p53 in cancer: ready for therapeutic targeting?

TP53 (p53) is one of the oldest and best studied genes implicated in cancer formation or progression. Although originally identified as an oncogene (1,2), it has been known for several decades that wild-type (WT) p53 functions as a tumor suppressor gene. Suppression of cancer formation involves the binding of p53 to specific DNA response elements, followed by the induction of genes involved in one or more of the following processes, apoptosis, cell cycle arrest, senescence, DNA repair, metabolism or reactive oxygen species (ROS) modulation (3). Because of its ability to prevent cancer formation, p53 has been referred to as the “Guardian of the Genome” (4).

In most if not all human cancer however, the “Guardian Angel” is transformed into a “Rebel Angel” (5), as p53 becomes dysfunctional and is unable to suppress carcinogenesis. Two main mechanisms are responsible for p53 inactivation; mutation and negative regulation of WT p53 mediated by MDM2, MDM4 or other proteins (3). Both these processes negate the ability of WT p53 to prevent cancer development or progression. Because inactivation of p53 is effectively universal in human malignancy, targeting this dysfunction is currently a highly active area of research. Although a variety of approaches for targeting p53 are undergoing investigation (Table 1), most work is focusing on blocking the degradation of WT p53, depleting mutant p53 and reactivation of mutant p53 to a WT-like form (6-8).

Table 1 Approaches used for targeting p53 in order to treat cancer
Full table

Blocking interaction between WT p53 and MDM2/MDM4

In many cancers that lack p53 mutations, the WT gene is maintained at low levels by interaction with negative regulators such as MDM2 and MDM4 (9). MDM2 negatively regulates p53 protein using 2 main mechanisms, i.e., by promoting its degradation and preventing it from activating its target genes. MDM4, although exhibiting strong sequence homology to MDM2, lacks ubiquitin ligase activity and is thus unable to target p53 for proteasomal degradation. However, like MDM2, MDM4 can bind to p53 and block it transcriptional activity. Furthermore, MDM4 can complex with MDM2 and thereby indirectly modulate levels of p53.

Inhibiting the binding of these proteins to p53 would thus be expected to block its degradation and maintain WT function. However, traditionally, preventing protein-protein interaction with low molecular weight compounds has proved difficult as their binding surfaces are frequently too flat or large for efficient blockage (9). However, in the case of p53-MDM2 interactions, structural studies have shown that the MDM2 N-terminal domain contains a deep hydrophobic pocket into which the transactivation domain of p53 can bind. Three amino acid residues in p53, i.e., Phe19, Trp23 and Leu26 appear to be primarily responsible for this protein-protein interaction (10). Based on this knowledge, several compounds (both low molecular weight organic compounds and peptides) were synthesised to inhibit the interaction between p53 and MDM2 (11,12).

Amongst the earliest investigated were the nutlins, especially nutlin 3a (11). Nutlin 3a mimics the 3 critical residues in p53 mentioned above that are necessary for interaction with MDM2. It thus acts as a competitive inhibitor of p53 binding to MDM2. Although preclinical studies showed that nutlin 3a increased p53 concentrations, enhanced apoptosis and decreased tumorigenicity in p53 WT cancer cells (11), this compound was not pursued for potential clinical use because of poor pharmacokinetic properties and limited efficacy. Rather, more potent p53-MDM2 inhibitors with superior pharmacokinetic properties and enhanced anticancer efficacy were developed, some of which recently entered clinical testing (Table 1) (13). Compared with the wide availability of p53-MDM2 inhibitors, fewer pure MDM4 or dual MDM2/MDM4 inhibitors have been described. To the author’s knowledge, only one dual MDM2/MDM4 inhibitor has entered clinical trials, i.e., the stapled peptide known as ALRN-6924 (Aileron Therapeutics) (NCT 02264613).

