(2007) 26, 2243–2254 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Reactivation of mutant p53: molecular mechanisms and therapeutic potential

G Selivanova1 and KG Wiman2

1Department of Microbiology, Tumor and , Karolinska Institutet, Stockholm, Sweden and 2Department of Oncology-Pathology, Center Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden

The p53 tumor suppressor is the most frequently domain, thereby abolishing specific DNA binding of mutated gene in cancer. Most p53 are missense p53. This prevents p53-dependent transcription and, point mutations that cluster in the DNA-binding core hence, p53-mediated tumor suppression. The exception- domain. This results in distortion of core domain folding ally high frequency of p53 mutations in human tumors and disruption of DNA binding and transcriptional of diverse types makes p53 unique among transactivation of p53 target genes. Structural studies involved in tumor development (see p53.free.fr and have demonstrated that mutant p53 core domain unfolding www-p53.iarc.fr; Be´ roud and Soussi, 1998; Olivier et al., is not irreversible. Mutant p53 is expressed at high levels 2002). Indeed, unbiased sequencing of whole of in many tumors. Therefore, mutant p53 is a promising breast and colon confirmed that p53 is the most target for novel cancer therapy. Mutant p53 reactivation commonly mutated gene in these tumors (Sjo¨ blom et al., will restore p53-dependent , resulting in efficient 2006). removal of tumor cells. A number of strategies for Accordingly, p53 is the focus of research aimed at targeting mutant p53 have been designed, including development of novel anticancer drugs. Several ap- peptides and small molecules that restore the active proaches that exploit p53 inactivation in tumors for conformation and DNA binding to mutant p53 and induce therapy are currently being pursued, such as selective p53-dependent suppression of tumor cell growth in vitro expression of killer genes in cells with nonfunctional p53 and in vivo. This opens possibilities for the clinical or replication-deficient that can propagate only application of mutant p53 reactivation in the treatment in p53-deficient cells (reviewed in Lane and Lain, 2002; of cancer. McCormick, 2001). Wild-type p53 reconstitution by Oncogene (2007) 26, 2243–2254. doi:10.1038/sj.onc.1210295 has been shown to inhibit tumor growth in clinical trials (reviewed by Wiman, 2006). However, the Keywords: mutant p53; apoptosis; cancer therapy absence of efficient delivery systems has precluded systemic administration so far. In addition, the immune response against viral vectors may reduce the clinical utility of gene therapy. A principally different line of attack on tumors is the Introduction rescue of p53 tumor suppressor functions by small molecules. Tumor cells should be particularly sensitive The p53 responds to a variety of to reconstitution of p53 function as mutations that stress conditions including oncogene activation, DNA inactivate p53 and ablate p53-induced apoptosis in damage and . Accumulation and activation of response to oncogenic stress have been selected during p53 upon such stress triggers growth arrest or apoptosis tumor evolution. Small molecule strategies for restora- through transcriptional regulation of specific target tion of p53 functions in tumors can either target wild- genes (see Vousden and Lu, 2002). This serves to type p53 or mutant p53. In wild-type p53-carrying eliminate cells carrying oncogenic lesions or damaged tumors, the aim is the identification of molecules DNA, thus preventing tumor development. Inactivation that can protect p53 from its own cellular destructor of p53 by occurs in around half of human Mdm-2 in many types of cancers or from degradation tumors. The great majority of mutations are missense by the human papilloma E6 in cervical point mutations that target the DNA-binding core carcinomas (reviewed in Chene, 2003; Zheleva et al., 2003). In tumors that carry p53 point mutations in the core domain, attempts have been made to restore wild-type functions to mutant p53. Since the first Correspondence: Dr KG Wiman, Department of Oncology-Pathology, evidence that the cryptic DNA binding of mutant Cancer Center Karolinska (CCK) and Dr G Selivanova, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, p53 could be restored by phosphorylation or antibody Stockholm, Sweden. binding (Hupp et al., 1993), results from several E-mails: [email protected] and [email protected] laboratories have provided proof-of-principle for Reactivation of mutant p53 G Selivanova and KG Wiman 2244 mutant p53 rescue. Specific DNA binding, transcrip- tional transactivation and, most importantly, the tumor suppressor functions of p53 have been restored by various strategies, including introduction of second-site mutations, antibody binding, short peptides and small molecules.

Advantages of mutant p53 rescue as a therapeutic strategy

Several features of p53 mutations make restoration of wild-type p53 functions to mutant p53 feasible. First, the vast majority of tumor-derived mutations target one domain of the protein, the so-called core domain, which harbors the specific DNA-binding activity. Around 80% of these mutations are missense point mutations resulting in substitution of only one Figure 1 General strategy for mutant p53 reactivation in tumor amino-acid residue (see p53.free.fr; www-p53.iarc.fr). cells. The target, mutant p53, is abundantly expressed but The so-called hot spots are residues where mutations functionally deficient in tumors cells. In addition, cellular stress, for example oncogenic stress, generates critical p53-activating occur with unusually high incidence. Substitutions signaling. In contrast, p53 is latent and expressed at low levels of residues at positions 175, 248, 273 and in normal cells in the absence of cellular stress. This provides the 282 represent around 20% of all mutations found basis for the selective induction of apoptosis in the mutant in tumors. Second, recent structural studies have p53-expressing tumor cells. revealed that the tumor-derived missense mutations in the core domain produce a common effect: destabi- lization of core domain folding at physiological tem- perature (for details, see below). This precludes This notion is supported by studies of p53ERTAM proper orientation of loops and helixes that form the knock-in (KI) mice (Christophorou et al., 2005). This DNA-binding interface and therefore disrupts DNA model makes it possible to switch p53 on and off in binding. However, the defect is not irreversible as many tissues in vivo. The p53ERTAM fusion protein can be mutants can bind DNA at lower temperature. Third, reliably and tightly regulated by 4-hydroxytamoxigen mutant p53 are usually overexpressed in (4-OHT) in vivo, faithfully reproducing various aspects tumors, which is in contrast to other tumor suppressors of p53 functions. Since the expressed p53ERTAM protein such as pRb and , the inactivation of which in is functionally competent only in the presence of 4-OHT tumors is achieved largely by loss of expression (Nevins, , the p53ERTAM KI mouse provides an excellent 2001). Mutant p53 proteins accumulate at high levels in tool for the analysis of p53 reactivation, both in mouse tumor cells mainly due to their inability to upregulate tissues in vivo and in explanted primary cells derived the expression of p53’s own destructor Mdm-2 (Midgley from such tissues in vitro. In the absence of 4-OHT, et al., 2000). Heat-shock proteins can probably con- p53ERTAM KI mice reproduce a classic p53 knockout tribute to mutant protein stabilization as well (Peng phenotype with a high incidence of spontaneous tumors, et al., 2001). mainly lymphomas. Systemic administration of 4-OHT Thus, around half of all human tumors overexpress to p53ERTAM KI mice rapidly restores p53 functions in a highly potent tumor suppressor, which is unable to all tissues. Notably, restoration of p53 functions in perform its functions because of defects in its folding. Is normal tissues is well tolerated in mice and produces it possible to reverse these folding defects and restore no visible toxic effects. This correlates with the absence wild-type functions to mutant p53 in tumors? This of p53-induced growth suppression in cultured cells would be an extremely efficient strategy for selective derived from p53ERTAM KI mice upon 4-OHT treatment elimination of tumor cells, because of the high expres- in vitro. However, restoration of p53 activity by 4-OHT sion of the target protein in most tumors. A small in irradiated cells or in cells expressing oncogenic Ras molecule targeting mutant p53 should not affect wild- leads to activation of p53 and its target genes, thus type p53 in normal cells because it is already properly unleashing the p53 growth suppressor function (Chris- folded and because of its low levels owing to continuous tophorou et al., 2005). These results provide compelling degradation by Mdm-2 in the absence of stress. evidence supporting the notion that oncogene expres- Importantly, tumor cells carrying mutant p53 are likely sion is required for the functional activity of p53 in cells. to preserve intact downstream apoptotic pathways. Importantly, recent results obtained using p53ERTAM Finally, owing to constitutive stress signaling in tumor KI mice demonstrate that re-establishment of active p53 cells, for example oncogene activation, DNA damage in fully developed -induced lymphomas confers and hypoxia, mutant p53 is probably already ‘activated’ significant tumor suppression (Christophorou et al., by post-translational modifications and partner pro- 2006). This indicates that tumor cells carrying oncogenic teins, whereas p53 is latent in normal cells in the absence mutations harbor persistent signals that can be engaged of stress (Figure 1). by p53 to suppress tumor outgrowth. Thus, only the

