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 2014 Macmillan Publishers Limited. All rights reserved 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 219 2014 Macmillan Publishers Limited. All rights reserved 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 220 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved Nature Reviews | Drug Discovery 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 221 2014 Macmillan Publishers Limited. All rights reserved Nature Reviews | Drug Discovery 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 222 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 223 2014 Macmillan Publishers Limited. All rights reserved 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 224 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 225 2014 Macmillan Publishers Limited. All rights reserved 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 226 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved Mutant p53 Mutant p53 Mutant p53 Mutant p53 Mutant p53 Mutant p53 p53-inducible genes Zn 2+ b a Nature Reviews | Drug Discovery 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 227 2014 Macmillan Publishers Limited. All rights reserved 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 228 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 229 2014 Macmillan Publishers Limited. All rights reserved 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 230 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved 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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 231 2014 Macmillan Publishers Limited. All rights reserved 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 232 | MARCH 2014 | VOLUME 13 www.nature.com/reviews/drugdisc 2014 Macmillan Publishers Limited. All rights reserved 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. 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