Accumulating evidence suggests that epigenetic pathways play an integral role in the sequential progression of cancer. It is shown that many epigenetic enzymes are targeted for mutation and deregulation in cancer patients. Inhibitors of several key epigenetic enzymes including DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) have proven to be instrumental for both basic understanding of the function of the respective activity of the enzymes and applications in cancer epigenetic therapy. However, efforts in development of inhibitors for histone methyltransferase inhibitors are still at its infancy. The overall goal of this project is to develop potent and selective inhibitors for histone methyltransferase MLL3. Furthermore, we will also develop a series of facile, sensitive and reproducible high-throughput screening assays ready to identify potent, selective inhibitors for other histone methyltransferase (HMT) activity. There are at least six MLL family HMTs. MLL proteins use the SAM cofactor to methylate histone H3 lysine residue 4 and work in concert with other histone modification enzymes to provide a mechanistic link between chromatin alteration and gene activation; and regulate different biological processes through recruitment of various H3K4 'reader' proteins. MLL mutations and dys-regulation have been correlated with several human diseases including various cancers (leukemia, lymphoma, colorectal, gastric and lung), obesity and human intelligent deficiency. Because of the interesting biology that is mediated by MLLs and their involvement to human diseases, it is of interest to develop novel small molecule inhibitors that might serve as biological probes as well as lead molecules for drug development. For this application, we will focus our screening efforts on the MLL3 protein, although the assay should be generally applicable to other MLL family HMTs. The specific aims of the proposal are to (1) Develop a high-throughput screen and counter screen for MLL3 inhibitors, (2) Develop an secondary assay to quantify the potency and to biochemically characterize candidate hit compounds identified in high-throughput screens, and (3) Assess compound on-target specificity in cellular assays and genome-wide studies. We expect these studies to define biochemical and cell based assays to screen large libraries of small molecules to identify potent and selective inhibitors of MLL3 histone methyltransferase, and to obtain some pan-MLL inhibitors that target global H3K4 methylation. We anticipate that these studies will result in the identification of potent and selective first-generation MLL3 inhibitors for further development as chemical probes.