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2245 environment of cancer cells provide the signals required with the aggregation of mutant proteins at 371C: the to unleash p53’s tumor suppressor effect. Therefore, higher the degree of unfolding, the faster the aggrega- restoration of p53 functions by small molecules should tion (Friedler et al., 2003). Interestingly, mutant p53 have a therapeutic effect at any point of tumor proteins are prone to loss of the Zn(2 þ ) atom that is development without harmful effects in normal cells. bound to the wild-type core. This also promotes One can therefore envisage abundant and activated aggregation of mutant p53 (Butler and Loh, 2003). mutant p53 as a ‘loaded gun’ present in the tumor cells High-resolution crystal structures of tumor-derived (Selivanova, 2001). However, the gun’s trigger is locked mutants provide deeper insight into the consequences of by a mutation. A small molecule that can reverse the mutations. Tumor-derived mutations might result in effect of the mutation will unlock the trigger and fire the loss of essential contacts with DNA, distortion of the gun, killing the tumor cells (Figure 1). Such a molecule DNA-binding interface, disruption of core–core inter- would be a most efficient, widely applicable and highly actions and loss of cooperativity of DNA binding, or selective anticancer drug. appearance of large internal crevices or cavities leading to a significant loss of thermodynamic stability (Joerger et al., 2005a, b, 2006; Ang et al., 2006). These findings provide a structural basis for rational design of Structural basis for mutant p53 reactivation molecules that can rescue the activity of mutant p53. For example, some mutants can potentially be rescued Structural consequences of oncogenic mutations in the p53 by a generic stabilizing drug, whereas a mutation- DNA-binding domain induced crevice represents a potential drug target in The cocrystal structure of the wild-type p53 core domain other mutants. bound to DNA together with more recent nuclear In summary, structural studies show that the extent of magnetic resonance (NMR) and X-ray studies on misfolding differs among mutants; therefore it appears tumor-derived core domain mutants have revealed the unlikely that different mutants possess a defined structural impact of amino-acid residues that are most alternative fold. Another important prediction from frequently mutated in human cancers (Cho et al., 1994; structural studies is that a ligand (i.e. protein, short Wong et al., 1999; Joerger et al., 2004, 2005a, b; Ang peptide or small molecule), which binds to the properly et al., 2006). The so-called DNA contact mutants, for folded fraction of the protein, is expected to shift the example R273H and R248Q, carry substitutions of equilibrium towards the native fold according to the law residues that directly contact DNA. Structural muta- of mass action. This suggests that a small molecule tions such as R175H and R249S target residues that are approach to reverse the effect of p53 mutation could be important for the proper orientation of the two loops successfully applied to a wide range of mutant forms. and loop-sheet-helix motif that form the DNA-binding surface of the core domain. NMR studies and quantitative assessment of folding Biochemical studies on conformational flexibilityof the and DNA-binding properties of mutant core domain core domain proteins have revealed that the major effect of oncogenic Numerous studies have demonstrated an intrinsic mutations is destabilization of the secondary structure conformational flexibility of wild-type p53 in cells. The of the core domain at physiological temperature. epitope recognized by the monoclonal p53 antibody Mutations result in lowering of the melting temperature PAb240 is either exposed or masked in cells depending by 5–101, which is sufficient to tip the balance towards on the growth culture conditions (Milner and Watson, the unfolded state at physiological temperature (Bullock 1990). PAb240 binds to residues 212–217 in the core et al., 1997). The extent of unfolding varies depending domain that are exposed in denatured wild-type p53 on the site and the nature of the mutation. For example, (Gannon et al., 1990; Vojtesek et al., 1995). This epitope 99% of the R273H mutant is folded at 371C, whereas is frequently displayed in tumor-derived p53 mutants, only 5% of the F134L mutant remains folded at this usually followed by the loss of reactivity with ano- temperature (Bullock et al., 2000). For some mutants, ther conformational core domain-specific antibody, such as G245S, R248Q and R249S, only localized PAb1620. This antibody recognizes a nonlinear epitope changes have been observed, suggesting that their in the p53 core domain which is present only in the overall tertiary folds are similar to that of the wild-type ‘wild-type’ active conformation and includes residues protein. Mutations located in the b-sandwich region, R156, L206, R209 and N210 (Cook and Milner, 1990; such as R175H and R282W, heavily destabilize the Wang et al., 2001). Induction of the PAb240 epitope was secondary structure, resulting in unfolding of 50% of shown to occur in embryonic stem cells upon differen- the protein at physiological temperature (for a review, tiation with a concomitant loss of PAb1620 epitope and see Joerger et al., 2005a, b). functional activity of the protein (Sabapathy et al., Thermodynamic instability correlates with the en- 1997). The mutually exclusive recognition by PAb240 hanced kinetics of unfolding at 371C: the heavily and PAb1620 led to the idea of two alternative destabilized mutants V143A, I195T, C242S and R249S conformations: PAb240 þ /PAb1620À ‘mutant’ non- unfold 10 times faster than wild type, whereas less functional p53 and PAb240À/PAb1620 þ ‘wild-type’ destabilized mutants such as R273H and G245S unfold active p53. However, as mentioned above, structural only slightly faster. Moreover, unfolding is associated studies do not support the idea of a defined ‘mutant’

