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Background: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells. Methods: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells. Results: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples – three NSCLC and one mesothelioma – were HRR defective and eight samples – one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers – were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1. Conclusions: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.

Background: Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. Methods: Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. Results: Rucaparib accumulation is carrier mediated (Km=8.4±1.2 μM, Vmax=469±22 pmol per 106 cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ≥50% for ≥72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg−1 i.p or 150 mg kg−1 p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg−1 p.o once a week was as effective as 10 mg kg−1 i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. Conclusions: Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered. Refereed/Peer-reviewed

Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. In conclusion, microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress—a phenomenon we term stress pathway interference—lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy., IMPORTANCE The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.

Introduction: BMN 673 is a novel, potent (IC50 < 1.0 nM) inhibitor of Poly(ADP-ribose) polymerase (PARP) 1 and PARP 2 in clinical development for the treatment of genetically defined cancers. Methods: The pharmacokinetics (PK) and pharmacodynamics (PD) of BMN 673 were evaluated in a Phase 1 dose-escalation study in patients with advanced solid tumors. Oral doses evaluated ranged from 0.025 to 1.1 mg/day, with 3-6 patients evaluated at each dose level in a standard 3+3 design. The first dose in Cycle 1 was followed for 7 days without dosing for collection of serial PK and PD samples. Thereafter BMN 673 was administered once daily on Days 8 to 35, with predose PK and PD samples collected on Days 15 and 22. Following the last dose in Cycle 1 on Day 35, serial PK and PD samples were collected for 7 and 3 days, respectively, without dosing. In subsequent cycles, BMN 673 was administered daily in contiguous 28-day cycles. The PD activity of BMN 673 was measured in peripheral blood mononuclear cells (PBMCs) using assay methods previously described (Clin Cancer Res 2008 14:7917023, Biochem J 2011 436:671-679). Correlations between systemic BMN 673 exposure and inhibition of PBMC PARP activity were investigated across dose levels. Results: BMN 673 demonstrated good oral bioavailability and a long half-life supporting daily dosing (ASCO 2013 Abstract 2580). While variable across and within patients, overall PBMC PARP activity decreased in a dose-dependent manner. Within individual patients at higher dose levels, PBMC PARP activity decreased soon after the first dose of BMN 673, and activity remained at suppressed levels with daily dosing. PBMC PARP activity rebounded when dosing was stopped, indicating the return of PARP function. Correlations between the mean percent baseline PARP activity with daily BMN 673 dosing and measures of steady-state BMN 673 exposures within individuals (i.e., Day 35 Cmin, Cmax, and AUC0-24) showed decreased PARP activity with increasing exposure. This correlation was well described using an Imax model with IC50 values based on an interim analyses of 1140 pg/mL, 3090 pg/mL, and 26700 pg-hr/mL for Cmin, Cmax, and AUC0-24, respectively. Conclusions: PBMC PARP activity was rapidly and continuously inhibited with daily dosing of BMN 673. A positive correlation between systemic BMN 673 exposure and inhibition of PARP activity in PBMCs was demonstrated. This effect on a relevant pharmacodynamic marker provides in vivo proof of an on-target effect of BMN 673 and may be an initial step with potential to inform BMN 673 dose selection. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A220. Citation Format: Joshua W. Henshaw, Huiyu Zhou, Ashleigh Herriott, Miranda Patterson, Evelyn W. Wang, Don Musson, Johann de Bono, Lida A. Mina, Ramesh K. Ramanathan, Charles O'Neill, Andrew Dorr, Nicola J. Curtin. Inhibition of PBMC PARP activity with the novel PARP 1/2 inhibitor BMN 673 in patients with advanced solid tumors. [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 A220.