Development of a new class of EZH2 inhibitors in ERa-negative breast cancer.

PROJECT SUMMARY/ABSTRACT The histone methyltransferase (HMTase) EZH2 was proved to drive malignant phenotypes in various types of cancer cells, including the estrogen receptor a-negative (ERa-) breast cancer. 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 blocking the HMTase-independent oncogenic function of EZH2, which may exhibit stronger antitumor potency, fewer side effects and wider clinical applications.

Recently, an N-terminal transactivation domain (TAD) was found to mediate the tumor-promoting effect of EZH2 via gene activation instead of the HMTase activity. In ERa- breast cancer cells, we demonstrated that malignant phenotypes such as proliferation and migration were unaffected upon tazemetostat treatment or

overexpression of the SET domain-deleted EZH2 but dramatically hindered when the TAD domain was truncated or when the critical residues for the proper protein folding of the TAD domain were mutated. More importantly, a group of genes that are essential for tumor migration was specifically activated by the functional TAD domain,

but not regulated by the SET domain. All these results suggest that this particular N-terminal TAD domain, rather than the methyltransferase activity that current EZH2 inhibitors block, 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. The lead compound directly binds to the TAD domain of EZH2, downregulates genes that promote cancer cell migration and abrogates the aggressive phenotypes of ERa- breast cancer cells without interfering

with the methyltransferase activity of EZH2. Computer-aided structural analysis suggests that the critical residues essential for the structure and functionality of the TAD domain may directly interact with this prototype compound. Based on these preliminary data, 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 in the proposed study. Under the instructions of Co-Investigator Dr. Daohong Zhou and Collaborator Dr. Yaxia Yuan, 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 carry out the pharmacokinetics studies of the new EZH2 inhibitors with the assistance of Consultant Dr. Nicholas Clanton, evaluate their efficacy and elucidate the

mechanism of action of these compounds in ERa- breast cancer cells as well as in xenograft mouse models of breast cancer in vivo by RNA-seq, ChIP-seq and proteomics analyses, which will be performed by Collaborator Dr. Zhao Zhang. Altogether, this study will yield first-in-class anticancer agents that target a different functional domain of

EZH2 to inhibit its oncogenic function in certain types of cancer cells that do not respond to current EZH2- targeting drugs.