Interaction Of P53 Research Articles

TRIM25 targets p300 for degradation.

p300 is an important transcriptional co-factor. By stimulating the transfer of acetyl residues onto histones and several key transcription factors, p300 enhances transcriptional initiation and impacts cellular processes including cell proliferation and cell division. Despite its importance for cellular homeostasis, its regulation is poorly understood. We show that TRIM25, a member of the TRIM protein family, targets p300 for proteasomal degradation. However, despite TRIM25's RING domain and E3 activity, degradation of p300 by TRIM25 is independent of TRIM25-mediated p300 ubiquitination. Instead, TRIM25 promotes the interaction of p300 with dynein, which ensures a microtubule-dependent transport of p300 to cellular proteasomes. Through mediating p300 degradation, TRIM25 affects p300-dependent gene expression.

Exploratory novel biomarker and resistance mechanism of milademetan, an MDM2 inhibitor, in amplified MDM2 intimal sarcoma from an open-label phase 1b/2 trial (NCCH1806/MK004).

11544 Background: Amplified Murine double minute 2 ( MDM2) is found in > 70% of intimal sarcoma, known as one of the ultra-rare sarcomas. Milademetan (DS3032, RAIN-32) is a novel, specific, small-molecule MDM2 inhibitor that disrupts MDM2 and the tumor suppressor protein p53 interactions in tumor cells. We conducted a phase 1b/2 trial (Trial registration No: JMA-IIA00402) in patients with amplified MDM2 wild-type TP53 intimal sarcoma as a sub-study under the nationwide large registry for rare cancers in Japan (MASTERKEY Project). Eleven patients were enrolled, and ten were included in the efficacy analysis. Two (20%) patients had durable responses for > 15 months. Milademetan provided clinical benefits in patients with amplified MDM2 intimal sarcoma. Predictive biomarkers other than amplified MDM2 and acquired resistance mechanisms for milademetan are unknown. Methods: Whole-exome and RNA sequencing analyses of pre-treatment tissue samples were conducted to identify determinants of response. Genomic alterations were analyzed for 10 patients, and gene expression was analyzed for 9 patients using their pre-treatment tissue samples. Targeted sequencing of cell-free DNA (cfDNA) samples (liquid biopsy) was also conducted sequentially at three points [before treatment with milademetan (baseline), at Cycle 2 Day1, and at the time of disease progression] to identify determinants of response and resistance. Results: From whole-exome and RNA sequencing analyses of pre-treatment tissue samples, we could not find any molecular pathways associated with the anti-tumor activity of milademetan. Focusing on 8 genes ( CDK4, CDKN2A, CDKN2B, EGFR, ERBB3, MDM2, PDGFRA, TP53) known to be frequently affected in intimal sarcoma and 10 genes ( AKT1, ATM, BBC3, CDKN1A, CDKN1C, CHEK2, MDM4, PMAIP1, PPM1D, TWIST1) reported to be associated with MDM2 inhibitor responses; we found that anti-tumor activity correlated with amplified TWIST1 ( p-value = 0.028) and negatively with CDKN2A loss ( p-value = 0.071). Eight of the 10 patients had their cfDNA collected sequentially at baseline, at Cycle 2 Day1 and disease progression; however, one did not consent to the exploratory analysis study using cfDNA, and one had cfDNA collected at baseline but not at disease progression due to ongoing treatment. Of the eight patients, TP53 mutations in cfDNA were detected in one and five patients at baseline and disease progression, respectively. The cfDNA allele frequency of TP53 mutations increased with disease progression. Conclusions: CDKN2A loss and amplified TWIST1 could be associated with the anti-tumor activity of milademetan in patients with amplified MDM2 intimal sarcoma. Acquired TP53 mutations were detected in sequential liquid biopsies as loss-of-function mutations, and these TP53 mutations might compromise the anti-tumor activity. Clinical trial information: JMA-IIA00402 .

Longitudinal mutational analysis of TP53 in plasma circulating tumor DNA (ctDNA) in patients (pts) with solid tumors in a phase I study of BI 907828, an MDM2–p53 antagonist.

