A global suppressor motif for p53 cancer mutants Timothy E. Baroni*†‡, Ting Wang*†§, Hua Qian*†¶, Lawrence R. Dearthʈ, Lan N. Truongʈ, Jue Zengʈ, Alec E. Denes*, Stephanie W. Chen*, and Rainer K. Brachmannʈ** *Department of Medicine, Division of Oncology, Washington University School of Medicine, 660 South Euclid Avenue, P.O. Box 8069, St. Louis, MO 63110; and ʈDepartment of Medicine, Division of Hematology͞Oncology, University of California, Medical Science I, C250, Zot Code 4075, Irvine, CA 92697 Communicated by Stanley J. Korsmeyer, Dana–Farber Cancer Institute, Boston, MA, February 19, 2004 (received for review December 4, 2003) The transcription factor and tumor suppressor protein p53 is fre- changes account for 30% and 50 amino acid changes for 55% of all quently inactivated in human cancers. In many cases, p53 gene core domain mutants with a single amino acid change (5). Thus, mutations result in high levels of inactive, full-length p53 protein with compounds active on a subset of p53 cancer mutants could benefit one amino acid change in the core domain that recognizes p53 a large number of patients. DNA-binding sites. The ability to endow function to mutated p53 Furthermore, as proof of principle, several compounds, amifos- proteins would dramatically improve cancer therapy, because it tine, ellipticine, CP-31398, and PRIMA-1, have already been would reactivate a central apoptotic pathway. By using genetic identified that endow function to a subset of p53 cancer mutants strategies and p53 assays in yeast and mammalian cells, we identified (11–14). The exact rescue mechanisms of and number of p53 a global suppressor motif involving codons 235, 239, and 240. These mutants rescued by these compounds are currently not known. intragenic suppressor mutations, either alone or in combination, CP-31398 and PRIMA-1 were isolated in large-scale drug screens restored function to 16 of 30 of the most common p53 cancer mutants that may be the key to this therapeutic challenge. However, screens tested. The 235–239–240 suppressor motif establishes that manipu- may also fail to identify compounds with the broadest activity, lation of a small region of the core domain is sufficient to activate a because, for example, such compounds were not represented in the large number of p53 cancer mutants. Understanding the structural screened libraries. We have taken a different approach of first basis of the rescue mechanism will allow the pursuit of small com- establishing the existence of a global rescue mechanism for p53 pounds able to achieve a similar stabilization of p53 cancer mutants. cancer mutants through the studies of intragenic suppressor mu- tations and then exploiting these findings for the design of small he transcription factor p53 exerts its tumor suppressor function, compounds. Intragenic suppressor mutations are very instructive, Tin part, by activating target genes after upstream stress signals, because they pinpoint key regions of the p53 core domain that, after such as DNA damage, have been relayed. Final outcomes of p53 modification, provide increased stability to p53 protein. activation are DNA repair, cell-cycle arrest, and͞or apoptosis (1–3). In a pilot study (15), we demonstrated that genetic strategies The 393-aa-long protein has an amino-terminal transactivation using a p53 yeast assay were feasible and yielded intragenic sup- domain, a core domain of Ϸ200 amino acids (amino acids 96–292) pressor mutations. This approach resulted in the rescue of p53 that recognizes p53 DNA-binding sites (DBSs), and a carboxyl- cancer mutant V143A by suppressor amino acid N268D, G245S by terminal tetramerization domain. Estimates suggest that up to half T123P, N239Y or S240N and R249S by the combination of T123A of all human cancers carry p53 gene mutations, of which many have and H168R. Two key questions, however, remained unanswered. been documented in large international databases (4, 5). The Are there additional suppressor regions within the p53 core do- International Agency for Research on Cancer (IARC) TP53 Mu- main? Are the identified suppressor mutations at codons 123, 168, tation Database contains 18,585 somatic p53 mutations (R8, 239͞240, and 268 important for the rescue of other frequent p53 www.iarc.fr͞p53; ref. 5). A total of 13,262, or 71%, of these cancer mutants? Improved tools allowed us to answer these central mutations result in full-length protein with one amino acid substi- questions, and we report here the identification of a global sup- tution within the p53 core domain. pressor motif involving amino acids 235, 239, and 240 that rescues The important functions of p53 and the high frequency of p53 more than half of the most common p53 cancer mutants tested. mutations have resulted in significant interest in exploiting the p53 pathway for novel cancer therapies (6, 7). One particularly appeal- Materials and Methods ing approach is the use of small compounds for the pharmacological PCR Mutagenesis