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Purpose Veliparib (V), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, potentiates effects of alkylating agents and topoisomerase inhibitors in preclinical tumor models. We conducted a phase I trial of V with iv cyclophosphamide (C) and V plus iv doxorubicin (A) and C. Methods Objectives were to establish the maximum tolerated dose (MTD) of the combinations, characterize V pharmacokinetics (PK) in the presence and absence of C, measure PAR in peripheral blood mononuclear cells (PBMCs) and γH2AX in circulating tumor cells (CTCs). In Group 1, dose escalations of V from 10 to 50 mg every 12 h Days 1-4 plus C 450 to 750 mg/m2 Day 3 in 21-day cycles were evaluated. In Group 2, V doses ranged from 50 to 150 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 in 21-day cycles. In Group 3, patients received AC Day 1 plus V Days 1-7, and in Group 4, AC Day 1 plus V Days 1-14 was given in 21-day cycles to evaluate effects on γH2AX foci. Results Eighty patients were enrolled. MTD was not reached for V and C. MTD for V and AC was V 100 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 every 21 days. V PK appears to be dose-dependent and has no effect on the PK of C. Overall, neutropenia and anemia were the most common adverse events. Objective response in V and AC treated groups was 22% (11/49). Overall clinical benefit rate was 31% (25/80). PAR decreased in PBMCs. Percentage of γH2AX-positive CTCs increased after treatment with V and AC. Conclusion V and AC can be safely combined. Activity was observed in patients with metastatic breast cancer.

NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.

// Jung-Min Lee 1 , John L. Hays 2 , Victoria L. Chiou 1 , Christina M. Annunziata 1 , Elizabeth M. Swisher 3 , Maria I. Harrell 3 , Minshu Yu 1 , Nicolas Gordon 1 , Tristan M. Sissung 4 , Jiuping Ji 5 , William D. Figg 4 , Lori Minasian 1 , Stanley Lipkowitz 1 , Bradford J. Wood 6 , James Doroshow 7 and Elise C. Kohn 1 1 Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 2 Division of Medical Oncology, Department of Internal Medicine, The James Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH, USA 3 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, USA 4 Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 National Clinical Target Validation Laboratory, Leidos Biomedical Research Inc, Frederick National Laboratory for Cancer Research, Frederick, MD, USA 6 Center for Interventional Oncology, Radiology, and Imaging Sciences, Clinical Center and National Cancer Institute, NIH, Bethesda, MD, USA 7 Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD, USA Correspondence to: Jung-Min Lee, email: // Keywords : triple negative breast cancer, olaparib, carboplatin Received : July 27, 2016 Accepted : March 13, 2017 Published : March 25, 2017 Abstract PURPOSE: To investigate the safety, activity, and potential biomarkers of response to olaparib and carboplatin combination in sporadic triple negative breast cancer (TNBC).EXPERIMENTAL DESIGN: Metastatic or recurrent TNBC patients with no germline BRCA mutation or with BRCAPro scores

Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations. Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism. Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD+), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD+, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival. Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 7301–11. ©2017 AACR.

Purpose: A phase I trial of veliparib in combination with topotecan plus carboplatin (T+C) demonstrated a 33% objective response rate in patients with hematological malignancies. The objective is to perform exposure–response analysis to inform the phase II trial design. Experimental Design: Pharmacokinetic, efficacy, and safety data from 95 patients, who were administered 10 to 100 mg b.i.d. doses of veliparib for either 8, 14, or 21 days with T+C, were utilized for exposure–efficacy (objective response and overall survival) and exposure–safety (≥grade 3 mucositis) analysis. Multivariate cox proportional hazards and logistic regression analyses were conducted. The covariates evaluated were disease status, duration of treatment, and number of prior therapies. Results: The odds of having objective response were 1.08-fold with 1,000 ng/hr/mL increase in AUC, 1.8-fold with >8 days treatment, 2.8-fold in patients with myeloproliferative neoplasms (MPN), and 0.5-fold with ≥2 prior therapies. Based on analysis of overall survival, hazard of death decreased by 1.5% for 1,000 ng/hr/mL increase in AUC, 39% with >8 days treatment, 44% in patients with MPN, while increased by 19% with ≥2 prior therapies. The odds of having ≥grade 3 mucositis increased by 29% with 1,000 ng.h/mL increase in AUC. Conclusions: Despite shallow exposure–efficacy relationship, doses lower than 80 mg do not exceed veliparib single agent preclinical IC50. Shallow exposure–mucositis relationship also supports the 80-mg dose. Based on benefit/risk assessment, veliparib at a dose of 80 mg b.i.d. for at least 14 days in combination with T+C is recommended to be studied in MPN patients. Clin Cancer Res; 23(21); 6421–9. ©2017 AACR.

