Nature Targeting Tp53 Cell Cycle

The tumour suppressor p53 is a transcription factor that
regulates several genes with a broad range of functions,

including DNA repair, metabolism, cell cycle arrest,
apoptosis and senescence (see BOX1 for cell fate deci-
sions after p53 activation, and BOX2 for the mechanisms
of p53-mediated tumour cell killing). Protein levels of
p53 within cells are tightly controlled and kept low by
its negative regulator, the E3 ubiquitin protein ligase
MDM2, which binds to the amino terminus of p53, tar-
geting it for ubiquitylation and subsequent degradation.
The interaction of p53 and MDM2 has been conserved
across 2.4 billion years of evolution
1
. MDM2 is a p53
target gene, thus creating an autoregulatory feedback
loop. MDMX (also known as MDM4), the structural
homologue of MDM2, has no ubiquitylation activity
but is able to bind to the N-terminus of p53 and inacti-
vate it directly or to aid MDM2 in ubiquitylating p53 by
hetero dimerization with MDM2 (REFS2,3).
p53 is inactivated by mutations in over 50% of all
cancers
4
. Such mutations can disrupt its direct binding
to DNA or lead to structural perturbations that prevent
the 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 p53
activity, 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 disorder
that is characterized by the early onset of several different
types of cancer, is caused by a mutant p53 loss-of-function
allele. Thus, p53 acts as a tumour suppressor in humans.
Conversely, normal p53 has an essential destructive role in
the killing of radiosensitive tissues after exposure to ion-
izing radiation. Various studies have separated these two
functions of p53. Therefore, as we discuss below, the prop-
erties of p53 that prevent tumour occurrence are distinct
from those that allow activated p53 to kill tumourcells.
Numerous strategies have been devised to correct a
dysfunctional p53-regulatory pathway. Small-molecule
inhibitors of the p53MDM2 interaction, p53 gene
therapies and drugs that act as chaperones by binding
to mutant p53 and restoring its function are some of the
approaches currently in clinical trials (TABLE1). A break-
through in the field was the development of nutlin, the
first small-molecule inhibitor of the p53MDM2 inter-
action
5
. Currently, the most advanced MDM2 inhibitors
include RG7112 (Roche), MI-773 (Sanofi) and DS-3032b
(Daiichi Sankyo), which are at various stages of PhaseI
clinical trials. Preclinical lead compounds have also been
published by Amgen and Novartis, and PRIMA-1
MET

(Aprea) which restores the activity of mutant p53
has also recently completed PhaseI trials. The literature
on the p53 system is growing very rapidly, so here we
focus on the latest insights into p53 function and the
developments and controversies in p53 drug discovery.
Genetic models of restoration of p53 function
A key issue in the development of any p53-based therapy
that is not exclusively targeted to tumour cells is to
understand the effects of p53 activation on normal
human tissues. Here, the use of genetically engineered
mouse models has been especially powerful.
1
p53 Laboratory (p53Lab),
Agency for Science,
Technology and Research
(A*STAR), 8A Biomedical
Grove, #06-06, Immunos,
138648 Singapore.
2
Bioinformatics Institute,
Agency for Science,
Technology and Research
(A*STAR), 30 Biopolis
Street #07-01, Matrix,
138671 Singapore.
3
School of Biological
Sciences, Nanyang
Technological University,
60 Nanyang Drive,
637551 Singapore.
4
Department of Biological
Sciences, National University
of Singapore, 14 Science
Drive 4, 117543 Singapore.
Correspondence to D.P.L.
e-mail:
dplane@p53Lab.a-star.edu.sg
doi:10.1038/nrd4236
Drugging the p53 pathway:
understanding the route to
clinical efficacy
Khoo Kian Hoe
1
, Chandra S.Verma
24
and David P.Lane
1
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 now
reached 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.
REVI EWS
NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 217
2014 Macmillan Publishers Limited. All rights reserved
A concern of therapies that aim to restore wild-
type p53 activity is that these might lead to widespread
apoptosis in normal tissues. -irradiation, for exam-
ple, has been shown to lead to p53 accumulation and
p53-dependent toxicity in tissues such as the spleen,
thymus, intestine and haematopoietic cells in the bone
marrow
6,7
. An important study in a lymphomagenesis
model using tamoxifen-regulated p53 showed that when
p53 was toggled on, whole-body -irradiation induced
p53-dependent apoptosis in radiosensitive tissues
but mice were protected from tumour development
8
.
Toggling p53 off at the time of -irradiation spared
mice from these radiation-induced toxicities but left
them vulnerable to tumour development. However, mice
were protected from radiation-induced cancers when p53
was transiently toggled on for 6days, starting at 8days
after irradiation. This protection required the action of
the small p53-activating protein ARF. The role of p53 in
radiation-induced toxicity, by contrast, did not require
ARF. 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-binding
sequence 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 arrest
151,211
. 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 alone
212,213
. 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 sites
214
. 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 functions
214,215
.
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 reached
151
.
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 BAX or p53-inducible protein 3 (PIG3), but not to
CDKN1A or MDM2 promoters, thus enhancing p53-induced apoptosis
216
. The binding of p53 to inhibitor of ASPP protein
(iASPP) can have the opposite effect
217
. iASPP has been found to be overexpressed in breast cancer
217
and acute myeloid
leukaemia
218
. Higher levels of iASPP have also been correlated with drug resistance in ovarian cancer
219
and reported in
melanoma
66
. 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 genes
220
. 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 damage
222
. 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 damage
210
. 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 line
226
.
The extrinsic as well as intrinsic apoptotic signalling pathways can have a key role in the response to p53 activation.
Nutlin
227
was 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.
REVI EWS
218 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc
b Senescence-induced phagocytosis
a Apoptotic threshold eect
Nature Reviews | Drug Discovery
Transcriptional
regulation by p53
Apoptosis Cell cycle arrest
Levels of p53 Levels of PUMA
and NOXA proteins
BCL-2, BCL-X and MCL1
p53
Nutlin
Macrophage Tumour cell
Eat me
signal
p21
NOXA
PUMA
activities of p53 do not need to be toxic to be effective
and that at least in this model the two pathways can
be distinguished by their differential requirement for the
ARF protein, which is required for tumour surveillance
but not for radiation-induced toxicity.
Induction of p53 function using the tamoxifen-
regulated p53 system described above in Mdm2-null
animals led to strong induction of apoptosis in radio-
sensitive tissues
9
, which demonstrates their suscepti-
bility towards p53-induced apoptosis. Conversely, in
radiation-resistant tissues the induction of p53 inhibited
cell proliferation.
Other studies suggested that more subtle changes in
the levels of p53 in mice can effectively prevent tumour
formation or lead to tumour regression. In one study,
mice that were engineered to express varying reduced
levels of MDM2 were found to have higher p53 activ-
ity across different tissues, which seemed to suppress
lymphoma formation
10
. The effects of strong p53 acti-
vation on normal tissues included thymic ablation dur-
ing development and increased levels of apoptotic cells
in the gut
11
. However, with a slightly lower level of p53
activation, effects on normal tissues were avoided, but
tumour suppression was still achieved.
It was also shown that a single nucleotide polymor-
phism (SNP) in the promoter of the MDM2 gene affects
MDM2 protein expression
12
. Higher levels of MDM2
expression as a result of the SNP309 polymorphism led
to a sustained reduction in p53 activity, which promoted
the formation of tumours in mice. By contrast, mice that
harbour an additional copy of the complete p53 locus
show improved longevity and a reduced tumour bur-
den
13
, 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 causing
toxicity to normal proliferative tissues. In theory, it should
be possible to control both the intensity and the dura-
tion of the p53 response through careful pharmaceutical
dosing of prospective p53-activatingdrugs.
The mechanism of tumour regression in response to
genetic restoration of p53 function was examined in dif-
ferent mouse models of cancer and found to be tumour-
type specific
1517
. Restoration of p53 in lymphomas led to
their widespread apoptosis, whereas restoration of p53
in sarcomas and hepatocarcinomas led to a senescence-
type response
1517
. 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 via
activated macrophages
17
.
Further studies dissected the individual functions
of p53 that are required for tumour suppression. Three
recent provocative studies examined the functional
requirements of p53 that confer resistance to the devel-
opment of the spontaneous lymphomas that occur in
p53-null mice. In the first study
18
, p53
3KR/3KR
knock-in
mutant mice were generated, in which three acetyla-
tion sites within the DNA-binding domain of p53 were
mutated. 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-X
L

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 the
pro-apoptotic proteins phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1;
also known as NOXA) and p53-upregulated modulator of apoptosis (PUMA); PUMA
seems to have a more important role in normal cells
22
. 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-X
L
, 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) is
reached owing to the inhibition of BCL-2, BCL-X
L
and 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 Puma and Noxa develop normally and do
not seem to be more prone to developing cancer
23,228
. Moreover, it has been shown that
thymocytes lacking Puma are protected from -irradiation-induced apoptosis to the
same extent as p53-deleted thymocytes
228
. In E-Myc-driven lymphoma cells, killing by
DNA-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 apoptosis
231,232
. The induction
of apoptosis in chronic lymphocytic leukaemia by nutlin, for example, has been shown to
involve both transcription-dependent and transcription-independent mechanisms
193
.
