Mutant p53 as a target for cancer treatment

Michael J. Duffy a,b,*, Naoise C. Synnott a, John Crown c

aUCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland
bUCD Clinical Research Centre, St. Vincent’s University Hospital, Dublin 4, Ireland
cDepartment of Medical Oncology, St Vincent’s University Hospital, Dublin 4, Ireland

Received 10 May 2017; accepted 20 June 2017

KEYWORDS p53;

Cancer; Inhibitor; APR-246
Abstract TP53 (p53) is the single most frequently altered gene in human cancers, with mu- tations being present in approximately 50% of all invasive tumours. However, in some of the most difficult-to-treat cancers such as high-grade serous ovarian cancers, triple-negative breast cancers, oesophageal cancers, small-cell lung cancers and squamous cell lung cancers, p53 is mutated in at least 80% of samples. Clearly, therefore, mutant p53 protein is an important candidate target against which new anticancer treatments could be developed. Although traditionally regarded as undruggable, several compounds such as p53 reactivation and induc- tion of massive apoptosis-1 (PRIMA-1), a methylated derivative and structural analogue of PRIMA-1, i.e. APR-246, 2-sulfonylpyrimidines such as PK11007, pyrazoles such as PK7088, zinc metallochaperone-1 (ZMC1), a third generation thiosemicarbazone developed by Critical Outcome Techonologies Inc. (COTI-2) as well as specific peptides have recently been reported to reactive mutant p53 protein by converting it to a form exhibiting wild- type properties. Consistent with the reactivation of mutant p53, these compounds have been shown to exhibit anticancer activity in preclinical models expressing mutant p53. To date, two of these compounds, i.e. APR-246 and COTI-2 have progressed to clinical trials. A phase I/IIa clinical trial with APR-246 reported no major adverse effect. Currently, APR-246 is undergo- ing a phase Ib/II trial in patients with advanced serous ovarian cancer, while COTI-2 is being evaluated in a phase I trial in patients with advanced gynaecological cancers. It remains to be shown however, whether any mutant p53 reactivating compound has efficacy for the treatment of human cancer.
ª 2017 Elsevier Ltd. All rights reserved.

 

 

 

* Corresponding author: Clinical Research Centre, St. Vincent’s University Hospital, Dublin 4, Ireland. Fax: þ353 1 2696018. E-mail address: [email protected] (M.J. Duffy).

http://dx.doi.org/10.1016/j.ejca.2017.06.023

0959-8049/ª 2017 Elsevier Ltd. All rights reserved.
1.Introduction

p53 (TP53) is one of the best studied genes involved in cancer formation and/or progression. Traditionally, p53 was believed to suppress cancer formation and pro- gression by inducing genes involved in cell cycle arrest, apoptosis or senescence or by participating in DNA repair. While these mechanisms of tumour suppression have been identified in several different model systems, more recent data suggests that p53 may also limit cancer formation via regulating metabolism, modulating reac- tive oxygen species (ROS) levels, altering expression of non-coding RNAs, enhancing autophagy or enhancing ferroptosis (for reviews, see refs. 1e3).
p53 accomplishes the above processes largely by acting as a homotetrameric transcription factor, binding to specific DNA sequences and regulating gene expres- sion. The specific genes and thus the specific processes altered as a result of binding to DNA appears to depend, at least in part, on the level of the p53 protein, its oligomerisation state, dynamics of its induction (slow, fast or pulsatile), presence of other transcriptional factors as well as the concentration of specific endoge- nous apoptotic-regulating proteins [4e7]. The specific genes altered may also depend on the presence and concentrations of p53 isoforms, some of which may be antagonistic to full-length p53 [8,9].
Impairment or loss of p53 function however, is widespread, if not universal in human malignancy. Impairment can occur either by mutation, gene deletion, protein sequestration by specific viral proteins, increased expression of negative regulators (e.g. MDM2 or MDM4) or alterations in upstream/downstream path- ways [1e3]. Of these inactivation mechanisms, mutation and interaction with the negative regulators MDM2 and MDM4 appear to be the most important. Since loss of function in p53 occurs in most cancers, reversing this process is an attractive strategy for the development of new treatments for the disease.
Several approaches have been investigated for restoring the lost function of p53 in cancer. These include reactivation of mutant p53 to a wild-type form, depletion of mutant p53, blocking the negative regula- tors MDM2 and MDM4, gene therapy with vectors containing wild-type p53, identification of synthetic le- thal partners for mutant p53 and treatment with com- pounds that promote readthrough of premature termination codons [10e12]. The aim of this article is to discuss recent developments with compounds that reactivate mutant p53 protein to a form exhibiting wild- type properties. Firstly, however, we briefly review the role of mutant p53 in malignancy.

