Function Of Poly ADP-ribose Polymerase Research Articles

Hyper-Active Non-Homologous End Joining Selects for Synthetic Lethality Resistant and Pathological Hematopoietic Stem Cells

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca-/- or Fancc-/- HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1-/-Parp1-/- but less effective in Fanca-/-Parp1-/- cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with "synthetic lethality" and reduces human engraftment. Our results suggest that hyper-active NHEJ may select for "synthetic lethality" resistant and pathological HSPCs. DisclosuresNo relevant conflicts of interest to declare.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Abstract LB-166: PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models

Abstract Background: Poly [ADP-ribose] polymerase (PARP) is an important mediator of DNA damage repair. Preclinical and clinical studies have indicated PARP inhibitors (i) as promising agents in DNA-repair-defective cancers, especially in the presence of BRCA1/2 alterations. Recent evidence also suggests a role for additional DNA damage-independent functions of PARP, involving transcriptional and epigenetic regulation. Particularly, PARP-1 can interact with the estrogen receptor (ER) and modulate its transcriptional activity in vascular smooth muscle cells, and PARPi can circumvent endocrine resistance in prostate cancer cells. However, the effect of PARPi on the efficacy of endocrine therapy (EndoTx) in ER+ breast cancer is unknown. Here we aimed to establish these effects using the PARPi olaparib (AZD2281, Olap) in our preclinical models. Methods: The effects of Olap (0.01-1μM) alone or in combination with EndoTx [estrogen deprivation (ED) or tamoxifen (Tam)] were tested in vitro in three ER+ parental cell lines (MCF7, ZR75-1, and T47D; BRCAwt) and their endocrine resistant (R) derivatives. Clonogenic assays were used to assess Olap-induced changes in cell growth. In vivo, ovariectomized nude mice bearing 200 mm3 tumors growing in the presence of estrogen supplementation (E2 pellets), were randomized to either continued E2 (control), ED, or ED+Tam; all ± oral Olap (100mg/kg daily). Results: In MCF7 parental cells in vitro, Olap, in a dose dependent manner, inhibited E2-stimulated growth and increased the growth inhibitory effect of EndoTx. In addition, at a clinically relevant dose (1μM), Olap significantly inhibited the growth of MCF7 EndoR cell derivatives. Similar results were also observed in the other 2 ER+ cell models. In vivo, long-term Olap-treatment in the presence of EndoTx (Tam ∼200days, and ED ∼300days) was well tolerated and no apparent drug-related toxicity was observed. Olap did not affect the growth of E2-stimulated MCF7 xenografts. In contrast, Olap enhanced sensitivity to Tam by delaying time to tumor progression (TTP; size doubling) from a median of 95 days to 155 days (p = 0.03), and numerically but not significantly reduced time to tumor regression (TTR; size halving) from 138 to 51 days (p = 0.2). Olap also numerically accelerated median time to tumor regression in the presence of ED (34 ED vs. 25 days ED+Olap; p = 0.23), but had no effect on TTP (P = 0.36). Conclusion: Our results suggest a potential role for Olap in enhancing EndoTx efficacy and circumventing resistance in the absence of BRCA mutations. The absence of any toxicity and negative interaction by adding Olap to EndoTx in the in vivo experiment supports the inclusion of ER+ breast cancer patients in clinical trials using PARPi. Further studies are warranted to better understand the biology of PARP and the efficacy of PARPi in ER+ breast cancer, especially in the presence of EndoTx and resistance. Citation Format: Agostina Nardone, Amit Goldstein, Martin J. Shea, Tamika Mithchell, Xiaoyong Fu, Carmine De Angelis, Huizhong Hu, Xiaowei Xu, Mahitha Rajendran, Mark O'Connor, Gershon Locker, Susan Hilsenbeck, Kent Osborne, Rachel Schiff. PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models. [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 LB-166. doi:10.1158/1538-7445.AM2015-LB-166

Inhibition of poly(ADP-ribose) polymerase 1 protects against acute myeloid leukemia by suppressing the myeloproliferative leukemia virus oncogene.

