Tetramerization Domain Of P53 Research Articles

Preclinical characterization of pretargeted radioimmunotherapy (PRIT) with GD2-SADA, a self-assembling and disassembling bispecific fusion protein.

e15101 Background: GD2-SADA is a bispecific fusion protein with binding domains for the tumor-associated antigen GD2 and Lutetium 177 (Lu-177)-DOTA complex, and a p53-derived domain that mediates tetramer self-assembly and disassembly (SADA). In Step 1 of PRIT, SADA tetramers bind to GD2+ tumor cells, while unbound protein disassembles into renally cleared monomers. In Step 2, the radioactive payload Lu-177-DOTA is delivered and binds to tumor-bound GD2-SADA. Here, we characterize the binding activities of GD2-SADA to a range of GD2+ tumors and Lanthanide (Ln) metal-DOTA complexes and report the dose responsive, anti-tumor efficacy of GD2-SADA with Lu-177-DOTA in mice. Methods: GD2-SADA binding to GD2+ cell lines (small-cell lung cancer [SCLC], melanoma, osteosarcoma) was evaluated by flow cytometry (FC). A colorimetric competition ELISA was used to investigate binding to Ln-DOTA complexes (terbium [Tb], europium [Eu], and lanthanum [La]), trace metal TM-DOTA complexes (iron [Fe], copper [Cu], or zinc [Zn]), and empty DOTA (used in excess during radiolabeling to ensure complete Lu-177 chelation). GD2 binding of tetrameric GD2-SADA and monomeric p53 mutant SADA was compared using ELISA and FC assays. Tumor response to GD2-SADA with Lu-177-DOTA was evaluated in mice with SC SCLC xenografts (NCI-H524). Mice received 3 weekly treatments with a fixed GD2-SADA dose (10 mg/kg) followed by a Lu-177-DOTA dose (32 mCi or 47mCi/kg; n=6-10 per group). Tumor volumes were monitored weekly for ≥60 days after treatment. Results: GD2+ cell lines showed dose-dependent binding of GD2-SADA, with half-maximal effective concentration (EC50) values between 1.6 and 5 μg/mL. In ELISA and FC assays, the monomeric p53 mutant SADA demonstrated 85-93% reductions in binding compared with GD2-SADA. Competition ELISA demonstrated comparable binding by all Ln-DOTA complexes (50% inhibition concentration, IC50 [nM]: Lu, 2.3; Tb, 1.0; Eu, 1.2; La, 1.9). TM-DOTA and empty DOTA showed negligible binding to GD2-SADA. In mouse SCLC models, median time to tumor volume for 10 mg/kg GD2-SADA with Lu-177-DOTA dosing at 32 mCi and 47 mCi/kg exceeded control groups by 30+ and 40+ days, respectively. Conclusions: The anti-GD2 domain of GD2-SADA mediated binding to GD2+ tumor cells, a highly avid interaction that required a functional p53 tetramerization domain. Selective binding of GD2-SADA to multiple Ln-DOTA complexes further supports the SADA platform’s theranostic applications. Negligible binding to TM-DOTA complexes or empty DOTA strongly suggests the absence of competitive antagonism, an important consideration for targeted delivery of the radioactive payload in patients. GD2-SADA Lu-177-DOTA showed potent and dose-responsive anti-tumor efficacy in mice. Clinical development of GD2-SADA with Lu-177-DOTA is underway in adults and adolescents with GD2+ tumors (Trial 1001).

Elevated expression of WSB2 degrades p53 and activates the IGFBP3-AKT-mTOR-dependent pathway to drive hepatocellular carcinoma.

