Effects Of PARP Inhibitors Research Articles

Abstract 2814: Antitumor effects of WEE1 inhibitor, AZD1775 alone or in combination with PARP inhibitor in triple negative breast cancer cells

Abstract Background: Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. Especially, recent reports have shown that DNA replication stress and double strand breaks are induced by the activation of CDK1 following WEE1 inhibitor treatment. We investigated the effects and underlying mechanisms of WEE1 inhibitor, AZD1775, in Triple Negative Breast Cancer (TNBC). In addition, we examined whether AZD1775 would enhance the anti-tumor effect of PARP inhibitor in TNBC cells by modulating DNA repair activity. Methods: MTT assay was performed to investigate the antitumor effect of WEE1 inhibitor, AZD1775 alone or with PARP inhibitor in breast cancer cell lines. The regulation of DNA damage response activity by AZD1775 or/and PARP inhibitor was accessed by the comet and western blotting. The status of the S phase was evaluated by EdU-BrdU dual pulse labeling, and the drug effect was also confirmed in vivo by mouse xenograft model of TNBC. Results: AZD1775 showed heterogeneous antitumor effects among TNBC cells regardless of TP53 mutation statues. WEE1 inhibition led to accelerated S phase progression and mitotic entry in the sensitive MDA-MB-231 cells. In addition, increased DNA damage and caspase-3 dependent apoptosis were observed in sensitive cells following AZD1775 treatment. Furthermore, AZD1775 enhanced cellular sensitivity to PARP inhibitor through increase apoptosis and DNA damage accumulation. In a MDA-MB-231 xenograft model, AZD1775 impedes tumor growth as well. Conclusions: In the TNBC, the WEE1 inhibitor has a heterogeneous cytotoxic effect regardless of the p53 mutation status. In MDA-MB-231 cell, which is a sensitive TNBC cells to WEE1 inhibitor AZD1775, the progression of the cell cycle was accelerated and the aberrant DNA content was increased. This resulted in DNA damage, which led to cell death, and this effect could be applied in vivo. In addition, we investigated that WEE1 inhibition can increase PARP inhibitor sensitivity in TNBC cells via increasing accumulation of DNA damage. Our results provide a rationale for the future clinical trials of PARP inhibitor combined with WEE1 inhibition in triple negative breast cancer. Citation Format: Dong Hyeon Ha, Arrum Min, Seongyeong Kim, So Hyeon Kim, Hyemin Jang, Yu Jin Kim, Daeun Jung, YoonJung Park, Koung Jin Suh, Kyung-Hun Lee, Tae-Yong Kiim Kim, Seock-Ah Im. Antitumor effects of WEE1 inhibitor, AZD1775 alone or in combination with PARP inhibitor in triple negative breast cancer cells [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 2814.

Abstract 626: Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response

Abstract Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit. Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines. Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid. Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages. Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona Yull. Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response [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 626.

Sex differences in the effects of PARP inhibition on microglial phenotypes following neonatal stroke

Neonatal acute ischemic stroke is a cause of neonatal brain injury that occurs more frequently in males, resulting in associated neurobehavioral disorders. The bases for these sex differences are poorly understood but might include the number, morphology and activation of microglia in the developing brain when subjected to stroke. Interestingly, poly (ADP-ribose) polymerase (PARP) inhibition preferentially protects males against neonatal ischemia. This study aims to examine the effects of PJ34, a PARP inhibitor, on microglial phenotypes at 3 and 8 days and on neurobehavioral disorders in adulthood for both male and female P9 mice subjected to permanent middle cerebral artery occlusion (pMCAo). PJ34 significantly reduced the lesion size by 78% and reduced the density of CX3CR1gfp-labeled microglial cells by 46% when examined 3 days after pMCAo in male but not in female mice. Eight days after pMCAo, the number of Iba1+/Cox-2+ cells did not differ between male and female mice in the cortical peri-infarct region. In the amygdala, Iba1+/Cox-2+ (M1-like) cell numbers were significantly decreased in PJ34-treated males but not in females. Conversely, Iba1+/Arg-1+ (M2-like) and Arg-1+/Cox-2+ (Mtransitional) cell numbers were significantly increased in PJ34-treated females. Regarding neurobehavioral disorders during adulthood, pMCAo induced a motor coordination deficit and a spatial learning deficit in female mice only. PJ34 prevented MBP fibers, motor coordination and learning disorders during adulthood in female mice. Our data show significant sex differences in the effects of PARP inhibition on microglia phenotypes following neonatal ischemia, associated with improved behavior and myelination during adulthood in females only. Our findings suggest that modulating microglial phenotypes may play key roles in behavior disorders and white matter injury following neonatal stroke.

MicroRNA‐203 regulates cell junction and adhesion proteins by modulating Zeb1/Vimentin axis in breast cancer cells after PARP1 inhibition.

