Pharmacological reactivation of p53 in the era of precision anticancer medicine.

p53, which is encoded by the most frequently mutated gene in cancer, TP53, is an attractive target for novel cancer therapies. Despite major challenges associated with this approach, several compounds that either augment the activity of wild-type p53 or restore all, or some, of the wild-type functions to p53 mutants are currently being explored. In wild-type TP53 cancer cells, p53 function is often abrogated by overexpression of the negative regulator MDM2, and agents that disrupt p53-MDM2 binding can trigger a robust p53 response, albeit potentially with induction of p53 activity in non-malignant cells. In TP53-mutant cancer cells, compounds that promote the refolding of missense mutant p53 or the translational readthrough of nonsense mutant TP53 might elicit potent cell death. Some of these compounds have been, or are being, tested in clinical trials involving patients with various types of cancer. Nonetheless, no p53-targeting drug has so far been approved for clinical use. Advances in our understanding of p53 biology provide some clues as to the underlying reasons for the variable clinical activity of p53-restoring therapies seen thus far. In this Review, we discuss the intricate interactions between p53 and its cellular and microenvironmental contexts and factors that can influence p53's activity. We also propose several strategies for improving the clinical efficacy of these agents through the complex perspective of p53 functionality.

Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene.

The retinoblastoma protein (Rb) is encoded by the RB1 tumor suppressor gene. Inactivation of RB1 by inherited or somatic mutation occurs in retinoblastoma and various other types of tumors. A significant fraction (25.9%) of somatic RB1 mutations are nonsense substitutions leading to a premature termination codon (PTC) in the RB1 coding sequence and expression of truncated inactive Rb protein. Here we show that aminoglycoside G418, a known translational readthrough inducer, can induce full-length Rb protein in SW1783 astrocytoma cells with endogenous R579X nonsense mutant RB1 as well as in MDA-MB-436 breast carcinoma cells transiently transfected with R251X, R320X, R579X or Q702X nonsense mutant RB1 cDNA. Readthrough was associated with increased RB1 mRNA levels in nonsense mutant RB1 cells. Induction of full-length Rb protein was potentiated by the cereblon E3 ligase modulator CC-90009. These results suggest that pharmacological induction of translational readthrough could be a feasible strategy for therapeutic targeting of tumors with nonsense mutant RB1.

Novel compounds that synergize with aminoglycoside G418 or eRF3 degraders for translational readthrough of nonsense mutant TP53 and PTEN.

The TP53 and PTEN tumour suppressor genes are inactivated by nonsense mutations in a significant fraction of human tumours. TP53 nonsense mutatant tumours account for approximately one million new cancer cases per year worldwide. We have screened chemical libraries with the aim of identifying compounds that induce translational readthrough and expression of full-length p53 protein in cells with nonsense mutation in this gene. Here we describe two novel compounds with readthrough activity, either alone or in combination with other known readthrough-promoting substances. Both compounds induced levels of full-length p53 in cells carrying R213X nonsense mutant TP53. Compound C47 showed synergy with the aminoglycoside antibiotic and known readthrough inducer G418, whereas compound C61 synergized with eukaryotic release factor 3 (eRF3) degraders CC-885 and CC-90009. C47 alone showed potent induction of full-length PTEN protein in cells with different PTEN nonsense mutations. These results may facilitate further development of novel targeted cancer therapy by pharmacological induction of translational readthrough.

Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine.

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

Evolutionary history of the p53 family DNA-binding domain: insights from an Alvinella pompejana homolog.

The extremophile Alvinella pompejana, an annelid worm living on the edge of hydrothermal vents in the Pacific Ocean, is an excellent model system for studying factors that govern protein stability. Low intrinsic stability is a crucial factor for the susceptibility of the transcription factor p53 to inactivating mutations in human cancer. Understanding its molecular basis may facilitate the design of novel therapeutic strategies targeting mutant p53. By analyzing expressed sequence tag (EST) data, we discovered a p53 family gene in A. pompejana. Protein crystallography and biophysical studies showed that it has a p53/p63-like DNA-binding domain (DBD) that is more thermostable than all vertebrate p53 DBDs tested so far, but not as stable as that of human p63. We also identified features associated with its increased thermostability. In addition, the A. pompejana homolog shares DNA-binding properties with human p53 family DBDs, despite its evolutionary distance, consistent with a potential role in maintaining genome integrity. Through extensive structural and phylogenetic analyses, we could further trace key evolutionary events that shaped the structure, stability, and function of the p53 family DBD over time, leading to a potent but vulnerable tumor suppressor in humans.

Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells.

