The MLL (Mixed Lineage Leukemia) gene codes for a histone methyltransferase, which functions as a transcriptional epigenetic regulatory factor for diverse subordinate genes of developmental and oncogenic importance. It is mutated by chromosomal translocations that result in the creation of chimeric fusion proteins whose gain of function is critically involved in the pathogenesis of a poor prognosis subset of acute leukemia. Recently, MLL oncoproteins have been shown to associate with menin, a product of the MEN1 tumor suppressor gene. Genetic analyses revealed that menin is required for MLL-mediated transcription and transformation, thus demonstrating an unusual and unprecedented role for a tumor suppressor protein serving as an essential cofactor for an oncoprotein in cellular transformation. The MLL- menin interaction is critically dependent on a 5 amino acid high affinity menin-binding motif, which is retained in all oncogenic forms of MLL, and therefore constitutes a potential target for pharmacologic manipulation of MLL function and possible therapeutic intervention. The overall objectives of the studies proposed in this application are to establish methodologies to isolate small molecules that specifically inhibit the MLL-menin interaction. In the first specific aim, a fluorescence polarization assay will be developed that is capable of quantitatively measuring recombinant menin binding in solution to a fluorescently labeled peptide corresponding to the MLL consensus binding sequence. In the second specific aim, the MLL-menin blocking assay will be configured and/or optimized for high throughput screening. Biochemical and cellular assays will also be devised and optimized for establishing the specificity and efficacy of small molecule inhibitors of MLL-menin interaction. A preliminary screen through a small molecule library of 1,280 compounds (LOPAC, Sigma) in a quantitative high throughput format will help identify technical problems and confirm the feasibility of a larger scale high throughput screen. Compounds that are capable of specifically interfering with MLL-menin interaction will provide valuable reagents for studying the unusual functional cooperative roles of these proteins in transcriptional epigenetic regulation and oncogenesis, and provide lead compounds for potential rational drug design in poor prognosis leukemia. Acute leukemias are an important cause of morbidity and mortality, and their effects are particularly significant because they often affect children and young adults. Despite improvements in therapy and outcome in recent years, the prognosis for long-term survival in most leukemia patients remains poor (1-4). Thus, there is an urgent need for novel therapeutic modalities based on rational drug design targeting the molecular abnormalities that underlie leukemia pathogenesis. [unreadable] [unreadable] [unreadable]