Inhibition Of PARP Activity Research Articles

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Anemonin ameliorates human diploid fibroblasts 2BS and IMR90 cell senescence by PARP1-NAD+-SIRT1 signaling pathway

ObjectiveAging becomes the most predominant risk factor for all age‐associated pathological conditions with the increase of life expectancy and the aggravation of social aging. Slowing down the speed of aging is considered an effective way to improve health, but so far, effective anti-aging methods are relatively lacking. MethodsAnemonin (ANE) was screened from eight existing small-molecule compounds by cell viability assay. The function of ANE was determined by the analysis of cell proliferation, β -galactosidase (SA-β -Gal) activity, cell cycle, SASP secretion, NAD+/NADH ratio, and other aging-related indicators. The targets of ANE were predicted by Drug Target Prediction System (DTPS) and Swiss Targe Prediction System. The effect of ANE on PARP-1-NAD+-SIRT1 signaling pathway was assessed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot, PARP1, NAD+ and SIRT1 activity detection. ResultsANE can delay cell senescence; PARP1 is one of the targets of ANE and plays a crucial role in ANE anti-aging; ANE release more NAD+ by inhibiting PARP1 activity, thereby conversely promoting the function of SIRT1 and delay cell senescence. ConclusionsOur study indicates that ANE can delay cellular senescence through the PARP1-NAD+-SIRT1 signaling pathway, which may be considered as an effective anti-aging strategy.

PARP inhibitor olaparib induced differential protein expression in cervical cancer cells

PARP inhibitors are a potential class of chemotherapeutic drugs but PARP inhibitor response has not been explored systematically. We lack a specific understanding of the subset of the proteome preferentially modified in various cancers by PARP inhibitors. Implications of PARP inhibitor and PARP1 in cervical cancer treatment and resistance are not fully elucidated. We conducted a mass spectrometry-based proteomic analysis of cervical cancer Hela cells treated with olaparib. We aimed to identify the alteration in the protein signaling pathway induced by PARP inhibitors beyond the DNA damage response pathway. Our data demonstrate a significant reduction in PARP activity and enhanced cell death after olaparib treatment. We further observed articulated proteomic changes with a significant enrichment of proteins in diverse cellular processes. The differentially expressed proteins were predominantly associated with RNA metabolism, mRNA splicing, processing, and RNA binding. Our data also identified proteins that could probably contribute to survival mechanisms resulting in resistance to PARP inhibitors. Hence, we put forth the overview of proteomic changes induced by PARP inhibitor olaparib in cervical cancer cells. This study highlights the significant proteins modified during PARP inhibition and thus could be a probable target for combination therapies with PARP inhibitors in cervical cancer. Significance•This study provides the overview of proteomic changes induced by PARP inhibitor olaparib in cervical cancer Hela cells.•We demonstrated that Olaparib inhibited PARP1 activity in Hela cells in a dose-dependent manner while no change was observed in the expression of PARP1.•PARP inhibition potentially regulated the RNA metabolism, RNA binding proteins, metastasis-related genes, mitochondrial proteins, transcription factors and regulators, and ubiquitination proteins.•We also identified increased expression of tumor-promoting and drug resistance proteins that could contribute to the resistance mechanism to PARPi therapy.•This approach will be helpful to generate insights into precision oncology for personalized treatment and generate data repositories for future artificial intelligence-based research.

Differences in Durability of PARP Inhibition by Clinically Approved PARP Inhibitors: Implications for Combinations and Scheduling.

