Trapping PARP-DNA Complexes Research Articles

The evolving landscape of predictive biomarkers of response to PARP inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPis) are DNA-damaging agents that trap PARP-DNA complexes and interfere with DNA replication. Three PARPis - olaparib, niraparib, and rucaparib - were recently approved by the FDA for the treatment of breast and ovarian cancers. These PARPis, along with 2 others (talazoparib and veliparib), are being evaluated for their potential to treat additional malignancies, including prostate cancers. While lack of PARP-1 confers high resistance to PARPis, it has not been established whether or not the levels of PARP-1 directly correlate with tumor response. In this issue of the JCI, Makvandi and coworkers describe an approach to address this question using [18F]FluorThanatrace, an [18F]-labeled PARP-1 inhibitor, for PET. The tracer was taken up by patient tumor tissue and appeared to differentiate levels of PARP-1 expression; however, future studies should be aimed at determining if this tracer can be used to stratify patient response to PARPi therapy.

Abstract 1781: EGFR amplification-A candidate predictive biomarker of PARP inhibitor Talazoparib sensitivity in gliomas

Abstract Poly (ADP-ribose) polymerases (PARP) are enzymes involved in DNA-damage repair. Inhibition of PARP is a promising strategy for targeting cancers with defective DNA-damage repair. Several PARP inhibitors are currently in trials in the adjuvant, neoadjuvant, and metastatic settings for the treatment of ovarian, BRCA-mutated breast, and other cancers. Glioma Stem Cells (GSCs) exhibit higher oxidative base damage, single-strand DNA breaks and genomic instability and is reliant on single-strand break repair/base excision repair to tolerate additional stress. PARP critical for maintaining genomic stability by regulating a variety of DNA damage repair mechanisms is constitutively activated in GSCs. In this study, we show that talazoparib – a unique PARP inhibitor exhibits a strong single agent activity to inhibit proliferation of GSCs in vitro and suppress tumor progression in GSCs xenografts. Talazoparib cytotoxic activity was through inhibiting PARylation and by trapping PARP-DNA complexes resulting in accumulation of DNA damage during replication and ultimately leading to apoptosis. More importantly, we found EFGR amplification, which occurs in about 45% of GBM as a biomarker of response to PARPi. Gene expression and RPPA data showed that GSCs with EGFR amplification were associated with increased ROS, basal expression of DNA repair proteins and more DNA damage following talazoparib treatment, which may partially explain the sensitivity of this group of GSCs to talazoparib. EGFR enzyme activity was important for PARPi sensitivity since kinase dead mutant of EGFR or EGFR knock down cell lines were resistant to PARPi as was also shown by decreased PARP/DNA complex formation. Talazoparib showed strong inhibition of tumor growth in subcutaneous glioma model and 20% increase in median survival in an intracranial model with a 10% blood brain penetration of talazoparib. These data provide mechanistic insights into the anti-cancer activity of PARP inhibitors with mechanistic rationale of PARP inhibition and potential EGFR amplification as biomarker in the development for precision cancer. Citation Format: Shaofang Wu, Feng Gao, Xiaolong Li, Jie Ding, Siyuan Zheng, Emmanuel Martinez-Ledesma, Ningping Feng, Erik Sulman, Roel Verhaak, John de Groot, Dimpy Koul, W.K.Alfred Yung. EGFR amplification-A candidate predictive biomarker of PARP inhibitor Talazoparib sensitivity in gliomas [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 1781. doi:10.1158/1538-7445.AM2017-1781

Abstract OT2-01-13: A phase 3, open-label, randomized, 2-arm international study of the oral dual PARP inhibitor talazoparib in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA)