PARP inhibitors are currently in trend to treat various type of cancers and they have shown promising results in homologous recombination deficient breast and ovarian cancers like those bearing BRCA1/2 mutations. There are many reports describing about the failure of PARP inhibitors due to resistance or unknown factors in clinical trials. It is crucial to look into in‐depth molecular mechanisms behind the role of PARP in cancer pathogenesis and long term effect of PARP inhibition. MicroRNAs (miRNAs) are small non‐coding RNAs, acting as post‐transcriptional regulators of gene expression and known to regulate variety of cellular processes, including the DNA damage response and regulation of cell junction and adhesion proteins. However, little is known about miRNAs mediated regulation of sensitivity to PARP inhibitors and its pathological significance in context to regulation of cell junction and adhesion proteins. Aberrant miRNA regulation of junction proteins is one of the leading causes of cancer pathogenesis. Keeping these views in consideration, we aimed to identify novel miRNAs involved in the regulation of cell junction and adhesion proteins in breast cancer malignancy after PARP inhibition. To investigate this, we performed miRNA microarray analysis of PARP inhibitor treated MDA‐MB‐231 cells and found upregulation of tumor suppressor miR‐203 after PARP inhibition. PARP1 inhibition led to reduced expression of vimentin and Zeb1 while ZO‐1, E‐cadherin and RhoGDI were upregulated in breast cancer cells. To confirm the whether PARP inhibitory effects were mediated by miR‐203, cells were transfected with miR‐203 mimic and anti‐miR. We observed that miR‐203 mimic resulted in inhibition of junction proteins Zeb1 and vimentin, coinciding with PARP inhibition. We also found that PARP inhibition caused reduced cell growth and proliferation of breast cancer cells which correlated with the overexpression of miR‐203. Our findings indicates that upregulation of miR‐203 due to PARP inhibition is involved in regulating cell junction proteins vimentin and zeb1 as well as cytosketelal RhoGTPase and thus regulating cell migration, invasion and proliferation. Taken together, these observations raise the possibility that these miRNAs could be used as biomarkers to identify patients that may benefit from treatment with PARP inhibitors.Support or Funding InformationJR acknowledge the support from Department of Biotechnology (DBT) for DBT Bio‐CARe fellowship. NS acknowledges the support by the senior research fellowship from the Indian Council of Medical Research (ICMR). This work was supported by the grants from Department of Biotechnology (GAP0187) to DP Mishra.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancer

PARP inhibitors have been widely tested in clinical trials, especially for the treatment of breast cancer and ovarian cancer, and were shown to be highly successful. Because PARP primarily functions in sensing and repairing DNA strand breaks, the therapeutic effect of PARP inhibition is generally believed to be attributed to impaired DNA repair. We here report that oxidative stress is also increased by PARP inhibition and mediates the antitumor effect. We showed that PARP1 is highly expressed in specimens of high grade serous ovarian carcinoma and its activity is required for unperturbed proliferation of ovarian cancer cells. Inhibition or depletion of PARP leads to not only an increase in DNA damage, but also an elevation in the levels of reactive oxygen species (ROS). Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation by PARP inhibition or depletion. We further showed that NADPH oxidases 1 and 4 were significantly upregulated by PARP inhibition and were partially responsible for the induction of oxidative stress. Depletion of NOX1 and NOX4 partially rescued the growth inhibition of PARP1-deficient tumor xenografts. Our findings suggest that in addition to compromising the repair of DNA damage, PARP inhibition or depletion may exert extra antitumor effect by elevating oxidative stress in ovarian cancer cells.

Asthma and poly(ADP-ribose) polymerase inhibition: a new therapeutic approach.

Asthma is a chronic lung disease affecting people of all ages worldwide, and it frequently begins in childhood. Because of its chronic nature, it is characterized by pathological manifestations, including airway inflammation, remodeling, and goblet cell hyperplasia. Current therapies for asthma, including corticosteroids and beta-2 adrenergic agonists, are directed toward relieving the symptoms of the asthmatic response, with poor effectiveness against the underlying causes of the disease. Asthma initiation and progression depends on the T helper (Th) 2 type immune response carried out by a complex interplay of cytokines, such as interleukin (IL) 4, IL5, and IL13, and the signal transducer and activator of transcription 6. Much of the data resulting from different laboratories support the role of poly(ADP-ribose) polymerase (PARP) 1 and PARP14 activation in asthma. Indeed, PARP enzymes play key roles in the regulation and progression of the inflammatory asthma process because they affect the expression of genes and chemokines involved in the immune response. Consistently, PARP inhibition achievable either upon genetic ablation or by using pharmacological agents has shown a range of therapeutic effects against the disease. Indeed, in the last two decades, several preclinical studies highlighted the protective effects of PARP inhibition in various animal models of asthma. PARP inhibitors showed the ability to reduce the overall lung inflammation acting with a specific effect on immune cell recruitment and through the modulation of asthma-associated cytokines production. PARP inhibition has been shown to affect the Th1–Th2 balance and, at least in some aspects, the airway remodeling. In this review, we summarize and discuss the steps that led PARP inhibition to become a possible future therapeutic strategy against allergic asthma.