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 necroptosis
152
.
The induction of senescence and macrophage engulfment
Another 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 cells
233
.
REVI EWS
to the reduced ability of the mutant p53 to activate the
transcription of some but not all p53-inducible
genes. These mice were not, however, more suscep-
tible to developing spontaneous tumours than wild-
type mice. This surprising result was attributed to the
metabolic 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 study
19,20
, knock-in mice were generated
in which the wild-type p53 gene Tp53 was replaced with
Tp53 genes that were partially defective owing to muta-
tions in the N-terminal transactivation domains TAD1
and/or TAD2. The p53
25,26
TAD1 mutant, which is defec-
tive with regard to the transactivation of many genes, can
nevertheless lead to tumour suppression in various types
of cancer. It cannot induce either G1 arrest or apoptosis
after DNA damage, but it is able to induce senescence
through p53 TAD2-driven transcription. Retention of
TAD1 in a TAD2 p53
53,54
mutant also provides effective
tumour protection. Conversely, the quadruple mutant
p53
25,26,53,54
is completely impaired in p53-transcriptional
activities and was shown to be unable to prevent tumour
development in several models and cell lineages. This
implies 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 but
that activation of the full complement of p53-inducible
genes is not required because either the TAD1- or the
TAD2-induced sets of genes are sufficient
19,20
.
The third and most recent study took a differ-
ent approach; here, mice with a triple knockout of
cyclin-dependent kinase inhibitor 1A (Cdkn1a; which
encodes 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 as Noxa) showed profound defects
in p53-driven induction of apoptosis, cell cycle arrest
and senescence. CDKN1A (encoding p21), PUMA and
NOXA are three of the most important p53-responsive
genes; p21 is required for growth arrest
21
, whereas PUMA
and NOXA are required for the apoptotic response to
radiation
22
. Nevertheless, even in this triple knockout
mouse model of the most important p53 effector genes,
p53 was still able to exert efficient tumour-suppressive
functions
23
. This, once again, suggests that other func-
tions of p53, such as its regulation of DNA repair genes
as well as genes involved in metabolism, have a crucially
important role in tumour suppression. In this context,
it is important to note that treatment of p53-null mice
by incorporating the antioxidant N-acetylcysteine into
their diet also prevents spontaneous tumour develop-
ment. This strongly supports the idea that the antioxidant
DNA repair response is crucial to the tumour-suppressive
surveillance activity of p53 (REF.24).
These studies examined the level and duration of the
p53 response that is necessary to prevent cancer formation
or to eliminate an established tumour. They also shed light
on the side effects in normal tissues that can be anticipated
from p53 therapy. Although the treatment of advanced
cancers may require an apoptotic or senescence response,
a DNA repair or antioxidant effect may be sufficient for
the treatment of pre-neoplasia (FIG.1). It will be important
to establish which tumours contain p53 mutations in the
acetylation sites
18
or the TADs
19,20
, and to assess the ability
of these partially defective p53 proteins to drive a thera-
peutic response to chemotherapy and radiation. These
results do, however, suggest that a limited non-toxic p53
response may be effective in tumour prevention, which
represents a very attractive goal for drug development.
Table 1 | p53 activators currently in clinical trials*
Compound Mechanism of
action
Status ClinicalTrials.gov
identifiers
Company
RG7112 (also known
as RO5045337)
Small-molecule
MDM2 antagonist
PhaseI trial in advanced
solid tumours, solid tumours,
haematological neoplasms and
liposarcomas (all completed)
NCT00559533
NCT01164033
NCT00623870
NCT01143740
Roche
RG7112 (also known
as RO5045337) with
cytarabine
Small-molecule
MDM2 antagonist
PhaseI in AML (completed) NCT01635296 Roche
RG7112 (also known
as RO5045337) with
doxorubicin
Small-molecule
MDM2 antagonist
PhaseI in soft tissue sarcoma
(completed)
NCT01605526 Roche
RO5503781 Small-molecule
MDM2 antagonist
PhaseI in advanced malignancies
(recruiting)
NCT01462175 Roche
RO5503781 with
cytarabine
Small-molecule
MDM2 antagonist
PhaseI in AML (recruiting) NCT01773408 Roche
MI-773 (also known
as SAR405838)
Small-molecule
MDM2 antagonist
PhaseI in malignant neoplasms
(recruiting)
NCT01636479 Sanofi
DS-3032b Small-molecule
MDM2 antagonist
PhaseI in advanced solid tumour
lymphoma (recruiting)
NCT01877382 Daiichi
Sankyo
PRIMA-1
MET
(also
known as APR-246)
Reactivation of
mutant p53
PhaseI in haematological and
prostatic neoplasms (completed)
NCT00900614 Aprea
AML, acute myeloid leukaemia. *Sourced from the ClinicalTrials.gov database.
REVI EWS
Induction of death and
senescence in tumour
cells
Prevention of tumour
occurrence
Protection of tumour
cells and normal cells
from cytotoxic therapy
Toxic eects of drugs
(such as doxorubicin) and
radiation 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 defects
in p53-regulatory pathways, such as overexpression or
amplification of MDM2 and MDMX or epigenetic silenc-
ing of the INK4AARF locus
25
, the major approach for the
restoration of p53 activity has been to inhibit the func-
tion of negative regulators of the p53 response
26
(FIG.2a).
Although a number of targets have been identified, such
as blocking the function of the viral E6 protein in inhibit-
ing p53 in human papillomavirus (HPV)-driven cancers
using bortezemib
27
, or inhibiting the enzymatic E3 ligase
activity of MDM2 (REF.28), the vast majority of current
efforts and current clinical trial activities are focused
around small-molecule drugs that block the N-terminal
proteinprotein interaction between p53 and MDM2 or
MDMX. In a more general approach, very large chemical
libraries have been screened in cell-based assays for small
molecules that can activate wild-type p53, but the targets
of these agents have only occasionally been identified
for example, the small-molecule compound tenovin 6,
which inhibits sirtuin 1 (SIRT1), thus enhancing the
acetylation and activity of p53 (REF.29).
In cases where the p53 protein is mutated, attempts
have been made to find molecules that act as p53
chaperones by binding to p53 and stabilizing its con-
formation. Genetic studies that demonstrate second-site
reversion of the common p53 mutations have supported
such an approach
30,31
because they show that alterations
in other parts of p53 can correct the defect induced by
the primary mutation. Detailed structural studies have
yielded candidate mutant p53-binding molecules, and
cell-based screening approaches for small molecules that
can act as chaperones of mutant p53 have also been pur-
sued. However, identifying the targets of the screening
compounds has proved to be challenging. About 8% of
p53 mutations result in early termination of translation
32
,
and read-through drugs are being sought that can allow
the bypassing of mutant stop codons and thus restore the
expression ofp53.
Attempts have also been made to manipulate the
p53 pathway using gene therapy or immunological
approaches. These have been recently reviewed (see
REFS33,34).
Targeting interactions between MDM2 and p53 with
small-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) and NOXA
22
,
but remarkable experiments have recently shown that apoptosis is not required for tumour suppression in mouse
models
18,19,23
. 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 suppression
23
.
Triple-knockout mice lacking Cdkn1a, Puma and Noxa still show a potent p53-dependent inhibition of tumour
development
23
. 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 protein4 (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 doxorubicin
234
, 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.
REVI EWS
p53 activators MDM2 inhibitors or
MDM2/MDMX dual inhibitors
E6 inhibitor
Tenovin 6
Downstream apoptotic or
cell cycle arrest genes
Wild-type p53
MDM2
MDM2
SIRT1
HPV E6
ARF
b
a
c d
Site 1
Site 1
Site 2
complexed with this peptide revealed the presence of a
deep hydrophobic cleft in MDM2, into which the p53
peptide was embedded
36
. The peptide interacted mainly
through three critical amino acids: Phe19, Trp23 and
Leu26. Using large phage display peptide libraries, another
study independently identified these three amino acids
as being essential for the interaction between p53 and
MDM2 (REF.37). Furthermore, it was found that peptides
with far higher affinities for MDM2 could be discovered,
thus supporting the concept that the pocket in MDM2
was druggable
38
. This was further established in assays
where the MDM2-binding peptides were displayed on the
surface of a thioredoxin protein scaffold, thus stabilizing
their expression in mammalian cells. Binding of MDM2
by such mini-proteins led to the induction of high levels
of active p53 protein and inhibited the cell cycle, demon-
strating that blocking the p53MDM2 proteinprotein
interaction was sufficient to activate the p53 response
37
.
