Targeting Actin-like 6A (ACTL6A) for Ewing Sarcoma (EwS) Treatment
Ewing sarcoma (EwS), the second most common bone cancer in children and adolescents, represents 16% of primary bone sarcoma. Over 80% of EwS is induced by the expression of fusion protein EWSR1-FLI1. EWSR1-FLI1 leads to EwS development by binding to chromatin, re-programming epigenetic status, and thereby activating oncogenic gene expression. However, the epigenetic regulators that facilitate EWS-FLI1-mediated epigenetic changes are not yet examined. To understand the epigenetic regulation in EwS, I developed an epigenetic regulator-focused CRISPR screening which targets 728 epigenetic regulators in the human genome. I found RuvB-like AAA ATPase 1 (RUVBL1) and actin-like 6A (ACTL6A) as essential regulators for EwS growth. Focusing on RUVBL1, I examined how RUVBL1 regulates EwS growth and proposed a combinatorial therapeutic strategy for EwS treatment. A manuscript describing these findings is under review at Cancer Discovery. Focusing on ACTL6A, I have found that ACTL6A is required for acetylation of H3K27 (H3K27ac) and methylation of H3K4 (H3K4me3), two epigenetic marks where fusion protein EWSR1-FLI1 binds. From an RNA-seq-based transcriptomics analysis, I found that ACTL6A inhibits the function of EWSR1-FLI1 in repressing gene expression and thus cell differentiation. Further, I found that, compared to other types of cancer, EwS has the strongest dependence on ACTL6A, indicating that characterizing how ACTL6A mediates cancer growth in EwS has the highest significance. To develop drugs to target ACTL6A, I used a high-density CRISPR gene body screening, in which the entire coding region was probed to identify important domains for its function. I found that the C-terminal region of ACTL6A forms a pocket to bind to its interacting proteins. This provided sites for the binding of small molecules and thereby inhibiting ACTL6A function. To further examine the roles of ACTL6A in mediating EwS growth and to develop therapeutic strategies by targeting ACTL6A, this proposed research aims to: 1. Using ChIP-seq, determine genes that are transcriptionally induced by ACTL6A directly and by ACTL6A-mediated H3K27ac/H3K4me3 indirectly. 2. Examine downstream regulators of ACTL6A using a secondary CRISPR screening that targets the entire human genome. 3. Study how ACTL6A represses EWSR1-FLI1 function and cell differentiation, and test the direct interaction between ACTL6A and EWSR1-FLI1. If no direct interaction exists, I will study how ACTL6A regulates EWSR1-FLI1-mediated gene expression using ChIP-seq. 4. Develop inhibitors targeting ACTL6A using a computational method (compounds virtual screening). I will use AutoDock to dock the repository available from the Division of Cancer Treatment and Diagnosis from the NIH, which contains more than 100,000 compounds. Collectively, I expect to provide a better understanding of epigenetic regulation in EwS, define the roles of ACTL6A for EwS treatment, and provide therapy strategies to treat EwS patients.