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The tumour suppressor p53 is a transcription factor thatregulates several genes with a broad range of functions,including DNA repair, metabolism, cell cycle arrest,apoptosis and senescence (see BOX 1for cell fate deci-sions after p53 activation, and BOX 2for the mechanismsof p53-mediated tumour cell killing). Protein levels ofp53 within cells are tightly controlled and kept lowbyits negative regulator, the E3 ubiquitin protein ligaseMDM2, which binds to the amino terminus of p53, tar-geting it for ubiquitylation and subsequent degradation.The interaction of p53 and MDM2 has been conservedacross 2.4 billion years of evolution1.MDM2is a p53target gene, thus creating an autoregulatory feedbackloop. MDMX (also known as MDM4), the structuralhomologue of MDM2, has no ubiquitylation activitybut is able to bind to the N-terminus of p53 and inacti-

vate it directly or to aid MDM2 in ubiquitylating p53 byheterodimerization with MDM2 (REFS 2,3).

p53 is inactivated by mutations in over 50% of allcancers4. Such mutations can disrupt its direct bindingto DNA or lead to structural perturbations that preventthe correct folding or oligomerization of the tumour sup-pressor. At other times, loss of p53 function is due to over-expression of p53-regulatory proteins that suppress p53activity, such as MDM2 and MDMX. In mouse models,the absence of p53 leads to the development of spontane-ous tumours, notably in the thymus. Li Fraumeni syn-drome, a rare autosomal dominant hereditary disorderthat is characterized by the early onset of several differenttypes of cancer, is caused by a mutant p53 loss-of-functionallele. Thus, p53 acts as a tumour suppressor in humans.

Conversely, normal p53 has an essential destructive role inthe killing of radiosensitive tissues after exposure to ion-izing radiation. Various studies have separated these twofunctions of p53. Therefore, as we discuss below, the prop-erties of p53 that prevent tumour occurrence are distinctfrom those that allow activated p53 to kill tumour cells.

Numerous strategies have been devised to correct adysfunctional p53-regulatory pathway. Small-moleculeinhibitors of the p53MDM2 interaction, p53 genetherapies and drugs that act as chaperones by bindingto mutant p53 and restoring its function are some of theapproaches currently in clinical trials (TABLE 1). A break-through in the field was the development of nutlin, thefirst small-molecule inhibitor of the p53MDM2 inter-action5. Currently, the most advanced MDM2 inhibitorsinclude RG7112 (Roche), MI-773 (Sanofi) and DS-3032b(Daiichi Sankyo), which are at various stages of Phase Iclinical trials. Preclinical lead compounds have also been

published by Amgen and Novartis, and PRIMA-1MET(Aprea) which restores the activity of mutant p53 has also recently completed Phase I trials. The literatureon the p53 system is growing very rapidly, so here wefocus on the latest insights into p53 function and thedevelopments and controversies in p53 drug discovery.

Genetic models of restoration of p53 function

A key issue in the development of any p53-based therapythat is not exclusively targeted to tumour cells is tounderstand the effects of p53 activation on normalhuman tissues. Here, the use of genetically engineeredmouse models has been especially powerful.

1p53 Laboratory (p53Lab),

Agency for Science,

Technology and Research

(A*STAR), 8A Biomedical

Grove, #06-06, Immunos,

138648 Singapore.2Bioinformatics Institute,

Agency for Science,

Technology and Research

(A*STAR), 30 BiopolisStreet #07-01, Matrix,

138671 Singapore.3School of Biological

Sciences, Nanyang

Technological University,

60 Nanyang Drive,

637551 Singapore.4Department of Biological

Sciences, National University

of Singapore, 14 Science

Drive 4, 117543 Singapore.

Correspondence to D.P.L.

e-mail:

[email protected]

doi:10.1038/nrd4236

Drugging the p53 pathway:understanding the route toclinical efficacyKhoo Kian Hoe1, Chandra S. Verma24and David P. Lane1

Abstract | The tumour suppressor p53 is the most frequently mutated gene in human cancer,

with more than half of all human tumours carrying mutations in this particular gene.

Intense efforts to develop drugs that could activate or restore the p53 pathway have nowreached clinical trials. The first clinical results with inhibitors of MDM2, a negative regulator

of p53, have shown efficacy but hint at on-target toxicities. Here, we describe the current

state of the development of p53 pathway modulators and new pathway targets that have

emerged. The challenge of targeting proteinprotein interactions and a fragile mutant

transcription factor has stimulated many exciting new approaches to drug discovery.

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NATURE REVIEWS |DRUG DISCOVERY VOLUME 13 |MARCH 2014 |217

2014 Macmillan Publishers Limited. All rights reserved
mailto:dplane%40p53Lab.a%E2%80%91star.edu.sg?subject=mailto:dplane%40p53Lab.a%E2%80%91star.edu.sg?subject=


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A concern of therapies that aim to restore wild-type p53 activity is that these might lead to widespreadapoptosis in normal tissues. -irradiation, for exam-ple, has been shown to lead to p53 accumulation andp53-dependent toxicity in tissues such as the spleen,thymus, intestine and haematopoietic cells in the bonemarrow6,7. An important study in a lymphomagenesismodel using tamoxifen-regulated p53 showed that whenp53 was toggled on, whole-body -irradiation inducedp53-dependent apoptosis in radiosensitive tissues

but mice were protected from tumour development 8.Toggling p53 off at the time of -irradiation sparedmice from these radiation-induced toxicities but leftthem vulnerable to tumour development. However, micewere protected from radiation-induced cancers when p53was transiently toggled on for 6 days, starting at 8 daysafter irradiation. This protection required the action ofthe small p53-activating protein ARF. The role of p53 inradiation-induced toxicity, by contrast, did not requireARF. These results suggest that the tumour-suppressive

Box 1 | Cell fate outcomes after p53 induction: apoptosis, cell cycle arrest or senescence?

Effective cancer therapy requires the irreversible elimination of tumour cells. Although it is still not clear which functions

of the tumour suppressor p53 are vital for tumour suppression and surveillance, harnessing the apoptosis and senescence-

inducing functions of p53 would be most useful for cancer therapy. It is also not clear why tumour cells are usually more

susceptible to cell death than normal cells. The concept of oncogene addiction, where tumour cells become dependent

on activated oncogene signalling for survival, is important in this context 16. The mechanisms by which p53 activation

controls cell fate outcomes apoptosis, cell cycle arrest or senescence are thought to be mediated by promoter

selectivity and response magnitude and duration. Promoter selectivity can either be defined by the p53 DNA-bindingsequence of the promoter or it can be induced by post-translational modifications of p53. Furthermore, it can be

determined by the absolute levels of p53 or by the presence of p53-interacting proteins. Moreover, the level of expression

of anti-apoptotic proteins in the target cell is crucial for cell fate outcomes after p53 activation.

p53 can be regulated by upstream signalling pathways in response to cellular stresses in many different ways.

The example of p53 phosphorylation on Ser46 (REF. 210)is discussed below. Moreover, degradation of p53 through the

E3 ubiquitin protein ligase MDM2 pathway can affect cell fate outcomes. The level of p53 protein itself, which is tightly

regulated, can determine promoter selectivity. Higher levels of p53 protein are known to lead to apoptosis, whereas

lower levels result in cell cycle arrest151,211. Although p53 recognition sites in the promoters of specific genes have

different binding affinities to the p53 protein, the decision between cell cycle arrest or apoptosis is not determined by

p53 protein concentration alone212,213. It seems that binding to apoptosis-inducing genes, as opposed to cell cycle arrest

or other pro-survival genes, requires the highly cooperative binding of p53 to multiple binding sites214. For example, a

single mutation in p53, E177R, which is located at the interaction interface between the DNA-binding domains of two

molecules in the p53 tetramer, was shown to hinder cooperative binding to DNA. This mutation abolishes the apoptotic

functions of p53 while retaining control of cell-cycle, senescence, metabolic and antioxidant functions214,215.

Using a p53-inducible system, it was shown that changes in the level of p53 correlated with the levels of transcriptional

activation of genes involved in cell cycle arrest and apoptosis. Apoptosis, however, only proceeded when a certain

threshold was reached151.

Another mechanism of regulation is the differential transcription of downstream genes owing to their binding to

cofactors. For example, p53 recruits cofactors such as ASPP1 (apoptosis-stimulating of p53 protein 1) or ASPP2, which

can promote the binding of p53 to the promoters of pro-apoptotic BAXor p53-inducible protein 3 (PIG3), but not to

CDKN1AorMDM2promoters, thus enhancing p53-induced apoptosis216. The binding of p53 to inhibitor of ASPP protein

(iASPP) can have the opposite effect217. iASPP has been found to be overexpressed in breast cancer217and acute myeloid

leukaemia218. Higher levels of iASPP have also been correlated with drug resistance in ovarian cancer219and reported in

melanoma66. High iASPP expression was significantly associated with a clear cell carcinoma subtype (P= 0.003), and with

chemoresistance to carboplatin and paclitaxel (P= 0.04)219.

p53 and its regulatory proteins can also be post-translationally modified via acetylation or phosphorylation to

modulate the transcription of different genes220. It has been proposed that p53 might be regulated by a dual signal on

MDM2, as there are two separate interaction sites between MDM2 and p53 that could be differentially disrupted by

phosphorylation events in the amino terminus of p53 and the carboxy-terminal RING finger domain of MDM2 (REF. 221).

For example, phosphorylation of MDM2 at Ser394 in the RING finger domain by ataxia telangiectasia mutated (ATM) has

been found to be important for modulating the p53 response after DNA damage222. MDM2 has also been shown to be

subjected to phosphorylation at other serine/threonine sites.

p53 is, itself, regulated by phosphorylation at Thr18, which disrupts the interaction between p53 and MDM2.

