Restoration of conformation of mutant p53
Authors: J.A. Green, M. Von Euler, L.B. Abrahmsen
Journal: Annals of Oncology (2018)

Compounds correcting the mutated antitumor protein p53 have been sought since its discovery almost 30 years ago. This transcription factor has a role in the regulation of DNA repair, metabolism, invasion and metastasis, cell cycle arrest and apoptosis. Between 50% and 60% of human tumors contain a mutated form of the p53 gene TP53, making it the most commonly mutated gene in human cancer [1.Muller P.A.J. Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities.Cancer Cell. 2014; 25: 304-317Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar] and almost 2000 mutations have been identified. In contrast to other tumor suppressor genes, TP53 is commonly inactivated by a missense mutation, leading to a single amino acid substitution which suppresses the transcriptional activation function of wild type p53. However, an increasing number of the resulting mutant proteins have been shown to have gain-of-function properties contributing to the oncogenic potential, including invasive and metastatic properties [1.Muller P.A.J. Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities.Cancer Cell. 2014; 25: 304-317Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar, 2.Cordani M. Pacchiana R. Butera G. Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: involvement in cancer invasion and metastasis.Cancer Lett. 2016; 376: 303-309Crossref PubMed Scopus (50) Google Scholar, 3.Kandoth C. McLellan M.D. Vandin F. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502: 333-339Crossref PubMed Scopus (2951) Google Scholar]. Hence, mutant p53 can be oncogenic despite wild-type (wt) p53 being a tumor suppressor. Mutant p53 protein may be a driver of tumorigenesis either by binding to DNA, to transcription factors or cofactors directly or as part of aggregates. There is also evidence for a complex set of interactions with oncogenes including PTEN, ERBB2 and Ras [1.Muller P.A.J. Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities.Cancer Cell. 2014; 25: 304-317Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar, 4.Bisio A. Ciribilli Y. Fronza G. et al.TP53 mutants in the tower of babel of cancer progression.Hum Mutat. 2014; 35Crossref PubMed Scopus (37) Google Scholar]. While mutations are most common in regions encoding the structural domains and hot-spot sites in the DNA-binding core domain of the molecule, the association between mutation site and biological effect is not fully established [4.Bisio A. Ciribilli Y. Fronza G. et al.TP53 mutants in the tower of babel of cancer progression.Hum Mutat. 2014; 35Crossref PubMed Scopus (37) Google Scholar, 5.Jackson J. Lozano G. The mutant p53 mouse as a pre-clinical model.Oncogene. 2013; 32: 4325-4330Crossref PubMed Scopus (48) Google Scholar]. However, in most cases, mutation is associated with impaired structure or folding of the p53 protein. This short article summarizes evolving clinical and preclinical data suggesting that mutant p53 is a druggable target for cancer therapy. The various approaches to targeting mutant p53 to date are summarized in Figure 1. Downstream targeting of p53 including synthetic lethality strategies will not be covered. Alkylation of thiols is an established mechanism for biophysical stabilization of wt and mutant p53 [6.Kaar J.L. Basse N. Joerger A.C. et al.Stabilization of mutant p53 via alkylation of cysteines and effects on DNA binding.Protein Sci. 2010; 19: 2267-2278Crossref PubMed Scopus (51) Google Scholar], and a number of thiol reactive compounds have been reported to restore the activity of mutant p53 including SCH529074 [7.Demma M. Maxwell E. Ramos R. et al.SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain, restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of HDM2-mediated ubiquitination of wild-type p53.J Biol Chem. 2010; 285: 10198-10212Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar], CP-31398 [8.Rippin T.M. Bykov V.J. Freund S.M. et al.Characterization of the p53-rescue drug CP-31398 in vitro and in living cells.Oncogene. 