Murine Double Minute Research Articles

Pre-clinical modeling of navtemadlin pharmacokinetics (PK), pharmacodynamics (PD), and efficacy in glioblastoma, IDH-wildtype.

2012 Background: Navtemadlin (nvtm) is a potent, selective, orally available small molecule inhibitor of murine double minute 2 homologue (MDM2), which has completed a Ph 0 surgical window of opportunity study in glioblastoma, IDH-wildtype (GBM; Lee SNO 2022). To optimally interpret the clinical data, an extensive evaluation of nvtm PK, PD, and efficacy was performed in GBM patient derived xenografts (PDXs). Methods: Response to nvtm +/- radiotherapy (RT) was characterized in vitro and in vivo across a panel of GBM PDXs with various TP53 and MDM2 alterations. Efficacy, PK, and PD were evaluated in flank or orthotopic PDX models. p53 pathway activation was evaluated by qPCR, Western blot, and IHC. Nvtm brain penetration was evaluated in immunodeficient abcb1a/b-/- (P-glycoprotein; P-gp), abcg2-/- (BCRP), or double-knockout mice. Binding was assessed by rapid equilibrium dialysis, and nvtm concentrations by LC/MS-MS and MALDI-MSI. Results: Nvtm decreased in vitro viability of TP53WT GBM PDX explant cultures with IC50s <100 nM, but was ineffective in TP53-mutant GBM. In flank PDX models, nvtm (100 mg/kg daily for 4 weeks) delayed tumor regrowth by 1.9 to 4.8-fold in TP53 WT PDXs without MDM2amplification (n=9 models), and more markedly by 20.1 to 29.4-fold in MDM2amplified models (n=4; 2 PDXs did not recur). In a flank PDX dose-response study, nvtm delayed the growth of MDM2amplified GBM108 at 25 mg/kg vs. placebo (61 vs. 11 days; p=0.014; mean intra-tumoral nvtm = 590 nM). In contrast, growth of MDM2 non-amplified GBM14 was delayed only at 100 mg/kg (37 vs. 15 days; p=0.082; mean intra-tumoral nvtm = 2990 nM). In both flank models, p53 target gene upregulation was similar between 25 and 100 mg/kg and did not correlate with regrowth. Nvtm was highly bound (fraction unbound = 0.090 in culture media and 0.014 in GBM tissue homogenate). In mice after oral dosing, distribution to CNS was limited by P-gp, with a brain to plasma ratio of 0.009 and unbound ratio of 0.005, whereas BCRP deficiency did not affect CNS distribution. With orthotopic GBM108, nvtm efficacy was evaluated in nude vs. efflux-deficient mice. In nude mice, nvtm was ineffective at doses up to 100 mg/kg; however, in efflux deficient mice, the mean intra-tumoral nvtm level at 25 mg/kg was similar to flank tumors (690 nM) and survival was significantly extended (134 vs. 62 days, p=0.0002) with additional benefit at 100 mg/kg (>300 vs. 62 days, p=0.002). Conclusions: Nvtm showed significant efficacy in TP53 WT GBM flank PDX models but efficacy in orthotopic PDX was highly dependent on CNS penetration. Targeting nvtm levels based on the in vitroIC50 may significantly underestimate the in vivo effective concentration . In vivo studies showed lower intra-tumoral nvtm levels are required to significantly inhibit growth of MDM2amplified compared with non-amplified tumors.

MDM2 accelerated renal senescence via ubiquitination and degradation of HDAC1.