An abnormal expression of poly(ADP-ribose) polymerase 1 (PARP-1) has been described in many tumors. PARP-1 promotes tumorigenesis and cancer progression by acting on different molecular pathways. PARP-1 inhibitors can be used with radiotherapy or chemotherapy to enhance the susceptibility of tumor cells to the treatment. However, the specific mechanism of PARP-1 in acute myeloid leukemia (AML) remains unknown. Our study showed that expression of PARP-1 was upregulated in AML patients. PARP-1 inhibition slowed AML cell proliferation, arrested the cell cycle, induced apoptosis in vitro and improved AML prognosis in vivo. Mechanistically, microarray assay of AML cells with loss of PARP-1 function revealed that the myeloproliferative leukemia virus oncogene (MPL) was significantly downregulated. In human AML samples, MPL expression was increased, and gain-of-function and loss-of-function analysis demonstrated that MPL promoted cell growth. Moreover, PARP-1 and MPL expression were positively correlated in AML samples, and their overexpression was associated with an unfavorable prognosis. Furthermore, PARP-1 and MPL consistently acted on Akt and ERK1/2 pathways, and the anti-proliferative and pro-apoptotic function observed with PARP-1 inhibition were reversed in part via MPL activation upon thrombopoietin stimulation or gene overexpression. These data highlight the important function of PARP-1 in the progression of AML, which suggest PARP-1 as a potential target for AML treatment.

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen.We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus.

Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone.

Poly [ADP-ribose] polymerase 1 (PARP-1) is a highly abundant chromatin-associated enzyme. It catalyzes the NAD(+)-dependent polymerization of long chains of poly-ADP ribose (PAR) onto itself in response to DNA damage and other cues. More recently, the enzymatic activity of PARP-1 has also been implicated in the regulation of gene expression. The molecular basis for the functional switch from chromatin architectural protein to transcription factor and DNA damage responder, triggered by PARP-1 automodification, is unknown. Here, we show that unmodified PARP-1 engages in at least two high-affinity binding modes with chromatin, one of which does not involve free DNA ends, consistent with its role as a chromatin architectural protein. Automodification reduces PARP-1 affinity for intact chromatin but not for nucleosomes with exposed DNA ends. Automodified (AM) PARP-1 has the ability to sequester histones (both in vitro and in cells) and to assemble nucleosomes efficiently in vitro. This unanticipated nucleosome assembly activity of AM-PARP-1, coupled with the fast turnover of the modification, suggests a model in which DNA damage or transcription events trigger transient histone chaperone activity.

Poly(ADP-ribose) polymerase inhibitors in Ewing sarcoma.

Purpose of reviewIn 2012, two publications revealed a particular sensitivity of Ewing sarcoma cells to the inhibition of poly(ADP-ribose) polymerase (PARP). This review updates the reader on PARP function, the development of PARP inhibitors (PARPi) and the evidence for targeting PARP in Ewing sarcoma. It concludes with a description of ongoing/emerging PARPi clinical trials in patients with Ewing sarcoma.Recent findingsPARP has a major role in DNA repair, and is a transcription regulator. The oncoprotein in Ewing sarcoma, EWS-FLI1, is proposed to interact with PARP-1, driving PARP-1 expression, which further promotes transcriptional activation by EWS-FLI1. Thus, there are two rationales for PARPi in the treatment of Ewing sarcoma: to disrupt the interaction between EWS-FLI1 and PARP, and for chemo-potentiation or radio-potentiation. The first clinical trial with a single agent PARPi failed to show significant responses, but preclinical evidence for combinations of PARPi with chemotherapy or radiotherapy is very promising.SummaryDespite initial excitement for the potential of PARPi as single agent therapy in Ewing sarcoma, the emerging preclinical data now strongly support testing PARPi in combination with chemo/radiotherapy clinically.