Purpose: To determine the dose limiting toxicities (DLT), maximum tolerated dose (MTD), and recommended phase II dose (RP2D) of veliparib in combination with weekly topotecan in patients with solid tumors. Correlative studies were included to assess the impact of topotecan and veliparib on poly(ADP-ribose) levels in peripheral blood mononuclear cells, serum pharmacokinetics of both agents, and potential association of germline repair gene mutations with outcome. Experimental Design: Eligible patients had metastatic nonhematologic malignancies with measurable disease. Using a 3 + 3 design, patients were treated with veliparib orally twice daily on days 1–3, 8–10, and 15–17 and topotecan intravenously on days 2, 9, and 16 every 28 days. Tumor responses were assessed by RECIST. Results: Of 58 patients enrolled, 51 were evaluable for the primary endpoint. The MTD and RP2D was veliparib 300 mg twice daily on days 1–3, 8–10, and 15–17 along with topotecan 3 mg/m2 on days 2, 9, and 16 of a 28-day cycle. DLTs were grade 4 neutropenia lasting >5 days. The median number of cycles was 2 (1–26). The objective response rate was 10%, with 1 complete and 4 partial responses. Twenty-two patients (42%) had stable disease ranging from 4 to 26 cycles. Patients with germline BRCA1, BRCA2, or RAD51D mutations remained on study longer than those without homologous recombination repair (HRR) gene mutations (median 4 vs. 2 cycles). Conclusions: Weekly topotecan in combination with veliparib has a manageable safety profile and appears to warrant further investigation.

Veliparib (ABT-888) is a novel oral poly-ADP-ribose polymerase (PARP) inhibitor that is being developed for the treatment of hematologic malignancies and solid tumors. Although the pharmacokinetics of veliparib have been studied in combination with cytotoxic agents, limited information exists regarding the pharmacokinetics (PK) of chronically dosed single-agent veliparib in patients with either BRCA 1/2-mutated cancer or PARP-sensitive tumors. The objectives of the current analysis were to characterize the population pharmacokinetics of veliparib and its primary, active metabolite, M8, and to evaluate the relationship between veliparib and M8 concentrations and poly-ADP-ribose (PAR) level observed in peripheral blood mononuclear cells (PBMCs). Seventy-one subjects contributed with veliparib plasma concentrations, M8 plasma concentrations, and PAR levels in PBMCs. Veliparib and M8 concentrations were modeled simultaneously using a population PK approach. A 2-compartment model with delayed first-order absorption and the elimination parameterized as renal (CLR /F) and nonrenal clearance (CLNR /F) adequately described veliparib pharmacokinetics. The pharmacokinetics of the M8 metabolite was described with a 2-compartment model. Creatinine clearance(CLCR ) and lean body mass (LBM) were identified as significant predictors of veliparib CLR /F and central volume of distribution, respectively. For a typical subject (LBM, 48 kg; CLCR , 95 mL/min), total clearance (CLR /F + CLNR /F), and central and peripheral volume of distribution for veliparib were estimated as 17.3 L/h, 98.7 L, and 48.3 L, respectively. At least 50% inhibition of PAR levels in PBMCs was observed at dose levels ranging from 50 to 500 mg.

Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.

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. Mol Cancer Ther; 13(2); 433–43. ©2013 AACR.

Inhibition of PARP is a promising therapeutic strategy for homologous recombination–deficient tumors, such as BRCA1-associated cancers. We previously reported that BRCA1-deficient mouse mammary tumors may acquire resistance to the clinical PARP inhibitor (PARPi) olaparib through activation of the P-glycoprotein drug efflux transporter. Here, we show that tumor-specific genetic inactivation of P-glycoprotein increases the long-term response of BRCA1-deficient mouse mammary tumors to olaparib, but these tumors eventually developed PARPi resistance. In a fraction of cases, this resistance is caused by partial restoration of homologous recombination due to somatic loss of 53BP1. Importantly, PARPi resistance was minimized by long-term treatment with the novel PARP inhibitor AZD2461, which is a poor P-glycoprotein substrate. Together, our data suggest that restoration of homologous recombination is an important mechanism for PARPi resistance in BRCA1-deficient mammary tumors and that the risk of relapse of BRCA1-deficient tumors can be effectively minimized by using optimized PARP inhibitors. Significance: In this study, we show that loss of 53BP1 causes resistance to PARP inhibition in mouse mammary tumors that are deficient in BRCA1. We hypothesize that low expression or absence of 53BP1 also reduces the response of patients with BRCA1-deficient tumors to PARP inhibitors. Cancer Discov; 3(1); 68–81. ©2012 AACR. See related commentary by Fojo and Bates, p. 20 This article is highlighted in the In This Issue feature, p. 1