Senescence, an intricate and inevitable biological process, characterized by the gradual loss of homeostasis and declining organ functions. The pathological features of cellular senescence, including cell cycle arrest, metabolic disruptions, and the emergence of senescence-associated secretory phenotypes (SASP), collectively contribute to the intricate and multifaceted nature of senescence. Beyond its classical interaction with p53, murine double minute gene 2 (MDM2), traditionally known as an E3 ubiquitin ligase involved in protein degradation, plays a pivotal role in cellular processes governing senescence. Histone deacetylase (HDAC), a class of histone deacetylases mainly expressed in the nucleus, has emerged as a critical contributor to renal tissues senescence. In this study we investigated the interplay between MDM2 and HDAC1 in renal senescence. We established a natural aging model in mice over a 2-year period that was verified by SA-β-GAL staining and increased expression of senescence-associated markers such as p21, p16, and TNF-α in the kidneys. Furthermore, we showed that the expression of MDM2 was markedly increased, while HDAC1 expression underwent downregulation during renal senescence. This phenomenon was confirmed in H2O2-stimulated HK2 cells in vitro. Knockout of renal tubular MDM2 alleviated renal senescence in aged mice and in H2O2-stimulated HK2 cells. Moreover, we demonstrated that MDM2 promoted renal senescence by orchestrating the ubiquitination and subsequent degradation of HDAC1. These mechanisms synergistically accelerate the aging process in renal tissues, highlighting the intricate interplay between MDM2 and HDAC1, underpinning the age-related organ function decline.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract CT066: Phase 1 study of MDM2 antagonist ASTX295 in patients with solid tumors with wild-type TP53

Abstract Background: ASTX295 is a novel small molecule antagonist of human Murine Double Minute 2 (MDM2), which tightly regulates the level and activity of the p53 tumor suppressor. To minimize potential thrombocytopenia previously seen with antagonism of this target, ASTX295 was designed to have a short half-life (t1/2) with decreased bone marrow (BM) exposure. ASTX295 has proapoptotic and tumor growth inhibitory activity in preclinical cancer (CA) models. We report here the safety and preliminary efficacy of the phase 1 and dose expansion portions of Study ASTX295-01. Methods: ASTX295-01 is a first-in-human, open-label, multicenter, phase 1/2 study to assess safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity of ASTX295 (NCT03975387). Eligible patients (advanced solid tumors with Wild-Type [WT] TP53, ECOG PS 0-2, adequate organ and bone marrow function, etc.) had oral administration of ASTX295 starting at a daily dose of 15 mg. Dose escalation was by standard 3 + 3 with cohorts testing multiple regimens. Safety was assessed by CTCAE and disease was assessed by RECIST 1.1. Safety and efficacy and dosing recommendations were overseen by the Data Safety Review Committee. Results:83 subjects were enrolled in over 14 cohorts, evaluating daily (QD) and intermittent dosing regimens and the effect of food. The mean age of subjects on study was 60.1 year, 56.9 were male, with 2.94 (0-15) prior chemotherapy regimens. For PK exposure, the total weekly AUC for 400 mg QD and 660 twice a week (BIW) are similar. The median tmax is 3 hours and mean t1/2 is 4-6 hours. PD demonstrates p53 pathway modulation. Nausea, vomiting, diarrhea, and fatigue were dose limiting. RP2D regimens were 660 mg BIW and 400 mg QD and these regimens were expanded with a total of 25 additional subjects. The most common treatment related AEs for both regimens were nausea, diarrhea, and vomiting at 76.2%,76.2%, and 33.3% respectively, for daily and 71.4%, 47.6%, and 33.3%, for intermittent. Grade 3 GI AEs were reported in 14.3% of subjects in the daily cohort vs 9.5% in the intermittent; none had Grade ≥ 4. One subject had grade 2 thrombocytopenia. Based on decreased incidence and duration of GI AEs, 660 mg BIW was selected as the RP2D.Multiple objective responses were seen, including 1 subject with non-small cell lung CA and 3 subjects with liposarcoma (LPS), a tumor with frequent MDM2 amplification. For LPS the ORR is 7.9% (de-differentiated 7.7%, well differentiated 8.0%), with a PFS of 7.95 and 9.66 months respectively, along with a 16-week disease control rate of 69.2% and 72.0%. Five subjects with GBM were enrolled, 4 with MDM2 amplification, 3 of whom showed evidence of tumor regression, with one subject remaining on study for > 34 cycles. Conclusion:ASTX295 was well tolerated at doses producing p53 pathway modulation with manageable toxicities, notably avoiding significant thrombocytopenia. Preliminary single agent efficacy was observed in heavily pretreated subjects across multiple solid tumor types with WT TP53. Citation Format: Ecaterina E. Dumbrava, Fabio M. Iwamoto, Mark Agulnik, Mohammed Milhem, Anthony W. Tolcher, Rashmi Chugh, Michael J. Demeure, Alain Mita, Kurt Demel, Mark Diamond, Beloo Mirakhur, Laksmi Wilson, Aram Oganesian, Danna Chan, Harold N. Keer, Jason Taylor, Martin J. Sims, Andrea Biondo, Simone Jueliger, Alexander Spira. Phase 1 study of MDM2 antagonist ASTX295 in patients with solid tumors with wild-type TP53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT066.