MDM2/4 antagonists would be expected to be of most value in malignancies with WT p53 and high expression of either MDM2 or MDM4. These include sarcomas, leukemias, neuroblastomas and retinoblastomas for MDM2 inhibitors and both melanomas and retinoblastomas for MDM4 inhibitors (9). Potential side effects from the administration of MDM2/4 inhibitors include the possibility that in addition to stabilising p53 in tumor cells, these compounds might also stabilise the protein in normal cells. Levels of WT p53 could thus accumulate in normal cells and induce inappropriate apoptosis and cell death. However, the reported side effects relating to the preclinical use of MDM2 inhibitors are minimal. This limited toxicity in normal cells may be due to p53 primarily inducing cell cycle arrest rather than apoptosis in non-malignant cells (14). MDM2 inhibitors may thus exhibit selective toxicity for malignant cells over their normal counterparts. A further concern with the use of MDM2 antagonists is that if mutant p53 is present in the malignancy undergoing treatment, its stabilisation and the subsequent increased concentrations could increase the risk of cancer progression (15). Indeed, the presence of mutant p53 has been shown to confer resistance to MDM2 inhibitors (16).

Also, in the context of potential side effects from the use of MDM2/MDM4 antagonists, it should be borne in mind that in addition to p53, MDM2 can ubiquitylate other proteins (e.g., estrogen receptor, androgen receptor, p21 and Rb), resulting in their degradation (9). Furthermore, MDM2 has p53-independent activities including the ability to regulate gene expression, participate in DNA repair and modify chromatin structure (17). The impact, if any, of the available MDM2 inhibitors on these processes is unclear.

Reactivation of mutant p53

Several low molecular weight compounds (PhiKan083, MIRA-1, STIMA-1, ZMC1, PK7088, PK11000, PK11007 and PK11011, PRIMA-1 and APR-246/PRIMA-1^MET) as well as specific peptides (ReAcp53, pCAP-250) have been shown to reactivate mutant p53 and restore its transcription activity (6-8). Of these, the most widely studied are PRIMA-1 and APR-246 (Aprea Therapeutics). Both PRIMA-1 and APR-246 are pro-drugs that must first be converted to methylene quinuclidinone (MQ) in order to bind to p53 (18). MQ acts by attaching to specific thiol groups in mutant p53, converting it to a WT-like conformation (18). As well as binding to p53, APR-246 has also been shown to inhibit thioredoxin reductase 1 and decrease levels of GSH (7). Both of these interactions result in increased levels of ROS. Thus, PRIMA-1 and APR-246 exhibit a dual mechanism of action, i.e., reactivation of mutant p53 and generation of ROS (7).

Both PRIMA-1 and APR-246 have been shown to exhibit anticancer activity in a wide variety of preclinical models (6-8). The preclinical findings with APR-246 led to a phase I/IIa clinical trial which was carried out in patients with refractory leukemia and prostate cancer (19). In this trial, APR-246 was administered as a 2-hour intravenous infusion once per day for 4 consecutive days. Overall, the treatment was well tolerated, the most common adverse effects being fatigue, dizziness, headache and confusion. Evidence that APR-246 acted via p53 activation included induction of cell cycle arrest, increased apoptosis and upregulation of several p53 target genes in leukemic cells recovered from the treated patients. In an extension of this trial, carried out in patients with CLL and AML, administration of APR-246 at a dose regimen of 67.5 mg/kg, given as a 6 h infusion on 4 consecutive days was found to be safe and well tolerated (20). APR-246 is currently undergoing a phase Ib/II clinical trial in patients with platinum sensitive relapsed high grade serous ovarian cancer (PISARRO trial) (21). In the phase Ib part of this study APR-246 is being administered in combination with carboplatin and pegylated liposomal doxorubicin.

Depletion of mutant p53

In order for mutant p53 protein to exert its oncogenic functions, it must be stabilized and accumulate in cancer cells (22). Stabilization is achieved by interaction with various chaperone proteins, the best known of which is HSP90. Preventing this stabilization would be expected to result in depletion of mutant p53 and thus the possibility of a reversion of the malignant phenotype. Proof of this principal emerged when it was demonstrated that inhibition of expression of HSP90 by RNAi led to accelerated MDM2 and CHIP-mediated degradation of mutant p53 (22,23).

