Kelly L. Bennett

Intracellular Trafficking of CTLA-4 and Focal Localization Towards Sites of TCR Engagement

T lymphocyte receptor CTLA-4 binds costimulatory molecules CD80 (B7-1) and CD86 (B7-2) with high avidity and negatively regulates T cell activation. CTLA-4 functions at the cell surface, yet is primarily localized in intracellular vesicles. Here, we demonstrate cycling of CTLA-4 between intracellular stores and the cell surface. Intracellular vesicles containing CTLA-4 overlapped with endocytic compartment(s) and with perforin-containing secretory granules. Cell surface expression of CTLA-4 was rapidly increased by raising intracellular calcium levels. During T cell activation, intracellular and cell surface CTLA-4 became focused towards sites of TCR activation. Cycling and directional control of CTLA-4 expression may regulate its functional interaction with APCs bearing peptide-MHC complexes of appropriate specificity and avidity.

Deleted in Colorectal Carcinoma (DCC) Binds Heparin via Its Fifth Fibronectin Type III Domain

DCC (deleted incolorectal carcinoma) is a broadly expressed cell-surface receptor. Netrin-1 was recently identified as a DCC ligand in brain, but the possibility of other DCC ligands was suggested by the finding that an anti-DCC antibody (clone AF5) neutralized netrin-1-dependent commissural axon outgrowth without blocking DCC/netrin-1 interactions. Here we have searched for alternative cell-surface DCC ligands. A DCC-Ig fusion protein bound to neural and epithelial derived cell lines, indicating that these lines express ligand(s) for DCC. The cell-surface binding activity was mediated by the loop between β-strands F and G of the fifth fibronectin type III repeat FNIII-D5. The loop included the sequence KNRR, which resembles heparin-binding motifs in other proteins. Heparinase and heparitinase treatment of cells reduced binding of DCC-Ig, suggesting that heparan sulfate proteoglycans are cell-surface DCC ligand(s). This was further supported by heparin blocking experiments and by binding of DCC-Ig to immobilized heparan sulfate. The interaction between DCC-Ig and heparan sulfate/heparin, both on the surface of cells and immobilized on plastic, was blocked by the same anti-DCC antibody that blocks netrin-1-dependent commissural axon outgrowth. Taken together, these findings suggest that the DCC-Ig/heparin interaction may contribute to the biological activity of DCC.

The extracellular matrix protein βIG-H3 is expressed at myotendinous junctions and supports muscle cell adhesion

Molecules of the extracellular matrix (ECM) play important roles in the development and maintenance of myotendinous junctions (MTJs), specialized regions of muscle to bone union. In this report we provide evidence that skeletal muscle cells synthesize the collagen- and fibronectin-binding ECM protein βIG-H3 and that βIG-H3 is localized to MTJs. In situ hybridization experiments revealed that during E16.5–E18.5 of murine development, βIG-H3 RNA transcripts were expressed where developing skeletal muscle fibers contact primordial cartilage and bone. Immunohistochemical analysis verified that the βIG-H3 protein itself localized distinctively at MTJs, and ultrastructural analysis suggested that βIG-H3 associates with extracellular fibers and the surface of cells. In vitro, recombinant βIG-H3 functioned as an adhesion substratum for skeletal muscle cells. Adhesion was significantly reduced by anti-integrin α7 and β1 antibodies, suggesting that βIG-H3 binds to skeletal muscle cells via α7β1 integrin. Localization of βIG-H3 to the termini of skeletal muscle fibers and the binding of βIG-H3 to cells and to molecules of the ECM suggests that βIG-H3 may play an organizational and structural role in developing MTJs, linking skeletal muscle to components of the ECM.

Amyloid and Pro501Thr-mutated βig-h3 gene product colocalize in lattice corneal dystrophy type IIIA

PURPOSE To assess the relative distribution in the cornea of amyloid and (beta)ig-h3 gene product in lattice corneal dystrophy type IIIA (LCD-IIIA). METHODS Serial sections from the cornea of a patient with LCD-IIIA were subjected to either Congo red staining or immunohistochemistry employing an antibody to (beta)ig-h3. Also, genomic DNA was isolated from peripheral blood and used as a template for polymerase chain reaction to amplify all exons of (beta)ig-h3. RESULTS Exon 11 of (beta)ig-h3 was mutated (Pro501Thr). Subepithelial and intrastromal congophilic deposits exhibited a birefringency characteristic of amyloid. These regions of the tissue were also highly immunoreactive with the antibody to the (beta)ig-h3 gene product. CONCLUSION Amyloid and Pro501Thr-mutated (beta)ig-h3 protein accumulate and colocalize in LCD-IIIA.

