Poly (ADP-ribose) Polymerase Trapping Research Articles

PARP trapping is governed by the PARP inhibitor dissociation rate constant

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. Yet, PARPi function mainly by trapping PARP1 onto DNA with a wide range of potency among the clinically relevant inhibitors. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon at sites of DNA damage that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA-binding protein recruitment. These results clarify how PARPi trap, shed new light on how PARPi function, and describe how PARPi properties correlate to trapping potency.

Real-time imaging of drug-induced trapping of cellular topoisomerases and poly(ADP-ribose) polymerase 1 at the single-molecule level.

Topoisomerases (TOP1, TOP2α, and β) are nuclear enzymes crucial for virtually all aspects of DNA metabolisms. They also are the targets of important anti-tumor chemotherapeutics that act by trapping the otherwise reversible topoisomerase-DNA covalent complex intermediates (TOPccs) that are formed during their catalytic reactions, resulting in long-lived topoisomerase DNA-protein crosslinks (TOP-DPCs) that interfere with DNA transactions. The Poly(ADP-ribose) polymerase (PARP) family protein PARP1 is activated by DNA damage to recruit DNA repair proteins, and PARP inhibitors are another class of commonly used chemotherapeutics, which bind and trap PARP molecules on DNA. To date, the trapping of TOPccs and PARP by their respective inhibitors can only be measured by immune-biochemical methods in cells. Here, we developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we calculated the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real time. This novel technique should help elucidate the molecular processes that repair TOPcc and PARP trapping and facilitate the development of novel topoisomerase and PARP inhibitor-based therapies.

Abstract 1581: KSQ-4279, a first-in-class USP1 inhibitor shows strong combination activity with multiple PARP inhibitors in BRCA mutant cancers

Abstract Tumors harboring BRCA1/2 mutations and other homologous recombination deficiencies are sensitive to agents targeting pathways involved in DNA repair including poly (ADP-ribose) polymerase (PARP) inhibitors, which have been approved for the treatment of BRCA mutant cancers. Despite the clinical benefit with these drugs, many patients achieve incomplete disease control and often develop resistance. By employing our CRISPRomics® technology to screen over 600 cancer cell lines, we identified the deubiquitinating enzyme USP1 as one of the top targets that displays selective anti-tumor activity in ovarian and triple negative breast cancers with homologous recombination deficiencies. Subsequent drug discovery efforts identified KSQ-4279 as a potent and highly selective first-in-class small molecule USP1 inhibitor that is now in clinical development. In preclinical models, we previously demonstrated that KSQ-4279 shows therapeutic potential in combination with olaparib for treating patients who are either intrinsically resistant, or have developed acquired resistance to PARP inhibitors. We performed studies to investigate the therapeutic potential of combining KSQ-4279 across a variety of PARP inhibitors with different ‘PARP trapping’ potencies. Clonogenic assays demonstrated synergistic effects of KSQ-4279 across multiple PARP inhibitors regardless of their PARP trapping potency. Second generation PARP1 selective inhibitors such as AZD5305 are currently in clinical development and may provide a safety and efficacy advantage for patients. We have investigated the potential of combining KSQ-4279 with AZD5305 in a PARP refractory patient-derived triple negative breast cancer (TNBC) xenograft model by evaluating tolerability and anti-tumor efficacy. We observed significantly greater and more durable anti-tumor activity, including regressions, with the combination therapy compared to single agents. Our data supports the clinical testing of KSQ-4279 in combination with PARP inhibitors. Citation Format: Erica Tobin, Pamela Sullivan, Morgan Murray, Hugh Gannon, Anne Dodson, Sol Shenker, Frank Stegmeier, Andrew Wylie, Louise Cadzow. KSQ-4279, a first-in-class USP1 inhibitor shows strong combination activity with multiple PARP inhibitors in BRCA mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1581.

151 (PB031) - Combination therapy with decitabine and olaparib in poly(ADP-ribose) polymerase inhibitor (PARPi) resistant high grade serous ovarian cancer

Background: Recurrent high grade serous ovarian cancer (HGSOC) is incurable and lethal. Therefore, novel drug combinations are critically needed for this patient population. DNA methyltransferase inhibitors (DNMTi) can cause DNA damage, which activates poly(ADP-ribose) polymerase (PARP) and leads to PARP trapping. Additionally, DNMTi are incorporated into replicating DNA, which causes DNMT-DNA adducts that are then degraded. Therefore, the novel drug combination of DNMTi decitabine and PARP inhibitor (PARPi) olaparib were tested in preclinical HGSOC models.

