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Depending on genetic and other factors, tumor cells exhibit replication stress (RS) to various extents. Low levels of RS contribute to genomic instability, an enabling hallmark of cancer, whereas high levels may be cytotoxic. The DNA damage response (DDR) checkpoint kinase 1, Chk1, is essential for managing RS by stabilizing replication forks, abrogating origin firing, pausing the cell cycle, and fostering homologous recombination DNA repair. Consequently, tumor cells become highly reliant on Chk1 to manage RS and maintain proliferation and survival. Certain chemotherapeutic drugs, including nucleoside analogs, DNA cross-linkers, and topoisomerase inhibitors, are effective extrinsic inducers of RS. Here we demonstrate that the potent, selective oral Chk1 inhibitor, SRA737, strongly synergizes with a range of extrinsic inducers of RS to kill tumor cells in vitro and in vivo. We find that clinically used drugs including the DNA crosslinking agents mitomycin C and cisplatin, as well as the nucleoside analog gemcitabine, exacerbate cellular RS markers (e.g. γ-H2A.X and p-RPA) and synergize with SRA737 to increase tumor cell death in vitro. Combination indices (CI) at various doses across a cell line panel were 0.46, 0.54, and 0.25 for mitomycin C, cisplatin, and gemcitabine, respectively. Similar results were observed with both type I and type II topoisomerase inhibitors (SN-38 and etoposide). Notably, gemcitabine potentiates SRA737 in several bladder cancer cell lines including 5637, TCCSUP, and J82 cells, as well as in human primary bladder cancer patient 3D organoids. Consistent with the observed cytotoxicity, the combination of SRA737 and gemcitabine induces γ-H2A.X and p-RPA 3- to 5-fold over gemcitabine alone in tumor xenografts. Moreover, the combination inhibits tumor growth by 64% following administration of both agents at 100 mg/kg in a gemcitabine-insensitive patient-derived xenograft (PDX) model of bladder cancer. Importantly, SRA737 also synergizes with subtherapeutic doses of gemcitabine (40 mg/kg) in xenograft models of colorectal adenocarcinoma and osteosarcoma, indications where gemcitabine is not standard of care. These findings support the clinical development of SRA737 in combination with gemcitabine in bladder cancer, as well as the broader application of SRA737 in combination with extrinsic inducers of RS. Citation Format: Ryan J. Hansen, Bryan Strouse, Kenna Anderes, Gregg Smith, Christian Hassig. The Chk1 inhibitor, SRA737, demonstrates chemical synthetic lethality with replication stress-inducing agents, including low-dose gemcitabine, in preclinical models of cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B181.

O(6)-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/- background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-proficient or -deficient mice (either Nf1+/+ or Nf1+/-) were given 6 weekly injections of a maximally tolerated dose of CP (250 mg/kg) or vehicle and followed for 15 months. CP-treated mice had more deaths than control mice but there was no difference in the long-term survival between Mgmt+/+ and Mgmt-/- mice (12 of 83 Mgmt+/+ mice died compared to 12 of 80 Mgmt-/- mice, disregarding Nf1 status). Lymphomas and adrenal tumours were the most frequent malignancies. Interestingly, CP-treated, Mgmt-deficient mice developed fewer tumours than controls. Ten of 71 (14%) Mgmt-proficient mice developed tumours after CP treatment compared to only 2 of 68 (3%) Mgmt-deficient mice (P = 0.02). Mgmt-/-, Nf1+/- mice developed fewer tumours (1 of 35, 3%) following CP compared to Mgmt+/+, Nf1+/- mice (7 of 37, 19%) (P = 0.03). Hypoxanthine-guanine phosphoribosyltransferase mutation assays showed no significant increases in mutant frequencies in Mgmt-/- (18.1 x 10(6)) compared to Mgmt+/+ mice (12.9 x 10(6)). These data indicate that MGMT deficiency does not protect against long-term toxicity or mutagenicity from CP and appears to attenuate the occurrence of CP-induced tumours in an Nf1+/- background.

Ondansetron, a 5-HT(3) receptor antagonist, is an effective anti-emetic in cats. The purpose of this study was to compare pharmacokinetics of subcutaneous (SQ) ondansetron in healthy geriatric cats to cats with chronic kidney disease (CKD) or liver disease using a limited sampling strategy. 60 cats participated; 20 per group. Blood was drawn 30 and 120 min following one 2 mg (mean 0.49 mg/kg, range 0.27–1.05 mg/kg) SQ dose of ondansetron. Ondansetron concentrations were measured by liquid chromatography coupled to tandem mass spectrometry. Drug exposure represented as area under the curve (AUC) was predicted using a limited sampling approach based on multiple linear regression analysis from previous full sampling studies, and clearance (CL/F) estimated using noncompartmental methods. Kruskal–Wallis anova was used to compare parameters between groups. Mean AUC (ng/mL·h) of subcutaneous ondansetron was 301.4 (geriatric), 415.2 (CKD), and 587.0 (liver). CL/F (L/h/kg) of SQ ondansetron was 1.157 (geriatric), 0.967 (CKD), and 0.795 (liver). AUC was significantly higher in liver and CKD cats when compared to geriatric cats (P < 0.05). CL/F in liver cats was significantly decreased (P < 0.05) compared to geriatric cats. In age-matched subset analysis, AUC and CL/F in liver cats remained significantly different from geriatric cats.

