Abstract 791: Targeting small domains of p53 to mitochondrial Bcl-XL for cancer therapy.

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Abstract The tumor suppressor p53 is involved in transcriptional induction or repression of various genes resulting in cell cycle arrest, senescence, DNA repair and apoptosis. While most of these functions can be linked to its role as a transcription factor in the nucleus, p53 also triggers apoptosis via the mitochondrial pathway. In response to cellular stress, p53 translocates to the mitochondria and directly interacts with Bcl-2 protein family members including anti-apoptotic Bcl-XL and Bcl-2 and pro-apoptotic Bak and Bax. Anti-apoptotic Bcl-XL forms inhibitory complexes with pro-apoptotic Bak and Bax preventing their homo-oligomerization. Upon translocation to the mitochondria p53 binds to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances their homo-oligomerization. Bak and Bax homo-tetramer formation disrupts the mitochondrial outer membrane, releases anti-apoptotic factors such as cytochrome c and triggers a rapid apoptotic response mediated by caspase induction. There are conflicting reports suggesting that the MDM2 binding domain (MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD) of p53 are the domains for interacting to Bcl-XL. The purpose of this work is to determine which subdomain of p53 binds optimally to Bcl-XL while maintaining the full apoptotic activity of full length p53. Different domains of p53 (MBD, PRD, DBD) were fused to the mitochondrial targeting signal from Bcl-XL to ensure Bcl-XL specific targeting. The designed constructs were tested for apoptotic activity (TUNEL, Annexin-V, and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231), in a cervical cancer cell line (HeLa), and in human leukemic cells (K562). Our results indicate that DBD-XL (p53 DBD fused to the Bcl-XL MTS) shows the same (in T47D and, K562 cells) or higher (in MCF-7, MDA-MB-231, and HeLa cells) apoptotic activity compared to p53-XL. Additionally, over-expression of Bcl-XL in T47D cells confirmed that DBD directly binds and inhibits Bcl-XL. Further, binding affinity of designed constructs to Bcl-XL will be explored via co-immunoprecipitation (co-IP) and mammalian two-hybrid assay. Taken together, our data highlights that DBD can be used instead of full length p53 for achieving apoptosis at the mitochondria. The benefit of decreasing the overall size of p53 while maintaining full apoptotic activity allows for better drug delivery options. Furthermore, DBD-XL-mediated apoptosis of multiple cell lines including such as breast cancer cells, cervical carcinoma cells, and leukemia cells highlighting the role of p53 as the ultimate target for cancer therapy. Citation Format: Karina J. Matissek, Mohanad Mossalam, Abood Okal, Carol S. Lim. Targeting small domains of p53 to mitochondrial Bcl-XL for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2013-791