Inhibitor Of Poly Research Articles

Abstract 5050: Pharmacodynamic and pharmacokinetic relationship of single agent E7449 in patients with advanced solid tumors or B-cell malignancies

Abstract E7449 is a small-molecule inhibitor of poly (ADP-ribose) polymerase (PARP). Preclinical studies reported antitumor activity of single-agent E7449 in BRCA-deficient in vivo models. An open-label, multicenter, phase 1 study was completed to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of single-agent E7449. The MTD was determined to be 600 mg/day (n = 8). Treatment-emergent adverse events led to drug withdrawal in 1 patient and dose interruption in 2 patients at this dose level. E7449 treatment at the MTD was associated with substantial and sustained dose-dependent PARP inhibition. The overall response rate was 7.1% (2 confirmed partial responses [PR]) and durable stable disease (≥23 weeks) was observed in 21.4% of patients. Here we report additional PD and PK results from this trial. Patients in this study (N=28) were ≥ 18 years and had measurable, confirmed, advanced solid tumors or B-cell lymphoma that had progressed after approved treatment. Patients received E7449 at 50, 100, 200, 600, or 800 mg/day. Food effect was examined in an MTD expansion cohort. PD assessments included measurement of PARP activity and comet assay to determine the extent of DNA damage. In the food-effect cohort (n=13), PARP inhibition (up to 90%) was sustained during treatment with the MTD of E7449 600 mg/day up to 24 hours post-dose. Peak E7449 plasma concentrations were delayed by 2 hours in fed patients compared with fasted patients. In the PK/PD analysis set, peak plasma concentrations of E7449 were observed at 2 hours following a single dose, and coincided with the lowest levels of poly (ADP-ribose) (PAR; up to 90% inhibition). Of note, while E7449 exposure was highest at the 800 mg dose, the lowest PAR levels were observed at the 600 mg dose. At the time of observed responses, the 2 patients with confirmed PR demonstrated greater than 90% PAR inhibition from baseline. E7449 did not cause a change in the level of DNA damage that could be detected by the comet assay. DNA damage levels remained similar to the damage seen in healthy donors. No significant changes in percent DNA in the electrophoresis comet tail by dose or E7449 plasma concentration were observed. In conclusion, continuous E7449 dosing at 600 mg/day was associated with sustained PARP inhibition. Dose-dependent PARP inhibition was observed and the greatest PARP inhibition occurred at the 600 mg dose. Peak E7449 plasma concentration appeared to coincide with maximal PARP inhibition. These results support E7449 dosing at 600 mg/day. Citation Format: Pallavi Sachdev, Shannon McGrath, Robert Shumaker, Jagadeesh Aluri, Claudio Savulsky. Pharmacodynamic and pharmacokinetic relationship of single agent E7449 in patients with advanced solid tumors or B-cell malignancies [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 5050. doi:10.1158/1538-7445.AM2017-5050

Inhibitors of Poly(ADP-Ribose)Polymerase-1 as Agents Providing Correction of Brain Dysfunctions Induced by Experimental Diabetes

The effects of 1,5-isoquinolinediol (IQD) and nicotinamide (NAm), inhibitors of poly-(ADP-ribose) polymerase-1 (PARP-1), on inflammatory processes and activation of PARP-1 under conditions of the development of experimental diabetic neuropathy, DN (a complication of streptozotocin-induced type-1 diabetes) in rats were studied. The content of IL-4 in blood serum in the case of DN was 50% higher, while that of monocyte-chemotactic protein-1 was 90% higher than those in the control. The content of gamma-interferon also increased, while the content of the granulocyte-macrophage colony-stimulating factor did not change. Against the background of activation of PARP-1 and a decrease in the content of the substrate of this enzyme nicotinamide adenine dinucleotide (NAD) in the brain, fragmentation of PARP-1 was intensified; an increase in the ratio of the contents of a 89 kDa fragment/intact enzyme molecules proved this fact. The mentioned two structurally dissimilar PARP-1 inhibitors partly or entirely normalized the above parameters under DN conditions. These results demonstrate that PARP-1 is one of the main functional targets in realization of the effects of IQD and NAm. At the same time, the spectrum of action of these inhibitors is wider. In particular, they affect the level of proinflammatory cytokines. The ability of the investigated PARP-1 inhibitors to prevent cell death in the brain by suppressing activation and fragmentation of the above-mentioned enzyme shows that other types of action of these agents at the molecular level are possible; these may be the maintenance of the genome integrity in the brain structures under DN conditions and preventing the development of inflammatory processes. Thus, the examined inhibitors can be used in the future in the treatment of brain dysfunctions that are complications of type-1 diabetes mellitus.

