Potent PARP Inhibitor Research Articles

18F]-SuPAR: A Radiofluorinated Probe for Noninvasive Imaging of DNA Damage-Dependent Poly(ADP-ribose) Polymerase Activity.

Poly(ADP ribose) polymerase (PARP) enzymes generate poly(ADP ribose) post-translational modifications on target proteins for an array of functions centering on DNA and cell stress. PARP isoforms 1 and 2 are critically charged with the surveillance of DNA integrity and are the first line guardians of the genome against DNA breaks. Here we present a novel probe ([18F]-SuPAR) for noninvasive imaging of PARP-1/2 activity using positron emission tomography (PET). [18F]-SuPAR is a radiofluorinated nicotinamide adenine dinucleotide (NAD) analog that can be recognized by PARP-1/2 and incorporated into the long branched polymers of poly(ADP ribose) (PAR). The measurement of PARP-1/2 activity was supported by a reduction of radiotracer uptake in vivo following PARP-1/2 inhibition with talazoparib treatment, a potent PARP inhibitor recently approved by FDA for treatment of breast cancer, as well as ex vivo colocalization of radiotracer analog and poly(ADP ribose). With [18F]-SuPAR, we were able to map the dose- and time-dependent activation of PARP-1/2 following radiation therapy in breast and cervical cancer xenograft mouse models. Tumor response to therapy was determined by [18F]-SuPAR PET within 8 h of administration of a single dose of radiation equivalent to one round of stereotactic ablative radiotherapy.

Exploring the Frequency of Homologous Recombination DNA Repair Dysfunction in Multiple Cancer Types.

Dysfunctional homologous recombination DNA repair (HRR), frequently due to BRCA mutations, is a determinant of sensitivity to platinum chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi). In cultures of ovarian cancer cells, we have previously shown that HRR function, based upon RAD51 foci quantification, correlated with growth inhibition ex vivo induced by rucaparib (a PARPi) and 12-month survival following platinum chemotherapy. The aim of this study was to determine the feasibility of measuring HRR dysfunction (HRD) in other tumours, in order to estimate the frequency and hence wider potential of PARPi. A total of 24 cultures were established from ascites sampled from 27 patients with colorectal, upper gastrointestinal, pancreatic, hepatobiliary, breast, mesothelioma, and non-epithelial ovarian cancers; 8 were HRD. Cell growth following continuous exposure to 10 μM of rucaparib was lower in HRD cultures compared to HRR-competent (HRC) cultures. Overall survival in the 10 patients who received platinum-based therapy was marginally higher in the 3 with HRD ascites (median overall survival of 17 months, range 10 to 90) compared to the 7 patients with HRC ascites (nine months, range 1 to 55). HRR functional assessment in primary cultures, from several tumour types, revealed that a third are HRD, justifying the further exploration of PARPi therapy in a broader range of tumours.

BMN 673 (talazoparib): A potent PARP inhibitor for triple negative breast cancer with different genetic profile.

The objective of the present study was to elucidate the effect of BMN 673 (talozoparib) on BRCA1 mutant (HCC1937) and wild-type (MDA-MB-231) triple negative breast cancer (TNBC). The in vitro cytotoxicity results indicated that BMN 673 had considerable inhibitory effects on HCC1937 and MDA-MB-231 cell lines by inducing apoptosis, multicaspase activity, G2/M arrest, and altering the expression levels of apoptosis-related genes (P < 0.01). Additionally, BMN 673 indicated no toxicity on MCF-10A control cells until a certain concentration and incubation time. However, BMN 673, a novel and selective poly ADP ribose polymerase inhibitor, was more potent in TNBC cells bearing BRCA1 mutant than those with wild-type BRCA1. In conclusion, our study, for the first time, demonstrated a molecular mechanism of the induction of apoptosis by BMN 673 in TNBC with different genetic profile. However, further investigations regarding the exact molecular mechanisms underlying BMN 673-inducing apoptotic death and gene-cell line associations are required.

A phase 3 trial comparing talazoparib, an oral PARP inhibitor, to physician’s choice of therapy in patients with advanced breast cancer and a germline BRCA mutation: EMBRACA subgroups by age

Background: Epidemiologic studies indicate that individuals with germline BRCA mutation (gBRCAm) status are diagnosed with breast cancer at a median age of 40-45, which is approximately 20 yrs younger than the overall breast cancer population. Talazoparib (TALA), a highly potent PARP inhibitor that causes cell death in BRCA1/2-mutated cells, may have treatment potential in this younger population.