of the Upstream and Downstream Regions of p53 restoration of p53 function to those cancers that carry full-length Cancer Mutations. PCR products covering the core domain regions p53 protein with one amino acid change in the DNA-binding core upstream or downstream of a cancer mutation were obtained under domain (8). This strategy, if realized, should have several significant mutagenic conditions (16, 17) and were transformed with appro- advantages. The number of patients that may potentially benefit is priately gapped mutant p53 expression plasmids into the p53 yeast quite large. Based on estimates by the IARC of new cancer cases reporter strain RBy377. RBy377 contains two copies of the p53- worldwide for the year 2000 (www-dep.iarc.fr͞globocan͞ dependent reporter gene 1cUAS53::URA and resulted from a globocan.html; ref. 9), estimates for the frequency of p53 mutations mating of RBy41 (18, 19) and RBy171 (MAT ␣ his3⌬200 leu2⌬1 in specific cancers (10) and the IARC TP53 Mutation Database (5), 1cUAS53::URA), selection for diploids on SC-His-Lys plates (lack- 10 million people are diagnosed with cancer every year, and 2.6 ing histidine and lysine) and segregation of pRB16. Yeast trans- million of them have cancers with a core domain missense muta- formants that had repaired the p53 plasmid by homologous recom- tion. In addition, p53 mutations are frequently found in solid tumors, such as lung, colon, head and neck, and pancreas cancers, that are quite resistant to conventional therapies (4, 5). Such Abbreviations: DBS, DNA-binding site; IARC, International Agency for Research on Cancer. compounds should, in theory, only have an effect on cancer cells, †T.E.B., T.W., and H.Q. contributed equally to this work. because the core domains of WT p53 in normal cells are already ‡Present address: Division of Genetic Disorders, Wadsworth Center, 120 New Scotland structurally intact. Furthermore, the approach promises systemic Avenue, Albany, NY 12084. delivery and lack of a host immune response. §Present address: Department of Genetics, Washington University School of Medicine, 660 The small compound strategy, however, poses formidable chal- South Euclid Avenue, P.O. Box 8232, St. Louis, MO 63110. lenges. Such compounds simply may not exist, or, if they do, may be ¶Present address: Department of Surgery, University of Western Ontario, 339 Windermere effective against only a small subset of the nearly 1,000 reported p53 Road, LHSC-UC 9L14, London, ON, Canada N6A 5A5. core domain mutants. However, from a clinical perspective, the **To whom correspondence should be addressed. E-mail: [email protected]. problem may not be quite as complex, because only 8 amino acid © 2004 by The National Academy of Sciences of the USA 4930–4935 ͉ PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401162101 Downloaded by guest on September 30, 2021 bination were selected on plates lacking histidine (circular plasmid Table 1. Intragenic suppressor mutations obtained with required) and uracil (expression of the URA3 reporter gene re- PCR mutagenesis quired). Single-colony-purified HisϩUraϩ yeast clones were tested ϩ Cancer for plasmid-dependency of the Ura phenotype. Retention of the mutation No. Suppressor mutations cancer mutation on the p53 plasmid was verified by yeast colony PCR, followed by digestion of the PCR product to identify the G245S 1 F113L unique restriction site provided by the cancer mutation codon. 2 L114V ϩ T123P* ϩ V172l ϩ A189V Positive plasmids were then rescued from yeast, were sequenced 3 T123P* ϩ A189V and were transformed again into RBy377 for phenotype confirma- 4 S227P ϩ N239Y* tion (15). Between 1 ϫ 106 to 1 ϫ 107 yeast transformants per region 5 N239Y* ϩ were analyzed (see Supporting Materials and Methods, which is R249S 6 T118M H168R* ϩ ϩ published as supporting information on the PNAS web site, for 7 V122I C124S H168R* ϩ ϩ more details, including the construction and characterization of 8 K139R H168R* N239Y* ϩ yeast and mammalian plasmids). 9 H168R* T231I R273C 10 T123A* ϩ S240R ϩ Oligonucleotide-Mediated Mutagenesis of p53 Codons 225–241. The 11 H178Y S240R ϩ ϩ ϩ saturation mutagenesis of codons 239 and 240 depended on our 12 D281G E285G G325R E343V ϩ ϩ p53 13 E285K E294G E346G ability to create a short gap in mutant plasmids by using ϩ ϩ restriction enzymes PflMI and NsiI. The second NsiIsiteinthe R273H 14 T81S A83V S240R 15 P87R ϩ Q100L ϩ Q104P ϩ Q144L ϩ S240R backbone of pRS413 was therefore destroyed, and all expression ϩ cassettes for p53 mutants were transferred into this new backbone 16 S183T S240R 17 E224G ϩ S240R by using ApaI–SacI. Thirty-eight annealed primer pairs were gen- 18 H233R ϩ S240R erated, each of them representing a different amino acid change at 19 S240R codon 239 or 240. Equal amounts of each primer pair were cloned separately into the same gapped p53 plasmid, and the number of All clones shown are derived from independent PCR-mediated mutational Escherichia coli clones was determined.