Accurate and precise targeted NGS with BRCA MASTR Plus Dx for BRCA1 and BRCA2 mutation detection in formalin-fixed paraffin-embedded tumor tissue-derived DNA.

e23116 Background: Targeted next-generation sequencing (NGS) has tremendous potential in clinical diagnostics as it allows oncogenetic profiling to steer therapy. Inhibitors of poly-(ADP-ribose) polymerase (PARPi) have emerged as a new class of targeted anti-cancer drugs, specifically for tumors showing homologous recombination repair deficiency, including BRCA1- and BRCA2-mutated ovarian and breast cancers. This multicentre study evaluated the performance of BRCA Tumor MASTR Plus Dx* (Multiplicom) to routinely diagnose somatic and germline BRCA mutations in formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived DNA. Methods: Three genetic centres participated in this performance evaluation study (PES) to detect single nucleotide variants (SNV) and small indels in the BRCA genes at a variant allele frequency down to 5%. The sample population comprised 54 FFPE-derived DNA extracts from 51 clinical and 3 reference samples. DNA extracts were subjected to quality control using Multiplicom’s QC plex assay. The clinical samples were characterized using an independent targeted NGS method and Integrative Genomics Viewer (IGV) analysis of the mapped raw reads. SNV calling was performed using third-party bioinformatics platforms in the BRCA coding regions +/- 2 intronic bp. Results: BRCA MASTR Plus Dx*showed a uniformity of 93.9%, i.e. the percentage of amplicons with at least 0.2x the mean amplicon coverage, and a target specificity of 99.1%. The limit of detection (LOD) proved to be as low as 1%. The diagnostic accuracy was ≥ 99.99% [95% CI ≥ 99.98%] (100% sensitivity [95% CI ≥ 99.02%] and ≥ 99.99% specificity [95% CI ≥ 99.98%]). Both repeatability and reproducibility were ≥ 99.99% [95% CI ≥ 99.98%]. Lot equivalence was 100% [95% CI ≥ 99.99%]. Conclusions: This multicentre study demonstrated that BRCA MASTR Plus Dx* can be routinely applied as an accurate and precise method with an LOD of 1%. The assay can be used to direct patients with somatic or germline BRCA mutations to PARPi therapy. Currently, a PES for BRCA MASTR Plus Dx* and Multiplicom’s MASTR Reporter software is ongoing. *Products described above are CE-IVD and not available for sale in the US

BRCA1 and BRCA2 mutations in ovarian cancer patients from China: Association of ethnic-specific mutations in BRCA1 with an increased risk of ovarian cancer.

1583 Background: BRCA1/2 are cancer predisposition genes involved in hereditary breast and ovarian cancer (HBOC). Mutation carriers display an increased sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP). Despite a number of small-size hospital-based studies being previously reported, there is not yet, precise data of BRCA1/2mutations among Chinese epithelial ovarian cancer (EOC) pts. Methods: We performed a multicenter cohort study including 916 unselected consecutive EOC pts from eastern China, to screen for BRCA1/2mutations using the next-generation sequencing approach. Results: 153 EOC pts were found to carry pathogenic germline mutations in BRCA1/2, accounting for an overall mutation incidence of 16.7% with the predominance in BRCA1 (13.1%) compared with BRCA2 (3.9%). We identified 53 novel pathogenic mutations, among which the c.283_286delCTTG and the c.4573C>T of BRCA1 were both found in two unrelated patients. More importantly, the most common mutation, c.5470_5477del8 was most likely to be Chinese population-related without an apparent founder origin. This hot-spot mutation was presumably associated with an increased risk of EOC. Taken together, germline BRCA1/2mutations were common in Chinese EOC pts with distinct mutational spectrum compared to Western populations. Conclusions: Our study contributes to the current understanding of BRCA1/2 mutation prevalence worldwide. We recommend BRCA1/2genetic testing to all Chinese women diagnosed with EOC in order to identify HBOC families, to provide genetic counselling and clinical management for at-risk relatives. Mutation carriers may also benefit from PARP-targeted therapies. [Table: see text]

Poly (ADP-ribose) polymerase inhibitor, an effective radiosensitizer in lung and pancreatic cancers.