Regulation of Amphiregulin mRNA by TGF-β in the Human Lung Adenocarcinoma Cell Line A549

Transforming growth factor beta is a strong growth inhibitor for many types of normal and transformed cells, although little is known on the mechanism of this anti-proliferative effect. The human lung adenocarcinoma cell line A549 is growth arrested by TGF-beta 1 and serves as a model for studying this effect. We describe that, concurrent with the inhibition of A549 cell growth, TGF-beta 1 treatment causes a dramatic reduction in the level of expression of the amphiregulin (AR) gene, a recently identified member of the EGF/TGF alpha family. Similar results were also observed with TGF-beta 2. Peak inhibition occurred at 24 hr of treatment and was reversible upon removal of TGF-beta 1. The level of AR protein secreted by A549 cells was also decreased by TGF-beta 1. In contrast, TGF-alpha mRNA was not detected in these cells regardless of TGF-beta 1 treatment. Another TGF-beta inhibited cell line, PC-3 (human prostatic adenocarcinoma) also exhibited a decrease in AR message levels following exposure to TGF-beta 1. The growth inhibitory effects of TGF-beta may in part be mediated by modulation of AR expression.

PS01.59: CheckMate 370: A Master Protocol of Phase 1/2 Studies of Nivolumab as Maintenance or First-Line ± Standard-of-Care Therapies in Advanced NSCLC: Topic: Medical Oncology

George Blumenschein Jr., Jason Chandler, Edward B. Garon, David Waterhouse, Jonathan W. Goldman, Vijay K. Gunuganti, Ralph Boccia, David Spigel, John Glaspy, Donald A. Berry, Beata Korytowsky, Jin Zhu, Wen Hong Lin, Kelly Bennett, Craig Reynolds The University of Texas MD Anderson Cancer Center, Houston, TX/United States of America, The West Clinic, PC, Memphis, TN/United States of America, David Geffen School of Medicine at UCLA/TRIO-US Network, Santa Monica, CA/United States of America, OncologyHematology Care (OHC), Cincinnati, OH/United States of America, US Oncology Research, Texas Oncology, Austin, TX/United States of America, Center for Cancer and Blood Disorders, Bethesda, MD/United States of America, Sarah Cannon Research Institute/Tennessee Oncology, PLLC, Nashville, TN/United States of America, Berry Consultants, LLC, Austin, TX/United States of America, Bristol-Myers Squibb, Princeton, NJ/United States of America, Ocala Oncology Center, Ocala, FL/United States of America

Abstract C18: Assessment of Gli1 expression during skin regeneration in mouse models and normal healthy volunteers

Background: Inhibition of the Hedgehog pathway using Smoothened antagonists is emerging as a promising targeted therapy for cancers with pathway activating mutations or dysregulated expression. In support of the clinical development of BMS‐833923 (XL139), an orally available, potent Smoothened antagonist, a pharmacodynamic assay was developed to measure Gli1 protein expression during the wound healing process. Results: Gli1 protein and mRNA expression were first examined by immunohistochemistry or RT‐PCR in mouse skin at baseline and in regenerating skin at various time points following the initial punch biopsy. The effects of BMS‐833923 treatment on pathway expression profiles were then determined. The results showed that in control mice, Gli1 protein levels were elevated at as early as 3h and sustained for up to 72h. The intensities of Gli1 staining in both hair follicles and epidermis were greatly enhanced. However, in the mice treated with BMS‐833923 at the time of initial biopsy, the magnitude and duration of Gli1 up‐regulation were greatly reduced. While the Gli1 protein level was elevated within 6h, it returned to nearbaseline levels at 24h and remained low thereafter. Gli1 mRNA expression data support these observations. To better define the timing of potential Hedgehog pathway activation in human skin, an experimental medicine study was conducted to examine the expression of Gli1 after skin wounding and during regeneration in eighteen normal healthy volunteers. The baseline skin was sampled at the nape of the neck and the regenerating skin sample completely encompassed the initial wound. The intensities of both nuclear and cytoplasmic staining, as well as the percent cell staining positivity were scored. The data indicated that Gli1 protein was significantly up‐regulated at 6h and 24h following wounding. Conclusion: Gli1 protein level at 24h post‐skin wounding assessed by immunohistochemistry is currently being used as a surrogate tissue pharmacodynamic measurement in a first‐in‐human multiple ascending dose trial in subjects with advanced or metastatic solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C18.