Introduction: Monoclonal antibodies (Mabs) that neutralize proprotein convertase subtilisin-kexin type 9 (PCSK9) have been shown to lower LDL-C in human trials. It is known that PCSK9 is cleaved by furin at Arg218 and that the cleaved PCSK9 is inactive in modulating LDLR. Antibodies whose epitopes are near the EGFA binding domain are more efficacious in increasing LDLR but may interfere with furin cleavage and thus normal PCSK9 clearance. Here we describe a unique antibody (LY) that both lowers LDL-C and allows for normal PCSK9 cleavage by furin. Hypothesis: An antibody with an epitope that permits furin cleavage of PCSK9 will show more durable LDL lowering compared to one with an epitope that interferes with cleavage. Methods: Furin cleavage of recombinant PCSK9 was evaluated by electrophoresis in the presence of antibodies. Antibodies were studied in mice expressing wild-type or a non-cleavable variant of human PCSK9. Results: We determined that LY3015014 (LY) permits furin cleavage of PCSK9 while Mab A, which shares the epitope of a Mab tested in humans, does not. LY’s epitope is completely N-terminal of the cleavage site, while the conformational epitope of Mab A spans the Arg218 cleavage site. To assess the impact of PCSK9 cleavage on antibody efficacy in vivo, we generated two human PCSK9 overexpressing mouse models; one with WT PCSK9 and one with a non-cleavable variant (R215A, R218A). In mice expressing WT human PCSK9, LY showed a longer duration of LDL lowering and a slower clearance rate than Mab A. Additionally, we found that PCSK9 did not accumulate in the serum of mice given LY, likely due to cleavage and release from the antibody. In mice expressing the non-cleavable variant PCSK9, the clearance and LDL durability benefits of LY compared to Mab A were lost. Additionally, in the non-cleavable model, PCSK9 accumulated in the serum of mice given LY, presumably due to lack of furin cleavage. Conclusions: LY allows normal cleavage and inactivation of bound PCSK9 while maintaining LDL-C lowering. This proteolysis reduces the antibody’s clearance rate and extends the therapeutic durability of LY in mice relative to Mab A which interferes with proteolytic degradation. We have shown that cleavage is the mechanism of durability through the use of non-cleavable PCSK9.

Two phase 1 studies evaluated the pharmacokinetics (PK), safety and biological activity of tabalumab, a human monoclonal antibody against B-cell activating factor (BAFF), administered intravenously (i.v.) or subcutaneously (s.c.) in subjects with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).In study A, subjects with RA (n = 23) or SLE (n = 6) received a single i.v. dose of tabalumab (RA 0.01, 0.04, 0.125, 0.5, 2.0, and 8.0 mg kg(-1) and SLE 0.125 or 2.0 mg kg(-1) ) or placebo. In study B, subjects with RA received a single tabalumab dose i.v. (10 mg) (n = 12) or s.c. (20 mg) (n = 12). Serum tabalumab and CD20+ B cells were evaluated and safety was assessed throughout both studies.Tabalumab PK were non-linear across the 0.01 to 8.0 mg kg(-1) dose range. Clearance (CL) decreased from 2.9 to 0.1 l day(-1) and terminal half-life (t1/2 ) increased from about 1.6 to 25 days. Subjects with RA or SLE had similar PK. After s.c. dosing, tabalumab time to maximal concentration (tmax ) was 5.5 days. Absolute bioavailability (F) was approximately 62%. Following tabalumab dosing, CD20+ B cells transiently increased from baseline followed by a progressive decrease below baseline.A single tabalumab dose administered i.v. or s.c. was well tolerated and had non-linear CL over the dose range investigated in subjects with RA and SLE. The non-linearity likely reflects target-mediated CL due to binding to BAFF. Tabalumab showed biological activity based on changes in peripheral CD20+ lymphocyte numbers in both subjects with RA and SLE.

Largazole is a potent class I-selective HDACi natural product isolated from the marine cyanobacteria Symploca sp. The purpose of this study was to test synthetic analogs of Largazole to identify potential scaffold structural modifications that would improve the drug-like properties of this clinically relevant natural product. The impact of Largazole scaffold replacements on in vitro growth inhibition, cell cycle arrest, induction of apoptosis, pharmacokinetic properties, and in vivo activity using a xenograft model was investigated. In vitro studies in colon, lung, and pancreatic cancer cell lines showed that pyridyl-substituted Largazole analogs had low-nanomolar/high-picomolar antiproliferative activity, and induced apoptosis and cell cycle arrest at concentrations equivalent to or lower than the parent compound Largazole. Using IV bolus delivery at 5 mg/kg, two compartmental pharmacokinetic modeling on the peptide isostere analog of Largazole indicated improved pharmacokinetic parameters. In an A549 non-small cell lung carcinoma xenograft model using a dosage of 5 mg/kg administered intraperitoneally every other day, Largazole, Largazole thiol, and Largazole peptide isostere demonstrated tumor growth inhibition (TGI %) of 32, 44, and 66 %, respectively. Largazole peptide isostere treatment was statistically superior to control (p = 0.002) and to Largazole (p = 0.006). Surprisingly, tumor growth inhibition was not observed with the potent pyridyl-based analogs. These results establish that replacing the depsipeptide linkage in Largazole with an amide may impart pharmacokinetic and therapeutic advantage and that alternative prodrug forms of Largazole are feasible.

O 6-alkylguanine-DNA alkyltransferase (AGT) protects from the mutagenic and toxic lesions induced by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and in many tumors, AGT overexpression provides a means of resistance. To circumvent this, O 6-benzylguanine, an inactivator of AGT, has been developed and is currently in clinical development with BCNU; however, the potential long-term toxicities associated with this treatment are unknown. With the inactivation of AGT by O 6-benzylguanine, a higher number of toxic and mutagenic O 6-alkylguanine lesions introduced by methylating or chloroethylating agents would be expected. In this study, cohorts of mice were treated with vehicle, O 6-benzylguanine (30 mg/kg), BCNU alone (low dose of 15 mg/kg or high dose of 50 mg/kg), or O 6-benzylguanine (30 mg/kg) plus BCNU (15 mg/kg) and followed for 12 months post-treatment. Mice treated with O 6-benzylguanine plus BCNU or high-dose BCNU died significantly earlier ( p < 0.0001) than mice in the other three cohorts with a median survival of 8.3 ( O 6-benzylguanine plus BCNU) and 7.9 months (high-dose BCNU). Histopathologic sections of tissues revealed that the most common morphological diagnosis in animals treated with O 6-benzylguanine plus BCNU (15 mg/kg) or BCNU (50 mg/kg) was cytomegaly in the lung with greater severity observed in mice receiving the combination O 6-benzylguanine plus BCNU. Four of five mice analyzed in this cohort had alveolar histiocytosis, with one also having alveolar edema. In contrast, liver and kidney toxicity was only observed in mice treated with BCNU (50 mg/kg). These results suggest that O 6-benzylguanine enhances long-term pulmonary toxicity associated with BCNU in mice.