The first non-peptidic molecule that demonstrated
the possibility of interrupting the p53MDM2 interface
was 4,5-dihydroimidazoline (nutlin; Roche)
5
. The crystal
structure of nutlin3a, an isomer of nutlin, when bound
to MDM2 (site 1 in FIG.2b), provided the template for
the design of better inhibitors
39
(TABLE2) such as the
benzodiazepinedione family of compounds (Johnson and
Johnson, 80 nM)
40
, chromenotriazolopyrimidine (Amgen,
1.2 M)
41
, terphenyls (Yale University, 182 nM)
42,43
and
chalcones (Max Planck Institute, Martinsried, Tbingen
University and Roche Diagnostics, 49 M)
44,45
; numbers
in parentheses refer to maximum affinities.
At the same time, structure-based screening of com-
pounds combined with molecular modelling enabled the
development 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 was
developed: the spirooxindole-based molecules (University
of Michigan)
46
. This led to the identification of MI-219,
which has a subnanomolar affinity for MDM2, is orally
available, has good pharmacokinetic and pharmacody-
namic (PK/PD) properties and is now in PhaseI clinical
trials. This compound led to an increase in p53 levels and
an associated increase in levels of the p53-targeted genes
CDKN1A and MDM2. It also selectively induced PUMA
in osteosarcoma and prostate cells but not in normal cells
invitro and in xenograft models
46
. However, complete
tumour 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 function
27
. 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) deacetylase
29
. b | The firstcrystal structure of a compound, nutlin3 (in coloured sticks), bound to the surface of
MDM2 (REF.235). The nutlin compound on the right binds to site 1, whereas thenutlin compound on the left binds to
site 2. c | Various MDM2 inhibitors (which bind to the surface and are shown in stick form) have beencrystallographically
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
hydantoinRO-2443. The two small molecules pack against each other and are shown instick form, along with the surface
of each MDMX molecule
72
.
REVI EWS
until recently, when the same group developed a series
of 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 in
severe combined immunodeficient (SCID) mice bear-
ing SJSA-1 osteosarcoma tumour xenografts, with mice
remaining tumour-free for 60days after treatment
47
.
Simultaneously, other drugs that have been reported
include pyrazole and imidazole compounds (University
of Pittsburgh, 20 nM)
48
, imidazole-indoles (Novartis,
2 nM)
49
, isoindolinones (University of Newcastle,
170 nM)
50
, pyrrolidinones such as PXN822 (Priaxon,
<10 nM)
51,52
, piperidines (Merck, 20 nM)
53
, spirooxin-
doles (University of Naples Federico II and the University
of Salerno, 50 nM)
54
and the sulphonamide NSC279287
(Virginia Commonwealth University, 32 M)
55
. More
recently, the rational design of inhibitors combining
structural, computational, biophysical and synthetic
techniques has been carried out by researchers at Amgen,
resulting in morpholinones and piperidinones (such as
AM-8553 and AMG232) that inhibit MDM2 at single-
digit nanomolar affinities and have good pharmacokinetic
profiles for clinical development
5658
.
Structurally, all of these compounds bind to MDM2
by mimicking the key residues of p53 (Phe19, Trp23 and
Leu26). These interactions are dominated by van der
Waals packing interactions and by a single hydrogen bond
between the side chain of Trp23 and the backbone of
Leu54. However, some of the more recent compounds lack
this key hydrogen bond and yet bind with high affinity,
which shows that this interaction is not essential to the
design of a tight binding ligand. The differences in the
specific interactions made with the amino acids that
constitute the peptide including Phe19, Trp23 and
Leu26 have been revealed by crystallography
36
, and
NMR studies have started to show additional differences
in the dynamics of the interactions between the com-
pounds and MDM2 (Ref.59), which have been exploited
to design ever more potent inhibitors
50
. The amide of the
spirooxindoles and the carboxylic acid of the piperi-
dinones give these compounds higher affinities through
additional interactions with the side chain nitrogen of
His96 in MDM2 (Ref.56). Recent NMR studies have
shown that the piperidinones also engage residues 1016
of MDM2, particularly through van der Waals contacts
with the residues Val14 and Thr16 (Ref.60).
The first 24 amino acids of MDM2 form a flexible
lid region and therefore cannot be resolved in X-ray
maps. Hence, they are missing from the crystal struc-
tures that have traditionally been used for the design of
inhibitors. Using several constructs of MDM2 with vary-
ing lengths of its N-terminal region, it has been shown
that although the thermodynamics of the binding of p53
peptides and nutlin3a remain unaffected, the piperidi-
nones gain significant enthalpy from the inclusion of
residues 616 of the MDM2 construct. This new NMR
study clearly shows that the inclusion of these residues
opens an opportunity for designing new inhibitors.
These studies will be aided by the recent discovery of
mutations in both the binding pocket and the lid region
of MDM2 that confer resistance to nutlin
61
.
Recently, a major drug discovery effort by Roche
combined the retention of important structural features
of MDM2 with a detailed optimization of the molecular
features of the small-molecule compounds; these efforts
yielded the molecule RG7112. RG7112 was resistant to
oxidation, which had been a problem with earlier mol-
ecules, and displayed good pharmacokinetics. This
molecule binds to MDM2 in a manner similar to nut-
lin3a
62
, with an affinity of 11 nM (compared to 90 nM for
nutlin3a). It achieved good tumour regression in ani-
mal models of various cancers and is currently in PhaseI
clinical trials. The same group also combined structural
data on the interactions of the pyrrolidines with denovo
design strategies to create RG7338, a small molecule that
achieved invivo efficacy against SJSA1 osteosarcoma
xenografts at one-fourth of the dosage needed for RG7112
(REF.63). However, these molecules are only active against
MDM2; some have argued that there is a need for dual
inhibitors of MDM2 and MDMX to expand the range
of tumours that can be treated
64
. For example, MDMX is
highly expressed in melanoma, although a recent paper
suggests that this may not be the only means by which p53
function is suppressed in this type of cancer
65,66
.
One advantageous property of these compounds, as
reported in preclinical studies, is the minimal toxicity
to both radiosensitive and radioresistant normal mouse
tissues. This appears to be related to the pharmacokinet-
ics of these compounds; as these compounds are only
present in the cell for a short time, they only induce a
short pulse of p53 activation. Indeed, in cell-based mod-
els the removal of the compounds immediately leads to
the sequestration and degradation of p53 (REF.67). This
supports the suitability of these molecules for carefully
titrating the level and duration of the p53 response.
As discussed above for the mouse models, the lack of
toxicity in preclinical models suggests that the right level
of p53 activation can be achieved. However, the toxicity
studies were carried out in mouse xenograft models, and
it is conceivable that subtle species-specific differences
in the protein sequence of MDM2 can result in different
affinities of the small molecules to mouse versus human
MDM2. The species specificity of the current MDM2
inhibitors has not been established. Nevertheless, these
drugs were optimized for human MDM2 and might
therefore have a lower toxicity towards healthy tissues
in mouse models owing to a lower binding affinity to
mouse MDM2, leading to an over-optimistic view of the
therapeutic index of the compound.
Several MDM2 inhibitors derived from natural prod-
ucts have also been identified, such as the prenylated
xanthones -mangostin (from the fruit of Garcinia man-
gostana L.) and gambogic acid (from the resin of Garcinia
hanburyi)
68
. These molecules are thought to bind in a
manner similar to nutlins and pave the way for the devel-
opment of new classes of inhibitors. As non-toxic natural
products, they also offer potential as chemopreventive
anticancer agents. Marine organisms are now also being
considered as a potential resource for a variety of lead
compounds. For example, the siladenoserinols, derived
from the marine invertebrate family Didemnidae, were
recently reported to inhibit the p53MDM2 interactions
REVI EWS
with low micromolar affinity
69
; these are large molecules,
and the structural mechanism underpinning the inhibition
remains 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 also
leading to the identification of new inhibitors; a recent
example is the report of a new affinity-based chemical
screen that has led to the identification of novel micro-
molar dual inhibitors of the p53MDM2 and p53
MDMX interactions
71
. There are three new avenues that
have paved the way for the discovery of next-generation
drugs. RO-2443 (Roche)
72
is an indolyl hydantoin that
inhibits both MDM2 and MDMX with low micromolar
affinity. Structural analysis showed that two molecules
of RO-2443 stack on top of each other and bring two
molecules of MDMX into a tetrameric complex (FIG.2d).
However, this compound displays poor solubility.
It served as a lead compound for the development of
RO-5963, which has a half-maximal inhibitory concen-
tration (IC
50
) of ~17 nM for MDM2 (the IC
50
of nutlin3a
is ~19 nM) and ~24 nM for MDMX (the IC
50
of nutlin3a is
~9 M). The molecule showed efficacy in wild-type
p53-containing cell lines from different lineages
72
.
