Targeting Bcl-x splicing for cancer treatment

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DESCRIPTION (provided by applicant): Progress has been made during the last two decades on treatment of cancers, however, there is still no effective approach for their cure. Many oncogenes as well as tumor suppressors have been identified. One class of these proteins forms a Bcl-2 protein family. Recent studies have shown that one of the Bcl-2 family member, Bcl-x is alternatively spliced in exon2. Alternative splicing of this exon generates isoforms either anti-apoptotic (L form) or apoptotic (S form), suggesting that regulation of L/S forms of Bcl-x has implication for cancer therapy. The goal of this application is to develop a cell-based assay for identification of biological molecules/drugs that modulate exon 2 splicing through usage of an alternative splicing site within exon 2 so that the ratio of Bcl-xL and Bcl-xS, and activities of Bcl-x can be regulated. These molecules are potentially used to reduce anti-apoptotic form of Bcl-xL in tumors, therefore treating cancers. The first aim of this project is to generate mini-gene constructs for Bcl-x splicing studies with a reporter gene luciferase attached so that efficiency of alternative usage of a splicing site residing within exon 2 of Bcl-x pre-mRNA can be measured by luciferase activity. The mini-gene constructs will be validated by RT-PCR and then stable cell lines will be established. The second aim of this project is to optimize stable cell lines and to identify biological reagents/small molecules that reduce production of Bcl-xL while increase the production of the Bcl-xS isoform in a low throughput setting (LTS) from libraries of small molecules. The compounds will be tested for activity on apoptotic cell death in cell lines such as PCS cells, a cell line that expresses an abnormal high level of anti-apoptotic Bcl-xL. Mechanisms of action of these compounds on regulation of exon2 alternative splicing will be investigated as a long-term goal. Relevance of this research to public health: Bcl-xL, the large form of Bcl-x protein that is elevated in many tumors, protects cells from chemotherapy. We plan in this project to establish a cell-based system for identification of drugs that reduce Bcl-xL. These compounds may be potentially used for cancer treatment with combination of other chemotherapies.