Altering Nitrogen Heterocycles of AZD2461 Affords High Affinity Poly(ADP-ribose) Polymerase-1 Inhibitors with Decreased P-Glycoprotein Interactions

Poly(ADP-ribose) polymerase inhibitors (PARPi) are targeted therapeutics with enhanced selectivity and cytotoxicity in BRCA1/2 mutant cancer cells. AZD2461, a congener of FDA approved olaparib, is a potent PARPi with high affinity for PARP-1 and nonsubstrate for P-glycoprotein (P-gp), an attractive characteristic for cancer therapeutics. Analogues of AZD2461 were synthesized and profiled in BRCA1 functional and nonfunctional cell lines, revealing compounds (2, 3, and 5) of low cytotoxicity and excellent PARP-1 affinities (∼4–8 nM). In comparison to AZD2461, these agents were found to be less stimulating of P-gp, suggesting that these compounds may be excellent candidates for neurological applications where blood brain barrier penetrance is sought.

Abstract PR02: Exploring the effects of PARP inhibition on CHK1 activation as a potential determinant of synergy with CHK1 inhibition

Abstract PARP inhibitors (PARPi) have improved the outcomes of patients with high-grade serous ovarian carcinomas (HGSOC) whose tumors harbor mutations in homologous recombination genes such BRCA1 and BRCA2. However, only 20% of cases with this aggressive subtype of ovarian cancer benefit from PARPi, thus highlighting the essential need to identify novel therapeutic strategies for the remainder of these patients. We have recently reported that off-target effects vary among two PARP inhibitors with different trapping potencies. The FDA-approved PARPi, Olaparib, activated the checkpoint kinase 1 (CHK1) protein at serine 345 and arrested cells in the G2/M phase; however, these effects were not observed after treatment with Veliparib, the least potent PARPi measured by the PARP trapping ability. In this study, we treated HGSOC cell lines with the five most clinically advanced PARP inhibitors and investigated the effect of varying PARP trapping potencies on the expression levels of major proteins that are involved in cell cycle pathways via Western blot and cell cycle analysis. The synergy between the most potent (Talazoparib) or least potent PARP trappers and a CHK1 inhibitor was also explored in HGSOC patient-derived xenograft (PDX) models. We hypothesize that PARP inhibitors that do not have an effect on the cell cycle will have greater synergy with CHK1 inhibitors. Our results reveal that the four most potent PARP trappers—olaparib, rucaparib, niraparib, and talazoparib--activate CHK1 protein and decrease total protein levels of CHK1’s downstream substrate CDC25C. However, we did not observe any significant changes in expression levels in these two proteins after treatment with veliparib. We also observed an arrest in the S/G2 phase of the cell cycle after treatment with the four most potent PARP inhibitors. Finally, the growth of our PDX model was significantly inhibited by combined treatment with veliparib and the CHK1 inhibitor MK-8776 compared to either single-agent therapy or talazoparib combined with MK-8776. These results suggest that there is a distinct off-target effect that differs among the least and most potent PARP inhibitors, with veliparib being more synergistic with CHK1 inhibition. This study will help to expand the use of PARP inhibitors in combination with CHK1 inhibitors and emphasizes the importance of single-agent off-target effects to develop optimal combination therapies. This abstract is also being presented as Poster A11. Citation Format: Monicka Wielgos-Bonvallet, Paulina Cybulska, Elke Van Oudenhove, Petar Jelinic, Douglas A. Levine. Exploring the effects of PARP inhibition on CHK1 activation as a potential determinant of synergy with CHK1 inhibition. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR02.

