Germline mutations in tumour suppressor p53 predispose individuals to a spectrum of cancers, most frequently sarcomas, gliomas, adrenocortical carcinoma and others. This hereditary cancer syndrome is termed Li-Fraumeni syndrome. Of the p53 mutations associated with Li-Fraumeni syndrome the most well studied are those that occur in the DNA-binding domain of p53. However, much less well known are how mutations in the oligomerization domain of p53 contribute to tumorigenesis.
We were specifically interested in the A347D mutation which causes p53 to be unable to form the active form, [inaudible] and instead, this protein preferentially forms dimers and is the only dimer-forming variance among the Li-Fraumeni syndrome associated oligomerization domain mutants. Ultimately, we wanted to understand from a basic science standpoint how the gain of function or loss of function activities of this mutant p53 contribute to tumorigenesis.
In order to study this mutation we used two experimental models. First we obtained fibroblasts from a Li-Fraumeni syndrome family and two sublims were heterozygous for the A347D mutation and one harboured wildtype p53. Our collaborators in the lab of Dung-Fang Lee at UT Health subjected these fibroblasts to the Yamanaka protocol to generate iPSCs. These iPSCs were then differentiated into osteoblasts which are the cell of origin for osteosarcomas which are commonly observed in Li-Fraumeni syndrome patients. In addition, we used CRISPR-Cas9 to knock out p53 from U-2 OS osteosarcoma cells which are wildtype for p53 and generate cells either heterozygous or homozygous for the A347D mutation or, as I mentioned, we knocked out p53 from these cells.
This gives us an isogenic allelic series to work cleanly to study the effects of p53 A347D. We used more global unbiased approaches such as RNA-Seq and ChIP-Seq to study this mutation and this p53 mutant as a transcription factor. We also used more targeted approaches such as immunoprecipitation and subcellular fractionation methods to study the interaction of p53 with other proteins.
We found that A347D is hyper-stable and yields a mutant p53 protein that preferentially forms dimers. Despite this hyperstability, we found that this mutant has completely lost canonical p53 transcriptional activity which is a loss of function that is likely tumour promoting. On the other hand, we found that this mutant p53 induces mitochondrial network changes and is able to directly associate with mitochondrial proteins to induce apoptosis in a transcriptionally independent manner. Additionally, we found that cells with this mutant are hypersensitive to topoisomerase 2 inhibition which may be exploited therapeutically.
This really suggests that this mutant p53 exhibits both loss of function and gain of function properties that give it almost a double-edged sword property in which loss of function is tumour promoting whereas the gain of function activity is almost paradoxically tumour suppressing.
How might these results impact future research?
To our knowledge, we are the first to demonstrate that a mutant p53 is able to induce cell death, to direct mitochondrial apoptosis after topoisomerase 2 inhibition. Although this is at a very preliminary stage using preclinical models, our work really suggests that topoisomerase 2 inhibitors might be more effective for Li-Fraumani syndrome patients with this mutation. Additionally, the study sets the stage to assess whether other more commonly occurring p53 variants may also exhibit this apoptotic ability that may be exploited therapeutically.
What are the next steps for this study?
As I mentioned previously, we are interested in how other hotspot mutants might be differentially sensitive to earn chemotherapeutic agents and may exhibit properties like A347D to directly induce mitochondrial apoptosis. Thus, we are examining other hotspot mutants that are associated with Li-Fraumeni syndrome to see whether they also retain the licence to kill.
I’d also like to mention that this work was performed primarily in the lab of Carol Prives at Columbia University and was just published last week in The Journal of Cancer Discovery. So if you’d like to learn more, please check our paper out.
