Citations

Abstract Pancreatic cancer is highly lethal with an estimated 5-year relative survival rate of less than 5%. A genome wide association study (GWAS) of pancreatic cancer (PanScan) conducted within the NCI Cohort Consortium and the Pancreatic Cancer Case-Control Consortium (PANC4) recently identified four common susceptibility loci for pancreatic cancer on chromosomes 1q32.1, 5p15.33, 9q34.2 and 13q22.1. To investigate how these germline variants influence pancreatic cancer risk, we are employing both genome wide and targeted approaches. For the former, we are characterizing the epigenome and transcriptome of pancreatic cell lines and tissue samples for genomic annotation and prioritization of methods to use in targeted approaches with the aim of directly linking pancreatic cancer risk variants to molecular phenotypes and mechanisms that underlie the association signals. As an example of our approach we will present results in a pancreatic cancer susceptibility locus on chromosome 1q32.2 marked by SNP (single nucleotide polymorphism) rs3790844. This SNP is located in the first intron of the NR5A2 gene that encodes a nuclear receptor of the Ftz-F1 family known to be involved in pancreatic development, cholesterol and bile acid homeostasis, steroidogenesis and cell proliferation. In ChIP-sequencing and MeDIP-chip experiments, we observed repressive histone modification marks (H3K27me3) and regions of DNA methylation in the NR5A2 gene locus in pancreatic cell lines whereas only a few weak positive histone modification marks were observed (H3K4me1 and H3K4me3). This pattern of epigenetic marks correlates well with low expression of NR5A2 in the same cell lines. Expression of the NR5A2 gene at the RNA level is high in normal pancreatic tissue samples as compared to stage I-III pancreatic adenocarcinoma, most islet cell and neuroendocrine tumors. Using immunohistochemistry, nuclear NR5A2 was detected in normal acinar cells and – at lower levels – in normal ductal cells. The protein was also expressed in low and high grade PanINs and in the majority of PDAC analyzed. Our analysis of the tissue specific expression of NR5A2 shows that it is mainly expressed in normal pancreas, colon, small intestine, liver and breast but detected at low levels or not at all in most other tissue types and cancer cell lines. Furthermore we have identified a novel NR5A2 transcript by rapid amplification of cDNA ends (RACE) and mapped a promoter in front of this transcript in the genomic region corresponding to first intron of the two annotated Refseq transcripts. Establishing a comprehensive catalog of epigenetic patterns coupled with targeted analysis to correlate risk variants to molecular phenotypes could provide functional plausibility for known pancreatic cancer association signals discovered by GWAS findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4965. doi:10.1158/1538-7445.AM2011-4965

Wiskott–Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E]3-10-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.

Abstract A phase I trial of ABT-888 (veliparib), a PARP inhibitor, in combination with topotecan, a topoisomerase I–targeted agent, was carried out to determine maximum tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of the combination in patients with refractory solid tumors and lymphomas. Varying schedules and doses of intravenous topotecan in combination with ABT-888 (10 mg) administered orally twice a day (BID) were evaluated. Plasma and urine pharmacokinetics were assessed and levels of poly(ADP-ribose) (PAR) and the DNA damage marker γH2AX were measured in tumor and peripheral blood mononuclear cells (PBMC). Twenty-four patients were enrolled. Significant myelosuppression limited the ability to coadminister ABT-888 with standard doses of topotecan, necessitating dose reductions. Preclinical studies using athymic mice carrying human tumor xenografts also informed schedule changes. The MTD was established as topotecan 0.6 mg/m2/d and ABT-888 10 mg BID on days one to five of 21-day cycles. Topotecan did not alter the pharmacokinetics of ABT-888. A more than 75% reduction in PAR levels was observed in 3 paired tumor biopsy samples; a greater than 50% reduction was observed in PBMCs from 19 of 23 patients with measurable levels. Increases in γH2AX response in circulating tumor cells (CTC) and PBMCs were observed in patients receiving ABT-888 with topotecan. We show a mechanistic interaction of a PARP inhibitor, ABT-888, with a topoisomerase I inhibitor, topotecan, in PBMCs, tumor, and CTCs. Results of this trial reveal that PARP inhibition can modulate the capacity to repair topoisomerase I–mediated DNA damage in the clinic. Cancer Res; 71(17); 5626–34. ©2011 AACR.

Abstract The diazeniumdiolate O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2diolate (JS-K, NSC 724771) is a nitric oxide (NO)-donating prodrug that was designed to release NO upon reaction with glutathione-S-transferase which is overexpressed in some tumors. We demonstrated that JS-K reacts with nucleophiles in biological fluids and has an extremely short half-life in mice. The purpose of the study was to test the hypothesis that liposomal encapsulation improves the in vivo half-life, tissue distribution and acute toxicity profile of JS-K. We investigated the pharmacokinetics of [3H]JS-K in CD mice administered the drug in DMSO or DMPC (dimyristoylphosphatidylcholine) liposomes (20:1 molar ratio of DMPC: JS-K) and in tumor-bearing athymic nu/nu NCr mice administered the radiolabeled drug in DMPC liposomes. The terminal elimination half-life of total radioactivity (2 hours) was similar for both formulations. We did not detect [3H]JS-K in any plasma or red blood cell samples from mice administered 6 mg/kg JS-K in DMSO and only trace amounts of [3H]JS-K were detected in samples collected at the earliest time-points from mice administered 6 mg/kg JS-K in DMPC liposomes. Small amounts of radioactivity, presumably associated with JS-K metabolites, were detected at the solvent front of HPLC chromatograms. Very little radioactivity and no JS-K was detected in tumor samples. Finally, we developed an LC/MS/MS assay to investigate formation of JS-K metabolites in biological samples and used that method to measure concentrations of JS-K and its metabolites in plasma, red blood cells, liver tissue and tumor tissue collected from tumor-bearing mice administered 3 mg/kg or 6 mg/kg JS-K in DMPC liposomes. We confirmed extremely rapid in vivo degradation of JS-K in the systemic circulation following intravenous administration and rapid formation of several expected metabolites including high concentrations of the glutathione conjugate of the dinitrophenol moiety and piperazine. Supported by NCI Contract NO1-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2662.

