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Separate p53 Mutations Share Gene Expression Signature, Could Predict Cancer Risk


NEW YORK – Two lesser-functioning p53 variants found in sub-Saharan West African populations share a common gene expression signature that associates with a higher cancer risk and can potentially inform therapeutic outcomes.

In a study published last week in the Proceedings of the National Academy of Sciences, researchers with the Molecular and Cellular Oncogenesis Program at the Wistar Institute identified and characterized two germline missense p53 variants, called P47S and Y107H, whose aberrant function triggers increased invasiveness and appears to explain complicated patterns of cancer occurrence in the families of carriers, most of whom cluster in sub-Saharan West Africa.

The missense p53 variants in this case are known as hypomorphs, or variants that result in only a partial loss of gene function.

P47S occurs in p53's transactivation domain and is currently classified as benign, while Y107H occurs in p53's DNA binding domain and is labeled a variant of unknown significance (VUS). According to the Wistar group's research, both may actually be likely pathogenic.

"We were studying these two African-specific variants and we started finding things in common between them, which was weird," said Maureen Murphy, the study's principal investigator. "One's in the transactivation domain of p53, one's in the DNA binding domain. Why would they have common attributes?"

Murphy hypothesized that protein instability could be the shared trait. Essentially, regardless of where they occurred in the protein, if the mutations triggered a conformational change toward similar metastable states, then they should share similar downstream effects.

"If this is true," she said, "we should see a [shared] gene signature."

Murphy and her team turned to machine learning to look at the genes expressed in the B cells of people with the two hypomorphs and identified a shared 143-gene signature that discriminated the hypomorphs from wild-type cells.

The computational analysis was then confirmed in B cell lines and mouse models through a combination of RNA sequencing and western blot analysis.

Pasquale Laise, a researcher at Columbia University specializing in cancer initiation and progression, and senior director of single-cell systems biology at precision health company DarwinHealth, commented that the study helps to fill a critical knowledge gap.

"While most of the published studies on p53 focus on mutations that are well known to be associated to cancer, our knowledge of germline missense variants of p53 and their functional role in cancer is still very limited," he said.

Laise found it interesting that both hypomorphs should share a common gene expression signature, as their respective mutations occur in distinct p53 domains.

"This suggests that, irrespective of which domain is affected, there is a canalization of the mutation-induced cellular signals which converges to the same set of transcriptional regulators," he said. "This finding significantly improves our understanding of the regulatory programs underlying p53 genetic hypomorphs."

Narrowing in on the genes whose activity was affected by each hypomorph, Murphy's team observed upregulated NF-kB and RRAD. Certain mutant forms of p53 recognized by the monoclonal antibody pAb240 are known to upregulate NF-kB, which in turn regulates RRAD.

Hypothesizing that the two hypomorphs might adopt a similar conformation to that mutant p53, Murphy and her team tested and confirmed that cells carrying either of the P47S or Y107H hypomorphs responded to treatment by pAb240.

Critically, the Wistar team discovered that treatment with arsenic trioxide (ATO) could reverse the biochemical properties of the two hypomorphs.

Arsenic trioxide, also marketed by Teva as Trisenox, is used to treat promyelocytic leukemia and is in clinical trials for other indications, such as ovarian and endometrial cancers, and for neuroblastoma. At least one trial, taking place in China, was investigating arsenic trioxide's potential for rescuing p53 structural mutations, but the status of this trial is currently unknown.

"No one knows why [ATO] works," Murphy said. "It just does. But we know that mutant forms of p53 misfold and are inactive, and hypomorphs misfold a little bit and are somewhat inactive. If we had a drug that could say, no, your protein is supposed to look like this and refold [it], that is the holy grail for the p53 field and it has been for 30 years."

Murphy pointed out that while promising, much work remains to be done with respect to the hypomorphs.

She noted that the regulation of RRAD appears to occur post-transcriptionally, and the mechanism by which that happens is not yet known. Additionally, the gene signature was established in immortalized cells and must be verified in primary peripheral blood cells.

As a step in that direction, Murphy is acquiring hundreds of B-cell lines from the MD Anderson Cancer Center, in an effort to more rigorously refine and test the signature.

DarwinHealth's Laise also noted that expanding future studies to include more cell lines for wet lab experiments and large, independent cohorts for data mining would be needed.

"The gene expression signature could be biased by idiosyncratic cellular interactions of the cell line models," he said. And because few cell lines were used in this study, he added that, "even if the predictive power of the gene expression signature identified by the authors is very high, it is based only on very few samples."

Murphy hopes to develop the hypomorphic gene expression signature into a liquid biopsy test. 

"[You] take a sample of a person’s blood and say this indicates that you have a mutation in p53 that is partially inactivating, [so] your cancer risk will be higher than the normal person."

Murphy noted that in addition to risk estimation, the gene expression signature could potentially be used to monitor therapy response.

"This signature is on resting cells that just differ in p53," she said. "We would want to have a test [of] people treated with, for example, a p53 refolder and nothing else, and see if we could refold that. My signature would be perfect for that."

The Wistar Institute has filed a provisional patent on the mutant p53 gene signature, for which Murphy and her colleague Andrew Kossenkov are listed as inventors, but Murphy said that it remains too early to contemplate licensing and commercialization plans.