483P Pre-clinical and early clinical assessment of the safety and anti-tumor activity of RP12146, a PARP1/2 inhibitor in solid tumors

RP12146 is an orally bioavailable, potent small-molecule inhibitor of poly (ADP-ribose) polymerase (PARP) 1 and PARP 2 with an IC50 of 0.62nM and 0.44nM respectively and with PARP trapping activity. A phase I/Ib study of RP12146 as a single agent is underway to determine the safety, pharmacokinetics, and anti-tumor activity.

Anticancer effects of PARP1 hyperactivation by a decoy oligodeoxynucleotide in vitro and in vivo.

e15060 Background: Poly (ADP-ribose) polymerase (PARP) inhibitors mediate significant anticancer effects against homologous recombination defective (HRD) tumors but have limited efficacy against homologous recombination proficient (HRP) neoplasms. Moreover, HRD tumors often become resistant to PARP inhibitors during treatment, constituting a major limitation to the clinical use of these agents. Here, we describe the metabolic, antineoplastic and immunomodulatory effects of a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy (OX413), resulting in constitutive hyperactivation of PARP1 and consequent exhaustion of the DNA damage response. Methods: OX413-induced PARP trapping and hyperactivation, NAD+ consumption and cell cytotoxicity were monitored using (HRP and HRD) breast, ovarian or prostate cancer cells as well as normal fibroblasts and leukocytes. DNA repair abrogation was monitored by analyzing repair protein recruitment to damage sites. Accumulation of cytoplasmic DNA fragments was monitored after DNA staining and microscopy analysis. Effects on the innate immune response was assessed by following STING pathway activation and T-cell mediated anti-tumor cytotoxicity. In a mouse xenograft model of breast cancer, OX413-induced PARP activation and tumor infiltration by immune cells were analyzed ex-vivo by flow cytometry. Antitumor efficacy of OX413 alone or combined to a PD1 blocker was assessed in MPA/DMBA-driven HR+ HER2- mouse mammary carcinomas. Results: At odds with conventional PARP inhibitors, OX413 bound to and hyperactivated PARP1 with high affinity and in a dose-dependent manner, resulting in elevated cytotoxicity in multiple cancer (but not normal) cells irrespective of HR status. OX413 impaired DNA repair due to PARP trapping and rapid NAD+ consumption, leading to the accumulation of cytoplasmic chromatin fragments (CCFs). Consistent with this, OX413 triggered activation of the CGAS/STING pathway, CCL5 secretion and potentiation of tumor-targeting T cell responses. PARP1 engagement and STING activation were corroborated in vivo in EMT6 mouse mammary carcinomas, correlating with anticancer effects coupled to tumor infiltration by innate and adaptive immune cells. In MPA/DMBA-driven tumors, OX413 mediated considerable anticancer effects that could be exacerbated by PD1 inhibition. Conclusions: Our results provide preclinical rationale for using OX413 to trigger metabolic exhaustion in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD as well as HRP tumors.

An investigator-initiated phase II trial of a PARP inhibitor niraparib monotherapy for patients with pre-treated, BRCA-mutated, unresectable/recurrent biliary tract, pancreatic, and other gastrointestinal cancers (NIR-B trial).

TPS4174 Background: Recent comprehensive genomic profiling tests have revealed therapeutic target molecules. However, because many targets are present in only a small fraction of patients, a large number of patients need to be screened for enrollment in a single study. In order to overcome this patient identification barrier, the SCRUM-Japan, nationwide large-scale genomic profiling platform, efficiently has been performed umbrella- and basket-type clinical trials. Among the platforms, we have reported that there are BRCA1/2-mutated patients in biliary tract, pancreatic, and other gastrointestinal cancers. Niraparib is an anticancer drug belonging to poly(ADP-ribose) polymerase (PARP) inhibitors. Niraparib has been shown to be selective for PARP1/2, to be more cytotoxic among other PARP inhibitor because of its PARP trapping activity. Methods: This is an investigator-initiated, multicenter, three-cohort phase 2 study. Main eligibility criteria are unresectable, advanced or recurrent biliary tract cancers (cohort A), pancreatic cancers (cohort B), and other gastrointestinal cancers (cohort C) with BRCA1/2 gene mutations identified by germline test or genomic profiling test with either circulating tumor DNA (ctDNA) or tumor tissue, refractory or intolerant to previous treatments, and adequate organ function. Primary endpoint is the investigator-assessed objective response rate in each cohort with a threshold-response rate of 10% and an expected response rate of 35%. Key secondary endpoints are progression-free survival, overall survival, disease control rate, duration of response, and safety. Patients with body weight of 77 kg or more and a platelet count of 150,000 /µL or more receive 300 mg of niraparib, and less than 77 kg or having a platelet count less than 150,000 /µL receive 200 mg of niraparib, orally once daily. Furthermore, pre-treatment tumor tissue and serial ctDNA will be collected and analyzed to investigate the resistance mechanisms and provide clinically meaningful biomarker which may be used for identifying and implementing treatment changes. The trial was initiated in January 2021 with enrollment being ongoing. Thirty-three out of planned 60 patients (cohort A/B/C; 25/25/10, respectively) have been enrolled as of December 2021. Funding: Takeda Pharmaceutical Co., Ltd. Clinical trial information: jRCT2011200023.

RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer.

Current targeted cancer therapies are largely guided by mutations of a single gene, which overlooks concurrent genomic alterations. Here, we show that RNASEH2B, RB1, and BRCA2, three closely located genes on chromosome 13q, are frequently deleted in prostate cancer individually or jointly. Loss of RNASEH2B confers cancer cells sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to impaired ribonucleotide excision repair and PARP trapping. When co-deleted with RB1, however, cells lose their sensitivity, in part, through E2F1-induced BRCA2 expression, thereby enhancing homologous recombination repair capacity. Nevertheless, loss of BRCA2 resensitizes RNASEH2B/RB1 co-deleted cells to PARP inhibition. Our results may explain some of the disparate clinical results from PARP inhibition due to interaction between multiple genomic alterations and support a comprehensive genomic test to determine who may benefit from PARP inhibition. Last, we show that ATR inhibition can disrupt E2F1-induced BRCA2 expression and overcome PARP inhibitor resistance caused by RB1 loss.

PARP Inhibition with Talazoparib Enhances DNA Damage and Anti-Leukemic Activity of Venetoclax in Preclinical Human Acute Myeloid Leukemia (AML) Models

Background: BCL-2 is an anti-apoptotic protein associated with chemotherapy resistance and survival of acute myeloid leukemia (AML) cells following cell stress or DNA damage. Venetoclax (Ven) is a BH3-mimetic that competitively binds to BCL-2, allowing BH3 proteins to bind to pro-apoptotic proteins (BAX, BAK) and induce cell death. The combination of Ven with Azacitidine (Aza), is now standard of care therapy for older patients with AML. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and preventing dissociation of PARP from the DNA (termed PARP trapping). The PARP inhibitor, talazoparib (Tala) displays 10,000-fold increased PARP trapping as compared to other clinical PARP inhibitor agents in tumor models. We hypothesized that Tala could enhance the anti-leukemic activity of Ven on AML cells specifically by increasing DNA damage.Methods: Human AML (Molm13) cells were continuously exposed to increasing doses of Ven (1nM - 10mM), Tala (1nM - 10mM), and Aza (0.1-5mM) alone and in combination. Synergy reports were generated using CompuSyn software. Cell viability was measured using a WST colorimetric assay. Apoptosis (annexin /PI), DNA damage (pH2AX), and cell cycle arrest (propidium iodide) were quantified by flow cytometric analysis. DNA damage was also evaluated using the Fast-Halo assay. In vivo efficacy was assessed in NSG mice systematically engrafted with stably luciferase transfected Molm13 BLIV cells via tail vein injection. Cohorts of mice (5-10) were treated with vehicle, Ven (100 mg/kg given 5 days/week for 3 weeks), Tala (0.33 mg/kg for 5 days/week over 3 weeks), Ven + Tala (same doses), and Ven+ Aza (1.25 or 2.5 mg/kg daily for 7 days). Treatment effects on leukemia burden, toxicity, and overall survival were determined using weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity, respectively.Results: Human AML cells treated with Ven displayed dose and time-dependent induction of DNA damage, apoptosis, and cell death. Similarly, treatment with single agent Tala resulted in dose-dependent anti-proliferative effects and PARP trapping. Combination in vitro therapy with Ven (0.1 - 1 μM) + Tala (10- 100 nM) or Ven (same doses) + Aza (1 μM) resulted in significant and synergistic anti-leukemic effects improving upon monotherapy with Ven, Tala, or Aza alone. However, in contrast to Ven+Aza, increased levels of DNA damage (as determined by phosphorylated H2AX and Fast Halo assays) were detected as well as induction of apoptosis, cell cycle arrest, decreased viability, and increased cell death vs vehicle and single agent therapy.We then evaluated the effects of in vivo treatment with vehicle, Ven, Tala, Ven + Tala, and Ven +Aza in NSG mice systemically engrafted with human luciferase-tagged AML cells (Molm13 BLIV). Single agent Ven or Tala therapy decreased systemic AML burden but did not prolong overall survival over vehicle treated mice. Combination Ven + Tala significantly prolonged overall survival of leukemia xenografted mice over vehicle, Ven, or Tala monotherapy. Of note, Ven + Tala treatment appeared to result in similar disease burden reduction and prolonged overall survival as Ven + Aza treated animals (Figure 1). Preliminary experiments of iv vivo triple therapy with Ven + Aza + Tala in AML xenografted mice resulted in further reduction in AML disease burden but was associated with marked weight loss leading to early morbidity and shortened overall survival (data not shown). Additional doses and schedules of this triplet approach are under investigation.Conclusions: Our results demonstrate that addition of the potent PARP inhibitor, talazoparib, to the BCL-2 inhibitor, venetoclax, enhances DNA damage and results in potent in vitro and in vivo activity in preclinical human AML models. Further evaluation of this combination in primary AML samples with varying genetic backgrounds as well as in confirmatory primary xenograft models is underway. These data strongly support future clinical investigation of PARP inhibitors as a novel class of agents with the potential to significantly enhance the efficacy of novel targeted therapies for acute leukemia. [Display omitted] DisclosuresWang: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; DAVA Oncology: Consultancy, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Advisory Board; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