Analysis of DNA damage response (DDR) genes and tumor mutational burden (TMB) across 17,486 carcinomas of the tubular GI tract: Implications for therapy.

43 Background: The DDR pathway is important in tumor biology, allowing cancer cells a mechanism to resist damage by chemotherapy and radiotherapy. BRCA1/2 are the most well described genes in the pathway, but several others ( ATM, ATR, PALB2, etc.) are involved with DDR and are mutated in many cancers. Tumors with a DDR defect are susceptible to PARP inhibition (PARPi) in some cases, but also potentially to immune checkpoint inhibitors (ICPIs), given immunomodulatory effects of PARPi and the hypermutated phenotype commonly associated with these genomic alterations (GA). We looked at the prevalence of select DDR defects in tubular GI cancers and the correlation between DDR and TMB. Methods: We prospectively analyzed samples from 17,486 unique patients with advanced colorectal (CRC), esophageal, gastric, or small bowel carcinomas using hybrid-capture based comprehensive genomic profiling. TMB (mutations/Mb) was calculated from 1.11 Mb of sequenced DNA. We looked at GA in 8 genes- ARID1A, ATM, ATR, BRCA1, BRCA2, CDK12, PALB2, RAD51 across these tumors types. Results: DDR GA were found in 16% of cases: gastric 464/1,750 (27%), small bowel 141/666 (21%), esophageal 441/2,501 (18%), and CRC 1,824/12,569 (15%). ARID1A (9.0%) and ATM (5%) were the most commonly altered DDR genes in this series, followed by BRCA2 (2.3%), BRCA1 (1.1%), ATR (0.8%), CDK12 (0.7%), PALB2 (0.6%), and RAD51 (0.1%), with 24% (675/2,870) of DDR-altered cases having GA in more than one DDR gene. Among DDR-altered cases, 21% had high TMB (≥20 mut/Mb) compared to just 1.4% high TMB in DDR-wild-type cases (p < 0.001). Microsatellite instability (MSI) status was available for a subset of cases and 19% (419/2,154) of those with a DDR GA were MSI high. CDK12 and ATR altered cases had the highest proportion of high TMB: CDK12 (55%, median TMB 31.5 mut/Mb) and ATR (55%, median TMB 35.1 mut/Mb), followed by cases with GA in BRCA2 (40%), BRCA1 (28%), ARID1A (27%), ATM (22%), and RAD51 (20%). Conclusions: DDR defects are relatively common across tubular GI tumor types and are associated with a hypermutated phenotype in over 20% of cases. This may be important to identify likely responders to both PARPi and ICPIs.

Restricted Delivery of Talazoparib Across the Blood–Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2-M arrest in vitroIn vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp-/- versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b-/- mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735-46. ©2017 AACR.

Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells

Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death. PARP-inhibitor-induced multinucleated cells fail clonogenic outgrowth, and high percentages of multinucleated cells are found in vivo in remnants of PARP inhibitor-treated Brca2−/−;p53−/− and Brca1−/−;p53−/− mammary mouse tumours, suggesting that mitotic progression promotes PARP-inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogates PARP-inhibitor-induced cytotoxicity. These findings provide insight into the cytotoxic effects of PARP inhibition, and point at combination therapies to potentiate PARP inhibitor treatment of HR-deficient tumours.

Abstract 5863: Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer

Abstract Ovarian cancer is the seventh-most common cancer with a high mortality rate among women. Radiation therapy (RT) is used in ovarian cancer for local relapse, but doses are limited due to concern for small bowel obstruction in patients who have had multiple surgeries and chemotherapeutic regimens. Therefore, it’s an ideal setting to improve the therapeutic window by combining RT and a radiosensitizer. Poly (ADP-ribose) polymerase-1 (PARP-1) is involved in the recognition of DNA damage and the facilitation of DNA repair by recognizing DNA single-strand breaks (SSB). Inhibition of PARP1, especially in tumors driven by BRCA1 or 2 mutations, has been shown to potentiate the DNA-damaging effects of radiation in vitro and in vivo and to increase tumor vasculature perfusion and oxygenation. Approximately half of ovarian cancers have germline or somatic BRCA gene mutation, epigenetic silencing or other mutations that affect homologous recombination (HR) competency resulting in DNA repair defects, making this tumor particularly susceptible to the radiation-sensitizing effects of PARP inhibitors. Here, we investigated the therapeutic effects of the PARP inhibitor olaparib in preclinical models of ovarian cancer. Basic BRCA1 and PARP1 protein and mRNA levels were characterized in a panel of ovarian cancer cell lines. The radiosensitizing effects of olaparib were tested on both HR-proficient and HR-deficient ovarian cancer cells by evaluating the colony formation, DNA damage, apoptosis, and PARP1 activity. Survival benefit of olaparib was also examined in xenograft models of ovarian cancer. Our results demonstrate that combination of olaparib with IR decreased colony formation, inhibited DNA damage (γH2AX) repair, and induced more apoptosis compared to olaparib or IR alone. In addition, HR-deficient cells were more sensitive to IR than HR-proficient cells in the presence of olaparib. Furthermore, olaparib inhibited PARP1 activity in vivo, significantly decreased tumor growth and increased overall survival when combined with IR in mice bearing subcutaneous xenografts of HR-deficient OVCAR8 cells while producing a relative modest effect on overall survival of mice bearing xenografts of HR-proficient SKOV3 cells. These results provide a preclinical rationale for improved treatment modalities using olaparib as an effective radiosensitizer in ovarian cancer, particularly in tumors with HR-deficiencies. Citation Format: Yue Bi, Ioannis Verginadis, Souvik Dey, linlang Guo, Lilie Lin, Yanfang Zheng, Constantinos Koumenis. Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5863. doi:10.1158/1538-7445.AM2017-5863