P300 cooperates with c-Jun and PARP-1 at the p300 binding site to activate RhoB transcription in NSC126188-mediated apoptosis

The anti-cancer agent NSC126188 induces apoptosis of stomach carcinoma NUGC-3 cells by inducing RhoB expression. Here, we present that the p300 binding site in the RhoB promoter is crucial for the binding of p300 and its partner transcription factors to activate RhoB transcription in NSC126188-mediated apoptosis. NSC126188 increased expression of p300 and c-Jun. Conversely, knockdown of p300 decreased RhoB expression in the presence of NSC126188. We found that poly(ADP-ribose) polymerase-1 (PARP-1) was associated with the p300 binding site and that PARP-1 knockdown inhibited NSC126188-mediated RhoB expression. In the cells treated with NSC126188, p300, PARP-1, and c-Jun interacted and bound the p300 binding site. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed strong p300 binding and weak c-Jun binding at the p300 binding site of RhoB promoter in cells treated with NSC126188. We also demonstrated that c-Jun played a crucial role in p300 binding. However, PARP-1 did not directly bind the p300 binding site, suggesting a bridging role between p300 and c-Jun. Electrophoretic mobility shift assays demonstrated a complex comprising p300/c-Jun/PARP-1 that bound wild type, but not a mutated, p300 binding site. In addition, overexpression of p300, PARP-1, or c-Jun dramatically enhanced RhoB promoter activity when it contained the wild type sequence but not mutated sequences, indicating the crucial role of the p300 binding site in NSC126188-induced transcription of RhoB. Taken together, these data suggest that p300 is recruited and cooperates with c-Jun and PARP-1 at the p300 binding site to activate RhoB transcription during NSC126188-mediated apoptosis.

Nicotinamide treatment reduces the levels of oxidative stress, apoptosis, and PARP-1 activity in Aβ(1–42)-induced rat model of Alzheimer's disease

The underlying mechanisms of Alzheimer's Disease (AD) are still unclear. It is suggested that poly(ADP-ribose) polymerase-1 (PARP-1) overactivation can cause neuroinflammation and cell death. In this study we searched the effects of nicotinamide (NA), endogenous PARP-1 inhibitor, on oxidative stress, apoptosis, and the regulation of PARP-1 and nuclear factor kappa B (NF-κB) in amyloid beta peptide (1–42) (Aβ(1–42))-induced neurodegeneration. Sprague–Dawley rats were divided into four groups as control, Aβ(1–42), Aβ(1–42) + NA(100 and 500 mg/kg). All groups were stereotaxically injected bilaterally into the hippocampus with Aβ(1–42) or saline. After surgery NA administrations were made intraperitoneally (ip) for 7 days. In order to investigate the effects of Aβ(1–42) and NA, protein carbonyls, lipid peroxidation, reactive oxygen species (ROS) production, glutathione (GSH) levels, activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), mitochondrial function, mRNA and protein levels of PARP-1, NF-κB, p53, Bax, and Bcl-2 were measured in specific brain regions such as cortex and hippocampus. Aβ(1–42) treatment only increased the oxidative stress parameters and caused decline in antioxidant enzyme activities, mitochondrial function, and GSH levels. Also, overexpression of PARP-1, NF-κB, p53, Bax, and the decreased levels of Bcl-2 were observed in Aβ(1–42)-treated group. NA treatments against Aβ(1–42)-upregulated Bcl-2 and downregulated PARP-1, NF-κB, p53, and Bax levels. NA treatments also decreased the oxidative stress parameters and elevated antioxidant enzyme activities, GSH levels, and mitochondrial function against Aβ(1–42) treatment. These data suggest that NA may have a therapeutic potential in neurodegenerative processes due to the decreased levels of oxidative stress, apoptosis, and PARP-1 activity.

Abstract A220: Inhibition of PBMC PARP activity with the novel PARP 1/2 inhibitor BMN 673 in patients with advanced solid tumors.