Design, Synthesis, and In vitro Anti-cervical Cancer Activity of a Novel MDM2-p53 Inhibitor Based on a Chalcone Scaffold.

Several novel fluorinated chalcone derivatives were synthesized, and their in vitro anticervical cancer activity and mechanism of action were investigated using the parent nucleus of licorice chalcone as the lead compound backbone and MDM2-p53 as the target. In this study, 16 novel chalcone derivatives (3a-3r) were designed and synthesized by molecular docking technology based on the licorice chalcone parent nucleus as the lead compound scaffold and the cancer apoptosis regulatory target MDM2-p53. The structures of these compounds were confirmed by 1H-NMR, 13C-NMR, and HR-ESI-MS. The inhibitory effects of the compounds on the proliferation of three human cervical cancer cell lines (SiHa, HeLa, and C-33A) and two normal cell lines (H8 and HaCaT) were determined by MTT assay, and the initialstructure-activity relationship was analyzed. Transwell and flow cytometry were used to evaluate the effects of target compounds on the inhibition of cancer cell migration and invasion, apoptosis induction, and cell cycle arrest. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were used to detect the effects of candidate compounds on mRNA, p53, and Murine double minute 2 (MDM2) protein expression. The binding characteristics of the target compounds to the MDM2 protein target in the p53-MDM2 pathway were evaluated by molecular docking technology. The target compounds had considerable inhibitory activity on the proliferation of three cervical cancer cell lines. Among them, compound 3k (E)-3-(4-(dimethylamino)phenyl)-2-methyl-1-(3-(trifluoromethyl)phenyl) prop-2-en-1-one) showed the highest activity against HeLa cells (IC50=1.08 μmol/L), which was better than that of the lead compound Licochalcone B, and 3k showed lower toxicity to both normal cells. Compound 3k strongly inhibited the migration and invasion of HeLa cells and induced apoptosis and cell cycle arrest at the G0/G1 phase. Furthermore, compound 3k upregulated the expression of p53 and BAX and downregulated the expression of MDM2, MDMX, and BCL2. Moreover, molecular docking results showed that compound 3k could effectively bind to the MDM2 protein (binding energy: -9.0 kcal/mol). These results suggest that the compounds may activate the p53 signaling pathway by inhibiting MDM2 protein, which prevents cancer cell proliferation, migration, and invasion and induces apoptosis and cell cycle arrest in cancer cells. This study provides a new effective and low-toxicity drug candidate from licochalcone derivatives for treating cervical cancer.

Cardiac intima sarcoma: a rapid review of case reports

Background and purpose: Cardiac intimal sarcoma (CIS) is a rare and often misdiagnosed clinical entity with notably high morbidity and mortality rates. Urgent efforts are required to comprehensively the disease and establish evidence-based management guidelines. Given the relative scarcity of data and the imperative for consensus on management strategies, this review assumes critical importance. Through a rapid review approach, we aims to synthesize and consolidate information gleaned from published case reports, thereby offering an up-to-date resource for CIS’s natural history. Methods: Utilizing inductive content analysis, we systematically searched for and synthesized case reports and series. Keywords related to CIS guided our search on Medline via Ovid, Scopus, and Web of Science from inception until March 17, 2024. Results: Our review identified 79 CIS, characterized by a mean age of 52 years, predominantly male (58%), with left-sided lesions. Approximately one-third of the patients presented with cardiovascular symptoms, and transthoracic echocardiography emerged as the preferred initial diagnostic modality in most cases. Fluorescence in situ hybridization (FISH) analysis on pre-operative tissue biopsies found murine double minute 2 (MDM2) amplification in 95% of cases tested. While surgery remained the cornerstone of treatment, recurrence occurred in 14% of cases postoperatively, with 28% succumbing to the disease despite therapeutic interventions. Conclusions: This synthesis of published case reports and series provides updated insights into the clinical profile CIS. Our findings highlight the need for increased clinical awareness, precise data collection, and further research to enhance diagnostic accuracy, treatment approaches, and patient outcomes in CIS.