The development of stereotactic body radiation therapy (SBRT) has revolutionized radiation therapy for lung cancers and is an emerging treatment option for pancreatic cancers. However, there are many questions on how to optimize its use in chemoradiotherapy. The most relevant addition to radiotherapy regimens are inhibitors of DNA repair and DNA damage response pathways. One such class of agents are inhibitors of poly (ADP-ribose) polymerase (PARP). In this study we examined the effects of the PARP inhibitor LT626 in combination with ionizing radiation in lung and pancreatic cancers. Our study demonstrated that combination treatment with LT626 and radiation effectively inhibited growth in lung and pancreatic cancer cell lines, better than individual treatment alone. Combination treatment also increased expression of γH2AX and 53BP1 foci and upregulated expression of phosphorylated ATM, ATR and their respective kinases. Using in vivo lung cancer xenograft models we demonstrated that LT626 functioned as an effective radiosensitizer during fractionated radiation treatment, leading to significant decrease in tumor burden and doubling the median survival compared to control group. Overall our in vitro and in vivo studies showed that PARP inhibitor LT626 acted synergistically with radiation in lung and pancreatic cancers.

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.

PARP inhibitors in ovarian cancer: evidence, experience and clinical potential.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are considered one of the most active and exciting new therapies for the treatment of ovarian cancer. The anticancer activity of PARP inhibitors is based on the DNA repair vulnerability of many ovarian cancer cells, and multiple mechanisms of action of PARP inhibitors have been identified. As single agents, PARP inhibitors have demonstrated their greatest activity in ovarian cancer cells that harbor mutations in BRCA genes. Additionally, recent phase III studies have shown that single-agent PARP inhibitor activity extends beyond BRCA-related cancers and can benefit patients with ovarian cancers that do not have known BRCA mutations, especially when clinical characteristics such as platinum sensitivity and high-grade serous histology are present. PARP inhibitors have also been combined with chemotherapy, however, overlapping myelosuppression observed with PARP inhibitor and chemotherapy combinations has hampered development of these combinations. Contrariwise, PARP inhibitor and biologic agent combinations, specifically antiangiogenic agents, appear well tolerated and show promising activity in both BRCA mutated (BRCAm) and BRCA wild-type (BRCAwt) cancers. Currently, multiple clinical trials are underway examining the antitumor activity of PARP inhibitor combination therapy.

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1. In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.2. Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC50 values of 20.3 μM and 27.1 μM) and OCT1 (IC50 37.9 μM), but limited inhibition of OATP1B3 (25% at 100 μM); inhibition of the renal uptake transporters OCT2 (IC50 19.9 μM) and OAT3 (IC50 18.4 μM), but limited inhibition of OAT1 (13.5% at 100 μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC50s 5.50 μM and 47.1 μM, respectively); inhibition of the efflux transporter MDR1 (IC50 76.0 μM), but limited inhibition of BCRP (47% at 100 μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.3. Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.

Adenoviral vectors for modulation of poly(ADP-ribose) polymerase-1 (PARP1) – dependent DNA repair as a predictive tool for chemotherapy

Loss of the DNA homologous recombination repair pathway (DHR) sensitizes cells to the effects of clastogenic chemotherapy agents when the alternative base excision DNA repair pathway (BER) is blocked by inhibitors of poly(ADP- ribose) polymerases (PARP). The mutation of BRCA is an example of defective DHR, where several PARP inhibitors are now in advanced stages of clinical trials due to their ability to form synthetic lethality with chemotherapy drugs in breast and ovarian cancer. Because the inability of cells to perform DHR is the determining factor for successful PARP inhibitor administration in a chemotherapy protocol, testing of patient cells for sensitization by PARP inhibitors would be advantageous for the prediction of therapy outcome. This proof-of-principle study set out to design recombinant adenoviruses as a simple tool for the functional block of PARP1-mediated DNA strand break repair important for the execution of BER that could potentially be used to test cultured cells from patients for the sensitizing effects of PARP inhibitors in a chemotherapy setting. Recombinant adenoviruses for the expression of the PARP1 DNA binding domain (DBD) as a dominant-negative PARP1 mutant, for full- length PARP1 expression and other control viruses were generated and used to infect normal human fibroblasts. Transgene expression and the modulation of poly(ADP-ribose) (PAR) were studied using immunofluorescence and immunoblotting techniques. Overexpression of PARP1 increased, but expression of the PARP1 DBD blocked PAR formation in response to methylnitronitrosoguanidine (MNNG) treatment of virally transduced cells. Ectopic DBD expression caused the formation of caspase 3 cleavage products of PARP1 protein, which are a marker of apoptotic cell death. Cells infected with control virus without PARP DBD did not induce caspase 3-mediated cleavage of PARP1. In conclusion, the set of adenoviral vectors presented in this study can be used to modulate PARP activity in normal human cells and may therefore be used as a tool for testing cellular sensitization by blocking DNA BER by administration of PARP inhibitors and to gain better understanding of potential PARP inhibitor resistance.