Translation is essential for cellular maintenance and growth, as well as survival in response to a variety of stresses. Translation factors are often overexpressed in cancers, and their experimental inhibition has been shown to retard cancer cell growth in a variety of pre-clinical models. Further, clinically relevant forms of cellular stress (e.g. ionizing radiation, hypoxia) result in altered translational programs. Inhibition of translation following such stress results in impaired cellular recovery. Taken together these observations suggest that modulation of translation, alone or in combination with genotoxic stress, is of potential therapeutic relevance in oncology. Bouvardin (BVD) is a plant-derived protein synthesis inhibitor that we identified in a screen for enhancers of ionizing radiation (IR) in D. melanogaster. We previously demonstrated that BVD functions as a translation elongation inhibitor by blocking dissociation of elongation factor 2 (eEF2) from the ribosome. In preclinical models of head and neck cancer (HNC) and glioma, two cancer indications for which IR is a standard of care, we demonstrated that BVD enhanced clonogenic death induced by IR. Further, BVD enhanced the growth inhibitory effects of IR in HNC tumor xenografts in mice. We show here that a fully synthetic derivative of BVD, SVC112, exhibits promising therapeutic properties in tissue culture and xenograft models of HNC. Like BVD, SVC112 inhibits general de novo protein synthesis in cell culture. In a panel of 19 HNC cell lines SVC112 exhibits antiproliferative activity with an average IC50 of ~160 nM, with no IC50 exceeding 500 nM. Combinations of SVC112 and IR dose-response curves in HNC cells yield average combination-index (CI) values generally lower than 1, especially in maximally tolerated dose ranges. CI values of 1 are indicative of additive interactions, while CI values 1 in HNC cells, when combined with a range of clinically relevant IR doses. DMF values of 1 are indicative of additive interactions, while DMF values >1 indicate synergistic enhancement of IR mediated clonogenic death. Additionally in clonogenic assays, SVC112 enhances cisplatin-IR mediated clonogenic death. Finally, SVC112 enhances the growth inhibitory effects of IR-cisplatin treatment (alone and in combination) in HNC tumor xenografts in mice. Taken together these data demonstrate that inhibition of translation elongation in combination with existing standards of care is a potentially promising treatment option for cancers of the head and neck. *This project has been funded in whole or in part with Federal funds from the NCI/NIH, Department of Health and Human Services, under Contract No. HHSN261201300021C Citation Format: Nathan Gomes, Barb Frederick, Ryan Hansen, Gan Zhang, Daniel Gustafson, Gijsbertus Pronk, Tin Tin Su. Development of a radiation modulator that functions via translation inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B24.

Buprenorphine and carprofen, 2 of the most commonly used analgesics in mice, must be administered every 8 to 12 h to provide sustained analgesia. Sustained-release (SR) formulations of analgesics maintain plasma levels that should be sufficient to provide sustained analgesia yet require less frequent dosing and thus less handling of and stress to the animals. The pharmacokinetics of SR formulations of buprenorphine (Bup-SR), butorphanol (Butp-SR), fentanyl (Fent-SR), carprofen (Carp-SR), and meloxicam (Melox-SR) were evaluated in mice over 72 h and compared with those of traditional, nonSR formulations. Bup-SR provided plasma drug levels greater than the therapeutic level for the first 24 to 48 h after administration, but plasma levels of Bup-HCl fell below the therapeutic level by 4 h. Fent-SR maintained plasma levels greater than reported therapeutic levels for 12 h. Therapeutic levels of the remaining drugs are unknown, but Carp-SR provided plasma drug levels similar to those of Carp for the first 24 h after administration, whereas Melox-SR had greater plasma levels than did Melox for the first 8 h. Butp-SR provided detectable plasma drug levels for the first 24 h, with a dramatic decrease over the first 4 h. These results indicate that Bup-SR provides a stable plasma drug level adequate for analgesia for 24 to 48 h after administration, whereas Carp-SR, Melox-SR, Fent-SR, and Butp-SR would require additional doses to provide analgesic plasma levels beyond 24 h in mice.

The absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic (PK) properties of inorganic nanoparticles with hydrodynamic diameters between 2 and 20 nm are presently unpredictable. It is unclear whether unpredictable in vivo properties and effects arise from a subset of molecules in a nanomaterials preparation, or if the ADME/PK properties are ensemble properties of an entire preparation. Here we characterize the ADME/PK properties of atomically precise preparations of ligand protected gold nanoclusters in a murine model system. We constructed atomistic models and tested in vivo properties for five well defined compounds, based on crystallographically resolved Au25(SR)18 and Au102(SR)44 nanoclusters with different (SR) ligand shells. To rationalize unexpected distribution and excretion properties observed for several clusters in this study and others, we defined a set of atomistic structure–activity relationships (SAR) for nanoparticles, which includes previously investigated parameters such as particle hydrodynamic diameter and net charge, and new parameters such as hydrophobic surface area and surface charge density. Overall we find that small changes in particle formulation can provoke dramatic yet potentially predictable changes in ADME/PK.

Objective To compare the pharmacokinetics and pharmacodynamics of propofol with or without 2% benzyl alcohol administered intravenously (IV) as a single induction dose in cats. Study design Prospective experimental study. Animals Six healthy adult cats, three female intact, three male castrated, weighing 4.8 ± 1.8 kg. Methods Cats received 8 mg kg −1 IV of propofol (P) or propofol with 2% benzyl alcohol (P28) using a randomized crossover design. Venous blood samples were collected at predetermined time points to 24 hours after drug administration to determine drug plasma concentrations. Physiologic and behavioral variables were also recorded. Propofol and benzyl alcohol concentrations were determined using high pressure liquid chromatography with fluorescence detection. Pharmacokinetic parameters were described using a 2-compartment model. Pharmacokinetic and pharmacodynamic parameters were analyzed using repeated measures anova ( p Results Plasma concentrations of benzyl alcohol were below the lower limits of quantification (LLOQ) at all time points for two of the six cats (33%), and by 30 minutes for the remaining four cats. Propofol pharmacokinetics, with or without 2% benzyl alcohol, were characterized by rapid distribution, a long elimination phase, and a large volume of distribution. No differences were noted between treatments with the exception of clearance from the second compartment (CLD2), which was 23.6 and 38.8 mL kg −1 minute −1 in the P and P28 treatments, respectively. Physiologic and behavioral variables were not different between treatments with the exception of heart rate at 4 hours post administration. Conclusions and clinical relevance The addition of 2% benzyl alcohol as a preservative minimally altered the pharmacokinetics and pharmacodynamics of propofol 1% emulsion when administered as a single IV bolus in this group of cats. These data support the cautious use of propofol with 2% benzyl alcohol for induction of anesthesia in healthy cats.