Revolutionizing pediatric neuroblastoma treatment: unraveling new molecular targets for precision interventions.

Neuroblastoma (NB) is the most frequent solid tumor in pediatric cases, contributing to around 15% of childhood cancer-related deaths. The wide-ranging genetic, morphological, and clinical diversity within NB complicates the success of current treatment methods. Acquiring an in-depth understanding of genetic alterations implicated in the development of NB is essential for creating safer and more efficient therapies for this severe condition. Several molecular signatures are being studied as potential targets for developing new treatments for NB patients. In this article, we have examined the molecular factors and genetic irregularities, including those within insulin gene enhancer binding protein 1 (ISL1), dihydropyrimidinase-like 3 (DPYSL3), receptor tyrosine kinase-like orphan receptor 1 (ROR1) and murine double minute 2-tumor protein 53 (MDM2-P53) that play an essential role in the development of NB. A thorough summary of the molecular targeted treatments currently being studied in pre-clinical and clinical trials has been described. Recent studies of immunotherapeutic agents used in NB are also studied in this article. Moreover, we explore potential future directions to discover new targets and treatments to enhance existing therapies and ultimately improve treatment outcomes and survival rates for NB patients.

Abstract 644: PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines

Abstract Ovarian cancer is the most lethal malignant disease of female. Treatment options for ovarian cancer are mostly the use of chemotherapeutics, surgery, or radiotherapy. However, due to resistance to drugs, relapse occurs and thus 5 years survival rate is only 47%. The tumor suppressor protein p53 works as a transcription factor to transcribe several proteins such as p21, p16 and PUMA that lead to halt cell cycle and also induce apoptosis when there is a damage in DNA. Mutation in several regions of p53 (mutp53) is quite common in cancers and can lead to the accumulation of p53 with loss of functions or sometimes gain of oncogenic functions. Thus there is a search for drugs that can degrade the mut53 and can restore the functions of normal p53. In this research, it was observed that the treatment of ovarian cancer cell lines (ES-2, HEY and OVCAR3) with atypical protein kinase C-iota (aPKCɩ) specific inhibitors ICA-1S can cause the degradation of mutp53 (53.6%, N=6 trials). Treatment with ICA-1S also lowered the level of phosphorylated p53 (ser15, 87%, N=3 trials). However, the level of MDM2 protein (murine double minute 2), a known p53 E3 ligase was not significantly (98%, N=2 trials) affected by ICA-1S in ES-2 cell line with mut p53 as well as p21 was also not affected. However level of PUMA was decreased both in ES-2 and HEY cell lines. Immunoprecipitation (IP) with aPKCɩ specific beads showed the aPKCɩ is associated with p53, though reverse IP with p53 beads was not able to show the association. Application of proteasome inhibitor MG-132 at a single dose of 0.5µM could not completely inhibit the degradation of mut53 and WTp53 in ICA-1S treated ES-2 cell line and HEY cell line, respectively, indicating the drug may be affecting the transcription of p53 as well. It is therefore postulated that aPKCɩ can phosphorylate p53, stabilizing it and may be affecting the transcription of p53 as well in those cell lines. Further research (kinase activity assay, immunoprecipitation, immunofluorescence) is going on to reveal the mode of this degradation. Citation Format: Mahfuza Marzan, Nuzhat Nowshin Oishee, Abigail Olatunji, Mildred Acevedo-Duncan. PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines [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 644.