Separately, it was found that synthetic analogues of
5-deazaflavins bound to the RING finger domain
of MDM2 with low micromolar affinity and prevented the
ubiquitylation of p53 invitro and invivo; these compounds
mimic the effects of ARF or ribosomal proteins such as L11
and provide the starting point for the development of novel
inhibitors
26
. Finally, a novel interaction has recently been
hypothesized to control the binding of nutlin-like drugs
and to also mediate the transmission of allosteric inter-
actions in the p53MDM2 system. The crystal structure
of MDM2 with nutlin shows two molecules of nutlin that
are closely associated with the surface of MDM2 (FIG.2b).
While one of the nutlin molecules binds to the site that
has been targeted for all small-molecule development
strategies (site 1 in FIG.2b), hydrogendeuterium exchange
and computer simulation studies suggest that the second
site (site 2 in FIG.2b) may actually be the site where nut-
lin first makes contact with MDM2 before tumbling into
the main binding pocket, and that this site of first contact
may be druggable
73,74
. Indeed, this hypothesis is further
strengthened by the demonstration that a mutation in the
second site renders MDM2 resistant to nutlin binding
74
. If
validated further, this will open a new window into drug
design efforts, signalling a need to incorporate kinetic
effects 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 and
piperidinones (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 68
SAH-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-1
MET
(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.
REVI EWS
Peptide and stapled peptide inhibitors of the p53MDM2
interaction. The p53 TAD1 region that is embedded in
the N-terminal domain of MDM2 adopts a helical motif
spanning residues Thr18 to Leu26 in the bound state.
It is clear that the helical motif displays an amphipathic
surface such that the residues Phe19, Trp23 and Leu26
are exposed on one side, and it is this side that is embed-
ded into the hydrophobic crevice in MDM2 (REF.36). This
insight has enabled the construction of many peptides
with amino acid substitutions at positions that are not
assumed to be crucial for the interaction with MDM2.
These changes alter the affinity for MDM2, as well as
stability, solubility and cell permeability. An increasing
number of high-affinity peptides that mimic p53 and sta-
bilize it by inhibiting MDM2 and/or MDMX have been
identified in recent years, including the high-affinity
peptides known as PMI
75
, pDIQ
76
and PMI-N8A
77
. These
studies have largely exploited sophisticated phage library
screens. Structural analyses have provided detailed
insights 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 the
peptides and the surface of MDM2 were found to con-
tribute to the altered affinities. The high-affinity peptide
PMI-N8A (with an affinity of 490 pM and 2.4 nM against
MDM2 and MDMX, respectively) was identified after an
alanine scan of the PMI peptide was performed, where
each amino acid was sequentially replaced by alanine and
the affinity for MDM2 or MDMX was measured to exam-
ine the contribution of the mutated amino acid to the
interaction
77
.
In addition to these designs, the crystallographic
resolution of a mutant form (pDIQ) of a phage-derived
high-affinity peptide (pDI)
76
showed that context
dependence must be taken into consideration. The pDIQ
peptide bound to MDM2 as a helix, whereas it bound to
MDMX as a distorted helix; the local loss of helicity in
the latter is associated with higher conformational plas-
ticity, which encourages the formation of compensatory
interactions between the peptide and MDMX.
A group at the University of Maryland designed
protease-resistant D-analogues of the PMI peptide
78
.
Theses peptides (termed
D
PMI- and
D
PMI-) could not
traverse the cell membrane, and upon conjugation to cati-
onic cell-penetrating peptides they were nonspecifically
cytotoxic in a p53-independent manner. However, when
encapsulated in liposomes that were targeted to integrins
through RGD signatures, the peptides were very active
against the human glioblastoma cell line U87 (which
expresses wild-type p53);
D
PMI- induced a dose-depend-
ent growth inhibition with an IC
50
of 1.9 M (compared to
3.8 M for nutlin3) and it also demonstrated growth inhi-
bition of U87 mouse xenograft models in a p53-dependent
manner. This important study implicitly warns against the
use of cationic peptides as delivery vehicles.
Molecular dynamics simulations have shown that
the interactions between MDM2 and the peptides
described above depend upon their mutual conforma-
tional modulation, leading to varying thermodynamic
signatures
79
. This insight resulted in the finding that even
single-amino-acid changes in the peptides can produce
a context-dependent effect. It is clear that a detailed
examination of the underlying conformational dynam-
ics is crucial for the design of peptides that bind with
high affinity
80
. Indeed, these findings were confirmed in a
study combining biophysics, crystallography and simula-
tions for the design of peptides against a different target,
eukaryotic translation initiation factor 4E (eIF4E), where
the plasticity of the interacting surfaces was observed to
yield synergistic positive effects on binding affinity
81
.
The development of techniques to stabilize the heli-
city of the peptides was given a major boost with the
introduction of the stapling technology
82
. Stapling entails
the introduction of a hydrocarbon linker between two
non-adjacent amino acids in the peptide. Recently, cell-
penetrating stapled peptides that inhibit both MDM2 and
MDMX have been developed, showing invitro and invivo
efficacy in cancer models
8386
. In an alternative chemical
approach to stabilizing helices, a synthetic spiroligomer
-helix mimic was described that can penetrate cells, bind
to MDM2 tightly and induce p53 pathway activation
87
.
Another approach to convert peptides into drug-like mol-
ecules involved the display of MDM2-interacting peptides
on a small, highly structured, disulphide-rich knotted
peptide scaffold called a cyclotide, which can bind to
MDM2 and activate the p53 response invitro
88
.
Nevertheless, the development of stapled peptides
as MDM2 inhibitors is controversial; moreover, some
of the early work
89,90
on stapled peptides that bind the B
cell lymphoma 2 (BCL-2) family of proteins and induce
apoptosis has recently been questioned, in particular the
cellular uptake of the stapled peptides and their activity
in cells
91
. However, more recent studies have estab-
lished that stapled peptides targeting both MDM2 and
MDMX are highly effective and able to enter every cell
in a variety of cell lines to activate the p53 response
85,86
.
In a detailed comparison with nutlin, the stapled pep-
tides sMTide02 and sMTide2A, which bind to MDM2
and MDMX, were shown to be more specific than nut-
lin. They showed less toxicity to p53-negative cells and
induced a higher level of p53 reporter gene response
than nutlin in several cell-based reporter assays
85
.
The stapled peptide ATSP-7041 (Aileron Therapeutics)
was also shown to bind to MDM2 and MDMX invitro,
and showed efficacy in various cancer cell lines as well
as in mouse tumour xenografts
86
. ATSP-7041 has been
shown to have a slower dissociation rate from MDM2
(43minutes) than small-molecule inhibitors of MDM2
(which typically have a dissociation rate of around 6min-
utes). Importantly, following drug removal, the activation
of p53, MDM2 and p21 protein levels in the MCF-7 cell
line appeared to persist for longer with ATSP-7041 (up
to 48hours) than with the small molecule RG7112 (up to
4 hours). The PK/PD properties of stapled peptides
were also investigated in mouse, rat and non-human
primate models using a labelled form of ATSP-7041.
Encouragingly, this novel class of molecules showed
excellent pharmaceutical properties with a very good dis-
tribution throughout all organs and a persistence in the
plasma that allowed therapeutic levels to be maintained
with once-a-day dosing
86
.
REVI EWS
Restoration of misfolded mutant p53. p53 seems to have
evolved to be thermodynamically and kinetically unsta-
ble at body temperature in humans, which may allow for
tighter control of p53 protein levels
92
. Many oncogenic
mutations inactivate p53 function by disrupting the direct
binding to specific DNA (known as contact mutants) or by
preventing the proper folding of the central DNA-binding
domain of p53 (known as structural mutants)
93
. Many
of these mutant p53 proteins are temperature-sensitive,
which has encouraged attempts to discover molecules
that can restore mutant p53 activity by acting as p53 chap-
erones (FIG.3a). This concept has recently proved to be
successful in Gauchers disease, in which small molecules
were found to restore the activity of the mutant glucocere-
brosidase enzyme by causing its stabilization
94,95
. These
observations have led to generic methods of identifying
protein 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 resistance
to denaturation induced by high temperatures
96
.
Small-molecule lead compounds have been discov-
ered by computational and fragment screening, and
shown to bind to a druggable surface crevice on p53
that is created by the Y220C hotspot mutation (FIG.3b),
which is found in approximately 75,000 new cancer cases
a year
32
. These compounds include the carbazole-based
PK083 (REF.97), the halogen-enriched PK5174, PK5196
(REF.98), PK7088 (developed at the MRC Laboratory of
Molecular Biology, Cambridge, UK)
99
and various ben-
zothiazoles
100
, and they have been characterized in detail
using biophysical and crystallographic techniques (FIG.3b).
