Ubiquitin Ligase MDM2 Research Articles

Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

BackgroundHigh expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets.MethodsKinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA.ResultsBy using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly.ConclusionOur research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer

BackgroundEndocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.MethodsBioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.ResultsRACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.ConclusionIn summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Sanggenol L induces ferroptosis in non-small cell lung cancer cells via regulating the miR-26a-1-3p/MDM2/p53 signaling pathway

Ferroptosis is a regulated cell death marked by iron-dependent lipid peroxidation. Tumor cells that survive by evading chemotherapy-induced apoptosis are vulnerable to ferroptosis. Therefore, it is particularly urgent to explore active ingredients that can selectively induce ferroptosis in cancer cells. Here, we revealed that sanggenol L, the active agent of Morus Bark, predisposed non-small cell lung cancer (NSCLC) cells to ferroptosis, evidenced by reactive oxygen species (ROS) accumulation, glutathione depletion, mitochondrial shrinkage, and lipid peroxidation. Furthermore, the ferroptosis-related miRNA array showed that sanggenol L treatment upregulated the level of miR-26a-1-3p, which directly targeted the E3 ubiquitin ligase MDM2. In addition, silencing MDM2 by miR-26a-1-3p resulted in a notable increase in p53 protein levels and decrease of its downstream target SLC7A11, ultimately triggered ferroptosis. The subcutaneous xenograft model and patient-derived tumor xenograft (PDX) model of NSCLC further confirmed the anti-tumor efficacy and safety of sanggenol L in vivo. Collectively, our data suggest that miR-26a-1-3p/MDM2/p53/SLC7A11 signaling axis plays a key role in sanggenol L-induced ferroptosis, which implies that sanggenol L can serves as an anticancer therapeutic arsenal for NSCLC.

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

Abstract 1743: Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered

Abstract Background: TP53 is the gene most frequently disrupted by somatic alterations in cancer. Mutations include single nucleotide variants resulting in functional alterations, as well as premature stop codons, splice variants, and cryptic alterations. TP53 gene dosage is disrupted by heterozygous and homozygous deletions, copy-neutral loss of heterozygosity, and other structural changes such as intragenic deletions and fusions. TP53 is altered by epigenetic events and viral oncogenes, such as E6 expressed by the human papillomavirus. The readout of altered TP53 includes decreased gene expression of either variant or wild-type alleles. Epistatic alteration of TP53 pathway components have also been described, with the E3 ubiquitin ligase MDM2 being the canonical example. Depending on the type of TP53 alteration, there may be differential signaling consequences, ranging from loss of function and dominant negative to oncogenic signaling. Hypothesis: Patterns of TP53 mutations will vary based on tumor type. Differential alterations of TP53 will suggest differential pathway dependence and downstream pathophysiology that may be relevant to signaling and therapeutic potential. Methods: We analyzed extensive cancer genomics repositories, such as the Pan-Cancer Atlas (10,000+ tumors, >30 cancer types) from The Cancer Genome Atlas project and the International Cancer Genome Consortium. Assessing genes in the TP53 pathway, we examined somatic mutations, copy number alterations, structural changes, and gene expression. Epigenetic data was considered when available. Results: A significant pattern amongst sarcomas, prostate cancers, and urothelial carcinomas emerged, revealing that certain tumors exhibited a predominant classic tumor suppressor gene genotype. This genotype featured frequent homozygous deletions, inactivating mutations, and relatively low TP53 gene expression, often accompanied by epistatic MDM2 amplification. By contrast, other tumors demonstrate an atypical pattern in which TP53 mutations were rarely if ever associated with loss of TP53 gene expression with low levels of homozygous TP53 gene deletion. Likewise, atypical TP53 mutant tumor types rarely demonstrated MDM2 amplifications, and in opposition to typical TP53 cases, MDM2 amplification was positively associated with TP53 mutation rather than anti-correlated. Head and neck squamous cell carcinoma, uterine carcinoma, and pancreatic adenocarcinoma followed this pattern. Investigation within selected tumor types such as lung squamous cell carcinoma suggested that both typical and atypical TP53 mutation patterns could be observed. Conclusions: TP53 mutation patterns were strongly associated with tumor type and may suggest differential pathway activation according to the somatic alteration status. Citation Format: Ubaid A. Tanveer, David N. Hayes, Julie George, Jeremiah R. Holt, Martin Peifer, Roman Thomas. Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered [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 1743.

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

METTL3-mediated N6-methyladenosine modification stimulates mitochondrial damage and ferroptosis of kidney tubular epithelial cells following acute kidney injury by modulating the stabilization of MDM2-p53-LMNB1 axis