Tumor Cell Recovery from Senescence Induced by Radiation with PARP Inhibition.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are clinically used as single-agent therapy for tumors with BRCA1 or BRCA2 mutations. One approach to expanding the use of PARP inhibitors to a wider range of tumors is to combine them with cytotoxic chemotherapy or radiotherapy. Preclinical studies in experimental animals and tumor cells in culture indicate that PARP inhibition modestly sensitizes most tumor cells to ionizing radiation. Studies of cell behavior after these combined treatments show that radiosensitization is manifested predominantly in an increase in prolonged growth arrest and senescence, with little or no contribution from apoptosis. The secretory phenotype associated with senescence can target these tumor cells for immune surveillance, and therefore increased senescence can effectively contribute to tumor control. However, the possible recovery of senescent cells and re-entry into cell cycle after prolonged arrest also needs to be considered. Such recovery could lead to tumor recurrence, yet may not be reflected in short-term assays commonly used to assess radiosensitization.

Abstract 3714: Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG)

Abstract The macrodomain protein poly(ADP ribose) glycohydrolase (PARG) has been shown to be a critical component in the repair of single stand DNA breaks and counteracts the function of the ARTD family of poly(ADP ribose) polymerases, commonly known as the PARPs. As PARG exists as a single protein, it presents an attractive target for therapeutic intervention in cancer cells with enhanced dependence upon DNA repair. Inhibitors of this enzyme have proved difficult to discover and develop. Moreover, intact cell-active tool compounds which have the propensity to be used as robust chemical probes to understand PARG pharmacology, are absent from the literature. This poster will describe our work in this emerging area, optimising a series of drug-like quinazolinedione derivatives to deliver molecules with the correct physicochemical and biochemical properties to function as in vitro cell probe compounds. These unprecedented agents display potent on-target biochemical (5 nM) and cell (10 nM) activity with a significant window to acute 3-day cytotoxicity. Moreover, these agents are selective against PARP family members and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of the scaffold will be described, alongside the outline pharmacology demonstrating on-target, selective inhibition of PARG in cells. Such tool compounds will be of value in revealing the detailed mechanisms of action of PARG in DNA repair and other PAR chain-mediated cellular processes, with the ultimate goal of delivering novel and clinically relevant therapeutic agents. Citation Format: Kate Smith, Ben Acton, Dominic James, Cliff Jones, Stuart Jones, Allan Jordan, Nicola Hamilton, Alison McGonagle, Daniel Mould, Helen Small, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3714.

Abstract 3718: Sensitization of human tumor cells to chemotherapy drugs by antisense downregulation of RAD51: Targeting DNA repair to induce synthetic lethality