Phosphorylation of Thr18 is also dependent on the prior phosphorylation of Ser15 of p53 (REF. 223). Conversely,

phosphorylation of Ser46 seems to favour the transcription of pro-apoptotic genes, such as p53-regulated

apoptosis-inducing protein (p53AIP), in response to DNA damage210. The Ser46 residue can be phosphorylated by

different kinases, including homeodomain-interacting protein kinase 2 (HIPK2)224,225. The p53 S46A mutation was

shown to render it unable to induce apoptosis in the HSC-2 human oral squamous cell carcinoma cell line226.

The extrinsic as well as intrinsic apoptotic signalling pathways can have a key role in the response to p53 activation.

Nutlin227was shown to induce p53-dependent cell cycle arrest, whereas 5-fluorouracil (5-FU) induced p53-dependent

apoptosis in the same cell type. Detailed biochemical analysis showed that this difference was due to the ability of 5-FU

to enhance the expression of TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as DR4) to a

greater extent than nutlin. The mechanism was, surprisingly, revealed to be due to the stabilization of the mRNA for DR4.

Thus, although both nutlin and 5-FU induced expression of the gene, only 5-FU led to the high-level accumulation of the

protein and to DR4-dependent apoptosis.

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b Senescence-induced phagocytosis

a Apoptotic threshold effect

Transcriptionalregulation by p53

ApoptosisCell cycle arrest

Levels of p53 Levels of PUMAand NOXA proteins

BCL-2, BCL-X and MCL1

p53

Nutlin

MacrophageTumour cell

Eat mesignal

p21

NOXA

PUMA

activities of p53 do not need to be toxic to be effectiveand that at least in this model the two pathways canbe distinguished by their differential requirement for theARF protein, which is required for tumour surveillancebut not for radiation-induced toxicity.

Induction of p53 function using the tamoxifen-regulated p53 system described above in Mdm2-nullanimals led to strong induction of apoptosis in radio-sensitive tissues9, which demonstrates their suscepti-bility towards p53-induced apoptosis. Conversely, inradiation-resistant tissues the induction of p53 inhibitedcell proliferation.

Other studies suggested that more subtle changes inthe levels of p53 in mice can effectively prevent tumourformation or lead to tumour regression. In one study,mice that were engineered to express varying reducedlevels of MDM2 were found to have higher p53 activ-ity across different tissues, which seemed to suppresslymphoma formation10. The effects of strong p53 acti-

vation on normal tissues included thymic ablation dur-ing development and increased levels of apoptotic cells

in the gut11. However, with a slightly lower level of p53activation, effects on normal tissues were avoided, buttumour suppression was still achieved.

It was also shown that a single nucleotide polymor-phism (SNP) in the promoter of theMDM2gene affectsMDM2 protein expression12. Higher levels of MDM2expression as a result of the SNP309 polymorphism ledto a sustained reduction in p53 activity, which promotedthe formation of tumours in mice. By contrast, mice thatharbour an additional copy of the complete p53 locusshow improved longevity and a reduced tumour bur-den13, which is dependent on the presence of ARF 14.These studies show that the level of p53 activity must be

very tightly controlled to achieve maximum tumour sur-veillance and tumour cell elimination without causingtoxicity to normal proliferative tissues. In theory, it shouldbe possible to control both the intensity and the dura-tion of the p53 response through careful pharmaceuticaldosing of prospective p53-activating drugs.

The mechanism of tumour regression in response togenetic restoration of p53 function was examined in dif-ferent mouse models of cancer and found to be tumour-type specific1517. Restoration of p53 in lymphomas led totheir widespread apoptosis, whereas restoration of p53in sarcomas and hepatocarcinomas led to a senescence-type response1517. In an orthotopic model of hepato-carcinoma, the senescence of tumour cells in response

to p53 reactivation was found to trigger an immune-inflammatory response that led to tumour clearance viaactivated macrophages17.

Further studies dissected the individual functionsof p53 that are required for tumour suppression. Threerecent provocative studies examined the functionalrequirements of p53 that confer resistance to the devel-opment of the spontaneous lymphomas that occur inp53-null mice. In the first study18, p533KR/3KRknock-inmutant mice were generated, in which three acetyla-tion sites within the DNA-binding domain of p53 weremutated. This led to defects in the cell-cycle, senes-cence and apoptosis-inducing functions of p53 owing

Box 2 | Mechanisms of p53-induced apoptosis

It is important to understand the downstream signalling mechanisms of tumour

suppressor p53, and to understand how the host of anti-apoptotic proteins such as

myeloid cell leukaemia sequence 1 (MCL1), B cell lymphoma 2 (BCL-2) and BCL-XL

might contribute towards resistance to apoptosis induced by agents (such as nutlin)

that promote p53 activation.

The apoptotic threshold effect

The tumour suppressor p53 induces apoptosis primarily via induction of thepro-apoptotic proteins phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1;

also known asNOXA) and p53-upregulated modulator of apoptosis (PUMA);PUMA

seems to have a more important role in normal cells22. The increase in levels of p53

protein leads to a corresponding accumulation of PUMA and NOXA. PUMA is able to

bind to mitochondrial anti-apoptotic proteins such as BCL-2, BCL-XL, BCL-W, MCL1 and

BCL-2-related protein A1 (BCL-2A1)230. By contrast, NOXA is only able to bind to MCL1

and BCL-2A1. Apoptosis occurs when an apoptotic threshold (see figure, part a) isreached owing to the inhibition of BCL-2, BCL-X

Land MCL1 by NOXA and PUMA.

Apoptosis, like blood clotting and complement fixation, is driven by a highly controlled

proteolytic cascade. These processes are characterized by highly cooperative

activation kinetics that are very sensitive to small concentration changes and are

irreversible. Interestingly, mice lacking both PumaandNoxadevelop normally and do

not seem to be more prone to developing cancer23,228. Moreover, it has been shown that

thymocytes lacking Puma are protected from -irradiation-induced apoptosis to the

same extent as p53-deleted thymocytes228. In E-Myc-driven lymphoma cells, killing byDNA-damaging drugs requires the p53-dependent induction of PUMA and NOXA, in

addition to the p53-independent induction of BCL-2-interacting mediator of cell death

(BIM)229.

Direct induction of cell death by p53

In addition to cellular responses that are dependent on p53 transcription, some studies

have suggested that a direct translocation of the p53 protein to the mitochondria, which

can activate pro-apoptotic BCL-2 family members, leads to apoptosis231,232. The induction

of apoptosis in chronic lymphocytic leukaemia by nutlin, for example, has been shown to

involve both transcription-dependent and transcription-independent mechanisms193.

In an exciting recent study it was shown that the inhibition of caspase activation blocks

nutlin-induced apoptosis but does not inhibit nutlin-induced cell death a finding that

strongly implies that p53 activation can induce caspase-independent forms of

programmed cell death such as necroptosis152.

The induction of senescence and macrophage engulfmentAnother mechanism by which p53 induction (for example, by nutlin) can lead to the

elimination of tumour cells is through the induction of senescence (mediated by the cell

cycle inhibitor p21) and an associated eat me (opsonization) signal (see figure, part b),which results in macrophage engulfment and killing of the senescent cells233.

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to the reduced ability of the mutant p53 to activate thetranscription of some but not all p53-induciblegenes. These mice were not, however, more suscep-tible to developing spontaneous tumours than wild-type mice. This surprising result was attributed to themetabolic and antioxidant functions of the mutant p53,achieved through its residual ability to induce transcrip-tional activation of the bisphosphatase enzyme TIGAR(TP53-induced glycolysis and apoptosis regulator).

In the second study19,20, knock-in mice were generatedin which the wild-type p53 gene Tp53 was replaced withTp53genes that were partially defective owing to muta-tions in the N-terminal transactivation domains TAD1and/or TAD2. The p5325,26TAD1 mutant, which is defec-tive with regard to the transactivation of many genes, cannevertheless lead to tumour suppression in various typesof cancer. It cannot induce either G1 arrest or apoptosisafter DNA damage, but it is able to induce senescencethrough p53 TAD2-driven transcription. Retention ofTAD1 in a TAD2 p5353,54mutant also provides effectivetumour protection. Conversely, the quadruple mutantp5325,26,53,54is completely impaired in p53-transcriptional

activities and was shown to be unable to prevent tumourdevelopment in several models and cell lineages. Thisimplies that the transcriptional activity of p53, as deter-mined by the N-terminal residues identified (25, 26,53 and 54), is important for tumour surveillance butthat activation of the full complement of p53-induciblegenes is not required because either the TAD1- or theTAD2-induced sets of genes are sufficient19,20.

The third and most recent study took a differ-ent approach; here, mice with a triple knockout ofcyclin-dependent kinase inhibitor 1A (Cdkn1a; whichencodes the p21 protein), p53-upregulated modu-lator of apoptosis (Puma; also known as Bbc3) and

phorbol-12-myristate-13-acetate-induced protein 1(Pmaip1; also known asNoxa) showed profound defectsin p53-driven induction of apoptosis, cell cycle arrestand senescence. CDKN1A (encoding p21), PUMAandNOXAare three of the most important p53-responsivegenes; p21 is required for growth arrest21, whereas PUMAand NOXA are required for the apoptotic response toradiation22. Nevertheless, even in this triple knockoutmouse model of the most important p53 effector genes,p53 was still able to exert efficient tumour-suppressivefunctions23. This, once again, suggests that other func-tions of p53, such as its regulation of DNA repair genesas well as genes involved in metabolism, have a cruciallyimportant role in tumour suppression. In this context,it is important to note that treatment of p53-null miceby incorporating the antioxidant N-acetylcysteine intotheir diet also prevents spontaneous tumour develop-ment. This strongly supports the idea that the antioxidantDNA repair response is crucial to the tumour-suppressivesurveillance activity of p53 (REF. 24).