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, regulated by poly(ADP-ribose) polymerase (PARP). Inhibition of PARP results in synthetic lethality in cells with a BRCA1/2 mutation because of accumulation of irreparable DNA damage; PARP inhibitors have the potential to be selectively toxic for BRCA-mutated cells. In addition to catalytic inhibition, it has been shown that some PARP inhibitors induce PARP trapping at sites of DNA damage. The capacity to trap PARP-DNA complexes varies widely across different PARP inhibitors and is not correlated with PARP catalytic inhibition. Preclinical models have shown trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone. Talazoparib is a dual-mechanism PARP inhibitor that both inhibits the PARP enzyme and effectively traps PARP on DNA, preventing DNA damage repair and resulting in cell death in BRCA1/2-mutated cells. In preclinical studies, talazoparib at nanomolar concentrations showed the highest efficiency at trapping PARP-DNA complexes relative to other PARP inhibitors. In a previous phase 1/2 clinical study, talazoparib as monotherapy (1 mg once daily) resulted in a 50% response rate and an 86% clinical benefit rate at 24 weeks in 14 patients with a germline BRCA1/2 mutation and advanced breast cancer (aBC). Methods: This open-label, randomized, 2-arm, international phase 3 trial (EMBRACA) compares the efficacy and safety of talazoparib with protocol-specific physician's choice (capecitabine, eribulin, gemcitabine or vinorelbine) in patients with aBC. The primary objective is progression-free survival by central imaging. Secondary objectives are objective response rate, overall survival, safety and pharmacokinetics of talazoparib. Exploratory objectives include health-related quality of life measurements and biomarker research in blood and tumor samples that may permit characterization of mechanisms involved in tumor sensitivity and resistance to talazoparib. Key patient eligibility criteria include aged ≥18 years with histologically/cytologically confirmed breast cancer; locally advanced and/or metastatic disease appropriate for systemic single-agent cytotoxic chemotherapy; deleterious or pathogenic germline BRCA1/2 mutations by central laboratory; ≤3 prior cytotoxic chemotherapy regimens for advanced disease (prior platinum is allowed provided patients did not relapse within 6 months in the adjuvant setting or did not progress on platinum therapy); prior treatment with a taxane and/or anthracycline unless medically contraindicated; and ECOG performance status ≤2. Patients (N=429) will be randomized 2:1 to receive either talazoparib capsules (1 mg/day, 21-day cycles) or physician's choice treatment. This trial is currently enrolling patients from the USA, Europe, Israel, Ukraine, Russia, Korea, Australia, Taiwan and Brazil (NCT01945775). This study is funded by Medivation, Inc. Citation Format: Litton J, Ettl J, Hurvitz SA, Mina LA, Rugo HS, Lee K-H, Yerushalmi R, Woodward N, Goncalves A, Moreno F, Roche H, Im Y-H, Martin M, Bhattacharya S, Peterson A, Hannah A, Eiermann W, Blum J. A phase 3, open-label, randomized, 2-arm international study of the oral dual PARP inhibitor talazoparib in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-13.

Abstract OT1-03-17: ABRAZO: An international phase 2 (2-stage, 2-cohort) study of the oral PARP inhibitor talazoparib (BMN 673) in BRCA mutation subjects with locally advanced and/or metastatic breast cancer

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, which is initiated by poly-(ADP-ribose) polymerase (PARP) [1-3]. In cells with deleterious BRCA1/2 mutations, PARP inhibition is synthetically lethal because of accumulation of irreparable DNA damage [1-3]. Talazoparib (BMN 673) exhibits a novel two-pronged approach in treating BRCA1/2-mutant tumors: 1) potent catalytic inhibition of the PARP enzyme; and 2) trapping of PARP at sites of DNA damage [4-7]. The capacity to trap PARP-DNA complexes varies widely across PARP inhibitors and is not correlated with catalytic inhibition potency [4-7]. In preclinical models, trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone [4,7]. Talazoparib is the most potent clinical-stage PARP inhibitor tested to date with the highest efficacy at trapping PARP-DNA complexes [7]. Talazoparib has shown single-agent antitumor efficacy in several solid tumor types and was generally well tolerated in a phase 1/2 clinical study [8]. Methods: This 2-stage, 2-cohort, phase 2 international study (ABRAZO) evaluates the safety and efficacy of talazoparib in patients with a deleterious germline BRCA1 or BRCA2 mutation with locally advanced and/or metastatic breast cancer. Eligible subjects will be assigned to one of two cohorts based on prior chemotherapy for metastatic disease. Cohort 1 (n=70) includes patients with a complete response (CR) or partial response (PR) to platinum-containing regimens for metastatic disease. Cohort 2 (n=70) includes patients who have received >2 prior chemotherapy regimens in the metastatic setting but have not had prior platinum therapy for locally advanced or metastatic disease (prior adjuvant or neoadjuvant therapy with a platinum is allowed). The primary objective is objective response rate (ORR). Secondary objectives include clinical benefit response (CBR) rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Health-related quality of life (QoL) assessments are an exploratory objective. Eligible subjects will receive oral talazoparib (1 mg/day, 21-day cycles) until disease progression or unacceptable toxicity. This trial is currently enrolling patients from the United States and Europe (NCT02034916). This study is funded by BioMarin Pharmaceutical Inc.