3036 Background: Inactivation of p53 can occur due to TP53 mutations or downregulation of wild-type p53 by its primary negative regulator, MDM2. Targeting the MDM2–p53 interaction may therefore restore p53 function. BI 907828, a highly potent MDM2–p53 antagonist, is being evaluated in a phase Ia/b study in pts with advanced solid tumors (NCT03449381). During dose escalation (phase Ia), BI 907828 demonstrated a manageable safety profile and early signs of efficacy. Here we present data from a longitudinal mutational analysis of TP53 using ctDNA. The objective of the analysis was to identify if mutations in TP53 can be associated with possible acquired resistance to BI 907828. Methods: Collection of blood samples for ctDNA analyses was optional and sampled longitudinally from pts at baseline and every cycle until end of treatment (EoT). ctDNA was purified from plasma and analyzed using tumor-specific next generation sequencing (NGS; custom-made 8-gene panel using KAPA HyperCap technology, including TP53) to identify tumor-derived somatic mutations that may be relevant to understanding resistance mechanisms to BI 907828. The limit of detection was determined to be 0.5% mutant allele frequency; common polymorphisms were filtered out. Results: Plasma samples for ctDNA analysis were available from 44/54 (81%) pts enrolled to phase Ia. Baseline plasma samples from 26/54 (48%) pts and EoT samples from 41/54 (76%) pts were analyzed. Mutations were found in 6/26 (23%) samples at baseline. In 2 pt samples, mutations were found in both tumor and ctDNA, but concordant results were limited to 1 pt. At EoT, no (0) mutations were found in 16/41 (39%) samples, 1 mutation in 11/41 (27%), 2 mutations in 6/41 (15%), 3 mutations in 2/41 (5%) and 4 mutations in 6/41 (15%). No sample had more than 4 mutations. The most frequent mutation at EoT (in 9/41 [22%] samples) was R175H, a known p53 loss of function (LOF) mutation, followed by R248Q/W LOF (in 6/41 [15%) samples). Mutations in non- TP53 genes were detectable independent of TP53 mutation status, suggesting ctDNA was present in all samples. No clear association of induction of mutations could be identified for any BI 907828 dose or dosing schedule, treatment duration, or tumor type. 9/20 (45%) pts with no detectable mutations at baseline maintained wild-type TP53 status at EoT. Signs of efficacy were observed in pts with acquired TP53 LOF mutations, suggesting that these mutations have no effect on efficacy. Analysis of TP53 mutational status at EoT in conjunction with correlative clinical efficacy will be presented. Conclusions: This mutation analysis represents one of the most comprehensive assessments of longitudinal ctDNA by NGS for TP53 from a clinical trial of an MDM2–p53 antagonist. Preliminary data suggest that BI 907828 does not systematically lead to broad acquisition of resistance by inducing alterations in TP53. Clinical trial information: NCT03449381 .

IL-13/IL-13RA2 signaling promotes colorectal cancer stem cell tumorigenesis by inducing ubiquitinated degradation of p53

Cancer stem cells (CSCs) are considered tumor-initiating cells and the main drivers of disease progression. Targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the up-regulation of IL-13RA2 expression in colorectal cancer (CRC) tissues and spheroid cells. The expression of IL-13RA2 was positively correlated with canonical stemness markers in CRC. We further demonstrated that the level of IL-13 was up-regulated in the serum of CRC patients. Biologically, recombinant IL-13 (rIL-13) stimulation promoted the sphere formation, proliferation, and migration of CRC cells in vitro and enhanced tumorigenesis in vivo. This phenotype could be reversed by knocking down IL-13RA2. Mechanistically, IL-13 activated autophagy by inducing LC3I/LC3II transformation in CRC-CSCs, which was crucial for the biological functions of IL-13. We further demonstrated that IL-13RA2 acted as a modular link of the E3 ligase UBE3C and the substrate p53 protein, enhancing the interaction of UBE3C and p53, thereby inducing the K48-linked ubiquitination of p53. In conclusion, the IL-13/IL-13RA2 signaling cascade promotes CRC-CSC self-renewal and tumorigenesis by inducing p53 ubiquitination, adding an important layer to the connection between IL-13 and p53, which can be translated into novel targeted therapies.

Rapid Evaluation of Staple Placement in Stabilized α Helices Using Bacterial Surface Display.

