P53 Tetramer Research Articles

Deciphering Internal Regulatory Patterns within the p53 Core Tetramer: Insights from Community Network Analysis.

Gene therapy is one of the most effective strategies for cancer treatment. The p53 protein, commonly known as the "guardian of the genome", plays a critical role in gene activation and tumor suppression. Tetramerization of the p53 core domain is an essential allosteric process that supports its suppression functions. This letter presents a framework to analyze the structure, function, and dynamic connectivity of the p53 tetramer, using community network analysis based on all-atom molecular dynamics simulations. The communities within the p53 monomer exhibit distinct functional roles, while interactions between molecules establish a symmetrical network structure. We identified direct evidence of single, double, and multiple pathway regulations within the p53 tetramer and crucial residue pairs involved in these connections. Our study provides a comprehensive framework to understand the community network of the p53 tetramer, offering new insights into the stable formation of the p53 core tetramer.

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).

Abstract 3025: Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis

Abstract TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations. Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3025.

Exploring the Role of Globular Domain Locations on an Intrinsically Disordered Region of p53: A Molecular Dynamics Investigation.

The pre-tetramerization loop (PTL) of the human tumor suppressor protein p53 is an intrinsically disordered region (IDR) necessary for the tetramerization process, and its flexibility contributes to the essential conformational changes needed. Although the IDR can be accurately simulated in the traditional manner of molecular dynamics (MD) with the end-to-end distance (EEdist) unhindered, we sought to explore the effects of restraining the EEdist to the values predicted by electron microscopy (EM) and other distances. Simulating the PTL trajectory with a restrained EEdist , we found an increased agreement of nuclear magnetic resonance (NMR) chemical shifts with experiments. Additionally, we observed a plethora of secondary structures and contacts that only appear when the trajectory is restrained. Our findings expand the understanding of the tetramerization of p53 and provide insight into how mutations could make the protein impotent. In particular, our findings demonstrate the importance of restraining the EEdist in studying IDRs and how their conformations change under different conditions. Our results provide a better understanding of the PTL and the conformational dynamics of IDRs in general, which are useful for further studies regarding mutations and their effects on the activity of p53.

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.

Full-atomistic molecular dynamics analysis of p53 active tetramer

p53 is a tumor suppressor protein that plays a crucial role in inhibiting cancer development and maintaining the genetic integrity. Within the cell nucleus, four p53 molecules constitute a stable tetrameric active structure through highly cooperative interactions, bind to DNA via its DNA-binding domain, and transcriptionally activate or inhibit their target genes. However, in most human tumor cells, there are numerous p53 mutations. The majority of these mutations are formed in the p53 DNA-binding domain, importantly, the p53 DNA-binding domain is critical for p53 to form the tetrameric active structures and to regulate the transcription of its downstream target genes. In this work, the all-atom molecular dynamics simulation is conducted to investigate the mechanism of interaction within the wild-type p53 tetramers. This study indicates that the symmetric dimers on either side of the DNA are stable ones, keeping stable structures before and after DNA binding. The binding of two monomers on the same side of the DNA depends on protein-protein interaction provided by two contact surfaces. DNA scaffold stabilizes the tetrameric active structure. Such interactions crucially contribute to the tetramer formation. This study clarifies the internal interactions and key residues within the p53 tetramer in dynamic process, as well as the critical sites at various interaction interfaces. The findings of this study may provide a significant foundation for us to further understand the p53’s anticancer mechanisms, to explore the effective cancer treatment strategies, and in near future, to develop the effective anti-cancer drugs.

Analysis of the Interaction of UBE2Q1 with B4GALT1 and P53: Experimental and Molecular Modeling Study.

UBE2Q1-dependent ubiquitination of key proteins including β 1,4- galactosyltransferase (GalT1), and P53 might play a pivotal role in cancer development. The present study aimed to evaluate the molecular analysis of possible interactions between UBE2Q1 with B4GALT1 and P53 proteins. We established SW1116 colorectal cancer cell line stably transfected with UBE2Q1. To verify the overexpression of UBE2Q1, we performed western blot and fluorescent microscopy analysis. Using the immunoprecipitation (IP) product of the over-expressed protein on the silver staining gel, we observed the potential interacting partners of UBE2Q1. The Molecular Operating Environment (MOE) software was also used to perform the molecular docking of the UBC domain of UBE2Q1 (2QGX) with B4GALT1 (2AGD), and P53 (tetramerization (1AIE) and DNA binding domains (1GZH)) proteins. Western blot and IP analysis detected a UBE2Q1-GFP band in transfected cells, while no band was detected for mock-transfected cells. Moreover, the overexpression of UBE2Q1 tagged with GFP was observed under fluorescent microscopy as well with about 60-70% shining. Silver staining of IP gel revealed several bands in colorectal cancer (CRC) with UBE2Q1 overexpression. Protein- Protein interaction (PPI) analysis also depicted a high affinity of the UBC domain of UBE2Q1 to the B4GALT1 and P53 (tetramerization and DNA binding domains). Molecular docking also revealed hot-spot regions for all poses. Our data suggest that UBE2Q1 as an E2 enzyme of ubiquitination system can interact with B4GALT1 and P53, and may contribute to the accumulation of misfolded important proteins and colorectal tumor development.

