Targeting the TP53/MDM2 axis enhances radiation sensitivity in atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT sub­groups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase­3­positive cells in the idasanutlin treatment group, confirming the induction of apoptosis in vivo. Using flow cytometry and western blot analysis we show that inhibition of MDM2 enhanced radiation sensitivity in vitro by potentiating DNA damage via the induction of the TP53/Bax/Puma proapoptotic axis. Furthermore, DNA damage was associated with increased mitochondrial reactive oxygen species accumulation. The present study demonstrated that MDM2 expression level was increased in ATRT patient samples and MDM2 inhibition suppressed ATRT cell growth in vitro, and leads to apoptosis in vivo. MDM2 inhibition potentiates DNA damage and sensitizes ATRT cells to radiation. These findings highlight the TP53/MDM2 axis as a rational therapeutic target in CNS ATRT.

A Slug-dependent mechanism is responsible for tumor suppression of p53-stabilizing compound CP-31398 in p53-mutated endometrial carcinoma.

Mutation in the tumor suppressor gene p53 is the most frequent molecular defect in endometrial carcinoma (EC). Recently, CP-31398, a p53-stabilizing compound, has been indicated to possess the ability to alter the expression of non-p53 target genes in addition to p53 downstream genes in tumor cells. Herein, we explore the alternative mechanisms underlying the restoration of EC tumor suppressor function in mutant p53 by CP-31398. A p53-mutated EC cell was constructed in AN3CA cells with restored or partial loss of Slug using lentiviral vectors, followed by treatment with 25 µM CP-31398. A p53-independent mechanism of CP-31398 was confirmed by the interaction between mouse double minute 2 homolog (MDM2) and Slug AN3CA cells treated with IWR-1 (inhibitor of Wnt response 1). Furthermore, the AN3CA cells were treated with short hairpin RNA against Slug, Wnt-specific activators (LiCl) or inhibitors (XAV-939) followed by CP-31398 treatment. Moreover, AN3CA cell proliferation and apoptosis were examined. A tumorigenicity assay was conducted in nude mice. CP-31398 could promote the apoptosis of p53-mutated EC cells, while Slug reversed this effect. Slug ubiquitination was found to occur via binding of Slug to MDM2 in AN3CA cells. We found that CP-31398 increased the GSK-3ß, p-Slug, Puma, Wtp53, and Bax expressions whereas Wnt, Mtp-53, Slug, Bcl-2, and Ki-67 expressions were decreased. However, these findings were reversed following the activation of the Wnt pathway and overexpression of Slug. Finally, the in vivo experimental evidence confirmed that CP-31398 with depleted Slug suppressed tumor growth by downregulating the Slug. Collectively, CP-31398-regulated Slug downregulation represses the p53-mutated EC via the p53/Wnt/Puma pathway.

Antitumor immunity augments the therapeutic effects of p53 activation on acute myeloid leukemia.

The negative regulator of p53, MDM2, is frequently overexpressed in acute myeloid leukemia (AML) that retains wild-type TP53 alleles. Targeting of p53-MDM2 interaction to reactivate p53 function is therefore an attractive therapeutic approach for AML. Here we show that an orally active inhibitor of p53-MDM2 interaction, DS-5272, causes dramatic tumor regressions of MLL-AF9-driven AML in vivo with a tolerable toxicity. However, the antileukemia effect of DS-5272 is markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. In relation to this, DS-5272 triggers immune-inflammatory responses in MLL-AF9 cells including upregulation of Hif1α and PD-L1, and inhibition of the Hif1α-PD-L1 axis sensitizes AML cells to p53 activation. We also found that NK cells are important mediators of antileukemia immunity. Our study showed the potent activity of a p53-activating drug against AML, which is further augmented by antitumor immunity.

Computer-Aided Identification and Lead Optimization of Dual Murine Double Minute 2 and 4 Binders: Structure-Activity Relationship Studies and Pharmacological Activity.