2.p53 mutations in malignancy

Overall, p53 is believed to be mutated in approximately 50% of all human malignancies. In contrast to most

tumour suppressor genes such as the adenomatous polyposis coli (APC) gene in colorectal cancer, PTEN gene (coding phosphatase and tensin homologue) and BRCA1/2 genes (coding breast cancer 1/2 proteins), which are usually inactivated by truncating or deletion- type mutations, mutations in p53 are predominantly missense [1]. Thus, the full-length form of mutant p53 is usually found in tumours. Furthermore, as the mutant protein in malignant cells is less susceptible than wild- type p53 to degradation, it accumulates, thereby providing a potential target for anticancer drugs.
Two broad types of p53 mutation have been described, i.e. contact mutations and conformational mutations [13]. Contact mutated proteins largely maintain the wild-type folded protein conformation but because of the specific residues that are mutated, are unable to bind to p53 specific DNA promoter sites. Conformation (also known as structural) mutations cause protein destabilisation, lowering of the melting temperature and unfolding at physiological tempera- tures. This abnormal folding is potentially reversible however, (see below). Functionally, mutations in p53 can result in loss of its tumour suppressive properties, exertion of a dominant-negative effect on the remain- ing wild-type allele or gain of an oncogenic activity. This gain in oncogenic activities is particularly found when missense mutations occur in the DNA-binding domain. Oncogenic activities acquired by mutant p53 include increased proliferation, enhanced meta- static potential and acquisition of resistance to specific therapies [1].
Although mutations in p53 have been detected in essentially all types of malignancy, the prevalence across tumour types is highly variable. In general, mutations are more prevalent in solid than in haematological ma- lignancies. In a recent study using exome sequencing in 12 common cancer types, p53 was found to be the most frequently mutated gene in 10 of the tumour types investigated, the exceptions being renal and colorectal cancers [14]. Indeed, p53 has been found to be the most frequently mutated gene in some of the most difficult-to- treat cancers such as lung cancer (squamous and small- cell types) [15,16], triple-negative breast cancer [17], high-grade serous ovarian cancer [18] and oesophageal (squamous type) cancer [19]. In these cancers, p53 is mutated in at least 80% of cases [14e19].
Although mutations can be found across the p53 gene, approximately 95% are located in the DNA-binding domain of p53 [1]. Most, if not all of these mutations, impair the ability of p53 to bind with high affinity to its cognate DNA binding sites. As mentioned above, missense mutant forms of p53 however, can exert a gain of oncogenic functions. One of the mechanisms by which mutant p53 gains function is by associating with specific transcriptional factors (p63, p73, SP1, sterol regulatory element-binding proteins (SREBPs), vitamin D receptor, ETS1/2, nuclear factor Y, Nrf2) or chromatin regulating
proteins such as SWItching/Sucrose Non Fermenting (SWI/SNF) [20e24].
Association with these transcription factors results in the binding of mutant p53 to their cognate DNA bind- ing sites and activation of their respective downstream pathways. This in turn leads to the expression of specific genes potentially important in cancer formation, pro- gression and metastasis [3]. Amongst the genes shown to be regulated by mutant forms of p53 are VEGFR2 [23], methyltransferases (MLL1, MML2), acetyltransferases (MOZ ) [24], SLC7A11 [25], EGFR [26], PDGFR [27]
and components of the proteasomal system [28]. Further studies however, are necessary to establish whether different mutant forms of p53 regulate the same or different genes. In this context, Walerych et al. [28] reported that activation of the proteasomal genes was mediated by several different mutant forms of p53 in different types of malignant cells.

preventing ROS accumulation, inducting ferroptosis, or promoting autophagy [3,12,35]. Thus, based on current evidence from a limited number of model tumour sys- tems, p53 appears to be able to suppress cancer forma- tion/progressions using multiple mechanisms.
The preclinical studies discussed above clearly show that mutant p53-containing cancers are potentially vulnerable to reversion following introduction of the wild-type protein. Consequently, in recent years, several different pharmacological approaches have been attempted to reactivate mutant p53 for the treatment of cancer (Table 1) [10e12]. The most widely investigated compounds for this purpose are discussed below.

