PROJECT SUMMARY Lung cancer is the leading cause of cancer-related deaths in both men and women in the U.S and worldwide. There are more than 400,000 deaths of lung cancer worldwide each year, among which lung squamous cell carcinoma (LUSC) accounts for about 30%. However, no effective treatments for LUSC are available. LUSC is one type of non-small cell lung cancer (NSCLC) characterized by numerous DNA alterations, including frequent amplification of the 3q26 chromosomal segment. The 3q26 segment is noteworthy because it contains the YEATS domain containing 2 (YEATS2) gene, a gene that is frequently amplified in a number of human cancers, including LUSC (~50%), ovarian (28%), head and neck (25%), and esophagus cancers (25%). High YEATS2 mRNA expression is associated with a poor prognosis of NSCLC patients, indicating that YEATS2 may have a tumor-promoting role. YEATS2 is a stoichiometric subunit of the Ada-Two-A-Containing (ATAC) complex, a conserved metazoan histone acetyltransferase (HAT) complex. YEATS2 contains an evolutionally conserved YEATS domain. We previously showed that the YEATS domain of YEATS2 functions as a reader of histone acetylation and other types of histone acylation such as crotonylation. Importantly, disrupting the YEATS histone reading activity impairs the normal functions of YEATS2 and the ATAC complex, resulting in reduced histone acetylation, decreased target gene expression, and inhibition of cell growth and survival of NSCLC. These data demonstrate that the YEATS domain of YEATS2 is a potential drug target, and that targeting YEATS2 may provide a therapeutic approach for treating NSCLC and other types of cancer characterized by YEATS2 amplification. The objective of this proposal is to develop potent and specific inhibitors targeting the histone acylation binding activity of the YEATS domain of YEATS2. For this, we will (1) conduct a high-throughput screen to identify YEATS2 small-molecule inhibitors, and (2) evaluate and characterize hit compounds in in vitro and cell-based assays. Through the proposed studies, we expect to identify potent, specific YEATS2 YEATS domain chemical probes of different chemotypes. These compounds will provide the basis for further development of small molecules for targeted therapies. Likewise, the research community will be able to use these new inhibitors as important tools to understand the functions and mechanisms of YEATS2 in human cancers.