The long-term objective of this Program Project Grant application is to advance our basic understanding of the epigenetic regulation of gene expression in cancer cells and to rapidly translate the basic discovery of the molecular mechanisms into clinical trials in patients with chronic lymphocytic leukemia (CLL). The Program consists of five highly interactive and interdependent Projects supported by three Cores. It is based on the hypothesis that aberrant methylation of specific genes plays a key role in tumor development and that reactivation of these genes can be used as an effective means to clinically arrest tumor growth. Project 1 focuses on early therapeutic trials using agents that target epigenetic modifications, and incorporate detailed pharmacokinetic and pharmacodynamic studies with the ultimate goal of re-activating genes suppressed in CLL. The development of this project is heavily dependent on the determination of target genes (Project 2 and 3), identification of histone modifications (Project 4) and importance of chromatin remodeling complexes in mediating the biologic effect of the proposed epigenetically targeted therapies. (Project 5). Project 2 is aimed at identification of novel genes in genetic subtypes of CLL silenced by methylation, correlation of the methylation profile of selected genes with clinical parameters, detailed study of the function of selected genes based upon potential importance to the pathogenesis and progression of CLL, and re-activation of these genes in CLL (interaction with Project 1). Project 3 will explore the methylation and suppression of the receptor-type protein tyrosine phosphatase (PTPRO) in CLL, PTPRO promoter methylation and reduced expression in genetic subtypes of CLL, the novel tumor suppressor function of PTPRO, elucidation of the molecular mechanisms by which DNA methylation of this gene leads to its suppression, and its re-activation in CLL (interaction with Project 1). Project 4 will focus on identification of novel sites of post-translational modifications on core histones by sensitive mass spectrometry, specific histone modifications that are associated with cancer, and characterization of the changes in histone modifications that result from the treatment of CLL patients with agents that target chromatin structure (interaction with Project 1). Project 5 will focus on biochemical characterization of the activity and substrate specificity of the Brg1 and hBrm-associated PRMT5, identification of common target genes regulated by PRMTS-containing Brg1 and hBrm chromatin remodeling complexes in CLL before and after treatment with the agents used in clinical trials (interaction with Project 1). In addition to the interdependence of all projects, the synergistic and interactive elements of the projects are also reflected in but not limited to the common use of a) reagents, b) well characterized primary CLL samples, and c) techniques & technologies (Restriction Landmark Genomic Sequencing, Bisulfite genomic sequencing, chIP assays, Real-time PCR) in order to accomplish the common goal of real-time translational research. ?
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