Abstract 3453: Progression through mitosis promotes PARP inhibitor induced cytotoxicity in homologous recombination deficient cancer cells

Abstract Mutations in homologous recombination (HR) DNA repair genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. The mechanisms underlying PARP inhibitor resistance are only partially understood. This lack of knowledge in part stems from incomplete understanding of how PARP inhibitors induce cytotoxicity in HR-deficient cancers. The aim of this study was to investigate the contribution of mitotic failure to PARP inhibitor-induced cytotoxicity. Using the DNA fiber assay we observed compromised replication fork protection upon BRCA2 depletion, which was aggravated upon PARP inhibition. In accordance with the loss of replication fork stability, we found increased FANCD2 foci in interphase cells upon BRCA2 depletion, and a significant further increase when BRCA2-depleted cells were treated with PARP inhibitor as assessed by immunofluorescence. Importantly, these replication lesions were not resolved prior to mitotic entry, as increased numbers of FANCD2 foci were observed in mitotic cells of BRCA2-depleted cells, which were further increased upon PARP inhibitor treatment. Subsequently we found that in mitosis, BRCA2 or BRCA1 and Brca2 or Brca1 deletion resulted in elevated levels of chromatin-bridges and lagging chromosomes which were greatly enhanced after PARP inhibition. Live-cell microscopy showed that chromatin bridges in anaphase often lead to cytokinesis failure with subsequent multinucleated progeny cells or cell death. Furthermore, PARP inhibitor-induced multinucleated cells failed clonogenic outgrowth and high percentages of multinucleated cells were found in vivo in the remnants of Brca2-/-;p53-/- and Brca1-/-;p53-/- mammary mouse tumors upon PARP inhibitor treatment, suggesting that mitotic progression promotes PARP inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogated the cytotoxic potential of PARP inhibitor sensitivity and indicates that progression through mitosis promotes cell death in response to PARP inhibitors in HR-defective cells. These findings add to our understanding how PARP inhibition induces cytotoxicity. Furthermore, these results provide starting points in tackling tumor resistance to PARP inhibitors, and point at combination therapies which could potentiate the effects of PARP inhibitors for the treatment of HR-deficient tumors. Citation Format: Pepijn M. Schoonen, Peter Bouwman, Floris Foijer, Madalena Tarsounas, Sohvi Blatter, Jos Jonkers, Sven Rottenberg, Marcel A. Vugt, van. Progression through mitosis promotes PARP inhibitor induced cytotoxicity in homologous recombination deficient cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3453. doi:10.1158/1538-7445.AM2017-3453

Abstract 1930: Evaluating the effect of PARP inhibitors in combination with nicotinamide phosphoribosyltransferase inhibitors in Ewing sarcoma