These studies examined the level and duration of thep53 response that is necessary to prevent cancer formation

or to eliminate an established tumour. They also shed lighton the side effects in normal tissues that can be anticipatedfrom p53 therapy. Although the treatment of advancedcancers may require an apoptotic or senescence response,a DNA repair or antioxidant effect may be sufficient forthe treatment of pre-neoplasia (FIG. 1). It will be importantto establish which tumours contain p53 mutations in theacetylation sites18or the TADs19,20, and to assess the abilityof these partially defective p53 proteins to drive a thera-peutic response to chemotherapy and radiation. Theseresults do, however, suggest that a limited non-toxic p53response may be effective in tumour prevention, whichrepresents a very attractive goal for drug development.

Table 1 | p53 activators currently in clinical trials*

Compound Mechanism ofaction

Status ClinicalTrials.govidentifiers

Company

RG7112 (also knownas RO5045337)

Small-moleculeMDM2 antagonist

Phase I trial in advancedsolid tumours, solid tumours,haematological neoplasms andliposarcomas (all completed)

NCT00559533NCT01164033NCT00623870NCT01143740

Roche

RG7112 (also knownas RO5045337) withcytarabine


Phase I in AML (completed) NCT01635296 Roche

RG7112 (also knownas RO5045337) withdoxorubicin


Phase I in soft tissue sarcoma(completed)

NCT01605526 Roche

RO5503781 Small-moleculeMDM2 antagonist

Phase I in advanced malignancies(recruiting)

NCT01462175 Roche

RO5503781 withcytarabine


Phase I in AML (recruiting) NCT01773408 Roche

MI-773 (also knownas SAR405838)


Phase I in malignant neoplasms(recruiting)

NCT01636479 Sanofi

DS-3032b Small-moleculeMDM2 antagonist

Phase I in advanced solid tumourlymphoma (recruiting)

NCT01877382 DaiichiSankyo

PRIMA-1MET(alsoknown as APR-246)

Reactivation ofmutant p53

Phase I in haematological andprostatic neoplasms (completed)

NCT00900614 Aprea

AML, acute myeloid leukaemia. *Sourced from the ClinicalTrials.gov database.

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Induction of death andsenescence in tumourcells

Prevention of tumouroccurrence

Protection of tumourcells and normal cellsfrom cytotoxic therapy

Toxic effects of drugs(such as doxorubicin) andradiation in normal cells

Radiation, MDM2 inhibitor Oncogene stress Radiation, cytotoxic drugs (such as doxorubicin)

NOXA

PUMA

CDKN1A

PHLDA3

ABHD4

TIGAR

NOXA

PUMA

p53 p53 p53 p53

Senescence

Apoptosis

a b c d

CDKN1A

Methods for restoring p53 activity

In tumours that retain wild-type p53 but have defectsin p53-regulatory pathways, such as overexpression oramplification of MDM2 and MDMX or epigenetic silenc-ing of the INK4AARFlocus25, the major approach for therestoration of p53 activity has been to inhibit the func-tion of negative regulators of the p53 response26(FIG. 2a).Although a number of targets have been identified, suchas blocking the function of the viral E6 protein in inhibit-ing p53 in human papillomavirus (HPV)-driven cancersusing bortezemib27, or inhibiting the enzymatic E3 ligase

activity of MDM2 (REF. 28), the vast majority of currentefforts and current clinical trial activities are focusedaround small-molecule drugs that block the N-terminalproteinprotein interaction between p53 and MDM2 orMDMX. In a more general approach, very large chemicallibraries have been screened in cell-based assays for smallmolecules that can activate wild-type p53, but the targetsof these agents have only occasionally been identified for example, the small-molecule compound tenovin 6,which inhibits sirtuin 1 (SIRT1), thus enhancing theacetylation and activity of p53 (REF. 29).

In cases where the p53 protein is mutated, attemptshave been made to find molecules that act as p53

chaperones by binding to p53 and stabilizing its con-formation. Genetic studies that demonstrate second-sitereversion of the common p53 mutations have supportedsuch an approach30,31because they show that alterationsin other parts of p53 can correct the defect induced bythe primary mutation. Detailed structural studies haveyielded candidate mutant p53-binding molecules, andcell-based screening approaches for small molecules thatcan act as chaperones of mutant p53 have also been pur-sued. However, identifying the targets of the screeningcompounds has proved to be challenging. About 8% of

p53 mutations result in early termination of translation32,and read-through drugs are being sought that can allowthe bypassing of mutant stop codons and thus restore theexpression of p53.

Attempts have also been made to manipulate thep53 pathway using gene therapy or immunologicalapproaches. These have been recently reviewed (seeREFS 33,34).

Targeting interactions between MDM2 and p53 withsmall-molecule inhibitors.A small peptide region of p53,located in its TAD, was found to bind to MDM2 (REF. 35).The crystal structure of the N-terminal domain of MDM2

Figure 1 | The multiple functions of p53 and their impact on therapy. The p53 protein acts as a potent tumour

suppressor that can mediate the induction of apoptosis and senescence in tumour cells and prevent tumour

recurrence; it also mediates the toxic effects of some chemotherapy drugs in normal and tumour cells. The apoptotic

response requires the p53-responsive genes p53-upregulated modulator of apoptosis (PUMA) andNOXA22,

but remarkable experiments have recently shown that apoptosis is not required for tumour suppression in mouse

models18,19,23

. Similarly the cell cycle inhibitor p21 (encoded by the cyclin-dependent kinase inhibitor 1A ( CDKN1A)gene), which is important for the senescence response to p53 induction, is not required for tumour suppression23.

Triple-knockout mice lackingCdkn1a, PumaandNoxastill show a potent p53-dependent inhibition of tumour

development23. Instead, other p53-induced changes seem to be crucial, such as the DNA repair, metabolic and

antioxidant responses to p53 activation. This suggests that the type of p53 response that needs to be induced by a

pharmaceutical activator varies with the intended use of the medicine. Where the medicine is designed to kill tumour

cells (part a), it will be essential that the drug induces PUMA, NOXA and, in some cases, p21. However, if the medicine is

intended to suppress tumour development induced by oncogene stress (part b), these responses may not be required

and could indeed be harmful. Instead, the induction of genes such as TP53-induced glycolysis and apoptosis regulator

(TIGAR), abhydrolase domain-containing protein 4 (ABHD4) and pleckstrin homology-like domain family A member 3

(PHLDA3), which appear to be exerting a tumour-suppressive effect, is desired. A third response to p53 activation

(part c) can actually be to protect cells from toxic stimuli through the induction of growth arrest (via p21) and the

induction of antioxidant states. Paradoxically, this can make some tumour cells resistant to radiation therapy and

some chemotherapeutic drugs such as doxorubicin234, so p53 inhibitors might also be of value in drug combinations.

The toxic effects (part d) of p53 induction in normal tissues might also be eliminated by such p53 inhibitors.

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p53 activators MDM2 inhibitors orMDM2/MDMX dual inhibitors

E6 inhibitor

Tenovin 6

Downstream apoptotic orcell cycle arrest genes

Wild-type p53MDM2

MDM2

SIRT1

HPV E6

ARF

b

a

c d

Site 1

Site 1

Site 2

complexed with this peptide revealed the presence of adeep hydrophobic cleft in MDM2, into which the p53peptide was embedded36. The peptide interacted mainlythrough three critical amino acids: Phe19, Trp23 andLeu26. Using large phage display peptide libraries, anotherstudy independently identified these three amino acidsas being essential for the interaction between p53 andMDM2 (REF. 37). Furthermore, it was found that peptideswith far higher affinities for MDM2 could be discovered,thus supporting the concept that the pocket in MDM2

was druggable38. This was further established in assayswhere the MDM2-binding peptides were displayed on thesurface of a thioredoxin protein scaffold, thus stabilizingtheir expression in mammalian cells. Binding of MDM2by such mini-proteins led to the induction of high levelsof active p53 protein and inhibited the cell cycle, demon-strating that blocking the p53MDM2 proteinproteininteraction was sufficient to activate the p53 response37.

The first non-peptidic molecule that demonstratedthe possibility of interrupting the p53MDM2 interfacewas 4,5-dihydroimidazoline (nutlin; Roche)5. The crystalstructure of nutlin 3a, an isomer of nutlin, when boundto MDM2 (site 1 in FIG. 2b), provided the template for

the design of better inhibitors39(TABLE 2)such as thebenzodiazepinedione family of compounds (Johnson andJohnson, 80 nM)40, chromenotriazolopyrimidine (Amgen,1.2 M)41, terphenyls (Yale University, 182 nM)42,43andchalcones (Max Planck Institute, Martinsried, TbingenUniversity and Roche Diagnostics, 49 M)44,45; numbersin parentheses refer to maximum affinities.