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2246 fold. Rather, p53 exists as a set of interconverting the folding and DNA binding of the core. The last 30 conformers. The lack of a rigid structure of the p53 residues of the C-terminal domain were proposed to protein may result in a number of p53 conformers negatively regulate DNA binding by an allosteric displaying different activity, allowing fine-tuning of the mechanism. This hypothesis was based on the observa- p53-mediated biological response depending on the type tion that the interaction of p53 with a short oligonucleo- of stress and cellular context. This notion is supported tide containing a consensus p53-binding site is greatly by recent studies of the solution structure of the p53 core enhanced either by the of the C-terminal basic domain that allowed the identification of several region (30 residues) or by binding of the antibody structural elements conferring flexibility to the core PAb421 to the same region (Hupp and Lane, 1994). This domain. This suggests that p53 has evolved to be was confirmed by a study showing that p53 transcrip- dynamic and conformationally unstable (Canadillas tional activity is activated by PAb421 in cells (Luand et al., 2006). Lane, 1993). Recent studies have demonstrated that Mutant p53 proteins exhibit greater conformational within the context of chromatin or supercoiled DNA, instability than the wild-type p53. Many mutants the C-terminal domain may actually facilitate binding of display the PAb240 epitope, although the extent of the core to its target DNA sequence by providing an PAb240 reactivity differs between the mutants, as well additional anchorage to specific DNA sites via non- as the loss of PAb1620 epitope and the ability to bind to specific DNA binding (Espinosa and Emerson, 2001; the heat-shock proteins Hsp70/ (Ory et al., 1994; McKinney and Prives, 2002; McKinney et al., 2004). Gaiddon et al., 2001). The differential ability to bind Along with its effect on DNA binding the C-terminal PAb240 and heat-shock proteins by p53 mutants lends domain confers conformational instability to the core further support to the idea that mutant proteins domain. Substitution of one of the phosphorylation sites expressed in cells display the whole spectrum of in the C terminus, S392E, by mutation mimics unfolded states. It is noteworthy that some mutations constitutive phosphorylation and increases DNA bind- can cause only slight changes in p53 conformation, ing and stability of the core (Nichols and Matthews, eliminating PAb1620 recognition, but not yet exposing 2002). Binding of the bacterial heat-shock protein DnaK the PAb240 epitope. or PAb421 antibody to the C-terminal basic region, and Importantly, the defect in folding produced by to a lesser extent deletion of this region, increases the substitution of one residue does not seem to be resistance of the core domain to thermal denaturation as irreversible: at least some p53 mutants maintain residual assessed by the retention of PAb1620 epitope upon DNA-binding ability. Mutants that fail to bind DNA at heating to 371C (Hansen et al., 1996). The N-terminal 371C can bind at subphysiological temperatures, for domain also appears to affect the folding of the core: the example 32 or 251C (Bargonetti et al., 1993; Zhang antibodies DO1 and PAb1801 that detect N-terminal et al., 1994), and several mutants can activate transcrip- epitopes (residues 20–25 and 46–55, respectively) protect tion from a p53-responsive promoter at 261C (Fried- p53 from thermal denaturation and inactivation of lander et al., 1996; Di Como and Prives, 1998). In DNA binding at 371C (Friedlander et al., 1996; Hansen addition, the isolated mutant core domain proteins et al., 1996). R245S, R282W, V143A and others were shown to have Some mutant p53 proteins were shown to bind and residual (30–60%) DNA-binding activity at 201C (Bul- inactivate the p53 homologs and p63 (Strano et al., lock et al., 2000). The transcriptional transactivation 2000; Gaiddon et al., 2001). Interestingly, p73 binding is activity of at least 10% of p53 mutants reported in p53 influenced not only by the nature of the mutation but databases is temperature-sensitive (Shiraishi et al., also by a in p53 codon 72, which is either 2004). Thus, many p53 mutants maintain the intrinsic Arg or Pro. Mutant p53 binding to p73 is enhanced ability to bind DNA. when codon 72 encodes Arg (Marin et al., 2000). As a The temperature sensitivity of DNA binding by p53 conformational change in the core has a major impact mutants is often linked to a temperature-dependent on binding to p73 (Strano et al., 2000; Gaiddon et al., variation in protein conformation. Even one of the most 2001; Bensaad et al., 2003), these results suggest that the heavily destabilized mutants, R175H, can adopt a native Pro/Arg residue at position 72 can affect the folding of fold at low temperature: R175H protein produced in the core. Yet another study has supported a role of the insect SF9 cells at 251C is predominantly PAb1620 þ / Pro-rich domain (residues 62–93) in core domain PAb240À, whereas the same mutant produced at 371Cis folding: deletion of this region resulted in attenuation mainly unfolded as it binds PAb240, but not PAb1620 of DNA binding similar to the effect produced by (Cohen et al., 1999). In conclusion, both biochemical certain tumor-derived point mutations in the core (Roth and structural studies suggest that the distortion of et al., 2000). In addition, a synthetic peptide spanning mutant p53 core domain folding and defects in mutant the Pro-rich region was shown to affect the DNA- p53 DNA binding are not irreversible. binding properties of the core domain (Mu¨ ller-Tiemann et al., 1998). Cross-talk between the different p53 domains has also Impact of the N- and C-terminal domains on the folding been indicated in earlier studies, showing that destabi- of the core lization of the core by mutation (R273H) inhibits the Accumulating experimental data suggest that the transactivation activity of the N-terminal domain carboxy- and amino-terminal domains are involved in (Fields and Jang, 1990). More recent NMR studies

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2247 confirm that the N- and C-terminal domains have an replaced by N/C-terminal interactions forming a dimer. impact on the thermodynamic instability of the p53 Further, tetramers are formed via the contacts between tetramer (Bell et al., 2002). core domains and N/C nodes in close proximity to the However, until recently it has remained unclear core domains (Figure 2). This provides a framework for whether and exactly how the cross-talk between p53 understanding existing biochemical data on the effect of domains occurs. Although the X-ray structures of p53 the N and C termini on the conformation and DNA domains, including the core domain, the oligomerization binding of the core domain. Since the N/C nodes of each domain and short stretches from both the C terminus dimer are in direct contact with the core domains of and the N terminus have been reported, the overall another dimer, any modification of the N or C termini, packing and mutual orientation of domains in p53 for example phosphorylation, , antibody tetramer is still unknown. The structure of the full- binding or small molecules, may affect the core, length p53 protein presents a formidable challenge to stabilizing and/or shifting the equilibrium to one state structural biologists and has eluded numerous attempts or another. Further structural studies of the full-length of crystallization, largely because of the intrinsic tetramer are required to understand the exact nature flexibility of the protein, leading to its aggregation at and consequences of the interactions between the p53 high concentration. domains. The first structure of the intact full-length p53 One important point that emerges from the studies tetramer has been solved at 13.7 A˚ resolution using mentioned above is that the binding of ligand(s) to the cryoelectron microscopy (cryoEM) (Okorokov et al., N- or C-terminal regions of the p53 protein can 2006). According to the proposed model, based on probably stabilize the conformation of the core domain, three-dimensional reconstruction of tetrameric p53, the and therefore, such ligands could be useful in therapeu- p53 molecule has a D2 symmetry and has a shape of a tic strategies aimed at stabilization of mutant p53 hollow skewed cube, in which the upper and lower layers conformation. are represented by dimers which are rotated approxi- mately 601 with respect to each other (Figure 2). The most surprising finding is that the interaction between residues 323–363 of the C termini, previously observed Current strategies for mutant p53 rescue using isolated C-terminal proteins, is suppressed and Reactivation of mutant p53 bystructural manipulations and peptides A significant proportion of mutants, including both DNA contact and structural hot-spot mutants such as R273 H, R273C, R248Q, R282W and G245S, can be activated for DNA binding by C-terminal manipula- tions, for example binding of PAb421 antibody, trunca- tion or phosphorylation (Hupp et al., 1993; Halazonetis and Kandil, 1993; Niewolik et al., 1995; for a review, see Selivanova and Wiman, 2001). Moreover, the transcrip- tional transactivation function of the common mutant R273H was restored by microinjection of PAb421 antibody or PAb421-derived Fv fragments in tumor cells (Abarzua et al., 1995; Caron de Fromentel et al., 1999). In addition, several monoclonal antibodies that recognize N-terminal epitopes can partially rescue the DNA binding of p53 mutants (Cohen et al., 1999). Finally, deletion of N-terminal residues 13–16 has been found to reverse the temperature-sensitive phenotype of the mouse mutant A135V (Liu et al., 2001). These Figure 2 Model for structural organization of p53 tetramers. results, taken together with the proposed new structure (a) Left: the upper density layer of the p53 3D map containing fitted structures of two core domains shown in green and orange. of the p53 tetramer discussed above, support the idea The positions of the N and C termini of two monomers forming a that regulatory regions at the N- and C-terminal ends of dimer are indicated by arrows. Nt1 and Ct1 represent the N and C the protein are suitable targets for the development of termini of one monomer; Nt2 and Ct2 represent the N and C p53-reactivating molecules. As both N- and C-terminal termini of the second monomer. (a) Right: schematic representa- tion of monomer interactions. N/C nodes are represented as blue/ manipulations can rescue several different mutants, magenta joints. Linkers representing N termini are in blue and these data indicate that a generic approach for mutant those representing C termini are in magenta. The core nodes are p53 reactivation is feasible. In other words, each shown as spheres colored as their corresponding core domains. (b) individual mutation will not require its own unique Left: core domain structures are fitted into the corresponding nodes reactivation strategy. of the tetramer. (b) Right: a schematic model of the p53 tetramer. The core nodes are colored as their corresponding core domains, Rational design and genetic selection has allowed whereas N and C termini and corresponding linkers are colored as the identification of secondary suppressor mutations in in (a). the core domain that rescue the DNA binding,