Some of the compounds seem to be able to restore varying
levels of p53 activity in cell lines harbouring the Y220C
mutation
99
.
PRIMA-1 (a 2,2-bis(hydroxymethyl)-3-quinuclidi-
none developed at the Karolinska Institute, Stockholm,
Sweden) and its structural analogue PRIMA-1
MET

(APR-246, developed by Aprea) have been shown to
restore mutant p53 (R273H and R175H) activity invitro
and invivo
101103
. PRIMA-1
MET
has also successfully com-
pleted a PhaseI clinical trial, showing some indication of
efficacy (ClinicalTrials.gov identifier: NCT00900614)
104
.
PRIMA-1
MET
seems to lead to the formation of covalent
adducts on mutant p53
R175H
and p53
R273H
proteins, but its
exact mechanism of action has yet to be fully understood
105
.
The formation of covalent adducts with alkylating
small 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 at
Cys124 and Cys141, which are most accessible
106
. Recent
computational and biochemical studies on PRIMA-1 also
suggest that its mechanism of action involves a covalent
interaction with Cys124 in a binding pocket of the core
domain of p53 (REF.107). A virtual screen of small mol-
ecules that bind to p53
R273H
identified stictic acid, which
was predicted to bind to this pocket. It was shown to
induce p21 activation in transcriptional gene reporter
experiments in cells
107
. In a separate study using top-down
Fourier transform ion cyclotron resonance (FTICR) mass
spectrometry and the alkylating agent N-ethylmaleimide,
Cys182 and Cys277 on p53 were identified to be most
susceptible to alkylation
108
. The OxMRM approach, which
combines oxidation through N-ethylmaleimide and mul-
tiple reaction monitoring (MRM) mass spectroscopy, was
also used to probe the oxidation status of the cysteine mol-
ecules of p53 in cells, and identified Cys182 as being very
susceptible to oxidation
109
.
PRIMA-1
MET
also seems to be able to restore the
function of mutant p63 (a p53 homologue)
110,111
. In the
congenital disease ectodermal dysplasia, mutations in
the DNA-binding domain of p63 are associated with
orofacial clefting and limb abnormalities. Some of these
mutations correspond to the hotspot oncogenic muta-
tions that are observed in the DNA-binding domain
of p53. In a transfection-based system using H1299
cancer cells, PRIMA-1
MET
was shown to also reactivate
other p53 homologues such as TAP73a, TAp73b and
TAP63g as well as inducing apoptosis
112
.
Other therapeutics that target mutant p53 via vari-
ous mechanisms have been described; for example,
NSC176327 and RETRA disrupt the binding interactions
between mutant p53 and p73, whereas MIRA-1 elimi-
nates mutant p53. These therapeutics have been reviewed
in detail elsewhere in the literature
52,113
. Some of these
drugs show good biological activity, but more studies are
required for a full biophysical characterization (BOX3).
Stabilizing p53 with zinc. Zinc is important for the
proper 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 of
p53 (REF.115). Mutations that affect zinc binding, such as
C242S, H179R, C176F and R175H, are frequently found
in human cancers
116
.
There have also been invitro studies showing that the
absence of homeodomain-interacting protein kinase 2
(HIPK2) leads to the accumulation of misfolded wild-
type p53 owing to the deregulation of zinc-binding met-
allothionieins
117
. However, supplementation with zinc
reversed this effect. More recent papers have shown that
the addition of zinc (invitro and in xenograft models)
restored DNA-binding activity to the contact mutant
p53
R273H
and the structural mutant p53
R175H
, thus allowing
p53-mediated cell killing by adramycin or cisplatin
118
. It is
surprising that both contact mutants and conformational
mutants of p53 are rescued by the same compounds;
however, this has been reported for several different
compounds
107,119
.
Zinc has also been found to be effective in promoting
cell killing in colon and breast cancer cell lines when
used in combination with the MDM2 inhibitor MI-219
(REF.120). One can assume that the efficacy of any drug
that restores the function of mutant p53 will be enhanced
by combining it with a nutlin-like MDM2-binding
inhibitor, as the immediate target of the transcriptional
function of p53 is the activation of MDM2 expression.
NSC319726, a thiosemicarbazone, was identified
in a screen of the NCI60 panel of human tumour cell
lines and reported to be selectively toxic to cell lines that
carry the p53
R175H
mutation. This compound appeared to
restore the transcriptional and apoptotic functions of the
R175H mutant and led to the loss of the PAb240 epitope
REVI EWS
Mutant p53
Mutant p53 Mutant p53
Mutant p53 Mutant p53
Mutant p53
p53-inducible genes
Zn
2+
b
a
Binding to DNA
Restoration of p53
conformation
p53 reactivators +
of p53, which is associated with the unfolded state of p53.
NSC319726 was also shown to inhibit the growth of a
tumour xenograft of the TOV112D cancer cell line in
a nude mouse model. TOV112D cells express high levels
of mutant p53
R175H
(REF.121). The activity of NSC319726
appears to depend on its ability to chelate zinc and
facilitate its transport to mutant p53. The most strik-
ing evidence for its selectivity for mutant p53 activation
as opposed to wild-type p53 activation comes from the
finding that NSC319726 is toxic to mice that carry the
germline R175H mutation but not to wild-type mice
121
.
p53 read-through drugs. About 8% of the cancer-associated
mutations found in p53 are nonsense mutations, and
recent developments in the isolation of small molecules
and drugs that promote the read-through of nonsense
codons could provide a novel approach for treating
tumours carrying this type of mutation
122,123
. The two most
frequent nonsense p53 mutations in the human mutation
database are R196X and R213X
32
. A recent study showed
that treatment of the human tumour cell line HDQ-P1,
which contains a homozygous nonsense mutation at
codon 213 (CGA-TGA), with the read-through-promoting
aminoglycoside antibiotic G418 led to a dramatic increase
in the level of TP53 mRNA and full-length p53 protein
124
.
The stabilization of the mRNA was due to inhibition of
the nonsense mutation, and the full-length protein was
shown to be transcriptionally active and able to induce
p53-dependent apoptosis. One would anticipate that the
restored full-length protein might re-establish the p53
MDM2 feedback loop, so a combination of G418 and
nutlin may be especially effective.
There are a number of read-through drugs in
development. The most advanced compound, PTC 124
(Ataluren; PTC Therapeutics), has completed PhaseIII
clinical trials for nonsense mutation cystic fibrosis
125,126
.
However, it is the subject of some controversy because of
its remarkable activity in directly binding to and stabiliz-
ing firefly luciferase, an enzyme used in the high-through-
put screen that originally identified the compound
127,128
.
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 model
of Duchenne muscular dystrophy (DMD)
129
. Most of the
research in this field has concentrated on the treatment of
genetic diseases such as cystic fibrosis and DMD, which
are highly demanding in terms of safety requirements as
the drugs would need to be administered for the lifetime
of the patient. Developing such compounds for cancer
may be easier as the treatment time would be limited,
which makes potential side effects more tolerable. Because
p53 mutations are so frequent, the number of patients
with a particular mutation can also be surprisingly high.
For example, the codon 213 nonsense mutation discussed
above is thought to be present in 1% of all human cancers
that is, roughly 220,000 cases worldwide
32
.
Other p53 activators. Cell-based screens for activators
of the p53 pathway have identified large numbers of
compounds, many of which have unknown targets and
incompletely defined mechanisms of action. The screens
that seem to be most informative have used reporter cell
lines that measure the activity of p53 as a specific tran-
scription factor. These cell lines show excellent assay
characteristics and are easily adapted to high-through-
put methods
29,130
. However, as DNA-damaging agents
and many cellular stress signals can activate p53, these
screens can have a high hit rate of compounds that are
not suitable for development. Surprisingly, a small reduc-
tion in the dose of the screening compound can lead
to very successful screens with more modest hit rates,
which implies that the reduced concentration eliminates
the detection of many of the DNA-damaging molecules
that are present in large compound collections.
In a few cases, the mechanisms by which these
compounds activate p53 have been determined. These
include the small molecule RITA
119
, which is reported to
target p53 itself, cyclin-dependent kinase (CDK) inhibi-
tors such as roscovitine (Cyclacel Pharmaceuticals)
131
,
RNA polymerase inhibitors such as actinomycin D
130
,
exportin 1 (XPO1; also known as CRM1)-binding
compounds such as leptomycin B and KPT-330 (also
known as selinexor; Karyopharm Therapeutics)
132
, the
NEDD1 (neural precursor cell expressed developmen-
tally downregulated protein 1) ligase inhibitor MLN4924
(Millennium Pharmaceuticals)
133
and sirtuin inhibitors
such as the tenovins (University of Dundee)
29
. In a recent
study using an invivo mouse model of chronic myeloid
leukaemia (CML), inhibition of SIRT1 by tenovin-6 or
knockdown of SIRT1 led to tumour regression and selec-
tive killing of leukaemia stem cells via p53-dependent
growth arrest and apoptosis
134
. 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 (surface
shown with the sphere representing the Zn
2+
atom) demonstrate that several molecules (in
sticks) that stabilize this mutant bind to the same region: that is, in the vicinity of thesite
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.
