Lori Clarke

S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors In Vitro and In Vivo

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.

ONCOLYTIC ADENOVIRAL VECTORS

It is important to analyze to what extent these random or designed mutations abrogate viral replication in normal cells because a tightly controlled vector could be injected at higher doses intratumorally or even systemically. On the other hand, it is also important to analyze to what extent these mutations affect the amount of virus produced per infected tumor cell (burst size) compared to wild-type virus because lower yields will result in a slower propagation throughout the tumor. Finally, as concluded from the clinical trials with wildtype.

Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening

5′-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.

Divergent roles for Clusterin in Lung Injury and Repair

Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU−/−) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.

Abstract 4328: Phenotypic selection for identification of functional antibodies and development of high throughput screening assays.

Discovery of therapeutic antibodies requires the continued identification of tractable targets, and we have employed a phenotypic screening strategy to this end. In this study, antibodies from phage display libraries were screened for their abilities to inhibit MDA-MB-231 (‘triple negative’) breast cancer cells. The screening cascade included tests for specific cell binding, antibody internalization, and cytotoxicity as an antibody-drug conjugate. Targets of these antibodies then were identified using immunoprecipitation and mass spectrometry, and confirmed by showing diminished antibody binding after siRNA-mediated knockdown of the putative target gene. One target was identified as NT5E (also known as CD73), a 5’-ectonucleotidase. NT5E catalyzes the conversion of adenosine monophosphate to adenosine, and its function has been linked with immunity, angiogenesis, and cancer. To screen for additional inhibitors of this target, a novel, high-throughput ectonucleotidase assay was developed. NT5E activity is typically assayed either by high performance liquid chromatography or by quantification of free phosphate using malachite green, and neither of these methods is suitable for high-throughput screening. One inhibitory antibody was found to significantly slow growth of MDA-MB-231 xenograft tumors in mice. In summary, our phenotypic screening approach provides a mechanism for rapidly discovering and evaluating new antibody targets and is being used to accelerate the discovery of new cancer drugs. Citation Format: Carl Hay, Steven Rust, Erin Sult, Lori Clarke, Kim Rosenthal, Sandrine Guillard, David Lowne, Matt Flynn, Lutz Jermutus, Ralph Minter, Robert Hollingsworth, Kris Sachsenmeier. Phenotypic selection for identification of functional antibodies and development of high throughput screening assays. [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 4328. doi:10.1158/1538-7445.AM2013-4328