At the same time, structure-based screening of com-pounds combined with molecular modelling enabled thedevelopment of new compounds (FIG. 2c). Using rational

engineering, guided by the interactions of the p53 pep-tide and nutlin with MDM2, a new class of inhibitors wasdeveloped: the spirooxindole-based molecules (Universityof Michigan)46. This led to the identification of MI-219,which has a subnanomolar affinity for MDM2, is orallyavailable, has good pharmacokinetic and pharmacody-namic (PK/PD) properties and is now in Phase I clinicaltrials. This compound led to an increase in p53 levels andan associated increase in levels of the p53-targeted genesCDKN1AandMDM2. It also selectively induced PUMAin osteosarcoma and prostate cells but not in normal cellsin vitroand in xenograft models46. However, completetumour regression in animal models remained elusive

Figure 2 | Mechanisms of activation of wild-type p53 to eliminate tumour cells. a| Strategies for activating

wild-type tumour suppressor p53. Tumours that retain wild-type p53 status often have dysfunctions in the regulatory

circuits that control p53 activity. These dysfunctions create targets for p53-activating therapies. The elevated expression

of the E3 ubiquitin protein ligase MDM2 and MDMX proteins or the loss of expression of the natural MDM2 inhibitor,

ARF, occurs commonly in tumours. Here, a dual MDM2 and MDMX inhibitor would be effective. In human papillomavirus

(HPV)-induced cancers, p53 is degraded by the viral protein host complex, and it has been shown that inhibition of the

viral protein E6 can restore p53 function27. More general p53 activators can be discovered by high-throughput screening;

for example, the small molecule tenovin-6 can activate p53 by promoting its acetylation through blockade of the sirtuin 1

(SIRT1) deacetylase29. b| The first crystal structure of a compound, nutlin 3 (in coloured sticks), bound to the surface of

MDM2 (REF. 235). The nutlin compound on the right binds to site 1, whereas the nutlin compound on the left binds to

site 2.c| Various MDM2 inhibitors (which bind to the surface and are shown in stick form) have been crystallographically

resolved and have been found to bind to site 1 (see above). Ligands include dihydroimidazothiazoles, oxopiperidine,

indole derivatives, imidazoles and oxomorpholins.d| The crystal structure of MDMX with the dimer-inducing indolyl

hydantoin RO-2443. The two small molecules pack against each other and are shown in stick form, along with the surfaceof each MDMX molecule72.

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until recently, when the same group developed a seriesof diastereomeric spirooxindoles (MI-888)47. These com-pounds bind to MDM2 with an inhibition constant (K

i)

of 0.44 nM and achieved complete tumour regression insevere combined immunodeficient (SCID) mice bear-ing SJSA-1 osteosarcoma tumour xenografts, with miceremaining tumour-free for 60 days after treatment47.

Simultaneously, other drugs that have been reportedinclude pyrazole and imidazole compounds (Universityof Pittsburgh, 20 nM)48, imidazole-indoles (Novartis,2 nM)49, isoindolinones (University of Newcastle,170 nM)50, pyrrolidinones such as PXN822 (Priaxon,


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with low micromolar affinity69; these are large molecules,and the structural mechanism underpinning the inhibitionremains to be determined.

There have also been attempts to develop small-molecule inhibitors against MDMX, such as SJ-172550,for use in combination with MDM2 inhibitors 70.Improvements in technological developments are alsoleading to the identification of new inhibitors; a recentexample is the report of a new affinity-based chemicalscreen that has led to the identification of novel micro-molar dual inhibitors of the p53MDM2 and p53MDMX interactions71. There are three new avenues that

have paved the way for the discovery of next-generationdrugs. RO-2443 (Roche)72is an indolyl hydantoin thatinhibits both MDM2 and MDMX with low micromolaraffinity. Structural analysis showed that two moleculesof RO-2443 stack on top of each other and bring twomolecules of MDMX into a tetrameric complex (FIG. 2d).However, this compound displays poor solubility.It served as a lead compound for the development ofRO-5963, which has a half-maximal inhibitory concen-tration (IC

50) of ~17 nM for MDM2 (the IC

50of nutlin 3a

is ~19 nM) and ~24 nM for MDMX (the IC50

of nutlin 3a is~9 M). The molecule showed efficacy in wild-typep53-containing cell lines from different lineages72.

Separately, it was found that synthetic analogues of5-deazaflavins bound to the RING finger domainof MDM2 with low micromolar affinity and prevented theubiquitylation of p53 in vitroand in vivo; these compoundsmimic the effects of ARF or ribosomal proteins such as L11and provide the starting point for the development of novelinhibitors26. Finally, a novel interaction has recently beenhypothesized to control the binding of nutlin-like drugsand to also mediate the transmission of allosteric inter-actions in the p53MDM2 system. The crystal structureof MDM2 with nutlin shows two molecules of nutlin thatare closely associated with the surface of MDM2 (FIG. 2b).

While one of the nutlin molecules binds to the site thathas been targeted for all small-molecule developmentstrategies (site 1 in FIG. 2b), hydrogendeuterium exchangeand computer simulation studies suggest that the secondsite (site 2 in FIG. 2b) may actually be the site where nut-lin first makes contact with MDM2 before tumbling intothe main binding pocket, and that this site of first contactmay be druggable73,74. Indeed, this hypothesis is furtherstrengthened by the demonstration that a mutation in thesecond site renders MDM2 resistant to nutlin binding 74. If

validated further, this will open a new window into drugdesign efforts, signalling a need to incorporate kineticeffects into the normally used thermodynamic signatures.

Table 2 | Compounds that bind to MDM2 or mutant p53

Compounds Company or institution Refs

Mechanism of action: binding to MDM2

Nutlin 3a, RG7112, RG7388, Ro-2443 Roche 5,62,63,72,152,153

MI-219, MI-713, MI-888 Ascenta Therapeutics, Sanofi 46-47

DS-3032b Daiichi Sankyo 236

Benzodiazepinediones (for example, TDP521252) Johnson & Johnson 40,237

Sulphonamides (for example, NSC279287) Virginia Commonwealth University 55

Chromenotriazolopyrimidine, morpholinone andpiperidinones (AM-8553)

Amgen 41,5658

Terphenyls Yale University 42,43

Chalcones Max Planck Institute of Biochemistry 44,45

Pyrazoles, imidazoles University of Pittsburg 48

Imidazole-indoles Novartis 49

Isoindolinone University of Newcastle 50

Pyrrolidinone (for example, PXN822) Priaxon 51,52

Piperidines Merck 53

Naturally derived prenylated xanthones Universidade do Porto 68SAH-8 (stapled peptides) Harvard University 83,84

sMTide-02, sMTide-02a (stapled peptides) LAB P53, A*STAR 85

ATSP-7041 (stapled peptide) Aileron Therapeutics 86

Spiroligomer (-helix mimic) Temple University 87

Mechanism of action: protein folding

PRIMA-1MET(also known as APR-246) Aprea 102105

PK083, PK5174, PK5196, PK7088, benzothiazoles Centre for Protein Engineering, MRC Laboratory of Molecular Biology 97100

Stictic acid University of California, Irvine 107

NSC319726 The Cancer Institute of New Jersey 121

A*STAR, Agency for Science, Technology and Research; MRC, UK Medical Research Council.

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Peptide and stapled peptide inhibitors of the p53MDM2interaction.The p53 TAD1 region that is embedded inthe N-terminal domain of MDM2 adopts a helical motifspanning residues Thr18 to Leu26 in the bound state.It is clear that the helical motif displays an amphipathicsurface such that the residues Phe19, Trp23 and Leu26are exposed on one side, and it is this side that is embed-ded into the hydrophobic crevice in MDM2 (REF. 36). Thisinsight has enabled the construction of many peptideswith amino acid substitutions at positions that are notassumed to be crucial for the interaction with MDM2.These changes alter the affinity for MDM2, as well asstability, solubility and cell permeability. An increasingnumber of high-affinity peptides that mimic p53 and sta-bilize it by inhibiting MDM2 and/or MDMX have beenidentified in recent years, including the high-affinitypeptides known as PMI75, pDIQ76and PMI-N8A77. Thesestudies have largely exploited sophisticated phage libraryscreens. Structural analyses have provided detailedinsights into the origins of these increased affinities,including unsurprisingly a correlation between

increased affinity and increased helicity of the peptides.In addition, interactions between the side chains of thepeptides and the surface of MDM2 were found to con-tribute to the altered affinities. The high-affinity peptidePMI-N8A (with an affinity of 490 pM and 2.4 nM againstMDM2 and MDMX, respectively) was identified after analanine scan of the PMI peptide was performed, whereeach amino acid was sequentially replaced by alanine andthe affinity for MDM2 or MDMX was measured to exam-ine the contribution of the mutated amino acid to theinteraction77.

In addition to these designs, the crystallographicresolution of a mutant form (pDIQ) of a phage-derivedhigh-affinity peptide (pDI)76 showed that contextdependence must be taken into consideration. The pDIQpeptide bound to MDM2 as a helix, whereas it bound toMDMX as a distorted helix; the local loss of helicity inthe latter is associated with higher conformational plas-ticity, which encourages the formation of compensatoryinteractions between the peptide and MDMX.

A group at the University of Maryland designedprotease-resistant D-analogues of the PMI peptide78.Theses peptides (termed DPMI- and DPMI-) could nottraverse the cell membrane, and upon conjugation to cati-onic cell-penetrating peptides they were nonspecificallycytotoxic in a p53-independent manner. However, whenencapsulated in liposomes that were targeted to integrins

through RGD signatures, the peptides were very activeagainst the human glioblastoma cell line U87 (whichexpresses wild-type p53); DPMI- induced a dose-depend-ent growth inhibition with an IC

50of 1.9 M (compared to

3.8 M for nutlin 3) and it also demonstrated growth inhi-bition of U87 mouse xenograft models in a p53-dependentmanner. This important study implicitly warns against theuse of cationic peptides as delivery vehicles.

Molecular dynamics simulations have shown thatthe interactions between MDM2 and the peptidesdescribed above depend upon their mutual conforma-tional modulation, leading to varying thermodynamicsignatures79. This insight resulted in the finding that even

single-amino-acid changes in the peptides can producea context-dependent effect. It is clear that a detailedexamination of the underlying conformational dynam-ics is crucial for the design of peptides that bind withhigh affinity80. Indeed, these findings were confirmed in astudy combining biophysics, crystallography and simula-tions for the design of peptides against a different target,eukaryotic translation initiation factor 4E (eIF4E), wherethe plasticity of the interacting surfaces was observed toyield synergistic positive effects on binding affinity81.

The development of techniques to stabilize the heli-city of the peptides was given a major boost with theintroduction of the stapling technology82. Stapling entailsthe introduction of a hydrocarbon linker between twonon-adjacent amino acids in the peptide. Recently, cell-penetrating stapled peptides that inhibit both MDM2 andMDMX have been developed, showing in vitroand in vivoefficacy in cancer models8386. In an alternative chemicalapproach to stabilizing helices, a synthetic spiroligomer-helix mimic was described that can penetrate cells, bindto MDM2 tightly and induce p53 pathway activation87.