Low-grade central osteosarcoma with extraosseous dedifferentiation: a rare case.

Low-grade central osteosarcoma (LGCOS), which arises from the intramedullary cavity of the metaphysis of long bones, occasionally exhibits extraosseous spread. Approximately 10-30% of patients with LGCOS exhibit dedifferentiation, but it is rare to experience a primary tumor with a dedifferentiated component. A 38-year-old female patient presented with right knee pain for two months. Imaging studies revealed a bone mass with extraosseous involvement. Wide resection was performed, and pathologic examination led to the diagnosis of LGCOS with a dedifferentiated extraosseous lesion. A single defect in the bone cortex constituted the boundary between the low- and high-grade components. The extraosseous high-grade component included more tumor cells with p53 overexpression and more murine double minute 2 (MDM2) copies compared with the low-grade component. These genetic mutations and copy number alterations can be associated with malignant transformation of LGCOS.

Impacts of Nutlin-3a and exercise on murine double minute 2-enriched glioma treatment.

JOURNAL/nrgr/04.03/01300535-202504000-00029/figure1/v/2024-07-06T104127Z/r/image-tiff Recent research has demonstrated the impact of physical activity on the prognosis of glioma patients, with evidence suggesting exercise may reduce mortality risks and aid neural regeneration. The role of the small ubiquitin-like modifier (SUMO) protein, especially post-exercise, in cancer progression, is gaining attention, as are the potential anti-cancer effects of SUMOylation. We used machine learning to create the exercise and SUMO-related gene signature (ESLRS). This signature shows how physical activity might help improve the outlook for low-grade glioma and other cancers. We demonstrated the prognostic and immunotherapeutic significance of ESLRS markers, specifically highlighting how murine double minute 2 (MDM2), a component of the ESLRS, can be targeted by nutlin-3. This underscores the intricate relationship between natural compounds such as nutlin-3 and immune regulation. Using comprehensive CRISPR screening, we validated the effects of specific ESLRS genes on low-grade glioma progression. We also revealed insights into the effectiveness of Nutlin-3a as a potent MDM2 inhibitor through molecular docking and dynamic simulation. Nutlin-3a inhibited glioma cell proliferation and activated the p53 pathway. Its efficacy decreased with MDM2 overexpression, and this was reversed by Nutlin-3a or exercise. Experiments using a low-grade glioma mouse model highlighted the effect of physical activity on oxidative stress and molecular pathway regulation. Notably, both physical exercise and Nutlin-3a administration improved physical function in mice bearing tumors derived from MDM2-overexpressing cells. These results suggest the potential for Nutlin-3a, an MDM2 inhibitor, with physical exercise as a therapeutic approach for glioma management. Our research also supports the use of natural products for therapy and sheds light on the interaction of exercise, natural products, and immune regulation in cancer treatment.

Association of MDM2 Overexpression in Ameloblastomas with MDM2 Amplification and BRAFV600E Expression.

Ameloblastoma is a rare tumor but represents the most common odontogenic neoplasm. It is localized in the jaws and, although it is a benign, slow-growing tumor, it has an aggressive local behavior and high recurrence rate. Therefore, alternative treatment options or complementary to surgery have been evaluated, with the most promising one among them being a targeted therapy with the v-Raf murine sarcoma viral oncogene homologue B (BRAF), as in ameloblastoma the activating mutation V600E in BRAF is common. Studies in other tumors have shown that the synchronous inhibition of BRAF and human murine double minute 2 homologue (MDM2 or HDM2) protein is more effective than BRAF monotherapy, particularly in the presence of wild type p53 (WTp53). To investigate the MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAFV600E and p53 expression. Forty-four cases of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin were examined for MDM2 overexpression and BRAFV600E and p53 expression by immunohistochemistry, and for MDM2 ploidy with fluorescence in situ hybridization. Sixteen of forty-four (36.36%) cases of ameloblastoma showed MDM2 overexpression. Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAFV600E positive and fifteen of sixteen MDM2-positive ameloblastomas (93.75%) were p53 negative. All MDM2 overexpressing tumors did not show copy number alterations for MDM2. Overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression. Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAFV600E-positive/WTp53 ameloblastomas.