Purpose. To determine the maximum tolerated dose per day of silybin phosphatidylcholine (Siliphos) in patients with advanced hepatocellular carcinoma (HCC) and hepatic dysfunction. Experimental Design. Patients with advanced HCC not eligible for other therapies based on poor hepatic function were enrolled in a phase I study of silybin phosphatidylcholine. A standard phase I design was used with 4 planned cohorts, dose escalating from 2, 4, 8, to 12 g per day in divided doses for 12 weeks. Results. Three participants enrolled in this single institution trial. All enrolled subjects consumed 2 g per day of study agent in divided doses. Serum concentrations of silibinin and silibinin glucuronide increased within 1 to 3 weeks. In all 3 patients, liver function abnormalities and tumor marker α-fetoprotein progressed, but after day 56 the third patient showed some improvement in liver function abnormalities and inflammatory biomarkers. All 3 participants died within 23 to 69 days of enrolling into the trial, likely from hepatic failure, but it could not be ruled out that deaths were possibly due to the study drug. Conclusion. Short-term administration of silybin phosphatidylcholine in patients with advanced HCC resulted in detectable increases in silibinin and its metabolite, silibinin glucuronide. The maximum tolerated dose could not be established. Since patients died soon after enrollment, this patient population may have been too ill to benefit from an intervention designed to improve liver function tests.

Purpose: The mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS-mutant colorectal cancer who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in colorectal cancer, indicating that rational combination strategies are needed. Experimental Design: In this study, we conducted unbiased gene set enrichment analysis and synthetic lethality screens with selumetinib, which identified the noncanonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we used shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in colorectal cancer cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and showed synergistic antiproliferative effects in in vitro and in vivo models of colorectal cancer. Results: Importantly, this combination not only showed tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of colorectal cancer. In mechanistic studies, we observed a trend toward increased markers of apoptosis in response to the combination of MEK and WntCa++ inhibitors, which may explain the observed synergistic antitumor effects. Conclusions: These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for patients with metastatic colorectal cancer. Clin Cancer Res; 19(15); 4149–62. ©2013 AACR.

Hydrogen sulfide is an important biological signalling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (λmax = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ρ = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated sub-micromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications.

There are currently no registered drugs that slow the progression of neurodegenerative diseases, in part because translation from animal models to the clinic has been hampered by poor distribution to the brain. The present studies examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM) compounds that display anti-inflammatory and neuroprotective efficacy in vitro. We postulated that the pharmacokinetic behavior of C-DIM compounds after oral administration would correlate with neuroprotective efficacy in vivo in a mouse model of Parkinson's disease. Pharmacokinetics and metabolism of 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5), 1,1-bis(3'-indolyl)-1-(phenyl)methane, 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1 mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and urine. C-DIM compounds given orally displayed the highest area under the curve, Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics of the compounds tested. Oral bioavailability of each compound ranged from 6% (C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for 14 days, a model of progressive neurodegeneration with a strong neuroinflammatory component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure to MPTP and probenecid prevented further loss of dopaminergic neurons in the substantia nigra pars compacta and striatal dopamine terminals, indicating that these compounds could be effective therapeutic agents to prevent neurodegeneration.

Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, preclinical studies in mouse models are equivocal with regard to HCQ exposure (pharmacokinetics) and inhibition of autophagy (pharmacodynamics) in various tissues. Understanding HCQ pharmacokinetics (PK) and pharmacodynamics (PD) in mice allows for a comparison to human PK as reported in recent trials and as such the subsequent PD associated with autophagy inhibition in tissues. In this study, female BALB/c mice were treated with a single intraperitoneal dose of 20, 40, or 80 mg/kg HCQ and tissues and whole blood collected at 3, 6, 12, 24, 48 and 72 hours. Levels of HCQ and desethylhydroxychloroquine (dHCQ), an active metabolite, in whole blood and tissues were determined via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Non-compartmental analysis (NCA) measured exposure, as determined by area under the drug concentration versus time curve (AUC0-inf), to both HCQ and dHCQ was dose proportional in whole blood, liver and brain. The maximum concentration (Cmax) of HCQ was 1.3, 3.1 and 4.7 μg/mL and 20.6, 41.7 and 114.7 μg/mL in whole blood and liver, respectively, following 20, 40 and 80 mg/kg. Brain exposure was far less with Cmax achieving 272, 717 and 2220 ng/mL. In general, the time of maximal concentration (Tmax) of HCQ and dHCQ was observed at 3 hr or 6 hr, regardless of dose and location measured. PD assessment for autophagy inhibition was determined by western blotting. The LC3 protein, which is involved in membrane dynamics during autophagy, was visualized and the ratio of LC3-II to tubulin was measured. In the liver, autophagy was inhibited approximately two-fold following all doses at 24 and 48 hr compared to controls. Autophagy was inhibited in both the gut and kidney at 24 and 48 hr at all doses. In the brain, autophagy inhibition was not dose related and may be a result of low HCQ levels. These results demonstrate that a dose of 20 mg/kg is sufficient to inhibit autophagy in most tissues investigated; however, while PK parameters appear to be dose proportional, increased tissue drug levels does not necessarily equate to increased PD effects/autophagy inhibition. In addition, the data establishes a dose of 40 mg/kg in mice results in Cmax and AUC0-inf of HCQ equivalent to that observed in human clinical trials targeting autophagy modulation following a dose of 600 mg daily. Studies are ongoing to assess this relationship in solid tumors. Citation Format: Kristen M. Jackson, Ryan J. Hansen, Daniel L. Gustafson. Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2084.