Six PARP inhibitors (PARPi) are approved for cancer therapy as monotherapy agents in daily or twice-daily continuous dosing schedules to maintain the necessary continuous suppression of PARP activity. Continuous PARP inhibition is required for single-agent anticancer activity. To investigate if such intense schedules are necessary, we determined the durability of PARP inhibition up to 72 h after a 1 h pulse of 1 µM of five of the approved PARPi, rucaparib, olaparib, niraparib, talazoparib and pamiparib, in IGROV-1 and ES-2 (human ovarian cancer) cells. Rucaparib caused the most persistent inhibition of PARP activity when maintained at ≥75% at 72 h after drug withdrawal in both IGROV-1 and ES-2 cells, but inhibition was more rapidly lost with the other PARPi. PARPi are also under clinical investigation with ATR inhibitors, and thus, we evaluated the implications for scheduling with an ATR inhibitor (VE-821). Rucaparib enhanced VE-821 cytotoxicity in co-exposure, sequential and delayed (24 h drug-free) schedules in IGROV-1 and ES-2 cells. Olaparib and niraparib enhanced VE-821 cytotoxicity only in co-exposed cells and not in sequential exposures. These data have clinical implications for the scheduling of PARPi as a monotherapy and in combination with ATR inhibitors and other cytotoxic drugs.

Synthesis and in vitro biological evaluation of 3-ethyl-1,5-naphthyridin-2(1H)-one derivatives as potent PARP-1 selective inhibitors and PARP-1 DNA trappers

Inhibition of poly (ADP-ribose) polymerase (PARP) has been applied with great success in the clinical treatment of homologous recombination-deficient malignancy. Recent study demonstrated that not only PARP-1 inhibition but also DNA trapping contributes to the efficacy in BRCA mutant tumors and the toxicities results from the poor selectivity of PARP-1 over PARP-2 as well as their DNA trapping. Herein, a series of 3-ethyl-1,5-naphthyridin-2(1H)-one derivatives (7a-7l, 8a-8n) were synthesized and identified as PARP-1 selective inhibitors and PARP-1 DNA trappers. Among them, compound 8m was found to be highly potent and selective. It inhibited PARP-1 activity and BRCA mutant DLD-1 cell activity with IC50 values of 0.49 nM and 4.82 nM, and the in vitro DNA trapping efficacy of compound 8m was 1.85 nM. Compared with AZD5305, compound 8m significantly improved the selectivity of PARP-1 over PARP-2 as well. Compound 8m was>1000-fold selective for PARP-1 DNA trapping over PARP-2.

202TiP A single-arm, open, multicenter and exploratory clinical study of fluzopari combined with apatinib in pts with platinum-sensitive relapsed ovarian cancer first-line treated with a PARP inhibitor

PARP inhibitors (PARPi) have been approved as maintenance therapy for patients with platinum-sensitve recurrent ovarian cancer. Fluzoparib, a novel, potent and orally available PARPi, significantly inhibited PARP1 activity. It had promising antitumor activity in patients with maintenance therapy and platinum-sensitive recurrent ovarian cancer with germline BRCA1/2 mutations who had received previous 2-4 lines of chemotherapy. However, the treatment consensus after PARPi has not been established.

NUPR1 protects against hyperPARylation-dependent cell death

Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.

Compartmentalized regulation of NAD+ by Di (2-ethyl-hexyl) phthalate induces DNA damage in placental trophoblast

Di (2-ethyl-hexyl) phthalate (DEHP) is a wildly used plasticizer. Maternal exposure to DEHP during pregnancy blocks the placental cell cycle at the G2/M phase by reducing the efficiency of the DNA repair pathways and affects the health of offsprings. However, the mechanism by which DEHP inhibits the repair of DNA damage remains unclear. In this study, we demonstrated that DEHP inhibits DNA damage repair by reducing the activity of the DNA repair factor recruitment molecule PARP1. NAD+ and ATP are two substrates necessary for PARP1 activity. DEHP abated NAD+ in the nucleus by reducing the level of NAD+ synthase NMNAT1 and elevated NAD+ in the mitochondrial by promoting synthesis. Furthermore, DEHP destroyed the mitochondrial respiratory chain, affected the structure and quantity of mitochondria, and decreased ATP production. Therefore, DEHP inhibits PARP1 activity by reducing the amount of NAD+ and ATP, which hinders the DNA damage repair pathways. The supplement of NAD+ precursor NAM can partially rescue the DNA and mitochondria damage. It provides a new idea for the prevention of health problems of offsprings caused by DEHP injury to the placenta.