Abstract OT1-03-16: EMBRACA: A phase 3, open-label, randomized, parallel, 2-arm international study of the oral PARP inhibitor talazoparib (BMN 673) versus physician's choice in BRCA mutation subjects with locally advanced and/or metastatic breast cancer

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, which is initiated by poly-(ADP-ribose) polymerase (PARP) [1-3]. In cells with deleterious BRCA1/2 mutations, PARP inhibition is synthetically lethal because of accumulation of irreparable DNA damage [1-3]. Talazoparib (BMN 673) exhibits a novel two-pronged approach in treating BRCA1/2-mutant tumors: 1) potent catalytic inhibition of the PARP enzyme; and 2) trapping of PARP at sites of DNA damage [4-7]. The capacity to trap PARP-DNA complexes varies widely across PARP inhibitors and is not correlated with catalytic inhibition potency [4-7]. In preclinical models, trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone [4,7]. Talazoparib is the most potent clinical-stage PARP inhibitor tested to date with the highest efficacy at trapping PARP-DNA complexes [7]. Talazoparib has shown single-agent antitumor efficacy in several solid tumor types and was generally well tolerated in a phase 1/2 clinical study [8]. Methods: This open-label, randomized, parallel, 2-arm, phase 3 international trial (EMBRACA) compares the safety and efficacy of talazoparib with physician's choice (capecitabine, eribulin, gemcitabine, or vinorelbine) in patients with advanced breast cancer. The primary objective is progression free survival (PFS). Secondary objectives include objective response rate (ORR), overall survival (OS), and safety. Exploratory objectives include duration of response (DOR) for objective responders and health-related quality of life measurements. Subject eligibility includes age ≥18 years with histologically/cytologically confirmed breast carcinoma, locally advanced and/or metastatic disease, germline BRCA1/2 mutations, ≤2 prior chemotherapy-inclusive regimens for locally advanced and/or metastatic disease, prior treatment with a taxane and/or anthracycline, and Eastern Cooperative Oncology Group (ECOG) performance status ≤1. Subjects (n=429) will be randomized 2:1 to receive either talazoparib oral capsules (1 mg/day, 21-day cycles) or physician's choice treatment. This trial is currently enrolling patients from the United States, Asia/Pacific, Europe, Israel, and South America (NCT01945775). This study is funded by BioMarin Pharmaceutical Inc.

Abstract B64: Schlafen 11 (SLFN11) irreversibly blocks cell cycle recovery independently of ATR following replicative damage by poly(ADPribose) polymerase inhibitors

