poly(ADP-ribose) Polymerase-1 Enzymatic Activity Research Articles

Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein involved in DNA repair, chromatin structure, and transcription. However, the regulation of its different functions remains poorly understood. Here, we report the role of PARP1 acetylation status in modulating its DNA repair and transactivation functions. We demonstrate that histone deacetylase 5 (HDAC5) determines PARP1 acetylation at Lys498 and Lys521 sites. HDAC5-mediated deacetylation at Lys498 site regulates PARP1 DNA damage response and facilitates efficient recruitment of DNA repair factors at damaged sites, thereby promoting cell survival. Additionally, HDAC5-mediated deacetylation at Lys521 site promotes PARP1 coactivator function, resulting in induction of proliferative and metabolic genes in an activating transcription factor 4-dependent manner. Thus, PARP1 induces metabolic adaptation to spur malignant phenotype. Our studies in mouse tumor models suggest that pharmacological inhibition of PARP1 enzymatic activity does not block tumor progression robustly as transactivation function remains unperturbed. These findings provide key mechanistic insights into PARP1 regulation and expand its role in tumor development.

Inhibition of host PARP1 contributes to the anti-inflammatory and antitubercular activity of pyrazinamide

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA’s host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA’s immune-modulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA’s bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA’s antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA’s mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB.

Abstract C034: Understanding the role of PARP activity in prostate cancer

Abstract Prostate cancer (PCa) is the second most fatal cancer in men in the United States. African American men have an incidence and death rate two times higher than that of European American men. The androgen receptor (AR) is the primary pro-oncogenic transcription factor responsible for PCa emergence and progression. AR directed therapies including androgen deprivation (ADT), direct receptor antagonism and inhibition of androgen synthesis are the current standard of care for advanced PCa. Adaptive resistance to anti-androgen therapy can occur resulting in castration-resistant prostate cancer (mCRPC). mCRPC remains universally fatal with no curative treatment options, which highlights the need for new treatment regimes. Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors are approved to treat mCRPC tumors that have homologous recombination (HR) mutations. PARP-1 is an enzyme that recruits DNA damage repair enzymes. PARP-1 enzymatic activity is elevated in mCRPC patient tumors compared to primary tumors. PARP1 enzymatic activity has been implicated in driving pro-survival mechanisms through recruitment of DNA-damage repair enzymes and regulation of oncogenic transcription factors, including AR. Although increased PARP activity has been associated with driving cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an area to be further explored. The biologically relevant doses of five PARPi were established in regular and steroid depleted growth conditions in CRPC models. PARP enzymatic activity was assessed by immunoblotting for changes in PARlyation, revealing that PARP protein remains stable, while enzymatic activity is attenuated upon challenge with PARPi. Transcriptomic analysis will assess changes in gene expression programs that are associated with inhibition of PARP enzymatic activity as it relates to key oncogenic pathways in PCa. It will be imperative to gain a deeper understanding of how PARP-1 manipulation modulates pro-oncogenic transcriptional networks to better inform clinical response. Patient derived explants (PDEs) from patients with distinct racial backgrounds will be used to examine differential PARPi responses as it relates to race. This will serve to unravel the mechanisms attributed to the increased lethality of PCa observed in African American populations. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Tessa Mulders, Matthew Schiewer. Understanding the role of PARP activity in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C034.

Pharmacodynamic effects of the PARP inhibitor talazoparib (MDV3800, BMN 673) in patients with BRCA-mutated advanced solid tumors

PurposeTalazoparib is an inhibitor of the poly (ADP-ribose) polymerase (PARP) family of enzymes and is FDA-approved for patients with (suspected) deleterious germline BRCA1/2-mutated, HER2‑negative, locally advanced or metastatic breast cancer. Because knowledge of the pharmacodynamic (PD) effects of talazoparib in patients has been limited to studies of PARP enzymatic activity (PARylation) in peripheral blood mononuclear cells, we developed a study to assess tumoral PD response to talazoparib treatment (NCT01989546).MethodsWe administered single-agent talazoparib (1 mg/day) orally in 28-day cycles to adult patients with advanced solid tumors harboring (suspected) deleterious BRCA1 or BRCA2 mutations. The primary objective was to examine the PD effects of talazoparib; the secondary objective was to determine overall response rate (ORR). Tumor biopsies were mandatory at baseline and post-treatment on day 8 (optional at disease progression). Biopsies were analyzed for PARylation, DNA damage response (γH2AX), and epithelial‒mesenchymal transition.ResultsNine patients enrolled in this trial. Four of six patients (67%) evaluable for the primary PD endpoint exhibited a nuclear γH2AX response on day 8 of treatment, and five of six (83%) also exhibited strong suppression of PARylation. A transition towards a more mesenchymal phenotype was seen in 4 of 6 carcinoma patients, but this biological change did not affect γH2AX or PAR responses. The ORR was 55% with the five partial responses lasting a median of six cycles.ConclusionIntra-tumoral DNA damage response and inhibition of PARP enzymatic activity were confirmed in patients with advanced solid tumors harboring BRCA1/2 mutations after 8 days of talazoparib treatment.

