E3 Ubiquitin Ligase MDM2 Research Articles

SUMO-specific protease SENP3 enhances MDM2-mediated ubiquitination of PARIS/ZNF746 in HeLa cells

The transcriptional repressor PARIS, a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α gene, and is involved in several pathological processes, including neurodegenerative disease and cancers. We have previously shown that SUMOylation of PARIS play an important role in its transcriptional repression activity. In addition, RNF4-mediated ubiquitination of SUMO2/3-conjugated PARIS is required for the control of PARIS-mediated transcriptional repression in HeLa cells that lack parkin expression. However, little is known about how PARIS ubiquitination and degradation are regulated in parkin-deficient cells. Here, we report that the deSUMOylase SENP3 interacted with PARIS and enhanced the ubiquitination of PARIS independently of its SUMOylation in HeLa cells. SENP3-enhanced PARIS ubiquitination mainly contributed to its proteasomal degradation, and required the oncogenic E3 ubiquitin ligase MDM2. MDM2 knockdown by small interfering RNA or expression of a dominant-negative MDM2 mutant inhibited the ubiquitination of PARIS. We further found that MDM2 activation via the PI3K/AKT pathway was involved in PARIS ubiquitination. Taken together, these results suggest that PARIS ubiquitination through SENP3-mediated MDM2 activation may control its functions in parkin-deficient cells.

SHANK1 facilitates non-small cell lung cancer processes through modulating the ubiquitination of Klotho by interacting with MDM2

SH3 and multiple ankyrin repeat domains 1 (SHANK1) is a scaffold protein, plays an important role in the normal function of neuron system. It has recently been shown to be a potential oncogene. In the present study, we report that the expression of SHANK1 is upregulated in non-small cell lung cancer (NSCLC), and is correlated with clinic pathological characteristics of NSCLC. Moreover, SHANK1 overexpression enhances the proliferation, migration and invasion of NSCLC cells. Mouse cell-derived xenograft model also confirmed the effects of SHANK1 on tumor growth in vivo. Furthermore, we found that SHANK1 increases the protein degradation of Klotho (KL), an important tumor suppressor, through ubiquitination-dependent pathway. In particular, we report discovery of KL as a SHANK1-interacting protein that acts as a new substate of the E3 ubiquitin ligase MDM2. SHANK1 can form a complex with KL and MDM2 and enhance the interaction between KL and MDM2. Our findings reveal an important oncogenic role and mechanism of SHANK1, suggesting SHANK1 can be a potential therapeutic target in NSCLC.

Treatment with b-AP15 to Inhibit UCHL5 and USP14 Deubiquitinating Activity and Enhance p27 and Cyclin E1 for Tumors with p53 Deficiency.

Background: TP53 protein is lost or mutated in about half of all types of human cancers and small molecules to regulate mutant p53 repair, or interrupt ubiquitination degradation of p53 induced by E3-ubiquitin ligase Mdm2 have a potential application in clinical application. Methods: To inhibit the deubiquitinase activity of 19S proteasome and restore the p53 protein level, in this study, we utilized p53 knockout mice to test the anti-cancer effect of a specific USP14 and UCH37 inhibitor b-AP15. Results: Our results show that UCHL5, USP14 and COPS5 are upregulated in p53-related tumors, and higher expression of these genes results in a shorter overall survival in patients with p53 deficiency. Treatment with b-AP15, a UCHL5 and USP14 deubiquitinating activity inhibitor in 19S regulatory subunit, induces tumor regression and prolong the survival period of tumor-loaded mice through down-regulation of COPS5 and its downstream AP-1 and E2F1, and up-regulation of the cell cycle-related proteins p27 and Cyclin E1. Conclusions: Thus, our results suggested that inhibition of UCHL5 and USP14 deubiquitinating activity in 19S proteasome may contribute an extensive approach to preventing tumor progress due to p53 deficiency.

Targeted β-catenin ubiquitination and degradation by multifunctional stapled peptides.