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2248 transcriptional activity and in some cases, the growth place; after that, CDB3 can be replaced by tighter suppressor function of mutant p53 (Wieczorek et al., binding cognate DNA (Friedler et al., 2002). CDB3 can 1996; Brachmann et al., 1998; Nikolova et al., 2000). enter cells and bind p53 in the context of cellular The major effects of second-site mutations include proteins. It restores the PAb1620 þ conformation and introduction of additional DNA contacts, correction transcriptional activity of two hot-spot p53 mutants, of local distortions and increase of overall stability of R273H and R175H, in human tumor cells (Issaeva et al., the core. These studies have established the important 2003). Interestingly, CDB3 induces accumulation of concept that a structural defect conferred by a p53 core both wild-type and mutant p53 in cells. Although CDB3 domain mutation can be fixed by manipulations outside does not trigger a complete biological response, it can the mutation site itself. serve as a lead for the further development of p53- A synthetic peptide derived from the p53 C-terminal reactivating molecules. domain (peptide 46) was shown to restore the specific p53 core domain stabilization may also be mediated DNA binding and transcriptional transactivation func- by binding to cellular chaperone proteins. p53 can bind tion of several hot-spot mutant p53 proteins and rescue to the chaperone proteins Hsp40, Hsp70 and Hsp90 the function of endogenous mutant p53 proteins (Walerych et al., 2004). Unfolded mutant p53 binds resulting in growth inhibition and cell death by Hsp70 with higher affinity than the wild-type protein apoptosis (Selivanova et al., 1997; Kim et al., 1999). (Rudiger et al., 2002). This has led to the idea that The observation that C-terminal peptides can restore the binding of Hsp proteins stabilizes p53 in an unfolded DNA-binding activity to isolated core domain proteins conformation and therefore, disruption of Hsp binding indicated that it might directly affect the p53 domain to mutant p53 should rescue p53 conformation. Several where tumor-derived mutations are clustered. It was antitumor fungal antibiotics including hypothesized that C-terminal peptides reactivate mutant have been shown to inhibit Hsp90-dependent unfold- p53 through stabilization of the core domain folding ing of p53 (Blagosklonny et al., 1996). Compounds and/or establishment of novel DNA contacts (Abarzu´ a targeting Hsp90 are already undergoing clinical trials et al., 1995; Selivanova et al., 1997, 1998, 1999). These (reviewed by Sharp and Workman, 2006). findings provided proof-of-principle for functional restoration of tumor-derived mutant p53 proteins by small molecules. Recent in vivo studies have demon- Small molecules that target mutant p53 strated that treatment of peritoneal carcinomas and The structural studies and work on short peptides lymphomas with cell-penetrating C-terminal peptides discussed above have clearly demonstrated that mutant can activate endogenous mutant p53, resulting in p53 proteins can be reactivated with regard to both disease-free animals and significantly increased lifespan DNA binding, transcriptional transactivation and in- (Snyder et al., 2004). duction of apoptosis in human tumor cells. In attempts The first rational design of a molecule capable of to exploit this knowledge for development of novel stabilizing p53 core domain folding was undertaken by anticancer drugs, screening efforts have been conducted Fersht and colleagues. The idea behind this approach is with the aim of identifying small molecules that that a ligand that binds with higher affinity to the reactivate mutant p53 with similar or higher efficiency properly folded state of p53, will shift the equilibrium of than for example the p53-derived C-terminal peptide. the mutant core conformation towards this state. An Small molecules offer certain important advantages as important requirement is that ligand binding should not therapeutic agents over peptides and larger macromo- interfere with specific DNA binding. A short peptide lecules. Most importantly, they are suitable for large- derived from the p53-interacting protein ASPP (pre- scale GMP synthesis and can potentially be used for viously referred to as 53BP2) (Samuels-Lev et al., 2001) systemic treatment, for example intravenous or oral served as a basis for the design of such a ligand. ASPP is administration. Systemic therapy is critical for efficient a p53-binding protein that interacts with the core treatment of metastases in patients with disseminated domain and enhances p53-dependent transactivation, tumors. This is particularly important as this group of specifically stimulating the apoptotic function of p53 patients generally has the lowest survival and thus (Vives et al., 2006). A nine-residue peptide, CDB3, was represents the greatest challenge for therapy. designed based on the crystal structure of the complex Screening for mutant p53 reactivating compounds has between the p53 core and ASPP/53BP2 (Gorina and been carried out using either protein assays or cellular Pavletich, 1996). NMR provided solid evidence that assays. Protein assays may enable the identification of CDB3 binds to the core domain and induces the compounds with a defined molecular mechanism, but refolding of mutant p53 core domain proteins (Friedler such hits may not necessarily be able to enter cells and/ et al., 2002). In vitro studies showed that CDB3 restores or may be too toxic owing to additional protein targets sequence-specific DNA binding to various p53 mutants, or induction of DNA damage. Cellular assays, on the including the highly destabilized I195T mutant. A other hand, are likely to yield hits with the desired chaperone mechanism was invoked by the authors: biological effect, such as cell death by apoptosis, but core-stabilizing factors like the CDB3 peptide probably investigators may face difficulties with elucidation of the have to bind p53 during or immediately after biosynth- exact molecular mechanism. esis to stabilize mutant p53 in an active conformation Novel compounds targeting mutant p53 have before rapid unfolding of the mutant protein takes been identified using both types of screening assays.