Abstract PARP inhibitors kill cancer cells by trapping PARP-DNA complexes, with talazoparib the inhibitor with the highest PARP trapping activity. Genomic analyses across the 60 cancer cell lines of the National Cancer Institute (NCI-60) revealed a highly significant correlation between SLFN11 expression and sensitivity to talazoparib. The causal role of SLFN11 was established after generating four SLFN11-knockout cell lines (prostate DU145, Ewing EW8, leukemia CCRF-CEM and MOLT4) by CRISPR/Cas9 system, and finding that SLFN11 inactivation conferred high resistance to both talazoparib and olaparib. Resistance was caused neither by impaired drug penetration nor by hyperactivation of homologous recombination, as demonstrated in BRCA2-inactivated cells. SLFN11-proficient cells were irreversibly arrested in S phase by talazoparib treatment. Although ATR is the key regulator of the intra-S phase checkpoint, the addition of the ATR inhibitor (VE-821) did not release the replication arrest in SLFN11-proficient cells. By contrast, SLFN11-deficient cells treated with talazoparib reached G2, and their cell cycle and replication arrest was reversed by ATR inhibition. The combination of VE-821 with talazoparib or olaparib was highly synergistic with the greater synergy in the SLFN11-deficient cells. Our study reveals that SLFN11 acts as a novel DNA damage response gene for replicative DNA damage, and that SLFN11 inactivation, which is common in cancer cells, is a novel mechanism of resistance for PARP inhibitors, which can be mitigated by combining PARP and ATR inhibitors. Citation Format: Junko Murai, Yves Pommier. Schlafen 11 (SLFN11) irreversibly blocks cell cycle recovery independently of ATR following replicative damage by poly(ADPribose) polymerase inhibitors. [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 B64.

Abstract 2849: The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells

Abstract Poly(ADP-ribose)polymerases (PARP) inhibitors work as catalytic inhibitors as well as PARP poisons by trapping PARP-DNA complexes (Murai et al., Cancer Res. 2012: 72:5588-99), and talazoparib is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping (Murai et al., Mol. Cancer Ther. 2014:13:433-43). Nevertheless, mining the NCI60 data shows that half of the 60 cell lines are tolerant to talazoparib. To elucidate the mechanisms of such tolerance, we searched for genes with expression levels correlated to talazoparib sensitivity using CellMiner (http://discover.nci.nih.gov/cellminer/). Schlafen 11 (SLFN11) turned out to be one of the most positively correlated genes whose expression is correlated with cellular sensitivity to talazoparib. Using genetically altered cells, we established that SLFN11 inactivation confers high resistance to talazoparib, revealing that SLFN11 inactivation, which is common in cancer cells (Zoppoli et al. PNAS 2014: 109:15030-5), is a novel mechanism of resistance for PARP inhibitors. Based on genetically engineered CRISPR/casp9 SLFN11-knockout cell lines and cell cycle analyses, we found that the parent SLFN11-proficient cells arrest in S phase without DNA replication, while SLFN11-knock out cells maintain their replication until entering G2 phase in the presence of talazoparib. Although ATR is the key regulator of intra-S phase check point, the addition of the ATR inhibitor (VE-821) (Josse et al., Cancer Res. 2014) does not rescue the replication arrest in SLFN11-proficient cells, suggesting the existence of a SLFN11-dependent replication arrest mechanism independently of ATR. On the other hand, the addition of the ATR inhibitor in SLFN11-knock out cells alters the arresting point from G2 to S phase, and eventually increases dead cells. This is probably because the prolonged replication block leads to the collapse of replication forks, resulting in DNA double-strand breaks. Consistent to these findings, the combination of the ATR inhibitor with talazoparib synergistically sensitizes SLFN11-knock out cells more efficiently than the parent SLFN11-proficient cells. Together, these results demonstrate that the combination of the ATR inhibitor with talazoparib can overcome the resistance of SLFN11-deficient cells to talazoparib. Citation Format: Junko Murai, Yves Pommier. The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2015-2849

Abstract 2948: Combination treatment of PARP inhibitor, BMN 673 and DNMT inhibitor, Azacytidine: A potential therapy for BRCA negative and positive, triple negative breast cancers

