1. Defining Met-driven Oncogenic Signaling Pathways in Urologic Malignancies. Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. We previously found that VHL loss allows robust RCC cell motility, invasiveness and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential, and showed that beta-catenin, a junctional protein and gene transactivator, was a critical intracellular mediator of these activities. These findings also identified beta-catenin as a potential therapeutic target and biomarker for RCC. In follow-up to this work, gene expression microarray analysis was performed on RCC cell lines that were engineered to express mutagenically activated beta-catenin or dominant-negative TCF and grown under invasive or non-invasive conditions (+/- HGF). Using this combination of pathway activating and silencing conditions and simple set theory, gene sets were filtered to derive a much smaller gene set most likely to represent causative common targets of HIF and beta-catenin/TCF. The functional characterization of these target genes is being carried out in collaboration with Dr. Irmgard Irminger-Finger of the University Hospitals Geneva. 2. Development and Evaluation of Met pathway antagonists through structural analysis of HGF/Met interaction, virtual and biological screening of compound libraries, and comparative analysis of available Met-targeting agents. HGF is a heparin-binding growth factor, a property that influences its biodistribution, stability, and biological activity. HGF pathway activation begins with HGF binding to the Met receptor tyrosine kinase and to cell surface heparan sulfate (HS) proteoglycans. The importance of HS binding for various HGF biological responses remains unclear. To better define the role of HS in HGF biology, HS binding residues in the N domain were replaced by residues of opposite charge in the context of the fully active truncated HGF isoform NK1. We then compared recombinant mutant (3M) and wild type NK1 proteins structurally, biochemically and biologically. Our results show that HS binding is critical for HGF-stimulated cell motility, proliferation and tumor growth, and reveal a novel strategy for the design of potent and selective antagonists of HS-dependent oncogenic growth factor signaling pathways. Preliminary PK and toxicology studies suggest that 3M has a long half-life no detectable toxicity. Preliminary efficacy studies show complete blockade of experimental metastasis. Comprehensive toxicology have been conducted in mice and not abnormalities were found. Preliminary pharmacokinetic and dose-response efficacy studies have been performed and indicate a plasma half-life of >24h and potency in animals of 5 nM. More recent work in this area extends our findings to VEGF, another heparin binding growth factor with a critical role in cancer progression. We developed a triple-substituted form of VEGF that antagonizes VEGFR activation and tumor angiogenesis in animals. The overall structure of the Met receptor is that of a typical RTK, with an extracellular ligand binding domain, a transmembrane helix, and an intracellular kinase domain. The latter has the standard kinase fold, with an amino-terminal beta-sheet-containing lobe and a carboxyl-terminal helical lobe connected through a hinge region. The ATP binding site is in a deep, narrow, coin-slot-like cleft between the two lobes. In prior work a virtual screen of a 13 million compound chemical library was used to identify 175 Met TK inhibitor candidates with little structural similarity to known kinase inhibitors. These were tested in cell-free and intact-cell based assay systems, leading to the identification of three compounds that inhibited Met kinase activity at micromolar or submicromolar levels. The three lead compounds are now published and patented and available for further preclinical and clinical development thorugh collaboration and/or technology transfer. 3. Development of HGF/Met-related reagents and methods for diagnosis, pharmacodynamics, patient selection and molecular imaging. Many proteins are proteolytically released from the cell surface by a process known as ectodomain shedding. Shedding occurs under normal physiologic conditions and can be increased in certain pathologies. We hypothesized that Met overexpression in cancer might result in increased ectodomain shedding and that its measure could be a useful biomarker of tumor progression. We developed a sensitive two site electrochemiluminescent immunoassay to quantitate Met protein in cell lysates, culture supernatants and biological samples. A survey of cultured cell lines revealed a direct correlation between malignant potential and Met shedding rate. Plasma and urine samples from mice harboring subcutaneous human tumor xenografts displayed soluble human Met levels that correlated directly with tumor volume, suggesting that Met ectodomain shedding may provide a reliable and practical indicator of malignant potential and overall tumor burden. This work has recently extended to bladder cancer, where it was found that urinary shed Met ectodomain levels varied directly with tumor grade and stage, suggesting that testing for urinary shed Met could be useful to identify those bladder cancer patients with a greater likelihood of responding to inhibitors of the HGF/Met pathway. A series of collaborations to measure soluble Met (sMet) in human urine and plasma samples obtained from patients with RCC, prostate and bladder cancer, as well as other malignancies have been established to follow up to these findings. We have measured plasma levels of sMet and those of HGF, VEGF and soluble VEGFR2 (sVEGFR2) of patients in phase 1 and 2 clinical trials of GSK1363089, a TK inhibitor of VEGFR2 and Met (GSK089). In a phase 2 study of patients with advanced gastric treated cancer GSK089 on an intermittent dosing schedule, plasma sMET and VEGF-A increased during the treatment periods and declined during the drug holidays. Follow-up cell-based studies show that inhibition of Met kinase activity blocks Met internalization resulting in increased exposure to cell surface Met sheddase(s). Median tumor burden did not change significantly over the course of the gastric study, but significant positive correlations between plasma levels of sMET, VEGF-A and tumor burden (sum of longest diameters) were observed at baseline and week 8, the termination of PD studies. Thus, the observed changes in plasma sMet and VEGF-A levels in the gastric study are likely to be a biological response of target inhibition. In collaboration with the CCR Molecular Imaging Program (Dr. Peter Choyke) we have recently obtained the recombinantly engineered one-armed monoclonal anti-Met antibody OA-5D5 (MetMab), as well as Met TK inhibitors as ancillary reagents, from Genentech to develop MetMab for PET imaging. Initial studies confirm the feasibility of using 76B-MetMab for micro-PET imaging of Met-expressing human tumor xenografts in mice. Future comparative studies between 76B and 124I should provide improved signal-to-noise ratio and increased sensitivity of Met detection. In parallel, we have obtained Met ectodomain-selective peptides from GE Healthcare for development as optical imaging agents. Preliminary studies using various human bladder cancer cell lines implanted as orthotopic xenografts show increased tumor labeling in direct proportion to Met expression level.
Showing the most recent 10 out of 38 publications