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2249 CP-31398 was identified by screening for molecules that The effects of PRIMA-1 on mutant p53 are clearly protect the isolated p53 core domain protein from different from those of CP-31398 and CDB3. Whereas thermal denaturation, as assessed by maintenance of CP-31398 confers protection from unfolding at phy- PAb1620 reactivity upon protein heating (Foster et al., siological temperature, PRIMA-1 converts already un- 1999). In contrast, PRIMA-1 and MIRA-1 were folded p53 into an active form independently of protein identified in a cellular screening for compounds that synthesis (Bykov et al., 2002a). Furthermore, PRIMA-1 induce apoptosis selectively in human tumor cells does not activate wild-type p53, in contrast to both CP- expressing exogenous mutant p53 (Bykov et al., 2002a, 31398 (see above) and CDB3 (Issaeva et al., 2003), and 2005b). The compounds were shown to rescue wild-type causes a decrease in the overall level of p53 in cells. conformation of mutant p53 proteins in vitro and induce The ability of compounds like CP-31398 and PRI- expression of p53 target genes such as , and/ MA-1 to rescue mutant p53 suggests that they may or PUMA. Moreover, both CP-31398 and PRIMA-1 increase sensitivity of tumor cells to chemotherapeutic can inhibit tumor growth in vivo in mice (for a review, drugs that are preferentially cytotoxic for tumor cells see Bykov et al., 2003). carrying wild-type p53. This idea has been confirmed The mechanism of action of CP-31398 remains in vitro and in vivo in several studies. Synergy has been unclear. NMR studies failed to detect any binding of observed between CP31398 and adriamycin or cisplatin CP-31398 to the p53 core domain (Rippin et al., 2002). (Takimoto et al., 2002) and between PRIMA-1MET Interestingly, CP-31398 increases the levels of wild-type and adriamycin, cisplatin or fludarabine (Nahi et al., p53 protein in cells by preventing its ubiquitination 2004; Bykov et al., 2005a). The synergy could result independently of Mdm-2 and p53 phosphorylation (Luu from enhanced expression of mutant p53 induced by et al., 2002; Takimoto et al., 2002; Wang et al., 2003). chemotherapeutic drugs (Bykov et al., 2005a). This CP-31398 affects and induces cell death indicates that PRIMA-1 may synergize with any agent both in a p53-dependent and -independent manner that increases expression of mutant p53 in tumor cells. (Takimoto et al., 2002; Wischhusen et al., 2003; Wang Clearly, the observed synergy may have important et al., 2003). Thus, it appears that CP-3138 has other implications for anticancer therapy in the future, as cellular targets than p53 that may account for its cellular combination treatment may allow lower doses of toxicity. conventional chemotherapy and therefore less severe PRIMA-1 (for p53 reactivation and induction of side effects. massive apoptosis), the methylated analog PRIMA- The list of mutant p53-targeting small molecules also 1MET and MIRA-1 (for mutant p53 reactivation and includes the aminothiol WR1065, a derivative of the induction of rapid apoptosis) have similar activity cytoprotective drug amifostine that protects normal cells profiles (Bykov et al., 2002a, b, 2005a, b). Yet they are from the toxic effects of irradiation through free-radical structurally unrelated and presumably affect mutant p53 scavenging and probably other unknown mechanisms through different mechanisms. So far, direct binding to without affecting the killing of tumor cells. WR1065 can mutant p53 has not been unequivocally demonstrated. at least partially restore wild-type conformation to the Further studies using NMR, X-ray crystallography and/ temperature-sensitive V272M p53 mutant at 371C, or mass spectrometry are needed to resolve this issue. stimulate its DNA binding activity and induce expres- An alternative mechanism could be targeting of cellular sion of the p53 target genes p21, GADD45 and MDM2, proteins that in turn bind p53 and restore its conforma- leading to cell-cycle arrest in G1 (North et al., 2002). tion and function. Indeed, PRIMA-1 induces expression WR1065 affects wild-type p53 as well; this effect of heat-shock protein 90 (Hsp90) and enhances its involves the JNK pathway and DNA damage-indepen- binding to mutant p53, suggesting that Hsp90 mediates dent p53 phosphorylation and stabilization. Moreover, mutant p53 refolding (Rehman et al., 2005). Moreover, WR1065-mediated activation of p53 relies, at least in PRIMA-1MET triggers redistribution of mutant p53 to part, on its ability to reduce thiol groups in p53 (Pluquet nucleoli, along with three other promyelocytic leukemia et al., 2003). protein (PML) body-associated proteins, PML, cAMP- Redox effects are apparently also involved in the responsive binding protein (CBP) and heat-shock action of MIRA-1 and related compounds. All MIRA protein 70 (Hsp70) (Ro¨ kaeus et al., 2006). PRIMA- compounds contain a maleimide group that could Dead, an inactive structural analog, failed to induce potentially react with thiol and amino groups in nucleolar redistribution of mutant p53 and other PML proteins. Interestingly, a reactive 3–4 double bond in body-associated proteins, consistent with the notion that the maleimide group seems to be required for the effect nucleolar translocation and perhaps interaction with on mutant p53 (Bykov et al., 2005b). Human p53 Hsp70 and other PML body proteins plays a role in contains 10 cysteine residues, all of which reside in the reactivation of mutant p53. PRIMA-1 may in fact core domain, and it is well established that p53 is subject induce apoptosis via multiple pathways, as it can also to redox regulation (Buzek et al., 2002; Seo et al., 2002). trigger mutant p53-dependent apoptosis in the absence Thus, it is conceivable that covalent modification of of transcription or de novo protein synthesis, or even a cysteine residues could have a role in conformational (Chipuk et al., 2003). This notion is further rescue and restoration of p53 functions. Modification of supported by studies showing that PRIMA-1 induces cysteine residues in the mutant p53 core could inhibit apoptosis via the c-Jun-NH2-kinase (JNK) pathway (Li intramolecular or intermolecular disulfide bond forma- et al., 2005). tion, which might promote proper core domain folding

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2250 and/or prevent protein aggregation. Further studies specifically affect tumor cells, as the p53 pathway is should address whether covalent modification of thiol intact in normal cells. According to this concept, there is groups in the core domain may stabilize mutant p53. no need to target and reconstitute multiple deficient pathways in the tumor cells; reconstitution of the p53 Screening for identification of novel mutant p53-targeting pathway will be sufficient to trigger apoptosis. Cellular compounds stress in the form of oncogene activation, DNA damage and hypoxia will provide critical constitutive signaling Although several low molecular compounds that re- that cooperates with mutant p53 reactivation and activate mutant p53 have been identified during recent sensitizes to p53-induced apoptosis. years, it is clearly important to perform additional An anticipated problem is development of screens of chemical libraries in order to identify resistance to therapy due to loss of mutant p53 compounds with even higher potency and selectivity for mutant p53-expressing cells. Cellular screening expression in tumors or ablation of downstream effectors in the p53 pathway. Resistance through assays should aim at the identification of compounds clonal selection is a fundamental problem in essentially that elicit the most desired outcome, that is, cell death by apoptosis. To allow a more precise analysis of all anticancer therapy. The observed synergistic effects between currently used chemotherapeutic the molecular mechanism, such cellular assays can be drugs and novel compounds like PRIMA-1 suggest combined with an analysis of p53-dependent transcrip- that combination treatment that targets multiple cellular tion using a p53 reporter system, for example luciferase pathways simultaneously will be a useful strategy or GFP, in the same cells. This may enable the for overcoming resistance to mutant p53-reactivating identification of two distinct classes of compounds that drugs. target mutant p53-expressing cells: those that act Although the effect of identified mutant p53-targeting through restoration of p53-dependent transcription molecules on cell growth and survival are relatively well and those that trigger p53-dependent apoptosis via characterized, their exact mechanisms of action remain transcription-independent mechanisms. This type of screening strategy was recently used to identify a series poorly understood. As discussed above, restoration of wild-type p53 functions may involve direct binding of of compounds that trigger a p53 response in both p53 a low molecular weight compound to mutant p53 in null and mutant p53-carrying colorectal carcinoma cells a manner that promotes correct folding of at least a and inhibit tumor growth in vivo (Wang et al., 2006). fraction of mutant p53 protein molecules in the cell To increase the screening throughput, a first round of (Figure 3). Alternatively, mutant p53-reactivating mo- screening should use cells carrying inducible exogenous lecules could act indirectly in various ways (Figure 4). mutant p53 only, and aim at the identification of potent Elucidation of the exact molecular mechanisms behind inducers of apoptosis, preferably at the nM range. In the next screening step, identified compounds can be tested mutant p53 reactivation by novel compounds is an for mutant p53 selectivity using the same cells lacking important goal. This will require further structural mutant p53 expression. The effect of the identified studies by NMR and other techniques, as well as global compounds should also be tested in cells carrying analysis of gene expression patterns by microarrays and endogenous mutant p53. In this case, the effect should proteomics. be compared with the effect in the same cells upon The successful identification of p53-targeting small silencing of mutant p53 expression by siRNA using an molecules during recent years should not discourage inducible knockdown system. In parallel, screening of chemical libraries should also be carried out using protein-based assays. Since misfolded mutant p53 does not bind specifically to DNA and fails to bind PAb1620 antibody, assays based on rescue of DNA and/or PAb1620 antibody binding could be designed and used for high-throughput screening. Hit molecules should be further tested in various cellular assays to confirm a mutant p53-dependent biological effect. Regardless of the type of assay, the mutant p53-dependent antitumor efficacy of identified hits should be assessed in vivo in suitable mouse tumor models.