Abstract Poly-ADP-ribose polymerase inhibitors (PARPis) represent one of the most exciting recent developments in cancer therapy and have shown efficacy in a minority (5-10%) of triple negative (estrogen, progesterone and herceptin receptor negative) breast cancers (TNBCs) with inherited mutations in breast cancer genes BRCA1/2 (BRCA-ve). In PARPi clinical trials, approximately of 60% BRCA-ve patients showed responses, while a significant proportion did not. In addition, sporadic TNBCs with intact BRCA1/2 have failed PARPi therapy. This suggests the urgent need for additional novel therapies for breast cancer. Our studies are underpinned by 4 important findings: (1) the development of a new class of potent PARPis exemplified by BMN 673, (2) the mechanism of cytotoxicity of PARPis, including BMN 673, is attributed to its ability to trap PARP-DNA complexes in chromatin, (3) unpublished findings by our group that PARP interacts with epigenetic factors such as DNMT1 in a high molecular weight complex on chromatin in response to DNA damage and (4) DNMT inhibitors (DNMTis) such as FDA approved Azacytidine (AzaC) covalently trap DNMTs in DNA as their mechanism of action. Therefore, the interaction of PARP and DNMT1 suggest a therapeutic opportunity for combining PARPis and DNMTis in an effort to enhance the effects of PARPi therapy. We first set out to determine whether a combination of these two drugs would enhance trapping of PARP-DNA complexes in chromatin, leading to increased cytotoxicity of TNBC. We treated MDA-MB-231 with BMN 673 (10-100nM) or AzaC (150nM) alone, or the two drugs in combination, and measured PARP trapping in chromatin extracts following induction of DNA damage by radiation (2Gy) after 72hrs. As expected, BMN 673 treatment leads to PARP trapping as determined by western blotting of proteins from chromatin extracts. Interestingly, treatment with AzaC treatment alone also leads to the presence of PARP in chromatin. Importantly, AzaC in combination with BMN 673 treatment shows an at least a 2-fold increase in PARP trapping, compared to the individual drug treatments. To determine whether PARP trapping correlates with cytotoxicity and cell death, BRCA+ve TNBC lines MDA-MB-231 and MDA-MB-468 and BRCA-ve TNBC lines SUM149PT and HCC1937 cells were treated with the drugs alone and in combination and placed in colony forming assays. Compared with single drug treatments, the combination treatment showed a significant (p<0.05) decrease in colony formation in both BRCA positive and negative cell lines indicating BMN 673 could potentially be used in combination with AzaC to improve treatment of TNBCs. In vivo xenograft studies are underway to determine the efficacy of the drug combination compared with single treatments and vehicle controls. Citation Format: Khadiza Chowdhury, Rena G. Lapidus, Eun Yong Choi, Stephen Baylin, Feyruz V. Rassool. Combination treatment of PARP inhibitor, BMN 673 and DNMT inhibitor, Azacytidine: A potential therapy for BRCA negative and positive, triple negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2948. doi:10.1158/1538-7445.AM2015-2948

Abstract OT1-1-14: A phase 2, 2-stage, 2-cohort study of the oral PARP inhibitor BMN 673 in patients with germline BRCA mutation and locally advanced and/or metastatic breast cancer (ABRAZO study)