An Overview of PARP Inhibitors for the Treatment of Breast Cancer.

Loss-of-function mutations in BRCA1 and BRCA2 are detected in at least 5% of unselected patients with breast cancer (BC). These BC susceptibility genes encode proteins critical for DNA homologous recombination repair (HRR). This review provides an update on oral poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of BC. Olaparib and talazoparib are PARP inhibitors approved as monotherapies for deleterious/suspected deleterious germline BRCA-mutated, HER2-negative BC. Olaparib is approved in the USA for metastatic BC and in Europe for locally advanced/metastatic BC. Talazoparib is approved for locally advanced/metastatic BC in the USA and Europe. In phase 3 trials, olaparib and talazoparib monotherapies demonstrated significant progression-free survival benefits compared with chemotherapy. Common toxicities were effectively managed by supportive treatment and dose interruptions/reductions. Veliparib combined with platinum-based chemotherapy has also shown promise for locally advanced/metastatic BC in a phase 3 trial. Differences in efficacy and safety across PARP inhibitors (olaparib, talazoparib, veliparib, niraparib, rucaparib) may relate to differences in potency of PARP trapping on DNA and cytotoxic specificity. PARP inhibitors are being investigated in early BC, in novel combinations, and in patients without germline BRCA mutations, including those with somatic BRCA mutations and other HRR gene mutations. Ongoing phase 2/3 studies include PARP inhibitors combined with immune checkpoint inhibitors for the treatment of triple-negative BC. Wider access to testing for BRCA and other mutations, and to genetic counseling, are required to identify patients who could benefit from PARP inhibitor therapy. The advent of PARP inhibitors has potential benefits for BC treatment beyond the locally advanced/metastatic setting.

Abstract A31: The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors

Abstract Background: Poly (ADP-ribose) polymerase (PARP) inhibitors have transformed treatment of ovarian cancer in the past decade. Most of the currently active PARP inhibitors have similar ability to inhibit PARP’s enzymatic activity. However, it has been previously reported that velipairb, rucaparib, olaparib, niraparib, and talazoparib vary in their PARP trapping potency levels. Veliparib was also characterized to be mainly a catalytic inhibitor while the other four drugs were determined to be both PARP trappers and catalytic inhibitors, with talazoparib being the most potent PARP trapper. Responses in many patients with HGSC tumors are limited, particularly those women without known mutations in BRCA1 or BRCA2. Recently, PARP inhibitors have been reported to influence innate immune responses. For these reasons, there are ongoing clinical trials testing the safety and efficacy of PARP inhibitors in combination with various immune checkpoint inhibitors. The objective of this study is to compare the effect of the five most clinically active PARP inhibitors on type 1 interferon regulated genes and the PD-L1 gene. Methods: Two high-grade serous ovarian cancer cell lines were used in this study: CAOV3 (TP53 mutant) and OVCAR3 (TP53 mutant, CCNE1-amplified, and EMSY-amplified). The cell lines were treated for 48 hours with the PARP inhibitors listed above as well as a vehicle control (DMSO). Gene expression was measured using TaqMan assays. CXCL10 (IP-10) protein expression levels were determined via an ELISA assay. Summary of the Data: Talazoparib upregulated CXCL10 gene expression in both OVCAR3 and CAOV3 cell lines. These results were translated to the protein level, as we observed an increase in IP-10 protein expression levels after talazoparib treatment in the OVCAR3 cell line. IP-10 protein levels were also significantly increased when treated with the second most potent PARP inhibitor, niraparib. A statistically significant effect was not observed after treatment with the three other PARPis. Futhermore, CD274 (PD-L1) gene expression levels were significantly increased after talazoparib and not veliparib treatment in the CAOV3 cell line. Conclusions: Our data suggest that there are distinct effects exhibited among PARP inhibitors on innate immune pathways. The two most potent PARP inhibitors, niraparib and talazoparib, had the greatest effect on these pathways, specifically in BRCA wild-type HGSCs, compared to the three other drugs. These finding support the idea that we need to define the best PARP inhibitor to use in future clinical trials when combining this group of anticancer agents with immune checkpoint inhibitors. Citation Format: Monica Wielgos-Bonvallet, Danhui Weng, Petar Jelinic, Douglas A. Levine. The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A31.