2002; 21: 2119-2129Crossref PubMed Scopus (147) Google Scholar], STIMA-1 [9.Zache N. Lambert J.M.R. Rökaeus N. et al.Mutant p53 targeting by the low molecular weight compound STIMA-1.Mol Oncol. 2008; 2: 70-80Crossref PubMed Scopus (86) Google Scholar], maleimide analogues such as MIRA-1 [10.Bykov V.J. Issaeva N. Zache N. et al.Reactivation of mutant p53 and induction of apoptosis in human tumor cells by maleimide analogs.J Biol Chem. 2005; 280: 30384-30391Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar] and PRIMA-1 [11.Bykov V.J. Issaeva N. Shilov A. et al.Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound.Nat Med. 2002; 8: 282-288Crossref PubMed Scopus (886) Google Scholar]. These differ in chemical structure and hence also in reactivity and predicted toxicity. The clinically most advanced mutant p53 targeting compound is APR-246, a methylated derivative of PRIMA-1, which was identified by screening a subset of the NCI compound library demonstrating functional restoration of mutant p53 in a cellular context [11.Bykov V.J. Issaeva N. Shilov A. et al.Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound.Nat Med. 2002; 8: 282-288Crossref PubMed Scopus (886) Google Scholar]. PRIMA-1 was shown to induce a wt-like structure of unfolded R175H mutant p53 in intact cancer cells and transactivate hot spot R175H and R273H mutants. Tumor growth was suppressed in vivo in xenografts [12.Jung C.L. Mun H. Jo S.Y. et al.Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo.Oncotarget. 2016; 7 (doi 1018632/oncotarget12758): 77664-77682Crossref PubMed Scopus (5) Google Scholar]. Furthermore, specific DNA binding was restored in 13 of 14 p53 mutants in bandshift assays. APR-246 is a prodrug forming the thiol binding 2-methylene-quinuclidin-3-one (MQ), which binds to cysteine residues [13.Lambert M.R. Gorzov P. Veprintzev D.B. et al.PRIMA-1 reactivates mutant p53 by covalent bindingS to the core domain.Cancer Cell. 2009; 15: 376-388Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar] and restores the structure of unfolded wt p53 and of mutant p63, which has significant homology with p53, sharing two hotspot mutations, and has a role in promotion of invasion [1.Muller P.A.J. Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities.Cancer Cell. 2014; 25: 304-317Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar, 2.Cordani M. Pacchiana R. Butera G. Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: involvement in cancer invasion and metastasis.Cancer Lett. 2016; 376: 303-309Crossref PubMed Scopus (50) Google Scholar, 14.Rökaeus N. Shen J. Eckhardt I. et al.PRIMA-1MET/APR-246 targets mutant forms of p53 family members p63 and p73.Oncogene. 2010; 29: 6442-6451Crossref PubMed Scopus (49) Google Scholar]. Both in vitro and in vivo the proapoptotic proteins Puma and Bax, as well as CDKN1A/p21 were induced [15.Bykov V.J.N. Wiman KG. Mutant p53 reactivation by small molecules makes its way to the clinic.FEBS Lett. 2014; 588: 2622-2627Crossref PubMed Scopus (138) Google Scholar]. A second effect of APR-246 is on platinum sensitivity mediated by depleting glutathione [16.Mohell N. Alfredsson J. Fransson Å et al.APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.Cell Death Dis. 2015; 6: e1794Crossref PubMed Google Scholar], and by conversion of thioredoxin reductase to an NADPH oxidase [17.Peng X. Zhang M.Q. Conserva F. et al.APR-246/Prima-1 inhibits thioredoxin reductase and converts the enzyme into a dedicated NADPH oxidase.Cell Death Dis. 2013; 4: e881Crossref PubMed Scopus (118) Google Scholar], together leading to a more oxidative environment. While this mechanism may be independent of p53 status, thiol pathway modulation will have the effect of further enhancing cell kill from all cross-linking agents including the platinum drugs and radiation. Thus, thiol alkylation by MQ is linked to alterations in the redox status of cells, which may contribute to both chemosensitization [16.Mohell N. Alfredsson J. Fransson Å et al.APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.Cell Death Dis. 2015; 6: e1794Crossref PubMed Google Scholar, 18.Liu D.S. Read M. Cullinane C. et al.APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma.Gut. 2015; 64: 1506-1516Crossref PubMed Scopus (75) Google Scholar] and radiosensitization [19.Supiot S. Zhao H. Wiman K. et al.PRIMA-1(met) radiosensitizes prostate cancer cells independent of their MTp53-status.Radiother Oncol. 