Asparaginase depletes extracellular asparagine in the blood and is an important treatment for acute lymphoblastic leukemia (ALL) due to asparagine auxotrophy of ALL blasts. Unfortunately, resistance occurs and has been linked to expression of the enzyme asparagine synthetase (ASNS), which generates asparagine from intracellular sources. Although TP53 is the most frequently mutated gene in cancer overall, TP53 mutations are rare in ALL. However, TP53 mutation is associated with poor therapy response and occurs at higher frequency in relapsed ALL. The mutant p53-reactivating compound APR-246 (Eprenetapopt/PRIMA-1Met) is currently being tested in phase II and III clinical trials in several hematological malignancies with mutant TP53. Here we present CEllular Thermal Shift Assay (CETSA) data indicating that ASNS is a direct or indirect target of APR-246 via the active product methylene quinuclidinone (MQ). Furthermore, combination treatment with asparaginase and APR-246 resulted in synergistic growth suppression in ALL cell lines. Our results thus suggest a potential novel treatment strategy for ALL.

Identification and functional characterization of new missense SNPs in the coding region of the TP53 gene.

Infrequent and rare genetic variants in the human population vastly outnumber common ones. Although they may contribute significantly to the genetic basis of a disease, these seldom-encountered variants may also be miss-identified as pathogenic if no correct references are available. Somatic and germline TP53 variants are associated with multiple neoplastic diseases, and thus have come to serve as a paradigm for genetic analyses in this setting. We searched 14 independent, globally distributed datasets and recovered TP53 SNPs from 202,767 cancer-free individuals. In our analyses, 19 new missense TP53 SNPs, including five novel variants specific to the Asian population, were recurrently identified in multiple datasets. Using a combination of in silico, functional, structural, and genetic approaches, we showed that none of these variants displayed loss of function compared to the normal TP53 gene. In addition, classification using ACMG criteria suggested that they are all benign. Considered together, our data reveal that the TP53 coding region shows far more polymorphism than previously thought and present high ethnic diversity. They furthermore underline the importance of correctly assessing novel variants in all variant-calling pipelines associated with genetic diagnoses for cancer.

A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR-246 (PRIMA-1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR-246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR-246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione-conjugated MQ (GS-MQ). Due to the reversibility of MQ conjugation, GS-MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53-targeted cancer therapy.

Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells.

Cancer-associated fibroblasts (CAFs) promote tumor growth and progression, and increase drug resistance through several mechanisms. We have investigated the effect of CAFs on the p53 response to doxorubicin in prostate cancer cells. We show that CAFs produce interleukin-6 (IL-6), and that IL-6 attenuates p53 induction and upregulation of the pro-apoptotic p53 target Bax upon treatment with doxorubicin. This is associated with increased levels of MDM2 mRNA, Mdm2 protein bound to p53, and ubiquitinated p53. IL-6 also inhibited doxorubicin-induced cell death. Inhibition of JAK or STAT3 alleviated this effect, indicating that IL-6 attenuates p53 via the JAK/STAT signaling pathway. These results suggest that CAF-derived IL-6 plays an important role in protecting cancer cells from chemotherapy and that inhibition of IL-6 could have significant therapeutic value.

Correction: APR-246 reactivates mutant p53 by targeting cysteines 124 and 277.

Since publication of this article, the authors have noticed that there was an error in Fig. 1d, third panel from left, "R273H + 200 µM MQ-H" should be "R273H + 200 µM MQ". Our corrections do not affect the original conclusions of this paper.

Functional characterization of novel germline TP53 variants in Swedish families.

Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively.

p53 as a hub in cellular redox regulation and therapeutic target in cancer.

The TP53 tumor suppressor gene encodes a DNA-binding transcription factor that regulates multiple cellular processes including cell growth and cell death. The ability of p53 to bind to DNA and activate transcription is tightly regulated by post-translational modifications and is dependent on a reducing cellular environment. Some p53 transcriptional target genes are involved in regulation of the cellular redox homeostasis, e.g. TIGAR and GLS2. A large fraction of human tumors carry TP53 mutations, most commonly missense mutations that lead to single amino acid substitutions in the core domain. Mutant p53 proteins can acquire so called gain-of-function activities and influence the cellular redox balance in various ways, for instance by binding of the Nrf2 transcription factor, a major regulator of cellular redox state. The DNA-binding core domain of p53 has 10 cysteine residues, three of which participate in holding a zinc atom that is critical for p53 structure and function. Several novel compounds that refold and reactivate missense mutant p53 bind to specific p53 cysteine residues. These compounds can also react with other thiols and target components of the cellular redox system, such as glutathione. Dual targeting of mutant p53 and redox homeostasis may allow more efficient treatment of cancer.

Correction: WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies.

[This corrects the article DOI: 10.1371/journal.pbio.1000521.].

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells.

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

Inhibition of the glutaredoxin and thioredoxin systems and ribonucleotide reductase by mutant p53-targeting compound APR-246.