Abstract Previous research has shown that Ewing sarcoma cells are sensitive to PARP inhibition, but in vivo studies and early phase clinical trials have failed to demonstrate meaningful responses to PARP inhibitors when used as single agents. For this reason, studies combining PARP inhibitors with other agents to enhance their effects in Ewing sarcoma are of particular interest. PARP, an enzyme involved in DNA damage repair, relies on NAD to function. In tumor cells, NAD production occurs primarily through the salvage pathway, in which nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme. Hence, the combination of PARP and NAMPT inhibition would be expected to enhance the activity of PARP inhibition through depletion of cellular NAD. The aim of this study was to assess the synergistic potential of PARP inhibition in combination with NAMPT inhibition in Ewing sarcoma cell lines and xenograft models. Synergistic drug combinations in Ewing sarcoma cells were initially identified using a high-throughput matrix drug screen. In vitro activity was further assessed using proliferation assays of multiple Ewing sarcoma cell lines treated with the PARP inhibitor niraparib and the NAMPT inhibitor GNE-618. In vitro measurement of PAR activity was performed to further understand the mechanism of action of the combination. For in vivo studies, female SCID-biege mice were orthotopically injected with Ewing sarcoma cells and randomized into treatment groups of vehicle, niraparib, GNE-618, or the combination. The initial screen revealed synergy between NAMPT inhibitors and PARP inhibitors using multiple combinations of different agents in each drug class. In vitro assays of the combination of niraparib with GNE-618 confirmed the results of the screen. Assays measuring PAR activity yielded results supporting the hypothesis that dual inhibition of NAMPT and PARP depletes PARP more than PARP inhibitors alone. In vivo studies with short term (5 day) dosing showed no activity of single agent nirapirib and only temporary disease stabilization with single agent GNE-618. However, mice treated with the combination underwent tumor regressions resulting in prolonged survival. When treatments were extended to 2 cycles (5 days each), the effect persisted for longer. Mice tolerated the agents well, with no signs of toxicity. Preclinical data suggest that PARP inhibition in combination with NAMPT inhibition may be a promising therapeutic strategy for Ewing sarcoma patients. Citation Format: Joshua T. Baumgart, Christine Heske, Mindy I. Davis, Kelli Wilson, Xiaoha Zhang, Rajarshi Guha, Marc Ferrer, Arnulfo Mendoza, Craig J. Thomas, Lee J. Helman. Evaluating the effect of PARP inhibitors in combination with nicotinamide phosphoribosyltransferase inhibitors in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1930. doi:10.1158/1538-7445.AM2017-1930

Abstract 1422: Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Treatment most commonly relies on ionizing radiation (IR) and platinum-based DNA damaging agents, but long-term survival is poor and patients tend to suffer chronic side-effects due to the high radiation dose to the surrounding normal tissues. Therefore, new treatments are needed that can be used in combination lower radiation doses. We have recently reported that low, non-cytotoxic doses Poly ADP ribose polymerase inhibitors (PARPi) Talazoparib in combination with DNA methyltransferase inhibitors (DNMTi) Decitabine (DAC) or azacytidine (AZA) significantly increase cytotoxicity in acute myeloid leukemia and breast cancer models in vitro and anti-tumor effects in vivo. Simultaneous administration of both inhibitors result in increased PARP binding in DNA, leading to higher levels of DNA double strand breaks (DSBs), yielding increased cytotoxicity, compared with each agent treatment alone. We first studied the efficacy of Talazoparib and AZA combination therapy in multiple NSCLC cell lines (A549, H358 and H838) in vitro through colony forming assays. Results showed, compared to single agent treatments, combination drug treatment significantly decreased colony formation. Cell viability was also significantly decreased with the drug combination in MTS assays (P&amp;lt;0.05). These results also showed synergistic activity between the two drugs, with a combination index of less than 1 for all tested NSCLC cell lines. Importantly, the drug combination increased PARP trapping in chromatin and DSB formation, as measured by immunofluorescent staining for γH2AX. We next determined whether AZA/Talazoparib in combination with a single dose of radiation (2Gy) had an increased anti-cancer effect compared to each modality alone in in vivo mouse xenografts of NSCLC A549. While the combination of PARPi+DNMTi with radiation treatment decreased tumor growth, compared to PARPi/DNMTi alone or RT alone, no significant enhancement with radiation was observed. We next determined whether low doses of fractionated IR (2Gy 3 fractions) would improve the efficacy of DNTMi and PARPi combination treatment. In vitro studies with colony forming assays of NSCLC cell line A549 show that IR doses in combination with PARPi and DNMTi decrease clonogenicity, compared with non-IR controls. Furthermore, A549 xenografts were treated with the drug combination and then irradiated 1 week later with 2Gy daily for 3 consecutive days. Mice treated with the drug combination followed by IR had significant decreases in tumor volume and survival (P&amp;lt;0.05). This suggests that low doses of PARPi and DNMTi therapy in combination with low dose IR can potentially target NSCLC tumors. This represents a novel treatment approach for NSCLC patients that may reduce chronic side-effects of high dose IR. Citation Format: Christopher Biondi, Daniel Fontaine, Lora Stojanovic, Pratik Nagaria, Rena Lapidus, Eun Yong Choi, Javed Mahmood, Stephen Baylin, Feyruz V. Rassool. Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1422. doi:10.1158/1538-7445.AM2017-1422

Cytotoxicity of anticancer drugs and PJ-34 (poly(ADP-ribose)polymerase-1 (PARP-1) inhibitor) on HL-60 and Jurkat cells.