2008; 86: 407-411Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar]. Both restored p53 activity and induction of oxidative stress are likely to contribute to this sensitization. The proof of principle of the thiol binding concept for mutant p53 restoration is currently under evaluation in phase II clinical studies in high-grade serous ovarian cancer (HGSOC), where p53 mutations are ubiquitous [20.The Cancer Genome Atlas Research Network Integrated genomic analysis of ovarian carcinoma.Nature. 2011; 474: 609-615Crossref PubMed Scopus (5418) Google Scholar]. Furthermore, as platinum compounds are the most active agents against this subtype, there are strong arguments for prioritizing development in this tumor. The phase Ib part of the study of APR-246 has shown combination with standard dose of carboplatin and liposomal doxorubicin is feasible with encouraging RECIST and CA125 response (GCIG) and progression-free survival [21.Basu B. Gourley C. Gabra H. et al.Pisarro: a phase 1b study of APR-246 with carboplatin and pegylated liposomal doxorubicin (PLD) in relapsed platinum-sensitive high grade serous ovarian cancer.Annal Oncol. 2016; 27: 386Google Scholar]. Of 21 evaluable patients by RECIST1.1, 3 had a confirmed complete response, 10 a confirmed partial response and 8 stable disease. Out of 23 patients evaluable by either CA125 criteria (GCIG) or RECIST, 17 responded (74%). COTI-2 is a compound undergoing clinical trial in p53 mutant gynecological malignancies and is currently in phase I (Clinicaltrials. Gov NCT 02433626) as a single agent and can be administered orally. The exact mechanism of action is unknown [22.Salim K.Y. Maleki Vareki S. Danter V.R. et al.COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo.Oncotarget. 2016; 7: 41363-41379Crossref PubMed Scopus (95) Google Scholar]. According to the company developing COTI-2 [23.www.criticaloutcome.com (accessed 4.01.2018)Google Scholar], the compound has an effect on the PI3K/AKT pathway via a direct gene level upregulation of PIK3IP, a specific protein that is known to inhibit PI3K. Based on preclinical data presented by the same group, COTI-2 is predicted to be effective in cancers with p53 mutations as well as those with mutations in the PI3K/AKT pathway [22.Salim K.Y. Maleki Vareki S. Danter V.R. et al.COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo.Oncotarget. 2016; 7: 41363-41379Crossref PubMed Scopus (95) Google Scholar, 23.www.criticaloutcome.com (accessed 4.01.2018)Google Scholar]. Unpublished data show that treatment of cells with COTI-2 can restore the conformation of mutant p53; the IC50 is stated to be ≤1 µM in HCT116 cells transfected with 15 different mutants of p53 [23.www.criticaloutcome.com (accessed 4.01.2018)Google Scholar]. Zinc ions play a crucial role in maintaining the p53 protein structure and an alternative approach for p53 stabilization is zinc ion supplementation, which has been shown to restore mutant p53 structure and function in several unstable mutants [24.Puca R. Nardinocchi L. Porru M. et al.Restoring p53 active conformation by zinc increases the response of mutant p53 tumor cells to anticancer drugs.Cell Cycle. 2011; 10: 1679-1689Crossref PubMed Scopus (91) Google Scholar]. Different zinc ion chelating/transporting compounds have been presented as candidate compounds for restoring p53 function, including derivatives of the herbal supplement curcumin [25.Garufi A. Pucci D. D'Orazi V. et al.Degradation of mutant p53H175 protein by Zn(II) through autophagy.Cell Death Dis. 2014; 5: e1271Crossref PubMed Scopus (70) Google Scholar] and the metallochaperone ZMC1/NSC319726 [26.Blanden A.R. Yu X. Wolfe A.J. et al.Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.Mol Pharmacol. 