Synthesis of Novel Dual Target Inhibitors of CDK12 and PARP1 and Their Antitumor Activities in HER2-Positive Breast Cancers.

Several anti-human epidermal growth factor receptor 2 (HER2) treatments have improved the landscape of HER2-positive breast cancer (BC) over the past few years; due to the heterogeneity of the disease itself, the drug resistance mechanisms and relapse are still the main issue in HER2-positive BC. Here, we intended to target simultaneous inhibition of both poly ADP-ribose polymerase 1 (PARP1) and cyclin-dependent kinase 12 (CDK12) that have had an impact on this disease up to their implementation in clinical practice. We successfully screened PARP1 inhibitors (PARPis) containing bicyclic tetrahydropyridine pyrimidines with antitumor activity. Most synthesized compounds with various alcohols were more effective at killing tumor cells than olaparib (ola), especially in HER2-positive cancer cells. Among them, compound 9 showed potent inhibitory effects on PARP1 enzymatic activity and the PAR protein level; moreover, the expression of CDK12 was inhibited by compound 9. Overall, compound 9 exhibited a significant antitumor effect by inhibiting DNA damage repair in tumors.

Homologous Recombination Deficiency (HRD) in Cutaneous Oncology

Skin cancers, including basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), and melanoma, are the most common malignancies in the United States. Loss of DNA repair pathways in the skin plays a significant role in tumorigenesis. In recent years, targeting DNA repair pathways, particularly homologous recombination deficiency (HRD), has emerged as a potential therapeutic approach in cutaneous malignancies. This review provides an overview of DNA damage and repair pathways, with a focus on HRD, and discusses major advances in targeting these pathways in skin cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed to exploit HRD in cancer cells. PARP inhibitors disrupt DNA repair mechanisms by inhibiting PARP enzymatic activity, leading to the accumulation of DNA damage and cell death. The concept of synthetic lethality has been demonstrated in HR-deficient cells, such as those with BRCA1/2 mutations, which exhibit increased sensitivity to PARP inhibitors. HRD assessment methods, including genomic scars, RAD51 foci formation, functional assays, and BRCA1/2 mutation analysis, are discussed as tools for identifying patients who may benefit from PARP inhibitor therapy. Furthermore, HRD has been implicated in the response to immunotherapy, and the combination of PARP inhibitors with immunotherapy has shown promising results. The frequency of HRD in melanoma ranges from 18% to 57%, and studies investigating the use of PARP inhibitors as monotherapy in melanoma are limited. Further research is warranted to explore the potential of PARP inhibition in melanoma treatment.