Abstract Background: Poly-ADP-ribose polymerase (PARP) represents a family of enzymes of which at least two (PARP1 and PARP2) play important roles in DNA repair. PARP inhibition induces synthetic lethality in tumor cells bearing mutations in the genes BRCA1 and BRCA2, both of which are key components in the homologous recombination DNA double-strand breaks repair pathway. BMN 673 is the most potent preclinical PARP inhibitor described to date with the highest efficiency at trapping PARP-DNA complexes (Murai et al, 2014). BMN 673 has shown promising single-agent anti-tumor efficacy in several tumor types in an ongoing Phase 1/2 clinical study. Methods: The purpose of this Phase 2 trial (ABRAZO) is to evaluate the safety and efficacy of BMN 673 in patients with locally advanced or metastatic breast cancer with a deleterious germline BRCA 1 or BRCA 2 mutation. This study is an open-label, 2-stage, 2-cohort Phase 2 study using a Southwest Oncology Group (SWOG) 2-stage design. Eligible subjects will be assigned to either Cohort 1 (n=70) or 2 (n=70) based on prior chemotherapy exposure for metastatic disease: Cohort 1) Subjects who have previously responded to a platinum-containing regimen for metastatic disease with disease progression > 8 weeks following the last dose of platinum; or Cohort 2) Subjects who have received > 2 prior chemotherapy regimens and who have had no prior platinum therapy for metastatic disease. The primary objective is to determine the objective response rate (ORR) of BMN 673 as a single agent for each cohort. The secondary objectives of the study are to determine the following for each cohort: clinical benefit rate (CBR), duration of response (DOR), progression free survival (PFS), and overall survival (OS). Health-related quality of life assessment is an exploratory objective. Patients may be eligible if they are 18 years or older, have histologically or cytologically confirmed carcinoma of the breast, locally advanced and/or metastatic disease, deleterious or pathogenic germline BRCA1 or BRCA2 mutation, prior chemotherapy for metastatic disease based on the above Cohort 1 or Cohort 2 inclusion criteria, ECOG performance status ≤ 1, and no central nervous system (CNS) metastasis except adequately treated brain metastasis documented by baseline CT or MRI scan that has not progressed since previous scans and that does not require corticosteroids for management of CNS symptoms. Eligible patients will receive BMN 673 oral capsules once daily (1.0 mg/day) continuously in 21-day cycles until disease progression or unacceptable toxicity. This trial is enrolling patients from the United States and European countries (NCT02034916). Citation Format: Nicholas C Turner, Judith Balmana, Susan M Domchek, Frances Visco, Charlie Zhang, Nathalie A Lokker, Debra L Lounsbury, Mark E Robson. A phase 2, 2-stage, 2-cohort study of the oral PARP inhibitor BMN 673 in patients with germline BRCA mutation and locally advanced and/or metastatic breast cancer (ABRAZO study) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-14.

Abstract OT1-1-12: A phase 3, open-label, randomized, parallel, 2-arm multi-center study of the oral PARP inhibitor BMN 673 versus physician's choice in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA study)

Abstract Background: Poly-ADP-ribose polymerase (PARP) represents a family of enzymes of which at least two (PARP1 and PARP2) play important roles in DNA repair. PARP inhibition induces synthetic lethality in tumor cells bearing mutations in the genes encoding breast cancer susceptibility gene 1 (BRCA 1) and breast cancer susceptibility gene 2 (BRCA 2), both of which are key components in the pathway of homologous recombination DNA repair. BMN 673 is the most potent preclinical PARP inhibitor described to date with the highest efficiency at trapping PARP-DNA complexes (Murai et al, 2014). BMN 673 is a novel and highly potent PARP inhibitor and has shown promising single-agent anti-tumor efficacy in several tumor types in an ongoing Phase 1/2 clinical study. Methods: The purpose of this multi-center, international, open-label, 2:1 randomized Phase 3 trial (EMBRACA) is to compare the safety and efficacy of BMN 673 versus protocol-specific physician’s choice treatment (capecitabine, eribulin, gemcitabine or vinorelbine) in subjects who have locally advanced and/or metastatic breast cancer with germline BRCA mutations. The primary objective of the study is to compare progression free survival (PFS) of subjects treated with BMN 673 as a monotherapy relative to those treated with protocol-specific physician’s choice treatment. Secondary objectives include objective response rate (ORR), overall survival (OS), safety and pharmacokinetics of BMN 673. Exploratory objectives include duration of response (DOR) and health-related quality of life assessment. Patients may be eligible if they are 18 years or older, have histologically or cytologically confirmed carcinoma of the breast, locally advanced and/or metastatic disease appropriate for systemic single cytotoxic chemotherapy, documentation of a deleterious or pathogenic germline BRCA1 or BRCA2 mutation, ≤ 2 prior chemotherapy-inclusive regimens for locally advanced and/or metastatic disease, prior treatment with a taxane and/or anthracycline in the adjuvant or metastatic setting, ECOG performance status ≤ 1, and no prior platinum treatment for metastatic disease. Patients (n=429) will be randomized 2:1 to receive either BMN 673 oral capsules once daily (1.0 mg/day) in 21-day cycles or protocol-specific physician’s choice treatment. All eligible subjects will receive study drug treatment until disease progression or unacceptable toxicity. This trial is enrolling patients from the United States, Europe, Israel, Asia/Pacific, and South America (NCT01945775). Citation Format: Jennifer K Litton, Joanne L Blum, Wolfgang Eiermann, Young-Hyuck Im, Miguel Martin, Lida Mina, Henri Roché, Hope S Rugo, Frances Visco, Charlie Zhang, Nathalie A Lokker, Debra L Lounsbury. A phase 3, open-label, randomized, parallel, 2-arm multi-center study of the oral PARP inhibitor BMN 673 versus physician's choice in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA study) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-12.