REVI EWS
described above (with the possible exception of RITA)
act on targets that will affect other pathways in addition
to the p53 pathway and will therefore require careful
evaluation in preclinicalmodels.
Considerations for therapy
As p53-based therapies are entering the clinic, a key issue
is the design of clinical trials that will demonstrate suf-
ficient efficacy to allow regulatory approval. There are
more than 65,000 papers published on p53, but these do
not yet provide an unequivocal prediction as to which
human tumours are most likely to respond to these
therapies with the best therapeuticindex.
The current crucial issues are efficacy, resistance
development and side effects in patients. The thera-
peutic index is determined by the relative sensitivity of
both tumour and normal tissues towards p53 activation.
Drugs may exhibit on-target or off-target side effects.
Small-molecule inhibitors of MDM2, for example, can
upregulate p53 in normal cells in an on-target effect
and this may lead to undesired toxicity. Off-target
effects of nutlin for example, at high doses have
been noted and are discussed below
135138
. The key issues
regarding p53 activation are the type of response this
induces whether it would lead to cell cycle arrest,
metabolic alteration, DNA repair, autophagy, senescence
or apoptosis and how it affects tumour cells versus
normal cells. Much of the preclinical cellular and invivo
data with drugs that activate p53 suggest that these dif-
ferent cell fate responses are highly dependent on the
intrinsic properties of the tumour or tissue type and may
vary greatly among patients, as known polymorphisms in
the p53 pathway for example, in the MDM2 promoter
12

can influence the outcome.
It is thus important to have carefully designed clinical
trials that take these factors into consideration. These tri-
als would select tumour types such as melanoma or acute
myeloid leukaemia (AML) in which p53 mutations are
very rare and where there is a very clear unmet clinical
need. It would be tremendously useful to understand the
intrinsic mechanisms of resistance towards p53-induced
apoptosis and how resistance is induced following pro-
longed treatments with p53-activating drugs. As dis-
cussed below, p53 mutations are expected to be a major
driver of resistance development. Therefore, searching
for tumours in which p53 mutations are not found but
which are still responsive to p53-activating drugs is an
attractive strategy. It is also possible to explore which
biomarkers may predict responsiveness towards the
clinical efficacy of p53 activation in patients.
Preclinical and clinical studies. Numerous early invitro
studies 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 (REFS5,139).
Nutlin and the MI drugs have been found to be effective
in killing a broad range of cell lines invitro and have also
shown invivo efficacy in mouse models
5
. Cancers such
as chronic lymphocytic leukaemia (CLL)
140
, acute lym-
phoblastic leukaemia (ALL), AML
139
, myeloma
141
, neuro-
blastoma
142
melanoma
66
and mantle cell lymphoma
143

often contain wild-type p53, which allows the targeting
of interactions between MDM2 and p53 in order to acti-
vate p53. MDM2 gene amplification is quite common in
sarcomas (occurring in about 20% of cases), which could
make these cancers good candidates for treatment with
MDM2 inhibitors
144145
.
The responses to these small-molecule drugs are,
however, dependent on cancer cell types. In an early
study of nutlins, carried out using a panel of cancer
cell lines from a variety of tumours, it was shown that
some cell lines were more susceptible to nutlin-induced
apoptosis than others
146147
. For example, the colorectal
cancer and lung cancer cell lines tested were shown to
undergo reversible cell cycle arrest without apoptosis,
whereas other cell lines such as the CML cell line
BV173 appeared to undergo apoptosis without cell
cycle arrest
147
. Testicular germ cell tumours were found
to be exquisitely sensitive to nutlin- or cisplatin-induced
p53-dependent apoptosis
148
. It was thought that this
high sensitivity to cell death was caused by mitochon-
drial priming owing to high levels of NOXA, which is
dependent on the transcription factor octamer-binding
protein 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 test
animal 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?
Invivo specificity: 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?
REVI EWS
proteins such as PUMA and NOXA (as discussed
below)
149
. Although the high level of curative testicular
germ cell tumour responses to cisplatin may make it an
unlikely choice for the development of MDM2 inhibi-
tors, the very high level of hearing damage induced by
cisplatin could provide a clinical opportunity for the
development of a less toxic drug. As polymorphisms that
predict adverse reactions to cisplatin become apparent,
patients with these polymorphisms may be better suited
to alternative therapies such as MDM2 inhibition
150
.
Nutlin
5
, 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 drugs
led to a robust accumulation of p53 and apoptosis in
xenograft tumours such as the osteosarcoma SJSA-1 cell
line and the prostate metastasis-derived LNCaP cell line,
with little toxicity observed in terms of weight loss or
in necroscopy studies
5,46,47,63
. There is also little toxicity
towards the normally radiosensitive tissues such as
small-intestine crypts and the thymus
46
, which show
only minimal accumulation of p53 protein. However, an
observed accumulation of the p21 protein suggests that
p53 is, nonetheless, activated in these tissues. This is in
contrast to the high levels of p53 accumulation observed
in these tissues after treatment with ionizing radiation.
It is known that ionizing radiation induces many other
signalling pathways independently of its effects on the
p53 pathway. Thus, although apoptosis induction by
irradiation is highly dependent on p53 activity, this
occurs in the context of the activation of many other
radiation-induced pathways. By contrast, in cells treated
with nutlin only the p53 pathway is activated. The
enhanced apoptotic response observed after radiation
treatment can therefore be explained as a consequence
of the integration of the pathways activated by radiation
7
.
As discussed above, the kinetics of the p53 response
are also crucial when considering p53-directed therapy;
for how long do tumour cells need to be exposed to the
p53-activating agent before they are eliminated? As
discussed elsewhere, some models suggest that a short
exposure to a p53-activating drug may activate a revers-
ible growth arrest, whereas prolonged exposure induces
apoptosis
151
.
Recent work investigating the effects of nutlin in an
axozymethane carcinogen-induced mouse model of colon
cancer showed that treatment with nutlin resulted in cell
cycle arrest in the colon cancer cells without causing a sig-
nificant increase in apoptosis. This suggests that nutlin has
to be used in combination with other drugs to elicit any
p53-dependent cell killing in colon cancer. Normal tissues
displayed limited toxicity with decreased cell proliferation
but without any induction of apoptosis
135
.
The second-generation nutlin, RG7112, was shown
to kill cancer cell lines with potent efficacy as a single
agent
152
. It also induced cell cycle arrest and apoptosis in
SJSA-1 and LNCAP xenografts in mice, leading to the
regression of tumours
152
. RG7112 is currently in clini-
cal trials for different types of cancer (TABLE 1). One of
the first published sets of clinical results revealed that
the treatment of liposarcoma patients with RG7112
increased p53 and p21 levels in biopsy specimens and
reduced proliferation in tumours. It was also shown that
macrophage inhibitory cytokine1 (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-dihydroxyvitaminD
3
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 240
Nutlin 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, tumour
suppressor p53; TRAIL, TNF-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.
REVI EWS
as GDF15) served as an effective serum biomarker of
p53 induction
153
. Liposarcomas were selected as these
tumours 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) showing
a partial response, and 14 showing stable disease, while
the remaining 5 patients had progressive disease
153
. But
more discouraging was the relatively high rate of adverse
reactions, including thrombocytopaenia and neutro-
penia. RG7112 is also in PhaseI clinical trials in patients
with AML, where it is hoped that the compound may
produce more promising results.
Side effects of p53 therapy. In the published clinical trial
of RG7112 in liposarcoma, patients were treated with
140 mg per m
2
of orally administered RG7112 daily for
10days on a 28-day cycle for up to three cycles, which
was followed by surgical resection
153
. As discussed, the
drug showed some toxicity, and so an important ques-
tion is whether a more pulsed approach to dosing would
have reduced this effect. The activation of p53 is thought
to induce cell cycle arrest in all proliferating tissues. Cell
cycle arrest perse is not necessarily toxic; for example, it
was shown that MYC inhibitor-induced cell cycle arrest
in mice was tolerable for 4weeks with no long-term side
effects
154
. However, the effects of cell division inhibitors
in humans have not yet been defined
154
.
The adverse effects observed in the liposarcoma
trial
153
were not predicted from the preclinical stud-
ies, in which there appeared to be a good therapeutic
window, with normal cells showing less susceptibility
towards drug-induced death than tumour cells
140,152,155
.
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 bone
marrow haematopoietic progenitors in colony out-
growth assays
140
. In vitro studies with the MI-219 com-
pound showed that it was effective in killing in cancer
cells but not primary normal prostate epithelial cells
46
.