Subsequently, several low molecular weight HSP90 inhibitors were identified (24). Most of the early compounds acted by binding to the N-terminal domain of HSP90, thus blocking ATP binding. This leads to inhibition of HSP90 which is followed by E3 ubiquitin ligase-mediated degradation of mutant p53 as well as several other HSP90 client proteins, see below. The original HSP90 inhibitors included the naturally occurring compounds, ansamycin, geldanamycin and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG). While these compounds showed proof-of-principle that targeting HSP90 could be used to inhibit cancer cell growth, their clinical potential was limited by excessive toxicity and modest clinical efficacy (24). Some HSP90 inhibitors however, were shown to exhibit preferentially growth inhibition for malignant versus non-malignant cells (24).

One of the most widely investigated of the cancer-selective compounds is ganetespib {also known as known as STA-9090 or 5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-4-1-methyl-1H-indol-5-yl-2,4-dihydro-1,2,4-triazol-3-one} (25). Indeed, in some of these experimental systems investigated, ganetespib was shown to exhibit enhanced cytotoxicity for mutant p53 versus p53-null or p53 WT cells (26). Ganetespib is currently undergoing clinical trials in a number of different cancers including non-small cell lung cancer. Other HSP90 inhibitors that have undergone or are currently undergoing clinical trials include onalespib (27) and luminespib (28).

Before concluding this section, it should be stated that HSP90 is responsible for the stabilization of several other proteins in addition to mutant p53. Amongst the other proteins stabilised by this chaperone are EGFR, HER2, AKT, CDK4 and estrogen receptor (22). Inhibition of HSP90 would thus be expected to results in the destabilization and degradation of several proteins involved in oncogenesis. HSP90 inhibitors, in contrast to other anti-cancer compounds, could thus simultaneously block multiple oncogenic pathways. However, this ability to block multiple pathways could also have detrimental effects in normal cells as HSP90 plays an essential role in normal cellular homeostasis (24). Treatment with HSP90 inhibitors might thus be expected to result in multiple toxicities, especially with long-term treatment. Whether such toxicity will emerge should soon become evident from the ongoing clinical trials (23). A further downside with HSP90 inhibitors is that although they eliminate mutant p53 (as well as other proteins) they are unable to reactivate the mutant protein.

Conclusions

Although originally discovered in the late 1970s (1,2), it has taken over 3 decades for clinical trials on targeting the p53 dysfunction in cancer to begin. The ongoing clinical trials are still at a preliminary stage. Thus, it is too early to conclude if targeting p53 will have efficacy for the treatment of cancer. If however, any of the compounds currently being evaluated in clinical trials exhibit potent anticancer activity, it is likely to usher in a new era in cancer treatment, especially as p53 dysfunction is so prevalent in human cancer.

