthesis, FK866. We found that PARP1 activation leads to a strong accumulation of glucose, likely as a secondary effect of HK1 inhibition. In addition, we observed a significant change in the level of other metabolites including an increase in inosine, inosine monophosphate (IMP), cytidine and uridine levels upon PARP1 activation, suggesting an indirect effect of PARP1 activation on purine and pyrimidine metabolism. Ongoing studies will use these global approaches to unravel the complete metabolic response of cancer cells to genotoxic treatment. Citation Format: Anna M. Wilk, Elise Fouquerel, Bobbie Johnston, Samuel A.J. Trammell, Lindsay Schambeau, Joel F. Andrews, Lewis Pannell, Sara J. Cooper, Charles Brenner, Robert W. Sobol. Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4.

Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells.

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.

Versatility in phospho-dependent molecular recognition of the XRCC1 and XRCC4 DNA-damage scaffolds by aprataxin-family FHA domains

Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 μM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 μM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability.

Development of Olaparib for BRCA-Deficient Recurrent Epithelial Ovarian Cancer.

The FDA approval of the PARP inhibitor olaparib for fourth-line therapy of germline BRCA1/2-mutated ovarian cancer represents the first registered indication for this class of drugs in any disease. PARP is a family of proteins involved in the repair of single-strand DNA breaks. High-grade serous ovarian carcinomas with BRCA deficiencies may be particularly vulnerable to both direct and indirect effects of PARP inhibition. This phenotype frequently arises as a consequence of defects in the repair of damaged DNA, rendering cancer cells susceptible to DNA-damaging platinum compounds and targeted therapies affecting homologous recombination repair (HRR). When cells already deficient in HRR are exposed to PARP inhibitors, apoptosis occurs by way of synthetic lethality. In this review, we trace the clinical development of olaparib for women with recurrent epithelial ovarian carcinoma harboring germline BRCA mutations, a biomarker for HRR deficiency present in 15% to 20% of cases. Clinical trials highlighted include not only those pivotal studies that have led to regulatory approval in the United States and in Europe, but also those in which olaparib was studied in novel combinations, including chemotherapy and antiangiogenesis agents.

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2–10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution.

Abstract 3481: Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment

Abstract Triple-negative breast cancer (TNBC) accounts for 15-25% of breast cancer cases. Effective targeted treatment for TNBC is lacking. Poly (ADP-ribose) polymerase (PARP) is a key enzyme involved in single-strand DNA break repair. PARP inhibitors have been used in combination with various chemotherapeutic agents in TNBC with limited success. The sigma-2 receptor is associated with cellular proliferation and quiescence and has been used as the target for imaging solid tumors by positron emission tomography. The sigma-2 receptor has been identified as progesterone receptor membrane component 1 (PGRMC1) protein complex. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target. In this study we looked for potential synergy between PARP inhibitors and sigma-2 ligands using a triple negative breast cancer cell line, MDA-MB231. We showed that the sigma-2 ligand, SW43, and a PGRMC1 ligand, AG205, induced cell toxicity and caspase 3 activation in a dose-dependent manner. The combination of SW43 and PARP inhibitor, Olaparib, resulted in additive cytotoxicity by cell viability assay and colony forming assay. Olaparib inhibited PARP activity in MDA-MB231 cell line, suggesting that the single-strand DNA break repair may be inhibited. SW43 and AG205 treatment decreased PGRMC1 and EGFR protein levels, suggesting that sigma-2/PGRMC1 ligands reduce growth factor signaling. In conclusion, SW43 and Olaparib induce cell death by separate signaling pathways. Sigma-2 ligands may selectively sensitize tumors to PARP inhibitor treatment in triple negative breast cancer patients. Citation Format: Chenbo Zeng, Julia Mankoff, Robert H. Mach. Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3481. doi:10.1158/1538-7445.AM2015-3481