Discovery of Novel Antitumor Small-Molecule Agent with Dual Action of CDK2/p-RB and MDM2/p53.

Cell cycle-dependent kinase 2 (CDK2) is located downstream of CDK4/6 in the cell cycle and regulates cell entry into S-phase by binding to Cyclin E and hyper-phosphorylating Rb. Proto-oncogene murine double minute 2 (MDM2) is a key negative regulator of p53, which is highly expressed in tumors and plays an important role in tumorigenesis and progression. In this study, we identified a dual inhibitor of CDK2 and MDM2, III-13, which had good selectivity for inhibiting CDK2 activity and significantly reduced MDM2 expression. In vitro results showed that III-13 inhibited proliferation of a wide range of tumor cells, regardless of whether Cyclin E1 (CCNE1) was overexpressed or not. The results of in vivo experiments showed that III-13 significantly inhibited proliferation of tumor cells and did not affect body weight of mice. The results of the druggability evaluation showed that III-13 was characterized by low bioavailability and poor membrane permeability when orally administered, suggesting the necessity of further structural modifications. Therefore, this study provided a lead compound for antitumor drugs, especially those against CCNE1-amplified tumor proliferation.

Design, synthesis and evaluation of the first DYRK1A degrader for promoting the proliferation of pancreatic β-cells

Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is the most promising target for diabetes treatment by promoting β-cell proliferation. The desmethylbellidifolin (DMB) as a DYRK1A inhibitor could facilitate β-cell proliferation in vivo and in vitro. However, DMB has the problem of weak binding affinity to DYRK1A, which means that continuous high concentration administration of DMB is effective for the diabetes. In order to solve this problem, we designed and synthesized a series of DMB-based proteolysis targeting chimeras (PROTACs) by taking advantage of the property of PROTAC that induce protein degradation in a cycle-catalytic manner. MDM2-based PROTAC X1–4P-MDM2 was identified as the most active PROTAC molecule. Mechanism research showed that X1–4P-MDM2 formed a ternary complex with DYRK1A and murine double minute 2 (MDM2), and induced the degradation of DYRK1A through the ubiquitin-proteasome system pathway. At a dose much lower than that of DMB, X1–4P-MDM2 still significantly enhanced β-cell proliferation by inhibiting transforming growth factor beta (TGF-β) and promoting the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which may provide a new strategy for the application of DMB in diabetes.

Ubiquitin ligase MDM2 mediates endothelial inflammation in Kawasaki disease vasculitis development.

Kawasaki disease (KD) often complicates coronary artery lesions (CALs). Despite the established significance of STAT3 signaling during the acute phase of KD and signal transducer and activator of transcription 3 (STAT3) signaling being closely related to CALs, it remains unknown whether and how STAT3 was regulated by ubiquitination during KD pathogenesis. Bioinformatics and immunoprecipitation assays were conducted, and an E3 ligase, murine double minute 2 (MDM2) was identified as the ubiquitin ligase of STAT3. The blood samples from KD patients before and after intravenous immunoglobulin (IVIG) treatment were utilized to analyze the expression level of MDM2. Human coronary artery endothelial cells (HCAECs) and a mouse model were used to study the mechanisms of MDM2-STAT3 signaling during KD pathogenesis. The MDM2 expression level decreased while the STAT3 level and vascular endothelial growth factor A (VEGFA) level increased in KD patients with CALs and the KD mouse model. Mechanistically, MDM2 colocalized with STAT3 in HCAECs and the coronary vessels of the KD mouse model. Knocking down MDM2 caused an increased level of STAT3 protein in HCAECs, whereas MDM2 overexpression upregulated the ubiquitination level of STAT3 protein, hence leading to significantly decreased turnover of STAT3 and VEGFA. MDM2 functions as a negative regulator of STAT3 signaling by promoting its ubiquitination during KD pathogenesis, thus providing a potential intervention target for KD therapy.

Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry.