3.2.Quinuclidines: PRIMA-1 and APR-246

Of the compounds listed in Table 1, the most widely studied are PRIMA-1 {2,2-bis(hydroxymethyl)quinu- clidin-3-one or APR-017} and APR-246 {2-(hydrox-

3.2.3.Targeting mutant p53 for cancer treatment

3.2.3.1.Proof of principle

Several studies in different animal models provide proof of principle that restoration of wild-type p53 function can suppress tumour growth [29e34]. The impact of p53 restoration on tumour growth however, appears to depend on the stage of cancer progression. Thus, in an animal model of pineoblastoma, Harajly et al. [33]
found that wild-type p53 restoration induced senes- cence in preinvasive but not in invasive lesions. The failure to induce senescence in the invasive tumours was reported to be due to high levels of mouse double minute homologue 2 (MDM2) which led to degradation of the reinstated p53. Indeed, administration of the MDM2 inhibitor, nutlin was found to result in respon- siveness to p53. In contrast to the findings in pineal tumours, restoration of p53 in murine lung cancers led to regression in the malignant but not in adenoma le- sions [32]. In this model, responsiveness to p53 was found to depend on p19arf signalling which upregulated levels of p53.
The mechanism by which introduction of wild-type p53 mediates tumour regression also appears to vary,
ymethyl)-2-(methoxymethyl)quinuclidin-3-one or PRIMA-1MET }. The 3-quinuclidinone derivative dub- bed PRIMA-1 was initially discovered following the screening of a library of low-molecular-weight com- pounds (NCI Diversity Set) for their ability to restore wild-type properties to mutant p53 such as; conversion to its active conformation, sequence-specific binding and the induction of apoptosis [36]. Although different types of missense mutation are found in p53, PRIMA-1 was found to restore specific DNA binding to 13 of the 14 different p53 mutant proteins tested, the exception being p53 possessing the Phe-176 mutation [36]. Thus, PRIMA-1 appeared to restore at least some wild-type p53 properties irrespective of the mutation location or whether mutations were of the structural or contact type [36]. The pro-apoptotic activity and membrane perme- ability of PRIMA-1 was subsequently enhanced by the addition of a methyl group, giving rise to the com- pound known as APR-246 or PRIMA-1MET [37]. Both PRIMA-1 and APR-246 have been shown to inhibit cell

Table 1
Compounds shown to reactivate mutant forms of p53. pCAP, p53 conformation activating peptides.
Compound Proposed mode of action

depending on the specific tumour model used. Thus, introducing wild-type p53 into mice lymphomas resulted in cell death by apoptosis [29,31]. In contrast, in liver and pineal tumours, it induced growth arrest and senescence [30,33]. In this liver cancer model, the in- duction of senescence gave rise to an immune response
PRIMA-1, APR-246,a MIRA1,
STIMA-1, COTI-2 PK7088, PK11000,
PK11007,a PK11010 ZMC1a
Convert mutant p53 to wild-type conformation
Increase thermal stability of mutant p53, thereby restoring wild-type activity
Restore zinc to zinc-deficient p53 mutant, thereby promoting