Purpose: Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, seeding, growth, and angiogenesis, as well as suppression of anti-tumor immunity. Migration of inflammatory monocytes to sites of inflammation or tumor metastasis is mediated primarily via the action of the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis through receptor or ligand blockade represents an attractive therapeutic target for the treatment of metastatic disease. Methods: Losartan, an angiotensin II type 1 receptor (AT1R) antagonist, has been previously reported to suppress tumor growth through mechanisms associated with tumor angiogenesis and inhibition of TGF-β signaling. However, our studies also suggest an important contribution to the anti-tumor effects of losartan from monocyte migration inhibition. Therefore, we conducted studies in murine breast (4T1) and colon (CT26) carcinoma experimental pulmonary metastasis models, to determine the degree to which losartan's anti-metastatic effect was predominantly dependent on disruption of CCL2-CCR2 mediated monocyte recruitment to tumor metastases. Results: We found that daily treatment with losartan prolonged survival, and significantly reduced metastatic burden, percentages of CD11b+/Ly6C+ lung monocytes, and tumor microvessel density in mice with 4T1 and CT26 pulmonary metastases. However, compared to pure CCR2 antagonists, our studies show that losartan does not act as a direct competitive antagonist to CCR2. Rather, in vitro assays and studies in CCR2 -\- mice indicated that losartan's anti-metastatic effect was primarily mediated via suppression of monocyte recruitment through indirect interruption of the CCL2-CCR2 axis. Importantly, losartan proved more effective at reducing monocyte recruitment and slowing CT26 pulmonary metastatic tumor growth compared to a specific CCR2 antagonist. Conclusions: We conclude therefore that losartan exerts significant anti-metastatic activity in aggressive murine metastasis models, and that losartan's major anti-metastatic effect is mediated by blockade of CCL2-CCR2 dependent monocyte recruitment. Thus, by virtue of its myeloid cell activity, losartan, in combination with conventional cancer therapeutics, has potential for use as an anti-metastatic agent in animals and humans at high risk for tumor metastasis. Citation Format: Daniel P. Regan, Amanda M. Guth, Ryan J. Hansen, Daniel L. Gustafson, Steven W. Dow. Angiotensin II type 1 receptor antagonism suppresses tumor metastasis through inhibition of CCL2-CCR2 mediated monocyte recruitment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A161.

The purpose of this study was to determine the impact of the non-steroidal anti-inflammatory drug tepoxalin on canine tumour cell growth and describe the changes associated with tepoxalin treatment. In vitro experiments were performed to assess tepoxalin-associated alterations in tumour cell growth. Clinically achievable tepoxalin concentrations did not significantly alter tumour cell growth in vitro. Vascular endothelial growth factor (VEGF) production and hypoxia-inducible factor-1α dose-dependently increased in vitro in the presence of tepoxalin. A canine osteosarcoma xenograft was used to determine in vivo effects of tepoxalin on tumour growth and angiogenesis. Despite increased VEGF in vitro, there was a significant growth delay associated with tepoxalin treatment. Normal dogs were administered tepoxalin to assess effects on systemic VEGF production, but not found to have significantly increased VEGF. These data suggest that tepoxalin may moderately inhibit tumour growth and may be administered as an analgesic to tumour-bearing dogs.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O(6)-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT(R128A)), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt-/- and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.

Background: Autophagy is a lysosomal degradation process that is characterized by cellular self-consumption. Autophagy allows for the recycling and removal of damaged cytoplasmic material. Many types of cancer have elevated autophagic activity in order to sustain a high rate of metabolism and circumvent stress-induced cell death. However, little is known of autophagy's role in metastasis. Therefore, we tested the effect of autophagy modulation in different mouse models and cell based assays that reflect different points along the metastatic cascade. Methods: Pharmacologic autophagy inhibition in the murine mammary carcinoma cell line 4T1 was achieved with 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1). Knockdown of autophagy critical genes Becn1 or Atg7 by lentiviral delivery of shRNA was also used as a means of inhibition. Proliferation, invasion, migration, and anchorage independent growth of cells were assessed in the presence or absence of active autophagy. For an experimental model of metastasis, Balb/c mice were challenged with 4T1 cells expressing luciferase (4T1-luc) injected via the tail vein. Mice were pre-treated with CQ (60 mg/kg, qd, i.p.) and monitored until the development of luciferase positive metastases. Mice were also challenged with 4T1 Becn1 knockdowns or cells pre-treated with CQ for 2h to assess effect of cellular autophagy impairment. An orthotopic model was also utilized by implanting 4T1-luc cells into the mammary fat pad, with tumors resected at 100-200 mm3 and mice followed until metastasis development. CQ was given either adjuvantly, 24h after surgery, or neoadjuvantly, 24h after implantation. Autophagy stimulation, 2% Trehalose in drinking water, was also given as a neoadjuvant therapy. Bone marrow derived cells (BMDCs), mediators of pre-metastatic niche formation, were measured by flow cytometry in the lungs and blood of treated mice prior to the arrival of tumor cells. Results: Autophagy inhibition was able to reduce cell proliferation, but did not significantly delay metastasis in the experimentally induced model. Similarly, CQ was not effective as an adjuvant therapy. Yet, as a neoadjuvant therapy both CQ and Becn1 knockdown were able to significantly delay metastases. Autophagy stimulation actually sped up metastasis development. Autophagy modulation had no effect on invasion, migration, or anchorage independent growth. Conversely, altering autophagy did impact the number of BMDCs present in the lung and blood. Trehalose treated mice had more than either vehicle or CQ. In the blood, CQ treated mice had even less BMDCs present than vehicle treated mice. Conclusions: Autophagy does not seem to be required for metastatic colonization and survival. Nor does it seem to affect the metastatic capability of cells. Rather, autophagy may be influencing the pre-metastatic microenvironment by impacting BMDCs. Thus autophagy appears to be most essential before the arrival of tumor cells to the site of metastasis. Citation Format: Rebecca A. Barnard, Daniel Regan, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Autophagy influences the development of the pre-metastatic niche. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2015-4121

Background Placement of ciclosporin (Atopica(®); Novartis Animal Health, Greensboro, NC, USA) capsules in a freezer prior to administration may reduce the incidence of vomiting in dogs. However, its impact on ciclosporin stability and pharmacokinetics is unknown. Hypothesis/objectives The purpose of this study was to evaluate the stability of Atopica(®) capsules and pharmacokinetics of ciclosporin in dogs after storage at -20°C in comparison with storage of capsules at 15-25°C. We hypothesized that there would be no difference in stability or pharmacokinetic parameters between freezer-stored and room-temperature Atopica(®) capsules. Animals Eight healthy research beagle dogs received one 5.0 mg/kg oral dose each of freezer-stored and room-temperature Atopica(®) capsules with a 1 week washout period between. Methods Ciclosporin concentrations of all available Atopica(®) capsule strengths were assessed for stability after -20°C storage at five time points over 30 days and at room temperature (15-25°C). A blinded, randomized cross-over study was also performed to compare blood concentrations of ciclosporin after capsule storage for 28 days at -20 versus 15-25°C. Blood samples were obtained over a 24 h period after administration. Capsule and whole-blood ciclosporin concentrations were assessed via high-performance liquid chromatography-tandem mass spectrometry. Results There was no significant difference in stability between freezer-stored and room-temperature Atopica(®) capsules at any time point. In the cross-over study, there were no significant differences in pharmacokinetic parameters assessed. Conclusions and clinical importance Placing Atopica(®) capsules in a -20°C freezer for 28 days does not affect stability or absorption in the dog.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) protects from toxicity and mutations incurred following alkylating agents by removing O(6)-alkylguanine lesions. Using Mgmt-/- mice, we examined MGMT's role in protecting from in vivo mutations induced by three different alkylating agents, temozolomide (TMZ), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide. Mutant frequencies were determined in the hypoxanthine-guanine phosphoribosyltransferase gene of splenic T-lymphocytes from C57BL/6 mice (Mgmt+/+ and Mgmt-/-) following TMZ, BCNU or cyclophosphamide. Following TMZ, the mutation frequency was significantly greater in Mgmt-/- mice (5.5 and 9.8 x 10(-6) for 7 and 10 mg/kg TMZ, respectively) compared with vehicle-treated mice (1.0 x 10(-6), P

