PROJECT SUMMARY The histone methyltransferase (HTMase) EZH2 was proved to drive malignant phenotypes in various types of cancer cells. Therefore, selective small-molecule inhibitors of EZH2 have been developed, all of which block its enzymatic activity. However, accumulating evidence suggests that HMTase activity of EZH2, which is executed by the C-terminal catalytic SET domain, may not represent an ideal target for anticancer drugs in certain situations. First, multiple genetic studies suggest that the HMTase activity of EZH2 plays a tumor suppressive role in some types of cancer. Second, EZH2 may promote tumorigenesis and cancer progression independent of its HMTase competence. These scientific discoveries explain why some cancer cells that require EZH2 for their tumorgenicity are insensitive to current EZH2 inhibitors and why secondary malignances were sometimes noted in patients receiving tazemetostat, the first FDA-approved HMTase inhibitor of EZH2. All these clinical failures warrant the development of new EZH2 inhibitors that may exhibit stronger antitumor potency, fewer side effects and wider clinical applications. Recently, an N-terminal transactivation domain (TAD) was found to mediate the HMTase-independent tumor-promoting effect of EZH2. In estrogen receptor a-negative (ERa-) breast cancer cells, we demonstrated that cell growth was unaffected upon tazemetostat treatment or overexpression of SET-deletion mutant EZH2 but was dramatically hindered when the TAD domain was truncated. More importantly, a group of genes that are essential for tumor migration was specifically activated by the TAD domain, but not regulated by the SET domain. All these results suggest that this particular N-terminal TAD domain, rather than the enzymatic activity that current EZH2 inhibitors target, is required for the oncogenic function of EZH2 in ERa- breast cancer cells. Based on these findings, we carried out a high-throughput screening for chemicals that specifically interact with this TAD domain and a candidate compound was thus identified. Computer-aided structural analysis clearly showed that this prototype compound binds to a cleft located at the center of the TAD domain. Most importantly, it significantly abrogated the growth of ERa- breast cancer cells that are insensitive to present HMTase inhibitors of EZH2. In the proposed research, we aim to develop a new class of EZH2 inhibitors that target the N- terminal TAD domain and to further characterize their chemical and biological features. Under the instructions of Co-Investigator Dr. Daohong Zhou and Collaborator Dr. Xin Liu, we will study the structure-activity relationship of our lead compound and further build its binding mode with the TAD domain. We will also work with Consultant Dr. Stanton McHardy to design and synthesize the analogs of the prototype compound. Finally, we will evaluate the efficacy and elucidate the mechanism of action of these compounds in normal and cancerous human breast e...