Targeting Deregulated Apoptotic and Transcriptional Programs in Lymphoma

Dana-Farber Cancer Inst, Boston MA

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Abstract

The primary goal of this project is to employ chemical strategies for generating highly specific and stable compounds that preserve both the primary and secondary structure of biologically-active apoptotic and transcriptional peptides, in order to maximize their potential as biological tools to study and treat germinal center lymphomas. Peptide helices that engage protein-binding pockets hold promise as a novel set of prototype therapeutics for cancer. Intracellular BCL-2 family, BCL-6, and Max-Myc protein interactions constitute major sites of deregulated signaling in lympoma, yet have proven difficult targets for small molecule chemistry, often reflecting binding interfaces that are shallow, extensive, and complex. The research proposed here aims to apply synthetic approaches such as "hydrocarbon-stapling''to reinforce natural peptide ligands relevant to deregulated BCL-2, BCL-6 and Myc pathways, and thereby provide alternative tool compounds to study and manipulate protein interactions integral to lymphomagenesis. Specifically, we will generate "Stabilized Alpha-Helix of BCL-2 Domains" (SAHBs) and "Stabilized Alpha- Helices of Transcription" (SAHTs) corresponding to the essential interaction domains of the BCL-2, BCL-6 and Max proteins. Using circular dichroism, protease resistance assays, FACS analysis and confocal imaging, and fluorescence polarization binding assays, the secondary structure, chemical stability, cell permeability, and binding specificity of SAHBs/SAHTs will be determined, respectively. The biophysical and biochemical properties of SAHBs/SAHTs will be optimized by evaluating diversified hydrocarbon-stapled libraries synthesized in high-throughput fashion. SAHBs/SAHTs will be screened for apoptotic and growth arrest activities in transcriptionally-defined human lymphoma cell lines. The molecular mechanism of SAHB/SAHT activities will be confirmed using cell-based assays and molecular targets validated by intracellular photoaffinity labeling and mass spectrometry. Xenograft and transgenic models of lymphoma will be established for evaluation of developmental SAHB/SAHT therapeutics and the in vivo pharmacologic profiles of the compounds determined. The therapeutic efficacy of SAHB-induced apoptotic activity and SAHT-induced transcriptional modulation will be evaluated in murine models of lymphoma using in vivo imaging technologies. Thus, we propose a multidisciplinary approach that combines synthetic chemistry, cancer biology, clinical pharmacology, and radiologic and pathologic diagnosis to generate a "chemical toolbox" to investigate and neutralize the aberrant signaling pathways that drive lymphoma. Extensive research into the origin of lymphoma has led to the identification of genetic mistakes that trigger the overproduction of specific cancer-causing proteins. Our research focuses on chemically optimizing natural peptides that target these proteins in order to study and treat human lymphoma.