N6-methyladenosine (m6A) and MELLT3 assume a role in the development of acute kidney injury (AKI). However, their mechanism in AKI remains under-explored. On this basis, this study explored the mechanism of MELLT3 in mitochondrial damage and ferroptosis of kidney tubular epithelial cells after AKI.HK-2 cells were induced by lipopolysaccharide (LPS) to simulate AKI, followed by gain and loss of function of genes, detection of mitochondrial damage and ferroptosis indicators, and analysis of gene interactions. An AKI mouse model was developed using the cecal ligation and puncture (CLP) method to investigate the effect of METTL3 knockdown on kidney injury.MDM2 and LMNB1 were upregulated and p53 was downregulated in LPS-treated HK-2 cells. Mechanistically, the E3 ubiquitin ligase MDM2 increased p53 ubiquitination to activate LMNB1. METTL3 knockdown decreased m6A methylation of MDM2, thus diminishing YTHDF1-mediated MDM2 mRNA stability and translation in LPS-treated HK-2 cells. Knockdown of LMNB1, MDM2, or METTL3 reduced NO, MDA, iron ion, and ROS levels as well as mitochondrial damage and raised SOD, GSH, XCT, GPX4, FPN1, and TFR1 levels in LPS-treated HK-2 cells. The in vivo results showed that METTL3 knockdown reduced renal injury and ferroptosis in CLP mice.METTL3 knockdown prevents mitochondrial damage and ferroptosis of kidney tubular epithelial cells after AKI via the MDM2-p53-LMNB1 axis.

GPCR targeting of E3 ubiquitin ligase MDM2 by inactive β-arrestin

E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2ABR peptide. The acidic residues of Mdm2ABR bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2ABR binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP6-induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.

Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif-mediated interactions.

The interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well-studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2-family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12-residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (KD around 0.1 μM). The affinity of the bay mussel p53TAD/MDM2 complex was lower (KD = 15 μM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (KD > 100 μM). Binding experiments with reconstructed ancestral p53TAD/MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/MDM2 affinity during speciation demonstrate high plasticity of motif-mediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD.

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1-uL18-uL5 and, upon further recruitment of the nucleolar factors Rpf2 and Rrs1, develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unravels how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.

CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation.

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

Cytoplasmic Localization of Mdm2 in Cells Expressing Mutated NPM is Mediated by p53.

Specific C-terminal nucleophosmin (NPM) mutations are related to the acute myeloid leukemia and cause mistargeting of mutated NPM (NPMmut) to the cytoplasm. Consequently, multiple NPM-interacting partners, e.g. the tumor suppressor p53, become also mislocalized. We found that ubiquitin ligase Mdm2 mislocalizes to the cytoplasm in the presence of NPMmut as well. Since p53 interacts with Mdm2, we searched for the NPMmut-p53-Mdm2 complex and interactions of its constituents in live cells and cell lysates using fluorescently tagged proteins, fluorescence lifetime imaging and immunoprecipitation. We proved existence of the ternary complex, which likely adopts a chain-like configuration. Interaction between Mdm2 and NPMmut was not detected, even under conditions of upregulated Mdm2 and p53 induced by Actinomycin D. We assume that p53 serves in the complex as a bridging link between Mdm2 and NPMmut. This conclusion was supported by disruption of the Mdm2-p53 interaction by Nutlin-3A, which resulted in a relocalization of Mdm2 to the nucleus, while both NPMmut and p53 remained in the cytoplasm. Importantly, silencing of p53 also prevented mislocalization of Mdm2 in the presence of NPMmut.

P90RSK Regulates p53 Pathway by MDM2 Phosphorylation in Thyroid Tumors

The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.

Upregulation of p53 through induction of MDM2 degradation: improved potency through the introduction of an alkylketone sidechain on the anthraquinone core

Overexpression of ubiquitin ligase MDM2 causes depletion of the p53 tumour-suppressor and thus leads to cancer progression. In recent years, anthraquinone analogs have received significant attention due to their ability to downregulate MDM2, thereby promoting p53-induced apoptosis. Previously, we have developed potent anthraquinone compounds having the ability to upregulate p53 via inhibition of MDM2 in both cell culture and animal models of acute lymphocytic leukaemia. Earlier work was focussed on mechanistic work, pharmacological validation of this class of compounds in animal models, and mapping out structural space that allows for further modification and optimisation. Herein, we describe our work in optimising the substituents on the two phenol hydroxyl groups. It was found that the introduction of an alkylketone moiety led to a potent series of analogs with BW-AQ-350 being the most potent compound yet (IC50 = 0.19 ± 0.01 µM) which exerts cytotoxicity by inducing MDM2 degradation and p53 upregulation.

Corosolic acid sensitizes ferroptosis by upregulating HERPUD1 in liver cancer cells

Primary liver cancer is the third leading cause of cancer death in the world, and the lack of effective treatments is the main reason for the high mortality. Corosolic acid (CA) has been proved to have antitumor activity. In this study, we found that CA can sensitize liver cancer cells to ferroptosis, which is a regulated form of cell death characterized by iron-dependent lipid peroxides reaching lethal levels. Here, we revealed that CA can inhibit glutathione (GSH) synthesis via HERPUD1, decreasing the cellular GSH level and causing liver cancer cells to become more sensitive to ferroptosis. Mechanistically, further studies found that HERPUD1 reduced the ubiquitination of the GSS-associated E3 ubiquitin ligase MDM2, which promoted ubiquitination of GSS, thereby inhibiting GSH synthesis to increase ferroptosis susceptibility. Importantly, a mouse xenograft model also demonstrated that CA inhibits tumor growth via HERPUD1. Collectively, our findings suggesting that CA is a candidate component for the development of treatments against liver cancer.

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function.

ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.

Discovery of a Potent and Selective Degrader for USP7

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.

Discovery of a Potent and Selective Degrader for USP7.

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.