The majority of the clinical trials with poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors were conducted or are ongoing in patients with solid tumors, while trials with leukemia patients are less frequent. Surprisingly scarce data is available on the combinatory effects of PARP inhibitors with DNA damaging antitumor drugs in leukemic cells (primary cells or established lines). The aim of the present study was to assess the effect of PJ-34 (PARP-1 inhibitor) on the cytotoxicity of different antileukemic drugs with different DNA damaging mechanisms and potency (doxorubicin, etoposide, cytarabine and chlorambucil) in human leukemic Jurkat and HL-60 cells. Different exposure scenarios were applied: 1) 72 h simultaneous incubation with PJ-34 (2.5 or 5 μM for Jurkat and HL-60 cells, respectively) and a drug used at a wide concentration range; 2) preincubation of the cells with PJ-34 for 24 h and then with a combination of PJ-34 + drug for an additional 48 h; 3) preincubation of the cells with the drug for 24 h with a subsequent incubation with a combination of PJ-34 + drug for an additional 48 h. Cytotoxicity was assessed using a WST-1 reduction test. It was determined that PJ-34, when used in all 3 scenarios, did not induce any significant enhancement of cytotoxicity of the drugs either in Jurkat or in HL-60 cells. Although the results do not confirm the beneficial effects of PARP inhibition in combination treatment of the leukemic cells, we propose that future studies including an additional step with the inhibition of DNA repair by homologous recombination should provide promising results.

Targeting the ATR/CHK1 Axis with PARP Inhibition Results in Tumor Regression in BRCA-Mutant Ovarian Cancer Models.

Purpose: PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression.Experimental Design: Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell cycle, genome instability, and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived xenograft (PDX) model.Results: PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum tolerated dose (MTD) in a BRCA2MUT PDX. PARPi treatment increased pATR and pCHK1, indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared with single-agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA2MUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission.Conclusions: PARPi causes increased reliance on ATR/CHK1 for genome stability, and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models. Clin Cancer Res; 23(12); 3097-108. ©2016 AACR.

A phase I study of carboplatin and talazoparib in patients with and without DNA repair mutations.

2527 Background: Talazoparib is a novel PARP inhibitor (PARPi) in clinical development. Synergistic anti-tumor effects of PARPi and chemotherapy have been observed in preclinical models. Overlapping toxicity may limit tolerability in patients with germline DNA repair defects. Methods: In a dose escalation Phase 1 trial, we tested the safety, tolerability, pharmacokinetics (PK), and efficacy of talazoparib and carboplatin in patients with and without germ line mutations. Results: 24 patients with solid tumors were enrolled in 4 cohorts evaluating talazoparib 0.75 or 1 mg daily and carboplatin AUC 1 or 1.5 mg/mL/min 2 or 3 weeks of a 3-week cycle. Dose-limiting toxicities included grade 3 fatigue and grade 4 thrombocytopenia. Other grade 3/4 toxicities included fatigue (13%), neutropenia (63%), thrombocytopenia (29%), and anemia (38%). Post cycle 2 hematologic toxicities required dose delays/reductions in all patients. One complete and two partial responses occurred in germline BRCA1/2 (gBRCA1/2) patients. Of the 4 patients with stable disease beyond 4 months, 3 had somatic BRCA mutations and 1 had a BRIP1 germline mutation suggesting greater benefit in tumors with DNA repair mutations. PK-toxicity modeling suggests that after 3 cycles of carboplatin AUC 1.5 weekly and talazoparib 1 mg daily, the percent decrease in neutrophil counts from baseline was significantly more pronounced in gBRCA carriers; -78% (95% CI: -87 to -68%) vs. non-carriers; -63% (95% CI: -72 to -55%), p-value &lt; 0.001. This modeling also showed that 2-4 fold dose reductions for each drug are needed to improve tolerability of this combination. Pulse dosing of talazoparib may be more tolerable in gBRCA carriers. Conclusions: The combination of carboplatin and talazoparib showed responses in gBRCA carriers but also had increased hematologic toxicity in gBRCA carriers. PK toxicity modeling was used to determine alternate dosing strategies based on carrier status. Clinical trial information: NCT02317874.

Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

In diabetes, matrix metalloproteinase-9 (MMP-9) is activated, which damages mitochondria, resulting in accelerated capillary cell apoptosis. Regulation of MMP-9 is controlled by multiple transcription factors including nuclear factor-kB (NF-kB) and activator protein-1 (AP-1). Binding of these transcription factors, however, can be regulated by poly(ADP-ribose) polymerase-1 (PARP-1), which forms a strong initiation complex at the promoter region and facilitates multiple rounds of gene transcription. This complex formation with the transcription factors is regulated by posttranslational acetylation of PARP-1, and in diabetes, the deacetylating enzyme, Sirt1, is inhibited. Our aim was to understand the role of PARP-1 in transcriptional regulation of MMP-9 in the development of diabetic retinopathy. Using human retinal endothelial cells, the effect of PARP-1 inhibition (pharmacologically by PJ34, 1μM; or genetically by its siRNA) on MMP-9 expression was investigated. The effect of PARP-1 acetylation on its binding at the MMP-9 promoter, and with NF-kB/AP-1, was investigated in the cells transfected with Sirt1. In vitro results were validated in the retinal microvessels from diabetic mice either administered PJ34, or overexpressing Sirt1. Inhibition of PARP-1 ameliorated hyperglycemia-induced increase in the binding of NF-kB/AP-1 at the MMP-9 promoter, decreased MMP-9 expression and ameliorated mitochondrial damage. Overexpression of Sirt1 attenuated diabetes-induced increase in PARP-1 binding at MMP-9 promoter or with NF-kB/AP-1. Thus, PARP-1, via manipulating the binding of NF-kB/AP-1 at the MMP-9 promoter, regulates MMP-9 expression, which helps maintain mitochondrial homeostasis.