Identification and preclinical characterization of a novel and potent poly (ADP-ribose) polymerase (PARP) inhibitor ZYTP1

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in the detection and repair of DNA damage. Studies have shown that inhibition of PARP and Tankyrase (TNKS) has significant antitumor effect in several types of cancers including BRCA-negative breast cancers. Identification of ZYTP1, a novel PARP inhibitor, through a battery of in vitro assays and in vivo studies. PARP and TNKS inhibitory activity of ZYTP1 was assessed in cell-free kinase assay. In vitro cell killing potency of ZYTP1 was tested in a panel of cell lines including BRCA-negative cells. ZYTP1 was also tested in xenograft models in combination with temozolomide (TMZ). The pharmacokinetic profile of ZYTP1 was determined in rodent and non-rodent preclinical species. Safety of ZYTP1 was assessed in Wistar rats and Beagle dogs upon repeated dosing. ZYTP1 inhibited PARP1, PARP2, Tankyrase-1 and Tankyrase-2 with IC50 of 5.4, 0.7, 133.3 and 289.8nM, respectively, and additionally trapped PARP1 onto damaged DNA. It also potentiated MMS-mediated killing of different cancer cell lines. Compound demonstrated good Caco-2 cell permeability. The oral bioavailability of ZYTP1 in mice, rats and dogs ranged between 40 and 79% and demonstrated efficacy in colon cancer xenograft model at a dose of 1-10mg/kg in combination with TMZ. In a 28-day repeat dosing, oral toxicity study in rats, it was found to show > 10× safety margin. ZYTP1 is a novel PARP inhibitor that showed potential for development as a treatment for various solid tumors.

Abstract 4845: Preclinical evaluation of HWH340, a novel PARP inhibitor

Abstract Objective: Poly-ADP-ribose polymerase (PARP) participates in DNA repair, replication and transcription, and is important to maintaining genome stability and integrity. PARP inhibitors have proven to be efficacious in the clinic to treat BRCA-deficient cancers. PARP inhibitors may also be used as sensitizing agent in combination with other antitumor agents. The purpose of this study is to investigate in preclinical models the antitumor activity of HWH340, a novel potent and selective PARP inhibitor currently undergoing clinical investigation. Method: HWH340 was evaluated for its PARP1/2 enzymatic activity in corresponding enzyme assays and its cellular PARylation inhibitory activity in HCC1937 cells. The anti-proliferative activity and the synergistic effect in combination with temozolamide (TMZ) were evaluated in vitro in a BRCA-deficient cell line MDA-MB-436 and BRCA proficient cell line MDA-MB-231. Antitumor activity was evaluated by daily oral administration of in xenograft tumor models. Synergistic effect in combination with TMZ was also determined. The animal models used were PARP highly expressed patient-derived xenograft (PDX) model of small cell lung cancer (SCLC) LU-01-0547 and BRCA1/2-deficient CDX models of breast cancer MDA-MB-436 and pancreatic cancer CAPAN-1. Result: HWH340 displayed potent PARP1/2 enzymatic inhibitory activity with IC50 1.14 nM and 1.72 nM respectively. It also potently inhibited cellular PARylation in HCC1937 cell with IC50 2.77 nM. In BRCA proficient cell line MDA-MB-231, HWH340 showed no synergistic effect when combined with TMZ and a weak anti-proliferative activity with IC50 5.83 uM and 4.86 uM. In contrast, it displayed potent anti-proliferative activity and a synergistic effect in combination with TMZ with IC50 of 71 nM and 7 nM in the BRCA1/2-deficient MDA-MB-436 cells, respectively. HWH340 showed excellent in vivo antitumor efficacy in PARP overexpressed SCLC LU-01-0547 PDX model (TGI=81.69%@50mpk, QD). In BRCA-deficient breast cancer MDA-MB-436 CDX model, HWH340 showed potent antitumor efficacy as single agent (TGI=122% @12.5mpk, QD). In BRCA-deficient pancreatic cancer CAPAN-1 CDX model, it showed potent antitumor efficacy in combination with TMZ (TGI=79.61% @25mpk+ TMZ 30mpk, QD). Conclusion: HWH340 is a potent PARP inhibitor. Its antitumor activity in the preclinical tumor models together with its excellent pharmaceutical properties provide confidence that HWH340 may have high potential in the clinic for treatment of BRCA deficient solid cancers. Citation Format: Wenjie Sun, Yang Yue, Hailiang Chen, Ronghua Tu, Jie Shen, Gang Li, Guoqiang Ma, Zhixiang Zhang, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of HWH340, a novel PARP inhibitor [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 4845.

