Abstract 790: An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression.

Abstract

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. Our data have shown that swapping the tetramerization domain (TD) of p53 with an alternative oligomerization domain enhances the utility of p53 for cancer gene therapy. Previously, we have swapped the tetramerization domain of p53 with the coiled-coil (CC) domain from breakpoint cluster region (Bcr). This alteration of the oligomerization motif of the tumor suppressor allowed for our construct, namely p53-CC, to evade hetero-oligomerization with endogenous mutant p53 commonly found in cancer cells. This proves to be critical since mutant p53 has a transdominant inhibitory effect over wild-type p53 upon hetero-oligomerization. A co-immunopreciptation (co-IP) experiment in human breast ductal carcinoma (T47D) cells validated our hypothesis that endogenous p53 interacts directly with exogenous wild-type p53, which is due to hetero-oligomerization via their TDs. In contrast, p53-CC, which lacks the TD, evaded binding to endogenous p53. Furthermore, overexpression of a potent mutant p53 (contains three hotspot mutations; R175H, R248W, and R273H) significantly impaired the function of exogenous wild-type p53, while the tumor suppressor activity of p53-CC was not affected. To further increase the apoptotic potential of p53-CC, rational design of mutations in the CC domain were investigated for two purposes: first to increase the dimerization affinity of CC, and second to prevent any potential interaction with endogenous Bcr. Leucine at residue 62 of the CC domain forms a critical hydrophobic pocket at the dimeric interface. Therefore, mutating this residue into the more hydrophobic isoleucine (with similar molecular weight),, should increase CC dimerization affinity. Results from a standard mammalian two-hybrid assay will be presented to validate the binding affinity of our mutant CC compared to the native CC. Furthermore, two additional mutations on the CC domain (R55E and E34K) were carried out to prevent any potential interaction of our construct with endogenous Bcr. These two mutations introduce a salt bridge that promotes homo-dimerization of mutant CC as well as preventing hetero-dimerization with native CC representative of endogenous Bcr. This will be confirmed using another co-IP experiment that shows no interaction between mutant CC and endogenous Bcr. Finally, two apoptotic assays were carried out to verify that introducing the aforementioned mutations did not cause any loss of tumor suppressor activity of our enhanced p53-CC variant. In summary, a p53 with altered TD has been designed for exclusive homo-dimerization and has potent activity as a tumor suppressor. Our ultimate goal is to use this version of p53 as a new gene therapeutic, capable of bypassing the dominant negative effect in cancers with mutated or mislocalized p53. Citation Format: Abood Okal, Mohanad Mossalam, Karina J. Matissek, Carol S. Lim. An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression. [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 790. doi:10.1158/1538-7445.AM2013-790