2015; 87: 825-831Crossref PubMed Scopus (53) Google Scholar]. The former was shown to reactivate p53 R175H, to induce autophagy and lead to cell death [25.Garufi A. Pucci D. D'Orazi V. et al.Degradation of mutant p53H175 protein by Zn(II) through autophagy.Cell Death Dis. 2014; 5: e1271Crossref PubMed Scopus (70) Google Scholar]. None of these compounds is yet under clinical investigation as an anticancer agent, although the original herbal compound curcumin is being evaluated in clinical trials, despite recent reservations about its role as a pharmaceutical [27.Nelson K.M. Dahlin J.L. Bisson J. et al.The essential medicinal chemistry of curcumin.J Med Chem. 2017; 60: 1620-1637Crossref PubMed Scopus (1048) Google Scholar]. Another preclinical compound suggested to target mutant p53 is NSC176327, which is a derivative of the plant alkaloid ellipticine, a known topoisomerase 2 inhibitor, which increases the expression of p73 and induces Noxa, Puma and p21 in cells lacking functional p53 [28.Lu C. Wang W. El-Deiry WS. Non-genotoxic anti-neoplastic effects of ellipticine derivative NSC176327 in p53-deficient human colon carcinoma cells involve stimulation of p73.Cancer Biol Ther. 2008; 7: 2039-2046Crossref PubMed Scopus (34) Google Scholar]. Ellipticine has been shown to rescue the conformation of mutant p53 by an indirect mechanism, requiring ongoing protein synthesis [29.Hong B. Prabhu V.V. Zhang S. et al.Prodigiosin rescues deficient p53 signaling and antitumour effects via upregulating p73 and disrupting its interaction with mutant p53.Cancer Res. 2014; 74: 1153-1165Crossref PubMed Scopus (58) Google Scholar] and a more recent study showed it to act as a modulator of p53 nuclear localization. PK7088 is a compound synthesized specifically for the binding pocket of the Y220C mutation and has not yet progressed to clinical trials. Several lines of evidence suggest that restoration of p53 may enhance the activity of DNA damaging agents and radiation treatment, which is known to elevate p53 activation signals. APR-246, based on preclinical data showing synergistic effects of the combination with cisplatin or doxorubicin, and potentiation of either drug [30.Mohell N. Alfredsson A. Uustalu M. et al.APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.Cell Death Dis. 2015; 6: e1794Crossref PubMed Scopus (126) Google Scholar], is being evaluated in combination with carboplatin and liposomal doxorubicin, an established second line regimen in HGSOC [21.Basu B. Gourley C. Gabra H. et al.Pisarro: a phase 1b study of APR-246 with carboplatin and pegylated liposomal doxorubicin (PLD) in relapsed platinum-sensitive high grade serous ovarian cancer.Annal Oncol. 2016; 27: 386Google Scholar]. In vitro studies have also confirmed synergy with PARP inhibitors, which may further aid development in the 50% of HGSOC which harbor DNA-repair defects [20.The Cancer Genome Atlas Research Network Integrated genomic analysis of ovarian carcinoma.Nature. 2011; 474: 609-615Crossref PubMed Scopus (5418) Google Scholar, 31.Deben C. Lardon F. Wouters A. et al.APR-246 (PRIMA-1MET) strongly synergizes with AZD 2281 (olaparib) induced PARP inhibition to induce apoptosis in non-small cell lung cancer cell lines.Cancer Lett. 2016; 375: 313-322Crossref PubMed Scopus (46) Google Scholar]. Drugs inhibiting the p53-MDM2 interaction have shown dose limiting thrombocytopenia [32.Zhao Y. Aguilar A. Bernard D. Wang S. Small molecule inhibitors of the MDM-2-p53 protein- protein interaction (MDM2 inhibitors) in clinical trials for cancer treatment.J Med Chem. 2015; 58: 1038-1052Crossref PubMed Scopus (337) Google Scholar] but more recent compounds including dual MDM2/MDM4 inhibitors are undergoing clinical trials [32.Zhao Y. Aguilar A. Bernard D. Wang S. Small molecule inhibitors of the MDM-2-p53 protein- protein interaction (MDM2 inhibitors) in clinical trials for cancer treatment.J Med Chem. 2015; 58: 1038-1052Crossref PubMed Scopus (337) Google Scholar]. In contrast, APR-246 targets misfolded p53 and not the feed-back control mechanism regulating the level of wt p53 in hematopoietic cell precursors, and as a single agent has not been shown to cause myelosuppression [33.