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements.

The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53’s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53’s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

Abstract LB361: Li-Fraumeni syndrome-associated dimer-forming mutant p53 promotes transactivation-independent mitochondrial cell death

Abstract Mutations that occur within the oligomerization domain (OD) of the tumor suppressor p53 generally abolish p53 tetramerization and are associated with increased cancer susceptibility in Li-Fraumeni syndrome (LFS). Despite their prevalence, the impact of OD-mutant p53 proteins in cancer, especially beyond a loss of canonical p53 activity, has not been well elucidated. We sought to delineate the gain-of-function (GOF) vs. loss-of-function (LOF) activities of OD-mutant p53, specifically focusing on the LFS tumor-derived p53(A347D) variant. We obtained LFS patient-derived dermal fibroblasts heterozygous for the mutation and generated an allelic series of U2OS cancer cell lines expressing wild-type p53, heterozygous (p53+/AD) or homozygous (p53AD/AD) mutant p53 or no p53 (p53 KO). In contrast to wild-type p53, mutant p53(A347D) exclusively forms dimers in both fibroblasts and cancer cells and has lost the ability to bind and transactivate the majority of canonical p53 target genes, which yields comparable tumorigenic properties between mutant p53 and p53 KO cells. Mutant p53(A347D), however, displays neomorphic activities. Glycolysis and oxidative phosphorylation pathways are enriched in cells bearing p53(A347D) relative to both wild-type p53 and p53 KO cells. Furthermore, dimeric mutant p53 induces striking mitochondrial network aberration and preferentially associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Ultimately, we describe dimeric mutant p53 as wielding a double-edged sword: driving tumorigenesis through LOF while gaining enhanced apoptogenic activity, thereby providing a potential basis for select therapeutic approaches. Citation Format: Joshua H. Choe, Tatsuya Kawase, An Xu, Asja Guzman, David R. Tong, Aleksandar Z. Obradovic, Ana M. Low-Calle, Bita Alaghebandan, Ananya Raghavan, Kaitlin Long, Paul M. Hwang, Joshua D. Schiffman, Yan Zhu, Ruiying Zhao, Dung-Fang Lee, Chen Katz, Carol L. Prives. Li-Fraumeni syndrome-associated dimer-forming mutant p53 promotes transactivation-independent mitochondrial cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB361.

C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo.

We previously demonstrated that a synthetic monomer peptide derived from the C-terminus of p53 (aa 361−382) induced preferential apoptosis in mutant p53 malignant cells, but not normal cells. The major problem with the peptide was its short half-life (half-life < 10 min.) due to a random coil topology found in 3D proton NMR spectroscopy studies. To induce secondary/tertiary structures to produce more stability, we developed a peptide modelled after the tetrameric structure of p53 essential for activation of target genes. Starting with the above monomer peptide (aa 361−382), we added the nuclear localization sequence of p53 (aa 353−360) and the end of the C-terminal sequence (aa 383−393), resulting in a monomer spanning aa 353−393. Four monomers were linked by glycine to maximize flexibility and in a palindromic order that mimics p53 tetramer formation with four orthogonal alpha helices, which is required for p53 transactivation of target genes. This is now known as the 4 repeat-palindromic-p53 peptide or (4R-Pal-p53p). We explored two methods for testing the activity of the palindromic tetrapeptide: (1) exogenous peptide with a truncated antennapedia carrier (Ant) and (2) a doxycycline (Dox) inducer for endogenous expression. The exogenous peptide, 4R-Pal-p53p-Ant, contained a His tag at the N-terminal and a truncated 17aa Ant at the C-terminal. Exposure of human breast cancer MB-468 cells and human skin squamous cell cancer cells (both with mutant p53, 273 Arg->His) with purified peptide at 7 µM and 15 µM produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361−382, termed p53p-Ant), at 15 µM and 30 µM induced 15% and 24% cell death, respectively. Compared to the p53p-Ant, the exogenous 4R-pal-p53p-Ant was over five-fold more potent for inducing apoptosis at an equimolar concentration (15 µM). Endogenous 4R-Pal-p53p expression (without Ant), induced by Dox, resulted in 43% cell death in an engineered MB468 breast cancer stable cell line, while endogenous p53 C-terminal monomeric peptide expression produced no cell death due to rapid peptide degradation. The mechanism of apoptosis from 4R-Pal-p53p involved the extrinsic and intrinsic pathways (FAS, caspase-8, Bax, PUMA) for apoptosis, as well as increasing reactive oxygen species (ROS). All three death pathways were induced from transcriptional/translational activation of pro-apoptotic genes. Additionally, mRNA of p53 target genes (Bax and Fas) increased 14-fold and 18-fold, respectively, implying that the 4R-Pal-p53p restored full apoptotic potential to mutant p53. Monomeric p53p only increased Fas expression without a transcriptional or translational increase in Fas, and other genes and human marrow stem cell studies revealed no toxicity to normal stem cells for granulocytes, erythrocytes, monocytes, and macrophages (CFU-GEMM). Additionally, the peptide specifically targeted pre-malignant and malignant cells with mutant p53 and was not toxic to normal cells with basal levels of WT p53.