Abstract B33: PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy

Abstract Introduction: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor treatment outcomes and prognoses. Treatment usually consists of surgery, radiation and chemotherapy, which often result in severe systemic side effects frequently accompanied by tumor recurrence of highly radioresistant tumor cell populations. PARP-1 is a nuclear enzyme crucial for DNA damage repair (DDR) response. PARP-1 inhibition is a highly studied adjuvant to many anti-cancer treatment strategies as this arrests DNA damage repair thus enhancing the efficacy of conventional chemoradiotherapy at lower doses. However, PARP inhibition also promotes anti-inflammatory responses. Cancer cells suppress the immune cells ability to recognize and eliminate the tumor and promote their release of cancer-supporting growth factors and promote angiogenesis. Microglia the immune cells of the brain, normally protect the brain from pathogens and help in tissue recovery, but when associated with GBM cells, microglia obtain anti-inflammatory state that can promote tumor growth. We are developing a novel bimodal immunotherapeutic/DNA repair suppressive approach to treating GBM through modulation of microglial PARP-1 activity while concurrently sensitizing GBM to DNA damaging therapeutics. We hypothesize that while PARP-1 inhibition can enhance efficacy of chemoradiotherapy it might further suppress microglial ability to promote tumor elimination. Furthermore PARP-1 inhibitors' efficacy in preventing DNA repair is partially linked to their ability to trap PARP-1 at DNA break-site, which might be detrimental for the viability of non-cancerous brain cells. Objectives: 1) to establish the effects of PARP-1 inhibition alone, and in combination with targeted microglial PARP-1 activation, on microglial ability to eliminate glioblastoma cells in vitro (tumor cells vs. healthy glial cells). 2) to identify how microglial PARP-1 modulation (on/off) impacts glioblastoma elimination in vivo (tumor vs. brain). Methods: We will use GBM cells co-cultured with primary microglial cells in the presence of PARP inhibitors and observe their effect on the proliferation and viability of GBM cells. Efficacy of PARP-DNA trapping and corresponding DNA damage repair activity utilizing a variety of PARP inhibitors will be tested by specific biochemical and genotoxicity assays which directly quantify the degree of PARP-trapping in the glial cells and their impact on tumor cell genotoxcity. These studies take advantage of newly-developed and unique high-throughput DNA damage repair assays that we have been optimized to determine potential synergy of PARP-inhibitors with existing chemotherapeutic agents. Results: DNA damage assays (gamma H2AX foci and alkaline comet analysis) show that co-treatment of GBM cells (U87 and U251) with the PARP-inhibitor Olaparib and the chemotherapeutic agent Camptothecin (CPT) significantly increased the level of DNA damage when compared to CPT treatment alone. In the co-culture experiments, microglia enhanced CPT-induced GBM cell death when treated with Olaparib at 50-100 fold lower doses. Real time co-culture experiments also showed that Olaparib does not jeopardize microglial ability to actively phagocytize GBM cells. Conclusion: Preliminary results show that Olaparib is effective in sensitizing GBM cells to chemotherapy while microglia appear to enhance this effect. Further studies linking this combination therapy with microglia-mediated GBM elimination will help to elucidate the mechanism of this bimodal effect. This study emphasizes the importance of: (1) identifying novel effects of PARP inhibition on microglia in association with GBM and (2) the importance of developing microglia-mediated immunotherapeutic intervention to overcome the limitations of current therapies to significantly improve patient outcome. Citation Format: Asha Sinha, Sachin Katyal, Tiina Kauppinen. PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B33.

Poly (ADP) Ribose Polymerase (PARP)‐1 Is A critical Determinant of Atopic Dermatitis through Control of Allergen‐Induced Th2 And T‐regulatory Responses And Expression of The Matricellular Protein Periostin