The function of p53 protein, also known as "genome guardian", might be impaired by the overexpression of its primary cellular inhibitor, the murine double minute 2 protein (MDM2). However, the recent finding that MDM2-selective inhibitors induce high levels of its homologue MDM4, prompt us to identify, through a receptor-based virtual screening on an in house database, dual MDM2/MDM4 binders. Compound 1 turned out to possess an IC50 of 93.7 and of 4.6 nM on MDM2 and MDM4, respectively. A series of compounds were synthesized to optimize its activity on MDM2. As a result, compound 12 showed low nanomolar IC50 for both targets. NMR studies confirmed the pocket of binding of 12 as predicted by the Glide docking software. Notably, 12 was able to cause concentration-dependent inhibition of cell proliferation, yielding an IC50 value of 356 ± 21 nM in neuroblastoma SHSY5Y cells and proved even to efficiently block cancer stem cell growth.

MDM2 promotes epithelial-mesenchymal transition and metastasis of ovarian cancer SKOV3 cells.

BACKGROUND: Metastasis accounts for the most lethal reason for the death of ovarian cancer patients, but remains largely untreated. Epithelial-mesenchymal transition (EMT) is critical for the conversion of early-stage ovarian tumours into metastatic malignancies. Thus the exploration of the signalling pathways promoting EMT would open potential opportunities for the treatment of metastatic ovarian cancer. Herein, the putative role of MDM2 in regulating EMT and metastasis of ovarian cancer SKOV3 cells was investigated. METHODS: The regulatory effects by MDM2 on cell motility was emulated by wound-healing and transwell assays. The effects on EMT transition and Smad pathway were studied by depicting the expression levels of epithelial marker E-cadherin as well as key components of Smad pathway. To evaluate the clinical relevance of our findings, the correlation of MDM2 expression levels with the stages of 104 ovarian cancer patients was investigated by immunohistochemistry assay. RESULTS: We demonstrate that MDM2 functions as a key factor to drive EMT and motility of ovarian SKOV3 cells, by facilitating the activation of TGF-ß-Smad pathway, which results in the increased transcription of snail/slug and the subsequent loss of E-cadherin levels. Such induction of EMT is sustained in either E3 ligase-depleted MDM2 or E3 ligase inhibitor HLI-373-treated cells, while being impaired by the N-terminal deletion of MDM2, which is also reflected by the inhibitory effects against EMT by Nutlin-3a, the N-terminal targeting agent. The expression levels of MDM2 is highly correlated with the stages of the ovarian cancer patients, and the higher expression of MDM2 together with TGFB are closely correlated with poor prognosis and predict a high risk of ovarian cancer patients. CONCLUSIONS: This study suggests that MDM2 activates Smad pathway to promote EMT in ovarian cancer metastasis, and targeting the N-terminal of MDM2 can reprogram EMT and impede the mobility of cancer cells.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Meeting Organocatalysis with Drug Discovery: Asymmetric Synthesis of 3,3'-Spirooxindoles Fused with Tetrahydrothiopyrans as Novel p53-MDM2 Inhibitors.

An organocatalytic enantioselective Michael-Michael cascade reaction is developed for the synthesis of chiral spirotetrahydrothiopyrans. This highly functionalized scaffold was assembled in moderate to good yield (55-74%) and excellent diastereo- and enantioselectivities (>30:1 dr, ≥ 99% ee) with the creation of four consecutive stereogenic centers. The novel spiro-oxindole scaffold is validated as a new class of p53-MDM2 protein-protein interaction inhibitors with good antitumor activity.

Violacein induces apoptosis in human breast cancer cells through up regulation of BAX, p53 and down regulation of MDM2.