Unsymmetric Cisplatin-Based Pt(IV) Conjugates Containing a PARP-1 Inhibitor Pharmacophore Tested on Malignant Pleural Mesothelioma Cell Lines.

Cisplatin is widely employed as a first-line chemotherapeutic agent for many solid tumors, including malignant pleural mesothelioma (MPM). However, its clinical use is limited by heavy side effects and acquired resistance, the latter being mainly related to enhanced DNA repair. Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPis) have been carried out, with the hope that such combinations might lead to improved therapeutic efficacy against tumors. Here, the synthesis and efficacy in reducing MPM cell viability of four cisplatin-based Pt(IV) prodrugs containing the PARPi 3-aminobenzamide (3-ABA) fragment are described. The most promising conjugate is more effective than cisplatin or cisplatin/3-ABA combination, administered in equimolar doses, in inhibiting PARP-1 activity and inducing apoptosis in BRCA1/2 wild type MPM cells, grown as monolayer or as multicellular spheroids.

GLP-1-Induced AMPK Activation Inhibits PARP-1 and Promotes LXR-Mediated ABCA1 Expression to Protect Pancreatic β-Cells Against Cholesterol-Induced Toxicity Through Cholesterol Efflux.

T2DM (Type 2 diabetes) is a complex, chronic disease characterized as insulin resistance and islet β-cell dysfunction. Bariatric surgeries such as Roux-en-Y gastric bypass (RYGB) surgery and laparoscopic sleeve gastrectomy (LSG) have become part of a critical treatment regimen in the treatment of obesity and T2DM. Moreover, GLP-1 increase following bariatric surgery has been regarded as a significant event in bariatric surgery-induced remission of T2DM. In this study, a high concentration cholesterol-induced lipotoxicity was observed in INS-1 cells, including inhibited cell viability and insulin secretion. Enhanced cell apoptosis and inhibited cholesterol efflux from INS-1 cells; meanwhile, ABCA1 protein level was decreased by cholesterol stimulation. Cholesterol-induced toxicity and ABCA1 downregulation were attenuated by GLP-1 agonist EX-4. GLP-1 induced AMPK phosphorylation during the protection against cholesterol-induced toxicity. Under cholesterol stimulation, GLP-1-induced AMPK activation inhibited PARP-1 activity, therefore attenuating cholesterol-induced toxicity in INS-1 cells. In INS-1 cells, PARP-1 directly interacted with LXR, leading to the poly(ADP-ribosyl)ation of LXRα and downregulation of LXR-mediated ABCA1 expression. In the STZ-induced T2DM model in rats, RYGB surgery or EX-4 treatment improved the glucose metabolism and lipid metabolism in rats through GLP-1 inhibition of PARP-1 activity. In conclusion, GLP-1 inhibits PARP-1 to protect islet β cell function against cholesterol-induced toxicity in vitro and in vivo through enhancing cholesterol efflux. GLP-1-induced AMPK and LXR-mediated ABCA1 expression are involved in GLP-1 protective effects.

Modulation of PARP-1 Activity in a Broad Time Window Attenuates Memorizing Fear.

The amygdala plays a critical role in the acquisition and consolidation of fear-related memories. Recent studies have demonstrated that ADP-ribosylation of histones, accelerated by PARPs, affects the chromatin structure and the binding of chromatin remodeling complexes with transcription factors. Inhibition of PARP-1 activity during the labile phase of re-consolidation may erase memory. Accordingly, we investigated the possibility of interfering with fear conditioning by PARP-1 inhibition. Herein, we demonstrate that injection of PARP-1 inhibitors, specifically into the CeA or i.p., in different time windows post-retrieval, attenuates freezing behavior. Moreover, the association of memory with pharmacokinetic timing of PARP inhibitor arrival to the brain enabled/achieved attenuation of a specific cue-associated memory of fear but did not hinder other memories (even traumatic events) associated with other cues. Our results suggest using PARP-1 inhibitors as a new avenue for future treatment of PTSD by disrupting specific traumatic memories in a broad time window, even long after the traumatic event. The safety of using these PARP inhibitors, that is, not interfering with other natural memories, is an added value.