Dysregulation of wild-type p53 turnover is a key cause of hepatocellular carcinoma (HCC), yet its mechanism remains poorly understood. Here, we report that WD repeat and SOCS box containing protein 2 (WSB2), an E3 ubiquitin ligase, is an independent adverse prognostic factor in HCC patients. WSB2 drives HCC tumorigenesis and lung metastasis in vitro and in vivo. Mechanistically, WSB2 is a new p53 destabilizer that promotes K48-linked p53 polyubiquitination at the Lys291 and Lys292 sites in HCC cells, leading to p53 proteasomal degradation. Degradation of p53 causes IGFBP3-dependent AKT/mTOR signaling activation. Furthermore, WSB2 was found to bind to the p53 tetramerization domain via its SOCS box domain. Targeting mTOR with everolimus, an oral drug, significantly blocked WSB2-triggered HCC tumorigenesis and metastasis in vivo. In clinical samples, high expression of WSB2 was associated with low wild-type p53 expression and high p-mTOR expression. These findings demonstrate that WSB2 is overexpressed and degrades wild-type p53 and then activates the IGFBP3-AKT/mTOR axis, leading to HCC tumorigenesis and lung metastasis, which indicates that targeting mTOR could be a new therapeutic strategy for HCC patients with high WSB2 expression and wild-type p53.

Single-molecule FRET and molecular diffusion analysis characterize stable oligomers of amyloid-β 42 of extremely low population

AbstractSoluble oligomers produced during protein aggregation have been thought to be toxic species causing various diseases. Characterization of these oligomers is difficult because oligomers are a heterogeneous mixture, which is not readily separable, and may appear transiently during aggregation. Single-molecule spectroscopy can provide valuable information by detecting individual oligomers, but there have been various problems in determining the size and concentration of oligomers. In this work, we develop and use a method that analyzes single-molecule fluorescence burst data of freely diffusing molecules in solution based on molecular diffusion theory and maximum likelihood method. We demonstrate that the photon count rate, diffusion time, population, and Förster resonance energy transfer (FRET) efficiency can be accurately determined from simulated data and the experimental data of a known oligomerization system, the tetramerization domain of p53. We used this method to characterize the oligomers of the 42-residue amyloid-β (Aβ42) peptide. Combining peptide incubation in a plate reader and single-molecule free-diffusion experiments allows for the detection of stable oligomers appearing at various stages of aggregation. We find that the average size of these oligomers is 70-mer and their overall population is very low, less than 1 nM, in the early and middle stages of aggregation of 1 µM Aβ42 peptide. Based on their average size and long diffusion time, we predict the oligomers have a highly elongated rod-like shape.

Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags.

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of VHH and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53’s conformational stability and function.

Abstract 829: p53 dimers elicit tumor suppressive activities through an altered metabolic program

Abstract p53 is the most frequently mutated gene in human cancer. As a tetrameric transcription factor, mutation of the p53 Tetramerization Domain (TD) is a mechanism by which cancers abrogate wild-type (WT) p53 function. p53 TD mutations result in a protein that preferentially forms monomers or dimers. Although it is accepted that tetrameric p53 is required for full tumor suppressive activities, the physiological relevance of monomeric and dimeric states of p53 is not well understood. We have established the first in vivo models for hot-spot p53 TD mutants, p53R339P and p53A344D (murine), that result in monomeric and dimeric p53, respectively. These models closely resemble Li-Fraumeni Syndrome (LFS) patients with germline p53 TD alterations and will be used to characterize their physiological activities. p53R339P and p53A344D proteins failed to form tetramers. These mutants were deficient in their transactivation ability for canonical p53 target genes, and unable to induce apoptosis following IR. Both p53 TD mutant mice rescued the p53-dependent embryo lethality caused by genetic deletion of Mdm2, suggesting these are loss-of-function alleles. Monomeric p53 bearing mice succumbed mainly to thymic lymphomas (~60%), with a median survival similar to p53-/- mice (~130 days). Dimeric p53 bearing mice displayed lower incidence of thymic lymphomas (~25%) and longer survival (~180 days). Analyses of the transcriptomes elicited after DNA damage in normal thymus revealed that the p53 signaling pathway is dampened in dimeric p53 thymi. However, under basal conditions, p53 dimers upregulate a lipid metabolic program that is associated with PPAR activation. Further RNA sequencing of common metabolic tissues of the mouse (pancreas, liver, and intestine) revealed that they upregulate lipid-related metabolic programs in p53 dimer samples. This study describes, for the first time, the consequences of altering p53 tetramerization in vivo. Monomer and dimer forms of p53 do not retain most WT p53 features. p53 dimers may exert tumor suppression as seen by extended survival and tumor spectra shift. p53 dimer-specific activities on PPAR signaling and lipid metabolism may explain these phenotypes. These activities are possible therapeutic targets for LFS patients. Citation Format: Jovanka Gencel-Augusto, Xiaoping Su, Guillermina Lozano. p53 dimers elicit tumor suppressive activities through an altered metabolic program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 829.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Stability of p53 oligomers: Tetramerization of p53 impinges on its stability

