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.

APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

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.

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.

Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound.

The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.

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.

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.

Extract from Asteraceae Brachylaena ramiflora induces apoptosis preferentially in mutant p53-expressing human tumor cells.

The p53 tumor suppressor gene is inactivated by point mutation in a large fraction of human tumors, allowing evasion of apoptosis and tumor progression. p53 mutation is often associated with increased resistance to therapy. Pharmacological reactivation of mutant p53 is an attractive therapeutic strategy. We previously identified p53 reactivation and induction of massive apoptosis, a low-molecular weight compound that suppresses the growth of cancer cells in a mutant p53-dependent manner. Here, we report the identification and characterization of an extract from the terrestrial plant Brachylaena ramiflora (Asteraceae) that preferentially induces apoptosis in human tumor cells expressing mutant p53. Further analysis of this extract and identification of active compounds may provide novel structural scaffolds for the development of mutant p53-targeting anticancer drugs.

The p53 tumor suppressor: a master regulator of diverse cellular processes and therapeutic target in cancer.

The tumor suppressor p53 has been implicated in a growing number of biological processes, including cell cycle arrest, senescence, apoptosis, autophagy, metabolism, and aging. Activation of p53 in response to oncogenic stress eliminates nascent tumor cells by apoptosis or senescence. p53 is regulated at the protein level by posttranslational modifications such as phosphorylation and acetylation. A p53 antisense gene, Wrap53, enhances p53 mRNA levels via the 5'UTR. Lack of Wrap53 transcripts that overlap with p53 abrogates the p53 DNA damage response. Around half of all human tumors carry p53 mutation that disrupt p53 specific DNA binding, and transcriptional transactivation of target genes. Reactivation of mutant p53 is a promising strategy for novel cancer therapy. The small molecule PRIMA-1 restores wild type conformation and DNA binding to mutant p53, induces mutant p53-dependent apoptosis, and inhibits tumor growth in vivo. The PRIMA-1 analog APR-246 is currently tested in a phase I clinical trial. Improved understanding of the p53 pathway should lead to better diagnosis and treatment of cancer in the future.

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.

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.

In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249.

Hepatocellular carcinoma (HCC) is highly lethal due to limited curative options. In high-incidence regions, such as parts of Africa and Southeastern Asia, >50% of cases carry an AGG to AGT mutation at codon 249 of the TP53 gene, considered as a 'signature' of mutagenesis by aflatoxins. The protein product, p53ser249, may represent a therapeutic target for HCC. The small molecule p53 reactivation and induction of massive apoptosis (PRIMA)-1 has been shown to induce apoptosis in tumour cells by reactivating the transactivation capacity of some p53 mutants. In this study, we have investigated the cytotoxic effects of PRIMA-1 on HCC cells expressing p53ser249. In p53-null Hep3B cells, over-expression of p53ser249 or p53gln248 by stable transfection increased the cytotoxicity of PRIMA-1 at 50 muM. Furthermore, PRIMA-1 treatment delayed the growth of p53ser249-expressing Hep3B cells xenografted in severe combined immunodeficiency mice. However, PRIMA-1 did not restore wild-type DNA binding and transactivation activities to p53ser249 or to p53gln248 in Hep3B cells. Moreover, in PLC/PRF/5, a HCC cell line constitutively expressing p53ser249, small interfering RNA (siRNA) silencing of the mutant increased the cytotoxic effect of PRIMA-1. These apparently contradictory effects can be reconciled by proposing that p53ser249 exerts a gain-of-function effect, which favours the survival of HCC cells. Thus, both inhibition of this effect by PRIMA-1 and removal of the mutant by siRNA can lead to the decrease of survival capacity of HCC cells.

PRIMA-1MET inhibits growth of mouse tumors carrying mutant p53.

Reactivation of the tumor suppressor activity to mutant p53 should trigger massive apoptosis and eliminate tumors. The low molecular weight compounds PRIMA-1 and the structural analog PRIMA-1MET reactivate human mutant p53 in vitro and suppress growth of human tumor xenografts in SCID mice. However, little is known about their effect on mouse mutant p53 in mouse tumor cells. We have examined the effect of PRIMA-1MET on mouse sarcomas, mammary carcinomas and chemically induced fibrosarcomas. PRIMA-1MET showed potent growth suppression in mutant p53-carrying mouse tumors in vitro and a significant anti-tumor effect in syngeneic mice in vivo. These results demonstrate that PRIMA-1MET targets mouse tumors carrying mutant p53 and provide strong support for the anti-tumor efficiency of PRIMA-1MET in vivo.

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.

Needling With 5-Fluorouracil (5-FU) After XEN Gel Stent Implantation: 6-Month Outcomes.

PURPOSE: The purpose of this study was to evaluate frequency, safety, and efficacy of needling in patients that underwent XEN Gel Stent implantation. METHODS: Retrospective case review of 19 eyes of 57 consecutive patients (61 eyes) with primary open-angle glaucoma or pseudoexfoliative glaucoma that previously underwent implantation of XEN45 alone or in combination with cataract surgery followed by needling procedure with 5-FU. Success was defined at 2 IOP levels: ≤21 mm Hg and ≤15 mm Hg, with or without additional glaucoma medications. Treatment failure was defined as IOP>21 mm Hg or <5 mm Hg, need for additional glaucoma surgery or loss of light perception. RESULTS: Totally 19 of 61 eyes that underwent XEN gel implantation had subsequent needling and were included. Preneedling IOP was 26.2±9.5 and postneedling IOP at last follow-up 15.4±3.7 mm Hg (P=0.0001). Overall success rates of 17 (90%) and 13 eyes (69%) were observed at the ≤21 mm Hg and ≤15 mm Hg level, respectively. Preneedling and postneedling visual acuity and number of medications remained unchanged (P>0.05). Two eyes (10%) were categorized as treatment failures. No major complications occurred. Mean follow-up was 203.8±142.2 (range, 22 to 456) days. CONCLUSIONS: Needling revision following XEN gel stent implantation showed a good IOP-lowering effect without significant increase in number of antiglaucoma medications, decrease in visual acuity, nor any major complications. Further studies with long-term follow-up and a larger number of patients are needed to fully assess the safety and efficacy of this procedure.

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.

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.