Concluding remarks

Mutant p53 reactivation by small molecules is a rapidly evolving approach with great potential for the deve- Figure 3 Proposed mechanism for targeting mutant p53-carrying tumor cells by a small molecule that binds directly to mutant p53. lopment of novel anticancer drugs. Inactivation of Such a compound may restore both p53-dependent transcription the p53 pathway is most likely an universal feature of and p53-mediated transcription-independent effects, resulting in tumor cells. Hence, p53-reactivating molecules should massive apoptosis and elimination of the tumor.

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2251 scaffolds for targeting the p53 pathway in human tumors. The molecules identified so far may prove unsuitable for clinical application for many reasons, including problems related to potency and toxicity. Further screening will increase the odds of finding compounds with robust clinical effect and low toxicity. The impressive body of information about the structure of wild-type p53 and the structural consequences of p53 mutations, along with improved tools for screening and drug development, will facilitate the design of additional mutant p53-targeting compounds in the future, many of which may act through entirely novel mechanisms. Thus, mutant p53 reactivation as a field is likely to provide both novel leads for drug development and important new insights into p53 function and regula- tion. Figure 4 Putative indirect mechanisms for targeting mutant p53- carrying tumor cells. A mutant p53-reactivating compound may act via cellular chaperones such as Hsp90 and restore wild-type Acknowledgements conformation to mutant p53. Alternatively, mutant p53-targeting compounds may inhibit binding of mutant p53 to the p53 family We thank the Swedish Cancer Society, EU FP6, the Cancer member protein p73 that unleashes p73-dependent apoptosis. Society of Stockholm, the Swedish Research Council and the Karolinska Institutet for generous support, and Andrej Okorokov for help with Figure 2. We apologize to all our investigators from initiating screening of chemical colleagues whose work could not be cited because of space libraries with the aim of identifying novel molecular limitations.

References

Abarzu´ a P, LoSardo JE, Gubler ML, Neri A. (1995). Bullock AN, Henckel J, Fersht AR. (2000). Quantitative Restoration of the transcription activation function to analysis of residual folding and DNA binding in mutant p53 mutant p53 in human cancer cells. Cancer Res 55: core domain: definition of mutant states for rescue in cancer 3490–3494. therapy. Oncogene 19: 1245–1256. Ang HC, Joerger AC, Mayer S, Fersht AR. (2006). Effects of Butler JS, Loh SN. (2003). Structure, function, and aggrega- common cancer mutations on stability and DNA binding of tion of the zinc-free form of the p53 DNA binding domain. full-length p53 compared with isolated core domains. J Biol Biochemistry 42: 2396–2403. Chem 281: 21934–21941. Buzek J, Latonen L, Kurki S, Peltonen K, Laiho M. (2002). Bargonetti J, Manfredi JJ, Chen X, Marshak DR, Prives C. Redox state of tumor suppressor p53 regulates its sequence- (1993). A proteolytic fragment from the central region of specific DNA binding in DNA-damaged cells by cysteine p53 has marked sequence-specific DNA-binding activity 277. Nucleic Acids Res 30: 2340–2348. when generated from wild-type but not from oncogenic Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, mutant p53 protein. Genes Dev 7: 2565–2574. Chumakov P et al. (2002a). Restoration of the tumor Bell S, Klein C, Muller L, Hansen S, Buchner J. (2002). p53 suppressor function to mutant p53 by a low-molecular- contains large unstructured regions in its native state. J Mol weight compound. Nat Med 8: 282–288. Biol 322: 917–927. Bykov VJN, Issaeva N, Selivanova G, Wiman KG. (2002b). Bensaad K, Le Braas M, Unsal K, Strano S, Blandino G, Mutant p53-dependent growth suppression distinguishes Tominaga O et al. (2003). Change of conformation of the PRIMA-1 from known anticancer drugs: a statistical DNA-binding domain of p53 is the only key element for analysis of information in the National Cancer Institute binding of and interference with p73. J Biol Chem 278: database. 23: 2011–2018. 10546–10555. Bykov VJN, Issaeva N, Zache N, Shilov A, Hultcrantz M, Be´ roud C, Soussi T. (1998). p53 gene mutation: software and Bergman J et al. (2005b). Reactivation of mutant p53 and database. Nucleic Acids Res 26: 200–204. induction of apoptosis in human tumor cells by maleimide Blagosklonny MV, Toretsky J, Bohen S, Neckers L. (1996). analogs. J Biol Chem 280: 30384–30391. Mutant conformation of p53 translated in vitro or in vivo Bykov VJN, Selivanova G, Wiman KG. (2003). Small requires functional HSP90. Proc Natl Acad Sci USA 93: molecules that reactivate mutant p53. Eur J Cancer 39: 8379–8383. 1828–1834. Brachmann RK, YuK, Eby Y, Pavletich NP, Boeke JD. Bykov VJN, Zache N, Stridh H, Westman J, Bergman J, (1998). Genetic selection of intragenic suppressor mutations Selivanova G, Wiman KG. (2005a). PRIMA-1(MET) that reverse the effect of common p53 cancer mutations. synergizes with cisplatin to induce tumor cell apoptosis. EMBO J 17: 1847–1859. Oncogene 24: 3484–3491. Bullock AN, Henckel J, DeDecker BS, Johnson CM, Canadillas JM, Tidow H, Freund SM, Rutherford TJ, Nikolova PV, Proctor MR et al. (1997). Thermodynamic Ang HC, Fersht AR. (2006). Solution structure of p53 core stability of wild-type and mutant p53 core domain. Proc domain: structural basis for its instability. Proc Natl Acad Natl Acad Sci USA 94: 14338–14342. Sci USA 103: 2109–2114.