Abstract 3715: Targeted nanotherapy using the PARP inhibitor talazoparib for breast cancer treatment

Abstract Introduction: PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells. These drugs are currently delivered orally, but Talazoparib, the most potent PARP inhibitor, exhibits greater toxicity than the others. Systemic administration of nanoparticles bypasses the first-pass metabolism of oral drugs and nanoparticles preferentially accumulate in tumors due to the leaky tumor vasculature. Additionally, nanoparticles can be actively targeted to tumors by conjugating different moieties such as antibodies that recognize overexpressed markers on the tumor cells. NanoTalazoparib (NanoTLZ) has been previously formulated and extensively characterized in breast, ovarian, and lung cancer models. Here we describe further characterization of NanoTLZ and the development of a next generation fluorescently labeled EPCAM targeted formulation of NanoTLZ for the treatment of triple negative breast cancer (TNBC). Methods: Animals with orthotopic TNBC xenografts were injected with NanoTLZ and at various time points tumor biopsies were taken to assay tumor PAR levels. Blood was collected at various time points and plasma separated for drug extraction and quantification. Pharmacokinetic modeling is underway. Fluorescently labeled NanoTLZ was developed with the addition of Cy5, followed by further improvement via conjugation of anti-EPCAM antibodies. Targeting capability was assessed via laser scanning confocal microscopy after treatment of the TNBC cell line MDA-MB-231 with either non-targeted or targeted NanoTLZ. Therapeutic efficacy studies with targeted NanoTLZ alone and in combination with radiation are underway in an MDA-MB-231 xenograft model. Results: Pharmacodynamics indicated PAR suppression in tumors within 30 minutes of NanoTLZ treatment. Tumor PAR levels began to increase 24 hours after a single dose but remained significantly lower than control levels up to 72 hours (P&amp;lt;0.005). Characterization of NanoTLZ after the addition of Cy5 and after anti-EPCAM targeting indicated that the formulation remained under 100 nm in diameter with a charge of ~10 mV and equivalent drug loading and release. Cy 5 labeling allowed for visualization of nanoparticle uptake and intracellular fluorescence was 70% greater in cells when treated with EPCAM targeted NanoTLZ compared to non-targeted. Conclusions: The sustained release of Talazoparib from the nanoformulation decreases tumor PAR levels for up to 72 hours after a single dose, allowing for less frequent administration than the current daily regime used for oral Talazoparib. The targeted formulation of NanoTLZ shares the same physicochemical properties as the untargeted formulation, but is taken up much faster in vitro, suggesting it will allow for greater accumulation at the tumor site making it more effective than the previously tested untargeted NanoTLZ. Supported by ARMY/W81XWH-16-1-0731 and Rivkin Foundation. Citation Format: Paige Baldwin, Rajiv Kumar, Srinivas Sridhar. Targeted nanotherapy using the PARP inhibitor talazoparib for breast cancer treatment [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 3715.

Abstract 410: Genome-wide and focused CRISPR screens to study PARP inhibitor resistance mediated by mutations in PARP1

Abstract To investigate mechanisms of PARP inhibitor (PARPi) cytotoxicity, we carried out genome wide CRISPR-Cas9 screens for mutants resistant to the potent PARPi talazoparib (BMN 673) in mouse ES cells and human breast cancer cells. Many talazoparib resistant clones had loss of function Parp1 mutations as expected from to the proposed role of trapped PARP1 in cytotoxicity. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARPi cytotoxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis. We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. Among these, we found mutants that display trapping despite conferring PARPi resistance, suggesting that PARP trapping is not sufficient for cytotoxicity. Mutations that confer resistance are restricted to DNA binding domains and a network of residues in the WGR and helical domains of PARP1 that may be involved in intramolecular activation of PARP1 upon DNA binding and thus affect trapping. We also established that PARP1 loss can be tolerated in some BRCA1 mutant breast and ovarian tumour cell lines, despite the well-described synthetic lethality of PARP1 and BRCA1 loss. Further investigation indicated that these cell lines that tolerate PARP1 loss of function mutations have exon 11 BRCA1 mutations, and may retain some function due to a splice variant that skips exon 11. siRNA knockdown of remaining BRCA1 function in SUM149 or COV362 cells with PARP1 mutations elicited the expected synthetic lethality. A PARP1 mutation identified in a PARPi resistant patient is localised to the WGR domain cluster identified in our screens and abolishes PARP1 trapping in a microirradiation assay. PARP1 mutation also caused talazoparib resistance in a SUM149 xenograft model. These results raise the possibility that PARP1 loss may be a clinical mechanism of PARP inhibitor resistance. Importantly, BRCA1 mutant cells with PARP1 mutations had distinct drug sensitivities to other known mechanisms of PARPi resistance - for example PARP1 mutation did not result in cross resistance to cisplatin or camptothecin, unlike BRCA1 reversion or 53BP1 loss. This suggests that studying the mechanism of acquired PARPi resistance may be important to inform subsequent therapy choice. Citation Format: Stephen J. Pettitt, Dragomir B. Krastev, Inger Brandsma, Amy Dréan, Feifei Song, Maria I. Harrell, Rachel Brough, Jessica Frankum, Jung-Min Lee, Elizabeth M. Swisher, Kosuke Yusa, Alan Ashworth, Christopher J. Lord. Genome-wide and focused CRISPR screens to study PARP inhibitor resistance mediated by mutations in PARP1 [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 410.