Viral infection is a significant risk factor for fertility issues. Here, we demonstrated that infection by neurotropic alphaherpesviruses, such as pseudorabies virus (PRV), could impair female fertility by disrupting the hypothalamus-pituitary-ovary axis (HPOA), reducing progesterone (P4) levels, and consequently lowering pregnancy rates. Our study revealed that PRV exploited the transient receptor potential mucolipin 1 (TRPML1) and its lipid activator, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), to facilitate viral entry through lysosomal cholesterol and Ca2+. P4 antagonized this process by inducing lysosomal storage disorders and promoting the proteasomal degradation of TRPML1 via murine double minute 2 (MDM2)-mediated polyubiquitination. Overall, the study identifies a novel mechanism by which PRV hijacks the lysosomal pathway to evade P4-mediated antiviral defense and impair female fertility. This mechanism may be common among alphaherpesviruses and could contribute significantly to their impact on female reproductive health, providing new insights for the development of antiviral therapies.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

EF1α-associated protein complexes affect dendritic spine plasticity by regulating microglial phagocytosis in Fmr1 knock-out mice.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. There is no specific treatment for FXS due to the lack of therapeutic targets. We report here that Elongation Factor 1α (EF1α) forms a complex with two other proteins: Tripartite motif-containing protein 3 (TRIM3) and Murine double minute (Mdm2). Both EF1α-Mdm2 and EF1α-TRIM3 protein complexes are increased in the brain of Fmr1 knockout mice as a result of FMRP deficiency, which releases the normal translational suppression of EF1α mRNA and increases EF1α protein levels. Increased EF1α-Mdm2 complex decreases PSD-95 ubiquitination (Ub-PSD-95) and Ub-PSD-95-C1q interaction. The elevated level of TRIM3-EF1α complex is associated with decreased TRIM3-Complement Component 3 (C3) complex that inhibits the activation of C3. Both protein complexes thereby contribute to a reduction in microglia-mediated phagocytosis and dendritic spine pruning. Finally, we created a peptide that disrupts both protein complexes and restores dendritic spine plasticity and behavioural deficits in Fmr1 knockout mice. The EF1α-Mdm2 and EF1α-TRIM3 complexes could thus be new therapeutic targets for FXS.

Investigation on the signaling pathways in the mechanism of hyperoxia-induced acute lung injury based on transcriptomics sequencing

To observe and verify the changes of transcriptome in hyperoxia-induced acute lung injury (HALI), and to further clarify the changes of pathways in HALI. Twelve healthy male C57BL/6J mice were randomly divided into normoxia group and HALI group according to the random number table, with 6 mice in each group. The mice in the normoxia group were fed normally in the room, and the mice in the HALI group was exposed to 95% oxygen to reproduce the HALI animal model. After 72 hours of hyperoxia exposure, the lung tissues were taken for transcriptome sequencing, and then Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis was performed. The pathological changes of lung tissue were observed under light microscope after hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to verify the key molecules in the signal pathways closely related to HALI identified by transcriptomics analysis. Transcriptomic analysis showed that hyperoxia induced 537 differentially expressed genes in lung tissue of mice as compared with the normoxia group including 239 up-regulated genes and 298 down-regulated genes. Further KEGG pathway enrichment analysis identified 20 most significantly enriched pathway entries, and the top three pathways were ferroptosis signaling pathway, p53 signaling pathway and glutathione (GSH) metabolism signaling pathway. The related genes in the ferroptosis signaling pathway included the up-regulated gene heme oxygenase-1 (HO-1) and the down-regulated gene solute carrier family 7 member 11 (SLC7A11). The related genes in the p53 signaling pathway included the up-regulated gene tumor suppressor gene p53 and the down-regulated gene murine double minute 2 (MDM2). The related gene in the GSH metabolic signaling pathway was up-regulated gene glutaredoxin 1 (Grx1). The light microscope showed that the pulmonary alveolar structure of the normoxia group was normal. In the HALI group, the pulmonary alveolar septum widened and thickened, and the alveolar cavity shrank or disappeared. RT-RCR and Western blotting confirmed that compared with the normoxia group, the mRNA and protein expressions of HO-1 and p53 in lung tissue of the HALI group were significantly increased [HO-1 mRNA (2-ΔΔCt): 2.16±0.17 vs. 1.00±0.00, HO-1 protein (HO-1/β-actin): 1.05±0.01 vs. 0.79±0.01, p53 mRNA (2-ΔΔCt): 2.52±0.13 vs. 1.00±0.00, p53 protein (p53/β-actin): 1.12±0.02 vs. 0.58±0.03, all P < 0.05], and the mRNA and protein expressions of Grx1, MDM2, SLC7A11 were significantly decreased [Grx1 mRNA (2-ΔΔCt): 0.53±0.05 vs. 1.00±0.00, Grx1 protein (Grx1/β-actin): 0.54±0.03 vs. 0.93±0.01, MDM2 mRNA (2-ΔΔCt): 0.48±0.03 vs. 1.00±0.00, MDM2 protein (MDM2/β-actin): 0.57±0.02 vs. 1.05±0.01, SLC7A11 mRNA (2-ΔΔCt): 0.50±0.06 vs. 1.00±0.00, SLC7A11 protein (SLC7A11/β-actin): 0.72±0.03 vs. 0.98±0.01, all P < 0.05]. HALI is closely related to ferroptosis, p53 and GSH metabolism signaling pathways. Targeting the key targets in ferroptosis, p53 and GSH metabolism signaling pathways may be an important strategy for the prevention and treatment of HALI.