Aberrant activation of the Wnt signaling pathway has been identified in numerous types of cancer. One common feature of oncogenic Wnt regulation involves an increase in the cellular levels of β-catenin due to interference with its constitutive ubiquitin-dependent degradation. Targeting β-catenin has therefore emerged as an appealing approach for the treatment of Wnt-dependent cancers. Here, we report a strategy that employs multifunctional stapled peptides to recruit an E3 ubiquitin ligase to β-catenin, thereby rescuing β-catenin degradation by hijacking the endogenous ubiquitin-proteasome pathway. Specifically, we designed, synthesized, and evaluated a panel of multifunctional stapled peptides that have a β-catenin binding moiety (StAx-35) covalently linked to a second stapled peptide moiety (SAH-p53-8), which is capable to interact with the E3 ubiquitin ligase MDM2. We found that in vitro these multifunctional peptides can recruit the MDM2 protein to β-catenin and induce poly-ubiquitination on β-catenin. In cellulo, treatment of the human colorectal cancer cell line SW480 with the multifunctional stapled peptides showed dose-dependent degradation of endogenous β-catenin levels. In addition, a luciferase reporter assay showed that the multifunctional stapled peptides can suppress β-catenin-mediated gene expression via the Wnt signaling pathway. Therefore, these multifunctional stapled peptides provide a unique research tool for examining the Wnt signaling pathway by targeted knockdown of β-catenin at the protein level, and may serve as leads for potential drug candidates in the treatment of Wnt-dependent cancers.

MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

Mice Deficient in the RNA-Binding Protein Zfp871 Are Prone to Early Death and Steatohepatitis in Part through the p53-Mdm2 Axis.

p53 transcription factor is activated upon exposure to various cellular stresses, leading to growth suppression. However, aberrant activation of p53 can lead to defects in embryonic development and other abnormalities. Here, we identified zinc finger protein Zfp871 as a p53 target gene. We showed that as an RNA-binding protein, Zfp871 binds to Mdm2 3'UTR and stabilizes Mdm2 mRNA, which in turn suppresses p53 expression through increased expression of Mdm2 E3 ubiquitin ligase. Consistently, Zfp871 deficiency increases p53 expression, leading to growth suppression in a p53-dependent manner. To determine the role of Zfp871 in the p53 pathway, we used Zfp871-deficient mouse model and found that Zfp871-null mice were prone to embryonic/pre-weaning lethality, which can be partially rescued by simultaneous deletion of Trp53. We also found that mice heterozygous for Zfp871 had a short lifespan and were susceptible to steatohepatitis but not to spontaneous tumors. To determine the underlying mechanism, RNA-seq analysis was performed and showed that an array of genes involved in development, lipid metabolism, and inflammation is regulated by Zfp871 in conjunction with p53. Taken together, we conclude that the Zfp871-Mdm2-p53 pathway plays a critical role in tumor-free survival and development. IMPLICATIONS: A fine equilibrium of p53 is required for preventing damaging effects of aberrant p53 expression. We identify the Zfp871-Mdm2-p53 pathway that plays a critical role in development of mice and steatohepatitis.

OMRT-6. Optimizing MDM2 inhibition for the treatment of high-grade glioma

Over 80% of high-grade gliomas have alterations in members of the p53 pathway, a central regulator of cell cycle progression and apoptosis that becomes activated in response to cellular stress and DNA damage. For tumors that retain wild-type p53, pathway deregulation frequently occurs through the amplification of negative regulators of p53, including the E3 ubiquitin ligase MDM2. The p53/MDM2 interaction axis has served as basis for the development of several classes of MDM2 inhibitors, with AMG232 being the most potent molecule currently undergoing clinical evaluation. As the effects of MDM2 inhibition (MDM2i) remain poorly understood in high-grade glioma, we performed genomic and transcriptomic analyses in patient-derived models to better characterize sensitive tumors and identify putative biomarkers of drug response. Treatment with AMG232 impaired the growth of cell lines with wild-type p53 status, particularly in tumors with additional amplification of MDM4 or PPM1D activating mutations. Treatment with AMG232 upregulated both cell cycle arrest and apoptotic cellular responses, as measured by annexin V/PI staining and immunoblotting. Interestingly, the dynamics of these two downstream p53 signaling axis were dependent on treatment duration across models. In addition to p53 pathway activation and apoptotic induction, RNA-sequencing revealed MDM2i to be associated with the activation of oncogenic MAPK and KRAS signaling as well as epithelial to mesenchymal transition markers. In most solid tumors, resistance to MDM2i is mainly mediated by acquisition of p53 inactivating mutations. We hypothesized that resistance mechanisms in glioma may be partially driven by transcriptional changes, as these tumors consist of subpopulations with diverse cell differentiation states. By chronic AMG232 treatment, we have developed in vitro and in vivo models of acquired MDM2i resistance that are not mediated by p53 inactivation. Ongoing work is focused on characterizing the transcriptional profile of these cells to identify transcriptional changes leading to decreased drug response.