Abstract The inherent genomic instability of cancer cells has been exploited as a tumor-selective drug target to treat tumors that are deficient in specific mechanisms of DNA repair. Inhibitors of poly(ADP-ribose) polymerase (PARP) are routinely used clinically against tumors deficient in BRCA1 or BRCA2, due to the poor capacity of these cells to undergo homologous recombination repair (HRR). This exploitation of a tumor cell deficiency to enhance selectivity to a particular drug is “synthetic lethality” (Nature 434: 913, 2005). To make use of this phenomenon in tumors that may not be inherently hypersensitive to a particular treatment, we have sought to induce synthetic lethality by down-regulating essential components of DNA repair, in particular BRCA2, to sensitize cells to chemotherapy drugs [Mol Oncol 8(8): 1429-1440, 2014]. Given that an important function of BRCA2 is to modulate the action of RAD51 in HRR, we determined whether antisense knockdown of RAD51 could enhance tumor cell sensitivity to the PARP inhibitor olaparib and the DNA-crosslinking agent cisplatin. Four different anti-RAD51 siRNA molecules (Dharmacon), targeting coding sequences, were tested against cell lines representative of different tumor types in an in vitro assay of proliferation (non-small cell lung cancer line A549b, colon carcinoma line HT-29, and prostate carcinoma lines DU145 and LNCaP). The siRNAs, as single agents, inhibited proliferation in a concentration-dependent fashion and to varying degrees, and sensitized tumor cells to olaparib. A sequence that targeted region 1169-1187 of the RAD51 cDNA (NM_002875.4) was utilized for further studies. At concentrations of anti-RAD51 siRNA 51a that inhibited proliferation of cell lines by less than 50%, 51a enhanced cytotoxicity of olaparib by over 90% and of cisplatin by 60-90%. In all cell lines except LNCaP (with mutant BRCA2) the combination of siRNAs against RAD51 and BRCA2 acted cooperatively to enhance cytotoxicity of olaparib and cisplatin. Notably, when used together, each siRNA down-regulated expression of its respective target mRNA, as demonstrated by quantitative RT-PCR, without interfering with the activity of the other. RAD51 can be exploited clinically as a target for inherent or induced synthetic lethality to DNA-damaging agents (e.g., cisplatin) or inhibitors of DNA repair (e.g., olaparib). Such treatment can include tumors with BRCA2-deficiency, either inherent or induced, to yield at least an additive anticancer effect. MR is a scholar of the CIHR Strategic Training Program in Cancer Research and Technology Transfer (CaRTT) and a recipient of the CIHR Banting and Best Canada Graduate Scholarship. Citation Format: Peter J. Ferguson, Mateusz Rytelewski, Mark D. Vincent, James Koropatnick. Sensitization of human tumor cells to chemotherapy drugs by antisense downregulation of RAD51: Targeting DNA repair to induce synthetic lethality. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3718.

Abstract 3715: Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG)

Abstract In recent years, many proteins involved in DNA repair, such as ATR, ATM and PARP, have received considerable attention as potential points of therapeutic intervention in cancer. Indeed, these efforts have recently delivered several agents into clinical evaluation or FDA regulatory approval. However, the DNA repair protein poly(ADP ribose) glycohydrolase (PARG), which plays an equally critical role in DNA single stand break repair, to successful drug discovery efforts. Through our innovative collaboration with AstraZeneca, we have discovered a novel PARG-binding pharmacophore and have employed this information to discover drug-like chemotypes, facilitating the development of potent and selective inhibitors. This poster will describe our emerging results in this area, where a novel benzimidazolone sulphonamide scaffold has been shown potently to inhibit PARG in both biochemical and cellular assays with potencies of 40 nM and 60 nM respectively. Moreover, these agents display pharmacology consistent with the anticipated mode of action, appropriate drug-like properties and are selective against PARP1 and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of this scaffold will be described, alongside the recent biological results obtained. Ultimately, this work has helped deliver tool compounds which may help to elucidate the true pharmacology and roles of PARG in cancer and other disease settings. Citation Format: Allan Jordan, Ben Acton, Nicola Hamilton, James Hitchin, Colin Hutton, Dominic James, Cliff Jones, Stuart Jones, Alison McGonagle, Helen Small, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3715.

Abstract 4352: Discovery of the first cell-active inhibitors of poly(ADP Ribose) glycohydrolase through high-throughput screening and computational approaches

Abstract DNA repair is a critical process for the survival and normal proliferation of healthy cells. However, given the enhanced levels of cellular stress and genomic instability, these repair processes are even more critical to the survival of malignant cells, where rates of DNA damage are considerably increased. Given this, inhibitors of DNA damage repair have seen a resurgence of interest in recent years in an effort to exploit tumour cell vulnerabilities. One such example of this approach has resulted in the recent approval of the PARP inhibitor olaparib (Lynparza™) for women with advanced ovarian cancer associated with defective BRCA genes. Olaparib acts against the poly(ADP-ribose)polymerase (PARP) enzymes, more recently re-defined as the ARTD (Diphtheria toxin-like human ADP-ribosyltransferase) enzyme class. Whilst PARP is widely known to play critical and well-understood roles in DNA repair, poly(ADP ribose) glycohydrolase (PARG) is less well known but equally essential for effective DNA repair, degrading PAR chains and facilitating effective DNA repair. However, its inhibition may offer several key advantages over PARP inhibition. Most critically, whilst there are 18 isoforms, there exists only a single PARG protein, offering a specific point of therapeutic intervention. However, due to the open nature of the PARG binding cleft and the nature of the binding site, this protein has been considered to be difficult to inhibit with small, drug-like small molecules, particularly in the cellular context. This poster will describe our efforts to overcome these challenges against this challenging target and report our early successes achieved through innovative computational chemistry strategies. These efforts have delivered several credible, drug-like startpoints for further medicinal chemistry optimisation. Citation Format: Bohdan Waszkowycz, Dominic James, Steven Durant, Nicola Hamilton, Cliff Jones, Stuart Jones, Allan Jordan, Alan Lau, Alison MGonagle, Mark O’Connor, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Donald Ogilvie. Discovery of the first cell-active inhibitors of poly(ADP Ribose) glycohydrolase through high-throughput screening and computational approaches. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4352.