Abstract Endoxifen (END), the active metabolite of tamoxifen (TAM), is currently being developed as a drug for the treatment of estrogen receptor positive breast cancer based on recent in vitro and clinical data demonstrating that TAM drug effect is substantially related to circulating plasma concentrations of END, a secondary metabolite produced by human CYP2D6 metabolism. Women with deficient CYP2D6 metabolism have lower END concentrations and a higher risk of breast cancer recurrence. We characterized the pharmacokinetics and oral bioavailability of END in female rats and dogs. We further compared END exposure following equivalent doses of END and TAM. Rats were administered 2 mg/kg i.v. END, 20 − 200 mg/kg oral END or 80 mg/kg oral TAM. Fasted dogs were administered 0.5 mg/kg i.v. END, 15 − 100 mg/kg oral END or 30 mg/kg oral TAM. Plasma END concentrations were also measured in rats or dogs after oral dosing once per day for 4 consecutive days. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. In the single-dose administration study, terminal elimination half-life and plasma clearance values in rats given 2 mg/kg i.v. END were 6.3 h and 2.4 L/h/kg, respectively. Plasma concentrations above 0.1 µM and 1 µM END were achieved with 20 mg/kg and 200 mg/kg doses, respectively, and those concentrations were maintained for longer than 24 h. Oral absorption of END was linear over the range of 20 − 140 mg/kg with a bioavailability greater than 67%. END exposure in rats following 80 mg/kg oral END was 100-fold greater than END exposure following 80-mg/kg oral TAM. Following 0.5 mg/kg i.v. END to dogs, terminal elimination half-life and plasma clearance values were 9.2 h and 0.4 L/h/kg, respectively. Plasma concentrations above 1 µM and 10 µM were achieved with the 15 mg/kg dose and 100 mg/kg, respectively, and those concentrations were maintained for longer than 24 h. Absorption was linear over the range of 15 − 100 mg/kg with a bioavailability greater than 50%. END exposure in dogs following 30 mg/kg oral END was 10-fold greater than END exposure following 30-mg/kg oral TAM. In the repeated-dose administration study, the day 4 END peak plasma concentration in rats increased from 1 to 9 µM as the daily END dose was increased from 20 to 200 mg/kg. The day 4 END peak plasma concentration in dogs exceeded 20 µM and plasma concentrations remained above 2 µM for 24 hours following 4 daily doses of 30 mg/kg END. Based on these data, we conclude that END has high oral bioavailability; high concentrations may be maintained after repeated dosing in dogs and rats; and substantially higher END concentrations are achievable following an oral dose of END in comparison to a similar dose of TAM. Supported by NCI contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2607.

We recently showed that the Wiskott–Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

Abstract Epidermal growth factor receptor (EGFR) vIII is a mutated EGFR that is frequently overexpressed in glioblastomas and implicated in response to receptor tyrosine kinase inhibitors. In this study, we investigate the effect of ZD6474 (ZACTIMA, vandetanib), a dual inhibitor for vascular endothelial growth factor receptor 2 and EGFR on growth and angiogenesis of gliomas expressing EGFRvIII. We used two glioma xenograft models, U87MG cells overexpressing EGFRvIII and short-term cultured primary glioma GBM8 cells with EGFRvIII. ZD6474 inhibited tumor growth and angiogenesis and induced cell apoptosis in various brain gliomas. Moreover, significant inhibition of EGFRvIII-expressing U87MG and GBM8 gliomas was observed compared with their controls. Magnetic resonance imaging analysis using the apparent diffusion coefficient and three-dimensional T2*weighed measurements validated ZD6474 inhibition on tumor growth and angiogenesis in EGFRvIII-expressing GBM8 gliomas. Mechanistically, ZD6474 shows better inhibition of cell growth and survival of U87MG/EGFRvIII, GBM6, and GBM8 cells that express EGFRvIII than U87MG or GBM14 cells that have nondetectable EGFRvIII through attenuation of activated phosphorylation of signal transducer and activator of transcription 3, Akt, and Bcl-XL expression. Albeit in lesser extent, ZD6474 also displays suppressions of U87MG/EGFR and GBM12 cells that overexpress wild-type EGFR. Additionally, ZD6474 inhibits activation of extracellular signal-regulated kinase 1/2 in both types of cells, and expression of a constitutively active phosphoinositide 3-kinases partially rescued ZD6474 inhibition in U87MG/EGFRvIII cells. Taken together, these data show that ZD6474 significantly inhibited growth and angiogenesis of gliomas expressing EGFRvIII by specifically blocking EGFRvIII-activated signaling mediators, suggesting a potential application of ZD6474 in treatments for glioblastomas that overexpress EGFRvIII. Mol Cancer Ther; 9(4); 929–41. ©2010 AACR.