These results suggest that more extensive studies of the
effects of p53 activation in normal tissues are required
and that preclinical studies should be extended to non-
human primates.
The thrombocytopaenia and neutropenia seen in
patients in the liposarcoma trial could be due to on-target
activation of PUMA and NOXA by p53, which would
induce 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 of
DNA damage
136,156
or the induction of differentiation
in a p53-independent manner
137
. Other studies have
suggested that nutlin can make direct interactions with
BCL-X
L
in 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 the
newer and more potent compounds in clinical trials. An
invitro study has shown that RG7112 can induce apop-
tosis in human megakaryocyte precursors and that it also
hinders the polyploidization step required for the end-
stage of platelet formation, forming an invitro model for
the thrombocytopaenia observed in patients
157
.
Mechanisms of resistance to MDM2 inhibitors
The rapid emergence of resistance has been demonstrated
for a number of molecularly targeted anticancer drugs.
In some cases for example, the BCRABL inhibi-
tor imatinib (Gleevec; Novartis) or the BRAF inhibitor
vemurafenib (Zelboraf; Roche/Plexxikon) the
resistance-conferring mutations have been shown to be
present before treatment so that their selection is inevita-
ble. As MDM2 inhibitors enter the clinic, it is important
to predict potential mechanisms of resistance
158
.
Various mutations have been shown to confer resist-
ance to MDM2 inhibitors in preclinical studies. The first
mechanism is a mutation in p53 that abrogates its tran-
scriptional activity, and this has been observed in cell
lines after prolonged nutlin treatment. The mutations
were found to occur in the DNA-binding domain of
p53, rendering it inactive as a transcription factor
159161
.
Recently, an invitro selection for MDM2 mutants that
can still interact with p53 in the presence of nutlin dem-
onstrated that subtle point mutations in the p53-binding
pocket of MDM2 can confer resistance to nutlin; these
mutant proteins are still able to bind to p53 and inactivate
it even when nutlin is present
61
.
Another study, using a short hairpin RNA library
to search for molecules that confer resistance to nutlin,
found that p53 and the DNA damage-sensing protein
p53BP1 (p53-binding protein 1) were essential for nut-
lin-induced growth arrest
162
. In a mouse model of glio-
blastoma, it was shown that resistance to p53-mediated
growth-inhibitory effects developed when the tumour
suppressor ARF was inactivated. This blocked the signal-
ling from the gliablastoma-inducing oncogene to p53
163
.
Such results support the use of small molecules like nut-
lin that act downstream of ARF. The glioblastoma model
also predicted that pulsed exposure to active p53, rather
than continuous exposure, was preferable as it reduced
the rate of induction of resistance
163
.
Intrinsic resistance to apoptosis has an important role
in determining the extent to which a cell line or tumour
responds to p53-activating treatment. The principal route
by which p53 induces apoptosis is via induction of the
pro-apoptotic proteins NOXA and PUMA (BOX2). The
level of induction of these proteins and the cellular levels
of their antagonistic anti-apoptotic proteins will deter-
mine whether BAX or BAK is activated and the apoptotic
threshold is crossed. One can therefore anticipate that
the cellular response to p53 induction by p53-activating
drugs 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 which
mimic the active BH3 domains of PUMA and NOXA
and antagonize anti-apoptotic mitochondrial proteins.
ABT-263, which has reached clinical trials
164,165
, acts by
neutralizing BCL-2, BCL-W and BCL-X
L
and activat-
ing BAX or BAK
166
. Some clinical efficacy was observed
in the PhaseI clinical trial against chronic lymphocytic
leukaemia (CLL)
165
, but the results from the PhaseII
clinical trial in relapsed small-cell lung cancer seemed
to be less promising, with little efficacy observed
164
. Side
effects such as thrombocytopaenia were a concern in
both studies.
REVI EWS
Resistance towards ABT-737 has been linked to the
overexpression of myeloid cell leukaemia sequence 1
(MCL1), BCL-2 related protein A1 (BCL-2A1; also
known as BFL-1) or BCL-2-like protein 10 (BCL-2L10;
also known as BCL-B)
166169
. Sensitivity towards
ABT-737-induced cell death has been found to cor-
relate well with MCL1 expression levels in a range of
AML cell lines (including HL60, KG1, NB4, U937
and 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 and
a range of other cytotoxic drugs
139,170
, which supports the
argument that downstream intrinsic resistance to apop-
tosis, such as that conferred by high levels of MCL1, may
have an important role in reducing the responsiveness to
p53-activating drugs and other cytotoxic drugs that lead
to apoptosis and celldeath.
Further evidence for intrinsic resistance to apoptosis
is provided by combination studies showing that either
nutlin or ABT-737 can synergise with a MAPK/ERK
kinase 1 (MEK1) inhibitor, PD-98059, which down-
regulates MCL1 and also suppresses BCL-2 phospho-
rylation, to induce apoptosis in OCL-AML3 cells
171,172
.
In a separate study, inactivation of MCL1 by inducible
expression of BCL-2-interacting mediator of cell death
(BIM)-derived peptides that bind to MCL1, induced
apoptosis in six out of seven AML-derived cell lines
(including OCL-AML3) and synergistically induced cell
death in combination with ABT-737 (REF.173).
The intrinsic susceptibility of cancer cells to chemo-
therapeutic drugs has also been studied in invitro BH3
profiling assays, in which cells were permeabilized with
digitonin 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 that
a comparison of the levels of apoptotic priming between
malignant myeloblasts and normal haematopoietic stem
cells allows an accurate prediction of the therapeutic index
for chemotherapy towards drugs such as etoposide and
daunorubicin
174
. In a study of 85 patients with AML, ALL
and ovarian cancer, the mitochondria of chemosensitive
tumours were found to be more primed towards apoptosis
than the mitochondria of chemoresistant or normal tis-
sues
175
. If this apoptotic threshold model is correct, then
one would predict that haematopoietic tumours that are
sensitive to MDM2 inhibitors and to BCL-2 inhibitors will
be the same tumours that are already sensitive to standard
chemotherapy. In a clinical trial setting this would make it
difficult to establish the advantage of these new targeted
therapies, except by virtue of their expected reduced toxicity
and lack of mutagenic activity.
Contribution of MDMX to intrinsic resistance. High
levels of MDMX are also known to confer resistance
to nutlin and MI drugs. The binding of MDMX to p53
inhibits apoptosis induction by p53, and nutlin and MI
drugs cannot interfere with this interaction
176,177
. MDMX
is often upregulated in tumours containing wild-type
p53, such as melanomas
65
. Restoration of p53 using
a tamoxifen-inducible allele in an MDMX-deficient
transgenic E-Myc mouse model of lymphoma led to
increased survival of the tumour-bearing mice com-
pared to control mice that expressed MDMX, which
suggests that MDMX is a valid target for inhibition
178
.
RO-5963 (Roche), a compound with dual specificity
against both MDM2 and MDMX, was recently shown
to have invitro and invivo efficacy against cell lines and
mouse xenografts that express high levels of MDMX
72
.
It is notable that the recently published stapled peptides
sMTide-02 and ATSP-7041 both show activity against
MDMX as well as MDM2, thus enhancing their attrac-
tiveness as preclinical candidates for development
85,86
.
Also encouraging for this class of molecules (stapled
peptides) 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 of
interactions between the peptide (compared to nutlin)
and its target, MDM2 (REF.179).
Drug combinations
The pharmacological activation of the p53 pathway can
be 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 combination
with other drugs (TABLE3), such as the mitotic inhibitor
vincristine
180
, CDK inhibitors such as roscovitine
181,182
,
Aurora kinase inhibitors
183,184
and DNA-damaging agents
such as doxorubicin
140,143,185
, as well as radiation ther-
apy
186,187
. Much work is required to further understand
how drug combinations with nutlin or other p53 acti-
vators can lead to synergistic enhancement of cell death
and improved therapeutic efficacy in the treatment of
cancer. These combination strategies may lead to greater
induction of p53 protein accumulation and transcrip-
tion of downstream pro-apoptotic genes or enhance cell
killing through the inhibition of anti-apoptotic proteins.
Examples are discussedbelow.
Drug combinations that induce higher expression of p53
downstream genes. As discussed above, in some cell types
p53 activation induces growth arrest but not apoptosis,
which may be due to an inadequate induction of p53
activity. A search has been conducted for drugs that will
synergize with MDM2 inhibitors to induce a stronger
p53 response. Different drug combinations have been
shown to enhance the expression of p53 target genes
through higher induction of p53 protein expression or via
co-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 to
inducing cell death in several cell lines. This was due to
enhanced transcription of the downstream p53-induced
pro-apoptotic genes PUMA and TP53I3 (p53-inducible
gene 3; also known as PIG3)
182
.