PARP Inhibitors as Therapeutics: Beyond Modulation of PARylation.

Poly (ADP-ribose) polymerase (PARP) 1 is an essential molecule in DNA damage response by sensing DNA damage and docking DNA repair proteins on the damaged DNA site through a type of posttranslational modification, poly (ADP-Ribosyl)ation (PARylation). PARP inhibitors, which inhibit PARylation through competitively binding to NAD+ binding site of PARP1 and PARP2, have improved clinical benefits for BRCA mutated tumors, leading to their accelerated clinical application. However, the antitumor activities of PARP inhibitors in clinical development are different, due to PARP trapping activity beyond blocking PARylation reactions. In this review, we comprehensively address the current state of knowledge regarding the mechanisms of action of PARP inhibitors. We will also discuss the different effects of PARP inhibitors in combination with cytotoxic chemotherapeutic agents regarding the mechanism of regulating PARylation.

Addition of the PARP Inhibitor, Talazoparib, to Gemtuzumab Ozogamicin Significantly Enhances Anti-Leukemic Activity in Human CD33+ Acute Myeloid Leukemia

Background CD33 (Siglec3) is a cell surface transmembrane receptor that is rapidly internalized and highly expressed on AML blasts but is absent on normal hematopoietic stem cells. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 antibody conjugated to a DNA strand scission inducing agent (calicheamicin) was recently FDA approved for the treatment of newly-diagnosed or relapsed/refractory CD33-positive acute myeloid leukemia (AML). GO has been shown to exert clinical activity in leukemia patients. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and by preventing the dissociation of PARP from the DNA (PARP trapping). Talazoparib is unique among clinical PARP inhibitors in displaying 10,000-fold increased PARP trapping as compared to other agents . We hypothesized that combination therapy using GO and Talazoparib would result in synergistic anti-leukemic effects on human CD33+ AML cells due to the ability of the PARP inhibitor to enhance levels of DNA damage induced by GO therapy. Materials and Methods Human AML cell lines were characterized for CD33 expression using flow cytometry after staining with antibody-linked fluorescent QuantiBrite Beads. Cells were continuously exposed to varying doses of GO (10pM - 100mM) and PARP inhibitors (1nM - 100mM) for 96h alone and in combination. Cell viability was measured immediately following treatment using a WST colorimetric assay. Treatment-induced apoptosis (annexin/PI) and DNA damage (H2AX) were quantified by flow cytometric assays. Synergy reports were generated using Compusyn software. In vivo efficacy was assessed in NSG mice systematically engrafted with luciferase labeled human CD33+ AML cells following tail vein injection. Animals were treated with varying doses of vehicle, GO (1 and 50ug/kg 1x/week for 3 weeks), or talazoparib (0.1 and 0.33mg/kg 5 days/week) either alone or in combination. Treatment effects on leukemia burden, toxicity, and survival were determined by weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity. Results Human AML cell lines (HEL, HL60) express high expression levels of CD33 molecules/cell (43,645 and 31,286 respectively) relative to negative controls. Continuous exposure to single agent GO and Talazoparib for 96h resulted in a dose dependent inhibition of human AML cell growth (HEL, HL60) . IC values for GO were 0.01 - 6.6μg/ml and for Talazoparib were 0.8-0.95μM. Combination in vitro therapy with GO (0.005 - 1μg/ml) and Talazoparib (fixed dose 100nM) resulted in synergistic anti-leukemic effects (p<0.01) significantly improving upon monotherapy. Software analyses yielded a combination Index (CI) <1 consistent with synergistic anti-leukemic effects. Combination GO and Talazoparib therapy also significantly enhanced AML cell apoptosis (p=0.0111) and levels of DNA damage (phosphorylated H2AX) (p=0.0054) over single agent activity. Evaluation of PARP trapping by western blot analysis is ongoing. In vivo administration of GO (1-50μg/kg) and Talazoparib (0.1-0.33mg/kg) in NSG mice with systemic engraftment of luciferase tagged human CD33+ AML cells was generally well tolerated with no significant weight loss or early morbidity. Single agent GO and Talazoparib therapy decreased systemic AML burden in a dose dependent manner and prolonged overall survival over vehicle treated mice (P<0.05). Concomitant GO (1μg/kg) and Talazoparib (0.33μg/kg) treatment was similarly well tolerated with no notable weight loss or toxicities. Combination GO and Talazoparib therapy significantly prolonged overall survival of leukemia xenografted mice over vehicle (p=0.0018) and single agent therapy with the same doses of GO (p=0.0018) and Talazoparib (p=0.0499), respectively). Conclusions In summary, our results demonstrate that the addition of the PARP inhibitor, Talazoparib, to the CD33 antibody drug conjugate, GO, results in potent in vitro and in vivo anti-tumor activity in human CD33+ AML preclinical models. Further studies investigating this novel combinatorial approach in AML are currently ongoing. Due to GO's FDA approval for CD33+ AML in 2018, this data strongly supports future clinical investigation using PARP inhibitors as a novel class of agents for combination therapy to significantly enhance the efficacy of ADCs. Figure 1 Disclosures Wang: Amgen: Other: Advisory role; Agios: Other: Advisory role; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau.