Abstract Introduction: BMN 673 is a novel, potent (IC50 < 1.0 nM) inhibitor of Poly(ADP-ribose) polymerase (PARP) 1 and PARP 2 in clinical development for the treatment of genetically defined cancers. Methods: The pharmacokinetics (PK) and pharmacodynamics (PD) of BMN 673 were evaluated in a Phase 1 dose-escalation study in patients with advanced solid tumors. Oral doses evaluated ranged from 0.025 to 1.1 mg/day, with 3-6 patients evaluated at each dose level in a standard 3+3 design. The first dose in Cycle 1 was followed for 7 days without dosing for collection of serial PK and PD samples. Thereafter BMN 673 was administered once daily on Days 8 to 35, with predose PK and PD samples collected on Days 15 and 22. Following the last dose in Cycle 1 on Day 35, serial PK and PD samples were collected for 7 and 3 days, respectively, without dosing. In subsequent cycles, BMN 673 was administered daily in contiguous 28-day cycles. The PD activity of BMN 673 was measured in peripheral blood mononuclear cells (PBMCs) using assay methods previously described (Clin Cancer Res 2008 14:7917023, Biochem J 2011 436:671-679). Correlations between systemic BMN 673 exposure and inhibition of PBMC PARP activity were investigated across dose levels. Results: BMN 673 demonstrated good oral bioavailability and a long half-life supporting daily dosing (ASCO 2013 Abstract 2580). While variable across and within patients, overall PBMC PARP activity decreased in a dose-dependent manner. Within individual patients at higher dose levels, PBMC PARP activity decreased soon after the first dose of BMN 673, and activity remained at suppressed levels with daily dosing. PBMC PARP activity rebounded when dosing was stopped, indicating the return of PARP function. Correlations between the mean percent baseline PARP activity with daily BMN 673 dosing and measures of steady-state BMN 673 exposures within individuals (i.e., Day 35 Cmin, Cmax, and AUC0-24) showed decreased PARP activity with increasing exposure. This correlation was well described using an Imax model with IC50 values based on an interim analyses of 1140 pg/mL, 3090 pg/mL, and 26700 pg-hr/mL for Cmin, Cmax, and AUC0-24, respectively. Conclusions: PBMC PARP activity was rapidly and continuously inhibited with daily dosing of BMN 673. A positive correlation between systemic BMN 673 exposure and inhibition of PARP activity in PBMCs was demonstrated. This effect on a relevant pharmacodynamic marker provides in vivo proof of an on-target effect of BMN 673 and may be an initial step with potential to inform BMN 673 dose selection. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A220. Citation Format: Joshua W. Henshaw, Huiyu Zhou, Ashleigh Herriott, Miranda Patterson, Evelyn W. Wang, Don Musson, Johann de Bono, Lida A. Mina, Ramesh K. Ramanathan, Charles O'Neill, Andrew Dorr, Nicola J. Curtin. Inhibition of PBMC PARP activity with the novel PARP 1/2 inhibitor BMN 673 in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A220.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology.

Abstract 2947: PARP-1 regulates death receptor 5-mediated apoptosis in pancreatic cancer.

Abstract Pancreatic cancer is a highly malignant tumor with limited therapy and poor prognosis. Activation of death receptors (DR)-induced apoptotic pathways using antibodies or agonists for the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy. However, most pancreatic tumors are resistant to TRAIL therapy. The present studies aimed to identify combination therapies that enhance the efficacy of TRAIL therapy by sensitizing resistant pancreatic cancer cells to TRAIL-induced apoptosis, and to investigate the underlying mechanisms. Using TRA-8, a monoclonal agonistic antibody specific for DR5, we demonstrated that TRA-8 induced apoptosis of 60% and 83% of BxPc-3 and MiaPaCa-2 pancreatic cancer cells in 24 hours. By contrast, only 20% and 24% of PANC-1 and Suit-2 cells underwent apoptosis in response to TRA-8 (n=3), despite the fact that they express high levels of the DR5 receptor. The expression of poly(ADP-ribose) polymerase-1 (PARP-1) was higher in the PANC-1 and Suit-2 cells than that BxPc-3 and MiaPaCa-2 cells, which may contribute to their resistance to TRA-8-induced apoptosis. Inhibition of PARP-1 with a pharmacologic inhibitor (PJ-34) sensitized PANC-1 to TRA-8 induced apoptosis in a dose-dependent manner. Furthermore, small interfering RNAs specifically knocking down PARP-1 in PANC-1 cells enhanced TRA-8-induced apoptosis in vitro by 2.5 fold, as well as the efficacy of TRA-8 therapy on tumorigenesis of PANC-1 cells in a mouse xenograft model by 3.8 fold (n=8, p<0.01). PARP-1 knockdown increased TRA-8-induced activation of caspase-8 in the death-induced signaling complex (DISC). Immuoprecipitation with DR5 antibody identified the recruitment of PARP-1 and PARP1-mediated protein poly-ADP-ribosylation(pADPr) modification in the DR5-associated DISC, which was associated with caspase 8 activation in the DISC. Our studies determined a novel function of PARP-1, independent of its known function in DNA repair, in regulating caspase 8 activation in the DISC. These results not only provide novel molecular insights into the function of PARP-1 in regulating the extrinsic apoptosis machinery, but also support the intervention of combining PARP inhibitors with death receptor agonists to enhance the efficacy of pancreatic cancer therapy. Citation Format: Kaiyu Yuan, Yong Sun, Tong Zhou, Jay McDonald, Yabing Chen. PARP-1 regulates death receptor 5-mediated apoptosis in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2947. doi:10.1158/1538-7445.AM2013-2947