Acknowledgments

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

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Zhi-Min Yuan) for the series “p53 Biology and Cancer” published in Translational Cancer Research. The article did not undergo external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2016.11.45). The series “p53 Biology and Cancer” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Lane DP, Crawford LV. T antigen is bound to a host protein in SV40-transformed cells. Nature 1979;278:261-3. [Crossref] [PubMed]
2. Clarke CF, Cheng K, Frey AB, et al. Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol Cell Biol 1988;8:1206-15. [Crossref] [PubMed]
3. Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer 2014;14:359-70. [Crossref] [PubMed]
4. Lane DP. Cancer. p53, guardian of the genome. Nature 1992;358:15-6. [Crossref] [PubMed]
5. Walerych D, Napoli M, Collavin L, et al. The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carcinogenesis 2012;33:2007-17. [Crossref] [PubMed]
6. Duffy MJ, Synnott NC, McGowan PM, et al. p53 as a target for the treatment of cancer. Cancer Treat Rev 2014;40:1153-60. [Crossref] [PubMed]
7. Bykov VJ, Zhang Q, Zhang M, et al. Targeting of Mutant p53 and the Cellular Redox Balance by APR-246 as a Strategy for Efficient Cancer Therapy. Front Oncol 2016;6:21. [Crossref] [PubMed]
8. Parrales A, Iwakuma T. Targeting Oncogenic Mutant p53 for Cancer Therapy. Front Oncol 2015;5:288. [Crossref] [PubMed]
9. Karni-Schmidt O, Lokshin M, Prives C. The Roles of MDM2 and MDMX in Cancer. Annu Rev Pathol 2016;11:617-44. [Crossref] [PubMed]
10. Kussie PH, Gorina S, Marechal V, et al. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996;274:948-53. [Crossref] [PubMed]
11. Klein C, Vassilev LT. Targeting the p53-MDM2 interaction to treat cancer. Br J Cancer 2004;91:1415-9. [PubMed]
12. Burgess A, Chia KM, Haupt S, et al. Clinical Overview of MDM2/X-Targeted Therapies. Front Oncol 2016;6:7. [Crossref] [PubMed]
13. Ribeiro CJ, Rodrigues CM, Moreira R, et al. Chemical Variations on the p53 Reactivation Theme. Pharmaceuticals (Basel) 2016;9:E25 [Crossref] [PubMed]
14. Lane DP, Brown CJ, Verma C, et al. New insights into p53 based therapy. Discov Med 2011;12:107-17. [PubMed]
15. Aziz MH, Shen H, Maki CG. Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3. Oncogene 2011;30:4678-86. [Crossref] [PubMed]
16. Jones RJ, Bjorklund CC, Baladandayuthapani V, et al. Drug resistance to inhibitors of the human double minute-2 E3 ligase is mediated by point mutations of p53, but can be overcome with the p53 targeting agent RITA. Mol Cancer Ther 2012;11:2243-53. [Crossref] [PubMed]
17. Jain AK, Barton MC. Outside the p53 RING: Transcription Regulation by Chromatin-Bound MDM2. Mol Cell 2016;62:805-7. [Crossref] [PubMed]
18. Lambert JM, Gorzov P, Veprintsev DB, et al. PRIMA-1 reactivates mutant p53 by covalent binding to the core domain. Cancer Cell 2009;15:376-88. [Crossref] [PubMed]
19. Lehmann S, Bykov VJ, Ali D, et al. Targeting p53 in vivo: a first-in-human study with p53-targeting compound APR-246 in refractory hematologic malignancies and prostate cancer. J Clin Oncol 2012;30:3633-9. [Crossref] [PubMed]
20. Deneberg S, Cherif H, Lazarevic V, et al. An open-label phase I dose-finding study of APR-246 in hematological malignancies. Blood Cancer J 2016;6:e447 [Crossref] [PubMed]
21. Gourley C, Gabra H, Vergote I, et al. EUTROC PiSARRO: A phase Ib study combining APR-246 with standard chemotherapy in platinum sensitive relapsed high grade serous ovarian carcinoma (HGSOC). J Clin Oncol 2015;33:abstr TPS5605.
22. Alexandrova EM, Marchenko ND. Mutant p53 - Heat Shock Response Oncogenic Cooperation: A New Mechanism of Cancer Cell Survival. Front Endocrinol (Lausanne) 2015;6:53. [Crossref] [PubMed]
23. Li D, Marchenko ND, Schulz R, et al. Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells. Mol Cancer Res 2011;9:577-88. [Crossref] [PubMed]
24. Butler LM, Ferraldeschi R, Armstrong HK, et al. Maximizing the Therapeutic Potential of HSP90 Inhibitors. Mol Cancer Res 2015;13:1445-51. [Crossref] [PubMed]
25. Proia DA, Bates RC. Ganetespib and HSP90: translating preclinical hypotheses into clinical promise. Cancer Res 2014;74:1294-300. [Crossref] [PubMed]
26. Alexandrova EM, Yallowitz AR, Li D, et al. Improving survival by exploiting tumour dependence on stabilized mutant p53 for treatment. Nature 2015;523:352-6. [Crossref] [PubMed]
27. Wagner AJ, Agulnik M, Heinrich MC, et al. Dose-escalation study of a second-generation non-ansamycin HSP90 inhibitor, onalespib (AT13387), in combination with imatinib in patients with metastatic gastrointestinal stromal tumour. Eur J Cancer 2016;61:94-101. [Crossref] [PubMed]
28. Seggewiss-Bernhardt R, Bargou RC, Goh YT, et al. Phase 1/1B trial of the heat shock protein 90 inhibitor NVP-AUY922 as monotherapy or in combination with bortezomib in patients with relapsed or refractory multiple myeloma. Cancer 2015;121:2185-92. [Crossref] [PubMed]