We aimed to explore the anticancer potential of violacein and its time, dose dependent mechanism of action in human MCF-7 breast cancer cells. We observed, violacein inhibit MCF-7 cells viability in a time and dose-dependent manner, IC50 value was 4.5 µM in 24 h, 1.7 µM in 48 h and 0.51 µM in 72 h. Violacein triggered generation of intra cellular ROS even from the lower doses, significant ROS production was observed from 0.25, 0.45 µM dose range and it is relative to higher doses. Further we fixed 0.45 µM and 4.5 µM as an experimental dose for relative dose dependent analysis. In nuclear staining, after 48 h 0.45 µM dose showed characteristic apoptotic morphological changes such as, 59% of cells in apoptosis and 11% of cells in necrotic stage, also in 72 h we found 68% in apoptosis and 12% in necrotic stage. However, 4.5 µM (IC50) dose of violacein, 78% of cells became apoptotic and 21% in necrotic after 48 h; but in 72 h only 61% cells are in apoptotic, necrosis was increased to 38%. Violacein increased both mitochondrial and extra mitochondrial apoptotic pathway related gene expressions; it was confirmed by increased CYP1A, GPX, GSK3ß and TNF-α gene. Further, 0.45 and 4.5 µM of violacein increased apoptotic genes, such as Bax, p53, caspase 3, Fas, FADD and markedly reduced Bcl-2 and MDM2 expression levels to two fold when compared to control. In addition violacein upregulated poly ADP-ribose polymerase (PARP), CDKN1A and caspase-9 significantly (p≤0.05) when compared to control. Relative quantification of caspase-8 was differently expressed; there were no changes in 0.45 µM, but in 4.5 µM we found two fold increased caspase-8 expression. In conclusion, lower dose of violacein treatment induced apoptosis in human breast cancer MCF-7 cells through TNF-α and p53 dependent mitochondrial pathways.

Combining targeted agents with modern radiotherapy in soft tissue sarcomas.

Improved understanding of soft-tissue sarcoma (STS) biology has led to better distinction and subtyping of these diseases with the hope of exploiting the molecular characteristics of each subtype to develop appropriately targeted treatment regimens. In the care of patients with extremity STS, adjunctive radiation therapy (RT) is used to facilitate limb and function, preserving surgeries while maintaining five-year local control above 85%. In contrast, for STS originating from nonextremity anatomical sites, the rate of local recurrence is much higher (five-year local control is approximately 50%) and a major cause of death and morbidity in these patients. Incorporating novel technological advancements to administer accurate RT in combination with novel radiosensitizing agents could potentially improve local control and overall survival. RT efficacy in STS can be increased by modulating biological pathways such as angiogenesis, cell cycle regulation, cell survival signaling, and cancer-host immune interactions. Previous experiences, advancements, ongoing research, and current clinical trials combining RT with agents modulating one or more of the above pathways are reviewed. The standard clinical management of patients with STS with pretreatment biopsy, neoadjuvant treatment, and primary surgery provides an opportune disease model for interrogating translational hypotheses. The purpose of this review is to outline a strategic vision for clinical translation of preclinical findings and to identify appropriate targeted agents to combine with radiotherapy in the treatment of STS from different sites and/or different histology subtypes.

Siladenoserinols A-L: new sulfonated serinol derivatives from a tunicate as inhibitors of p53-Hdm2 interaction.

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 µM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 µM.

Ultraviolet B-induced LGI3 secretion protects human keratinocytes.

Leucine-rich glioma inactivated 3 (LGI3) is known to be expressed mainly in the brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In this study, it was found for the first time that LGI3 is expressed mostly by normal human keratinocytes. Furthermore, ELISA analysis showed that HaCaT human keratinocytes increased LGI3 secretion after exposure to ultraviolet B (UVB) in a time- and dose-dependent manner. We next investigated the possible role of LGI3 in keratinocytes. LGI3 (50 ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150 mJ/cm(2) ). It was also found that LGI3 stimulates the phosphorylation of Akt, which is involved in the cell survival-signalling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, the data suggest that LGI3 may regulate p53 levels and that keratinocyte-derived LGI3 may act as a novel cytokine for skin homoeostasis.

[Pharmacological targeting of Mdm2: rationale and perspectives for radiosensitization]. / Ciblage pharmacologique de Mdm2 : bases biologiques et perspectives de radiosensibilisation.

The central role of p53 after exposure to ionizing radiation has been widely demonstrated. Mdm2, the main cellular regulator of p53, is a promising target for radiosensitizing purposes. In this article, we review the most recent data on the pharmacological targeting of Mdm2, with focus on strategies of radiosensitization. Antitumor activity of Mdm2 inhibitors has been related with activation of p53-dependant apoptosis, action on DNA repair systems, and antiangiogenic activity. Preliminary data suggested a synergic interaction between Mdm2 inhibitors and ionizing radiations. However, no clinical data has been published yet on the pharmacological targeting of Mdm2. Given their new mechanisms of action, these new molecules should be subject to careful clinical assessment. Although promising, these strategies expose to unexpected toxicities.