BackgroundAtopic dermatitis (AD) is a complex and multi‐factorial chronic inflammatory skin disorder. Individuals with AD have limited treatment options and, thus, identifying the key molecular mechanisms that participate in the pathogenesis of the disease will increase the likelihood of establishing precise strategies to prevent/combat the debilitating symptoms of AD. Immune system defects, including deregulated Th2 responses and hyperproduction of IgE, play a key role in the progression of the disease. Increasing evidence shows that the extracellular matrix (ECM) protein periostin plays an equally important role in AD. Our laboratory has extensively examined the role of PARP‐1 in the pathogenesis of several inflammatory diseases including chronic allergic asthma. PARP‐1 inhibition genetically or pharmacologically by Olaparib, a drug that is currently being used as a monotherapy against human ovarian cancer, provides a significant protection against the onset of the disease by blocking Th2 cytokines and IgE production and several other related factors.ObjectiveTo examined the efficacy of PARP‐1 inhibition by gene deletion or by Olaparib against AD traits using both an in vitro and an animal model of the disease.MethodsWild type and PARP‐1−/− mice were epicutaneously exposed to ovalbumin (OVA) 3 times a week for 3 weeks; each week was separated by a 2‐week resting period. Olparib (5mg/kg) was injected to WT mice. At the end of the protocol, the severity of dermatitis was evaluated using a clinical skin scoring (CSS) system. Mice were euthanized; skin biopsies were analyzed for immunohistopathologic features. Serum were collected and assessed for OVA‐specific IgE and cytokines levels. Spleen‐derived cells were assessed by FACS. WT or PARP‐1−/− skin fibroblasts were treated with LPS or IL‐13 for different time intervals then subjected to RT‐PCR or immunoblot analysis.ResultsPARP inhibition by gene knockout or pharmacologically by Olaparib provided a robust reduction in the manifestation of all traits of the disease including Redness, thickness, scratching and lichenification as assessed by the Eczema Area and Severity Index (EASI). The protective effects of PARP inhibition may be mediated by a differential regulation of Th1/Th2 cytokines production. Indeed, while PARP inhibition suppressed production of Th2 cytokines (e.g. IL‐4/IL‐5/GM‐CSF) and allergen‐specific IgE, it exerted no effect on production of the anti‐inflammatory cytokine IL‐10 or the Th1 cytokine, IFN‐γ. Such differential effects occurred with a statistically significant increase in T‐regulatory cell population. Remarkably, PARP‐1 appeared to play a critical role in periostin protein expression both in vitro using skin fibroblasts and in the skin of mice using the AD animal model. PARP‐1 inhibition may not only alter the function and ability of T cells to produce Th2 cytokines but also regulate the production of the ECM proteins as periostin.ConclusionOur results show for the first time that PARP‐1 plays a critical role in atopic dermatitis. These findings provide evidence for the potential of PARP‐1 inhibitors as a novel therapeutic strategy and Olaparib as a likely candidate to be tested in human clinical trials.Support or Funding InformationNIH

Abstract A38: Targeting Microenvironment Damage Responses via PARP inhibition to Enhance Prostate Cancer Therapy

Abstract Introduction: Radiation and chemotherapeutic drugs have significantly improved the long-term outlook for patients diagnosed with prostate cancer. However, in men with metastatic prostate cancer, the development of radio and chemo resistance is almost universal. As a result, understanding and circumventing chemo/radiotherapy resistance has become a research priority. In addition to damaging neoplastic cells, radiation and chemotherapy also exert potent “bystander” effects on the tumor microenvironment (TME) by induction of a DNA Damage Secretory Program (DDSP). In this study we sought to identify and modulate master regulators of the DDSP in order to augment the effectiveness of conventional cancer therapeutics. The Nelson group and others have shown that NFkB is a master regulator of the DDSP, but pharmacological inhibition of NFkB has been challenging. As PARP can activate NFkB and PARP inhibitors (PARPi) have received clinical approval for cancer therapy, we evaluated the effects of PARPi to modulate the DDSP. Results: We used prostate (PF) and bone fibroblasts (BF) as our experimental model as this cell type comprises a substantial component of the TME in bone. We determined that ionizing radiation (IR) induced DDSP in PFs and BFs that was suppressed by PARPi, and observed significant reductions in transcripts encoding several key tumor promoting DDSP factors such as IL8, Wnt16b. Fractionated IR was more effective in increasing the transcript levels of various DDSP factors compared to single dose of IR. PARPi also suppressed DDSP-induced nuclear translocation of NFkB in fibroblasts and modulated the DDSP via the function/modification of NEMO. Conditioned media from irradiated PFs and BFs or co-culture with them induced growth and therapy resistance in prostate cancer (PCa) cells, indicative of the tumor promoting role of DDSP, which was suppressed after PARPi addition either later or in the cells treated with both IR and PARPi. Another PARP family member, PARP5 inhibition in irradiated BFs showed significant reduction in DDSP marker's transcript level, which was also reflected by inhibited growth of PCa cells when radiated BF's condition media was added along with PARP5 inhibitor. Conclusions: PARP inhibitors, developed primarily to target vulnerabilities in tumor cells directly, may provide additional anti-tumor effects via DDSP and repurposing PARPi to suppress the DDSP could augment responses to radiation and chemotherapy via microenvironment modulation. In addition to PARP1, our ongoing studies are designed to evaluate other PARP family members including PARP5 and PARP10 that may influence damage responses. Citation Format: Payel Chatterjee, Peter Nelson. Targeting Microenvironment Damage Responses via PARP inhibition to Enhance Prostate Cancer Therapy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A38.