The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors

Y-box-binding protein 1 (YB-1) is a multifunctional cellular factor overexpressed in tumors resistant to chemotherapy. An intrinsically disordered structure together with a high positive charge peculiar to YB-1 allows this protein to function in almost all cellular events related to nucleic acids including RNA, DNA and poly(ADP-ribose) (PAR). In the present study we show that YB-1 acts as a potent poly(ADP-ribose) polymerase 1 (PARP1) cofactor that can reduce the efficiency of PARP1 inhibitors. Similarly to that of histones or polyamines, stimulatory effect of YB-1 on the activity of PARP1 was significantly higher than the activator potential of Mg2+ and was independent of the presence of EDTA. The C-terminal domain of YB-1 proved to be indispensable for PARP1 stimulation. We also found that functional interactions of YB-1 and PARP1 can be mediated and regulated by poly(ADP-ribose).

Improved Reperfusion and Vasculoprotection by the Poly(ADP-Ribose)Polymerase Inhibitor PJ34 After Stroke and Thrombolysis in Mice.

Benefits from thrombolysis with recombinant tissue plasminogen activator (rt-PA) after ischemic stroke remain limited due to a narrow therapeutic window, low reperfusion rates, and increased risk of hemorrhagic transformations (HT). Experimental data showed that rt-PA enhances the post-ischemic activation of poly(ADP-ribose)polymerase (PARP) which in turn contributes to blood-brain barrier injury. The aim of the present study was to evaluate whether PJ34, a potent PARP inhibitor, improves poor reperfusion induced by delayed rt-PA administration, exerts vasculoprotective effects, and finally increases the therapeutic window of rt-PA. Stroke was induced by thrombin injection (0.75UI in 1μl) in the left middle cerebral artery (MCA) of male Swiss mice. Administration of rt-PA (0.9mgkg-1) or saline was delayed for 4h after ischemia onset. Saline or PJ34 (3mgkg-1) was given intraperitoneally twice, just after thrombin injection and 3h later, or once, 3h after ischemia onset. Reperfusion was evaluated by laser Doppler, vascular inflammation by immunohistochemistry of vascular cell adhesion molecule-1 (VCAM-1) expression, and vasospasm by morphometric measurement of the MCA. Edema, cortical lesion, and sensorimotor deficit were evaluated. Treatment with PJ34 improved rt-PA-induced reperfusion and promoted vascular protection including reduction in vascular inflammation (decrease in VCAM-1 expression), HT, and MCA vasospasm. Additionally, the combined treatment significantly reduced brain edema, cortical lesion, and sensorimotor deficit. In conclusion, the combination of the PARP inhibitor PJ34 with rt-PA after cerebral ischemia may be of particular interest in order to improve thrombolysis with an extended therapeutic window.

An 18F-Labeled Poly(ADP-ribose) Polymerase Positron Emission Tomography Imaging Agent.

Poly(ADP-ribose) polymerase (PARP) is involved in repair of DNA breaks and is over-expressed in a wide variety of tumors, making PARP an attractive biomarker for positron emission tomography (PET) and single photon emission computed tomography imaging. Consequently, over the past decade, there has been a drive to develop nuclear imaging agents targeting PARP. Here, we report the discovery of a PET tracer that is based on the potent PARP inhibitor olaparib (1). Our lead PET tracer candidate, [18F]20, was synthesized and evaluated as a potential PARP PET radiotracer in mice bearing subcutaneous glioblastoma xenografts using ex vivo biodistribution and PET–magnetic resonance imaging techniques. Results showed that [18F]20 could be produced in a good radioactivity yield and exhibited specific PARP binding allowing visualization of tumors over-expressing PARP. [18F]20 is therefore a potential candidate radiotracer for in vivo PARP PET imaging.