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: e447Crossref PubMed Scopus (51) Google Scholar]. Therefore, combination of several established anticancer treatments with restoration of p53 activity is possible from a toxicological perspective and may also be synergistic. An emerging body of work applicable to a number of tumor suppressors is targeting protein aggregates which have been demonstrated to occur in several malignancies. Some mutant p53 proteins have a reduced intrinsic structural stability which makes them more prone to aggregation than wt p53 [34.Yang-Hartwich Y. Soteras M.G. Lin Z.P. et al.p53 protein aggregation promotes platinum resistance in ovarian cancer.Oncogene. 2015; 34: 3605-3616Crossref PubMed Scopus (102) Google Scholar]. Aggregation may occur with wt p53, p63 and p73, and has been shown to promote platinum resistance in ovarian cancer cell lines [35.Soragni A. Janzen D.M. Johnson L.M. et al.A designed inhibitor of p53 aggregation rescues p53 tumor suppression in ovarian carcinomas.Cancer Cell. 2016; 29: 90-103Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar]. A recent in vitro and in vivo study in HGSOC has shown that a designed peptidic inhibitor of p53 aggregation (ReACp53) induces cell cycle arrest, cell death and degradation of mutant p53 [36.Xu J. Reumers J. Couceiro J.R. et al.Gain of function of mutant p53 by co-aggregation with multiple tumor suppressors.Nat Chem Biol. 2011; 7: 285-295Crossref PubMed Scopus (375) Google Scholar]. Dissociation of the mutant p53-heat shock protein (HSP) complex, leading to degradation of mutant p53 protein has been evaluated as a therapeutic strategy [37.Alexandrova E.M. Yallowitz A.R. Li D. et al.Improving survival by exploiting tumour dependence on stabilized mutant p53 for treatment.Nature. 2015; 523: 352-356Crossref PubMed Scopus (233) Google Scholar]. HSP90 contributes to accumulation of mutant p53 by inactivating p53 ubiquitin ligases, MDM2 and CHIP. The HSP90 inhibitor ganetespib has been shown to sensitize non-small-cell lung cancer (NSCLC) cells to radiation [38.Wang Y. Liu H. Diao L. et al.Hsp90 inhibitor ganetespib sensitizes non-small cell lung cancer to radiation but has variable effects with chemoradiation.Clin Cancer Res. 2016; 22: 5876-5886Crossref PubMed Scopus (24) Google Scholar] and progressed to clinical trials in ovarian and NSCLC. However, further development has been discontinued following a negative phase II trial in NSCLC [39.Ramalingam S. Goss G. Rosell R. et al.A randomized phase 2 study of ganetespib, a heat shock protein 90 inhibitor, in combination with docetaxel in second line non-small-cell lung cancer (GALAXY-1).Ann Oncol. 2015; 26: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Kevetrin (thiobutyronitrile) has several effects, one of which is HDAC inhibition. It might also have direct effect on mutant p53 and has been shown to lead to phosphorylation of p53 at serine 15 and G2/M cell division arrest with a decline in Wee1, p21 and PUMA, and tumor cell death [40.Kumar A. Hiran T. Holden S.A. et al.Kevetrin activates p53, enhances expression of p21 induces cell cycle arrest and apoptosis in a human cancer cell line.Cancer Res. 2011; 71 (Abstract 4470)Google Scholar, 41.Kumar A. Brennan D.P. Chafai-Fadela K. et al.Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies.Cancer Res. 2017; 77 (Abstract 3221)Google Scholar]. A possible mechanism is induction of mutant p53 degradation by downregulation of HSP90 through inhibition of HDAC6. A phase I trial has now been completed with IV administration as a single agent in ovarian cancer and a phase II study in drug resistant HGSOC was started in 2017 (Table 1).Table 1Summary of mutant p53-directed clinical trials with preliminary efficacy data reportedDrugSponsorMechanismPhaseTumorNumberStatusReferenceNCTAPR-246Aprea TherapeuticsConformation restorationCysteine alkylatingIOvary HGSCarboplatinDoxorubicin28CR 3/22PR 9/22GCIG[21.Basu B. Gourley C. Gabra H. et al.Pisarro: a phase 1b study of APR-246 with carboplatin and pegylated liposomal doxorubicin (PLD) in relapsed platinum-sensitive high grade serous ovarian cancer.Annal Oncol. 