Modulation of PARP activity by Monomethylarsonous (MMA+3) acid and uranium in mouse thymus

Arsenic exposure is well established to impair the function of zinc finger proteins, including PARP-1. Previous studies from our lab show that early developing T cells in the thymus are very sensitive to arsenite (As+3)-induced genotoxicity mediated through PARP-1 inhibition. Additionally, it has been shown that uranium (in the form of uranyl acetate, UA) also suppresses PARP-1 activity in HEK cells. However, very little is known about whether the As+3 metabolite, monomethylarsonous acid (MMA+3), also inhibits PARP-1 activity and if this is modified by combined exposures with other metals, such as uranium. In the present study, we found that MMA+3 significantly suppressed PARP-1 function, whereas UA at high concentrations significantly increased PARP-1 activity. To evaluate whether the effects on PARP-1 activity were mediated through oxidative stress, we measured the induction of hemoxygenase-1 (Hmox-1) expression by qPCR. MMA+3, but not UA, significantly induced oxidative stress; however, the inhibition of PARP-1 produced by MMA+3 was not reversed by the addition of the antioxidant, Tempol. Further evaluation revealed minimal interactive effects of MMA+3 and UA on PARP-1 function. Collectively, our results show that contrary to As+3, the suppressive effects of MMA+3 on PARP-1 were not substantially driven by oxidative stress. in mouse thymus cells. Results for this study provide important insights into the effects of MMA+3 and uranium exposures on PARP-1 function, which is essential for future studies focused on understanding the effects of complex environmentally relevant metal mixtures.

Novel poly (ADP-ribose) polymerases inhibitor DHC-1 exhibits in vitro and in vivo anticancer activity on BRCA-deficient pancreatic cancer cells

Poly (ADP-ribose) polymerases (PARPs) play a key role in DNA repair. In this study we designed a novel small-molecular compound, (E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (DHC-1), which was a potent and selective PARP-1 inhibitor. DHC-1 selectively inhibited PARP-1 activity with an IC50 value of 41.12 ± 13.28 nM. Cytotoxicity results showed that DHC-1 selectively inhibited the proliferation of BRCA1-deficient breast cancer HCC-1937 and BRCA2-deficient pancreatic cancer Capan-1 cells. Mechanism studies found that DHC-1 stabilized PARP-1–DNA complexes and inhibited PAR formation in BRCA2−/− Capan-1 cells. Further experiments found that DHC-1 induced DNA double-strand damage in BRCA2−/− Capan-1 cells, which was demonstrated by accumulation of γ-H2AX foci. Flow cytometry experiments revealed that DHC-1 induced G2/M phase arrest and activate mitochondrial-induced apoptotic pathways. Interestingly, we also found that DHC-1 enhanced cell proliferation inhibitory effect of oxaliplatin (OXA). The further in vivo nude mouse studies showed that DHC-1 inhibited the growth of Capan-1 xenografts and showed a similar mechanism to that in vitro. Collectively, our results demonstrate that DHC-1 may be an excellent candidate for treatment of BRCA-deficient pancreatic cancers.

Establishing the promising role of novel combination of triple therapeutics delivery using polymeric nanoparticles for Triple negative breast cancer therapy.

