Generation of a Novel D. melanogaster Platform to Elucidate Oncogenic Activity of Common Human p53 Missense Mutants

The tumor suppressor p53 prevents uncontrolled cell growth by three separate mechanisms: inducing apoptosis, initiating cell-cycle arrest, and activating DNA repair mechanisms in response to cell damage. Due to its central role in tumor eradication, it is unsurprising that p53 mutations are found in over half of human cancers. Unlike all other tumor suppressors however, 75% of these are missense mutations, with just six of them accounting for a third of all mutations found in the DNA binding domain of p53. Recent findings indicate that mutations in these "hotspot" locations may encode gain of function oncogenic activity to p53. Given their high prevalence, these mutations suggest a previously underappreciated selective advantage. We sought to decode this novel oncogenic activity of human p53 mutations by exploiting the Drosophila model system. This organism shares a similar p53 regulatory network with humans, as well as many of the same DNA repair and pro-apoptotic target genes. We recently showed that human p53, despite millions of years of evolutionary distance, complements loss of function mutations in the native fly p53 gene. We used six humanized p53 Drosophila strains previously generated in the lab; these contain a human p53 gene insertion, each with one of the six most commonly found missense mutations in patients. To study these mutations, we first profiled the expression patterns of wild type and mutant hp53 in the fly and their ability to rescue dp53 function. Expression levels of p53 were determined by immunofluorescence, while biological function was determined by the use of a GFP biosensor that specifically reports dp53 activity and acridine orange staining to identify dying cells in irradiated embryos. Expression studies demonstrate that the reporter is activated within stem cells in region 1 of the germarium, while its activation was absent in p53 null mutants. This phenotype was recovered with a dp53 insertion rescue. Additionally, two separately generated hp53+ strains show unusually elevated levels of expression compared to the wild type strains, whereas all mutant strains show diminished reporter activation in the region 1 stem cells. Functional studies in the embryo and the wing disc demonstrate that both wild type flies and the dp53 rescue promote cell death after irradiation, while the p53 null mutant does not. The two hp53+ strains rescued the wild type phenotype in the embryo; however, one of the hp53+ strains, named B2, was unable to induce cell death in the wing disc. The missense mutant strains do not exhibit IR-induced apoptosis in the embryo, but preliminary imaging shows they may be able to in the wing disc. We also discovered that, unlike the six hotspot mutants, wild type human p53 localizes to unidentified subnuclear compartments. Importantly, this may allow us to stratify and characterize p53 mutations according to functional differences.