Background In tumors carrying BRCA mutations, DNA damage caused by standard cytotoxic chemotherapy can be potentiated by poly [ADP-ribose] polymerase (PARP) inhibitors, leading to increased cell death through synthetic lethality. Individuals carrying mutations in BRCA have an increased incidence of triple negative breast cancer (TNBC). In order to assess the role of PARP inhibition in the treatment of TNBC, we conducted a randomized phase II trial of the combination of veliparib, a small molecule PARP inhibitor, with the cytotoxic agent cyclophosphamide versus cyclophosphamide alone in patients with refractory TNBC. Methods Adult patients with TNBC were randomized to receive oral cyclophosphamide 50 mg once daily with or without oral veliparib at 60 mg daily in 21-day cycles. Patients on the cyclophosphamide arm could crossover to the combination arm at disease progression. Results Forty-five patients were enrolled; 18 received cyclophosphamide alone and 21 received the combination as their initial treatment regimen. Lymphopenia was the most common grade 3/4 toxicity noted in both arms. One patient in the cyclophosphamide alone arm, and 2 in the combination arm had objective responses. Response rates and median progression free survival did not significantly differ between both treatment arms. Conclusion The addition of veliparib to cyclophosphamide, at the dose and schedule evaluated, did not improve the response rate over cyclophosphamide treatment alone in patients with heavily pre-treated triple-negative breast cancer.

Purpose: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor–mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. Experimental Design: Patients with advanced solid tumors were treated with 100 mg/m2 irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10–50 mg) occurred on days 3 to 14 (cycle 1) and days −1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). Results: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m2 irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days −1–14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. Conclusions: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227–37. ©2016 AACR.

Inhibition of poly (ADP-ribose) polymerase (PARP) sensitizes tumor cells to DNA damage that would normally be repaired through the base excision repair pathway. PARP inhibitors are active clinically against BRCA-deficient ovarian cancers. The PARP inhibitor talazoparib produces cytotoxicity in human cancer cell lines and animal models of tumors that harbor mutations that compromise DNA repair pathways. In this study, single agent talazoparib (1000 µg/day) was administered to patients with deleterious BRCA1 or BRCA2 mutations and advanced solid tumors in 28 day cycles. The primary objective of the trial was to examine pharmacodynamic (PD) effects of talazoparib; the secondary objective was to determine response rate in patients whose tumors carry BRCA mutations. Mandatory paired tumor biopsies were obtained pre-treatment and 3-6 hrs post-treatment on cycle 1 day 8. Optional biopsies were collected at the time of progression. One core from each time point was analyzed for PARP inhibition by a validated ELISA assay while the other core was used for IFA analysis of γH2AX. A total of 9 patients (pts) were enrolled and treated before this trial was closed due to lack of drug availability: [prostate (3), ovarian (2), breast (2), uterine sarcoma (1), pancreatic (1)]. Median age was 63 (range: 33-73 yrs); male-to-female ratio was 4:5; and the median number of prior treatments was 6 (range: 1-12). All 9 pts were evaluable for PD endpoints. One pt progressed during the first cycle of treatment; 8 pts were evaluable for clinical response. Mean time on study for evaluable pts was 8 cycles (range: 2-18); 5 of 8 (62%) pts experienced a documented partial responses [ovarian (2), prostate (2), breast (1)] lasting between 4 and 12 cycles (median: 6 cycles). Two pts had stable disease for 4 to 6 cycles, and one progressed after 2 cycles. The agent was well tolerated; the most frequent adverse events were hematologic including grade (gr) 4 anemia (1) and thrombocytopenia (1), and gr 3 anemia (2), neutropenia (1), lymphopenia (1). Decreases in PAR levels (>75%) were observed in all cycle 1 day 8 biopsy pairs, documenting a primary PD effect. Increased γH2AX expression was observed for 4/6 pts in post-dose biopsies; pre-treatment γH2AX levels, measured as %Nuclear Associated Protein (NAP), increased from a mean ± SD of 1.33 ± 1.08 to a post-treatment %NAP mean of 5.60 ± 0.78; (p=0.018), supporting a role for drug-enhanced DNA double strand breaks in the mechanism of action of talazoparib for BRCA mutant tumors. In summary, talazoparib demonstrated significant clinical activity as a single agent in patients with BRCA-deficient tumors and produced substantial reductions in tumor PAR levels in matched pre and post-treatment tumor biopsies. Citation Format: Robert S. Meehan, Alice P. Chen, Geraldine O’Sullivan Coyne, Shivaani Kummar, Jiuping Ji, Rasa Vilimas, Lamin Juwara, Robert J. Kinders, Katherine Ferry-Galow, Deborah Wilsker, Yiping Zhang, Angie B. Dull, Tony Navas, Lihua Wang, Ralph E. Parchment, James H. Doroshow. Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations [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 4678. doi:10.1158/1538-7445.AM2017-4678

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC). Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression. Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit. Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival. Clin Cancer Res; 21(7); 1574–82. ©2015 AACR.