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis) show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR). However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi), become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi)). Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG)–a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF) 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

New Targeted Agents in Gynecologic Cancers: Synthetic Lethality, Homologous Recombination Deficiency, and PARP Inhibitors.

Inhibitors of poly (ADP-ribose) polymerase (PARP) have emerged as a new class of anti-cancer drugs, specifically for malignancies bearing aberrations of the homologous recombination pathway, like those with mutations in the BRCA 1 and BRCA 2 genes. Olaparib, a potent PARP1 and PARP2 inhibitor, has been shown to significantly increase progression-free survival (PFS) in women with recurrent ovarian cancer related to a germline BRCA mutation and is currently approved fourth-line treatment in these patients. PARP inhibitors (PARPi) target the genetic phenomenon known as synthetic lethality to exploit faulty DNA repair mechanisms. While ovarian cancer is enriched with a population of tumors with known homologous recombination defects, investigations are underway to help identify pathways in other gynecologic cancers that may demonstrate susceptibility to PARPi through synthetically lethal mechanisms. The ARIEL2 trial prospectively determined a predictive assay to identify patients with HRD. The future of cancer therapeutics will likely incorporate these HRD assays to determine the best treatment plan for patients. While the role of PARPi is less clear in non-ovarian gynecologic cancers, the discovery of a predictive assay for HRD may open the door for clinical trials in these other gynecologic cancers enriched with patients with HRD. Identification of patients with tumors deficient in homologous repair or have HRD-like behavior moves cancer treatment towards individualized therapies in order to maximize treatment effect and quality of life for women living with gynecologic cancers.

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity.

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment.

Structural modeling of NAD+ binding modes to PARP-1

The nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) plays an important role in the signaling and repair of DNA. PARP-1 catalyses the covalent binding of poly (ADP-ribose) polymers to its subunit, as well as to other acceptor proteins, using NAD+ as a donor of ADP-ribose. Inhibitors of poly (ADP-ribose) polymerase have been shown to be effective in improving radiation therapy and chemotherapy of cancer in clinical testing. Development of new poly (ADP-ribose) polymerase-1 inhibitors based on derivatives of natural compounds such as NAD+ represents a novel and promising strategy. The structure of the complex of human poly (ADP-ribose) polymerase-1 with NAD+ can be a starting point for the rational design of small molecule inhibitors based on NAD+ derivatives. Moreover, there is no crystal structure of the complex of poly (ADP-ribose) polymerase-1 with nicotinamide adenine dinucleotide (NAD+) available yet. In this work, using molecular modeling approaches, we have predicted NAD+ binding modes to PARP-1 at the donor binding site of the catalytic domain. Using structures of PARP-1 homologs in a complex with NAD+, we predict the pharmacophore restraints of NAD+ binding to PARP-1. Based on the clustering of PARP-1 conformations in a complex with cocrystallized inhibitors and the predicted pharmacophore restraints, we propose several possible models of NAD+ binding to PARP-1 at the donor binding site of the catalytic domain. According to the predicted models, two conformations for the pyrophosphate group of NAD+ in complex with PARP-1 at the donor binding site are possible. The proposed models of NAD+ binding to PARP-1 can be validated by the quantitative structure–activity analysis of NAD+ derivatives. We designed two NAD+ derivatives, which can be used to validate the predicted NAD+ binding models.