The tumor suppressor p53 is commonly inactivated in human tumors, allowing evasion of p53-dependent apoptosis and tumor progression. The small molecule APR-246 (PRIMA-1Met) can reactive mutant p53 in tumor cells and trigger cell death by apoptosis. The thioredoxin (Trx) and glutaredoxin (Grx) systems are important as antioxidants for maintaining cellular redox balance and providing electrons for thiol-dependent reactions like those catalyzed by ribonucleotide reductase and peroxiredoxins (Prxs). We show here that the Michael acceptor methylene quinuclidinone (MQ), the active form of APR-246, is a potent direct inhibitor of Trx1 and Grx1 by reacting with sulfhydryl groups in the enzymes. The inhibition of Trx1 and Grx1 by APR-246/MQ is reversible and the inhibitory efficiency is dependent on the presence of glutathione. APR-246/MQ also inhibits Trxs in mutant p53-expressing Saos-2 His-273 cells, showing modification of Trx1 and mitochondrial Trx2. Inhibition of the Trx and Grx systems leads to insufficient reducing power to deoxyribonucleotide production for DNA replication and repair and peroxiredoxin for removal of ROS. We also demonstrate that APR-246 and MQ inhibit ribonucleotide reductase (RNR) in vitro and in living cells. Our results suggest that APR-246 induces tumor cell death through both reactivations of mutant p53 and inhibition of cellular thiol-dependent redox systems, providing a novel strategy for cancer therapy.

Role of Thiol Reactivity for Targeting Mutant p53.

Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target thiol groups in mutant p53. Here we have investigated the relationship between thiol reactivity, p53 thermostabilization, mutant p53 refolding, mutant p53-dependent growth suppression, and induction of cell death. Analysis of the National Cancer Institute database revealed that Michael acceptors show the highest selectivity for mutant p53-expressing cells among analyzed thiol-reactive compounds. Further experimental testing demonstrated that Michael acceptors, aldehydes, imines, and primary alcohols can promote thermodynamic stabilization of mutant p53. Moreover, mild thiol reactivity, often coupled with combined chemical functional groups, such as in imines, aldehydes, and primary alcohols, can stimulate mutant p53 refolding. However, strong electrophile activity was associated with cellular toxicity. Our findings may open possibilities for rational design of novel potent and selective mutant p53-reactivating compounds.

APR-246 reactivates mutant p53 by targeting cysteines 124 and 277.

The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain. We identified cysteine 277 as a prime binding target for MQ in p53. Cys277 is also essential for MQ-mediated thermostabilization of wild-type, R175H and R273H mutant p53, while both Cys124 and Cys277 are required for APR-246-mediated functional restoration of R175H mutant p53 in living tumor cells. These findings may open opportunities for rational design of novel mutant p53-targeting compounds.

Genetic landscape of hepatitis B virus-associated diffuse large B-cell lymphoma.

Hepatitis B virus (HBV) infection is endemic in some parts of Asia, Africa, and South America and remains to be a significant public health problem in these areas. It is known as a leading risk factor for the development of hepatocellular carcinoma, but epidemiological studies have also shown that the infection may increase the incidence of several types of B-cell lymphoma. Here, by characterizing altogether 275 Chinese diffuse large B-cell lymphoma (DLBCL) patients, we showed that patients with concomitant HBV infection (surface antigen positive [HBsAg+]) are characterized by a younger age, a more advanced disease stage at diagnosis, and reduced overall survival. Furthermore, by whole-genome/exome sequencing of 96 tumors and the respective peripheral blood samples and targeted sequencing of 179 tumors from these patients, we observed an enhanced rate of mutagenesis and a distinct set of mutation targets in HBsAg+ DLBCL genomes, which could be partially explained by the activities of APOBEC and activation-induced cytidine deaminase. By transcriptome analysis, we further showed that the HBV-associated gene expression signature is contributed by the enrichment of genes regulated by BCL6, FOXO1, and ZFP36L1. Finally, by analysis of immunoglobulin heavy chain gene sequences, we showed that an antigen-independent mechanism, rather than a chronic antigenic simulation model, is favored in HBV-related lymphomagenesis. Taken together, we present the first comprehensive genomic and transcriptomic study that suggests a link between HBV infection and B-cell malignancy. The genetic alterations identified in this study may also provide opportunities for development of novel therapeutic strategies.

Targeting mutant p53 for efficient cancer therapy.

The tumour suppressor gene TP53 is the most frequently mutated gene in cancer. Wild-type p53 can suppress tumour development by multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumour cell death and rapid tumour progression. The high frequency of TP53 mutation in tumours has prompted efforts to restore normal function of mutant p53 and thereby trigger tumour cell death and tumour elimination. Small molecules that can reactivate missense-mutant p53 protein have been identified by different strategies, and two compounds are being tested in clinical trials. Novel approaches for targeting TP53 nonsense mutations are also underway. This Review discusses recent progress in pharmacological reactivation of mutant p53 and highlights problems and promises with these strategies.