Abstract 2925: Targeting histone acetyltransferase (HAT) function for synthetic cytotoxicity in CREBBP/EP300 mutant tumors
Authors: Pickering Curtis , Kumar Manish , Bridges Kathleen , Xie Tongxin , Molkentine David , Sheth Aakash , Yang Liang , Frederick Mitchell , Heffernan Tim , Seth Sahil , Myers Jeffrey , Skinner Heath D. 
Published: August 2019

CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300) share significant homology and are collectively mutated in ~14% of head and neck cancers (HNSCC). Recently we have identified CREBBP/EP300 mutations being associated with poor outcome. Thus, tumors which harbor these mutations may be an ideal subset to study for agents that lead to synthetic cytotoxicity. We have performed in vivo shRNA screening combined with radiation in tumors derived from human HNSCC cell lines (CREBBP/EP300 mutant: Cal27, UMSCC22A, UMSCC47; CREBBP/EP300 wild type: UPCISCC152, HN31) to identify targets that are synthetically cytotoxic when combined with radiation in the CREBBP/EP300 mutant background. This identified CREBBP and EP300 proteins as the most significant targets. Additionally, inhibition of either CREBBP or EP300 with shRNA, combined with radiation, led to increased γ-H2AX, decreased BRCA1, increased apoptosis and increased cell death in CREBBP/EP300 mutant, but not wild type cells. In vivo studies of CREBBP mutant tumors using both HPV (+) and HPV (-) xenograft models showed dramatically increased tumor growth delay (TGD) following radiation in CREBBP knockdown tumors, but not controls, with 40-80% of tumors cured after low doses of XRT. This was accompanied by increased TUNEL staining in shCREBBP tumors following radiation (p&lt;0.001), as well as an increase in pro-apoptotic proteins on RPPA. We then evaluated chemical inhibitors of CREBBP and EP300. A CREBBP specific inhibitor led to significant in vitro radiosensitization, irrespective of HPV status, but only in CREBBP mutant HNSCC cell lines. A similar finding was seen using shRNA-mediated inhibition, namely that specific inhibition of either CREBBP or EP300 led to synthetic cytotoxicity only in HNSCC lines harboring the individual mutant. We next turned our attention to agents that affect specific functions of EP300 and CREBBP. We initially used a bromodomain inhibitor in vitro in combination with radiation and observed minimal effect at therapeutic doses. We next examined a HAT inhibitor specific for CREBBP and EP300. This agent, but not its inactive sister compound, led to a profound radiosensitization on clonogenic assay in a panel of EP300 and/or CREBBP mutant HNSCC cell lines (p&lt;0.001 for each of 6 HNSCC lines), but not in 3 double wild type lines. This effect was accompanied by increased apoptosis and decreased BRCA1 foci following radiation. We also observed decreased H3K18 and H3K27 acetylation following HAT inhibitor treatment, but only in CREBBP/EP300 mutant cell lines. In conclusion, the observed synthetic cytotoxicity of radiation and CREBBP/EP300 inhibition in mutated background is seen both in vitro and in vivo, largely due to increased apoptosis, and may be primarily due to the HAT function of the protein. As HAT inhibitors are being developed for clinical use, these observations could have a direct positive clinical impact.

Citation Format: Curtis Pickering, Manish Kumar, Kathleen Bridges, Tongxin Xie, David Molkentine, Aakash Sheth, Liang Yang, Mitchell Frederick, Tim Heffernan, Sahil Seth, Jeffrey Myers, Heath D. Skinner. Targeting histone acetyltransferase (HAT) function for synthetic cytotoxicity in CREBBP/EP300 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2925.