Introduction: Triple-negative breast cancer (TNBC) is a lethal tumor with an advanced degree of metastasis and poor survivability as compared to other subtypes of breast cancer. TNBC which consists of 15 % of all types of breast cancer is categorized by the absence of expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER2). This is the main reason for the failure of current hormonal receptor-based therapies against TNBCs, thus leading to poor patient outcomes. Therefore, there is a necessity to develop novel therapies targeting this devastating disease. Methods: In this study, we have targeted TNBC by simultaneous activation of apoptosis through DNA damage via cytotoxic agent such as paclitaxel (PAC), inhibition of PARP activity via PARP inhibitor, olaparib (OLA) and inhibiting the activity of FOXM1 proto-oncogenic transcription factor by using RNA interference technology (FOXM1-siRNA) in nanoformulations. Experiments conducted in this investigation include cellular uptake, cytotoxicity and apoptosis study using MDA-MB-231 cells. Results: The present study validates that co-delivery of two drugs (PAC and OLA) along with FOXM1-siRNA by cationic NPs, enhances the therapeutic outcome leading to greater cytotoxicity in TNBC cells. Conclusion: The current investigation focuses on designing a multifunctional drug delivery platform for concurrent delivery of either PAC or PARP inhibitor (olaparib) and FOXM1 siRNA in chitosan-coated poly(D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with the ability to emerge as a front runner therapeutic for TNBC therapy.

A phase I study of olaparib in combination with capecitabine-based chemoradiation (CRT) in patients (pts) with locally advanced pancreatic cancer (LAPC).

709 Background: Olaparib is a potent inhibitor of PARP-1, which has a critical role in signalling DNA single strand breaks (SSB) as part of the base excision repair pathway, and may have radio-sensitizing effects due to impaired resolution of radiation induced SSB. We hypothesize that O may potentiate the effects of X-CRT in pts with LAPC. Methods: Eligible pts with LAPC, ECOG < 1, tumor diameter < 6cm, with stable disease (SD) or response after 12 weeks’ induction chemotherapy, were treated with 1 of 4 escalating doses of O given bid po starting on day -3, and then in combination with X (830 mg/m2 bid) and radiation (50·4 Gy in 28 fractions) all administered Mon-Fri. Dose limiting toxicities (DLT) were determined on clinical and lab toxicity assessments (NCI-CTC AE v4.03) performed weekly from the start of O until completion of O plus X-CRT (i.e. 6 weeks). Dose escalation continued with a rolling-six design until the Maximum Tolerated Dose (MTD) was reached. Blood samples for PK analyses of O and PD measurement (inhibition of PARP activity) were collected on day -3 (O monotherapy) and during week 1 of O + X-CRT. Results: 18 pts, (9 m, 9 f, ECOG 0/1 [n=6/12]), age range 49-81 (median=70) years, with histologic (14) or cytologic (4) proven LAPC, had received induction chemotherapy with gemcitabine [GEM] (n=2), GEM + X (12), or FOLFIRINOX (3) with partial response (n=4) or stable disease (14). Pts received 50 (3), 100 (4), 150 (6), or 200 (5) mgs bid of O with X+CRT. DLTs were observed in 2 pts (both at 200mgs bid): 1 pt with grade 3 nausea (on optimal anti-emetics) and grade 3 fatigue, 1 pt with grade 3 anorexia. 6 pts were subsequently recruited at 150mgs bid with no DLTs. No pts had complete response, 2 pts had partial response (1 pt each at 100 and 150 mgs bid) and 1 pt (at 100 mgs bid) had progressive disease; the remaining 14 pts had SD. Conclusions: The recommended dose (RP2) of O is 150mgs bid when given in combination with X + CRT in LAPC. Recruitment of up to 12 pts with borderline operable LAPC at the RP2 is ongoing. PK analyses of O, PD studies (PARP inhibition – PBMCs; cytokeratin 18 – serum; γH2AX foci – hair follicles), and exploratory predictive marker studies (tumor – NGS; RNA exome sequencing) are ongoing. Clinical trial information: ISRCTN10361292.

Treatment with the poly(ADP-ribose) polymerase inhibitor PJ-34 improves cerebromicrovascular endothelial function, neurovascular coupling responses and cognitive performance in aged mice, supporting the NAD+ depletion hypothesis of neurovascular aging.

Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) plays an important role in the maintenance of healthy cognitive function. Strong evidence demonstrates that age-related cerebromicrovascular endothelial dysfunction and consequential impairment of NVC responses contribute importantly to cognitive decline. Recent studies demonstrate that NAD+ availability decreases with age in the vasculature and that supplemental NAD+ precursors can ameliorate cerebrovascular dysfunction, rescuing NVC responses and improving cognitive performance in aged mice. The mechanisms underlying the age-related decline in [NAD+] in cells of the neurovascular unit are likely multifaceted and may include increased utilization of NAD+ by activated poly (ADP-ribose) polymerase (PARP-1). The present study was designed to test the hypothesis that inhibition of PARP-1 activity may confer protective effects on neurovascular function in aging, similar to the recently demonstrated protective effects of treatment with the NAD+ precursor nicotinamide mononucleotide (NMN). To test this hypothesis, 24-month-old C57BL/6 mice were treated with PJ-34, a potent PARP inhibitor, for 2weeks. NVC was assessed by measuring CBF responses (laser speckle contrast imaging) in the somatosensory whisker barrel cortex evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. Treatment with PJ-34 improved NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory. PJ-34 treatment also improved endothelium-dependent acetylcholine-induced relaxation of aorta rings. Thus, PARP-1 activation, likely by decreasing NAD+ availability, contributes to age-related endothelial dysfunction and neurovascular uncoupling, exacerbating cognitive decline. The cerebromicrovascular protective effects of pharmacological inhibition of PARP-1 highlight the preventive and therapeutic potential of treatments that restore NAD+ homeostasis as effective interventions in patients at risk for vascular cognitive impairment (VCI).

PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H2O2, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H2O2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.

Modulating the tumor microenvironment to enhance efficacy of PARP inhibitors.

e14715 Background: PARP inhibitors (PARPi) have demonstrated activity in HR deficient tumors through synthetic lethality. However, their clinical efficacy has not been highly promising, with response rates ranging between 30-60% for Olaparib, Talazoparib and Veliparib (1). This highlights the necessity to understand the mechanisms of resistance and how these agents work in the context of the tumor microenvironment. In this study, we explore the effect of hypoxia on the efficacy of PARPi in a range of HR-deficient tumors. Hypoxia is known to downregulate genes of homologous recombination (2) but its role in HR deficiency in still unknown. Methods: HR deficient tumor cell lines were treated with PARPi under both aerobic and hypoxic conditions. DNA damage markers such as γH2AX, 53BP1 and RIF1 were detected by immunofluorescence. Tumor xenografts were grown to 100 mm3 prior to treatment with vehicle, PARPi, hypoxia activated cytotoxin, or the combination and tumor volumes were measured. Co-localization of apoptosis and hypoxia was detected by IHC. Finally, hypoxic signature in breast PDX models was correlated with their sensitivity to PARPi. Results: We found that hypoxic cells are significantly more resistant to PARPi in cell culture models. Efficiency of inhibition of PARP activity by these agents was similar under both air and hypoxia. The DNA damage marker γH2AX increased in cells treated in air, but not under hypoxia. There was no loss of 53BP1 protein that regulate engagement of the c-NHEJ pathway, indicating that HR has not been restored in these cells (3). However, it’s binding partner RIF1 was lost in hypoxia upon treatment indicating deficient signaling downstream of 53BP1. RIF1 is known to inhibit 5’ resection and its loss leads to resistance to PARPi in BRCA-deficient cells (4). In vivo, combining PARPi with hypoxia activated prodrugs led to substantial tumor growth delay in three xenograft models. DNA damage and apoptosis in these tumors was only detected outside of hypoxic regions positive for pimonidazole. In the breast PDX models, there was a strong correlation between the level of hypoxia in these tumors and their sensitivity to Olaparib. Conclusions: The significant increase in anti-tumor effects with the combination of PARPi and hypoxia-targeting cytotoxins leads us to propose a new strategy for tumors which are not responsive to PARPi alone. 1. Brown JS et al. Br J cancer 2016, 114, 713-5 2. Chan N et al. Cancer Res 2010, 70,8045-54 3. Bunting SF et al. Cell 2010, 141, 243-54 4. Chapman JR et al. Mol Cell 2013, 49, 858-71.