DNA repair is essential for cells to survive damage caused by ambient environmental toxins, chemotherapy, and other treatments (1,2). Homologous recombination (HR) is a predominantly error-free mechanism to repair double-strand DNA break (DSB). Key components of DSB repair are the tumor-suppressor proteins BRCA1 and BRCA2. Dysfunctional HR leads to activation of the base excision repair pathway, a single-strand DNA break repair pathway requiring polyADPribose polymerase (PARP) (3). Increased PARP-1 expression and/or activity in tumor cells has been demonstrated in many tumor types (4–6). Active base excision repair stalls replication to allow single-strand DNA break repair. The absence of single-strand DNA break repair causes DNA helix strain at transcription forks and leads to DSBs requiring HR and, therefore, BRCA1 and BRCA2 (7). Development of DSBs in the absence of HR activates nonhomologous end joining, a poor-fidelity DSB repair pathway. PARP inhibition has been shown to induce phosphorylation of DNA-dependent protein kinase and permit error-prone nonhomologous end joining in HR-deficient cells (8). Thus, HR dysfunction sensitizes cells to PARP inhibition, leading to further chromosomal instability, cell cycle arrest, and apoptosis (9). Germline HR deficiency improves the therapeutic window for PARP inhibition (10–12). The interrelationship between DNA repair pathways led to the development of PARP inhibitors (PARPis) as therapeutic agents. Olaparib, an oral PARP1/2 inhibitor, is tolerated at single-agent continuous doses up to 400mg twice daily (capsules) (13). It has been active as monotherapy in tumors with defective HR, specifically germline BRCA1 or BRCA2 mutations (gBRCAm). Clinical activity with prolonged disease stabilization and tumor burden reduction has been reported in gBRCAm-associated breast and/or ovarian cancer (gBRCAm BrCa/OvCa) (14–18). Phase II studies have confirmed the activity of olaparib monotherapy with response rates of 41% in gBRCAm patients with advanced BrCa (17) and 31% to 40% in those with OvCa (13,15). The optimal application of PARPis in treatment of gBRCAm BrCa/OvCa has not yet been determined. gBRCAm carriers with OvCa have increased susceptibility to platinums (19) and often receive multiple lines of platinum-based chemotherapy and can have longer overall survival than nonmutation carriers (20,21). It has been demonstrated preclinically that concomitant use of olaparib increases cytotoxic activity of cisplatin in cisplatin-resistant OvCa cell lines (22), and an intermittent schedule of olaparib with carboplatin yields antitumor activity in a BRCA2-mutated human ovarian cancer xenograft model (23). Thus, we hypothesized addition of olaparib to carboplatin as a stress to the DNA repair machinery would improve clinical benefit of carboplatin and could be safely given to women with gBRCAm-associated cancers. We conducted a phase I/Ib study to evaluate the safety, tolerability, and activity of olaparib administered with carboplatin. Our translational aim was to discover potential predictive biomarkers of response to the carboplatin/PARPi combination.

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.

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

Purpose: To determine the safety and tolerability of olaparib with cisplatin and gemcitabine, establish the maximum tolerated dose (MTD), and evaluate the pharmacodynamic and pharmacokinetic profile of the combination. Experimental Design: We conducted a phase I study of olaparib with cisplatin and gemcitabine in patients with advanced solid tumors. Treatment at dose level 1 (DL1) consisted of olaparib 100 mg orally every 12 hours on days 1 to 4, gemcitabine 500 mg/m2 on days 3 and 10, and cisplatin 60 mg/m2 on day 3. PAR levels were measured in peripheral blood mononuclear cells (PBMC). Results: Dose-limiting toxicities (DLT) in two of three patients at DL1 included thrombocytopenia and febrile neutropenia. The protocol was amended to enroll patients treated with ≤2 prior severely myelosuppressive chemotherapy regimens and treated with olaparib 100 mg once daily on days 1 to 4 (DL−1). No DLTs were seen in six patients at DL−1. Because of persistent thrombocytopenia and neutropenia following a return to DL1, patients received 100 mg olaparib every 12 hours on day 1 only. No hematologic DLTs were observed; nonhematologic DLTs included gastrointestinal bleed, syncope, and hypoxia. Of 21 patients evaluable for response, two had partial response. Olaparib inhibited PARP in PBMCs and tumor tissue, although PAR levels were less effectively inhibited when olaparib was used for a short duration. Conclusions: Olaparib in combination with cisplatin and gemcitabine is associated with myelosuppression even at relatively low doses. Modified schedules of olaparib in chemotherapy naive patients will have to be explored with standard doses of chemotherapy. Clin Cancer Res; 18(8); 2344–51. ©2012 AACR.