Purpose: Intravenous delivery of chemotherapy is an indisputable pillar of cancer therapy, but associated with unfavorable systemic toxicity. Locally delivered chemotherapy – via subcutaneous, surgical placement, or intracavitary routes – is effective in animal models of breast cancer and in client-owned dogs receiving treatment for bone tumors. There are unexplained beneficial effects of chemotherapy delivered via nontraditional routes resulting in decreased systemic toxicity and positive tumor control at dosages less than traditionally given. One role of the lymphatic system is to return large particulate matter and proteins in the interstitial space to the vascular system which cannot be absorbed by adjacent blood capillaries. This unidirectional circulatory pathway for particles deposited in areas outside of blood capillaries has not been characterized to date for nontraditionally delivered chemotherapy agents. Experimental Design: In this study the pharmacokinetics of docetaxel and carboplatin in the blood and lymph following subcutaneous (SQ) versus intravenous (IV) delivery was determined in a surgical lymphatic and hemovascular cannulated rat model. In anesthetized adult male Sprague Dawley rats, fixed length polyurethane catheters were surgically placed simultaneously into a jugular vein and the lymphatic thoracic duct in each animal. Either docetaxel (5 mg/kg) or carboplatin (14 or 28 mg/kg) were delivered IV via tail vein catheter or SQ into the mammary fat pad. Rats were permitted free movement and access to water and food ad libitum. Paired blood and lymph samples were serially collected in animals at 0, ½, 1, 2, 3, 4, 12, and 24 hrs. Drug levels were determined via LC/MS/MS or ICP/MS for docetaxel and carboplatin, respectively. Pharmacokinetic parameters were determined via noncompartmental analysis. Results: The maximum concentration measured (Cmax) and the area under the time-concentration curve (AUC0-24hr) in the plasma and lymph for docetaxel delivered IV were 2.86 µg/ml and 1160 hr*ng/ml, and 172 ng/ml and 1020 hr*ng/m, respectively. When administered SQ, the Cmax and AUC0-24hr were 0.644 µg/ml and 815 hr*ng/ml, and 107 ng/ml and 1650 hr*ng/ml, for plasma and lymph respectively, which demonstrates preferential lymphatic accumulation when delivered SQ. The terminal half-life (t1/2) in lymph for docetaxel is greater with either IV or SQ delivery (18.3 hr and 22.6 hr, respectively) as compared to t1/2 in blood for docetaxel with either IV or SQ delivery (9.6 hr and 6.0 hr, respectively), possibly suggesting lipophilic docetaxel resides in the lymph for longer periods. The Cmax and AUC0-24hr in the plasma and lymph for total platinum following delivery of carboplatin (14 mg/kg) IV were 23.0 µg/ml and 28.8 hr*µg/ml, and 16.4 µg/ml and 36.1 hr*µg/m, respectively. When delivered SQ, the Cmax and AUC0-24hr for the plasma and lymph were 6.99 µg/ml and 16.6 hr*µg/ml, and 9.39 µg/ml and 24.3 hr*µg/ml, respectively. While a dose response was observed in the plasma regardless of route of administration with the Cmax in the plasma following carboplatin (28 mg/kg) of 40.0 µg total platinum/ml when given IV and 12.9 µg total platinum/ml when given SQ, the AUC0-24hr did not when administered IV. Additionally, the AUC0-24hr in the lymph was 31.0 hr*µg/ml following IV dosing at 28 mg/kg which is similar to that observed in the plasma. However, when dosed SQ at 28 mg/kg, the Cmax and AUC0-24hr for total platinum in the lymph responded in a dose dependent manner with 17.3 µg/ml and 76.7 hr*µg/ml, respectively. This demonstrates preferential lymphatic accumulation of carboplatin delivered SQ and that there is a dose response with regards to the level of total platinum in the lymph when delivered SQ. Conclusions: Nontraditional SQ delivery of docetaxel and carboplatin achieves targeted lymphatic accumulation greater than with traditional IV delivery. This study has broad implication for dosing strategies of chemotherapeutics for cancers metastasizing predominantly via lymphatic pathways and for cancer patients susceptible to toxicity resulting from peak maximum hemovascular concentrations that may be lessened via SQ delivery. Citation Format: Deanna R. Worley, Ryan J. Hansen, Laura S. Chubb, Daniel L. Gustafson. Subcutaneous delivery of docetaxel and carboplatin accumulate preferentially in lymphatic circulation as compared to intravenous delivery in rats with surgically created lymph and venous fistulae. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B48.