The p53 protein has been known to exist structurally in three different forms inside the cells. Earlier studies have reported the predominance of the lower oligomeric forms of p53 over its tetrameric form inside the cells, although only the tetrameric p53 contributes to its transcriptional activity. However, it remains unclear the functional relevance of the existence of other p53 oligomers inside the cells. In this study, we characterize the stability and conformational state of tetrameric, dimeric and monomeric p53 that spans both DNA Binding Domain (DBD) and Tetramerization Domain (TD) of human p53 (94–360 amino acid residues). Intriguingly, our studies reveal an unexpected drastic reduction in tetrameric p53 thermal stability in comparison to its dimeric and monomeric form with a higher propensity to aggregate at physiological temperature. Our EMSA study suggests that tetrameric p53, not their lower oligomeric counterpart, exhibit rapid loss of binding to their consensus DNA elements at the physiological temperature. This detrimental effect of destabilization is imparted due to the tetramerization of p53 that drives the DBDs to misfold at a faster pace when compared to its lower oligomeric form. This crosstalk between DBDs is achieved when it exists as a tetramer but not as dimer or monomer. Our findings throw light on the plausible reason for the predominant existence of p53 in dimer and monomer forms inside the cells with a lesser population of tetramer form. Therefore, the transient disruption of tetramerization between TDs could be a potential cue for the stabilization of p53 inside the cells.

Implementing a method for engineering multivalency to substantially enhance binding of clinical trial anti-SARS-CoV-2 antibodies to wildtype spike and variants of concern proteins

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

Analysis of Adenoviral p53 Gene Therapy Clinical Trials in Recurrent Head and Neck Squamous Cell Carcinoma.

BackgroundWe conducted an analysis of previous adenoviral p53 (Ad-p53) treatment data in recurrent head and neck squamous cell carcinoma (HNSCC) patients to identify optimal Ad-p53 treatment methods for future clinical trials.MethodsThe analysis involved recurrent HNSCC patients treated with Ad-p53 for whom p53 genotyping and immunohistochemistry tumor biomarker studies had been performed (n = 70). Ad-p53 tumor treatment responses defined by RECIST 1.1 criteria were correlated with Ad-p53 dose and tumor p53 biomarkers. Gene expression profiles induced by Ad-p53 treatment were evaluated using the Nanostring IO 360 panel.ResultsAd-p53 dose based upon the injected tumor volume had a critical effect on tumor responses. All responders had received Ad-p53 doses greater than 7 × 1010 viral particles/cm3 of tumor volume. There was a statistically significant difference in tumor responses between patients treated with greater than 7 × 1010 viral particles/cm3 compared to patients treated at lower Ad-p53 doses (Tumor Response 31% (9/29) for Ad-p53 > 7 × 1010 viral particles/cm3 versus 0% (0/25) for Ad-p53 < 7 × 1010 viral particles/cm3; p = 0.0023). All responders were found to have favorable p53 biomarker profiles defined by less than 20% p53 positive tumor cells by immunohistochemistry (IHC), wild type p53 gene sequence or p53 deletions, truncations, or frame-shift mutations without functional p53 tetramerization domains. Preliminary gene expression profiling results revealed that Ad-p53 treatment increased interferon signaling, decreased TGF-beta and beta-catenin signaling resulting in an increased CD8+ T cell signature which are associated with increased responses to immune checkpoint blockade.ConclusionsOur findings have important implications for future p53 targeted cancer treatments and identify fundamental principles to guide Ad-p53 gene therapy. We discovered that previous Ad-p53 clinical trials were negatively impacted by the inclusion of patients with unfavorable p53 biomarker profiles and by under dosing of Ad-p53 treatment. Future Ad-p53 clinical trials should have favorable p53 biomarker profiles inclusion criteria and Ad-p53 dosing above 7 × 1010 viral particles/cm3 of injected tumor volume. Preliminary gene expression profiling identified p53 mechanisms of action associated with responses to immune checkpoint blockade supporting evaluation of Ad-p53 in combination with immune checkpoint inhibitors.