Abstract B059: Understanding the role of poly (ADP-ribose) polymerase 1 (PARP-1) activity in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer in men in the United States. African American men have two times higher incidence rate and death rate from PCa than European American men. In PCa, the androgen receptor (AR) acts as a pro-oncogenic transcription factor. Androgen deprivation therapy (ADT) and AR antagonists are the current standard of care for advanced PCa. Late stage of disease is recognized as metastatic castrate resistant prostate cancer (mCRPC) due to adaptive resistance to ADT. mCRPC remains universally fatal with no durable therapeutic options. Poly (ADP-ribose) polymerase 1 (PARP-1) has been identified as a driver of cancer cell survival through the recruitment of DNA-damage-repair enzymes and regulation of oncogenic transcription factors, such as AR, in PCa. PARP-1 enzymatic activity is elevated in mCRPC patient tumors as compared to primary tumors. Although increased PARP activity in these tumors has been shown to be a driver of cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an active research program in our lab, especially regarding PARP-responsive transcriptional networks. PARP inhibitors (PARPi) have been approved for use in mCRPC with alterations in homologous recombination (HR) genes. The experiments below are designed to better understand the mechanisms by which PARP enzymes may transcriptionally regulate and promote pro-tumorigenic functions. Five different PARP inhibitors were analyzed in a CRPC cell line model to assess their relative potencies and develop IC50 values. A cell growth assay using trypan blue exclusion was used to compare the effectiveness of this IC50 value in regular growth conditions as it compares to steroid depleted growth conditions used to mimic ADT. Immunoblot analyses show that there is differential impact on PARP activity (PARylation) in response to the PARPi tested at the respective IC50 concentrations. It will be imperative to better understand how PARP-1 manipulation regulates gene expression. Tissue microarray (TMA) data was analyzed to assess the level of PARP-1, PAR & gH2AX levels in tissue derived from African American versus European American patients. Although not statistically significant, higher PARylation levels were present in African American TMAs compared to European American prostate tumors. It is essential to obtain TMAs representing a more diverse patient demographic to further assess this difference in PARylation levels As well as TMA data, Patient-Derived Explants (PDEs) from patients with different racial backgrounds were assessed for differential impact of PARPi based on race. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Matthew Schiewer. Understanding the role of poly (ADP-ribose) polymerase 1 (PARP-1) activity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B059.

Abstract 1566: DNA damage generated by transcription-replication conflicts explains the synthetic lethality of PARP inhibitors with homologous recombination deficiency

Abstract An important advance in cancer therapy in the last decade has been the development of poly ADP-ribose polymerase (PARP) inhibitors for the treatment of select ovarian and breast cancers. The clinical benefit stems from the synthetic lethality of PARP inhibitors with homologous recombination (HR) deficiency, which deficiency is prevalent in the cancer types listed above. The current model to explain the synthetic lethality is based on the observation that PARP inhibitors trap PARPs on DNA: the trapped PARPs block progression of the replisome, leading to the formation of DNA double-strand breaks (DSBs), which require HR for repair. Here, we propose a novel mechanism to explain the synthetic lethality between PARP inhibitors and HR deficiencies. We show that PARP1 functions together with the proteins TIMELESS and TIPIN to protect the replisome from transcription-replication conflicts (TRCs). In the absence of any one of these proteins, TRCs evolved into DNA DSBs that required HR for repair, explaining the observed synthetic lethality. In further support of this model, when we inhibited transcription elongation, which prevents the emergence of transcription-replication conflicts, then the HR-deficient cancer cells became resistant to PARP inhibitors. We further observed that trapping of PARPs on DNA was not required for the synthetic lethality with HR deficiency, since we could observe strong synthetic lethality simply by depleting PARP1 and PARP2 by siRNA. Rather, trapping of PARPs on DNA correlated with the ability of the various PARP inhibitors to inhibit PARP enzymatic activity in cells; the strongest trappers were also the most potent inhibitors of PARP1 in cells. In vitro, all PARP inhibitors tested were almost equipotent in their ability to inhibit the enzymatic activity of PARP1, meaning that the potency of PARP1 inhibitors in vitro did not reflect their inhibitory potency in cells. Our model provides a new framework for understanding the mechanism of action of PARP inhibitors in the clinic and the mechanisms by which resistance can emerge. Citation Format: Thanos D. Halazonetis, Michalis Petropoulos, Giacomo G. Rossetti, Angeliki Karamichali, Alena Freudenmann, Luca Iacovino, Vasilis Dionellis, Sotirios K. Sotiriou. DNA damage generated by transcription-replication conflicts explains the synthetic lethality of PARP inhibitors with homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1566.

NAD+ DEPLETION IN THE INTESTINAL EPITHELIUM RESULTS IN MITOCHONDRIAL DYSFUNCTION AND INFLUENCES THE PATHOGENESIS OF EXPERIMENTAL COLITIS

