2004 MARF Grant Winners (9)

Richard Kornbluth - Veterans Medical Research Foundation, San Diego, CA

Title: IMMUNOTHERAPY WITH SOLUBLE, MULTIMERIC CD40L AND OTHER TNFSF LIGANDS

Precis: Immune clearance of mesotheliomas has been achieved in mouse models using CD40 ligand (CD40L), a member of the TNF superfamily (TNFSF) of ligands. A new technology for administering CD40L and other TNFSF as a soluble, multimeric protein will be optimized in the mouse mesothelioma model in anticipation of a clinical trial in humans.

Specific Aims: 1: To prepare expression plasmids encoding soluble, multimeric forms of TNFSF ligands, including pSP-D-CD40L, pSP-D-RANKL, pSP-D-CD27L/CD70, pSP-D-4- 1BBL, pSP-D-LIGHT, and pSP-D-GITRL. 2: To evaluate the effects of these soluble, multimeric TNFSF ligands in an established mesothelioma model in mice. Plasmid DNA for the soluble, multimeric TNFSF ligands will be tested both individually and in combinations. After peritumoral injection, the mice will be followed for tumor rejection, delayed tumor growth, and/or prolongation of survival.

I. David Goldman - Albert Einstein Cancer Center AE College of Medicine of Yeshiva University, Bronx, NY

Title: PEMETREXED: CELLULAR PHARMACOLOGY AND MECHANISMS OF RESISTANCE IN MESOTHELIOMA; ROLE OF METHYLTHIOADENOSINE PHOSPORYLASE

Precis: The proposed studies are directed to developing an understanding the basis for the cytotoxicity of pemetrexed in mesothelioma and the mechanisms by which this cancer develops resistance to this agent.

Specific Aims: 1. To characterize the membrane transport and polyglutamation of PMX in mesothelioma cell lines. 2. To develop and characterize mesothelioma cell lines with acquired resistance to PMX. 3. To assess the impact of methylthioadenosine phosphorylase on PMX activity in mesothelioma cell lines A. To assess PMX sensitivity in the NCI-H28 cell line that lacks MTAP expression and following restoration of MTAP function by transfection of the cDNA. B. To assess PMX sensitivity in the NCI-H2052 mesothelioma cell line with intact MTAP activity and the impact of silencing this gene by siRNA. To assess the impact of pharmacologic suppression of MTAP on PMX activity.

Bin Liu - University of California, San Francisco, San Francisco, CA

Title: DEFINING AND TARGETING MESOTHELIOMA-SPECIFIC INTERNALIZING CELL SURFACE RECEPTORS FOR NOVEL THERAPEUTICS

Precis: A panel of novel mesothelioma-specific, internalizing human monoclonal antibody will be identified through selection of phage antibody library on live mesothelioma cells. Targeted therapeutics will be developed based on the newly acquired ability to deliver payload to the interior of mesothelioma cells.

Specific Aims: 1: To identify and characterize a panel of mesothelioma-specific phage antibodies. We will select phage antibody library on mesothelioma cell lines as well as primary mesothelioma cells under conditions that allow receptor-mediated endocytosis. We will characterize selected phage antibodies with regards to binding specificity, internalization kinetics, and clinical relevance. Aim 2. To develop anti-mesothelioma therapy based on highly efficient payload delivery. (a) To construct immunoliposomes using a panel of mesothelioma-specific scFvs. (b) To deliver cytotoxic payloads specifically to mesothelioma cells.

Ravi Salgia - University of Chicago, Chicago, IL

Title: STUDIES OF C-MET IN MESOTHELIOMA

Precis: To determine the biological role of c-Met in mesothelioma. As well, determine the therapeutic targeting of c-Met.

Specific Aims: Specific Aim 1. Determine the levels of c-Met expression and its functional importance in mesothelioma cell lines and tumor tissue. Specific Aim 2. Determine the mutation or amplification of the c-Met gene and its functional importance in mesothelioma. Specifically determine if the mutation(s) alter cell growth and viability in response to HGF, alter cell survival/inhibition of apoptosis, alter transformation ability, and alter biochemical downstream signal transduction pathways. Specific Aim 3. Determine the in vitro effect of c-Met inhibition in mesothelioma by siRNA and selective small molecule inhibitors of c-Met.

Gregory Ottersen - The Ohio State University Research Foundation, Columbus, OH

Title: EPIGENETIC CHANGES IN MESOTHELIOMA

Precis: We will explore epigenetic/methylation profiles of malignant mesothelioma using Restriction Landmark Genomic Scanning (RLGS) to identify novel gene targets of methylation and amplification. We will then utilize the information and genes identified in this scanning technique to explore possible epigenetic therapy using in vitro cell culture models with mesothelioma cell lines.