Xinnaotongluo liquid protects H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3.

Xinnaotongluo liquid has been used to improve the clinical symptoms of patients with myocardial infarction. However, the molecular mechanism of Xinnaotongluo liquid is not completely understood. H9c2 cells exposed to hypoxia/reoxygenation (H/R) was used to simulate damage to cardiomyocytes in myocardial infarction in vitro. The biological indicators of H9c2 cells were measured by cell counting kit-8, enzyme linked immunoabsorbent assay, and western blot assay. In H/R-induced H9c2 cells, a markedly reduced murine double minute 2 (MDM2) was observed. However, the addition of Xinnaotongluo liquid increased MDM2 expression in H/R-induced H9c2 cells. And MDM2 overexpression strengthened the beneficial effects of Xinnaotongluo liquid on H9c2 cells from the perspective of alleviating oxidative damage, cellular inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells. Moreover, MDM2 overexpression reduced the protein expression of p53 and Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3). Whereas, STEAP3 overexpression hindered the function of MDM2-overexpression in H/R-induced H9c2 cells. Our results insinuated that Xinnaotongluo liquid could protect H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3, which provide a potential theoretical basis for further explaining the working mechanism of Xinnaotongluo liquid.

Progress in deciphering the role of p53 in diffuse large B-cell lymphoma: mechanisms and therapeutic targets.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, accounting for 30%-40% of non-Hodgkin lymphoma in adults. The mechanisms underlying DLBCL occurrence are extremely complex, and involve the B-cell receptor (BCR) and Toll-like receptor (TLR) signaling pathways, as well as genetic abnormalities and other factors. With the development of high-throughput sequencing, an increasing number of abnormal genes have been identified in DLBCL. Among them, the tumor protein p53 (TP53/p53) gene is important in DLBCL occurrence. Patients with DLBCL carrying TP53 gene abnormalities generally have poor prognosis and studies of p53 have potential to provide a better basis for their treatment. Normally, p53 is maintained at low levels through its interaction with murine double minute 2 (MDM2), and prevents tumorigenesis by mediating cell cycle arrest, apoptosis, and repair of damaged cells, among other processes. Therefore, the prognosis of patients with DLBCL harboring TP53 gene abnormalities (mutations, deletions, etc.) is poor, and targeting p53 for tumor therapy has become a research hotspot, following developments in molecular biology technologies. Current treatments targeting p53 mainly act by restoring the function or promoting degradation of mutant p53, and enhancing wild-type p53 protein stability and activity. Based on the current status of p53 research, exploration of existing therapeutic methods to improve the prognosis of patients with DLBCL with TP53 abnormalities is warranted.