Purpose: Oral administration of the alkylating agent cyclophosphamide at low doses, metronomic dosing, is well tolerated, with efficacy in multiple tumor types. PARP inhibition potentiates effects of cyclophosphamide in preclinical models. We conducted a phase I trial of the PARP inhibitor veliparib and metronomic cyclophosphamide in patients with refractory solid tumors and lymphoid malignancies. Experimental Design: Objectives were to establish the safety and maximum tolerated dose (MTD) of the combination; characterize veliparib pharmacokinetics (PK); measure poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies and peripheral blood mononuclear cells (PBMC); and measure the DNA-damage marker γH2AX in PBMCs and circulating tumor cells (CTC). Cyclophosphamide was administered once daily in 21-day cycles in combination with veliparib administered once daily for 7, 14, or 21 days. Results: Thirty-five patients were enrolled. The study treatment was well tolerated, and the MTD was established as veliparib 60 mg with cyclophosphamide 50 mg given once daily. Seven patients had partial responses; an additional six patients had disease stabilization for at least six cycles. PAR was significantly decreased in PBMCs (by at least 50%) and tumor biopsies (by at least 80%) across dose levels (DL); γH2AX levels were increased in CTCs from seven of nine patients evaluated after drug administration. Conclusions: The combination of veliparib with metronomic cyclophosphamide is well tolerated and shows promising activity in a subset of patients with BRCA mutations. A phase II trial of the combination compared with single-agent cyclophosphamide is ongoing in BRCA-positive ovarian cancer, triple-negative breast cancer, and low-grade lymphoma. Clin Cancer Res; 18(6); 1726–34. ©2012 AACR.

Exposure to oxygen-rich environments can lead to oxidative damage, increased body iron stores, and changes in status of some vitamins, including folate. Assessing the type of oxidative damage in these environments and determining its relationships with changes in folate status are important for defining nutrient requirements and designing countermeasures to mitigate these effects. Responses of humans to oxidative stressors were examined in participants undergoing a saturation dive in an environment with increased partial pressure of oxygen, a NASA Extreme Environment Mission Operations mission. Six participants completed a 13-d saturation dive in a habitat 19 m below the ocean surface near Key Largo, FL. Fasting blood samples were collected before, twice during, and twice after the dive and analyzed for biochemical markers of iron status, oxidative damage, and vitamin status. Body iron stores and ferritin increased during the dive (P

A forward mutation assay was developed to study mutagenic specificity induced by temperature-sensitive alleles of bacteriophage T4 gene 42, which encodes a thermolabile deoxycytidylate hydroxymethylase. Thymidine kinase (tk) mutations induced by T4 ts B3 at a semi-permissive temperature (34°C) were selected under near-ultraviolet light on synthetic agar plates containing bromodeoxyuridine, and sequenced after PCR amplification of the tk gene. 21 of 23 tk− mutations identified were C → T transitions, while the ramainder were C → A transversions. Analyses of the DNA sequence around each mutant site suggest that the mispairing of thymine with guanine in the template is suppressed when the next nucleotide is dGTP. The 5′ neighbor nucleotide of the mismatch may influence mutation frequency as well; no mutations with dAMP residues on the upstream side were seen. Our observations with the forward mutation assay here at consistent with previous results from an rII reversion assay, supporting our model that the mutator phenotype displayed by tsLB3 is a consequence of perturbation of dNTP supplies to replication sites due to partial impairment of thermolabile deoxytidylate hydroxymethylase at a semi-permissive temperature. The forward mutation assay described here is readily adapted for other studies of mutagenesis in T4 phage.