Abstract 4490: Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo

Abstract The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers and this overexpression is frequently correlated with poor prognosis. Selective Inhibitors of Nuclear Export (SINE) compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 to inhibit nuclear export. This results in nuclear retention of major tumor suppressor proteins, such as p53, pRB, FOXO3A and other critical proteins in cancer biology, which leads to selective cancer cell death. Selinexor is the most advanced SINE with >500 hematological and solid tumor cancer patients treated to date in a number of Phase I/II clinical trials. Blocking XPO1 function also blocks nuclear transport of key DNA damage repair (DDR) proteins and we therefore hypothesized that combination of selinexor with radiation therapy (RT) would lead to synergistic anti-tumor activity. Radiation-chemotherapy combination is an established therapeutic option for patients with medically inoperable non-small cell lung cancer (NSCLC), but its use is limited due to tolerability and adverse effects. Moreover, this treatment strategy typically has short-term benefits and the patients relapse with progressive disease. Here we report on studies designed to test whether selinexor could interfere with DDR and synergize with radiation to enhance anti-tumor potency relative to either treatment alone. Cell cycle analysis and clonogenic assays were conducted in the presence or absence of selinexor in combination with escalating doses of RT on the radiation-sensitive and radiation-insensitive H1299 and A549 cell lines, respectively. Interestingly, Selinexor-treatment induced G1 cell cycle arrest in the p53-deficient cell line H1299 and a prominent G2 cell cycle arrest in p53-wild type A549 cells 24 hrs post treatment. Furthermore, clonogenic assays revealed that selinexor + RT treatment act synergistically to produce a dose-dependent growth inhibition in both cell lines. In vivo combination treatment in the A549 NSCLC xenograft model with low doses of selinexor and RT displayed a strong synergistic effect for reduction of tumor volume. In addition, microscopic and immunohistochemical analysis of the resected tumors showed an overall reduction in tumor cell numbers, increased fibrosis and induction of apoptosis in the selinexor-RT treated tumors compared to controls. Finally, mechanistic studies revealed that selinexor does not induce DNA damage like RT, but downregulates CHK1, RAD51 and other DDR proteins. Together, our results suggest that selinexor treatment sensitizes cells to RT by preventing single-stranded DNA break repair via downregulation of DDR protein expression. In contrast, selinexor induces cell cycle arrest at G1 in p53 deficient H1299 cells, allowing DNA damage accumulation and induction of apoptosis. These results provide a rationale basis for combining selinexor with RT in clinical trials studies. Citation Format: Tami Rashal, Sivan Elloul, Marsha Crochiere, Trinayan Kashyap, William Senapedis, Ryan George, Sharon Friedlander, Maya Ilouze, Yosef Landesman, Robert Carlson, Nir Peled, Michael Kauffman, Sharon Shacham, Yaacov Lawrence. Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2015-4490

The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function.

The nuclear expression of poly (ADP-ribose) polymerase-1 (PARP1) in invasive primary breast tumors is associated with chemotherapy sensitivity

It has been reported that expression levels of DNA repair genes are frequently associated with chemotherapy sensitivity and prognosis in breast cancer (BC) subtypes. The poly (ADP-ribose) polymerase-1 (PARP1), one of the major DNA single-strand break (SSBs) repair proteins, has been demonstrated a role in BC development. Because many of the chemotherapeutic agents target the tumor cell DNA, a DNA damage repair protein function is expected to impact therapeutic responses. However, the predictive effect of PARP1 in neoadjuvant chemotherapy (NC) treated BC is still controversial. To investigate whether PARP1 expression in BC is a possible biomarker to predict chemotherapeutic response, we assessed PARP1 expression in BC specimens based on collagen gel droplet embedded culture-drug sensitivity test (CD-DST) (in vitro) results and chemotherapeutic response of NC (in vivo). The surgical specimens from 108 patients with BC were recruited for CD-DST and PARP1 immunohistochemistry. We found that higher nuclear PARP1 (nPARP1) expression correlated with increased in vitro chemosensitivity against docetaxel (p=0.001) and epirubicin (p=0.022) based on CD-DST results. We also found that tumors with high nPARP1 expression were more sensitive to anthracycline/taxane based chemotherapy and associated with pathologic responses to NC using univariate and multivariate analyses (p=0.019 and p=0.037, respectively). Taken together, we conclude that nuclear expression of PARP1 is a useful marker to predict BC therapeutic responses.

PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels.

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells' poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

Abstract 2402: Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma cell lines

Abstract The loss of PTEN is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in the nucleus. Poly (ADP-ribose) polymerase (PARP) plays a key role in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in a panel of endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN introduction on the sensitivity of cells to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN+ (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. Results The SF50 values were 100 nM or less in six of the 16 (38%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in five of the 16 (31%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 995 ± 1,096 nM; Wild-type [n = 4]: 144 ± 91 nM, p = 0.17 by Student'st test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN+ cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN+ cells. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. The inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. Citation Format: Aki Miyasaka, Katsutoshi Oda, Yuji Ikeda, Tomoko Kashiyama, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Chinami Makii, Kayo Asada, Reiko Kurikawa, Osamu Wada Hiraike, Kenbun Sone, Yuriko Uehara, Yoko Matsumoto, Takahide Arimoto, Hiroyuki Kuramoto, Tetsu Yano, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii. Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2402. doi:10.1158/1538-7445.AM2014-2402

Abstract SY42-03: Novel DNA repair inhibitors for radiosensitization and cancer therapy

Abstract There is growing interest in the development of molecules that can inhibit DNA repair or disrupt DNA damage response pathways for application to cancer therapy. Such inhibitors offer the potential to achieve synthetic lethality in the context of DNA repair deficient cancers and/or to sensitize tumors to chemotherapy and radiation. We have identified a series of structurally related small molecules that demonstrate increased toxicity to human cancer cells deficient in BRCA2, PTEN, ATM, and the Fanconi pathway. These compounds are not PARP inhibitors but rather impact DNA damage response pathways at upstream steps, causing impairment of both homology-dependent DNA repair (HDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks. The molecules also reduce the formation of DNA repair foci by several damage response factors, including 53BP1, BRCA1, and DNA-PK, following ionizing radiation treatment of cancer cells. Efforts to identify the molecular target of these compounds are underway. In addition, we have found that a highly unusual lupus-derived, cell-penetrating autoantibody, 3E10, preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we find that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low dose doxorubicin. These findings identify a new antibody that may have clinical application in cancer, particularly in BRCA-related malignancies. Because lupus is associated with circulating autoantibodies reactive against host DNA, the work also suggests that lupus autoantibodies may be a valuable source of other potential therapeutic agents. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the unexpectedly low rates of breast and ovarian cancers observed in lupus patients. Citation Format: Peter M. Glazer. Novel DNA repair inhibitors for radiosensitization and cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY42-03. doi:10.1158/1538-7445.AM2014-SY42-03

Abstract 2411: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) have been developed and tested in a context of combining them with drugs that inhibit double-stranded (ds) DNA repair, because PARPi impair single-stranded (ss) DNA break repair, resulting in activation of dsDNA break repair machinery. Rapamycin, an mTOR inhibitor, has been widely prescribed for more than a decade, and recent studies revealed its ability to inhibit dsDNA break repair. The combination of the PARPi, olaparib, and rapamycin synergistically inhibited cell proliferation in non-small cell lung cancer (NSCLC) cells, as well as triple negative breast cancer (TNBC) cells that have BRCA1 mutations. Rad51, which forms a polymer on ssDNA upon dsDNA breaks, plays an essential role in homologous recombination. Olaparib induced Rad51 focus formation and rapamycin successfully inhibited it both in vivo and in vitro, suggesting this combination worked through blocking both ssDNA break repair and dsDNA break repair, resulting in cells that cannot go through G2/M checkpoint. The protein level of PARP was predictive for both PAR activity and Rad51 focus formation with this combination. Collectively, these data suggest that this combination could have a therapeutic potential to inhibit cancer with high PARP expression, or in combination with cytotoxic chemotherapy or radiation. Citation Format: Atsushi Osoegawa, Joell J. Gills, Shigeru Kawabata, Kenji Sugio, Phillip A. Dennis. Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2411. doi:10.1158/1538-7445.AM2014-2411