Small Molecule p53-MDM2 Inhibitors As Potential Regulators of p53-Dependent Genes Expression in Multiple Sclerosis

Transcription factor p53 plays an important role in numerous processes such as apoptosis, cell cycle regulation, metabolism, etc. This protein is believed to regulate apoptosis and neuronal death in various disorders such as multiple sclerosis (MS), Parkinson's and Alzheimer's diseases. In many cases p53 inactivation is caused by overexpression of its negative regulator - E3 ubiquitin ligase MDM2 - that exerts its regulatory functions on p53 by poly-ubiquinating it for proteasomal degradation. Under normal conditions p53 and MDM2 are constituted in an interdependent negative-feedback loop that controls their levels. There are multiple examples in the literature that describe inhibition of MDM2 using small molecules that activate p53 and induce p53-mediated molecular processes, primarily in tumor cells. However, currently the range of potential applications of MDM2 inhibitors is not restricted to cancer, but also includes autoimmune diseases. Therefore, suppression of MDM2 by small molecules is considered as one of the major strategies for activation of p53 and its transcriptional functions, further leading to expression of p53-dependent genes.To evaluate the effect of small molecule p53-MDM2 inhibitors (Nutlin-3a and RG-7112) we have isolated peripheral blood mononuclear cells (PBMCs) of MS patients and healthy donors. First, using colorimetric MTS assay we determined cytotoxicity of the compounds in PBMCs for 0.001-100 uM concentration range and incubation time of 24, 48, 72 hours. It was found that both compounds have only minor effect on cell viability in tested conditions.Finally, we employed Taqman real-time reverse transcription PCR (RT-PCR) to quantitatively analyze gene expression with the use of fluorescence-based detection system BioRad CFX-96 Touch. Freshly isolated cells were treated with both compounds at 5-20 uM concentrantion range. Total RNA was isolated and used to synthesize cDNA via reverse transcription reaction and then real-time PCR was carried out. Relative normalized expression was calculated by normalization to beta-actin, data analyzed using CFX Manager software. All PCR reactions were performed in triplicates. The effect of PBMC treatment was investigated on expression levels of key p53 target genes such as p21, Mdm2, PUMA, GADD45A, BAX, Bcl2 . We observed concentration-dependent expression of p21, Mdm2, Puma, GADD45A and Bax genes. Interestingly, Nutlin-3a demonstrated a more profound effect on these genes compared to RG-7112. Neither Nutlin-3a nor RG-7112 displayed any noticeable effect on the expression of Bcl2 . Importantly, we also noticed that gradual increase in Nutlin-3a concentration leads to higher expression levels of p53-dependent genes in MS patient samples compared to healthy donor samples. In conclusion, we expect that specific MDM2 inhibitors such as Nutlin-3a and RG-7112 can be used for regulation of p53-mediated processes in MS.Further experiments are currently ongoing to examine the effect of p53 activation on expression of various cytokines in stimulated and non-stimulated cells from both MS patients and healthy donors.The study was funded by RFBR research grant 16-34-60213 mol_а_dk. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SETD2 Non-Genomic Loss of Function and p53 Functional Inactivation in Advanced Systemic Mastocytosis (SM): Pathogenetic and Therapeutic Implications