2016; 27: 386Google Scholar]2098343IIOvary HGS25017/22RecruitingCOTI-2Critical Outcomes TechnologiesConformation restorationPI3KithiosemicarbazoneIGynecologicalRecruitingPhase I MD Anderson[23.www.criticaloutcome.com (accessed 4.01.2018)Google Scholar]2433626GanetespibSyntaHsp90iIIRNSCLC381HR 0.82P = 0.078[40.Kumar A. Hiran T. Holden S.A. et al.Kevetrin activates p53, enhances expression of p21 induces cell cycle arrest and apoptosis in a human cancer cell line.Cancer Res. 2011; 71 (Abstract 4470)Google Scholar]GanetespibSynta (closed)Hsp90iIIBreast51OR 2/6 Hormone resistantOR 2/38 TNBCD/C01677455GanetespibSynta (closed)Hsp90iIIIOvary HGSPlatinum resistantpaclitaxel15Discontinued1962948KevetrinIpharamceuticsSer15 phosphorylationCaspase activatorThiobutyonitrileIOvary48CompletedDatabase lock June 2016www.ipharminc.com[41.Kumar A. Brennan D.P. Chafai-Fadela K. et al.Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies.Cancer Res. 2017; 77 (Abstract 3221)Google Scholar]01664000 01798485IIPlatinum resistant HGSOC10Started February 201703042702 Open table in a new tab Clinical studies of APR-246 have shown encouraging preliminary data in HGSOC and are likely to be extended to evaluation in other tumors, where p53 is mutated and likely to be a driver, including TNBC, NSCLC and esophageal cancers [43.Malkin D. Li-Fraumeni syndrome.Genes Cancer. 2011; 2: 475-484Crossref PubMed Scopus (279) Google Scholar]. Current studies have used selection based on p53 immunohistochemistry as a surrogate for mutation [21.Basu B. Gourley C. Gabra H. et al.Pisarro: a phase 1b study of APR-246 with carboplatin and pegylated liposomal doxorubicin (PLD) in relapsed platinum-sensitive high grade serous ovarian cancer.Annal Oncol. 2016; 27: 386Google Scholar, 42.Tamborero D. Gonzalez-Perez A. Perez-llamas C. et al.Comprehensive identification of mutational cancer driver genes across 12 tumour types.Sci Rep. 2013; 3: 2650Crossref PubMed Scopus (350) Google Scholar], but these are likely to be replaced by sequencing either of the tumor biopsy or by liquid technologies based on cell-free DNA, as has been developed in NSCLC mutation detection for standard of care. Longer term, there are exciting opportunities for prevention strategies such as in the Li–Fraumeni syndrome where there are germ line p53 mutations and a high incidence of early presenting tumors [44.Purvis J.E. Karhohs K.W. Mock C. et al.p53 dynamics control cell fate.Science. 2012; 336: 1440-1444Crossref PubMed Scopus (542) Google Scholar]. Prolonged scheduling for prevention strategies presents formidable challenges in respect of toxicity but preclinical data have shown intermittent scheduling of p53 targeted agents may lead to more effective and less toxic regimens appropriate for long-term use to maintain the proapoptotic state maintained by p53 [45.Quartuccio S.M. Karthikeyan S. Eddie S.L. et al.Mutant p53 expression in fallopian tube epithelium drives cell migration.Int J Cancer. 2015; 137: 1528-1538Crossref PubMed Scopus (34) Google Scholar]. Extrapolation of this approach could be applied to prevention of transformation in premalignant foci such as STIC lesions in the fallopian tube to invasive cancer [46.Wu R. Baker S. Hu T.C. et al.Type I to Type II ovarian carcinoma progression: mutant Trp53 or Pik3ca confers a more aggressive tumor phenotype in a mouse model of ovarian cancer.Am J Pathol. 2013; 182: 1391-1399Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar], and of low- to high-grade tumors where this has been shown to be due to acquisition of p53 mutations [47]. Decades of research on this enigmatic molecule now seems likely to yield important clinical progress. Gain-of-function properties of p53 including acquisition of invasion and drug resistance have shed new light on how the complexities of its interactions can be modified for therapeutic benefit. However, it is the restoration of the conformation of the mutated protein and its associated functional effects which has provided an opportunity to target a wide range of cancers and premalignant states. None declared.