Purpose We conducted the first phase 0 clinical trial in oncology of a therapeutic agent under the Exploratory Investigational New Drug Guidance of the US Food and Drug Administration. It was a first-in-human study of the poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888 in patients with advanced malignancies. Patients and Methods ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT-888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre- and postdrug administration for evaluation of PARP activity and pharmacokinetics. A novel statistical approach was developed and utilized to study pharmacodynamic modulation as the primary end point for trials of limited sample size. Results Thirteen patients with advanced malignancies received the study drug; nine patients underwent paired tumor biopsies. ABT-888 demonstrated good oral bioavailability and was well tolerated. Statistically significant inhibition of poly (ADP-ribose) levels was observed in tumor biopsies and peripheral blood mononuclear cells at the 25-mg and 50-mg dose levels. Conclusion Within 5 months of study activation, we obtained pivotal biochemical and pharmacokinetic data that have guided the design of subsequent phase I trials of ABT-888 in combination with DNA-damaging agents. In addition to accelerating the development of ABT-888, the rapid conclusion of this trial demonstrates the feasibility of conducting proof-of-principle phase 0 trials as part of an alternative paradigm for early drug development in oncology.

Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by a dCMP deaminase-deficient T4 mutant, shrinks to near-normal levels as a consequence of dTTP accumulation, and ribonucleotide reductase is the only apparent control point. Second, analysis of mutagenesis by 5-bromodeoxyuridine suggests that most induced mutations result from localized pool depletion of 5-hydroxymethyl-dCTP at replication sites, as if 5-bromo-dUTP were behaving like dTTP in inhibiting the CDP reductase activity of the phage enzyme. We found that CDP reductase activity in crude extracts of T4 phage-infected bacteria is sensitive to inhibition by either dTTP or 5-bromo-dUTP, at concentrations as low as 0.01 mM. However, in partially purified enzyme preparations that sensitivity is lost. Although we don't know the basis for this loss of feedback sensitivity, the results suggest that kinetic properties of enzymes in intact cells are determined by the cellular milieu in ways not apparent from analysis of purified enzymes.

Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009). Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals. Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs. Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03). Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment. Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. Figure 1. Figure 1. Disclosures Off Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

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.

BRCA 1/2 dysfunction sensitizes cells to inhibition of PARP enzymatic activity, due to the persistence of DNA breaks normally repaired by homologous recombination (HR), thus cancer pts carriers of germ line BRCA deleterious mutations have been targeted for treatment with PARP inhibitors. An example of HR repair is the FA pathway where BRCA 1/2 collaborate with several other genes resulting in FancI and FancD2 activation and nucleus repair foci formation. We have developed a triple stain immunofluorescence based method to evaluate FA pathway functionality (foci formation) (FancD2/DAPI/Ki67) (FATSI) in paraffin embedded tumor tissues, and hypothesized that among patients with sporadic tumors, a significant proportion with tumoral dysfunctional FA pathway can be identified. A two steps clinical trial (NCT01017640) was designed to 1- screen solid tumor patients for absence of FA tumor foci formation, and 2- evaluate safety and recommended doses in these pts of the PARPi veliparib, either alone or in combination with chemotherapy (MMC). 607 pts were consented, 532 had their tumor tissue screened, 142 (27%) had absent foci. All major solid tumors were represented in FA dysfunction, notably 36 of 105 (34%) colorectal and 40 of 135 (30%) breast cancers. 52 pts received 192 cycles of veliparib, 29 pts as monotherapy in escalating dose cohorts (starting at 50 mg PO BID continuously), and 23 following MMC 10 mg-sqm every 4 weeks (40mg-sqm cumulative cap) at 50 mg BID at increased duration cohorts (1, 2 and 3 wks) every 4 wks (100 and 200 mg BID for 3 wks in subsequent cohorts). Recommended dose levels are: veliparib 300 mg BID as monotherapy (2DLT/3 pts at 400 BID:G3 fatigue) and MMC/veliparib 10mg-sqm/200 mg BID three weeks duration. Analysis for germ line mutations (BRCA1 and 2 sequencing and for the 5 most common BRCA1 large rearrangements) in PBMC among 50 pts showed 7 pts (3 breast, 1 ampullary, 2 ovarian) with BRCA abnormalities: 6 known deleterious mutations and 1 polymorphism. Five RECIST criteria responses occurred (1CR, 4 PR) (1 lung, 2 breast, 1 ovarian, 1 endometrial), all in the combination arm. Two breast cancer pts (39 and 13 cycles) and 1 ovarian (6 cycles) on monotherapy, plus 1 breast (15 cycles), 1 colon (7 cycles) on the combination, had prolonged stability. rH2AX (n=49) and PAR (n=17) analysis in PBMC suggests veliparib dose dependency (blunting of rH2AX induction, lower PAR level and slower recovery for higher doses). The trial was amended to enroll an expanded cohort of 10 FATSI negative colorectal patients who will undergo fresh biopsy for whole exome sequencing (RNAseq) and FA/DNA repair gene panel analysis prior to treatment at the recommended dose in the combination arm, with the goal of identifying the specific genetic defect mediating the FA dysfunction, as well as partner mutations to be targeted in subsequent trials. Supported by R01CA152101 & N01CM62207 Citation Format: Miguel A. Villalona-Calero, Wenrui Duan, Weiqiang Zhao, Konstantin Shilo, Jiuping Ji, Jennifer Thurmond, Adam Norris, Jeffrey Rose, Rachel Layman, John Marshall, Tanios Bekaii-Saab, Alice Chen. Phase I trial of veliparib or mitomycin (MMC) + veliparib in patients with sporadic solid tumors screened for somatic deficiency in the Fanconi Anemia (FA) pathway. [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 LB-139. doi:10.1158/1538-7445.AM2013-LB-139

