Bypassing transdominant inhibition with chimeric p53 for cancer gene therapy

It is well documented that more than 50% of all human cancers have a mutated p53 gene status, rendering it inactive. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain. Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain (CC) from breakpoint cluster region (Bcr). This approach led to the hypothesis that swapping the oligomerization domain of p53 with an alternative oligomerization domain will prevent hetero-oligomerization and transdominant inhibition by mutant p53 in cancer cells. The tumor suppressor activity of the chimeric p53-CC was evaluated in vitro and found to be similar to that of wt-p53 regardless of cancer type or endogenous p53 status. However, co-immunoprecipitation and viral transduction of p53-CC and wt-p53 into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53. Following proof-of-concept studies, the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model was demonstrated in vivo. In addition, the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model were investigated. Finally, since domain swapping to create p53-CC could result in p53-CC interacting with endogenous Bcr, which is ubiquitous in cells, modifications on the CC domain were necessary to minimize potential interactions with Bcr. Hence, the possible design of mutations that will improve homo-dimerization of CC mutants and disfavor hetero-oligomerization with wild-type CC (CCwt) were investigated, with the goal of minimizing potential interactions with endogenous Bcr in cells. Indeed, the resulting lead candidate p53-CCmutE34K-R55E avoided binding to endogenous Bcr and retained p53 tumor suppressor activity. Although breast cancer was the main focus of this dissertation, the application of this research extends to many other types of cancer, including the deadliest cancers (pancreatic, lung, and ovarian), which currently lack effective treatments.