Veliparib in ovarian cancer: a new synthetically lethal therapeutic approach.

Epithelial ovarian cancer (EOC) accounts for nearly 90% of all ovarian malignancies. The standard therapeutic strategy includes cytoreductive surgery and neo (adjuvant) platinum-based chemotherapy. Relapse of advanced high grade serous ovarian cancer (HGSOC) is related to the development of drug resistance. A defective DNA damage response is a defining hallmark of HGSOC. Poly (ADP-ribose) polymerase (PARP) inhibitors exploit this deficiency through synthetic lethality and have emerged as promising anticancer therapies, especially in breast cancer gene (BRCA1 or BRCA2) mutation carriers. Apart from inducing synthetic lethality, PARP inhibitors have also been shown to trap PARP1 and PARP2 on DNA, leading to PARP-DNA complexes. This "PARP trapping" potentiates synergism between PARP inhibition and both alkylating agents and platinum-based chemotherapy. However, there are remarkable differences in the ability of PARP inhibitors to trap PARP, based on the size and structure of each separate molecule. Since monotherapy with PARP inhibitors is unlikely to induce cancer cell death in BRCA-proficient tumors, the efficacy of PARP inhibitors could be potentially optimized when combined with DNA-damaging agents, or with molecular targeted agents that also impair mechanisms of DNA repair. Olaparib, rucaparib, and niraparib have all obtained US Food and Drug Administration (FDA) and/or European Medicines Agency (EMA) approval in ovarian cancer in different settings. Veliparib does not yet have an approved label; nevertheless, there are currently promising results available in preclinical and early clinical settings. This comprehensive review summarizes the mechanism of action of veliparib and provides an overview of its early and ongoing clinical investigations.

NCI9782: A phase 1 study of talazoparib in combination with carboplatin and paclitaxel in patients with advanced solid tumors.

e14640 Background: Poly(ADP-ribose) polymerase (PARP) enzymes are involved in DNA repair and activated by DNA strand breaks. DNA damage from carboplatin is associated with activation of PARP. Preclinical data indicate that PARP inhibition and trapping potentiates the anti-tumor effect of platinum chemotherapy. Talazoparib (T) is an oral, selective PARP inhibitor. This phase I study combines T with carboplatin (C) and paclitaxel (P). Methods: Two dosing schedules are being investigated. C is administered on day 1 and P on days 1, 8, and 15 of a 21-day cycle. T (100-1000mcg) is dosed once daily for days 1-7 (schedule A) or days 1-3 (schedule B). Dose escalation is by 3+3 design. Patients (pts) must have tumor type that is expected to respond to C + P or have BRCA germline or somatic mutation and adequate organ function. After 4-6 cycles of combination therapy, pts may continue the combination, change to C and intermittent T without P or change to T alone. Each schedule will have a 6 pt dose expansion at the MTD. The dose level (DL) 1 for schedule B is the previously reported MTD from schedule A (T 250mcg with C AUC 6 + P 80mg/m2). Results: Schedule B results are reported: 15 pts (median age 56 yrs [range 43-76]) have been enrolled. Primary malignancies include colorectal (4), pancreas (4), prostate (2), urothelial (2), and other (3). Dose was initiated at Schedule A MTD. DL2 (T 350mcg with C AUC 6 + P 80mg/m2) exceeded the MTD with 2 of 6 pts experiencing hematologic dose limiting toxicities (DLTs). DL1 is the confirmed schedule B MTD. Dose expansion to 6 pts is ongoing. Of the 11 pts with measurable disease, 3 (27%) had PR and 5 (45%) had SD. Pts were on study a median of 10 weeks (range 5-36+). Most common grade 3/4 AEs include leukopenia (53%), neutropenia (47%), and anemia (47%). One grade 5 AE of intracranial hemorrhage occurred, possibly related to therapy in the setting of grade 3 thrombocytopenia and concern for CNS disease. Conclusions: The schedule B MTD and RP2D is T 250 mcg with C AUC 6 and P 80mg/m2. Data from the full dose expansion will be presented. This combination was tolerated with prolonged responses seen at lower dose T in combination with C+P. Clinical trial information: NCT02317874.