Another approach to convert peptides into drug-like mol-ecules involved the display of MDM2-interacting peptideson a small, highly structured, disulphide-rich knottedpeptide scaffold called a cyclotide, which can bind toMDM2 and activate the p53 response in vitro88.

Nevertheless, the development of stapled peptidesas MDM2 inhibitors is controversial; moreover, someof the early work89,90on stapled peptides that bind the Bcell lymphoma 2 (BCL-2) family of proteins and induceapoptosis has recently been questioned, in particular thecellular uptake of the stapled peptides and their activityin cells91. However, more recent studies have estab-lished that stapled peptides targeting both MDM2 andMDMX are highly effective and able to enter every cellin a variety of cell lines to activate the p53 response85,86.In a detailed comparison with nutlin, the stapled pep-tides sMTide02 and sMTide2A, which bind to MDM2and MDMX, were shown to be more specific than nut-lin. They showed less toxicity to p53-negative cells andinduced a higher level of p53 reporter gene responsethan nutlin in several cell-based reporter assays85.

The stapled peptide ATSP-7041 (Aileron Therapeutics)was also shown to bind to MDM2 and MDMX in vitro,and showed efficacy in various cancer cell lines as wellas in mouse tumour xenografts86. ATSP-7041 has beenshown to have a slower dissociation rate from MDM2(43 minutes) than small-molecule inhibitors of MDM2

(which typically have a dissociation rate of around 6 min-utes). Importantly, following drug removal, the activationof p53, MDM2 and p21 protein levels in the MCF-7 cellline appeared to persist for longer with ATSP-7041 (upto 48 hours) than with the small molecule RG7112 (up to4 hours). The PK/PD properties of stapled peptideswere also investigated in mouse, rat and non-humanprimate models using a labelled form of ATSP-7041.Encouragingly, this novel class of molecules showedexcellent pharmaceutical properties with a very good dis-tribution throughout all organs and a persistence in theplasma that allowed therapeutic levels to be maintainedwith once-a-day dosing86.

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Restoration of misfolded mutant p53.p53 seems to haveevolved to be thermodynamically and kinetically unsta-ble at body temperature in humans, which may allow fortighter control of p53 protein levels92. Many oncogenicmutations inactivate p53 function by disrupting the directbinding to specific DNA (known as contact mutants) or bypreventing the proper folding of the central DNA-bindingdomain of p53 (known as structural mutants)93. Manyof these mutant p53 proteins are temperature-sensitive,which has encouraged attempts to discover moleculesthat can restore mutant p53 activity by acting as p53 chap-erones (FIG. 3a). This concept has recently proved to besuccessful in Gauchers disease, in which small moleculeswere found to restore the activity of the mutant glucocere-brosidase enzyme by causing its stabilization94,95. Theseobservations have led to generic methods of identifyingprotein targets of small-molecule drugs via their chaperon-ing function, where the binding of a small-molecule chap-erone to its target protein selectively increases its resistanceto denaturation induced by high temperatures96.

Small-molecule lead compounds have been discov-

ered by computational and fragment screening, andshown to bind to a druggable surface crevice on p53that is created by the Y220C hotspot mutation (FIG. 3b),which is found in approximately 75,000 new cancer casesa year32. These compounds include the carbazole-basedPK083 (REF. 97), the halogen-enriched PK5174, PK5196(REF. 98), PK7088 (developed at the MRC Laboratory ofMolecular Biology, Cambridge, UK)99and various ben-zothiazoles100, and they have been characterized in detailusing biophysical and crystallographic techniques (FIG. 3b).Some of the compounds seem to be able to restore varyinglevels of p53 activity in cell lines harbouring the Y220Cmutation99.

PRIMA-1 (a 2,2-bis(hydroxymethyl)-3-quinuclidi-none developed at the Karolinska Institute, Stockholm,Sweden) and its structural analogue PRIMA-1MET(APR-246, developed by Aprea) have been shown torestore mutant p53 (R273H and R175H) activity in vitroand in vivo101103. PRIMA-1METhas also successfully com-pleted a Phase I clinical trial, showing some indication ofefficacy (ClinicalTrials.govidentifier: NCT00900614)104.PRIMA-1METseems to lead to the formation of covalentadducts on mutant p53R175Hand p53R273Hproteins, but itsexact mechanism of action has yet to be fully understood105.

The formation of covalent adducts with alkylatingsmall molecules can affect the binding of p53 to DNA.The alkylation of cysteine molecules on p53 by these small

molecules proceeds in a progressive manner, starting atCys124 and Cys141, which are most accessible106. Recentcomputational and biochemical studies on PRIMA-1 alsosuggest that its mechanism of action involves a covalentinteraction with Cys124 in a binding pocket of the coredomain of p53 (REF. 107). A virtual screen of small mol-ecules that bind to p53R273Hidentified stictic acid, whichwas predicted to bind to this pocket. It was shown toinduce p21 activation in transcriptional gene reporterexperiments in cells107. In a separate study using top-downFourier transform ion cyclotron resonance (FTICR) massspectrometry and the alkylating agent N-ethylmaleimide,Cys182 and Cys277 on p53 were identified to be most

susceptible to alkylation108. The OxMRM approach, whichcombines oxidation through N-ethylmaleimide and mul-tiple reaction monitoring (MRM) mass spectroscopy, wasalso used to probe the oxidation status of the cysteine mol-ecules of p53 in cells, and identified Cys182 as being verysusceptible to oxidation109.

PRIMA-1METalso seems to be able to restore thefunction of mutant p63 (a p53 homologue)110,111. In thecongenital disease ectodermal dysplasia, mutations inthe DNA-binding domain of p63 are associated withorofacial clefting and limb abnormalities. Some of thesemutations correspond to the hotspot oncogenic muta-tions that are observed in the DNA-binding domainof p53. In a transfection-based system using H1299cancer cells, PRIMA-1METwas shown to also reactivateother p53 homologues such as TAP73a, TAp73b andTAP63g as well as inducing apoptosis112.

Other therapeutics that target mutant p53 via vari-ous mechanisms have been described; for example,NSC176327 and RETRA disrupt the binding interactionsbetween mutant p53 and p73, whereas MIRA-1 elimi-

nates mutant p53. These therapeutics have been reviewedin detail elsewhere in the literature52,113. Some of thesedrugs show good biological activity, but more studies arerequired for a full biophysical characterization (BOX 3).

Stabilizing p53 with zinc.Zinc is important for theproper folding of the central core domain of p53(REF. 114), and the absence of a zinc molecule in the cen-tral core of p53 can lead to unfolding and aggregation ofp53 (REF. 115). Mutations that affect zinc binding, such asC242S, H179R, C176F and R175H, are frequently foundin human cancers116.

There have also been in vitrostudies showing that theabsence of homeodomain-interacting protein kinase 2(HIPK2) leads to the accumulation of misfolded wild-type p53 owing to the deregulation of zinc-binding met-allothionieins117. However, supplementation with zincreversed this effect. More recent papers have shown thatthe addition of zinc (in vitro and in xenograft models)restored DNA-binding activity to the contact mutantp53R273Hand the structural mutant p53R175H, thus allowingp53-mediated cell killing by adramycin or cisplatin118. It issurprising that both contact mutants and conformationalmutants of p53 are rescued by the same compounds;however, this has been reported for several differentcompounds107,119.

Zinc has also been found to be effective in promoting

cell killing in colon and breast cancer cell lines whenused in combination with the MDM2 inhibitor MI-219(REF. 120). One can assume that the efficacy of any drugthat restores the function of mutant p53 will be enhancedby combining it with a nutlin-like MDM2-bindinginhibitor, as the immediate target of the transcriptionalfunction of p53 is the activation of MDM2 expression.

NSC319726, a thiosemicarbazone, was identifiedin a screen of the NCI60 panel of human tumour celllines and reported to be selectively toxic to cell lines thatcarry the p53R175Hmutation. This compound appeared torestore the transcriptional and apoptotic functions of theR175H mutant and led to the loss of the PAb240 epitope

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Mutant p53

Mutant p53 Mutant p53

Mutant p53 Mutant p53

Mutant p53

p53-inducible genes

Zn2+

ba

Binding to DNA

Restoration of p53conformation

p53 reactivators +

of p53, which is associated with the unfolded state of p53.NSC319726 was also shown to inhibit the growth of atumour xenograft of the TOV112D cancer cell line ina nude mouse model. TOV112D cells express high levelsof mutant p53R175H(REF. 121). The activity of NSC319726appears to depend on its ability to chelate zinc andfacilitate its transport to mutant p53. The most strik-ing evidence for its selectivity for mutant p53 activationas opposed to wild-type p53 activation comes from thefinding that NSC319726 is toxic to mice that carry thegermline R175H mutation but not to wild-type mice121.

p53 read-through drugs.About 8% of the cancer-associatedmutations found in p53 are nonsense mutations, andrecent developments in the isolation of small moleculesand drugs that promote the read-through of nonsensecodons could provide a novel approach for treatingtumours carrying this type of mutation122,123. The two mostfrequent nonsense p53 mutations in the human mutation

database are R196X and R213X32. A recent study showedthat treatment of the human tumour cell line HDQ-P1,which contains a homozygous nonsense mutation atcodon 213 (CGA-TGA), with the read-through-promotingaminoglycoside antibiotic G418 led to a dramatic increasein the level of TP53mRNA and full-length p53 protein124.The stabilization of the mRNA was due to inhibition ofthe nonsense mutation, and the full-length protein wasshown to be transcriptionally active and able to inducep53-dependent apoptosis. One would anticipate that therestored full-length protein might re-establish the p53MDM2 feedback loop, so a combination of G418 andnutlin may be especially effective.