Combinations such as selumetinib (AZD6244), a
MEK inhibitor, with nutlin also had a synergistic effect
in inducing cell death in AML cells invitro owing to the
induction of high levels of PUMA and BIM proteins and
the downregulation of MCL1 (REF.188). The increase in
PUMA protein levels after treatment with the drug com-
bination was thought to be regulated not only by the p53
REVI EWS
protein but also by the effects of the forkhead box O3A
(FOXO3A) transcription factor on the transcriptional
regulation of the PUMA promoter.
A combination of nutlin with 1,25-dihydroxyvita-
minD
3
also accelerated cell death in AML cell lines owing
to the increased transcription of pro-apoptotic proline
dehydrogenase 1 (PRODH; also known as PIG6) and
downregulation of BCL-2 (REF.189). Combination studies
were 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 apoptosis
190
. This was due
to a higher accumulation of p53 protein and higher lev-
els of p53 acetylation, and thereby greater induction of
downstream p53-responsive genes. Acetylation of p53
can inhibit its ubiquitylation by MDM2 and enhance its
DNA-binding activity, thus leading to an increase in the
levels and activity of the p53 protein
191
.
Targeting anti-apoptotic genes and sensitizing cells
towards death. The growth arrest induced by p53 in some
cancer cell types is transient and reversible, whereas in
other cell lines it is permanent and involves either senes-
cence or apoptotic mechanisms. One way to shift this
balance and achieve tumour elimination is to sensitize
the cells to the apoptotic proteins PUMA and NOXA.
Drug combinations can sensitize cells to apoptosis by
targeting anti-apoptotic genes or proteins. For example,
the combination of nutlin with ABT-737, which binds to
BCL-2 and BCL-X
L
, synergistically targets the balance of
pro-apoptotic and anti-apoptotic proteins at the mito-
chondrial level, thereby promoting cell death
151,192,193
.
It has been shown that p53 activity in melanomas is
inhibited by phosphorylated nuclear inhibitor of ASPP
(iASPP; also known as RAI) protein. The CDK1 inhibi-
tor JNJ-7706621 inhibits iASPP phosphorylation and
nuclear entry. The combination of JNJ-7706621 and
nutlin was shown to restore p53 activity, leading to syn-
ergistic activation of apoptosis in melanomas invitro and
invivo. Additionally, it was found that vemurafenib, a
small-molecule BRAF inhibitor that selectively inhibits
tumour cells containing the BRAF
V600E
mutation (which
is found in about 50% of melanomas), could be used
concurrently with the drug combination of JNJ-7706621
and nutlin to achieve an additive suppression of human
melanoma cell growth invitro and invivo
66
.
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 in
AML cells invitro
155
. 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 downregulation
of survivin
194
.
Synergism between p53 activation and microenvironmen-
tal signals. There are various examples where extracellular
signals can work with or against p53-activating drugs to
induce cell death. For example, patients with advanced
prostate cancer often receive androgen deprivation
therapy; however, resistance development to this class
of drugs is a challenge. The combination of nutlin with
androgen depletion has been shown to enhance the pro-
apoptotic activity of p53 invitro and in a mouse xenograft
model of prostate cancer
195
. In other cases, the tumour
microenvironment might modulate susceptibility towards
apoptosis. For example, co-culturing AML cells with stro-
mal cells has been shown to lead to the upregulation of
anti-apoptotic proteins
196
. Similarly, CLL cells that were
co-cultured with stromal cells or primary CLL cells that
have been derived from the bone marrow were found
to be less primed towards apoptosis
197
. This suggests that
it might be possible to combine p53-activating drugs with
therapeutics that attenuate pro-survival signalling from
the tumour microenvironment.
Other mechanisms of action of drug combinations. It is
important to note that pharmacological activators of the
p53 pathway, such as nutlin, can also work in combination
with other pathways to promote cell death via p53-inde-
pendent mechanisms. For example, inhibition of ataxia
telangiectasia mutated (ATM) or MET either by genetic
knockdown or via small-molecule-mediated pharma-
cological inhibition (KU-55933 for ATM and crizotinib
for MET) has been found to exert synergistic effects with
nutlin in the killing of various cancer cell lines invitro
198
.
Inhibition of ATM or MET does not affect the expres-
sion levels of p53 target genes induced by nutlin, such as
PUMA, BAX or CDKN1A, yet it promotes cell death when
used in combination withnutlin.
Dasatinib (Sprycel; Bristol-Myers Squibb) in B-CLL
199

or sorafenib (Nexavar; Bayer/Onyx) in AML
200
have also
shown synergism with nutlin to induce cytotoxic killing
in both wild-type and mutant p53 cell lines. Dasatinib,
which inhibits various BCRABL and SRC family tyros-
ine kinases, works in combination with nutlin to down-
regulate AKT in both p53wild-type and p53-mutant
B-CLL cell lines. Sorafenib, which also inhibits multiple
kinases, displays synergistic cytotoxicity with nutlin by
enhancing the transcriptional induction of the BAX gene
in p53 wild-type OCL-AML3 cell lines or by upregulat-
ing the BAK protein in the p53-null HL60 cellline.
The synergistic combination of radiation with nut-
lin, which was observed in hypoxic prostate cancer
cells, seems to be independent of the p53 status
187
but
is dependent on the p53 status in lung cancer cells and
prostate cancers cells
186,201
.
The conclusion from these studies is that the new
generation of MDM2 inhibitors may have limited effi-
cacy when used as monotherapies, but may find wider
application in combination therapies. This can compli-
cate clinical development, and so clinical indications with
strong responses to MDM2 inhibitors as monotherapies
which would allow early registration are actively
being pursued. The dramatic results observed in invivo
models with the latest compounds, such as RG7112 and
MI-773, suggest that this may be possible
46,47
; however,
based on the current preclinical data, an attractive alter-
native strategy to using these compounds as monothera-
pies would be to use these drugs in combination with
REVI EWS
androgen ablation in advanced prostate cancer, where
there is a high unmet need and the combination seems
to be highly effective in preclinical models
195
.
Cyclotherapy
The concept of cyclotherapy
202205
, in which drugs are
used to selectively protect normal tissues but not
tumour tissues from a cytotoxic drug, is very attrac-
tive as it offers the patient a reduction in the side effects
of chemotherapy without the loss of efficacy. Indeed,
the concept was first suggested in 1992 (REF.206). For
cyclotherapy to work, the chemoprotectant drug must
have very low toxicity and a high selectivity for normal
cells. Owing to the high frequency of p53 mutations in
human cancer and the ability of p53 activation to induce
a reversible protective cell cycle arrest, the p53 system is
an ideal test case within which to pursue this concept.
Tissue culture experiments have shown the p53-depend-
ent selectivity of tumour cell killing in cyclotherapy com-
binations
207
(reviewed in REF.208), but the more relevant
and challenging invivo demonstration has so far only
been reported once
209
. In this study it was shown that the
neutropenia induced by a POLO-like kinase inhibitor
(BI2536) could be inhibited by prior exposure to nutlin.
Importantly, the cell cycle arrest induced by nutlin did not
have an impact on the antitumour effect ofBI2536.
Conclusion
The translation of basic research findings in biomedical
science into effective medicines is often a long journey.
In the case of p53, details of its structure and function, as
well as its regulation, were elucidated by the mid-1990s,
but the pharmaceutical targets revealed by these studies
were very challenging, requiring drugs that could block
proteinprotein interactions or chaperone mutant mis-
folded proteins to re-establish activity. This challenge has
been met as a result of enormous efforts driven by the
compelling realization that such drugs could be useful
in the treatment of nearly all human malignancies. Now,
a plethora of proteinprotein interaction inhibitors that
block the p53MDM2 interaction are being developed,
which include highly novel molecular entities such as
stapled peptides. The field is at a critical juncture while
the therapeutic efficacy of these molecules is deter-
mined. One lesson that can be learned is that more of the
basic research efforts should have been dedicated to core
studies that would have more precisely determined the
likely side effects of such treatment and identified which
tumour types might show the most selective response.
The availability of excellent small-molecule compounds
is perhaps somewhat belatedly enabling these studies.
The next frontier is the development of drugs that act as
molecular chaperones to restore the function of mutant
p53 proteins. This truly represents the cutting edge
of drug development and is an exceptionally exciting
area where the increasing appreciation of the dynam-
ics of protein structure is complementing approaches
informed by X-ray structure in the development of new
medicines. Even within these highly developed fields,
new approaches and new targets are emerging, such as
the concept of read-through drugs, and the next dec-
ade offers the exciting prospect of these bold scientific
ventures reaping real clinical benefit for many patients
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Competing interests statement
The authors declare no competing interests.
DATABASES
ClinicalTrials.gov website: http://www.clinicaltrials.gov
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The tumour suppressor p53 is a transcription factor that

regulates several genes with a broad range of functions,

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