The human fragile-X syndrome, a major cause of inherited mental retardation, is associated with expansion of the trinucleotide repeat GGC:GCC. Repetitive sequences in DNA are subject to slippage during catalysis by DNA polymerases. We characterized the extent of slippage of synthetic GGC:GCC repeats by various DNA polymerases: Taq DNA polymerase, Klenow fragment of DNA polymerase I, DNA Sequence, DNA polymerase-alpha and polymerase-beta, as well as HIV reverse transcriptase. All of these enzymes were found to expand GGC:GCC repeats, with the most extensive expansion exhibited by Taq DNA polymerase. Starting with a template and primer, each 15 nucleotides (nt) in length, the product of one round of synthesis by Taq polymerase is as long as 250 nt. Sequence analysis of cloned DNA fragments expanded by Taq polymerase indicates that expansion involves multiple triplet additions and that it is asymmetric. The asymmetric distribution of terminal nucleotides in the expanded product is consistent with active expansion of the GCC strand and passive additions onto the GGC strand. The preferential elongation and expansion of the GCC strand was confirmed in studies utilizing longer repeats within a single-stranded M-13 template.

Background Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. Methodology/Principal Findings We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. Conclusions/Significance Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.

The unusually high mutation frequency exhibited by the human immunodeficiency virus (HIV) is a major impediment to developing effective vaccines against the virus and to designing nucleotide analogs that inhibit viral replication. To investigate the molecular basis of HIV hypermutability, we established cell-free assays to measure the fidelity of HIV-1 reverse transcriptase (RT) in copying either DNA or both RNA and DNA templates that contain the hypervariable region 1 of the HIV-1 env gene (V-1). The fidelity of DNA synthesis was measured by repetitively copying the envelope gene (V-1) DNA by HIV-1 RT, followed by cloning and sequencing these newly synthesized DNA products. We found that the error rate of HIV RT copying either RNA or DNA of the env V-1 region is about one misincorporation per 5 kb polymerized. This rate is similar to that found with the M13mp2 forward mutation assay using the lacZ α gene as a template. This similarity suggests that the HIV env hypervariable sequence is not inherently hypermutable. The high error rate of HIV RT suggests that misincorporation by this enzyme is a major source of mutations throughout the viral genome and a determinant for rapid viral evolution. The spectrum of mutations produced by HIV RT in vitro partially correlates with the spectrum of HIV mutations observed in AIDS patients. The differences between these spectra highlight the contribution of phenotypic selection during HIV-1 infection. The overall uniformity of misincorporation of HIV-1 RT further suggests an alternative anti-HIV strategy based on increasing viral mutagenesis by nucleotide analogs.

The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.

2520 Background: The PARPi O has single-agent activity against BRCA1/2 mutation carriers (Br/Ov mut+) Br/OvCa. We hypothesized addition of C to O to stress the DNA repair machinery would further im...

3000 Background: The nuclear enzyme PARP is essential in recognition and repair of DNA damage. In preclinical models, PARP inhibitors work as sensitizing agents for DNA-damaging agents such as Ir. V is an orally bioavailable PARP 1 and 2 inhibitor. This phase I study determined the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), PK and PD of V together with Ir. Methods: Eligibility included patients with performance status 0-2; ≥ age 18; adequate bone marrow, hepatic and renal function. Cycles were 21 days. Ir was given i.v. 100 mg/m2 over 90 min on Days 1 and 8. Twice daily (BID) oral dosing of V (10-50mg) occurred Days 3-14 (Cycle 1) and Days -1-14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Tumor biopsies and matched peripheral blood mononuclear cells (PBMCs) were collected for PD evaluation of PAR, γ-H2AX, PARP1, TOPO1, and ERCC1 after Ir alone and after the combination. Results: 32 pts were treated (2 lung, 14 breas...

