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Molecular & Cellular Oncology

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Functional characterization of the p53 “mutome”

Eran Kotler, Eran Segal & Moshe Oren

To cite this article: Eran Kotler, Eran Segal & Moshe Oren (2018) Functionalcharacterization of the p53 “mutome”, Molecular & Cellular Oncology, 5:6, e1511207, DOI:10.1080/23723556.2018.1511207

To link to this article: https://doi.org/10.1080/23723556.2018.1511207

Published online: 25 Sep 2018.

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Functional characterization of the p53 “mutome”Eran Kotler a,b, Eran Segal a,b, and Moshe Oren a

aDepartment of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; bDepartment of Computer Science and AppliedMathematics, Weizmann Institute of Science, Rehovot, Israel

ABSTRACTPhenotypic characterization of mutations in the tumor protein p53 (TP53) gene has so far focused on ahandful of relatively frequent “hotspot” mutations, accounting for only ~ 30% of cases. We expanded thescope and quantitatively measured the impact of thousands of distinct TP53 mutations in vitro and invivo, providing insights into the connections between structure, function, evolutionary conservation andclinical impact.

ARTICLE HISTORYReceived 19 July 2018Revised 7 August 2018Accepted 9 August 2018

KEYWORDSmutant p53; deepmutational scan;massively-parallel reporterassay (MPRA); phenotypiccatalogue; gain of function(GOF)

The tumor protein p53 (TP53) gene, encoding the p53 tran-scription factor, is the most frequently mutated gene inhuman cancer1. Unlike other tumor suppressors, the majorityof cancer-associated mutations in p53 are missense mutationsresiding in its DNA-binding domain (DBD)2,3, leading to lossof tumor suppressive activity and, in some cases, also to gainof novel oncogenic functions (gain-of-function, GOF;reviewed in4). However, while thousands of distinct p53mutations have been reported and variant-specific phenotypeshave been observed, cancer-related research has so far focusedalmost exclusively on 6 relatively common hotspot mutations,which altogether account for only ~ 30% of the p53-mutatedcases.

Despite the emerging understanding that p53 mutationsare not all alike, personalized therapeutics lag behind andmutant-specific effects are widely overlooked, as highlightedby Sabapathy and Lane5. It is therefore important to deter-mine the functional consequences of all distinct p53 muta-tions; this might be particularly valuable for the personalizedtreatment of cancer. We therefore sought to systematicallystudy the impact of the entire spectrum of p53 DBD muta-tions, accounting for over 90% of tumor-associated mutations,in human cancer-derived cells. To quantitatively measure theanti-proliferative capacity of thousands of mutant p53(mutp53) variants, we devised a massively-parallel mutationalscan that allows to simultaneously measure the relative impactof a plethora of mutations. Using this assay, we measured theanti-proliferative functional capacity of a synthetically-designed library of ~ 10,000 distinct p53 variants (Figure 1)6.

Specifically, we computationally designed a genomic libraryincluding nearly all p53 DBD mutations reported in tumorsamples3, the majority of which were previously unstudied, aswell as combinations of mutations with single-nucleotide poly-morphisms (SNPs) found across human individuals (aimed to

identify possible genetic interactions). Additionally, to unbiasedlycharacterize the effect of mutations across the entire DBD, wesystematically generated all single-nucleotide alterations withinthe DBD, as well as single amino acid substitutions, prematuretermination codons and in-frame deletions. Technically, ourapproach employs a designed microarray-synthesized oligonu-cleotide library to generate a mutp53 lentiviral library. Theseviruses were then transduced into human cancer-derived cells,resulting in a mixed population of cells, each expressing a singlemutp53 variant. Sampling of these cells at different time pointspost-infection allowed us tomeasure the dynamics of each variantalong time and deduce a relative fitness score (RFS), indicative ofeach mutant’s functional outcome.

Using this library, we first examined how proteinsequence variations affect wild-type p53 (wtp53)-likeanti-proliferative capacity. Expectedly, regardless of posi-tion along the DBD, premature termination codons andframeshift mutations strongly disrupted p53 functionality,whereas synonymous mutations retained full wtp53 func-tionality. In contrast, the effects of substituting or deletinga single amino acid were strongly dependent on its posi-tion within the DBD, corresponding to well-defined struc-tural motifs, and on the biochemical properties of thesubstituting amino acid. Interestingly, the majority ofp53 mutations discretely segregated bimodally as eitherretaining wtp53 functionality or strongly disrupting it.Moreover, most residues dichotomized either as tolerant(retaining wtp53 functionality regardless of particular sub-stitution) or highly intolerant (functionality is disruptedby nearly any mutation at that position).

