Investigations of Personalized Medicine in Mesothelioma

Malignant mesothelioma is a neoplasm that involves lesions on the pleural linings of the lung or the peritoneal lining of the abdominal cavity. Current standard of care involves a multi-targeted antifolate drug Pemetrexid and an alylating agent, Cisplatin. This regimen results in regression of tumors in 25% of patients. As the genetics lesions that cause mesothelioma have been elucidated, our understanding of possible targets of therapeutics has grown. Rational drug design (personalized medicine) would dictate that we consider genetic alterations specific to mesothelioma tumor cells and use those to target our therapy. The most common genetic alterations in human mesotheliomas are the loss of function of three tumor suppressor genes, PTEN, NF2, and CDKN2A.In this project, we propose to induce genomic deletion of the PTEN locus by using CRISPR/Cas9 nuclease-generating lentiviral vectors targeting this site in a “normal” mesothelial cell model, LP9/hTert. As we complete the establishment of this model system with various genomic alterations, the model system will be used to test a small set of frontline chemotherapeutic agents for any increased therapeutic index, the ratio of tumor cell killing to toxicity of normal cells. Theoretically, genes and proteins downstream of these genetic lesions will make some cancer cells more susceptible to specific inhibitors. Among the drugs to be tested are novel PI3Kinase and mTOR inhibitors. Our hypothesis is that PTEN knockout by CRISPR/Cas9 editing will result in an increase in sensitivity of suppressed cells to novel inhibitors directed at the pathways affected. To confirm genomic alteration, sequencing of the affected locus has been completed, other assays are planned to complete the confirmation of PTEN knockout. These include, quantitative PCR of PTEN mRNA, Immunoblotting to assess protein status, and functional analysis of PTEN function