Abstract 3634 PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7. Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T.Dose levelVeliparib Oral Daily Days 1–8Topotecan Continuous IV Days 3–7Carboplatin Continuous IV Days 3–7Clinical Responses/#Pts1A10 mg p.o. BID1.0 mg/m2/day—1/3(CRi)1B10 mg p.o. BID1.3 mg/m2/day—1/3(CR)2A10 mg p.o. BID1.3 mg/m2/day150 mg/m2 IV0/42C10 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV2/6(CRi, PR)2D10 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV1/6(PR)2E10 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CRi)3C20 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV0/63D20 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV2/6(CR, PR)3E20 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)440 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)5§80 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/4(CR, CRi) Of 56 pts treated at doses of V 10–80 mg orally BID Days1–8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 25 (45%) have experienced > grade 3 non-hematologic toxicity, 6(11%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 27% (8CR, 4 CRi, 3 PR), with 7 in relapsed AML, 7 in refractory AML and 1 refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V is detectable in marrow supernatant and cells, does not accumulate with multiple dose administrations in plasma but does in marrow, and is not altered by T or C administration. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to Disclosures: No relevant conflicts of interest to declare.

3027 Background: Poly (ADP-ribose) polymerase (PARP) group of NAD+-dependent enzymes participate in multiple DNA- related functions. Increased PARP activity can lead to drug resistance. Olaparib (O), a potent oral inhibitor of PARP-1 and PARP-2 may partially overcome resistance to C and G when used in combination. Methods: Eligibility: histologically confirmed metastatic or unresectable solid tumors, PS 0–2, normal organ functions. Treatment at dose level 1 (DL1): O 100 mg orally bid on days (D) −2 to 2, G 500 mg/m2/h on D1 and D8, C 60 mg/m2 on D1 after G; q21 days ×6. Doses (same schedule) at DL −1: O 100 mg once daily, G 400 mg/m2/h, C 50 mg/m2. Peripheral blood mononuclear cells (PBMCs) were collected and processed within 2 hours and poly (ADP-ribose) (PAR) concentration determined by immunoassay. Results: From 5/2008 to 11/2009, 23 patients (pts) enrolled; 14 males, median age 52 yrs (25- 88). Tumor types: 8 NSCLC; 3 ovarian; 3 pancreatic; 2 esophageal; 2 thymic ca; 1 mesothelioma; 1 adrenocortical c...

The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.

To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34 degrees C). One such marker, rII SN103, which showed the highest stimulation at 34 degrees C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34 degrees C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34 degrees C cultures showed no significant deviations from values obtained at 30 degrees C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the mutator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.

10565 Background: DNA double-strand breaks (DSBs) caused by exposure to DNA damaging agents initiate phosphorylation of histone H2AX to form gamma-H2AX, which is considered a surrogate marker of DSBs. However, it is a challenge to quantitatively measure gamma-H2AX in clinical samples such as tumor biopsies. The aim of this study was to develop an immunohistochemical detection method for gamma-H2AX and to quantitatively evaluate the levels of gamma-H2AX in paraffin-embedded tumor samples. Methods: Human breast cancer MCF-7 cells were treated with the topoisomerase I inhibitor irinotecan at 1 μM or vehicle for 1 h, and fixed in 10% neutral buffered formalin and embedded in paraffin. Staining with gamma-H2AX antibody was performed on sections of treated MCF-7 cells, tumor specimens, and biopsies at baseline and after doxorubicin-containing chemotherapy from cancer patients. Numbers of foci and level of gamma-H2AX expression per tumor nucleus were determined by manual counting under a light microscope and an Automated Cellular Imaging System. Results: There was a rise in the mean numbers of nuclear foci and intensity of gamma-H2AX in MCF-7 cells treated with irinotecan versus vehicle (19.9 ± 2.7 vs. 9.95 ± 3.6; P < 0.0001 and 61.2 ± 8.5 vs. 16.2 ± 13.6; P < 0.0001 by Wilcoxon rank sum test). The level of gamma-H2AX foci in human tumor samples was 18.8 ± 13.1, 44.8 ± 14.5, 51.2 ± 20.8, or 69.7 ± 21.2 in carcinomas of the breast, colon, ovary, or prostate. In a patient with stable disease, levels of gamma-H2AX foci were 62.7 ± 26.9 at baseline and 67.2 ± 25.3 after doxorubicin-containing regimen chemotherapy. Conclusions: Our data suggest that the quantitative immunohistochemical detection of gamma-H2AX levels is facilitated by a digital imaging system, and is a reliable method to measure the effects of DNA damaging agents in cells and paraffin-embedded human tumor samples. Its application may help evaluate tumor response to various DNA damaging agents currently in the clinic and those presently undergoing clinical development. No significant financial relationships to disclose.

Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis — if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.