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2252 Caron de Fromentel C, Gruel N, Venot C, Debussche L, tetrameric DNA binding domain and restore DNA binding Conseiller E, Dureuil C et al. (1999). Restoration of to select p53 mutants. EMBO J 12: 5057–5064. transcriptional activity of p53 mutants in human tumour Hansen S, Hupp TR, Lane DP. (1996). Allosteric regulation cells by intracellular expression of anti-p53 single chain Fv of the thermostability and DNA binding activity of human fragments. Oncogene 18: 551–557. p53 by specific interacting proteins. J Biol Chem 271: Chene P. (2003). Inhibiting the p53-MDM2 interaction: 3917–3924. an important target for cancer therapy. Nat Rev Cancer 3: Hupp TR, Lane DP. (1994). Allosteric activation of latent p53 102–109. tetramers. Curr Biol 4: 865–875. Chipuk JE, Maurer U, Green DR, Schuler M. (2003). Pharma- Hupp TR, Meek DW, Midgley CA, Lane DP. (1993). cologic activation of p53 elicits Bax-dependent apoptosis in Activation of the cryptic DNA binding function of mutant the absence of transcription. Cancer Cell 4: 371–381. forms of p53. Nucleic Acids Res 21: 3167–3174. ChristophorouMA, Martin-Zanca D, SoucekL, Lawlor ER, Issaeva N, Friedler A, Bozko P, Wiman KG, Fersht AR, Brown-Swigart L, Verschuren EW et al. (2005). Temporal Selivanova G. (2003). Rescue of mutants of the tumor dissection of p53 function in vitro and in vivo. Nat Genet 37: suppressor p53 in cancer cells by a designed peptide. Proc 718–726. Natl Acad Sci USA 100: 13303–13307. ChristophorouMA, RingshausenI, Finch AJ, Swigart LB, Joerger AC, Allen MD, Fersht AR. (2004). Crystal structure Evan GI. (2006). The pathological response to DNA of a superstable mutant of human p53 core domain. Insights damage does not contribute to p53-mediated tumour into the mechanism of rescuing oncogenic mutations. J Biol suppression. Nature 443: 214–217. Chem 279: 1291–1296. Cho Y, Gorina S, Jeffrey PD, Pavletich NP. (1994). Crystal Joerger AC, Ang HC, Fersht AR. (2006). Structural basis for structure of a p53 tumor suppressor-DNA complex: under- understanding oncogenic p53 mutations and designing standing tumorigenic mutations. Science 265: 346–355. rescue drugs. Proc Natl Acad Sci USA 103: 15056–15061. Cohen PA, Hupp TR, Lane DP, Daniels DA. (1999). Joerger AC, Ang HC, Veprintsev DB, Blair CM, Fersht AR. Biochemical characterization of different conformational (2005a). Structures of p53 cancer mutants and mechanism of states of the Sf9 cell-purified p53His175 mutant protein. rescue by second-site suppressor mutations. J Biol Chem FEBS Lett 463: 179–184. 280: 16030–16037. Cook A, Milner J. (1990). Evidence for allosteric variants of Joerger AC, Friedler A, Fersht AR. (2005b). Wild type p53 wild-type p53, a tumour suppressor protein. Br J Cancer 61: conformation, structural consequences of p53 mutations 548–552. and mechanisms of mutant p53 rescue. In: Hainaut P, Di Como CJ, Prives C. (1998). Human tumor-derived p53 Wiman KG (Eds). 25 years of p53 Research pp 377–397. proteins exhibit binding site selectivity and temperature Kim AL, Raffo AJ, Brandt-Rauf PW, Pincus MR, Monaco R, sensitivity for transactivation in a yeast-based assay. Abarzua P et al. (1999). Conformational and molecular Oncogene 16: 2527–2539. basis for induction of apoptosis by a p53 C-terminal peptide Espinosa JM, Emerson BM. (2001). Transcriptional regulation in human cancer cells. J Biol Chem 274: 34924–34931. by p53 through intrinsic DNA/chromatin binding and site- Lane DP, Lain S. (2002). Therapeutic exploitation of the p53 directed cofactor recruitment. Mol Cell 8: 57–69. pathway. Trends Mol Med 8: S38–S42. Fields S, Jang SK. (1990). Presence of a potent transcrip- Li Y, Mao Y, Brandt-Rauf PW, Williams AC, Fine RL. tion activating sequence in the p53 protein. Science 249: (2005). Selective induction of apoptosis in mutant p53 1046–1049. premalignant and malignant cancer cells by PRIMA-1 Foster BA, Coffey HA, Morin MJ, Rastinejad F. (1999). through the c-Jun-NH2-kinase pathway. Mol Cancer Ther Pharmacological rescue of mutant p53 conformation and 4: 901–909. function. Science 286: 2507–2510. LiuWL, Midgley C, Stephen C, Saville M, Lane DP. (2001). Friedler A, Hansson LO, Veprintsev DB, Freud SM, Rippin TM, Biological significance of a small highly conserved region in Nikolova PV et al. (2002). A peptide that binds and the N terminus of the p53 tumour suppressor protein. J Mol stabilizes p53 core domain: chaperone strategy for rescue Biol 313: 711–731. of oncogenic mutants. Proc Natl Acad Sci USA 99: 937–942. LuX, Lane DP. (1993). Differential inductionof transcrip- Friedler A, Veprintsev DB, Hansson LO, Fersht, A R. (2003). tionally active p53 following UV or : Kinetic instability of p53 core domain mutants: implica- defects in instability syndromes? Cell 75: tions for rescue by small molecules. J Biol Chem 278: 765–778. 24108–24112. Luu Y, Bush J, Cheung Jr KJ, Li G. (2002). The p53 stabilizing Friedlander P, Legros Y, Soussi T, Prives C. (1996). compound CP-31398 induces apoptosis by activating the Regulation of mutant p53 temperature-sensitive DNA intrinsic Bax/mitochondrial/caspase-9 pathway. Exp Cell binding. J Biol Chem 271: 25468–25478. Res 276: 214–222. Gaiddon C, Lokshin M, Ahn J, Zhang T, Prives C. (2001). A Marin MC, Jost CA, Brooks LA, Irwin MS, O’Nions J, subset of tumor-derived mutant forms of p53 down-regulate Tidy JA et al. (2000). A common polymorphism acts as an p63 and p73 through a direct interaction with the p53 core intragenic modifier of mutant p53 behaviour. Nat Genet 25: domain. Mol Cell Biol 21: 1874–1887. 47–54. Gannon JV, Greaves R, Iggo R, Lane DP. (1990). Activating McCormick F. (2001). Cancer gene therapy: fringe or cutting mutations in p53 produce a common conformational effect. edge? Nat Rev Cancer 1: 130–141. A monoclonal antibody specific for the mutant form. McKinney K, Mattia M, Gottfredi V, Prives C. (2004). p53 EMBO J 9: 1595–1602. linear diffusion along DNA requires its C terminus. Mol Gorina S, Pavletich NP. (1996). Structure of the p53 tumor Cell 16: 413–424. suppressor bound to the ankyrin and SH3 domains of McKinney K, Prives C. (2002). Efficient specific DNA binding 53BP2. Science 274: 1001–1005. by p53 requires both its central and C-terminal domains as Halazonetis TD, Kandil AN. (1993). Conformational shifts revealed by studies with high-mobility group 1 protein. Mol propagate from the oligomerization domain of p53 to its Cell Biol 22: 6797–6808.