Exposure of HepG2 cells to low levels of PAH-containing extracts from contaminated soils results in unpredictable genotoxic stress responses.

Contaminated soil is a serious environmental problem, constituting a risk to humans and the environment. Polycyclic aromatic hydrocarbons (PAHs) are often present at contaminated sites. However, risk levels are difficult to estimate because of the complexity of contaminants present. Here, we compare cellular effects of extracts from contaminated soils collected at six industrial settings in Sweden. Chemical analysis showed that all soils contained complex mixtures of PAHs and oxy-PAHs. Western blotting and immunocytochemistry were used to investigate DNA damage signaling in HepG2 cells exposed to extracts from these soils. The effects on phosphorylated Mdm2, p53, Erk, H2AX, 53BP1, and Chk2, cell cycle regulating proteins (cyclin D1 and p21), and cell proliferation were compared. We found that most soil extracts induced phosphorylation of Mdm2 at the 2A10 epitope at low concentrations. This is in line with previous studies suggesting that this endpoint reflects readily repaired DNA-damage. However, we found concentration- and time-dependent gammaH2AX and 53BP1 responses that were sustained for 48 hr. These endpoints may reflect the presence of different types of persistent DNA-damage. High concentrations of soil extracts decreased cyclin D1 and increased p21 response, indicating cell cycle arrest. Phosphorylation of Mdm2 at Ser166, which attenuates the p53 response and is induced by many tumor promoters, was induced in a time-dependent manner and was associated with Erk phosphorylation. Taken together, the PAH extracts elicited unpredictable signaling responses that differed between samples. More polar compounds, i.e., oxy-PAHs, also contributed to the complexity.

Targeting Mdm2 and Mdmx in cancer therapy: better living through medicinal chemistry?

Genomic and proteomic profiling of human tumor samples and tumor-derived cell lines are essential for the realization of personalized therapy in oncology. Identification of the changes required for tumor initiation or maintenance will likely provide new targets for small-molecule and biological therapeutics. For example, inactivation of the p53 tumor suppressor pathway occurs in most human cancers. Although this can be due to frank p53 gene mutation, almost half of all cancers retain the wild-type p53 allele, indicating that the pathway is disabled by other means. Alternate mechanisms include deletion or epigenetic inactivation of the p53-positive regulator arf, methylation of the p53 promoter, or elevated expression of the p53 regulators Mdm2 and Mdmx. This review discusses current models of p53 regulation by Mdm2 and Mdmx and presents the rationale for design of future Mdmx-specific therapeutics based on our knowledge of its structure and biological functions.

Effect of a single nucleotide polymorphism in the murine double minute 2 promoter (SNP309) on the sensitivity to topoisomerase II-targeting drugs.

A single nucleotide polymorphism (SNP) SNP309 (T-->G) in the murine double minute 2 (MDM2) promoter creates a high-affinity Sp1 binding site and increases the expression of MDM2 mRNA and protein. Approximately 40% of the populations harbor at least one variant allele and 12% to 17% are homozygous G/G at codon 309. This MDM2 SNP increases susceptibility to cancer and decreases the response of cancer cells to certain forms of treatment, such as radiation therapy and DNA-damaging drugs. Topoisomerase II (TopoII)-targeting agents are commonly used chemotherapeutic drugs with a broad spectrum of activity. However, resistance to TopoII poisons limits their effectiveness. We show that MDM2 SNP309 rendered a panel of cancer cell lines that are homozygous for SNP309 selectively resistant (approximately 10-fold) to certain TopoII-targeting chemotherapeutic drugs (etoposide, mitoxantrone, amsacrine, and ellipticine). The mechanism underlying this observation was Mdm2-mediated down-regulation of TopoII; on drug exposure, MDM2 bound to TopoII and resulted in decreased cellular enzyme content. Knockdown of MDM2 by RNA interference stabilized TopoIIalpha and decreased resistance to TopoII-targeting drugs. Thus, MDM2 SNP309 (T-->G) may represent a relatively common, previously unappreciated determinant of drug sensitivity. Given the frequency of SNP309 in the general population (40% in heterozygous T/G and 12% in homozygous G/G condition), our observation may have important implications for the individualization of cancer chemotherapy.