There are a wealth of proteins involved in disease that cannot be targeted by current therapeutics because they are inside cells, inaccessible to most macromolecules, and lack small-molecule binding pockets. Stapled peptides, where two amino acids are covalently linked, form a class of macrocycles that have the potential to penetrate cell membranes and disrupt intracellular protein-protein interactions. However, their discovery relies on solid-phase synthesis, greatly limiting queries into their complex design space involving amino acid sequence, staple location, and staple chemistry. Here, we use stabilized peptide engineering by Escherichia coli display (SPEED), which utilizes noncanonical amino acids and click chemistry for stabilization, to rapidly screen staple location and linker structure to accelerate peptide design. After using SPEED to confirm hotspots in the mdm2-p53 interaction, we evaluated different staple locations and staple chemistry to identify several novel nanomolar and sub-nanomolar antagonists. Next, we evaluated SPEED in the B cell lymphoma 2 (Bcl-2) protein family, which is responsible for regulating apoptosis. We report that novel staple locations modified in the context of BIM, a high affinity but nonspecific naturally occurring peptide, improve its specificity against the highly homologous proteins in the Bcl-2 family. These compounds demonstrate the importance of screening linker location and chemistry in identifying high affinity and specific peptide antagonists. Therefore, SPEED can be used as a versatile platform to evaluate multiple design criteria for stabilized peptide engineering.

Abstract 287: Novel p62 interaction suggests novel role in glutamate metabolism

Abstract Prostate cancer (PCa) is the second most common cause of cancer-related death among American men. Androgen Receptor (AR) transcriptional activity is required for PCa tumor growth. Thus, androgen deprivation therapy (ADT) is a first line PCa therapy. However, 10-20% of patients will inevitably develop lethal castration-resistant PCa (CRPC). Thus, we must uncover the mechanisms that drive CPRC. Chronic inflammation is a known hallmark of cancer initiation and progression. Using the LNCaP human PCa cell line, we have shown that chronic exposure to the inflammatory cytokine, interleukin-1 (IL-1) can transform castration-sensitive PCa cells into castration-resistant PCa cells. IL-1 is elevated in PCa patient tissue and serum and is associated with disease progression and metastasis. IL-1 is clinically relevant, but the role of IL-1 in CRPC development is not fully elucidated. To mimic ADT, we serum-starved LNCaP and the chronic IL-1 sublines, LNas1 and LNbs1, and performed mass spectrometry to identify chronic IL-1-induced changes in protein-protein interactions. Specifically, we looked at p62 protein interactors. p62 is a multi-domain, multifunctional pro-survival protein that mediates autophagic flux in response to cellular stress and serum starvation, promotes NF-κB inflammatory signaling and induces NRF2 antioxidant signaling. We discovered that p62 forms a constitutive interaction with KEAP1 under serum starved and normal growth conditions in the chronic IL-1 sublines. KEAP1 inhibits NRF2 nuclear translocation. In response to antioxidant stress, p62 binds and sequesters KEAP1 from NRF2 to activate NFR2 signaling. Surprisingly, we do not observe constitutive canonical NRF2 signaling in the chronic IL-1 sublines. However, in response to serum starvation, we do see both high basal levels and upregulation, specifically, of the NRF2-induced gene, GCLC. GCLC is a glutamate-cysteine ligase that synthesizes the antioxidant, glutathione. Therefore, using mass spectrometry and immunoprecipitation western blotting, we looked at p62 interactions under serum starvation conditions. While we did not find p62 interaction with GCLC in the chronic IL-1 sublines, we do find that p62 interacts with GLUL. GLUL is a glutamate-ammonia ligase that plays a role in cellular detoxification. These data suggests that chronic IL-1 exposure induces novel p62 interactions that have a role in glutamate metabolism. Citation Format: Haley Christine Dahl, Anisha R. Konakalla, Preethi Kanumuri, Basir Mansoor, Anastasia Kelley, Riya Shipurkar, Nikki A. Delk. Novel p62 interaction suggests novel role in glutamate metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 287.