We have previously shown that mitochondrial dysfunction and disruption of mitochondrial biogenesis contribute to the pathogenesis of IBD. Peroxisome Proliferator-activated Receptor–gamma Coactivator 1-alpha (PGC1α) is the primary regulator of mitochondrial biogenesis. In patients with ulcerative colitis (UC) and mice undergoing experimental colitis, the transcript and protein levels of PGC1α are reduced, which is concomitant with a decrease in mitochondrial function, derangement of mitochondrial structure, and an increase in oxidative stress. Further, mice lacking PGC1α in the intestinal epithelium develop severe disease during acute and chronic colitis models. PGC1α is primarily activated via deacetylation by the nicotinamide adenine dinucleotide (NAD+)-dependent Sirtuin 1 (SIRT1). Thus, we hypothesized that decreased NAD+ in the intestinal epithelium during inflammation renders SIRT1 unable to activate PGC1α, resulting in decreased mitochondrial biogenesis and subsequent mitochondrial dysfunction. Here, we show that levels of deacetylated PGC1α (active) are decreased within the intestinal epithelium of patients with UC (n=4), implying that Sirt1 may be inactivated within diseased tissue. We also found a decrease in the intestional NAD+ levels in UC patients (n=10; p<0.001)). Similar to findings in humans, we show that not only are the levels of deacetylated PGC1α (active) decreased within the intestinal epithelium during murine models of DSS colitis (n=10; p<0.001) and infectious (Citrobacter rodentium-induced) colitis (n=10; p<0.001), but both the transcript and protein levels of Sirt1 are also decreased in mice undergoing experimental colitis (n=10 per group per model). We further found a decrease in intestinal NAD+ levels in colitic mice (n=8; p<0.0001). Although we found no significant decrease in the mRNA levels of NAD+-synthesizing enzymes in diseased intestinal tissue (p>0.05), there was a 1.5-fold increase in the intestinal mRNA levels of the NAD+-consuming DNA-repair enzyme Poly(ADP-ribose) polymerase-1 (PARP1) in mice undergoing experimental colitis (n=8; p<0.001), as well as a dramatic increase in the enzymatic activity of PARP1 in DSS-subjected intestinal tissue relative to controls. Treatment of mice undergoing experimental colitis with an NAD+ precursor [nicotinamide riboside (NR), 500 mg/kg/day] decreased the severity of colitis (n=10; p<0.001), restored mitochondrial function (p<0.001), and increased active PGC1α levels (p<0.05). NR treatment did not benefit villincre;PGCfl/fl mice (mice that lack PGC1α specifically in the intestinal epithelium; n=10; p>0.05) undergoing experimental colitis, suggesting that the therapeutic effects of NR require active PGC1α. Thus, future therapeutic approaches targeting the activity of PGC1α, and in turn mitochondrial health, may complement the treatment for IBD and improve outcomes in patients.

NAD+ DEPLETION IN THE INTESTINAL EPITHELIUM RESULTS IN MITOCHONDRIAL DYSFUNCTION AND INFLUENCES THE PATHOGENESIS OF EXPERIMENTAL COLITIS

Abstract We have previously shown that mitochondrial dysfunction and disruption of mitochondrial biogenesis contribute to the pathogenesis of IBD. Peroxisome Proliferator-activated Receptor–gamma Coactivator 1-alpha (PGC1α) is the primary regulator of mitochondrial biogenesis. In patients with ulcerative colitis (UC) and mice undergoing experimental colitis, the transcript and protein levels of PGC1α are reduced, which is concomitant with a decrease in mitochondrial function, derangement of mitochondrial structure, and an increase in oxidative stress. Further, mice lacking PGC1α in the intestinal epithelium develop severe disease during acute and chronic colitis models. PGC1α is primarily activated via deacetylation by the nicotinamide adenine dinucleotide (NAD+)-dependent Sirtuin 1 (SIRT1). Thus, we hypothesized that decreased NAD+ in the intestinal epithelium during inflammation renders SIRT1 unable to activate PGC1α, resulting in decreased mitochondrial biogenesis and subsequent mitochondrial dysfunction. Here, we show that levels of deacetylated PGC1α (active) are decreased within the intestinal epithelium of patients with UC (n=4), implying that Sirt1 may be inactivated within diseased tissue. We also found a decrease in the intestional NAD+ levels in UC patients (n=10; p&amp;lt;0.001)). Similar to findings in humans, we show that not only are the levels of deacetylated PGC1α (active) decreased within the intestinal epithelium during murine models of DSS colitis (n=10; p&amp;lt;0.001) and infectious (Citrobacter rodentium-induced) colitis (n=10; p&amp;lt;0.001), but both the transcript and protein levels of Sirt1 are also decreased in mice undergoing experimental colitis (n=10 per group per model). We further found a decrease in intestinal NAD+ levels in colitic mice (n=8; p&amp;lt;0.0001). Although we found no significant decrease in the mRNA levels of NAD+-synthesizing enzymes in diseased intestinal tissue (p&amp;gt;0.05), there was a 1.5-fold increase in the intestinal mRNA levels of the NAD+-consuming DNA-repair enzyme Poly(ADP-ribose) polymerase-1 (PARP1) in mice undergoing experimental colitis (n=8; p&amp;lt;0.001), as well as a dramatic increase in the enzymatic activity of PARP1 in DSS-subjected intestinal tissue relative to controls. Treatment of mice undergoing experimental colitis with an NAD+precursor [nicotinamide riboside (NR), 500 mg/kg/day] decreased the severity of colitis (n=10; p&amp;lt;0.001), restored mitochondrial function (p&amp;lt;0.001), and increased active PGC1α levels (p&amp;lt;0.05). NR treatment did not benefit villincre;PGCfl/flmice (mice that lack PGC1α specifically in the intestinal epithelium; n=10; p&amp;gt;0.05) undergoing experimental colitis, suggesting that the therapeutic effects of NR require active PGC1α. Thus, future therapeutic approaches targeting the activity of PGC1α, and in turn mitochondrial health, may complement the treatment for IBD and improve outcomes in patients.