We next compared the functional impact of proteinsequence alterations to the relative evolutionary represen-tation of each amino acid substitution (i.e. the percent ofspecies in which that amino acid is present at a given

CONTACT Eran Kotler erankotler@gmail.com Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, IsraelColor versions of one or more of the figures in the article can be found online at www.tandfonline.com/kmco.

MOLECULAR & CELLULAR ONCOLOGY2018, VOL. 5, NO. 6, e1511207 (3 pages)https://doi.org/10.1080/23723556.2018.1511207

© 2018 Taylor & Francis Group, LLC

position). This revealed that the mean relative representa-tion of variants retaining wtp53 functionality is dramati-cally higher than that of non-functional variants. Thus, thefunctional effects measured in our assay faithfully repro-duce the constraints that shape the DBD sequence alongevolution. These associations between structure, functionand conservation, underscore p53’s anti-proliferative capa-city as a fundamental property under strong evolutionaryselection.

We further observed high concordance between the func-tional outcome of each mutation and its prevalence in humantumors. This reinforces the notion that loss of anti-prolifera-tive capacity is a key selective force in shaping the landscapeof p53 mutations in cancer. Thus, tumor-associated mutationsretaining wtp53-like anti-proliferative functionality are unli-kely to be driver mutations. This was further demonstrated bycomparing the age at tumor diagnosis in individuals harbor-ing germline p53 mutations (Li-Fraumeni syndrome), whenstratified by their specific mutation’s RFS.

Importantly, our in vitro measurements could not identify agrowth advantage for commonly occurring “hotspot”mutations,compared to equally disruptive mutations observed less fre-quently in tumors. Therefore, we sought to pursue evidence forGOF under selective pressures operating in vivo, within a grow-ing tumor. We injected library-expressing cells into immuno-compromised mice and quantified the relative enrichment ofeach variant in the formed tumors. Interestingly, mutations thatequally disrupted p53 functionality in vitro were found to span abroad phenotypic spectrum in the tumors; reassuringly, hotspotmutations now exhibited selective in vivo GOF.

Finally, we took advantage of our experimental plat-form to evaluate the significance of SNPs within theDBD. Thus, we measured the combinatorial effects ofbackground SNPs with an additional acquired mutation.We found that, when presented on a 217M (Methionine atcodon 217) background (rs35163653), missense mutationsexhibited a more disruptive phenotype compared to theoutcomes of the same mutations on a wild-type (217V)p53 background, highlighting the importance of experi-mental assessment of combinatorial genetic interactions.

Altogether, our data provides a comprehensive pheno-typic catalogue of p53 mutations (Figure 1), quantifyingthe relative fitness effect of each mutation in distinctbiological contexts and providing information regardingthe effects of thousands of variants of unknown signifi-cance. This paves the way to studying the entire “p53mutome” and supports the applicability of similar large-scale systematic scans to broaden our understanding ofadditional mutation-driven phenotypic landscapes. Ourdataset might serve as a resource for subsequent clinicaland in-depth mechanistic studies of the effects of specificp53 mutations. Furthermore, this library of p53 mutantsmay serve as a tool for further phenotypic studies in vitroand in vivo and for high-throughput drug screening.

Funding

This work was supported in part by the Dr. Miriam and Sheldon G.Adelson Medical Research Foundation and a Center of Excellence grantfrom the Israel Science Foundation (to M. Oren) and the EuropeanResearch Council (to E. Segal).

ORCID

Eran Kotler http://orcid.org/0000-0002-1463-7462Eran Segal http://orcid.org/0000-0002-6859-1164Moshe Oren http://orcid.org/0000-0003-4311-7172

References

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Figure 1. Comprehensive characterization of the functional outcomes of p53 mutations in vitro and in vivo. A synthetic library of 9,833 mutations in thetumor protein p53 (TP53) DNA-binding domain was expressed in human cells enabling quantitative measurements of the effect of distinct variants in cell culture andin mouse xenografts, providing a comprehensive phenotypic catalogue for p53 mutations. SNP – Single nucleotide polymorphism.

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