Ganoderic Acid A and Its Amide Derivatives as Potential Anti-Cancer Agents by Regulating the p53-MDM2 Pathway: Synthesis and Biological Evaluation

The mechanisms of action of natural products and the identification of their targets have long been a research hotspot. Ganoderic acid A (GAA) is the earliest and most abundant triterpenoids discovered in Ganoderma lucidum. The multi-therapeutic potential of GAA, in particular its anti-tumor activity, has been extensively studied. However, the unknown targets and associated pathways of GAA, together with its low activity, limit in-depth research compared to other small molecule anti-cancer drugs. In this study, GAA was modified at the carboxyl group to synthesize a series of amide compounds, and the in vitro anti-tumor activities of the derivatives were investigated. Finally, compound A2 was selected to study its mechanism of action because of its high activity in three different types of tumor cell lines and low toxicity to normal cells. The results showed that A2 could induce apoptosis by regulating the p53 signaling pathway and may be involved in inhibiting the interaction of MDM2 and p53 by binding to MDM2 (KD = 1.68 µM). This study provides some inspiration for the research into the anti-tumor targets and mechanisms of GAA and its derivatives, as well as for the discovery of active candidates based on this series.

HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma

BackgroundTumor-derived exosomes are critical elements of the cell–cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes.MethodsWe observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components.ResultsWe found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size.ConclusionsOur present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity.Graphical Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma

CopA3 peptide inhibits MDM2-p53 complex stability in colorectal cancers and activates p53 mediated cell death machinery

The diverse expression patterns of the tumor suppressor p53 in cancer cells reflect the regulatory efficiency of multiple cellular pathways. By contrast, many human tumors are reported to develop in the presence of wild-type p53. Recently, several oncogene inhibitors have been used clinically to suppress tumor development by functionally reactivating other oncoproteins. On the other hand, p53 reactivation therapies have not been well established, as few of the p53-MDM2 complex inhibitors such as Nutlin-3 induces mutation in p53 gene upon prolonged usage. Therefore, in this study CopA3, a 9-mer dimeric D-type peptide with anticancer activity against the human colorectal cancer cells, was used to explore the efficacy of p53 reactivation in-vitro and in-vivo. The anticancer activity of CopA3 was more selective towards the wild-type p53 expressing cells than the p53 deficient or mutant colorectal cancer cells. In response to this, this study investigated the signaling pathway in vitro and validated its anti-tumor activity in-vivo. The protein-peptide interaction and molecular docking efficiently provided insight into the specific binding affinity of CopA3 to the p53-binding pocket of the MDM2 protein, which efficiently blocked the p53 and MDM2 interaction. CopA3 plays a crucial role in the binding with MDM2 and enhanced the nuclear translocation of the p53 protein, which sequentially activated the downstream targets to trigger the autophagic mediated cell death machinery through the JNK/Beclin-1 mediated pathway. Collectively, CopA3 affected the MDM2-p53 interaction, which suppressed tumor development. This study may provide a novel inhibitor candidate for the MDM2-p53 complex, which could ultimately suppress the growth of colorectal cancer cells without being cytotoxic to the healthy neighboring cells present around the tumor microenvironment.

Efficacy and safety of the MDM2–p53 antagonist BI 907828 in patients with advanced biliary tract cancer: Data from two phase Ia/Ib dose-escalation/expansion trials.