Overexpressed ABCB1 Induces Olaparib-Taxane Cross-Resistance in Advanced Prostate Cancer

Castration-resistant prostate cancer remains as an incurable disease. Exploiting DNA damage repair defects via inhibition of poly (ADP-ribose) polymerase (PARP) is becoming an attractive therapeutic option. The TOPARP-A clinical trial demonstrated that the PARP inhibitor olaparib may be an effective strategy for treating prostate cancer. However, several unanswered questions regarding the use of olaparib remain: 1) How do we best stratify patients for olaparib treatment? 2) Where do we place olaparib in the treatment sequence paradigm? 3) Is there cross-resistance between olaparib and currently used therapies? Here, we tested putative cross-resistance between current therapies and olaparib in treatment-resistant castration-resistant prostate cancer models. Docetaxel-resistant cells exhibited robust resistance to olaparib which could be attributed to blunted PARP trapping in response to olaparib treatment. Upregulated ABCB1 mediates cross-resistance between taxanes and olaparib, which can be overcome through decreasing ABCB1 expression or inhibiting ABCB1 using elacridar or enzalutamide. We also show that combining olaparib with enzalutamide is more effective in olaparib-sensitive cells than either single agent. Our results demonstrate that cross-resistance between olaparib and other therapies could blunt response to treatment and highlight the need to develop strategies to maximize olaparib efficacy.

Olaparib: A Novel Therapy for Metastatic Breast Cancer in Patients With a BRCA1/2 Mutation

Breast cancer is the most frequently diagnosed cancer in women globally. Genetic mutations can increase the risk of developing breast cancer. Inherited germline mutations in BRCA1 and BRCA2 tumor suppressor genes (gBRCAm) account for 5% to 10% of breast cancer cases. The recent approval of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in HER2-negative, metastatic breast cancer provides an additional treatment option for patients with a gBRCAm. Inhibition of PARP results in the trapping of the PARP-DNA complex at replication forks, causing single-strand breaks to become double-strand breaks (DSBs). PARP trapping and the accumulation of DSBs ultimately leads to cell apoptosis. Cells deficient in BRCA1/2 are particularly sensitive to the effects of PARP inhibition, as cells lacking these functional proteins are unable to repair DSBs, resulting in synthetic lethality. The phase III OlympiAD trial showed a progression-free survival benefit but no overall survival benefit, leading to the US Food and Drug Administration approval of olaparib. The purpose of this article is to describe current data regarding the use of olaparib in metastatic breast cancer, its role in the treatment of patients with a gBRCAm, and the clinical implications of its approval for oncology advanced practitioners.

PARP trapping by Talazoparib is a Potent Mechanism of Radiosensitization in Small Cell Lung Cancer Cell Lines and Patient-Derived Xenografts

Small cell lung cancer (SCLC) is an aggressive malignancy with a critical need for novel molecular therapies. We hypothesized that poly(ADP)-ribose polymerase (PARP) inhibitors could effectively sensitize SCLC cells to ionizing radiation (IR). We further hypothesized that PARP trapping, a cytotoxic mechanism of action distinct from inhibition of the PARP enzyme and the DNA damage response, contributes to radiosensitization by PARP inhibitors. Short-term viability assays and clonogenic survival assays (CSA) were used to assess radiosensitization in six SCLC cell lines. PARP trapping and PARP enzymatic inhibition were analyzed by quantifying the chromatin-bound fraction of PARP1 and the total cell PAR polymer, respectively, using western blot. Doses of veliparib, a weak PARP trapper, and talazoparib, a potent PARP trapper, that had equivalent enzymatic inhibitory activity but differing PARP trapping activity were identified and compared in CSAs. The number of g-H2AX foci induced by veliparib and talazoparib were compared with and without IR using immunofluorescence assays. Talazoparib, IR, and their combination were tested in three patient-derived xenograft (PDX) models, and time for the tumors to grow to 1000 mm3 was compared using the log-rank test. Radiosensitization by talazoparib was seen in 5 of 6 cell lines in short term viability assays: one cell line at 0.2 nM (Dose Modification Factor [DMF] 1.56), two cell lines at 2 nM (DMF 1.34 – 1.86), and four cell lines at 20 nM (DMF 1.61 – 2.88). Using CSAs, sensitization to IR was seen in 3 of 3 cell lines, with two cell lines sensitized at 20 nM (DMF 1.40-2.13) and one at 200 nM (DMF 2.20). Doses of 200 nM talazoparib and 1600 nM veliparib similarly inhibited PAR polymerization; however, as expected, talazoparib exhibited greater PARP trapping activity. This dose of veliparib was insufficient for radiosensitization, while the greater PARP trapping observed with 200 nM talazoparib was associated with significant radiosensitization (DMF 3.3). This further correlated with more double-stranded DNA breaks (g-H2AX foci) seen with talazoparib compared to veliparib both in the absence of IR (mean foci per cell: 8.4 DMSO, 19.3 veliparib, 58.7 talazoparib) and with IR (22.4 DMSO, 32.8 veliparib, 75.6 talazoparib). Finally, a dose of 0.2 mg/kg talazoparib in vivo led to statistically significant tumor growth inhibition in combination with IR but not as a single agent in three SCLC PDX models. PARP inhibition effectively sensitizes SCLC cell lines and PDXs to IR, and PARP trapping correlates with radiosensitization. PARP inhibitors, especially those with high PARP trapping activity, may provide a powerful tool to improve the efficacy of radiation therapy in SCLC.