In vitro analysis of PARP inhibitor nanoformulations.

PARP-l is a DNA repair protein that plays a role in a number of repair pathways and also helps in transcriptional regulation; thus PARP inhibitors (PARPi), such as olaparib and BMN-673, act by inhibiting DNA damage repair. This leads to an accumulation of deleterious mutations leading to genetic instability as a result of a number of cell replications. Currently, olaparib is only available in an oral form and has poor bioavailability, consequently leading to poor accumulation in the tumor due to first-pass metabolism. Therefore, in the present study, an injectable nanoparticle formulation of olaparib was created that offers a delivery route in which the drug would be fully bioavailable in the vasculature, suggesting greater tumor accumulation. Our results illustrated that injectable nanoformulations of olaparib and BMN-673, a next generation PARPi, could be developed, and an efficacy test indicated that BMN-673 is a much more potent PARPi than olaparib. The success of these molecular inhibitors as a monotherapy in inhibiting colony formation suggests enhanced efficacy of these treatments in combination with other therapies, even in tumors which have developed resistance.

PARP inhibitor olaparib sensitizes cholangiocarcinoma cells to radiation.

Cholangiocarcinoma (CCA) is a highly malignant tumor with resistance to radiotherapy alone. Olaparib, a highly potent poly(ADP‐ribose) polymerase (PARP) inhibitor, has been shown to sensitize many types of tumor to radiotherapy. However, the effect of olaparib, either as monotherapy or as combination therapy with radiotherapy, on CCA is not known, and our study aimed to explore this. To assess radiosensitization in three CCA cell lines (QBC939, HuH28 and TFK‐1), viability and clonogenic assays were conducted. The absorbed radiation doses were 0 Gy, 2 Gy, 4 Gy, and 6 Gy; olaparib concentrations were 0 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/L, 1000 nmol/L, 2500 nmol/L, 5000 nmol/L and 10 000 nmol/L. The mechanism of olaparib radiosensitization was explored by Western blotting. Immunofluorescence staining and flow cytometry were conducted to explore DNA damage and apoptosis. The radiosensitivity of CCA cells was enhanced by olaparib, which alone had little effect on the CCA cell lines without BRCA mutations. The degree of radiosensitization increased with increasing doses of olaparib by viability and clonogenic assays in vitro. Olaparib was able to enhance the effect of radiation by inhibiting PARP1, inducing DNA lesions and apoptosis. These findings emphasize the role of olaparib in the radiosensitization of CCA cells.

TRITON3: An international, randomized, open-label, phase III study of the PARP inhibitor rucaparib vs. physician’s choice of therapy for patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD).

TPS389 Background: Recent data have shown that up to 25% of patients with advanced prostate cancer, including mCRPC, have a deleterious germline or somatic mutation in BRCA1, BRCA2, ATM, or another homologous recombination DNA repair gene. Such mutations can be used as a molecular marker to select patients for targeted treatment with poly(ADP-ribose) polymerase inhibitors (PARPis), which are lethal to cells with HRD. Treatment with PARPis has shown preliminary evidence of antitumor activity in patients with mCRPC and a mutation in a homologous recombination DNA repair gene (Mateo et al. N Engl J Med. 2015;373:1697-708). These data provide a compelling rationale for evaluating rucaparib, a potent inhibitor of PARP1, PARP2, and PARP3, in patients with mCRPC associated with HRD. Methods: TRITON3 (NCT02975934) is a randomized, phase 3 study evaluating rucaparib 600 mg BID vs physician’s choice of abiraterone, enzalutamide, or docetaxel in patients with mCRPC and a deleterious germline or somatic BRCA1, BRCA2, or ATM mutation (identified by prior local testing or central testing during screening). Patients must have progressed on androgen receptor signaling–directed therapy in the mCRPC setting; prior PARPi treatment or chemotherapy for mCRPC are exclusion criteria. Patients will be randomly assigned in a 2:1 ratio to either rucaparib or physician’s choice, with the possibility for cross over from the comparator treatment to rucaparib upon radiographic progression confirmed by independent radiology review. The primary endpoint is radiographic progression-free survival (modified RECIST v1.1/PCWG3 criteria) assessed by independent radiology review. Secondary endpoints include objective response rate, duration of response, patient-reported outcomes, overall survival, and safety. Pretreatment blood samples collected from all patients will enable development of a noninvasive plasma-based companion diagnostic to select patients who may benefit from rucaparib treatment. Patients (≈400) will be enrolled at &gt; 100 sites worldwide. Clinical trial information: NCT02975934.