that subsequently led to tumour regression [30]. As mentioned above, although induction of apoptosis, cell cycle arrest and senescence are the classical mecha- nisms through which p53 is believed to suppress cancer formation/maintenance, newer identified mechanisms by which it may do so include altering metabolism,
wild-type folding and function ReACp53 Prevent mutant p53 aggregation
pCAP-250 and related peptides Stabilise wild-type conformation of mutant p53 and restore its sequence-specific DNA binding
a These compounds have also been shown to reduce GSH and in- crease ROS levels.
proliferation and promote apoptosis in a wide range of cancer cell lines and to exhibit anticancer activity in several different xenograft tumour systems [36e47].
To reactivate mutant p53, both PRIMA-1 and APR- 246 are first converted to the active metabolite, methy- lene quinuclidinone (MQ) which in turn covalently re- acts with specific thiol groups in the mutant protein [48]. Using computational analysis of p53 structural models, a transiently open pocket close to Cys124, Cys135 and Cys141 was identified as a potential binding site for MQ [49]. Binding of MQ caused refolding of the mutant protein. Evidence for this refolding was the finding of a differential interaction between APR-246 pre-treated and post-treated p53 with conformation-specific anti- p53 antibodies. Thus, following the binding of MQ, reactivity with the mutant-specific antibody, PAb240 decreased, while interaction with the wild-type antibody, PAb1620 increased [48].
Although most studies reported that PRIMA-1 and APR-246 selectively inhibited cancer cells expressing mutant p53, some reports concluded that these com- pounds can act independently of the mutant protein. Thus, in addition to reactivating mutant p53, APR-246 has also been reported to bind to and activate the p53- homologous proteins, p63 and p73 [50e53]. Other mechanisms by which APR-246/MQ has been reported to exert anticancer activity include reducing glutathione formation and increasing ROS production [53e57]. Thus, in oesophageal cancer cells, MQ was found to react with glutathione (GSH) which resulted in ROS accumulation [22]. The mitochondrial accumulation of ROS led to lipid peroxidation, mitochondrial rupture, release of cytochrome C and triggering of apoptosis.
Although both PRIMA-1 and APR-246 display anticancer activity when used alone, this activity can be enhanced when combined with different conventional and experimental systemic cancer treatments (Table 2). The extent of enhancement however, depends on the specific cell line investigated and specific compound added to PRIMA-1 or APR-246. Thus, using breast cancer cell lines, Synnott et al. [44] found synergistic growth inhibition when APR-246 was combined with eribulin in the two different cell lines investigated, i.e. in

MDA-MB-453 and MDA-MB-468 cells. In contrast, combined treatment with docetaxel plus APR-246 gave an additive effect in MDA-MB-453 cells but provided no enhanced inhibition in MDA-MB-468 cells. On the other hand, combined addition of carboplatin and APR- 246 was additive in MDA-MB-468 cells but not in MDA-MB-453 cells. No enhanced benefit was seen when APR-246 was combined with cisplatin in these two cell lines.
APR-246 was the first mutant p53 reactivating com- pound that progressed to clinical trials. In a phase I clinical trial, APR-246 was administered as a 2- h intravenous infusion once per day for 4 consecutive days to 22 patients with either haematological malig- nancies or prostate cancer [63]. Overall, the inhibitor was found to be well tolerated, the most frequent adverse effects being fatigue, dizziness, headache and confusion. Malignant cells obtained from patients with leukaemia undergoing treatment with APR-246 were found to exhibit cell cycle arrest, increased apoptosis, and increased expression of p53 target genes (PUMA, NOXA, BAX ). These finding are consistent with reac- tivation of mutant p53 in the APR-246-treated patients.
Currently, APR-246 is undergoing a phase Ib/II study trial (PiSARRO) in patients with recurrent, high-grade serous ovarian cancer (clinical trial code, NCT02098343). Patients for the trial are selected based on tumour cell positivity for p53 protein staining which is used as a surrogate biomarker for the presence of mutant p53. In this trial, APR-246 is being administered in combina- tion with carboplatin and pegylated liposomal doxoru- bicin every 4 weeks for 6 cycles. End-points for the trial include determination of the appropriate APR-246 dose, safety, pharmacokinetics, progression-free survival, response rate and overall survival. Preliminary finding from this trial suggest that APR-246 is well tolerated with an acceptable safety profile [64,65].
Although multiple preclinical studies have shown that APR-246 exhibits anticancer activity and early clinical trials suggest that it is relatively non-toxic [63e65], several questions regarding its use remain to be addressed. These include its relative reactivity for the different p53 mutated forms; how exactly its active form, MQ, binds to mutant p53; its potential for binding to molecules other than those mentioned above; possible

Table 2
Anticancer agents shown to synergise with PRIMA-1 or APR-246 in inhibiting cancer cell growth in model systems.
Cancer type Drug Refs.
adverse effects of long-term treatment and most importantly, whether it has clinical efficacy as an anti- cancer agent. Answers to some of these questions should emerge from the ongoing clinical trial in patients with

Lung (NSC) Doxorubicin, cisplatin, camptothecin, 5-FU, olaparib
37,58,59
ovarian cancer.