Abstract C61: Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors

Abstract Background: Veliparib, an inhibitor of Poly(ADP-ribose) polymerase (PARPi), in combination with carboplatin showed efficacy in triple negative breast cancer (TNBC) patients in the I-SPY 2 TRIAL. However ∼42% of TNBC did not achieve pathologic complete response. Insufficient uptake of drug in TNBC may lead to inadequate response to PARPi. As a first step toward testing this hypothesis in patients, we quantified veliparib penetration in mouse xenograft models of TNBC. Methods: MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Veliparib at low dose (20mg/kg) or high dose (60mg/kg) and carboplatin (60mg/kg) was given three times daily for three days. MR images were taken at day 1. Plasma, fresh frozen and OCT embedded tissues were analyzed using Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI) Liquid chromatography–mass spectrometry (LC-MS). Drug penetration was compared among doses and cell lines. Results: Ex vivo veliparib concentrations quantified by LC-MS differed significantly among the tumors derived from the three cell lines. Liver and plasma concentrations were uniformly high in all mice compared to tumor and muscle tissues. Plasma pharmacokinetics in mice exhibited non-linear clearance resulting in prolonged high plasma levels at higher doses, while tumor and plasma concentrations were linearly correlated. MALDI-MSI images of tumor and muscle in 12 mice showed higher veliparib concentrations in necrotic areas compared to areas with viable tumor cells (P = 0.126, Table) and higher concentrations at the rim then in the center of the tumor (P = 0.046). Lower concentrations were found in MDA-MB-231 than in other cell lines (0.008). Contrast agent and veliparib accumulated near the rim of the tumors and a fast elimination of the contrast agent from the tumor correlated with relatively low veliparib tumor concentrations. Conclusions: The spatial distribution of veliparib in TNBC depends on the dose and tumor cell biology. We demonstrated that MALDI-MSI can be used to measure veliparib penetration tumor samples, which may have potential to monitor response to PARPi therapies. Table: Veliparib concentrations by LC-MS and its spatial distribution by MALDI varies by tissue, drug dose and TNBC cell line of origin. TissueNMedian conc. (mg/L)RSEPMethodPlasma low/high dose9/60.19 / 0.8610%/11%0.001 (low/high dose)LC-MSTNBC xenograft tissue19/170.05 / 0.3356% / 12%0.322 (tumor/plasma)LC-MSMuscle5/60.12 / 0.4770% / 47%0.882 (muscle/plasma)LC-MSLiver6/60.47 / 1.7720% / 14%<0.001 (liver/plasmaLC-MSnecrotic tissue/ cellular tumor tissue19/191.63 / 1.0170% / 85%0.126MALDIrim/ center19/191.54 / 1.1389% / 87%0.046MALDIHCC70 / MDA-MB-231 / MDA-MB-4366/7/60.31/ 0.12/0.1418% / 22% / 49%0.008LC-MS Citation Format: Imke H. Bartelink, Brendan Prideaux, Gregor Krings, Lisa Wilmes, Pei R.E. Lee, Byron Hann, Jean-Philippe Coppé, Diane Heditsian, Lamorna Swigart-Brown, Ella F. Jones, Sergey Magnitsky, Ron Keizer, Laura Esserman, Weiming Ruan, Alan Wu, Douglas Yee, Veronique Dartois, Denise Wolf, Rada Savic, Laura vantVeer. Non-homogeneous drug penetrance of veliparib measured in triple negative breast tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C61.

BCRP and P-gp relay overexpression in triple negative basal-like breast cancer cell line: a prospective role in resistance to Olaparib.

The triple negative basal-like (TNBL) breast carcinoma is an aggressive and unfavorable prognosis disease. Inhibitors of poly(ADP-ribose) polymerase such as Olaparib could represent a promising targeted therapy but their sensitivity against Multidrug Resistance proteins (MDR), which causes resistance, is not well defined. Thus, our work focused on the analysis of P-gp and BCRP coexpression in the SUM1315 TNBL human cell line, in correlation with Olaparib intracellular concentration. Western blot analyses showed a clear coexpression of P-gp and BCRP in SUM1315 cells. A low cytotoxic Olaparib treatment clearly led to an increased expression of both BCRP and P-gp in these cells. Indeed, after 1.5 h of treatment, BCRP expression was increased with a 1.8 fold increase rate. Then, P-gp took over from 3 h to 15 h with an average increase rate of 1.8 fold, and finally returned to control value at 24 h. HPLC-UV analyses showed that, in the same treatment conditions, the intracellular Olaparib concentration increased from 1 h to 3 h and remained relatively stable until 24 h. Results suggest that the resistance mechanism induced by Olaparib in TNBL SUM1315 cell line may be overpassed if a cytotoxic and stable intracellular level of the drug can be maintained.