Theoretical Computational Analysis Predicts Interaction Changes Due to Differences of a Single Molecule in DNA

Theoretical methods, such as molecular mechanics and molecular dynamics, are very useful in understanding differences in interactions at the single molecule level. In the life sciences, small conformational changes, including substituent modifications, often have a significant impact on function in vivo. Changes in binding interactions between nucleic acid molecules and binding proteins are a prime example. In this study, we propose a strategy to predict the complex structure of DNA-binding proteins with arbitrary DNA and analyze the differences in their interactions. We tested the utility of our strategy using the anticancer drug trifluoro-thymidine (FTD), which exerts its pharmacological effect by incorporation into DNA, and confirmed that the binding affinity of the BCL-2-associated X sequence to the p53 tetramer is increased by FTD incorporation. On the contrary, in p53-binding sequences extracted from FTD-resistant cells, the binding affinity of DNA containing FTD was found to be greatly reduced compared to normal DNA. This suggests that thymidine randomly substituted for FTD in resistant cells may acquire resistance by entering a position that inhibits binding to DNA-binding proteins. We believe that this is a versatile procedure that can also take energetics into account and will increase the importance of computational science in the life sciences.

Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans-intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.

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.

Abstract 3309: Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA

Abstract Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models.

P53 oligomerization status as an indicator of sensitivity of p53-wildtype neuroblastomas to the combination of DNA damaging agent and Chk1 inhibitor.

Neuroblastomas are one of the most common types of solid tumors in infants and children and are responsible for approximately 15% of childhood cancer deaths. Neuroblastomas rarely have mutations in p53, with less than 2% of NB containing mutations in p53, compared to up to 60% for other tumor classes. Previous studies on the therapeutic combination of a DNA damaging agent and checkpoint kinase 1 (Chk1) inhibitor have shown that DNA damage-induced cell cycle arrest can be specifically abrogated in p53-defective tumors. However, some p53-wildtype tumors have also been shown to be sensitive to this therapeutic combination, suggesting that these cells have other defects in the p53 response that can be exploited for therapeutic purposes. In the current study, we investigated the response to the combination of a DNA damaging agent (SN38) and a Chk1 inhibitor (UCN-01) of four p53-wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, SK-N-AS, and Lan-5. When the cells were treated with concentrations of SN38 ranging from 0–30 ng/ml, all four cell lines accumulated p53 which was phosphorylated on serines 15 and 20. However, only the SK-N-SH were found to activate p21waf1 and repress cyclin B. In order to assess sensitivity to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 10 ng/ml SN38 for 24 h, followed by 25 nM UCN-01 for 6 and 24 h. The SK-N-SH showed no sensitivity to UCN-01 treatment whereas the SH-SY5Y, SK-N-AS, and Lan-5 abrogated G2 arrest within 24 h. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers, so we analyzed the oligomerization status of p53 in all four cell lines using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53-wildtype MCF10A cells. The SH-SY5Y, SK-N-AS, and Lan-5 however, had extremely low to undetectable levels of dimers and tetramers. Our study also showed no cytoplasmic accumulation of p53 in these cells contrary to some previous reports. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53-wildtype tumors to the therapeutic combination of DNA damaging agent and Chk1 inhibitor.

Low-frequency collective motion of DNA-binding domain defines p53 function.

Most mutations in the DNA-binding domain (DBD) of p53 inactivate or rescue the protein function interacting with the minor groove of DNA. However, how the conformation changes propagating from the mutation sites result in distinct molecular recognition is still not well understood. As the protein mobility is an intrinsic property encrypted in its primary structure, we examined if different structures of wild-type and mutant p53 core domains display any unique patterns of intrinsic mobility. Normal mode calculation was employed to characterize the collective dynamics of DBD in p53 monomer and tetramer as well as their mutants. Intriguingly, the low-frequency collective motions of DBD show similar patterns between the wild-type protein and the rescued mutants. The analysis on atomic backbone fluctuations and low-frequency vibration mode statistics does further support the correlation between the intrinsic collective motion of DBD and the p53 protein function. The mutations in the DBD influence the low-frequency vibration of the p53 tetramer via the change of the collective motions among its four monomers. These findings thus provide new insights for understanding the physical mechanism of p53 protein structure-function relationship and help find the small molecule drug to modulate protein dynamic for disease therapy.

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.