MUC1-C Integrates Chromatin Remodeling and PARP1 Activity in the DNA Damage Response of Triple-Negative Breast Cancer Cells.

The oncogenic MUC1-C protein is overexpressed in triple-negative breast cancer (TNBC) cells and contributes to their epigenetic reprogramming and chemoresistance. Here we show that targeting MUC1-C genetically or pharmacologically with the GO-203 inhibitor, which blocks MUC1-C nuclear localization, induced DNA double-strand breaks and potentiated cisplatin (CDDP)-induced DNA damage and death. MUC1-C regulated nuclear localization of the polycomb group proteins BMI1 and EZH2, which formed complexes with PARP1 during the DNA damage response. Targeting MUC1-C downregulated BMI1-induced H2A ubiquitylation, EZH2-driven H3K27 trimethylation, and activation of PARP1. As a result, treatment with GO-203 synergistically sensitized both mutant and wild-type BRCA1 TNBC cells to the PARP inhibitor olaparib. These findings uncover a role for MUC1-C in the regulation of PARP1 and identify a therapeutic strategy for enhancing the effectiveness of PARP inhibitors against TNBC. SIGNIFICANCE: These findings demonstrate that targeting MUC1-C disrupts epigenetics of the PARP1 complex, inhibits PARP1 activity, and is synergistic with olaparib in TNBC cells.

PO-484 Development of a two-step screening-and-confirmation approach to efficiently identify synergistic drug combinations with the PARP inhibitor niraparib

IntroductionPoly (ADP-ribose) polymerases (PARPs) are important players in DNA damage repair. Inhibition of PARP activity is highly effective against cancers deficient in homologous recombination repair due to, a.o., BRCA1/2 mutations. Clinically, PARP inhibitors (PARPi) are used to treat, a.o., ovarium cancers. Although effective, repeated treatment with PARPi may lead to resistance. To identify new synergistic drug combinations, we developed a two-step approach of screening and confirmation using two of our platform technologies SynergyScreen and SynergyFinder. We first screen for synergy in presence of a fixed concentration of compound (e.g., PARPi), followed by confirmation using dose response curves. The PARPi niraparib was used as a proof-of-concept combinatorial drug at a fixed concentration, and combined with over 150 anti-cancer agents.Material and methodsProliferation of cell lines and patient derived primary ovarium cancer cells was measured using ATPLite 1 Step after 120 hours compound incubation. Growth was calculated relative to vehicle treated cells; relative IC50s were calculated from a four parameter logistics curve. The fixed concentration niraparib used in the SynergyScreen represented 80% viability. Anti-cancer agents were screened in absence and presence of niraparib, followed by analysis of IC50 shifts. Combinations with IC50shifts>2 fold indicated a synergistic hit and were re-examined using equipotent mixtures and calculation of Combination Index (CI) within our SynergyFinder platform.1 CI <1 indicates synergy. As a control, the combinatorial drug was tested against itself.Results and discussionsResults of the SynergyScreen showed various synergistic drug combinations from the high-throughput setup, i.e. niraparib in combination with the topoisomerase I inhibitor irinotecan (CI0.5=0.70) and the DNA alkylating agent temozolomide (CI0.5=0.42). These and other synergies were confirmed in patient samples. Independently from niraparib, the bromodomain inhibitor JQ1 and the histone acetyltransferase inhibitor anacardic acid showed synergy (CI0.5 0.42), which was not demonstrated before.ConclusionOur Synergy platforms can identify novel and reproducible synergistic drug combinations in an unbiased and efficient manner. Proof-of-concept was demonstrated and new synergistic drug combinations were found. Our method is therefore excellently suited for the in vitro discovery and validation of synergistic combinations.ReferenceUitdehaag, et al. PLoS ONE 2015;10(5): e0125021.