There are a number of read-through drugs indevelopment. The most advanced compound, PTC 124(Ataluren; PTC Therapeutics), has completed Phase IIIclinical trials for nonsense mutation cystic fibrosis125,126.However, it is the subject of some controversy because ofits remarkable activity in directly binding to and stabiliz-ing firefly luciferase, an enzyme used in the high-through-put screen that originally identified the compound127,128.Newer read-through compounds such as RTC13(University of Los Angeles, California) also look promis-ing and have been shown to be able to restore partial dys-trophin expression and muscle strength in a mouse modelof Duchenne muscular dystrophy (DMD)129. Most of theresearch in this field has concentrated on the treatment ofgenetic diseases such as cystic fibrosis and DMD, whichare highly demanding in terms of safety requirements asthe drugs would need to be administered for the lifetimeof the patient. Developing such compounds for cancermay be easier as the treatment time would be limited,which makes potential side effects more tolerable. Becausep53 mutations are so frequent, the number of patients

with a particular mutation can also be surprisingly high.For example, the codon 213 nonsense mutation discussedabove is thought to be present in 1% of all human cancers that is, roughly 220,000 cases worldwide32.

Other p53 activators.Cell-based screens for activatorsof the p53 pathway have identified large numbers ofcompounds, many of which have unknown targets andincompletely defined mechanisms of action. The screensthat seem to be most informative have used reporter celllines that measure the activity of p53 as a specific tran-scription factor. These cell lines show excellent assaycharacteristics and are easily adapted to high-through-put methods29,130. However, as DNA-damaging agentsand many cellular stress signals can activate p53, thesescreens can have a high hit rate of compounds that arenot suitable for development. Surprisingly, a small reduc-tion in the dose of the screening compound can leadto very successful screens with more modest hit rates,which implies that the reduced concentration eliminatesthe detection of many of the DNA-damaging moleculesthat are present in large compound collections.

In a few cases, the mechanisms by which thesecompounds activate p53 have been determined. Theseinclude the small molecule RITA119, which is reported totarget p53 itself, cyclin-dependent kinase (CDK) inhibi-tors such as roscovitine (Cyclacel Pharmaceuticals)131,

RNA polymerase inhibitors such as actinomycin D130,exportin 1 (XPO1; also known as CRM1)-bindingcompounds such as leptomycin B and KPT-330 (alsoknown as selinexor; Karyopharm Therapeutics)132, theNEDD1 (neural precursor cell expressed developmen-tally downregulated protein 1) ligase inhibitor MLN4924(Millennium Pharmaceuticals)133and sirtuin inhibitorssuch as the tenovins (University of Dundee)29. In a recentstudy using an in vivomouse model of chronic myeloidleukaemia (CML), inhibition of SIRT1 by tenovin-6 orknockdown of SIRT1 led to tumour regression and selec-tive killing of leukaemia stem cells via p53-dependentgrowth arrest and apoptosis134. All of the compounds

Figure 3 | Mechanisms of mutant p53 reactivation. a| Strategies for restoring wild-type

activity to mutant tumour suppressor p53. p53 is often inactivated by oncogenic

mutations, which can prevent the proper folding of the transcription factor or directly

disrupt its binding to DNA as a functional tetramer. p53 reactivators are currently being

developed to bind to mutant p53 and restore its binding activity to DNA, leading to

transcription of downstream genes. These reactivators act as molecular chaperones

by preferentially binding to the correctly folded form of the protein and stabilizing its

functional conformation. b| The crystal structures of the Y220C-mutant p53 (surfaceshown with the sphere representing the Zn2+atom) demonstrate that several molecules (in

sticks) that stabilize this mutant bind to the same region: that is, in the vicinity of the site

of the Y220C mutation (shown in yellow). These molecules include PK7242, PK5116,

PK5174, PK784, PK5086, PK5176, PK5196 and 2-amino substituted benzothiazole,4-

(trifluoromethyl)benzene-1,2-diamine, 5,6-dimethoxy-2-methylbenzothiazole.

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described above (with the possible exception of RITA)act on targets that will affect other pathways in additionto the p53 pathway and will therefore require carefulevaluation in preclinical models.

Considerations for therapy

As p53-based therapies are entering the clinic, a key issueis the design of clinical trials that will demonstrate suf-ficient efficacy to allow regulatory approval. There aremore than 65,000 papers published on p53, but these donot yet provide an unequivocal prediction as to which

human tumours are most likely to respond to thesetherapies with the best therapeutic index.

The current crucial issues are efficacy, resistancedevelopment and side effects in patients. The thera-peutic index is determined by the relative sensitivity ofboth tumour and normal tissues towards p53 activation.Drugs may exhibit on-target or off-target side effects.Small-molecule inhibitors of MDM2, for example, canupregulate p53 in normal cells in an on-target effect and this may lead to undesired toxicity. Off-targeteffects of nutlin for example, at high doses havebeen noted and are discussed below135138. The key issuesregarding p53 activation are the type of response this

induces whether it would lead to cell cycle arrest,metabolic alteration, DNA repair, autophagy, senescenceor apoptosis and how it affects tumour cells versusnormal cells. Much of the preclinical cellular and in vivodata with drugs that activate p53 suggest that these dif-ferent cell fate responses are highly dependent on theintrinsic properties of the tumour or tissue type and may

vary greatly among patients, as known polymorphisms inthe p53 pathway for example, in the MDM2 promoter12 can influence the outcome.

It is thus important to have carefully designed clinicaltrials that take these factors into consideration. These tri-als would select tumour types such as melanoma or acutemyeloid leukaemia (AML) in which p53 mutations are

very rare and where there is a very clear unmet clinicalneed. It would be tremendously useful to understand theintrinsic mechanisms of resistance towards p53-inducedapoptosis and how resistance is induced following pro-longed treatments with p53-activating drugs. As dis-cussed below, p53 mutations are expected to be a majordriver of resistance development. Therefore, searching

for tumours in which p53 mutations are not found butwhich are still responsive to p53-activating drugs is anattractive strategy. It is also possible to explore whichbiomarkers may predict responsiveness towards theclinical efficacy of p53 activation in patients.

Preclinical and clinical studies.Numerous early in vitrostudies suggested that the cell-killing efficacy of small-molecule inhibitors of MDM2, such as nutlin and MI-219,was dependent on the wild-type status of p53 (REFS 5,139).Nutlin and the MI drugs have been found to be effectivein killing a broad range of cell lines in vitroand have alsoshown in vivoefficacy in mouse models5. Cancers suchas chronic lymphocytic leukaemia (CLL)140, acute lym-phoblastic leukaemia (ALL), AML139, myeloma141, neuro-blastoma142melanoma66and mantle cell lymphoma143often contain wild-type p53, which allows the targetingof interactions between MDM2 and p53 in order to acti-

vate p53.MDM2 gene amplification is quite common insarcomas (occurring in about 20% of cases), which couldmake these cancers good candidates for treatment withMDM2 inhibitors144145.

The responses to these small-molecule drugs are,however, dependent on cancer cell types. In an earlystudy of nutlins, carried out using a panel of cancercell lines from a variety of tumours, it was shown thatsome cell lines were more susceptible to nutlin-induced

apoptosis than others146147. For example, the colorectalcancer and lung cancer cell lines tested were shown toundergo reversible cell cycle arrest without apoptosis,whereas other cell lines such as the CML cell lineBV173 appeared to undergo apoptosis without cellcycle arrest147. Testicular germ cell tumours were foundto be exquisitely sensitive to nutlin- or cisplatin-inducedp53-dependent apoptosis148. It was thought that thishigh sensitivity to cell death was caused by mitochon-drial priming owing to high levels of NOXA, which isdependent on the transcription factor octamer-bindingprotein 4 (OCT4). This makes these cells highly sus-ceptible to small increases in BCL-2 homology 3 (BH3)

Box 3 | Criteria for validation of p53 pathway drugs

Many small-molecule drugs have been described as E3 ubiquitin protein ligase MDM2

inhibitors or mutant tumour suppressor p53 reactivators, but stringent criteria need to

be applied in making this assignation.

Criteria for validation of an MDM2 or MDMX drug

Structure of drug interaction with MDM2 or MDMX: does the drug bind to MDM2 or

MDMX as characterized by a range of structural and biophysical methods?

Evidence for biological activity in tissue culture: does the drug induce growth arrest

and apoptosis in wild-type p53 cells but not in p53-mutant cells over a broad range of

concentrations?

Evidence for biological activity in animal models: does the drug induce biomarkers of

p53 activation, such as an increase in serum levels of the macrophage inhibitory

cytokine 1 (MIC1) protein in mice expressing wild-type p53 but not in p53-mutant or

p53-null mice?

Therapeutic index: is it effective in killing wild-type p53-expressing cancer cells

without resulting in toxicity to normal tissues?

Absence of off-target toxicity: does the drug induce DNA damage in p53-null or

p53-mutant cells? Is it toxic in p53-null mice?

On-target toxicity: does the drug induce p53-dependent cell death in normal cells

such as thymocytes or megakaryocytes?

Species specificity: does the drug interact equally well with human as well as testanimal MDM2 or MDMX?

Criteria for validation of a drug that reactivates mutant p53

Biophysical interaction: does it interact with mutant p53? Is there a structure

(X-ray or NMR) of the drug with p53? What is the binding affinity?

Biological activity: is the activity in cells dependent on mutant p53? Is there no

activity in p53-null cells or p53 wild-type cells over a range of concentrations?

Mutant specificity: is the biological activity mutant-specific? Does it work in all cell

lines that have the particular p53 mutation?

In vivospecificity: is the drug selectively toxic to mice harbouring the p53 mutation

in their germline (for example, R172H) but not to wild-type or p53-null mice?