KIT constitutive activation is thought to be necessary but not sufficient for the development of aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). The identification and dissection of cooperating pathogenetic pathways may set the basis for the development of novel therapeutic strategies.We have recently found that the HMC-1.1 and -1.2 MCL cell lines and ASM/MCL patients (pts) display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non-genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity, homologous recombination (HR), mismatch repair (MMR). Inhibition of proteasome-mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3. Reversing the detrimental effects of SETD2 and H36K36Me3 deficiency may be a promising therapeutic strategy.In this study, we aimed to investigate the effects of SETD2 loss of function in ASM and MCL and to assess the in vitro efficacy of second-generation proteasome inhibitors as therapeutic agents. To investigate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting and immunofluorescence to assess phosphorylated histone 2A.X (γH2AX), which marks ongoing DNA damage signaling and DNA double strand breaks and Rad51, which is a surrogate for active HR. Compared to cells from healthy controls, cells from SETD2- and H3K36Me3-deficient MCL pts had significantly higher levels of γH2AX and Rad51. To assess whether the increased DNA damage may translate into a greater number of submicroscopic genomic lesions, and whether the degree of SETD2 down-modulation may be inversely correlated with the degree of genomic complexity, SNP-array data from 13 pts were examined. Structural complexity score (SCS) and genome instability index (GII) were not significantly different (SCS, 387 vs 376; GII, 0.293 vs 0.337) between pts with > or <50% residual SETD2 and H3K36Me3 levels, suggesting that the activity of HR repair may counteract DNA damage events. Moreover, SETD2 down-modulation was not associated with reduced MMR proficiency, as assessed by lack of microsatellite instability. On the other hand, RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras and intron retention not observed in healthy donors.In renal cell carcinoma cell lines, SETD2 has been shown to bind and activate p53. After proteasome inhibition, SETD2 was indeed found to immunoprecipitate with p53 in HMC-1.1 and -1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with SP-141, a new class of MDM2 inhibitors that promote MDM2 auto-ubiquitination and degradation, also rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses for 24-48-72-96 hours induced cytostatic but not cytotoxic effects as shown by cell growth curves. Cytostatic effects resulted from a G2/M block of cell cycle induced by Gadd45a overexpression. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells (LD50=0.133µM and 0.050µM respectively).Finally, treatment with the second-generation proteasome inhibitors carfilzomib and ixazomib potently induced apoptosis and reduced colony growth in HMC-1.1 (LD50=0.10nM and 0.66nM respectively) and -1.2 (LD50=0.13nM and 0.72nM respectively) cells.In conclusion: MDM2-mediated ubiquitination contributes to SETD2 non-genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for TP53 gene mutations. Approaches aimed to inhibit MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies worth to be further explored.Supported by AIL and AIRC (16996) DisclosuresZanotti:Deciphera: Consultancy. Ciceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Valent:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Blueprint: Research Funding; Teva: Honoraria. Martinelli:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Roche: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy. Soverini:Incyte Biosciences: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

In Vitro andpre-Clinical In Vivo evidence Support MDMX/MDM2 As Common Vulnerabilities across TP53 -Wild-Type T-Cell Lymphomas That Are Targetable with the α-Helical p53 Stapled Peptide Alrn-6924