Ovarian Cisplatin, doxorubicin 58
Pancreatic Gemcitabine, erlotinib, bortezomib, nutlin-3 60 Oesophageal Cisplatin, 5-FU, sulfasalazine 22,61
Breast Eribulin, olaparib, carfilzomib 28,44
AML Daunorubicin 62 NSC, non-small cell; 5-FU, 5-fluorouracil; AML, acute myeloid
leukaemia.
3.3.Pyrazoles: PK7088

Unlike PRIMA-1 and APR-246 which apparently reactivate several different mutant forms of p53 protein, PK7088 (1-methyl-4-phenyl-3-(1H-pyrrol-1-yl)-1H-pyr- azole) is thought to bind specifically to the Y220C
mutated form of the protein [66]. The Y220C mutation destabilises p53 and creates a surface crevice into which PK7088 can attach. Binding of PK7088 to the Y220C mutated protein was found to increase its melting tem- perature (Tm). This increase in Tm led to increased levels of folded mutant p53 with wild-type like confor- mation, and furthermore restored at least some of its wild-type transcriptional activity. Consistent with the restoration of wild-type transcriptional activity, treat- ment of Y220C mutant cells with PK7088 led to increased expression of the p53 target genes, p21 and NOXA as well as induction of cell-cycle arrest and apoptosis [66]. The potential anticancer activity of PK7088 does not appear to have been investigated in an animal model.

3.4.2-Sulfonylpyrimidines: PK11000, PK11007 and PK11101

Like PK7088, PK11000 (5-chloro-2-methanesulfonyl- pyrimidine-4-carboxylic acid) was originally identified by its ability to increase the Tm of the Y220C p53 DNA binding domain [67]. Further work however, showed that PK11000 increased the Tm not only of the mutant Y220C form but also of the full length p53 protein in the absence of the Y220C mutation, indicating that stabili- sation did not occur by binding to the mutant-associated crevice. Rather, using NMR spectroscopy, Cys182 and Cys277 in the p53 protein were found to be the most reactivate sites for the binding of PK11000. Subse- quently, 2 structural analogues of PK11000, i.e. PK11007 and PK11010 were synthesised and found to react with p53 faster than PK11000. Both these com- pounds were also found to be more potent in inducing p53 stability than the parent molecule [67]. Although all 3 compounds inhibited cancer cell viability in vitro, PK11007 was shown to be the most potent [68]. Further work showed that PK11007 preferentially inhibited the viability of gastric and breast cancer cell lines with mutationally compromised p53 compared to cell lines containing wild-type p53 [67,68]. However, similar to APR-246, PK11007 has also been found to act inde- pendently of mutant p53, i.e. by depleting GSH and increasing concentration of ROS [67].

3.5.Zinc-metallochaperones: ZMC1

Wild-type p53 protein requires critical amounts of zinc binding for protein folding, stabilization and interaction with DNA response elements [13]. Consequently, impaired zinc binding leads to protein destabilisation and loss of sequence-specific DNA binding. Zinc met- allochaperones are low molecular weight molecules that function by transporting zinc into cells, donating it to zinc-deficient proteins such as specific mutant forms of p53. Following attachment to specific mutant forms of p53, wild-type folding and function are restored [69].

One of the most detailed studied metallochaperones is the thiosemicarbazone compound, ZMC1 (also known as NSC319726) [70,71]. In an earlier study, Yu et al. [70] showed that ZMC1 inhibited cell viability and induced apoptosis in tumour cells containing the zinc- deficient R175H mutant form of p53. Following administration to a xenograft model, ZMC1 was found to exhibit anticancer activity specifically in tumours possessing the p53 R175H mutation. In contrast, no growth inhibition was observed in tumours with wild- type p53 or in tumours expressing the p53 R273H mu- tation (a mutation that does not impair zinc binding). As with APR-246, ZMC1 appeared to mediate its anti- cancer effects by inducing wild-type conformation and restoring DNA-binding to the R175H mutant form of p53. Also, similar to APR-246 [54e57] and PK11007 [67], ZMC1 was found to decrease cellular GSH levels and increase ROS levels. A subsequent report showed that ZMC1 also reactivated other p53 mutant proteins with impaired zinc-binding, i.e. proteins possessing the C238S, C242F and C176F mutations [71].

3.6.Thiosemicarbazone: COTI-2

COTI-2 is a thiosemicarbazone-related compound identified using a computational platform known as CHEMAS [72]. CHEMAS is an in silico machine learning system that predicts target biological activities from molecular structures [72]. COTI-2 appears to act both by reactivating mutant p53 and inhibiting the [phosphatidylinositol 3-kinase (PI3K)/Akt, a serine- threonine kinase /mammalian target of rapamycin (mTOR)] PI3K/AKT/mTOR pathway [73]. It is unclear however, whether COTI-2 targets multiple or specific mutant forms of p53. Although COTI-2 was found to inhibit the growth of a diverse range of cancer cell lines [72], mutant p53 cell lines were found to be preferentially inhibited by the compound compared to p53 wild-type cells [73]. Similar to the situation in vitro, COTI-2 was found to exhibit anticancer activity in different animal model systems. In vivo, the compound was reported to be well tolerated with no evidence of morbidity or weight loss [72]. Currently, COTI-2 is undergoing eval- uation for the treatment of gynaecological cancers in a phase I clinical trial (NCT02433626).

