Summary of Top Scoring Grant Proposals

Dr. Mitchell Ho [ In Honor of Craig Kozicki]

National Cancer Institute, NIH

Title: Targeting Mesothelin by Human Antibodies for Mesothelioma Treatment

Specific Aim: Aim 1: Determine the CA125 functional binding domain in mesothelin. Based on the results that will be presented under Preliminary Studies, the working hypothesis here is that CA125 binds a site located at the N-terminal of mature membrane-bound mesothelin. Antibodies with high affinity for the CA125-binding site will inhibit the interaction of mesothelin with CA125. 
Aim 2: Identify human Fv inhibiting the interaction of mesothelin and CA125. As shown under Preliminary studies, we have successfully identified a number of human antibody Fv specific for human mesothelin and cross-reactive for mouse mesothelin. Several human Fv antibodies bind the same region of the CA125-binding site in mesothelin and may likely inhibit the interaction of mesothelin and CA125. Such antibodies will be further improved by innovative hotspot mutagenesis and mammalian cell display on human HEK 293T cells. We will test the selected human antibodies in heterotypic cell adhesion and inhibition assays by flow cytometry. 
Aim 3: Inhibit the peritoneal metastasis of mesothelioma in a mouse model. We postulate that human antibodies blocking the mesothelin-CA125 interaction and cross-reactive for mouse mesothelin will inhibit peritoneal metastasis of CA125-positve human mesothelioma in an intraperitoneal xenograft model in nude mice. The data generated in the MARF proposal will be used as a foundation to develop a new program studying antibody therapy to treat mesothelioma.

Lay Description: Mesothelin and CA125 are highly expressed in most malignant mesotheliomas. Their interaction plays an important role in peritoneal tumorigenesis. We aim to generate a panel of human monoclonal antibodies that will effectively neutralize human and mouse mesothelins, blocking the mesothelin-CA125 interaction in vitro and inhibiting tumor growth in a peritoneal mesothelioma mouse model.

Dr. Robert Kratzke [Christopher Stoeckler Memorial Grant]

University of Minnesota

Title: Targeting Cap-Mediated Translation for Mesothelioma Therapy

Specific Aim: Aim 1: Test a novel small molecule inhibitor of cap-mediated translation for reversion of increased cell proliferation, aberrant translational activity, and eIF4E cap-binding in mesothelioma cells. 
Aim 2: Analyze expression of previously identified biomarker proteins preferentially enhanced in mesothelioma cells following administration of the novel small molecule inhibitor of eIF4E mediated activity. 
Aim 3: Demonstrate that small molecule inhibitors of cap-mediated translation will inhibit both translation and proliferation in cultured human mesothelioma cells, as well as enhance chemosensitivity.

Lay Summary: The cellular processes that regulate the synthesis of proteins are disrupted in mesothelioma resulting in the unregulated production of cancer related proteins. The restoration of this process to its normal state results in the inhibition of mesothelioma. We are developing a new class of chemotherapy drugs that restore regulation of protein synthesis and reverse mesothelioma. This study will test the capability of this new class of treatments to halt mesothelioma and increase chemotherapeutic sensitivity.

Dr. Ravi Salgia [Jeffrey P. Hayes Memorial Grant]

University of Chicago

Title: Protein Kinase C as a Novel Therapeutic Target in Mesothelioma

Specific Aims: Aim 1: Determine the expression, amplification and mutation status of protein kinase C beta (PKCß) in MPM. Correlate these variables with clinical parameters, and arrive at useful prognostic information in MPM. Hypothesis: PKCβ will be overexpressed in MPM, and will correlate with clnical outcome. We will also be able to identify unique “gain-of-function” single nucleotide polymorphisms (SNPs)/mutations in a subset of MPM. Finally, there will also be certain subsets of MPM with amplification. 
Aim 2: Determine the inhibition of PKCß in MPM cell lines, and in combination with cytotoxic chemotherapies. Determine biological and biochemical functions with respect to PKC (stimulation and inhibition). Hypothesis: There will be specific inhibition of PKCβ in MPM cell lines, and there will be subsets of MPM cell lines that will respond greater to PKCβ inhibition (such as amplified cells as well as those harboring SNPs/mutations). There will be synergism with therapies such as cisplatin and pemetrexed with PKCβ inhibition. The inhibition will effect not only cell survival/cell growth, and cell motility/migration, but also downstream signaling through various molecules including the focal adhesion proteins. 
Aim 3: Determine the activation and inhibition of PKCß in C. elegans, and develop the C. elegans system as a high throughput model for MPM. Hypothesis: C. elegans will serve as a novel high-throughput screen for: 1. Determining the function of PKCβ (wild-type and variant); 2. Determining the effects of asbestos fibers on the function of PKCβ; 3. Determining the effects of PKCβ inhibition.