T-cell lymphomas (TCL) are a heterogeneous group of non-Hodgkin lymphomas with poor outcomes. Biological heterogeneity across TCLs has hampered the development of targeted therapies. Thus, we sought to identify common vulnerabilities that could be targeted across multiple TCL subtypes. We performed transcriptome and exome sequencing of 21 TCL cell lines and 8 TCL patient-derived xenografts (PDX), and a genome-wide CRISPR-Cas9 pooled screening in 7 TCL lines. Sequencing identified multiple genomic alterations of clinical significance and disease-specific recurrence, like activating JAK1 and STAT3 mutations in cell lines and a PDX-model of anaplastic large cell lymphoma (ALCL) or mutations of TET2, DNMT3A and RHOA in PDX-models of angioimmunoblastic TCL (AITL). Unsupervised hierarchical clustering of transcriptomes identified disease-specific gene-expression profiles across cell lines and PDX models. Ranking the genetic vulnerabilities of the CRISPR-Cas9 screen by z-score and recurrence across multiple cell lines, we identified both previously defined (e.g. IRF4) and novel vulnerabilities (e.g. PTPN2) within multiple TCL subtypes. The most consistent dependence in TP53- wild-type (WT) lines was the E3 ubiquitin ligase MDM2, which polyubiquitinates and targets p53 for degradation. Multiple TCL lines harbored copy number gains or amplifications of MDM2 and/or MDMX that correlated with high transcript levels. To address the therapeutic potential for targeting MDMX/MDM2 in TCL we tested ALRN-6924, a p53-derived stapled peptide that inhibits both MDMX and MDM2, and that is currently in multicenter phase I/Ib and phase II trials. ALRN-6924 was recently shown to induce objective responses in a variety of cancers as a single agent in phase 1 dose-escalation (Meric-Bernstam, F. et al, ASCO 2017). Our results showed that ALRN-6924 potently induced G1 cell cycle arrest and apoptotic cell death in WT TP53 TCL lines (n=8; IC50 100 nm-1 µM) while inducing expression of p21 and Annexin V. ALRN-6924 was inactive in TP53 -mutant lines (n=11; IC50 > 5 µM). Next, we performed a phase II-like trial in eight WT TP53 PDXs: hepatosplenic (HS-TCL), cutaneous (CTCL), NK/T, AITL (n=2), T-cell prolymphocytic leukemia (TPLL) and Alk+ ALCL (n=2). At the time of extensive lymphoma engraftment (e.g. >20% splenic involvement), mice were randomized to treatment with vehicle, ALRN-6924 or the 2nd line standard-of-care agent Romidepsin (n=3/arm/model) on days 1, 4 and 7, and sacrificed on day 8. ALRN-6924 had a favorable safety profile and significantly reduced tumor burden compared to vehicle in all 8 models, with superior efficacy to Romidepsin in the majority of models (Figure). Residual tumor cells collected from ALRN-6924-treated mice consistently had increased AAD/Annexin V staining and p21 induction, consistent with on-target p53 activation and apoptosis.In summary, we identified MDM2/MDMX as a targetable vulnerability that is effectively targeted by the stapled peptide ALRN-6924 in TP53 WT TCLs with promising activity across multiple subtypes, supporting further clinical development of ALRN-6924 in TCL for patients requiring 2nd or frontline treatment. A Phase IIa clinical trial of ALRN-6924 in PTCL patients is currently enrolling (NCT02264613). [Display omitted] DisclosuresHorwitz:Millenium/Takeda: Consultancy, Research Funding; HUYA: Consultancy; Forty-Seven: Consultancy, Research Funding; BMS: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Mundipharma: Consultancy; ADCT Therapeutics: Research Funding; Aileron Therapeutics: Research Funding; Infinity/Verastem: Consultancy, Research Funding. Santiago:Aileron: Employment. Ren:Aileron: Employment. Guerlavais:Aileron: Employment. Annis:Aileron: Employment. Aivado:Aileron: Employment.

The Role of p53 Signaling in Colorectal Cancer.