3.7.Small peptides

In addition to the low molecular weight molecules dis- cussed above, several different peptides have been re- ported to reactivate mutant p53 [74e78]. These peptides were discovered using different approaches; have different molecular sizes; display different amino acid sequences and bind to different regions of the p53 pro- tein (Table 3). Furthermore, they reactivate mutant p53 using different mechanisms (Table 3). Thus, using a rational design approach, Soragni et al. [77] identified a
Table 3
Peptides reported to reactivate mutant p53.
Peptide Discovery method Sequence p53 binding region Mode of action Refs.

Peptide 46 In silico GSRAHSSHLKSKKGQSTSRHKK DBD and C-terminal Restoration of WT-folding and
DNA-specific binding
74

CDB3
In silico
REDEDEIEW
DBD
Stabilisation and restoration of DNA-specific binding
76

ReACp53 In silico LTRITLE DBD Blocks aggregation 76

pCAP Phage display
DBD, DNA binding domain; pCAP, p53 conformation activating peptides.
DBD Restoration of WT-folding 75
peptide dubbed ReACp53 which was found to block the amyloid-like aggregation of mutant p53 proteins con- taining either the R248Q or R175H mutation. By inhibiting this aggregation, ReACp53 rescued p53 wild- type properties such as; induction of its canonical genes, promotion of apoptosis and reduction of cell prolifera- tion. Administration of conjugated ReACp53 to mice bearing ovarian carcinomas expressing aggregation- prone p53 mutations resulted in decreased prolifera- tion and tumour shrinkage.
Rather than using an in silico approach, Tal et al. [75]
investigated the potential of small peptides generated by phage display to reactivate mutant p53. Amongst the large number of peptides identified, several converted mutant p53 to a form with wild-type-like conformation, restored DNA sequence-specific binding and induced expression of p53 target genes. Consistent with the reactivation of mutant p53, some of these peptides were found to induce apoptosis selectively in mutant p53 containing cancer cells. To establish whether the pep- tides exhibited anticancer activity, mixtures were injec- ted into a number of different murine cancer models expressing mutant p53. Following their administration, tumour regression was seen in breast cancers, ovarian cancers and colorectal cancers.
The disadvantage of using peptides for therapeutic purposes is their intrinsic chemical and physical insta- bility. Furthermore, depending on their amino acid composition, they may exhibit low membrane perme- ability [78]. The problem of instability however, can be reduced by chemical modification such as stapling or conjugation to carriers such as liposomes or magnetic nanoparticles [78,79]. Peptides however, are potentially more specific for their target than non-peptide low molecular weight compounds. A further advantage of using peptides is they are less likely to be excluded from cells by multiple drug resistance mechanisms [75].

4. Conclusion

Because of its high mutation frequency and critical role in driving cancer formation/progression, mutant p53 is a high-priority target for anticancer therapy. However, as mentioned in the Introduction above, until recently, mutant p53 was regarded as undruggable. As discussed above, this situation has now clearly changed, as several

compounds have recently become available that can reactivate mutant p53 to a form with wild-type prop- erties. Several questions however, need to be addressed in order to progress the available compounds for po- tential cancer treatment. These include, their detailed mechanism of action; their specificity for mutant p53 including their specificity for different mutations; their ability to interact and synergise with available treat- ments; possible complications of long-term treatment and most important, their efficacy for treating cancer. Answers to the last two questions should soon emerge, at least for APR-246, from the ongoing phase Ib/II clinical trial in ovarian cancer.

Conflict of interest statement

JC received honoraria and research funding from Eisai Ltd.

Acknowledgements

We thank Science Foundation Ireland, Strategic Research Cluster Award (08/SRC/B1410) to Molecular Therapeutics for Cancer Ireland (MTCI), the BREAST- PREDICT (CCRC13GAL) program of the Irish Cancer Society and the Clinical Cancer Research Trust for funding this work.

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