Modification of autophagy to enhance cancer treatment is being aggressively pursued in the laboratory and clinic; however, it remains to be seen if inhibition or induction of autophagy is going to result in better outcomes. In addition, the advantages of autophagic modification with neoadjuvant chemotherapy vs. surgical adjuvant chemotherapy have yet to be reported. The 4T1-luc metastatic mammary mouse cell line modified to express luciferase was used to induce lung metastases, both from orthotopic growth of primary tumors or experimentally induced via tail vein injection. Both pharmacologic inhibition (chloroquine (CQ) administered 60 mg/kg daily to mice) and genetic knockdown of a key protein necessary for autophagy alone (4T1-luc-shBeclin-1 cells) did not alter time to metastasis (as determined by luciferase positive lungs) when 4T1-luc cells were injected via tail vein. In addition, inhibiting autophagy in 4T1-luc cells with CQ in vitro prior to injection via the tail did not alter time to metastasis. Similar results were observed with pharmacologic inhibition on experimentally induce lung metastases using the metastatic mouse melanoma B16-F10 cell line. In vitro work, by us and others, has lead us to conclude that pharmacologic inhibition plus cisplatin leads to greater growth inhibition and cell kill in vitro than either alone. Effect of autophagy inhibition in combination with cisplatin on metastatic formation in a neoadjuvant setting was tested by treating mice with daily with CQ and cisplatin (q14d) one day post tumor cell injection in the mammary fat pad. Additional experiments were conducted to determine if autophagy inhibition plus cisplatin in a surgical adjuvant setting delays metastasis formation. In each instance, primary tumors were removed at ~100mm3 and mice were followed until luciferase positive lungs were observed. From these results we conclude that autophagy inhibition, using CQ, does not alter metastatic growth alone and appears to slightly enhance the efficacy of cisplatin on treating metastases. It remains to be seen if autophagy inhibition with other compounds would alter results. Citation Format: Rebecca Barnard, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Role of autophagy inhibition in metastatic disease utilizing mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A37.

Intravenous immunoglobulin (IVIG) has been an important therapy of immune thrombocytopenic purpura (ITP) for more than 20 years; however, questions still remain regarding the mechanisms of IVIG action in ITP. Recent reviews have focused on the hypotheses that IVIG effects are mediated via inhibition of Fc-receptor mediated platelet phagocytosis, suppression of anti-platelet antibody production, and anti-idiotypic inhibition of anti-platelet antibodies; however, new research suggests that a significant portion of IVIG benefit may be due to IVIG-mediated acceleration of the elimination of anti-platelet antibodies. It is anticipated that promising new therapies of ITP, and other autoimmune conditions, will be developed based on a better understanding of IVIG mechanism of action.

O(6)-Benzylguanine (O(6)-BG), a potent inactivator of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), is presently in clinical trials combined with alkylating agents that modify the O(6) position of DNA guanine residues, i.e., 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Previous work demonstrated that O(6)-BG also enhances the cytotoxicity of cyclophosphamide, ifosfamide, and nitrogen mustards in Chinese hamster ovary cells. We have extended this study to include other clinically relevant agents that form interstrand and intrastrand cross-links including cisplatin and carboplatin. Pretreatment of a series of head and neck tumor cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon tumor cells with O(6)-BG (100 micro M for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED(50) of cisplatin and a concomitant increase in the percentage of cells undergoing apoptosis. The enhancement was independent of AGT activity. Similar enhancement was observed with carboplatin, but no enhancement was seen in AGT-deficient cell lines with radiation or temozolomide, demonstrating the dependence of the effect on bifunctional, cross-linking agents. Furthermore, levels of platinum on DNA after treatment with cisplatin increased 1.4-fold in SQ20b cells and 4.5-fold in JSQ3 cells immediately after treatment with O(6)-BG plus cisplatin and remained elevated for 48 h. Consistent with greater cytotoxicity and apoptosis is the approximately 2-fold higher amount of DNA damage when cells are treated with O(6)-BG plus cisplatin compared with cisplatin alone. Modulation of cisplatin therapy with O(6)-BG might improve the prognosis of patients with head and neck, ovarian, testicular, or lung cancer who are treated with this drug.

We have recently shown that intravenous immunoglobulin (IVIG) therapy leads to an increased rate of anti-platelet antibody clearance in an animal model of immune thrombocytopenia. The present study was performed to confirm the importance of the FcRn receptor in mediating this effect of IVIG. The pharmacokinetics of an anti-platelet antibody, 7E3, were studied in mice lacking expression of FcRn and in control mice, both in the presence and absence of IVIG. IVIG increased the clearance of 7E3 in mice with functioning FcRn receptors, with an average clearance value of 14.4 +/- 1.4 ml/d/kg in IVIG treated mice vs. 5.2 +/- 0.3 ml/d/kg in controls (P0.001). In mice lacking expression of FcRn, IVIG treatment did not increase 7E3 clearance (61.0 +/- 3.6 ml/d/kg vs. 71.5 +/- 4.0 ml/d/kg in controls). Thus, these data support the hypothesis that IVIG increases antibody elimination via saturation of FcRn.

Experiments were conducted to investigate the effects of intravenous immunoglobulin (IVIG) in a rat model of immune thrombocytopenia (ITP). Rats were pretreated with 0 to 2 g/kg IVIG and then challenged with an antiplatelet antibody (7E3, 8 mg/kg). IVIG effects on 7E3-induced thrombocytopenia and on 7E3 pharmacokinetics were determined. IVIG pretreatment led to significant changes in the degree and time-course of 7E3-induced thrombocytopenia (P =.031). Nadir percent platelet counts were 121% to 279% greater in animals treated with IVIG (0.4-2 g/kg) than in animals receiving 7E3 alone. IVIG treatment also led to dose-dependent increases in 7E3 clearance (P.001), with more than 2-fold increases in 7E3 clearance seen following the highest dose of IVIG. In vitro experiments showed that IVIG effects on platelet count are not likely due to anti-idiotypic inhibition of 7E3-platelet binding and that IVIG did not directly bind to 7E3. Consequently, IVIG-7E3 binding cannot explain the increase of 7E3 clearance following IVIG treatment. We propose that the observed increase in 7E3 clearance with IVIG therapy is due to saturation of the FcRn salvage receptor for IgG. The importance of the effect of IVIG on 7E3 clearance to the prevention of thrombocytopenia in these animals is unclear at present; nonetheless, these data provide experimental support for a new mechanism of IVIG action in ITP (ie, IVIG-mediated increases in antiplatelet antibody elimination). This model of ITP will be useful for further investigations of IVIG mechanism of action and for development of new therapies for ITP.

We have investigated the sensitivity to DNA-damaging agents of a strain of Saccharomyces cerevisiae containing a deletion of the RAD27 gene. The mutant strain is sensitive to a number of alkylating agents that modify DNA at a variety of positions, including one that produces primarily phosphotriesters. In contrast, the mutant strain is not sensitive to the oxidizing agent hydrogen peroxide. The introduction of a plasmid containing the FEN-1 gene (the human ortholog of the RAD27 gene) can substantially complement the sensitivity to alkylating agents observed in the mutant strain.