Simple SummaryThe transcription factor p53 is a crucial tumor suppressor that regulates diverse cellular responses to protect against cancer development. Deactivating p53 signaling either by altering p53 regulators or by p53 mutations occurs frequently in human colorectal carcinoma (CRC). Forty-three percent of CRCs harbor p53 mutations that reduce wild-type p53 tumor suppressor activity and often provide neo-morphic functions, which contribute to tumorigenesis. In this review, we summarize wild-type p53 signaling, how it can be deregulated in CRC, and the functional and phenotypical effects of p53 mutations. We also discuss current therapeutic strategies of targeting p53.The transcription factor p53 functions as a critical tumor suppressor by orchestrating a plethora of cellular responses such as DNA repair, cell cycle arrest, cellular senescence, cell death, cell differentiation, and metabolism. In unstressed cells, p53 levels are kept low due to its polyubiquitination by the E3 ubiquitin ligase MDM2. In response to various stress signals, including DNA damage and aberrant growth signals, the interaction between p53 and MDM2 is blocked and p53 becomes stabilized, allowing p53 to regulate a diverse set of cellular responses mainly through the transactivation of its target genes. The outcome of p53 activation is controlled by its dynamics, its interactions with other proteins, and post-translational modifications. Due to its involvement in several tumor-suppressing pathways, p53 function is frequently impaired in human cancers. In colorectal cancer (CRC), the TP53 gene is mutated in 43% of tumors, and the remaining tumors often have compromised p53 functioning because of alterations in the genes encoding proteins involved in p53 regulation, such as ATM (13%) or DNA-PKcs (11%). TP53 mutations in CRC are usually missense mutations that impair wild-type p53 function (loss-of-function) and that even might provide neo-morphic (gain-of-function) activities such as promoting cancer cell stemness, cell proliferation, invasion, and metastasis, thereby promoting cancer progression. Although the first compounds targeting p53 are in clinical trials, a better understanding of wild-type and mutant p53 functions will likely pave the way for novel CRC therapies.

Circadian clock protein CRY1 prevents paclitaxel‑induced senescence of bladder cancer cells by promoting p53 degradation.

Bladder cancer is a common tumor type of the urinary system, which has high levels of morbidity and mortality. The first-line treatment is cisplatin-based combination chemotherapy, but a significant proportion of patients relapse due to the development of drug resistance. Therapy-induced senescence can act as a ‘back-up’ response to chemotherapy in cancer types that are resistant to apoptosis-based anticancer therapies. The circadian clock serves an important role in drug resistance and cellular senescence. The aim of the present study was to investigate the regulatory effect of the circadian clock on paclitaxel (PTX)-induced senescence in cisplatin-resistant bladder cancer cells. Cisplatin-resistant bladder cancer cells were established via long-term cisplatin incubation. PTX induced apparent senescence in bladder cancer cells as demonstrated via SA-β-Gal staining, but this was not observed in the cisplatin-resistant cells. The cisplatin-resistant cells entered into a quiescent state with prolonged circadian rhythm under acute PTX stress. It was identified that the circadian protein cryptochrome1 (CRY1) accumulated in these quiescent cisplatin-resistant cells, and that CRY1 knockdown restored PTX-induced senescence. Mechanistically, CRY1 promoted p53 degradation via increasing the binding of p53 with its ubiquitin E3 ligase MDM2 proto-oncogene. These data suggested that the accumulated CRY1 in cisplatin-resistant cells could prevent PTX-induced senescence by promoting p53 degradation.

PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2Δ/Δ) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1+/-Ezh2Δ/Δ) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1+/-Ezh2Δ/Δ mice. Cdkn2a, a major PRC2 target encoding p19Arf, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1+/-Ezh2Δ/Δ erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1+/-Ezh2Δ/Δ mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

Neddylation modification of ribosomal protein RPS27L or RPS27 by MDM2 or NEDP1 regulates cancer cell survival.

Neddylation plays a distinct role in stabilization of a subset of ribosomal proteins. Whether the family of ribosomal proteins S27 (RPS27 and RPS27-like) is subjected to neddylation regulation with associated biological consequence is totally unknown. Here, we report that both family members are subjected to neddylation by MDM2 E3 ubiquitin ligase, and deneddylation by NEDP1. Blockage of neddylation with MLN4924, a small molecule inhibitor of neddylation-activating enzyme, destabilizes RPS27L and RPS27 by shortening their protein half-lives. Biologically, knockdown of RPS27L and RPS27 sensitizes, whereas ectopic expression of RPS27L and RPS27 desensitizes cancer cells to MLN4924-induced apoptosis. Taken together, our study demonstrates that neddylation stabilizes RPS27L and RPS27 to confer the survival of cancer cells.