A tetravalent single-chain variable fragment antibody for the detection of staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) synthesized by Staphylococcus aureus is a foodborne and heat-stable toxin, which is a great threat to human health (Pexaraet al., 2010). Highly sensitive antibodies are a key factor in the immunological detection of SEA, which is one of the most effective ways to detect SEA because of its accuracy, agility, and efficiency (Nouri et al., 2018). In this study, we constructed a tetravalent anti-SEA antibody gene by linking the tetramerization domain of human p53 to the C-terminus of the anti-SEA single-chain variable fragment (scFv), which was then transformed into Escherichia coli BL21 (DE3) for the production of a SEA-specific tetravalent antibody. Successful expression of the tetravalent antibody was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. An indirect non-competitive enzyme-linked immunosorbent assay (ELISA) revealed that the tetravalent antibody exhibited SEA-specific binding activity. A sandwich ELISA demonstrated that compared to the scFv monomer, the tetravalent antibody was more sensitive in detecting SEA. Molecular docking analysis revealed that the SEA interacted with the scFv mainly on the opposite side of the residue linked to p53. Thus, this study indicated that genetically engineered tetramerization is a potential way to improve the sensitivity of SEA-specific scFv.

Targeting Triple Negative Breast Cancer with a Nucleus-Directed p53 Tetramerization Domain Peptide.

Triple negative breast cancer (TNBC) has no targeted detection or treatment method. Mutant p53 (mtp53) is overexpressed in >80% of TNBCs, and the stability of mtp53 compared to the instability of wild-type p53 (wtp53) in normal cells makes mtp53 a promising TNBC target for diagnostic and theranostic imaging. We generated Cy5p53Tet, a novel nucleus-penetrating mtp53-oligomerization-domain peptide (mtp53ODP) to the tetramerization domain (TD) of mtp53. This mtp53ODP contains the p53 TD sequence conjugated to a Cy5 fluorophore for near-infrared fluorescence imaging (NIRF). In vitro co-immunoprecipitation and glutaraldehyde cross-linking showed a direct interaction between mtp53 and Cy5p53Tet. Confocal microscopy and flow cytometry demonstrated higher uptake of Cy5p53Tet in the nuclei of TNBC MDA-MB-468 cells with mtp53 R273H than in ER-positive MCF7 cells with wtp53. Furthermore, depletion of mtp53 R273H caused a decrease in the uptake of Cy5p53Tet in nuclei. In vivo analysis of the peptide in mice bearing MDA-MB-468 xenografts showed that Cy5p53Tet could be detected in tumor tissue 12 min after injection. In these in vivo experiments, significantly higher uptake of Cy5p53Tet was observed in mtp53-expressing MDA-MB-468 xenografts compared with the wtp53-expressing MCF7 tumors. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold shows promise for modification in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.

NMR spectroscopy uncovers direct interaction between BAF60A and p53

The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.

Predicting Peptide Oligomeric State Through Chemical Artificial Intelligence

Oligomerization plays a crucial role in the structure and function of peptides and proteins, wherein sequence variations can affect the oligomeric stability of the biomolecule. In this study, an artificial neural network classifier that can predict the oligomeric state of peptides is presented, using the p53 tetramerization domain and associated mutants as the model system. The FASGAI vectors were utilized as the peptide descriptors, and the resulting binary classifier exhibits satisfactory predictive ability as demonstrated by a test set accuracy of 86%.

Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

P53 tetramerization: at the center of the dominant-negative effect of mutant p53.