Lay Summary: Malignant mesothelioma is a cancer of the pleura, which has a poor prognosis and few therapeutic options. Our research focuses on identifying drugs that may significantly improve quality of life as well as patient survival. Protein kinase C is a molecule known to cause cells to be more invasive and to divide faster, and we have shown that this molecule is present in human mesothelioma. We propose to target this molecule in mesothelioma cells with a promising new drug, enzastaurin, which specifically acts upon it. Our plan is to test the biology and response of tumor cells to drugs in isolation and in a microscopic worm model (C. Elegans). C. elegans is a novel model system which is able to produce answers to our scientific questions in a much shorter time span and at lower cost. Our aim is to translate our findings into useful new insights for the treatment of this devastating disease.

Dr. Ramasamy Jagadeeswaren

University of Chicago

Title: Paxillin is a Potential Molecular Therapeutic Target in Malignant Pleural Mesothelioma (MPM)

Specific Aims: In order to have a significant impact on the therapy for mesothelioma, novel targets have to be identified. Biologically, mesothelioma cells migrate and tend to invade quite frequently, and mechanisms can involve the cytoskeleton. We have recently identified that the paxillin is an important cytoskeletal protein in MPM. We have previously shown that c-Met receptor is an important in MPM, and now show that a significant downstream target for Met activation is paxillin. Since c-Met is a known oncogene that contributes to mesothelioma cell proliferation and invasion, it is also possible that a cytoskeletal protein like paxillin mediates some of the functions of c-Met (proliferation, invasion, and angiogenesis). Our hypothesis is that paxillin is an important therapeutic target in MPM. Our goal is to arrive at effective novel targeted therapy for MPM that will improve the survival from this devastating illness.

Lay Summary: Targeting paxillin is likely to be more effective than targeting a single receptor tyrosine kinase. Importantly, we believe paxillin will ultimately become a potential therapeutic target and the studies proposed in this award will give us a novel target against this devastating illness. The proposed work will certainly define the role of paxillin mutations and amplification in mesothelioma and aid in the development of novel therapeutics, and thus highly innovative.

Dr. David I. Goldman

Albert Einstein Collge of Medicine of Yeshiva University

Title: The Role of the Proton-Coupled Folate Transporter (PCFT) as a Determinant of the Activity of Pemetrexed in Mesothelioma

Specific Aims: Aim 1: To determine the expression of the proton-coupled folate transporter (PCFT) in fresh-frozen human mesothelioma tissues by quantitative RT-PCR and immunohistochemical analysis. In parallel studies, expression of folate receptor-α, the reduced folate carrier, folylpolyglutamate synthetase, thymidylate synthase, glycinamide ribonucleotide transformylase will also be assessed by RT-PCR.
Aim 2: To assess the impact of PCFT over-expression on pemetrexed activity in mesothelioma cell lines.
Aim 3: To determine the impact of the loss of PCFT expression by siRNA on pemetrexed activity in mesothelioma cell lines in which RFC is, and is not, functional.
Aim 4: To dissect the mechanisms that account for why PCFT is highly expressed in some, but only modestly expressed in other tumor cell lines and tissues.

Lay Summary: This proposal is based upon the recent discovery in this laboratory of a novel transport system (PCFT), present in most human cancers, that drives pemetrexed into tumor cells. The aims of this proposal are to assess the extent to which PCFT and other transporters are present in human mesothelioma specimens and to determine the impact of PCFT relative to other transporters on the antitumor activity of pemetrexed when PCFT is generically engineered into mesothelioma cell lines.

Dr. Alain Borczuk

Columbia University Medical Center

Title: Poor Prognosis Gene Expression Signatures Suggest Molecular Pathways of Mesothelioma Progression and Potential Therapeutic Targets.

Specific Aims: None Submitted

Lay Summary: Abdominal malignant mesothelioma (AMM) is a subtype of mesothelioma that involves intra-abdominal surfaces. Several factors are associated with increased chance of therapeutic success and prolonged survival. Using gene expression profiling, our prior work identified factors in AMM that were predictive of poor prognosis that have been confirmed by other groups in the more common pleural mesothelioma. We have subsequently determined a 31-gene signature that segregates AMM into favorable prognosis and poor prognosis subgroups. The proposal seeks to validate this signature on an independent test group of AMM and pleural mesothelioma and to further study pathways of aggressive AMM. The function of one of the genes (called EZH2) found to be increased in the poor prognosis AMM suggests a therapeutic strategy in patients with aggressive AMM.

