Targeting p53 and its domains for cancer gene therapy

The tumor suppressor p53 is one of the most frequently mutated proteins in human cancer and has been extensively targeted for cancer therapy. This resulted in wild type p53 gene therapeutic approval for the treatment of head and neck cancer in China. p53 mainly functions as a transcription factor and stimulates a variety of genes involved in the intrinsic and extrinsic apoptotic pathway by binding to p53 responsive elements as a tetramer. In cancer cells, mutations in p53 typically occur in its DNA binding domain (DBD), while its tetramerization domain remains intact. Therefore, mutant p53 can heterotetramerize with wt p53 and abolish its transcriptional activity (dominant negative effect). While transcriptionally active wt p53 is used for gene therapy, mitochondrial p53 has not been fully exploited yet. Targeting p53 to the mitochondria causes a direct rapid apoptotic response by directly interacting with pro-and anti- apoptotic proteins at the mitochondrial outer membrane. Because the monomeric from is sufficient to interact with pro-and anti-apoptotic proteins, mitochondrial p53 is not affected by the dominant negative inactivation. To ensure mitochondrial targeting of p53, we targeted p53 to different mitochondrial compartments; mitochondrial outer membrane, inner membrane and matrix. We have demonstrated that MTSs from the mitochondrial outer membrane are optimal for p53-specific activation. In addition, we discovered the minimal domain of p53, its DNA binding domain (DBD), which is needed for apoptosis induction. Further studies have shown that fusing p53 or DBD to MTS from Bcl-XL causes p53/Bcl-XL specific apoptosis while fusing them to Bak results in p53/Bak specific apoptosis, emphasizing that mitochondrial targeting of p53 is highly dependent on the MTS used. Further, we have shown that DBD fused to the MTS from Bcl-XL can overcome dominant negative inhibition in vitro, but it was unable to shrink dominant negative MDA-MB-468 tumors in an orthotopic mouse model at one dosing regimen attempted. The main theme of this thesis was to design apoptotic proteins based on p53 domains to create modified versions of p53. We focused mainly on optimizing mitochondrial targeting of p53 for cancer therapy but also redesigned the TD of p53 to overcome the dominant negative effect. We substituted the oligomerization domain of p53 with the coiled-coil (CC) domain from BCR to bypass dominant negative inhibition of mutant p53. Our chimeric p53 (p53-CC) can translocate into the nucleus, transactivate genes and cause apoptosis in a similar manner as wt p53. Unlike wt p53, p53-CC does not interact with endogenous mutant p53 and retains apoptotic activity in cancer cells harboring dominant negative mutant p53 in vitro and in vivo. In summary this dissertation focuses on new p53 gene therapeutics, with the potential to overcome current limitations with wt p53 therapy.