The p53 tumor suppressor functions as a tetrameric transcription factor to regulate hundreds of genes-many in a tissue-specific manner. Missense mutations in cancers in the p53 DNA-binding and tetramerization domains cement the importance of these domains in tumor suppression. p53 mutants with a functional tetramerization domain form mixed tetramers, which in some cases have dominant-negative effects (DNE) that inactivate wild-type p53. DNA damage appears necessary but not sufficient for DNE, indicating that upstream signals impact DNE. Posttranslational modifications and protein-protein interactions alter p53 tetramerization affecting transcription, stability, and localization. These regulatory components limit the dominant-negative effects of mutant p53 on wild-type p53 activity. A deeper understanding of the molecular basis for DNE may drive development of drugs that release WT p53 and allow tumor suppression.

The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.

No Significant Association between Radiation Therapy and Subsequent Malignancies in Patients with Li-Fraumeni Syndrome: A Multi-Institutional Hereditary Cancer Registry Study

Li-Fraumeni Syndrome (LFS) is a rare cancer-predisposing disease caused by germline mutations in a potent tumor suppressor gene calledTP53. Accordingly, LFS patients carry a high lifetime risk of developing multiple primary cancers. Conventional wisdom and prior work have suggested that the risk of developing a subsequent malignancy is increased by the use of radiation therapy (RT) during primary or secondary cancer treatment. This risk, however, is not well characterized. Here we describe the risk of subsequent malignancy and cancer-related death in LFS patients after undergoing RT for a first or second primary cancer. We reviewed a cohort of patients in a multi-institution hereditary cancer registry with germline TP53 mutations. We assessed the rate of subsequent malignancy and death in patients who received RT (RT group) as part of their cancer treatment against those who did not (non-RT group). We further evaluated outcomes after RT with respect to p53 mutation type. Among 41 LFS patients (14 males and 28 females from 28 different families), 22 TP53mutations were present. 61% (25) and 7% (3) of patients had missense mutations in the DNA binding domain and tetramerization domain of p53, respectively, and 27% (11) had truncating mutations caused by nonsense (3), frameshift (1), splice site (2), or deletion (5) mutations. The median age of first cancer diagnosis was 22 years. 13 patients received RT with curative intent as part of their cancer treatment which included 3 for locally recurrent disease. The median time to follow up after RT was 5.0 years. 61% of patients in the RT group compared to 40% of patients in the non-RT group developed a subsequent malignancy (P=0.31). The median time to subsequent malignancy in the RT group and non-RT group was 3.2 years and 4.3 years respectively. Although 4 patients developed a subsequent malignancy within the radiation field, all were of the same initial histology and none of the cancers had features of an RT-induced secondary malignancy. To date, 8 deaths have occurred in the RT group (median age 36.5 years) and 2 deaths in the non-RT group (median age 30.5 years). The 5-year overall survival (OS) for the RT group was 54% compared to 95% in the non-RT group (P=0.033). Notably, there was no association between the type of p53 mutation and the rate of subsequent malignancy after RT. LFS patients who received RT for a primary cancer suffered from subsequent malignancies and death at a higher rate than those who did not receive RT. However, these results must be interpreted with caution as the majority of patients within the RT-treated group had more aggressive cancers with a higher risk of recurrence. We found that all 4 in-field malignancies in the RT-group likely represent locally recurrent disease and not RT-induced malignancies. Accordingly, the higher rate of mortality in the RT-group is likely a reflection of more aggressive cancer diagnoses and not their treatment.

P32 is a negative regulator of p53 tetramerization and transactivation.

p53 is a sequence‐specific transcription factor, and proper regulation of p53 transcriptional activity is critical for orchestrating different tumor‐suppressive mechanisms. p32 is a multifunctional protein which interacts with a large number of viral proteins and transcription factors. Here, we investigate the effect of p32 on p53 transactivation and identify a novel mechanism by which p32 alters the functional characteristics of p53. Specifically, p32 attenuates p53‐dependent transcription through impairment of p53 binding to its response elements on target genes. Upon p32 expression, p53 levels bound at target genes are decreased, and p53 target genes are inactivated, strongly indicating that p32 restricts p53 occupancy and function at target genes. The primary mechanism contributing to the observed action of p32 is the ability of p32 to interact with the p53 tetramerization domain and to block p53 tetramerization, which in turn enhances nuclear export and degradation of p53, leading to defective p53 transactivation. Collectively, these data establish p32 as a negative regulator of p53 function and suggest the therapeutic potential of targeting p32 for cancer treatment.