543 Background: Standard-of-care treatment for advanced biliary tract cancer (BTC) is chemotherapy and outcomes remain poor; hence, there is a clinical need for effective targeted treatments. As MDM2 is a negative regulator of the tumor suppressor p53, blocking the MDM2–p53 interaction is a potential antitumor strategy. Further, preliminary data indicate that MDM2 amplification is a negative prognostic marker in BTC. The MDM2–p53 antagonist BI 907828 has shown preclinical antitumor activity in a range of malignancies and is currently being assessed in two phase Ia/Ib dose-escalation/expansion trials in patients with advanced solid tumors: as monotherapy (NCT03449381) and in combination with an anti-PD-1 antibody, ezabenlimab (NCT03964233). Here, we present data for patients with advanced BTCs in these trials. Methods: Patients in the monotherapy trial received escalating doses of BI 907828 on day 1 of 21-day cycles (q3w). Patients in the combination trial received escalating doses of BI 907828 and 240 mg ezabenlimab q3w (doublet); one patient also received the anti-LAG-3 antibody BI 754111 (a drug that has since been discontinued; triplet). Results: A total of 8 patients with BTC were enrolled in the 2 trials, 4 in the monotherapy trial and 4 in the combination trial. In the monotherapy trial, 2 patients had ampullary carcinoma (both received BI 907828 45 mg q3w), and 2 had cholangiocarcinoma (CC; 1 received BI 907828 45 mg q3w and 1 with intrahepatic CC [iCC] received 80 mg q3w). In the combination trial, 3 patients with iCC received 30 mg/45 mg BI 907828 doublet or 45 mg triplet, and 1 with gallbladder carcinoma (GBC) received BI 907828 45 mg doublet. Across both trials, 5 patients achieved PR, 2/4 in the monotherapy trial and 3/4 in the combination trial. In the monotherapy trial, the responding patients had iCC (80 mg q3w; MDM2-amplified; 73% tumor shrinkage; PFS event at 404 days) and ampullary adenocarcinoma (45 mg q3w; MDM2-amplified; 51% tumor shrinkage; ongoing, PFS censored at 255 days). In the combination trial, all 3 responding patients had MDM2-amplified biliary tract adenocarcinoma (2 iCC, 1 GBC); tumor shrinkage was 49–54%; PFS was 162–241 days. A further 2 patients (1 in each trial) achieved stable disease (SD). In the monotherapy trial, most common grade ≥3 treatment-related AEs (TRAEs) were thrombocytopenia, decreased white blood cell (WBC) count and neutropenia (2 patients each). In the combination trial, most common grade ≥3 TRAEs were anemia and decreased WBC count (3 patients each), neutropenia and thrombocytopenia (2 patients each). Conclusions: The MDM2–p53 antagonist BI 907828 has shown a manageable safety profile and encouraging preliminary efficacy in patients with BTC, with 5 PRs and 2 SD in 8 patients. A phase IIa/IIb trial of BI 907828 (45 mg q3w) in patients with BTC is planned to start recruitment at the end of 2022 (NCT05512377). Clinical trial information: NCT03449381 and NCT03964233 .

In-Cell Chemical Crosslinking Identifies Hotspots for SQSTM-1/p62-IκBα Interaction That Underscore a Critical Role of p62 in Limiting NF-κB Activation Through IκBα Stabilization

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB–activation cycle. In p62−/−-cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47, and K67) and C-terminal (K238/C239) residues in its fifth ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα-cotransfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα–protein-protein interactions. Interestingly, consistent with p62’s capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα′s interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IκBα on an interface opposite to that of its NF-κB–interacting interface, p62 failed to significantly affect IκBα–NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling leads us to speculate that it may be involved in “piggy-back” nuclear transport of IκBα following its NF-κB–elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB–activation cycle. Consequently, mice carrying a liver-specific deletion of p62-residues 68 to 252 reveal age-dependent–enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

Sodium butyrate inhibits high glucose-induced inflammation by controlling the acetylation of NF-κB p65 in human monocytes.

Hyperglycemia is a major cause of diabetes and diabetes-related diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells. THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0-25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay. HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α. These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

Novel piperazine-1,2,3-triazole leads for the potential treatment of pancreatic cancer.

From lead 1, (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl)sulfonyl)-phenyl)acetamide), a S100A2-p53 protein-protein interaction inhibitor based on an in silico modelling driven hypothesis, four focused libraries were designed and synthesised. Growth inhibition screening was performed against 16 human cancer cell lines including the pancreatic cell lines MiaPaCa2, BxPC3, AsPC-1, Capan-2, HPAC, PANC-1 and the drug resistant CFPAC1. Modification of 1's phenylacetamide moiety, gave Library 1 with only modest pancreatic cancer activity. Modification of the 3-OCH3Ph moiety (Library 2) gave 4-CH3 (26), 4-CH2CH3 (27), 4-CF3 (31) and 4-NO2 (32) with sterically bulky groups more active. A 4-CF3 acetamide replacement enhanced cytotoxicity (Library 3). The 4-C(CH3)336 resulted in a predicted steric clash in the S100A2-p53 binding groove, with a potency decrease. Alkyl moieties afforded more potent analogues, 34 (4-CH3) and 35 (CH2CH3), a trend evident against pancreatic cancer: GI50 3.7 (35; BxPC-3) to 18 (40; AsPC-1) μM. Library 4 analogues with a 2-CF3 and 3-CF3 benzenesulfonamide moiety were less active than the corresponding Library 3 analogues. Two additional analogues were designed: 51 (4-CF3; 4-OCH3) and 52 (4-CF3; 2-OCH3) revealed 52 to be 10-20 fold more active than 51, against the pancreatic cancer cell lines examined with sub-micromolar GI50 values 0.43 (HPAC) to 0.61 μM (PANC-1). MOE calculated binding scores for each pose are also consistent with the observed biological activity with 52. The obtained SAR data is consistent with the proposed interaction within the S100A2-p53 bonding groove.