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2253 Midgley CA, Desterro JM, Saville MK, Howard S, Sparks A, mutant p53 and PML nuclear body-associated proteins. Hay RT et al. (2000). An N-terminal peptide Oncogene Epub online. blocks Mdm2-dependent ubiquitination in vitro and can Roth J, Koch P, Contente A, Dobbelstein M. (2000). Tumor- activate p53 in vivo. Oncogene 19: 2312–2323. derived mutations within the DNA-binding domain of p53 Milner J, Watson JV. (1990). Addition of fresh medium that phenotypically resemble the deletion of the -rich induces and conformation changes in p53, a domain. Oncogene 19: 1834–1842. tumour suppressor protein. Oncogene 5: 1683–1690. Rudiger S, Freund SM, Veprintsev DB, Fersht AR. (2002). Mu¨ ller-Tiemann BF, Halazonetis TD, Elting JJ. (1998). CRINEPT-TROSY NMR reveals p53 core domain bound Identification of an additional negative regulatory region in an unfolded form to the chaperone Hsp90. Proc Natl for p53 sequence-specific DNA binding. Proc Natl Acad Sci Acad Sci USA 99: 11085–11090. USA 95: 6079–6084. Sabapathy K, Klemm M, Jaenisch R, Wagner EF. (1997). Nahi H, Lehmann S, Mollgard L, Bengtzen S, Selivanova G, Regulation of ES cell differentiation by functional and Wiman KG et al. (2004). Effects of PRIMA-1 on chronic conformational modulation of p53. EMBO J 16: 6217–6229. lymphocytic leukaemia cells with and without hemizygous Samuels-Lev Y, O’Connor DJ, Bergamaschi D, Trigiante G, p53 deletion. Br J Hematol 127: 285–291. Hsieh JK, Zhong S et al. (2001). ASPP proteins specifically Nevins JR. (2001). The Rb/ pathway and cancer. Hum stimulate the apoptotic function of p53. Mol Cell 8: Mol Genet 10: 699–703. 781–794. Nichols NM, Matthews KS. (2002). Human p53 phosphoryla- Selivanova G. (2001). Mutant p53: the loaded gun. Curr Opin tion mimic, S392E, increases nonspecific DNA affinity and Invest Drugs 2: 1136–1141. thermal stability. Biochemistry 41: 170–178. Selivanova G, Iotsova V, Okan I, Fritsche M, Strom M, Niewolik D, Vojtesek B, Kovarik J. (1995). p53 derived from Groner B et al. (1997). Restoration of the growth suppres- human tumour cell lines and containing distinct point sion function of mutant p53 by a synthetic peptide derived mutations can be activated to bind its consensus target from the p53 C-terminal domain. Nat Med 3: 632–638. sequence. Oncogene 10: 881–890. Selivanova G, Kawasaki T, Ryabchenko L, Wiman KG. Nikolova PV, Wong KB, DeDecker B, Henckel J, Fersht AR. (1998). Reactivation of mutant p53: a new strategy for (2000). Mechanism of rescue of common p53 cancer cancer therapy. Semin Cancer Biol 8: 369–378. mutations by second-site suppressor mutations. EMBO J Selivanova G, Ryabchenko L, Jansson E, Iotsova V, Wiman KG. 19: 370–378. (1999). Reactivation of mutant p53 through interaction of North S, Pluquet O, Maurici D, El-Ghissassi F, Hainaut P. a C-terminal peptide with the core domain. Mol Cell Biol 19: (2002). Restoration of wild-type conformation and 3395–3402. activity of a temperature-sensitive mutant of p53 (p53 Selivanova G, Wiman KG. (2001). Functional rescue of (V272M)) by the cytoprotective aminothiol WR1065 mutant p53 as a strategy to combat cancer. In: Maruta H in the esophageal cancer cell line TE-1. Mol Carcinog 33: (ed). Tumor-suppressing viruses, genes and drugs. Academic 181–188. Press: San Diego, CA, USA, pp 397–415. Okorokov AL, Sherman MB, Plisson C, Grinkevich V, Seo YR, Kelley MR, Smith ML. (2002). Selenomethionine Sigmudsson K, Selivanova G et al. (2006). The structure regulation of p53 by a ref1-dependent redox mechanism. of p53 tumour suppressor protein reveals the basis for its Proc Natl Acad Sci USA 99: 14548–14553. functional plasticity. EMBO J 25: 5191–5200. Sharp S, Workman P. (2006). Inhibitors of the HSP90 Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, molecular chaperone: current status. 95: 323–348. Hainaut P. (2002). The IARC TP53 database: new online Shiraishi K, Kato S, Han SY, LiuW, OtsukaK, Sakayori M mutation analysis and recommendations to users. Hum et al. (2004). Isolation of temperature-sensitive p53 muta- Mutat 19: 607–614. tions from a comprehensive missense mutation library. Ory K, Legros Y, Auguin C, Soussi T. (1994). Analysis of the J Biol Chem 279: 348–355. most representative tumour-derived p53 mutants reveals Sjo¨ blom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD that changes in protein conformation are not correlated with et al. (2006). The consensus coding sequences of human loss of transactivation or inhibition of cell proliferation. breast and colorectal cancers. Science 314: 268–274. EMBO J 13: 3496–3504. Snyder EL, Meade BR, Saenz CC, Dowdy SF. (2004). Peng Y, Chen L, Li C, LuW, Chen J. (2001). Inhibition of Treatment of terminal peritoneal carcinomatosis by a MDM2 by hsp90 contributes to mutant p53 stabilization. transducible p53-activating peptide. PLoS Biol 2: E36. J Biol Chem 276: 40583–40590. Strano S, Munarizz E, Rossi M, Castagnoli L, Shaul Y, Sacchi A, Pluquet O, North S, Richard MJ, Hainaut P. (2003). Oren M et al. (2000). Physical and functional interaction Activation of p53 by the cytoprotective aminothiol between p53 mutants and different isoforms of p73. JBiol WR1065: DNA-damage-independent pathway and redox- Chem 275: 29503–29512. dependent modulation of p53 DNA-binding activity. Takimoto R, Wang W, Dicker DT, Rastinejad F, Lyssikatos J, Biochem Pharmacol 65: 1129–1137. El-Deiry WS. (2002). The mutant p53-conformation modi- Rehman A, Chahal MS, Tang X, Bruce JE, Pommier Y, fying drug, CP-31398, can induce apoptosis of human Daoud SS. (2005). Proteomic identification of heat shock cancer cells and can stabilize wild-type p53 protein. Cancer protein 90 as a candidate target for p53 mutation reactiva- Biol Ther 1: 47–55. tion by PRIMA-1 in cells. Breast Cancer Vives V, Slee E, LuX. (2006). ASPP2: a gene that controls life Res 7: R765–74. and death in vivo. Cell Cycle 5: 2187–2190. Rippin TM, Bykov VJ, Freund SM, Selivanova G, Wiman KG, Vojtesek B, Dolezalova H, Lauerova L, Svitakova M, Havlis P, Fersht AR. (2002). Characterization of the p53-rescue drug Kovarik J et al. (1995). Conformational changes in p53 CP-31398 in vitro and in living cells. Oncogene 21: analysed using new antibodies to the core DNA binding 2119–2129. domain of the protein. Oncogene 10: 389–393. Ro¨ kaeus N, Klein G, Wiman KG, Szekely L, Mattsson K. Vousden K, Lu X. (2002). Live or let die: the cell’s response to (2006). PRIMA-1(MET) induces nucleolar accumulation of p53. Nat Rev Cancer 2: 594–604.

Oncogene Reactivation of mutant p53 G Selivanova and KG Wiman 2254 Walerych D, Kudla G, Gutkowska M, Wawrzynow B, Muller L, Wiman KG. (2006). Strategies for therapeutic targeting King FW et al. (2004). Hsp90 chaperones wild-type p53 tumor of the p53 pathway in cancer. Cell Death Differ 13: suppressor protein. JBiolChem279: 48836–48845. 921–926. Wang PL, Sait F, Winter G. (2001). The ‘wildtype’ conforma- Wischhusen J, Naumann U, Ohgaki H, Rastinejad F, Weller M. tion of p53: epitope mapping using hybrid proteins. (2003). CP-31398, a novel p53-stabilizing agent, induces p53- Oncogene 20: 2318–2324. dependent and p53-independent cell death. Oncogene Wang W, Kim S-H, El-Deiry WS. (2006). Small-molecule 22: 8233–8245. modulators of p53 family signaling and antitumor effects in Wong KB, DeDecker BS, Freund SM, Proctor MR, Bycroft M, p53-deficient human colon tumor xenografts. Proc Natl Fersht AR. (1999). Hot-spot mutants of p53 core domain Acad Sci USA 103: 11003–11008. evince characteristic local structural changes. Proc Natl Acad Wang W, Takimoto R, Rastinejad F, El-Deiry WS. (2003). Sci USA 96: 8438–8442. Stabilization of p53 by CP-31398 inhibits ubiquitination Zhang W, Guo XY, Hu GY, Liu WB, Shay JW, Deisseroth AB. without altering phosphorylation at serine 15 or 20 or (1994). A temperature-sensitive mutant of human p53. MDM2 binding. Mol Cell Biol 23: 2171–2181. EMBO J 13: 2535–2544. Wieczorek AM, Waterman JL, Waterman MJ, Halazonetis TD. Zheleva DI, Lane DP, Fischer PM. (2003). The p53-Mdm2 (1996). Structure-based rescue of common tumor-derived p53 pathway: targets for the development of new anticancer mutants. Nat Med 2: 1143–1146. therapeutics. Mini Rev Med Chem 3: 257–270.

Oncogene