C15-162: Phase IB trial of radium 223 and niraparib in patients with castrate resistant prostate cancer (NiraRad).

TPS385 Background: PARP1 is a key regulator of the transition to lethal castrate resistant prostate cancer (CRPC); niraparib is a potent and selective PARP-1 and PARP-2 inhibitor that has shown single agent clinical activity in CRPC. Radium-223 is an alpha particle emitter that causes DNA double strand breaks and is FDA approved for use in CRPC. The addition of a PARP inhibitor (niraparib) may further enhance the clinical benefit of Radium-223 through inhibition of DNA repair and modifications in the bone microenvironment. We hypothesize that targeting the PARP1/androgen receptor (AR) axis in combination with radiopharmaceutical therapy will be safe and may improve clinical outcomes for men with advanced CRPC. Furthermore, combining clinical and pathologic features with correlative studies on tumor tissue, blood, and circulating tumor cells (CTCs) may help us develop biomarkers for future use in the treatment of CRPC. Methods: This is aphase IB dose finding study of patients with metastatic CRPC using a time-to-event continual reassessment (TITE-CRM) design to identify the maximum tolerated dose (MTD) of niraparib (100, 200, or 300 mg QD) in combination with standard doses of Radium-223 in patients with and without prior chemotherapy. Niraparib dosing will continue after 6 cycles of radium-223 until disease progression or intolerable adverse events. Correlative studies include the assessment of tumor biopsy tissues using DNA and RNA next-generation sequencing to assay for the presence of DNA repair aberrations in genes; targeted gene expression profiling of serial RNA (baseline and after cycles 1 and 3) performed on whole blood using the Nanostring PanCancer panel; and evaluation of pre- and post-therapy bone marrow biopsies in a subcohort to characterize the effects of the combined therapy on the bone microenvironment. Additional correlative studies that will be performed on CTC include: assay for nuclear AR expression; immunostaining for the presence of persistent g-H2AX foci, (markers of unrepaired DNA double strand breaks); NFkB activity assayed by immunostaining for p-p65). Enrollment to Dose Level 1 began in October 2017. Clinical trial information: NCT03076203.

TRITON2: An international, multicenter, open-label, phase II study of the PARP inhibitor rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD).

TPS388 Background: Up to 25% of patients with advanced prostate cancer, including mCRPC, have a deleterious germline or somatic mutation in BRCA1, BRCA2, ATM or another homologous recombination (HR) DNA repair gene that can serve as a molecular marker to select those who may respond to poly(ADP-ribose) polymerase inhibitors (PARPis). PARPis are lethal to cells with HRD, and PARPi treatment has shown preliminary evidence of an antitumor effect in patients with mCRPC who harbor a mutation in an HR DNA repair gene (Mateo et al. N Engl J Med. 2015;373:1697-708). These data provide a compelling rationale for evaluating rucaparib, a potent PARP1, PARP2 and PARP3 inhibitor, in patients with mCRPC associated with HRD. Methods: TRITON2 (NCT02952534) is a phase 2 study evaluating rucaparib 600 mg BID in patients with mCRPC. Patients with a deleterious germline or somatic BRCA1, BRCA2 or ATM mutation (per prior local test or central test during screening) will be enrolled into 1 of 2 cohorts based on the presence or absence of measurable visceral and/or nodal disease. An exploratory cohort will enroll patients with an alteration in any of 12 other prespecified HR genes (eg, RAD51C, RAD51D and PALB2), with or without measurable visceral and/or nodal disease. Patients must have progressed on androgen receptor signaling–directed therapy and 1 prior taxane-based chemotherapy for mCRPC. Patients who received prior treatment with a PARPi, mitoxantrone, cyclophosphamide or platinum-based chemotherapy are excluded. The primary endpoint is objective response rate measured using modified RECIST v1.1/PCWG3 for patients with soft-tissue disease and prostate-specific antigen response for patients with nonmeasurable disease. Secondary endpoints include duration of response, radiographic progression-free survival, overall survival, clinical benefit rate and safety. Pretreatment blood samples collected from all patients will enable development of a plasma-based companion diagnostic to select patients who may benefit from rucaparib treatment. Patients (≈160) will be enrolled at &gt; 100 sites worldwide. Clinical trial information: NCT02952534.