Abstract 1718: Schlafen 11 (SLFN11) is a critical determinant of cellular sensitivity to PARP inhibitors

Abstract Poly(ADP-ribose)polymerases (PARP) are DNA damage sensors and major repair factors for DNA single-strand breaks. Since the discovery of the synthetic lethality of PARP inhibitors in homologous recombination-deficient cells, the mechanism by which PARP inhibitors exert their cytotoxicity has been dominantly interpreted as an accumulation of unrepaired single-strand breaks resulting from catalytic PARP inhibition. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes (1). BMN 673 is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping potency (2). Nevertheless, the NCI60 data show that half of the 60 cell lines are tolerant to BMN 673. To elucidate the mechanisms of tolerance, we searched for genes with expression levels correlated to BMN 673 sensitivity, and found that Schlafen 11 (SLFN11) is one of the most positively correlated genes. Most cells without SLFN11 expression were tolerant to BMN 673. To examine the involvement of SLFN11 in BMN 673 sensitivity, we disrupted the SLFN11 gene in DU145 prostate cancer cells and EW8 Ewing's sarcoma cells where SLFN11 expression levels and BMN 673 sensitivities are high. Knocking out SLFN11 in DU145 and EW8 did not affect their cell growth or cell cycle progression under normal conditions, but diminished their hypersensitivity to BMN 673 and another PARP inhibitor, olaparib. Cell cycle analyses revealed that SLFN11-proficient cells arrested at S phase while SLFN11 deficient cells were able to progress to G2 phase in the presence of BMN 673. Interestingly, arresting points at S phase in SLFN11 proficient cells were earlier when more PARP-DNA complexes were induced, suggesting that SLFN11 inhibits DNA replication under DNA damage. Differential kinetics of DNA repair and PARP-DNA complex formation were observed between SLFN11 proficient versus deficient cells. Our data suggest that the expression of SLFN11 may be a novel genomic biomarker for PARP inhibitor sensitivity. 1. Murai J, Huang S-yN, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Research. 2012;72:5588-99. 2. Murai J, Huang SN, Renaud A, Zhang Y, Ji J, Takeda S, et al. Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib. Mol Cancer Ther. 2014; Submitted. Citation Format: Junko Murai, Rozenn Josse, James H. Doroshow, Yves Pommier. Schlafen 11 (SLFN11) is a critical determinant of cellular sensitivity to PARP inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1718. doi:10.1158/1538-7445.AM2014-1718

Rationale for Poly(ADP-ribose) Polymerase (PARP) Inhibitors in Combination Therapy with Camptothecins or Temozolomide Based on PARP Trapping versus Catalytic Inhibition

We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD(+)-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors.

Stereospecific PARP Trapping by BMN 673 and Comparison with Olaparib and Rucaparib

Anti-PARP drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacologic concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP-DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib, and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as single agent than olaparib, whereas olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also approximately 100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP-DNA complexes.

Abstract A257: Stereospecific trapping of PARP-DNA complexes by BMN 673 and comparison with olaparib and rucaparib.