[Therapeutic agents targetting protein-protein interactions: myth or reality?]. / Des agents thérapeutiques ciblant des interactions protéine-protéine: mythe ou réalité?

Protein-protein interactions have a key role in transduction pathways that regulate many cellular functions. Structural and functional properties of protein-protein interface are now better understood, therefore offering attractive opportunities for therapeutic intervention. Developping small molecules that modulate protein-protein interactions is challenging. Nethertheless, significant progress in this endeavour has been made on several fronts. Here, we use few illustrative examples to summarize recent work in this emerging field.

Synthesis of 5-deazaflavin derivatives and their activation of p53 in cells.

A family of 5-deazaflavin derivatives has been synthesised using a two-step convergent strategy. The biological activity of these compounds was evaluated in cells, by assessing their ability to stabilize and activate p53. These compounds may act as low molecular weight inhibitors of the E3 activity of HMD2 in tumours that retain wild-type p53. Importantly, we have demonstrated that the nitro group present in all three of the original lead compounds [1-3 (HL198C-E)] is not essential for observation of this biological activity.

Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells.

Human cytomegalovirus (HCMV) infection results in endothelial dysfunction, typically known as dysregulated apoptosis, and aberrant expression and sub-cellular localization of p53, a tumor suppressor that accumulates at the late stage of infection. In this study, we examined three hypotheses that could be responsible for HCMV-induced cytoplasmic p53 accumulation at the later stage of infection: hyperactive nuclear export, cytoplasmic p53 tethering and delayed p53 degradation. Leptomycin B treatment, a nuclear export inhibitor, was unable to reduce cytoplasmic p53, thereby eliminating the hyperactive nuclear export mechanism. The findings that nascent p53 still entered nuclei after the nuclear export inhibition indicated that cytoplasmic tethering may play a minor role. Cytoplasmic p53 was still observed after the translation activities were blocked by cycloheximide. There was more than an eight-fold increase in the cytoplasmic p53 half-life with abnormal p53 ubiquitination. Taken together, these results suggest that delayed degradation could be responsible for the cytoplasmic p53 accumulation. The general slow-down of the proteasomal activity and the dysregulated p53 ubiquitination process at the later stage of infection could contribute to the reduced cytoplasmic p53 degradation and might be relevant to dysregulated endothelial apoptosis. The HCMV-induced changes in p53 dynamics could contribute to endothelial dysfunction.

Interferon-inducible protein IFIXalpha1 functions as a negative regulator of HDM2.

The 200-amino-acid repeat (HIN-200) gene family with the hematopoietic interferon (IFN)-inducible nuclear protein encodes highly homologous proteins involved in cell growth, differentiation, autoimmunity, and tumor suppression. IFIX is the newest member of the human HIN-200 family and is often downregulated in breast tumors and breast cancer cell lines. The expression of the longest isoform of IFIX gene products, IFIXalpha1, is associated with growth inhibition, suppression of transformation, and tumorigenesis. However, the mechanism underlying the tumor suppression activity of IFIXalpha1 is not well understood. Here, we show that IFIXalpha1 downregulates HDM2, a principal negative regulator of p53, at the posttranslational level. IFIXalpha1 destabilizes HDM2 protein and promotes its ubiquitination. The E3 ligase activity of HDM2 appears to be required for this IFIXalpha1 effect. Importantly, HDM2 downregulation is required for the IFIXalpha1-mediated increase of p53 protein levels, transcriptional activity, and nuclear localization, suggesting that IFIXalpha1 positively regulates p53 by acting as a negative regulator of HDM2. We found that IFIXalpha1 interacts with HDM2. Interestingly, the signature motif of the HIN-200 gene family, i.e., the 200-amino-acid HIN domain of IFIXalpha1, is sufficient not only for binding HDM2 but also for downregulating it, leading to p53 activation. Finally, we show that IFIX mediates HDM2 downregulation in an IFN-inducible system. Together, these results suggest that IFIXalpha1 functions as a tumor suppressor by repressing HDM2 function.

Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo.

The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.