Species specificity: does the drug interact equally well in human as well as test

animal mutant p53?

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proteins such as PUMA and NOXA (as discussedbelow)149. Although the high level of curative testiculargerm cell tumour responses to cisplatin may make it anunlikely choice for the development of MDM2 inhibi-tors, the very high level of hearing damage induced bycisplatin could provide a clinical opportunity for thedevelopment of a less toxic drug. As polymorphisms thatpredict adverse reactions to cisplatin become apparent,patients with these polymorphisms may be better suitedto alternative therapies such as MDM2 inhibition150.

Nutlin5, MI-219 (REF. 46) and the very recently pub-lished follow-on drugs RG7388 (REF. 63)and MI-888(REF. 47)were shown to be very potent in xenograft mod-els of human cancer with wild-type p53. These drugsled to a robust accumulation of p53 and apoptosis inxenograft tumours such as the osteosarcoma SJSA-1 cellline and the prostate metastasis-derived LNCaP cell line,with little toxicity observed in terms of weight loss orin necroscopy studies5,46,47,63. There is also little toxicity

towards the normally radiosensitive tissues such assmall-intestine crypts and the thymus46, which showonly minimal accumulation of p53 protein. However, anobserved accumulation of the p21 protein suggests thatp53 is, nonetheless, activated in these tissues. This is incontrast to the high levels of p53 accumulation observedin these tissues after treatment with ionizing radiation.It is known that ionizing radiation induces many othersignalling pathways independently of its effects on thep53 pathway. Thus, although apoptosis induction byirradiation is highly dependent on p53 activity, thisoccurs in the context of the activation of many otherradiation-induced pathways. By contrast, in cells treated

with nutlin only the p53 pathway is activated. Theenhanced apoptotic response observed after radiationtreatment can therefore be explained as a consequenceof the integration of the pathways activated by radiation7.

As discussed above, the kinetics of the p53 responseare also crucial when considering p53-directed therapy;for how long do tumour cells need to be exposed to thep53-activating agent before they are eliminated? Asdiscussed elsewhere, some models suggest that a shortexposure to a p53-activating drug may activate a revers-ible growth arrest, whereas prolonged exposure inducesapoptosis151.

Recent work investigating the effects of nutlin in anaxozymethane carcinogen-induced mouse model of coloncancer showed that treatment with nutlin resulted in cellcycle arrest in the colon cancer cells without causing a sig-nificant increase in apoptosis. This suggests that nutlin hasto be used in combination with other drugs to elicit anyp53-dependent cell killing in colon cancer. Normal tissues

displayed limited toxicity with decreased cell proliferationbut without any induction of apoptosis135.

The second-generation nutlin, RG7112, was shownto kill cancer cell lines with potent efficacy as a singleagent152. It also induced cell cycle arrest and apoptosis inSJSA-1 and LNCAP xenografts in mice, leading to theregression of tumours152. RG7112 is currently in clini-cal trials for different types of cancer (TABLE 1). One ofthe first published sets of clinical results revealed thatthe treatment of liposarcoma patients with RG7112increased p53 and p21 levels in biopsy specimens andreduced proliferation in tumours. It was also shown thatmacrophage inhibitory cytokine 1 (MIC1; also known

Table 3 | Summary of published experimental drug combinations with nutlin

List of drug combinations Tumour types (xenograft models) Refs

Nutlin with doxorubicin; nutlin with cytarabine AML and B-CLL 140,143,185

Nutlin with vincristine Neuroblastoma, rhabdomyosarcoma and melanoma 180

Nutlin with roscovitine Various cancers 181,182

Nutlin with valproic acid AML 190

Nutlin with Aurora kinase inhibitors Various cancers 183,184

Nutlin with 1,2,5-dihydroxyvitamin D3

AML 189

Nutlin with XIAP inhibitor AML 155

Nutlin with cisplatin Ovarian cancer 194

Nutlin with androgen-depleting agent Prostate cancer 195

Nutlin with CDK1 inhibitor; JNJ-7706621 Melanoma 66

Nutlin with TRAIL Haematological malignancies 238

Nutlin with sorafenib Renal cell carcinoma 239

Nutlin with ABT-737 Various cancers 151,192,193

Nutlin with selumetinib (AZD6244) AML 188

Nutlin with KPT-185 AML 240Nutlin with sorafenib (independent of p53 status) AML 200

Nutlin with dasatinib (independent of p53 status) B-CLL 199

Nutlin with radiation Lung and prostate cancer 186,187,201

AML, acute myeloid leukaemia; B-CLL, B cell chronic lymphocytic leukaemia; CDK1, cyclin-dependent kinase 1; p53, tumoursuppressor p53; TRAIL, TNF-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.

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as GDF15) served as an effective serum biomarker ofp53 induction153. Liposarcomas were selected as thesetumours commonly retain wild-type p53 and have ampli-fied MDM2 (REF. 144). Clinical benefit in the study, how-ever, was modest, with only 1 patient (out of 20) showinga partial response, and 14 showing stable disease, whilethe remaining 5 patients had progressive disease153. Butmore discouraging was the relatively high rate of adversereactions, including thrombocytopaenia and neutro-penia. RG7112 is also in Phase I clinical trials in patientswith AML, where it is hoped that the compound mayproduce more promising results.

Side effects of p53 therapy.In the published clinical trialof RG7112 in liposarcoma, patients were treated with140 mg per m2of orally administered RG7112 daily for10 days on a 28-day cycle for up to three cycles, whichwas followed by surgical resection153. As discussed, thedrug showed some toxicity, and so an important ques-tion is whether a more pulsed approach to dosing wouldhave reduced this effect. The activation of p53 is thought

to induce cell cycle arrest in all proliferating tissues. Cellcycle arrest per se is not necessarily toxic; for example, itwas shown that MYC inhibitor-induced cell cycle arrestin mice was tolerable for 4 weeks with no long-term sideeffects154. However, the effects of cell division inhibitorsin humans have not yet been defined154.

The adverse effects observed in the liposarcomatrial153were not predicted from the preclinical stud-ies, in which there appeared to be a good therapeuticwindow, with normal cells showing less susceptibilitytowards drug-induced death than tumour cells140,152,155.Nutlin was found to be effective in killing B cell CLL(B-CLL) cells, with less toxicity towards CD19+B lym-phocytes, peripheral blood mononuclear cells and bonemarrow haematopoietic progenitors in colony out-growth assays140. In vitrostudies with the MI-219 com-pound showed that it was effective in killing in cancercells but not primary normal prostate epithelial cells46.These results suggest that more extensive studies of theeffects of p53 activation in normal tissues are requiredand that preclinical studies should be extended to non-human primates.

The thrombocytopaenia and neutropenia seen inpatients in the liposarcoma trial could be due to on-targetactivation of PUMA and NOXA by p53, which wouldinduce apoptotic depletion of the relevant cell popula-tions. Of note, other reports show that nutlin may have

off-target biological effects owing to direct induction ofDNA damage136,156or the induction of differentiationin a p53-independent manner137. Other studies havesuggested that nutlin can make direct interactions withBCL-X

Lin addition to MDM2 (REF. 138). It is not clear,

however, to what extent these nonspecific effects con-tribute to the toxicities seen in patients treated with thenewer and more potent compounds in clinical trials. Anin vitrostudy has shown that RG7112 can induce apop-tosis in human megakaryocyte precursors and that it alsohinders the polyploidization step required for the end-stage of platelet formation, forming an in vitromodel forthe thrombocytopaenia observed in patients157.

Mechanisms of resistance to MDM2 inhibitors

The rapid emergence of resistance has been demonstratedfor a number of molecularly targeted anticancer drugs.In some cases for example, the BCRABL inhibi-tor imatinib (Gleevec; Novartis) or the BRAF inhibitor

vemur afenib (Zelb oraf; Roche/Ple xxikon) theresistance-conferring mutations have been shown to bepresent before treatment so that their selection is inevita-ble. As MDM2 inhibitors enter the clinic, it is importantto predict potential mechanisms of resistance158.

Various mutations have been shown to confer resist-ance to MDM2 inhibitors in preclinical studies. The firstmechanism is a mutation in p53 that abrogates its tran-scriptional activity, and this has been observed in celllines after prolonged nutlin treatment. The mutationswere found to occur in the DNA-binding domain ofp53, rendering it inactive as a transcription factor 159161.Recently, an in vitroselection for MDM2 mutants thatcan still interact with p53 in the presence of nutlin dem-onstrated that subtle point mutations in the p53-bindingpocket of MDM2 can confer resistance to nutlin; these

mutant proteins are still able to bind to p53 and inactivateit even when nutlin is present61.

Another study, using a short hairpin RNA libraryto search for molecules that confer resistance to nutlin,found that p53 and the DNA damage-sensing proteinp53BP1 (p53-binding protein 1) were essential for nut-lin-induced growth arrest162. In a mouse model of glio-blastoma, it was shown that resistance to p53-mediatedgrowth-inhibitory effects developed when the tumoursuppressor ARF was inactivated. This blocked the signal-ling from the gliablastoma-inducing oncogene to p53163.Such results support the use of small molecules like nut-lin that act downstream of ARF. The glioblastoma modelalso predicted that pulsed exposure to active p53, ratherthan continuous exposure, was preferable as it reducedthe rate of induction of resistance163.