Specific Aims: Aim 1: Characterize the methylation pattern in malignant mesothelioma using Restriction Landmark Genomic Scanning (RLGS). Objective 1: Establish RLGS profiles from mesothelioma samples procured through the Cooperative Human Tissue Network (CHTN). We will plan to generate profiles from 10 patients with malignant mesothelioma, examining the tumor and normal control from each patient. Objective 2: Analyze the data to determine the overall extent and patterns of aberrant methylation in mesothelioma. Particular attention in this analysis will be given to two stratifying factors: a) epithelioid and sarcomatoid histologic variants of mesothelioma and b) SV40 positive and negative mesothelioma. Objective 3: Altered RLGS fragments identified in objectives 1 and 2 will be prioritized for cloning and analysis based upon frequency of loss and differences noted between sarcomatoid and epithelioid variants as well as differences between SV40 positive and negative tumors. Aim 2: Investigate the ability of agents that alter DNA methylation and histone acetylation to induce changes in phenotype and change expression of novel methylated genes (identified in aim1) in vitro. Objective 1: The ability of DNMT inhibitors alone and in combination with Histone Deacetylase (HDAC) inhibitors to alter phenotype of mesothelioma cells in culture will be investigated. These phenotypic changes include cell cycle alterations, apoptosis, growth inhibition and sensitivity to chemotherapeutic agents. Objective 2: The effect of DNMT and HDAC inhibitors to alter expression of genes identified in Aim 1 will be analyzed in vitro. We will analyze DNA, RNA and protein changes using MS-PCR, COBRA analysis, RT-PCR, Real-time RT-PCR and Western immunoblot analysis.

Bernadette Scott - Monash Institute of Reproduction and Development, Clayton, VIC AUSTRALIA

Title: PRE-CLINICAL STUDIES OF A NOVEL THERAPEUTIC STRATEGY FOR MALIGNANT MESOTHELIOMA

Precis: Macrophages infiltrate many solid tumors, including malignant mesothelioma, and often aid in tumor growth by providing tumor-growth factors or by subverting the immune system. We propose that the modulation of their function or removal of these macrophages from the tumor will be beneficial in malignant mesothelioma and other solid cancers.

Specific Aims: 1. To determine the efficacy by which the depletion of tumor infiltrating macrophages prevents malignant mesothelioma growth and whether macrophage depletion causes regression of established tumors. 2.To determine whether embryonic-derived macrophages cells, transduced with anti-tumorigenic molecules, will traffic to tumors and lead to regression.

Paul Baas - Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, the Netherlands

Title: Malignant Mesothelioma and Thalidomide; a Translational Research Project.

Precis: Can our conditional mouse models with mesothelioma be used as a model to predict outcome of specific therapies in mesothelioma patients?

Specific Aims: Validate the mouse - mesothelioma model by comparing the mRNA expression and CGH Bac array profiles of the different murine mesotheliomas with those of human mesotheliomas. Determine the DLT for thalidomide in mice and compare the antitumor activity of thalidomide in mice with its activity in humans at comparable serum levels of thalidomide. Follow the effects of thalidomide on tumor growth both in vivo with luciferase imaging and on cell proliferation in vitro. Identify the characteristics of tumors that show a response when on thalidomide, and subsequently compare these characteristics in human mesotheliomas that either are responders or became resistant to thalidomide intervention during in vitro selection. Perform a detailed analysis of mouse tumors or available cell lines from these tumors that have become refractory to thalidomide to identify factors that confer resistance to Thalidomide.

Cecilia Camacho Hübner - St Bartholomew's Hospital, William Harvey Research Institute Queen Mary, University of London, London, UK

Title: Development of Novel Therapeutic Strategies for Malignant Pleural Mesothelioma: Targeting the IGF System

Precis: Malignant pleural mesotheliomas have resisted treatment with surgery, chemotherapy and prolonged radiotherapy. Insulin-like growth factors (IGF), their binding proteins and the IGF1 receptor have been implicated in cancer pathophysiology. This study will target the IGF system by using specific inhibitors of IGF1-R and determine their effect on mesothelioma cell growth and survival.

Specific Aims: To characterize the IGF system (genes and proteins) in malignant pleural mesotheliomas cell lines, in primary MPM tumor specimens and compare the profiles to normal human mesothelial cells (reference cells) in vitro. To evaluate the effect of agents that will block IGF action by either inhibiting the IGF-1 receptor or by altering IGFBP (i.e. rescuing the loss or absence of IGFBP-3) expression in MPM, which consequently will impair tumor cell proliferation and survival. ! To determine if pro-IGF-II is synthesized by different MPM cell lines in vitro and in isolated primary MPM cells and to further delineate its effect on MPM cell growth and survival.

Brad Black - Center for Asbestos Related Disease, Libby, MT

Title: SMRP SERUM LEVELS IN ASBESTOS-EXPOSED LIBBY MONTANA AREA RESIDENTS

Precis: SMRP levels will be measured in clients of the CARD in Libby Montana. The levels will define the range of values for exposed and non-exposed individuals in this cohort.

Specific Aims: 1 - To evaluate serum mesothelin-related protein (SMRP) as an early marker of MM. By 1.1) Defining the normal ranges for SMRP in serum for age and sex matched asbestos exposed and non-asbestos individuals in Libby, Montana using the newly formulated SMRP ELISA kit. 1.2) Validating thse "normal ranges" by defining sensititvity, specificity, and predictive values for SMRP in a blinded fashion. 1.3) Monitoring the stability of SMRP levels over a 12 month period in a high-risk for mesothelioma cohort.

*** POSTED APRIL 12, 2005 ***