Dr. Sergey Ivanov

NYU School of Medicine

Title: ROMA for the Identification of Genes Associated with Early Stages and Progression of Asbestos-Induced Pleural Malignant Mesothelioma

Specific Aims: Aim 1: Perform Affymetrix expression array analysis on 50 mesothelioma specimens and 10 normal tissue controls (60 specimens in total) for identification of genes differentially expressed in two groups of patients with different recurrence terms and in tumors versus controls. Aim 2: Assess clinical importance of genes selected by both ROMA and Affymetrix studies via correlation of their expression with recurrence, asbestos exposure, smoking status, tumor histology, stage, and age. Aim 3: Validate tumorigenic potential of the genes selected in specific aim B using functional assays of proliferation, adhesion, migration, and invasion.

Lay Summary: We studied the genetic history of asbestos-induced mesothelioma using a novel technology (ROMA) and computational approaches. The critical chromosomal regions and hundreds of genes contained within were identified as a result. We are planning now to separate mesothelioma-associated genes from “by-standers” through global analysis of gene activity (expression array study). The genes selected by both methods will then be thoroughly tested in functional assays to identify therapeutically important targets.

Dr. Adi Gazdar

University of Texas Southwestern Medical Center

Title: Establishment of Immortalized Mesothelial Cell Lines: A Unique Resource for the Study of Mesothelioma

Specific Aims: Aim 1: Immortalize a further five mesothelial cell cultures from patients with benign pleural effusions without the use of viral oncogenes. One such cell line already exists. We will introduce Cdk4 and hTERT into short term cultures in order to immortalize them. Data from mesothelial and bronchial epithelial cells immortalized by such means indicate that the cells become permanent cultures and have minimal genetic changes compared to tumor lines.
Aim 2: Characterize the cells so established. We will perform comparative genomic hybridization (for total genomic profiling) as well as study gene expression for markers used to separate mesothelial from epithelial cells by qPCR and immunostains. These markers include high molecular weight cytokeratins, vimentin, CEA, B72.1, Ber-Ep4, calretinin, WT1, osteopontin and mesothelin. In addition, DNA fingerprinting and mycoplasma testing will be performed.
Aim 3: Distribute the cells to the scientific community. We will advertise the availability of the cultures and their characterization on our web site. We will deposit the cells in public cell depositories. We will also announce their availability in publications and presentations. They will be freely available to the scientific community, although we may ask for courier charges and a small handling fee. We have assembled an outstanding team of scientists/clinicians uniquely qualified to

Lay Summary: Mesothelioma research has been hampered by the lack of suitable laboratory systems to study the development of mesothelioma. Normal mesothelial cells survive for relatively short periods of time and are not suitable for studying the lengthy tumor process which takes several years. We have developed a simple method to immortalize normal mesothelial cells while retaining their basic genomic properties. We will initiate several such cultures and distribute them freely to the scientific community. These reagents will be unique and invaluable for the study of mesothelioma development

Dr. Marc de Perrot

University of Toronto

Title: Development of a Novel Therapeutic Strategy with Pemetrexed and Regulatory T cell Depletion for Malignant Pleural Mesothelioma: A Translational Research Project

Specific Aims: We propose that the depletion of CD4+CD25+ T cells at the time of chemotherapy with pemetrexed changes the tumor microenvironment of established tumor in a mouse model of intrapleural mesothelioma by switching the tumor macrophages and CD4+ tumor infiltrating lymphocytes from an anti-inflammatory phenotype (M2 and Th2) towards a pro—inflammatory phenotype (M1 and Th1) with the release of pro-inflammatory cytokines that enable recruitment and activation of CD8+ cytotoxic T cells within the tumor.

Lay Summary: Immunotherapy is a promising option for the treatment of mesothelioma. Our preliminary data in a mice model of intrapleural mesothelioma suggests that the combination of chemotherapy and immunotherapy with depletion of regulatory T cells can have a synergistic impact on outcome. These results may offer new therapeutic avenues in the treatment of this devastating tumor.

Dr. Carmen J. Marsit

Brown University, Division of Biology and Medicine

Title: Array-Based miRNA Expression and Methylation Profiling of Normal and Tumorigenic Pleural Mesothelium

Specific Aims: 1. Examine miRNA expression profile of human normal pleural mesothelium, and tumorigenic mesothelium using an array-based approach that can simultaneously examine over 325 human miRNAs.

a. Test the hypothesis that normal pleura and tumorigenic mesothelium have distinct miRNA

expression profiles.

b. Test the hypothesis that miRNA expression profiles are predictive of tumor histology, asbestos

exposure, and survival.

2. Examine the promoter CpG hypermethylation profile of normal pleura and tumorigenic mesothelium using a novel bead-array based technology that can simultaneously examine >800 cancer-related gene promoters to determine their methylation status.

Lay Summary: This research hopes to discover novel, specific, diagnostic and prognostic biomarkers of disease and will greatly enhance an ongoing study. In addition, this project aims to describe new markers of disease that may translate to novel treatments, more peronalized medicine, and improved patient outcomes.