Inhibitory Effects of 7-Methylguanine and Its Metabolite 8-Hydroxy-7-Methylguanine on Human Poly(ADP-Ribose) Polymerase 1.

Previously, we have found that a nucleic acid metabolite, 7-methylguanine (7mGua), produced in the body can have an inhibitory effect on the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, an important pharmacological target in anticancer therapy. In this work, using an original method of analysis of PARP1 activity based on monitoring fluorescence anisotropy, we studied inhibitory properties of 7mGua and its metabolite, 8-hydroxy-7-methylguanine (8h7mGua). Both compounds inhibited PARP1 enzymatic activity in a dose-dependent manner, however, 8h7mGua was shown to be a stronger inhibitor. The IC50 values for 8h7mGua at different concentrations of the NAD+ substrate were found to be 4 times lower, on average, than those for 7mGua. The more efficient binding of 8h7mGua in the PARP1 active site is explained by the presence of an additional hydrogen bond with the Glu988 catalytic residue. Experimental and computational studies did not reveal the effect of 7mGua and 8h7mGua on the activity of other DNA repair enzymes, indicating selectivity of their inhibitory action.

Design, synthesis, and bioactivity study on Lissodendrins B derivatives as PARP1 inhibitor

Poly(ADP-ribose) polymerase-1 (PARP1) is an enzyme that catalyzes the polymerization of ADP-ribose units to target proteins, and it is a potential target for anti-cancer drug discovery, especially for BRAC1/2 mutated tumors. In this study, a series of 2-aminoimidazole Lissodendrins B derivatives were designed, synthesized, and evaluated as PARP1 inhibitors. We found that compound D3 is better due to its PARP enzyme inhibitory activity and in vitro anti-cancer activity compared with other tested compounds. It could inhibit PARP1 enzymatic activity (IC50 = 17.46 µM) in the non-cell system and BRCA1-deficient HCC1937 and MDA-MB-436 cells growth (IC50 = 17.81 and 12.63 µM, respectively). Further study demonstrated that compound D3 inhibits tumor growth through multiple mechanisms, such as reduction of PARylation, accumulation of cellular DNA double-strand breaks, induction of G2/M cell cycle arrest, and subsequent apoptosis of BRCA1-deficient cells. Besides, the molecular docking study also confirmed that compound D3 could effectively occupy the active pocket of PARP1. Our findings provide a new skeleton structure for PARP1 inhibitor, and the results suggested that the compound D3 may serve as a potential lead compound to develop novel PARP1 inhibitors for cancer therapy.