The chameleonic behavior of p53 in health and disease: the transition from a client to an aberrant condensate scaffold in cancer.

In 1972, the Weber statement, "The multiplicity of interactions and the variety of effects that follow from them show that multimer proteins are unlikely to be limited to a minimal number of allowed conformations," first addressed the dynamic nature of proteins. This idea serves as a foundation for understanding why several macromolecules, such as p53, exhibit the properties of a molecular chameleon. Functionally competent states comprise a myriad of p53 three-dimensional arrangements depending on the stimuli. For instance, the interaction of p53 with nuclear components could induce liquid-liquid phase separation (LLPS) and the formation of membraneless organelles. The functional or deleterious role of p53 in liquid droplets is still unclear. Functional aspects display p53 interconverting between droplets and tetramer with its functional abilities maintained. In contrast, the aberrant phase separation is likely to fuel the aggregation path, usually associated with the onset and progression of age-related neurodegenerative diseases and cancer. Here, we gathered the most relevant aspects that lead p53 to phase separation and the resulting structural effects, attempting to understand p53's functional and disease-relevant processes. Aberrant phase separation and aggregation of mutant p53 have become important therapeutic targets against cancer.

MDMX elevation by a novel Mdmx–p53 interaction inhibitor mitigates neuronal damage after ischemic stroke

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein–Protein Interaction (PPI) inhibitors, K-181, on Mdmx–p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

In-silico and in-vitro evaluation of docetaxel and berberine as potential p53 modulating apoptotic inducers in oral squamous cell carcinoma

Objective: To investigate the interaction of p53 with docetaxel and berberine and their anticancer activities against oral squamous cell carcinoma. Methods: The interaction between p53 with docetaxel and berberine was investigated and their mechanisms of action against oral squamous cell carcinoma were studied. Toxicity studies were performed to determine any toxic impact of the drugs on the vital organs of tested animals. Results: In silico results revealed the molecular interaction of docetaxel and berberine with p53 and the molecules were found to be potential p53 inducers. Docetaxel and berberine inhibited the proliferation of cancer cells in a concentration-dependent manner. Flow cytometry analysis revealed that docetaxel and berberine at IC50 concentrations upregulated the expression of p53 in oral squamous cell carcinoma cells, thus triggering apoptotic cell death. In addition, no toxicity was observed in the liver and kidney tissues of mice after docetaxel and berberine treatment. Conclusions: Docetaxel and berberine significantly suppressed the proliferation of oral cancer cells by activating p53 expression and causing apoptotic cell death. Both compounds can be potential agents for the treatment of oral cancer, with little to no toxicity at the tissue level.

Targeting the MDM2-p53 pathway in dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is an aggressive adipogenic cancer with poor prognosis. DDLPS tumors are only modestly sensitive to chemotherapy and radiation, and there is a need for more effective therapies. Genetically, DDLPS is characterized by a low tumor mutational burden and frequent chromosomal structural abnormalities including amplification of the 12q13-15 chromosomal region and the MDM2 gene, which are defining features of DDLPS. The MDM2 protein is an E3 ubiquitin ligase that targets the tumor suppressor, p53, for proteasomal degradation. MDM2 amplification or overexpression in human malignancies is associated with cell-cycle progression and worse prognosis. The MDM2-p53 interaction has thus garnered interest as a therapeutic target for DDLPS and other malignancies. MDM2 binds p53 via a hydrophobic protein interaction that is easily accessible with synthetic analogues. Multiple agents have been developed, including Nutlins such as RG7112 and small molecular inhibitors including SAR405838 and HDM201. Preclinical in vitro and animal models have shown promising results with MDM2 inhibition, resulting in robust p53 reactivation and cancer cell death. However, multiple early-phase clinical trials have failed to show a benefit with MDM2 pathway inhibition for DDLPS. Mechanisms of resistance are being elucidated, and novel inhibitors and combination therapies are currently under investigation. This review provides an overview of these strategies for targeting MDM2 in DDLPS.