Background: Autophagy is an intracellular process that involves the sequestration of cytosolic proteins and organelles into double-membraned vesicles called autophagosomes and their subsequent degradation in the lysosome. This process is induced during cellular stress and is crucial for maintaining proper homeostasis. Recently, autophagy has been identified as a potential mechanism of chemoresistance. Thus, autophagy inhibition has become an attractive anti-cancer therapy. However, autophagy is a multi-step process and it is still unclear if blocking autophagy at one particular stage is more advantageous than another. Therefore, we inhibited autophagy at various points in the pathway using genetic and pharmacologic means in multiple cancer models. Material and Methods: Murine cell lines used were 4T1, mammary carcinoma, B16-F10, melanoma, DLM8, osteosarcoma, and A20, lymphoma. Autophagy was inhibited by multiple methods. The first method was pharmacologic inhibition using 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1), doses sufficient to inhibit autophagy. The second method was genetic knockdown by decreasing expression of critical autophagy proteins Beclin-1 (Bcn-1) or Atg7 by shRNA lentiviral delivery. Cells were assessed for protein knockdown by western blot and qPCR analysis. Proliferation of cells in the presence or absence of active autophagy was assessed using an Alamar Blue assay. For cell cycle analysis, cells were grown for 48h, stained with propidium iodine and analyzed by flow cytometry. Results and Conclusions: A significant decrease in expression of Bcn-1 and Atg7 was observed after lentiviral transduction. Autophagy was also successfully inhibited as indicated by a failure to induce elevations in autophagy marker LC3 after serum starvation, a positive autophagy control. CQ and Baf A1, which inhibit late stage autophagy, had little effect on the 4T1 cells, but CQ did inhibit the other three lines. Bcn-1 knockdown, preventing nucleation of the autophagosome, was able to inhibit proliferation in all lines at a level that was even more pronounced than CQ inhibition. In addition, the percentage of cells in G1 significantly increased in the Bcn-1 knockdown cells. Continued study with the Atg7 cells and Baf A1 will reveal if the effects of CQ and Bcn-1 knockdown are merely off target effects or truly autophagy inhibition. The inclusion of Vps 34 inhibitors and late stage mediator knockdown lines will also be useful in determining if the early, nucleation stage of autophagy is the critical step(s) to target. Lastly, it will be important to determine if these effects differ when cells experience stress, such as the presence of chemotherapy. These studies suggest that the sensitivity toward autophagy inhibition may depend on the stage of the pathway that is targeted and cancer type. Acknowledgements: Role of Autophagy in Tumor Cell Death National Cancer Institute 1R01CA150925 Citation Format: Rebecca A. Barnard, Paola Maycotte, Ryan J. Hansen, Daniel L. Gustafson, Andrew Thorburn. Differing sensitivity to stage-specific autophagy inhibition. [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 1863. doi:10.1158/1538-7445.AM2013-1863

Background: Autophagy is an intracellular process that involves the sequestration of cytosolic proteins and organelles into double-membraned vesicles called autophagosomes and their subsequent degradation in the lysosome. This process is induced during cellular stress and is crucial for maintaining proper homeostasis. Autophagy has also been shown to play a role in tumorigenesis, yet the role of autophagy in metastasis progression is poorly understood. Some of the key steps in metastasis progression are the ability of tumor cells to detach from the extracellular matrix, enter circulation, and seed a distal site. Non-tumorigenic cells undergo apoptosis upon detachment, referred to as anoikis. Some evidence suggests that autophagy is induced after detachment and facilitates anchorage independent growth and anoikis evasion. Therefore, we studied the effect of autophagy inhibition on anchorage independent growth using multiple cancer models both in vitro and in vivo. Material and Methods: Murine cell lines used were 4T1, mammary carcinoma, B16-F10, melanoma, and DLM8, osteosarcoma. Autophagy was inhibited by multiple methods. The first method was pharmacologic inhibition using 10uM chloroquine (CQ) or 1nM bafilomycin A1 (BafA1). The second method was genetic knockdown by decreasing expression of critical autophagy proteins Beclin-1 (Bcn-1) or Atg7 by shRNA lentiviral delivery. Cells were assessed for Bcn-1 or Atg7 knockdown by western blot and qPCR analysis. Proliferation of cells in the presence or absence of active autophagy was assessed using an Alamar Blue assay. To determine anchorage independent proliferation, PolyHEMA coated plates were used to prevent attachment. For cell cycle analysis, cells were grown for 48h, stained with propidium iodine and analyzed by flow cytometry. For the in vivo model, mice were pretreated with CQ for 72h at 60mg/kg daily IP, allowing for systemic autophagy inhibition. 4T1 cells transfected with a luciferase reporter, or B16-F10 cells were injected via the tail vein of syngeneic Balb/c or C57Bl/6J mice. Mice continued to receive CQ or vehicle daily and were monitored thrice weekly until the development of luciferase positive metastases or 10% weight loss. Blood and liver samples were also collected to assess autophagy inhibition by measuring LC3 expression as determined by flow cytometry or Western blot. Results and Conclusions: A significant decrease in expression of Bcn-1 and Atg7 was observed after lentiviral transduction. Autophagy was also successfully inhibited as indicated by a failure to induce elevations in autophagy marker LC3 after serum starvation, a positive autophagy control. Pharmacologic inhibition had no significant effect on 4T1 proliferation under any culture condition and failed to delay metastasis development in vivo. However, CQ did inhibit proliferation for B16-F10 and DLM8 cells, and this effect was more pronounced in B16-F10 cells grown in suspension. To demonstrate if a similar affect can be seen in vivo, mice will also be challenged with B16-F10 cells and treated with CQ. Conversely, Bcn-1 knockdown was able to significantly inhibit proliferation for all cell types and under all culture conditions, but there was no difference whether cells were attached or suspended. Further study with the Atg7 cells and BafA1 will reveal if this effect is merely an off target effect of Bcn-1 or truly autophagy inhibition. These studies suggest that the role autophagy plays in anchorage independent growth and survival of tumor cells is context dependent and thus broad use of autophagy inhibitors in cancer treatment is a questionable strategy. Acknowledgements: Role of Autophagy in Tumor Cell Death. National Cancer Institute 1R01CA150925 Citation Format: Rebecca A. Barnard, Paola Maycotte, Ryan J. Hansen, Daniel L. Gustafson, Andrew Thorburn. The effect of autophagy inhibition on anchorage-independent cell growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C19.