Beyond DNA Repair: Additional Functions of PARP-1 in Cancer.

Poly(ADP-ribose) polymerases (PARPs) are DNA-dependent nuclear enzymes that transfer negatively charged ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) to a variety of protein substrates, altering protein–protein and protein-DNA interactions. The most studied of these enzymes is poly(ADP-ribose) polymerase-1 (PARP-1), which is an excellent therapeutic target in cancer due to its pivotal role in the DNA damage response. Clinical studies have shown susceptibility to PARP inhibitors in DNA repair defective cancers with only mild adverse side effects. Interestingly, additional studies are emerging which demonstrate a role for this therapy in DNA repair proficient tumors through a variety of mechanisms. In this review, we will discuss additional functions of PARP-1 – including regulation of inflammatory mediators, cellular energetics and death pathways, gene transcription, sex hormone- and ERK-mediated signaling, and mitosis – and the role these PARP-1-mediated processes play in oncogenesis, cancer progression, and the development of therapeutic resistance. As PARP-1 can act in both a pro- and anti-tumor manner depending on the context, it is important to consider the global effects of this protein in determining when, and how, to best use PARP inhibitors in anticancer therapy.

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.

Targeted Therapy for Brain Tumours: Role of PARP Inhibitors

The prognosis of malignant glioma and metastatic brain tumours is still extremely poor, despite recent advances in therapeutic strategies with molecular-targeted agents. Poly(ADP-ribose) polymerase (PARP) inhibitors are a promising, novel class of anticancer drugs to be used either as single agents or in combination with chemotherapy and radiotherapy. PARP-1 and PARP-2 are the only PARP proteins that bind to DNA single strand breaks (SSBs), facilitating the repair process by the base excision repair. For this reason, PARPs have been extensively investigated as targets of novel drugs that may be used to enhance the antitumour activity of SSBs inducing agents, such as the methylating compound temozolomide, which is the drug of choice for glioblastoma, or ionizing radiations. Moreover, PARP inhibitors exert cytotoxic effects in monotherapy in BRCA mutated tumours, which are defective in the homologous recombination (HR) repair. Finally, recent studies have shown that inhibition of PARP function might also induce anti-angiogenic effects which might contribute to impair tumour growth. Many clinical trials with PARP inhibitors are ongoing for the treatment of a variety of advanced solid tumours, including primary or secondary brain tumours. This review discusses the implications of targeting PARP on the design of new treatment regimens.

Abstract IA9: Cross talk of the androgen receptor and DNA damage pathways: Molecular and translational prostate cancer relevance