Intrinsic resistance to apoptosis has an important rolein determining the extent to which a cell line or tumourresponds to p53-activating treatment. The principal routeby which p53 induces apoptosis is via induction of thepro-apoptotic proteins NOXA and PUMA (BOX 2). Thelevel of induction of these proteins and the cellular levelsof their antagonistic anti-apoptotic proteins will deter-mine whether BAX or BAK is activated and the apoptoticthreshold is crossed. One can therefore anticipate thatthe cellular response to p53 induction by p53-activatingdrugs will be similar to the response to newly developed

small molecules such as ABT-737 and the orally avail-able ABT-263 (also known as navitoclax), both of whichmimic the active BH3 domains of PUMA and NOXAand antagonize anti-apoptotic mitochondrial proteins.ABT-263, which has reached clinical trials164,165, acts byneutralizing BCL-2, BCL-W and BCL-X

Land activat-

ing BAX or BAK166. Some clinical efficacy was observedin the Phase I clinical trial against chronic lymphocyticleukaemia (CLL)165, but the results from the Phase IIclinical trial in relapsed small-cell lung cancer seemedto be less promising, with little efficacy observed164. Sideeffects such as thrombocytopaenia were a concern inboth studies.

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Resistance towards ABT-737 has been linked to theoverexpression of myeloid cell leukaemia sequence 1(MCL1), BCL-2 related protein A1 (BCL-2A1; alsoknown as BFL-1) or BCL-2-like protein 10 (BCL-2L10;also known as BCL-B)166169. Sensitivity towardsABT-737-induced cell death has been found to cor-relate well with MCL1 expression levels in a range ofAML cell lines (including HL60, KG1, NB4, U937and OCL-AML3), with the most resistant cell line,OCL-AML3, having high levels of MCL1 (REF. 167).Incidentally, OCL-AML3 is also resistant to nutlin anda range of other cytotoxic drugs139,170, which supports theargument that downstream intrinsic resistance to apop-tosis, such as that conferred by high levels of MCL1, mayhave an important role in reducing the responsiveness top53-activating drugs and other cytotoxic drugs that leadto apoptosis and cell death.

Further evidence for intrinsic resistance to apoptosisis provided by combination studies showing that eithernutlin or ABT-737 can synergise with a MAPK/ERKkinase 1 (MEK1) inhibitor, PD-98059, which down-

regulates MCL1 and also suppresses BCL-2 phospho-rylation, to induce apoptosis in OCL-AML3 cells171,172.In a separate study, inactivation of MCL1 by inducibleexpression of BCL-2-interacting mediator of cell death(BIM)-derived peptides that bind to MCL1, inducedapoptosis in six out of seven AML-derived cell lines(including OCL-AML3) and synergistically induced celldeath in combination with ABT-737 (REF. 173).

The intrinsic susceptibility of cancer cells to chemo-therapeutic drugs has also been studied in in vitroBH3profiling assays, in which cells were permeabilized withdigitonin and exposed to BH3 peptides. Loss of mito-chondrial potential was then measured by flow cytom-etry using a radiometric dye. For AML, it was shown thata comparison of the levels of apoptotic priming betweenmalignant myeloblasts and normal haematopoietic stemcells allows an accurate prediction of the therapeutic indexfor chemotherapy towards drugs such as etoposide anddaunorubicin174. In a study of 85 patients with AML, ALLand ovarian cancer, the mitochondria of chemosensitivetumours were found to be more primed towards apoptosisthan the mitochondria of chemoresistant or normal tis-sues175. If this apoptotic threshold model is correct, thenone would predict that haematopoietic tumours that aresensitive to MDM2 inhibitors and to BCL-2 inhibitors willbe the same tumours that are already sensitive to standardchemotherapy. In a clinical trial setting this would make it

difficult to establish the advantage of these new targetedtherapies, except by virtue of their expected reduced toxicityand lack of mutagenic activity.

Contribution of MDMX to intrinsic resistance. Highlevels of MDMX are also known to confer resistanceto nutlin and MI drugs. The binding of MDMX to p53inhibits apoptosis induction by p53, and nutlin and MIdrugs cannot interfere with this interaction176,177. MDMXis often upregulated in tumours containing wild-typep53, such as melanomas65. Restoration of p53 usinga tamoxifen-inducible allele in an MDMX-deficienttransgenic E-Mycmouse model of lymphoma led to

increased survival of the tumour-bearing mice com-pared to control mice that expressed MDMX, whichsuggests that MDMX is a valid target for inhibition 178.RO-5963 (Roche), a compound with dual specificityagainst both MDM2 and MDMX, was recently shownto have in vitroand in vivoefficacy against cell lines andmouse xenografts that express high levels of MDMX72.It is notable that the recently published stapled peptidessMTide-02 and ATSP-7041 both show activity againstMDMX as well as MDM2, thus enhancing their attrac-tiveness as preclinical candidates for development 85,86.Also encouraging for this class of molecules (stapledpeptides) is the very recent finding that MDM2 muta-tions that confer resistance to nutlin do not confer resist-ance to sMTide-02. This is due to the larger number ofinteractions between the peptide (compared to nutlin)and its target, MDM2 (REF. 179).

Drug combinations

The pharmacological activation of the p53 pathway canbe exploited to work in combination with other therapeu-

tic agents to promote clinical efficacy. Nutlin, for exam-ple, has also been shown to work well in combinationwith other drugs (TABLE 3), such as the mitotic inhibitor

vincristine180, CDK inhibitors such as roscovitine181,182,Aurora kinase inhibitors183,184and DNA-damaging agentssuch as doxorubicin140,143,185, as well as radiation ther-apy186,187. Much work is required to further understandhow drug combinations with nutlin or other p53 acti-

vators can lead to synergistic enhancement of cell deathand improved therapeutic efficacy in the treatment ofcancer. These combination strategies may lead to greaterinduction of p53 protein accumulation and transcrip-tion of downstream pro-apoptotic genes or enhance cellkilling through the inhibition of anti-apoptotic proteins.Examples are discussed below.

Drug combinations that induce higher expression of p53downstream genes.As discussed above, in some cell typesp53 activation induces growth arrest but not apoptosis,which may be due to an inadequate induction of p53activity. A search has been conducted for drugs that willsynergize with MDM2 inhibitors to induce a strongerp53 response. Different drug combinations have beenshown to enhance the expression of p53 target genesthrough higher induction of p53 protein expression or viaco-regulation with other p53-independent mechanisms.For example, the combination of nutlin with CDK inhibi-

tors such as the nucleoside analogue DRB and roscovi-tine was shown to have a synergistic effect with regard toinducing cell death in several cell lines. This was due toenhanced transcription of the downstream p53-inducedpro-apoptotic genes PUMAand TP53I3(p53-induciblegene 3; also known as PIG3)182.

Combinations such as selumetinib (AZD6244), aMEK inhibitor, with nutlin also had a synergistic effectin inducing cell death in AML cells in vitroowing to theinduction of high levels of PUMA and BIM proteins andthe downregulation of MCL1 (REF. 188). The increase inPUMA protein levels after treatment with the drug com-bination was thought to be regulated not only by the p53

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protein but also by the effects of the forkhead box O3A(FOXO3A) transcription factor on the transcriptionalregulation of the PUMApromoter.

A combination of nutlin with 1,25-dihydroxyvita-min D

3also accelerated cell death in AML cell lines owing

to the increased transcription of pro-apoptotic prolinedehydrogenase 1 (PRODH; also known as PIG6) anddownregulation of BCL-2 (REF. 189). Combination studieswere also carried out in AML cell lines, in which co-inhi-bition of histone deacetylase (HDAC) and MDM2 with

valproic acid (a HDAC inhibitor) and nutlin, respectively,led to synergistic activation of apoptosis190. This was dueto a higher accumulation of p53 protein and higher lev-els of p53 acetylation, and thereby greater induction ofdownstream p53-responsive genes. Acetylation of p53can inhibit its ubiquitylation by MDM2 and enhance itsDNA-binding activity, thus leading to an increase in thelevels and activity of the p53 protein191.

Targeting anti-apoptotic genes and sensitizing cellstowards death.The growth arrest induced by p53 in some

cancer cell types is transient and reversible, whereas inother cell lines it is permanent and involves either senes-cence or apoptotic mechanisms. One way to shift thisbalance and achieve tumour elimination is to sensitizethe cells to the apoptotic proteins PUMA and NOXA.Drug combinations can sensitize cells to apoptosis bytargeting anti-apoptotic genes or proteins. For example,the combination of nutlin with ABT-737, which binds toBCL-2 and BCL-X

L, synergistically targets the balance of

pro-apoptotic and anti-apoptotic proteins at the mito-chondrial level, thereby promoting cell death151,192,193.

It has been shown that p53 activity in melanomas isinhibited by phosphorylated nuclear inhibitor of ASPP(iASPP; also known as RAI) protein. The CDK1 inhibi-tor JNJ-7706621 inhibits iASPP phosphorylation andnuclear entry. The combination of JNJ-7706621 andnutlin was shown to restore p53 activity, leading to syn-ergistic activation of apoptosis in melanomas in vitroandin vivo. Additionally, it was found that vemurafenib, asmall-molecule BRAF inhibitor that selectively inhibitstumour cells containing the BRAFV600Emutation (whichis found in about 50% of melanomas), could be usedconcurrently with the drug combination of JNJ-7706621and nutlin to achieve an additive suppression of humanmelanoma cell growth in vitroand in vivo66.

Synergistic activity has also been shown for the com-bination of nutlin with an X-linked inhibitor of apopto-

sis protein (XIAP) antisense oligonucleotide inhibitor inAML cells in vitro155. XIAP is a potent inhibitor of apop-tosis and is found to be overexpressed in many cancers.

Nutlin has also been shown to be useful for sensitiz-ing the chemoresistant ovarian cancer cell line A2780cis,as well as primary ovarian tumours, towards cisplatin-induced cell killing, possibly through the downregulationof survivin194.

Synergism between p53 activation and microenvironmen-tal signals.There are various examples where extracellularsignals can work with or against p53-activating drugs toinduce cell death. For example, patients with advanced

prostate cancer often receive androgen deprivationtherapy; however, resistance development to this classof drugs is a challenge. The combination of nutlin withandrogen depletion has been shown to enhance the pro-apoptotic activity of p53 in vit

nature targeting tp53 cell cycle

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