Variable Regions of p53 Isoforms Allosterically Hard Code DNA Interaction.

Allosteric regulation of protein activity pervades biology as the "second secret of life." We have been examining the allosteric regulation and mutant reactivation of the tumor suppressor protein p53. We have found that generalizing the definition of allosteric effector to include entire proteins and expanding the meaning of binding site to include the interface of a transcription factor with its DNA to be useful in understanding the modulation of protein activity. Here, we cast the variable regions of p53 isoforms as allosteric regulators of p53 interactions with its consensus DNA. We implemented molecular dynamics simulations and our lab's new techniques of molecular dynamics (MD) sectors and MD-Markov state models to investigate the effects of nine naturally occurring splice variant isoforms of p53. We find that all of the isoforms differ from wild type in their dynamic properties and how they interact with the DNA. We consider the implications of these findings on allostery and cancer treatment.

Heterochiral dipeptide d-phenylalanyl- l-phenylalanine (H-D Phe-L Phe-OH) as a potential inducer of metastatic suppressor NM23H1 in p53 wild-type and mutant cells.

In recent years, significant progress has been made to the use-case of small peptides because of their diversified edifice and hence their versatile application scope in cancer therapy. Here we identify the heterochiral dipeptide H-D Phe-L Phe-OH (F1) as a potent inducer of the metastatic suppressor NM23H1. We divulge the effect of F1 on the major EMT/metastasis-associated genes and the implications on the invasion and migration ability of cancer cells. The anti-invasive potential of F1 was directly correlated with NM23H1 expression. Mechanistically, F1 treatment elevated p53 levels as validated by localization and transcriptional studies. In the NM23H1 knockdown condition, F1 failed to induce any p53 expression/nuclear localization, indicating that the upregulation in p53 expression by F1 is NM23H1 dependent. We also demonstrate how the antimetastatic potential of F1 is primarily mediated through NM23H1 irrespective of the p53 status of the cell. However, both NM23H1 and a functional p53 protein in conjunction govern the apoptotic and cytostatic potential of F1. Coimmunoprecipitation studies unraveled the augmentation of the p53 and NM23H1 interaction in p53 wild-type cells. However, in p53 mutated cells, no such enrichment was evidenced. We employed mouse isogenic cell lines (4T-1 and 4T-1 p53) to determine the in vivo efficacy of F1 (spontaneous and experimental models). Decreased tumor volume in the cohort injected with 4T-1 p53 cells demonstrated that while the antimetastatic potential of F1 was reliant on NM23H1, p53 activation was required for ablation of primary tumor burden. Our findings unravel that F1 treatment induces significant abrogation of the migration, invasion and metastatic potential of both p53 wild-type and p53 deficient cancers mediated through NM23H1.

Identification of a new member of Mortaparib class of inhibitors that target mortalin and PARP1.

Mortalin, a heat shock family protein enriched in cancer cells, is known to inactivate tumor suppressor protein p53. Abrogation of mortalin-p53 interaction and reactivation of p53 has been shown to trigger growth arrest/apoptosis in cancer cells and hence, suggested to be useful in cancer therapy. In this premise, we earlier screened a chemical library to identify potential disruptors of mortalin-p53 interaction, and reported two novel synthetic small molecules (5-[1-(4-methoxyphenyl) (1,2,3,4-tetraazol-5-yl)]-4-phenylpyrimidine-2-ylamine) and (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) called Mortaparib and MortaparibPlus, respectively. These compounds were shown to possess anticancer activity that was mediated through targeting mortalin and PARP1 proteins, essential for cancer cell survival and proliferation. Here, we report characterization of the third compound, {4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine}, isolated in the same screening. Extensive computational and molecular analyses suggested that the new compound has the capability to interact with mortalin, p53, and PARP1. We provide evidence that this new compound, although required in high concentration as compared to the earlier two compounds (Mortaparib and MortaparibPlus) and hence called MortaparibMild, also downregulates mortalin and PARP1 expression and functions in multiple ways impeding cancer cell proliferation and migration characteristics. MortaparibMild is a novel candidate anticancer compound that warrants further experimental and clinical attention.