Synthesis of novel dual target inhibitors of PARP and EGFR and their antitumor activities in triple negative breast cancers

The therapeutic strategy of poly (ADP-ribose) polymerase (PARP) inhibition of BRCA1/2 mutant cancers has been overwhelmingly successful, however, the highly aggressive triple negative breast cancers (TNBC) that receptor protein tyrosine kinase (RTKs) is known to be overexpressed are not sensitive to PARP inhibitors. Our research focused on exploring PARP inhibitors incorporating a bicyclic tetrahydropyridine pyrimidine. All synthesized compounds were more potent than Olaparib (ola) in killing tumor cells, especially in TNBC. Furthermore, compound 7 exhibited strong inhibitory effects on PARP enzymatic activity, moreover, the expression of EGFR and phosphorylated EGFR was inhibited by compound 7. Therefore, compound 7 can effectively inhibit TNBC cells with high expression of EGFR. In addition, significant synergistic effect of anti-tumor effect of new PARP inhibitors and adriamycin was also observed.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1’s role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

Synthesis, chemical characterization, PARP inhibition, DNA binding and cellular uptake of novel ruthenium(II)-arene complexes bearing benzamide derivatives in human breast cancer cells

Inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) showed remarkable clinical efficacy in BRCA-mutated tumors. Based on the rational drug design, derivatives of PARP inhibitor 3-aminobenzamide (3-AB), 2-amino-4-methylbenzamide (L1) and 3-amino-N-methylbenzamide (L2), were coordinated to the ruthenium(II) ion, to form potential drugs affecting DNA and inhibiting PARP enzyme. The four conjugated complexes of formula: C1 [(ƞ6-toluene)Ru(L1)Cl]PF6, C2 [(ƞ6-p-cymene)Ru(L1)Cl]PF6, C3 [(ƞ6-toluene)Ru(L2)Cl2] and C4 [(ƞ6-p-cymene)Ru(L2)Cl2], have been synthesized and characterized. Colorimetric 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay showed the highest antiproliferative activity of C1 in HCC1937, MDA-MB-231, and MCF-7 breast cancer cells. Efficiency of inhibition of PARP-1 enzymatic activity in vitro decreased in order: C2 > C4 > 3-AB>C1 > C3. ICP-MS study of intracellular accumulation and distribution in BRCA1-mutated HCC1937 revealed that C1-C4 entered cells within 24 h. The complex C1 showed the highest intracellular accumulation, nuclear-targeting properties, and exhibited the highest DNA binding (39.2 ± 0.6 pg of Ru per μg of DNA) that resulted in the cell cycle arrest in the S phase.

A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.

Adaptation of glioblastoma to caloric restriction induces compensatory changes in tumor metabolism that are incompletely known. Here we show that in human glioblastoma cells maintained in exhausted medium, SHC adaptor protein 3 (SHC3) increases due to down-regulation of SHC3 protein degradation. This effect is reversed by glucose addition and is not present in normal astrocytes. Increased SHC3 levels are associated to increased glucose uptake mediated by changes in membrane trafficking of glucose transporters of the solute carrier 2A superfamily (GLUT/SLC2A). We found that the effects on vesicle trafficking are mediated by SHC3 interactions with adaptor protein complex 1 and 2 (AP), BMP-2-inducible protein kinase and a fraction of poly ADP-ribose polymerase 1 (PARP1) associated to vesicles containing GLUT/SLC2As. In glioblastoma cells, PARP1 inhibitor veliparib mimics glucose starvation in enhancing glucose uptake. Furthermore, cytosol extracted from glioblastoma cells inhibits PARP1 enzymatic activity in vitro while immunodepletion of SHC3 from the cytosol significantly relieves this inhibition. The identification of a new pathway controlling glucose uptake in high grade gliomas represents an opportunity for repositioning existing drugs and designing new ones.

Evidence for Decreased Nucleolar PARP-1 as an Early Marker of Cognitive Impairment.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that regulates gene expression through poly(ADP)-ribosylation, resulting in the loosening of chromatin structure. PARP-1 enzymatic activity has been shown to be necessary for the expression of several genes required for memory formation and consolidation. Previously, we showed that nucleolar PARP-1 is significantly decreased in hippocampal pyramidal cells in Alzheimer's disease (AD). We proposed that the displacement of PARP-1 from the nucleolus results in downregulation of new rRNA expression and ribosome biogenesis, leading to cognitive impairment. To further investigate the relationship between nucleolar PARP-1 and memory impairment, we examined PARP-1 expression in the hippocampi of individuals with mild cognitive impairment (MCI) compared to control and AD cases. We used immunohistochemical techniques to examine the nucleolar distribution of PARP-1 in the Cornu Ammonis (CA region) of the hippocampus. PARP-1 positive cells were then scored for the presence or absence of PARP-1 in the nucleolus. We found a significant decrease of PARP-1 staining in the nucleolar compartment of hippocampal pyramidal cells in MCI compared with Control and AD. When the four CA (CA1-4) regions were considered separately, only the CA1 region showed significant differences in nucleolar PARP-1 with Control > AD > MCI cases. Categorization of nucleolar PARP-1 into “distinct” and “diffuse” groups suggest that most of the changes occur within the distinct group. In addition, measurements of the nucleolar diameter of nucleolar PARP-1 positive cells in CA2 and CA4 showed Control > MCI. Thus, MCI cases had a lower percentage of PARP-1 nucleolar positive cells in CA1 and smaller nucleolar diameters in CA2 and CA4, compared to Control. Our data suggest that disruption of nucleolar form and function is an early and important step in the progression of cognitive impairment.