A phase 1b study of the anti-PD-1 monoclonal antibody BGB-A317 (A317) in combination with the PARP inhibitor BGB-290 (290) in advanced solid tumors.

48 Background: The release of tumor-associated antigens may enhance the response to immunotherapy. BGB-A317, a humanized IgG4 variant monoclonal antibody engineered to have no Fc gamma receptor binding, targets the programmed cell death-1 (PD-1) receptor. It is being developed in solid and hematologic malignancies at a dose of 200 mg IV Q3W. BGB-290, a potent inhibitor of PARP 1/2, is hypothesized to promote neoantigen release that may potentially increase the efficacy of BGB-A317. A phase 1 study identified 60 mg BID as the recommended Phase 2 dose (RP2D) for BGB-290. This study consists of initial dose escalation to determine the maximum-tolerated dose (MTD), safety, pharmacokinetic (PK) profile, and preliminary antitumor activity of the combination, followed by expansion into ovarian, breast, prostate, gastric, bladder, pancreatic and small cell lung cancers. Methods: Cohorts of 6–12 patients with advanced solid tumors were treated in each of 5 planned dose levels (DLs). In DLs 1–3, BGB-290 doses ranged between 20–60 mg PO BID with BGB-A317 2 mg/kg IV Q3W. In DLs 4–5, BGB-290 doses were 40 or 60 mg BID; A317 was given at 200 mg IV Q3W based on PK data from a single agent Phase 1 study. Results: As of 31 March 2017, 43 patients [median age 63 years (34–75)] were treated in DLs 1–5. Three patients experienced four dose-limiting toxicities: grade 2 nausea (DL4), grade 2 nausea and grade 2 vomiting (DL5), and grade 4 autoimmune hepatitis (DL5). MTD was identified as BGB-A317 200 mg IV Q3W + BGB-290 40 mg PO BID. The most common adverse event (AE) considered related to both study drugs was fatigue. Immune-related AEs of Grade ≥3 were elevated alanine aminotransferase/aspartate aminotransferase (n = 3), autoimmune hepatitis (n = 3), and hepatitis (n = 1). Complete or partial response was observed in 11 patients, 4 of whom had confirmed PR or CR. Plasma/serum exposure of BGB-290 and BGB-A317 were consistent with those in single-agent trials. Conclusions: The combination of BGB-A317 and BGB-290 was generally well tolerated in patients with advanced solid tumors. These results support the continuation of this trial with enrollment into the disease-specific cohorts. Clinical trial information: NCT02660034.

Development of novel synthesized phthalazinone-based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer

Herein we report the synthesis of two series of 4-phenylphthalazin-1-ones 11a-i and 4- benzylphthalazin-1-ones 16a-h as anti-lung adenocarcinoma agents with potential inhibitory activity against PARP-1. All the newly synthesized phthalazinones were evaluated for their anti-proliferative activity against A549 lung carcinoma cell line. Phthalazinones 11c-i and 16b, c showed significant cytotoxic activity against A549 cells at different concentrations (0.1, 1 and 10 μM) for two time intervals (24 h and 48 h). These nine phthalazinones were further examined for their inhibitory activity towards PARP-1. Compound 11c emerged as the most potent PARP-1 inhibitor with IC50 value of 97 nM, compared to that of Olaparib (IC50 = 139 nM). Furthermore, all these nine phthalazinones passed the filters of Lipinski and Veber rules, and predicted to have good pharmacokinetics properties in a theoretical kinetic study. On the other hand, western blotting in A549 cells revealed the enhanced expression of the cleaved PARP-1, alongside, with the reduced expression of pro-caspase-3 and phosphorylated AKT. In addition, ELISA assay confirmed the up-regulation of active caspase-3 and caspase-9 levels compared to the control, suggesting the activation of the apoptotic machinery in the A549 cells. Finally, molecular docking of 11c into PARP-1 active site (PDB: 5WRZ) was performed to explore the probable binding mode.