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

SummaryThe observation that BRCA1- and BRCA2-deficient cells are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors spurred their development into cancer therapies that target homologous recombination (HR) deficiency1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1–4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here were present a high-confidence set of 73 genes whose mutation causes increased PARP inhibitor sensitivity. In addition to an expected enrichment for HR-related genes, we discovered that mutation in all three genes encoding RNase H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP inhibitor hypersensitivity of RNase H2-deficient cells is impaired ribonucleotide excision repair (RER)5. Embedded ribonucleotides, abundant in the genome of RER-deficient cells, are substrates for topoisomerase 1 cleavage, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukemia could provide an opportunity to exploit these findings therapeutically.

Abstract LB-273: A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles

Abstract Four poly(ADP-ribose) polymerase (PARP) inhibitors have now presented phase 3 monotherapy data showing compelling benefit of targeting tumours enriched with DNA damage response (DDR) pathway deficiencies, including BRCA gene mutations. Indirect treatment comparisons using the published clinical data from these late stage trials suggest similar levels of monotherapy efficacy are observed in spite of reported differences in PARP trapping potency. However, there is greater diversity in the observed safety profiles. To try and understand these observations, we have carried out a head-to-head comparison of these four PARP inhibitors (olaparib, niraparib, rucaparib and talazoparib) as well as veliparib, which recently reported phase 3 chemotherapy combination data. In our studies, we included an assessment of molecular mechanism of action that included PAR inhibition, PARP trapping and synthetic lethality in isogenic BRCA mutant and wild type models. In addition, an assessment of selectivity in terms of both inhibition of PARP family members using a novel chemoproteomic approach, as well as secondary (off-target) activities was performed. Finally, effects on human haematopoietic stem cell viability and bio-distribution to bone marrow in the rat were tested and compared. A detailed correlation of our datasets with the observed clinical results, including adverse events, suggests these preclinical experiments provide an excellent predictor of clinical response and could be used to assess emerging as well as novel PARP inhibitors. OlaparibVeliparibRucaparibNiraparibTalazoparibCompanyAZAbbVieClovisTesaroPfizerPhaseApprovedIIIApprovedApprovedIIIPARP1 SPR Kd (µM)0.0010.0070.0010.0130.002PARP2 SPR Kd (µM)0.0010.0140.0230.0430.005PARPs with Proteomic Kd <1 µM1,2,3,4,131,2,3,4,131,2,3,4,10,131,2,131,2,3,4,5a,13, 16Sec. Pharm. #, top hit µM0/855/85 5HT7, 0.513/85 5HT4, 0.517/84 DAT, 0.040/85Monotherapy dose (mg)300 bd (tablet)500 bd600 bd300 od1 od Citation Format: Elisabetta Leo, Jeffrey Johannes, Giuditta Illuzzi, Andrew Zhang, Paul Hemsley, Michal J. Bista, Jonathan P. Orme, Verity A. Talbot, Ana J. Narvaez, Elizabeth Underwood, Andrew Pike, Jenni K. Nikkila, Lucy Riches, Sinbad Sweeney, Frida Gustafsson, Anna Cronin, Piero Ricchiuto, Debora A. Roaquin, Fiona Pachl, Eric Miele, Ruth MacDonald, Glen Hawthorne, Andrew N. Mead, Mark J. O'Connor. A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles [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 LB-273.