Tau affects P53 function and cell fate during the DNA damage response

Cells are constantly exposed to DNA damaging insults. To protect the organism, cells developed a complex molecular response coordinated by P53, the master regulator of DNA repair, cell division and cell fate. DNA damage accumulation and abnormal cell fate decision may represent a pathomechanism shared by aging-associated disorders such as cancer and neurodegeneration. Here, we examined this hypothesis in the context of tauopathies, a neurodegenerative disorder group characterized by Tau protein deposition. For this, the response to an acute DNA damage was studied in neuroblastoma cells with depleted Tau, as a model of loss-of-function. Under these conditions, altered P53 stability and activity result in reduced cell death and increased cell senescence. This newly discovered function of Tau involves abnormal modification of P53 and its E3 ubiquitin ligase MDM2. Considering the medical need with vast social implications caused by neurodegeneration and cancer, our study may reform our approach to disease-modifying therapies.

MAGEA9: Driver or Passenger in CARcinogenesis

Type I Melanoma Antigen Genes (MAGEs) are a family of genes that are normally expressed in the spermatogonial cells of the testis and abnormally reactivated in a broad variety of cancers. Due to this expression pattern and the immune response they elicit when abnormally expressed, type I MAGEs are also known as Cancer Testis Antigens (CTAs). Melanoma Antigen Genes‐A (MAGE‐A) are CTAs whose expression in cancers have been correlated with poor patient prognosis. The biological function of several members of the MAGE‐A gene family in cancer remains unclear. Ectopic expression of type I MAGEA9 in normal cells results in increased cellular proliferation. We therefore hypothesize that MAGEA9 is needed for sustenance of proliferative phenotypes. Our goals are to elucidate the molecular mechanism by which MAGEA9 increases cellular proliferation and the mechanism(s) that regulate the expression of MAGEA9. We have employed RT‐qPCR array for cancer‐associated genes to determine signaling pathways modulated by expression of MAGEA9. Results from our RT‐qPCR array indicate that MAGEA9 expression causes an upregulation in the DNA mismatch repair genes MSH6 and MSH2 and the E3 ubiquitin ligase MDM2. To identify MAGEA9 binding partners we have used IP‐mass spectrometry, and our data indicate that MAGE‐A9 binding partners are involved in invasion. We will use a combination of in silico promoter analyses and chromatin immunoprecipitation (ChIP) to determine the transcription factor (s) regulating expression of MAGEA9. Collectively, these data provide a clearer understanding for the function of MAGE‐A9 in cancer. Elucidating the role of MAGEs in cancer may provide insight for the development of novel therapeutic interventions.Support or Funding InformationNSF Research Initiation Award to S.R. #HRD1764201Department of Education Award # P382G170104 (TITLE‐VII)

The interaction between p53 and Mdm2 is independent of MEG3–p53 association

The ability of the long noncoding RNA MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has previously been shown to bind to p53 in vitro, which led us to hypothesize that MEG3 functions by disrupting the interaction of p53 and its E3 ubiquitin ligase Mdm2. To test this hypothesis in vivo, we built a cross‐nearest neighbor/Monte Carlo analytical method based on two color direct stochastic optical reconstruction microscopy (dSTORM), a single‐molecule localization microscopy (SMLM) technique. Our data support the interaction of MEG3 and p53. Surprisingly, this association had no effect on the binding of p53 and Mdm2, distinct from the most commonly proposed model for the mechanism of MEG3 action. Additionally, our mathematical approach to analyzing SMLM data has general applicability to assessing molecular interactions in a native cellular context.Support or Funding InformationRJS: NIH R01CA193520, NIH R01DK062472, NIH S10RR027931; NCB: NIH T32DK007540; AK and YZ: NIH R01CA193520; and The Jarislowsky Foundation

Mdm2 enhances ligase activity of parkin and facilitates mitophagy

Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.

E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

The respiratory lineage initiates from the specification of NKX2-1+ progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2, which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size.

Regulation of the MDM2-p53 pathway by the ubiquitin ligase HERC2.

The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53‐response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53‐MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.