Genetic Modifiers of the Anti-apoptotic Functions of Bcl-2

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This proposal is designed to provide the candidate a period of mentored research in the laboratory of Dr. A. T. Look, a pioneer in the field of hematopoietic malignancies and the use of zebrafish as a model system to study these diseases. The goal of this application is to use the zebrafish as a model to study novel genes and pathways necessary for the anti-apoptotic functions of Bcl-2, a gene whose aberrant expression is highly associated with follicular B-cell lymphoma and other hematological malignancies. The zebrafish, with its close synteny to the human genome and its conserved molecular pathways regulating the development of tissues and organs, offers a powerfultool with which to conduct such research. A transgenic zebrafish line that specifically expresses an EGFP-tagged zbcl-2 fusion protein in lymphoid cells has been employed in a genetic modifier screen, and the candidate has identified four zebrafish mutant lines that demonstrate suppression of the anti-apoptotic functions of Bcl-2 in vivo. The underlying hypothesis is that knowledge of the zebrafish genes able to suppress Bcl-2-mediated apoptosis in lymphoid cells with damaged DMAwill implicate novel pathways through which human BCL-2 exerts its anti-apoptotic activity in cancer cells. In Aim 1,the mutated genes will be mapped and positionally cloned. Mutations will be analyzed using a rag2- EGFP-mMyc zebrafish model of T-ALL to determine their ability to resensitize Bcl-2-expressing leukemic T cells to radiation-induced cell death. In Aim 2, functional and molecular analyses will be used to investigate the mechanisms by which each mutant gene suppresses the function of Bcl-2. The long-term goal of this application is to restore normal cell death pathways in B-cell follicular lymphoma and other hematological cancers by targeting pivotal molecules with small molecule inhibitors or antibodies. As part of their regulated life cycle, normal cells undergo programmed cell death (apoptosis) in response to DNA damage. Cancer cells derived from B-cell follicular lymphoma, as well as many other blood diseases, have aberrantly high levels of Bcl-2, a protein that causes cell survival in the face of apoptosis- inducing cancer therapies. The goal of this application is to use the zebrafish model system to identify targets for small molecule inhibitors of Bcl-2 function in order to restore normal cell death pathways in cancer cells.