Abstract Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. Yet, several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes; both olaparib and niraparib act as PARP poisons at pharmacological concentrations (Murai et al., Cancer Res, 2012). Here, we evaluate the novel PARP inhibitor, BMN 673, and compare its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. We show that BMN 673, olaparib, and rucaparib are similarly potent at inhibiting PARP catalytic activity. At the same time, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as a single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient, and that BMN 673 is more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor to date with the highest efficiency at trapping PARP-DNA complexes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A257. Citation Format: Junko Murai, Shar-yin N. Huang, Amelie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H. Doroshow, Yves Pommier. Stereospecific trapping of PARP-DNA complexes by BMN 673 and comparison with olaparib and rucaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A257.

Abstract CN05-01: PARP inhibitors: Trapping of PARP and rational for combinations.

Abstract Poly(ADPribose) polymerases (PARPs) are promising targets of several anticancer drugs in clinical development. We will review the genetic and biochemical evidence that PARP inhibitors act as cytotoxic anticancer agents by trapping PARP-DNA complexes (1). We will also show that PARP inhibitors differ from each other by their ability to trap PARP. They can be ranked by decreasing potency for PARP trapping: BMN-673 ≫ Niraparib > Olaparib = Rucaparib ≫ Veliparib, while all the PARP inhibitors are relatively similar with respect to PARP catalytic inhibition. We propose that PARP trapping results from allosteric effect whereas catalytic inhibition results from competition with NAD+. We will also discuss the rationale for combining PARP inhibitors with topoisomerase I inhibitors, and its dependence on PARP catalytic inhibition, and the rationale for combination with temozolomide, which depends both on catalytic inhibition and PARP trapping. 1. Murai J, Huang S-yN, Das BB, Renaud A, Zhang Y, Doroshow JH, Ji J, Takeda S, Pommier Y. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Research 2012; 72: 5588-99. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN05-01. Citation Format: Yves G. Pommier, Junko Murai, Joel Morris, James H. Doroshow. PARP inhibitors: Trapping of PARP and rational for combinations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN05-01.

Abstract 3421: Differential potentiation of temozolomide and camptothecin by the PARP inhibitors olaparib and veliparib in relationship with their PARP-DNA trapping abilities, and lack of impact of PARP inhibitors on cisplatin activity.

Abstract We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors act by a novel allosteric mechanism of action that involves the trapping of PARP-DNA complexes in addition to their previously established NAD-competitive catalytic inhibitory mechanism). We also demonstrated that PARP inhibitors differ in their ability to trap PARP-DNA complexes, with olaparib being more effective than veliparib in spite of their comparable potency as catalytic PARP inhibitors). Here, by using PARP knockout cells, olaparib and veliparib, we tested the role of PARP trapping in the potentiating effect of PARP inhibitors with the alkylating agent temozolomide and the topoisomerase I inhibitor, camptothecin. Using isogenic chicken DT40 cells, we show that PARP-dependent potentiation of temozolomide is markedly greater for olaparib than veliparib, and goes beyond knocking out PARP-1. On the other hand, in the case of camptothecin, knocking out PARP-1 has a greater effect than veliparib or olaparib. In human DU145 prostate cancer and SF295 glioblastoma cells, olaparib is markedly more potent than veliparib in potentiating the cytotoxicity of temozolomide whereas both olaparib and veliparib are almost comparable in the case of camptothecin. By measuring cellular PARP-DNA complexes), we find that in the case of temozolomide, olaparib is a least 10-fold more potent than veliparib at trapping PARP-DNA complexes. On the other hand, in the case of camptothecin, neither olaparib nor veliparib produced detectable trapping of PARP-DNA complexes. Finally, olaparib fails to sensitize DT40, DU145 or SF295 cells to cisplatin and does not induce detectable PARP-DNA complexes in cisplatin-treated cells. These results suggest that, in the case of temozolomide, olaparib trapping of PARP-DNA complexes is more toxic than inactivating PARP catalytic activity, which is not the case for topoisomerase I-induced DNA damage, where catalytic inactivation of PARP appears to be the sole enhancing mechanism. Citation Format: Yves G. Pommier, Junko Murai. Differential potentiation of temozolomide and camptothecin by the PARP inhibitors olaparib and veliparib in relationship with their PARP-DNA trapping abilities, and lack of impact of PARP inhibitors on cisplatin activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2013-3421