Abstract Prostate cancers (PCa) are exquisitely dependent on the action of the androgen receptor (AR) for cell survival and proliferation, and there is a significant need to develop new means for targeting recurrent AR activity in both locally advanced and castration-resistant PCa(1, 2). PARP1 (Poly ADP-ribose polymerase 1) is an enzyme that modifies a subset of nuclear proteins by poly (ADP-ribose)-ylation, and is known to play a critical role in base excision repair(3). This function of PARP1 has been cultivated as a therapeutic target for tumors that harbor alterations of specific DNA repair pathways(4, 5). Multiple enzymatic inhibitors of PARP1 function are in clinical trial; while little dose limiting toxicity has been observed, suppressing PARP1-mediated DNA damage repair in BRCA1/2 deficient tumors leads to synthetic lethality and heightened clinical response to chemotherapy. Recently, it has been revealed that PARP1 has a second major cellular function on chromatin as a transcriptional coregulator, capable of modulating chromatin structure and selected transcription factor activity(6–8). New observations in our laboratory point toward PARP1 inhibitors as a means to simultaneously dampen AR activity and sensitize PCa cells to genotoxic insult. This premise is based on three major arms of investigation. First, abrogation of PARP1 activity results in sensitization of both androgen deprivation-therapy (ADT) naïve and castration-resistant PCa cells to ionizing radiation, thus indicating that PARP1 activity plays a significant role in the cellular response to radiotherapy. Second, PARP1 activity was found to be increased as a function of tumor progression in model systems of human disease, suggesting that gain of PARP1 activity may promote resistance to combined ADT and radiotherapy. Third, robust molecular analyses indicate that PARP1 is recruited to sites of AR activity on chromatin, and therein serves as a requisite cofactor for AR activity. The dependence of AR on PARP1 activity is conserved in cells that failed hormone therapy, thus indicating that the requirement for PARP1 is maintained or enhanced during the process of tumor progression. Together, these data strongly support a model wherein the dual functions of PARP1 in controlling AR activity and the response to radiotherapy can be leveraged to improve treatment of locally advanced prostate cancer. Citation Format: Matthew J. Schiewer, Adam P. Dicker, Jonathan R. Brody, John M. Pascal, Karen E. Knudsen, Jonathan F. Goodwin, J. Chad Brenner, Michael A. Augello, Fengzhi Liu, Jamie L. Planck, Randy S. Schrecengost, Felix Y. Feng, Arul M. Chinnaiyan. Cross talk of the androgen receptor and DNA damage pathways: Molecular and translational prostate cancer relevance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr IA9.

Poly(ADP-Ribose) Polymerase 1 (PARP-1) Regulates Ribosomal Biogenesis in Drosophila Nucleoli

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

Pleiotropic cellular functions of PARP1 in longevity and aging: genome maintenance meets inflammation.

Aging is a multifactorial process that depends on diverse molecular and cellular mechanisms, such as genome maintenance and inflammation. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1), which catalyzes the synthesis of the biopolymer poly(ADP-ribose), exhibits an essential role in both processes. On the one hand, PARP1 serves as a genomic caretaker as it participates in chromatin remodelling, DNA repair, telomere maintenance, resolution of replicative stress, and cell cycle control. On the other hand, PARP1 acts as a mediator of inflammation due to its function as a regulator of NF-κB and other transcription factors and its potential to induce cell death. Consequently, PARP1 represents an interesting player in several aging mechanisms and is discussed as a longevity assurance factor on the one hand and an aging-promoting factor on the other hand. Here, we review the molecular mechanisms underlying the various roles of PARP1 in longevity and aging with special emphasis on cellular studies and we briefly discuss the results in the context of in vivo studies in mice and humans.

Novel poly (ADP-ribose) polymerase 1 binding motif in hepatitis B virus core promoter impairs DNA damage repair

It is well-established that hepatitis B virus (HBV) infection is associated with the development of hepatocellular carcinoma (HCC), but patients with high viral DNA load have significantly higher risk. As host factors are required for efficient viral replication and may, therefore, contribute to high viral DNA load, we screened for host factors that can transcriptionally activate the HBV core promoter (HBVCP). We report here that poly (ADP-ribose) polymerase 1 (PARP1), which is known for its DNA repair activity, binds prominently to an octamer motif in the HBVCP and increases transcriptional efficiency. By utilizing a series of single base substitutions at each nucleotide position of the octamer, the PARP1 binding motif can be defined as "RNNWCAAA." Intriguingly, introduction of a vector construct bearing tandem repeats of the octamer motif was able to impair the DNA repair function of PARP1. This finding suggests that HBV viral DNA contains specific sequence motifs that may play a role in disrupting the DNA repair pathways of infected hepatocytes. This study has identified a novel octamer motif in the HBVCP that binds PARP1, and this interaction increases the replication efficiency of HBV. The presence of this octamer motif in hepatocytes was shown to inhibit the DNA repair capacity of PARP1, potentially contributing to the development of HCC.