New perspectives on the plant PARP family: Arabidopsis PARP3 is inactive, and PARP1 exhibits predominant poly (ADP-ribose) polymerase activity in response to DNA damage

BackgroundPoly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates DNA repair, gene transcription, stress responses and developmental processes in multicellular organisms. Poly (ADP-ribose) polymerase (PARP) catalyzes PARylation by consecutively adding ADP-ribose moieties from NAD+ to the amino acid receptor residues on target proteins. Arabidopsis has three canonical PARP members, and two of these members, AtPARP1 and AtPARP2, have been demonstrated to be bona fide poly (ADP-ribose) polymerases and to regulate DNA repair and stress response processes. However, it remains unknown whether AtPARP3, a member that is highly expressed in seeds, has similar biochemical activity to that of AtPARP1 and AtPARP2. Additionally, although both the phylogenetic relationships and structural similarities indicate that AtPARP1 and AtPARP2 correspond to animal PARP1 and PARP2, respectively, two previous studies have indicated that AtPARP2, and not AtPARP1, accounts for most of the PARP activity in Arabidopsis, which is contrary to the knowledge that PARP1 is the predominant PARP in animals.ResultsIn this study, we obtained both in vitro and in vivo evidence demonstrating that AtPARP3 does not act as a typical PARP in Arabidopsis. Domain swapping and point mutation assays indicated that AtPARP3 has lost NAD+-binding capability and is inactive. In addition, our results showed that AtPARP1 was responsible for most of the PARP enzymatic activity in response to the DNA damage-inducing agents zeocin and methyl methanesulfonate (MMS) and was more rapidly activated than AtPARP2, which supports that AtPARP1 remains the predominant PARP member in Arabidopsis. AtPARP1 might first become activated by binding to damaged sites, and AtPARP2 is then poly (ADP-ribosyl) ated by AtPARP1 in vivo.ConclusionsCollectively, our biochemical and genetic analysis results strongly support the notion that AtPARP3 has lost poly (ADP-ribose) polymerase activity in plants and performs different functions from those of AtPARP1 and AtPARP2. AtPARP1, instead of AtPARP2, plays the predominant role in PAR synthesis in both seeds and seedlings. These data bring new insights into our understanding of the physiological functions of plant PARP family members.

Significance of NF-kappaB signaling and PARP1 activity in the TNF-induced inhibition of PHEX gene expression in human osteoblasts.

Although loss of bone mineral density is a common symptom of chronic inflammatory diseases, its mechanisms are still poorly understood. The PHEX gene encodes a Zn-endopeptidase expressed in osteoblasts and contributes to bone mineralization. Data derived from rodents has indicated co-repression of the PHEX gene by the NF-κB pathway and poly(ADP-ribose) polymerase 1 (PARP1). The aim of this study was to determine the molecular mechanism involved in TNF-mediated downregulation of PHEX expression in human osteoblasts and human osteosarcoma cell line. We observed that activation of the NF-κB pathway by TNF was manifested as a nuclear increase in RELA and NFKB1 heterodimer. We found that TNF reduced PHEX expression and the proteasome inhibitor reversed this effect in osteosarcoma cell line. Contrary to the effects seen in rodents, inhibition of PARP1 enzymatic activity did not significantly reverse the effect of TNF on the human PHEX gene expression. EMSA studies showed that the number of adenines in the PHEX proximal promoter is crucial for the transcription factors' interactions within that region. The obtained results support the hypothesis indicating the existence of a molecular mechanism of gene repression that involves a poly adenine-rich region of the proximal gene promoters and PARP1 transcriptional activity.

Abstract LB-333: PARP-1 supports HPV infection and HPV-associated head and neck cancer

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [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 LB-333.