Rational design, synthesis, and biophysical characterization of a peptidic MDM2-MDM4 interaction inhibitor.

In recent years, the restoration of p53 physiological functions has become an attractive therapeutic approach to develop novel and efficacious cancer therapies. Among other mechanisms, the oncosuppressor protein p53 is functionally regulated by MDM2 through its E3 ligase function. MDM2 promotes p53 ubiquitination and degradation following homodimerization or heterodimerization with MDM4. Recently, we discovered Pep3 (1, Pellegrino et al., 2015), a novel peptidic inhibitor of MDM2 dimerization able to restore p53 oncosuppressive functions both in vitro and in vivo. In this work, we were able to identify the key interactions between peptide 1 and MDM2 RING domain and to design peptide 2, a truncated version of 1 that is still able to bind MDM2. Integrating both computational and biophysical techniques, we show that peptide 2 maintains the conserved peptide 1-MDM2 interactions and is still able to bind to full-length MDM2.

Long-Read MDM4 Sequencing Reveals Aberrant Isoform Landscape in Metastatic Melanomas.

MDM4 is upregulated in the majority of melanoma cases and has been described as a "key therapeutic target in cutaneous melanoma". Numerous isoforms of MDM4 exist, with few studies examining their specific expression in human tissues. The changes in splicing of MDM4 during human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Compounding this, studies relying on short reads lose "connectivity" data, so full transcripts are frequently only inferred from the presence of splice junction reads. To address this problem, long-read nanopore sequencing was utilized to read the entire length of transcripts. Here, MDM4 transcripts, both alternative and canonical, are characterized in a pilot cohort of human melanoma specimens. RT-PCR was first used to identify the presence of novel splice junctions in these specimens. RT-qPCR then quantified the expression of major MDM4 isoforms observed during sequencing. The current study both identifies and quantifies MDM4 isoforms present in melanoma tumor samples. In the current study, we observed high expression levels of MDM4-S, MDM4-FL, MDM4-A, and the previously undescribed Ensembl transcript MDM4-209. A novel transcript lacking both exons 6 and 9 is observed and named MDM4-A/S for its resemblance to both MDM4-A and MDM4-S isoforms.

SNRPB2 promotes triple-negative breast cancer progression by controlling alternative splicing of MDM4 pre-mRNA.

Alternative splicing generates cancer-specific transcripts and is now recognized as a hallmark of cancer. However, the critical oncogenic spliceosome-related proteins involved in triple-negative breast cancer (TNBC) remain elusive. Here, we explored the expression pattern of spliceosome-related proteins in TNBC, non-TNBC, and normal breast tissues from The Cancer Genome Atlas breast cancer (TCGA-BRCA) cohort, revealing higher expression of nearly half of spliceosome-related proteins in TNBC than their counterparts. Among these TNBC-specific spliceosome-related proteins, the expression of SNRPB2 was associated with poor prognosis in patients with TNBC. In TNBC cells, the knockdown of SNRPB2 strongly suppressed cell proliferation and invasion and induced cell cycle arrest. Mechanistically, transcriptome data showed that SNRPB2 knockdown inactivated E2F1 signaling, which regulated the cell cycle. We further validated the downregulation of several cell cycle genes in SNRPB2 knockdown cells. Moreover, the analysis showed that SNRPB2 knockdown triggered the alteration of many alternative splicing events, most of which were skipping of exon. In TNBC cells, it was found that SNRPB2 knockdown led to the skipping of exon 6 in MDM4 pre-mRNA, generating MDM4-S transcript and downregulating MDM4 protein expression. More importantly, downregulation of MDM4 decreased retinoblastoma 1 (Rb1) protein expression, which is a target of MDM4 and a regulator of E2F1 signaling. In summary, the current study revealed an SNRPB2/MDM4/Rb axis in promoting the progression of TNBC, providing novel insights and novel targets for combating TNBC.

The nuclear GYF protein CD2BP2/U5-52K is required for T cell homeostasis.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.

RPL22 is a tumor suppressor in MSI-high cancers and a splicing regulator of MDM4.

Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

A defective splicing machinery promotes senescence through MDM4 alternative splicing.

Defects in the splicing machinery are implicated in various diseases, including cancer. We observed a general reduction in the expression of spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced, and telomere uncapping-induced senescence. Supporting the view that defective splicing contributes to senescence, splicing inhibitors herboxidiene, and pladienolide B induced senescence in normal and cancer cell lines. Furthermore, depleting individual spliceosome components also promoted senescence. All senescence types were associated with an alternative splicing transition from the MDM4-FL variant to MDM4-S. The MDM4 splicing shift was reproduced when splicing was inhibited, and spliceosome components were depleted. While decreasing the level of endogenous MDM4 promoted senescence and cell survival independently of the MDM4-S expression status, cell survival was also improved by increasing MDM4-S. Overall, our work establishes that splicing defects modulate the alternative splicing of MDM4 to promote senescence and cell survival.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

SETBP1 activation upon MDM4-enhanced ubiquitination of NR3C1 triggers dissemination of colorectal cancer cells.

Colorectal cancer (CRC) presents a growing concern globally, marked by its escalating incidence and mortality rates, thus imposing a substantial health burden. This investigation delves into the role of nuclear receptor subfamily 3 group C member 1 (NR3C1) in CRC metastasis and explores the associated mechanism. Through a comprehensive bioinformatics analysis, NR3C1 emerged as a gene with diminished expression levels in CRC. This finding was corroborated by observations of a low-expression pattern of NR3C1 in both CRC tissues and cells. Furthermore, experiments involving NR3C1 knockdown revealed an exacerbation of proliferation, migration, and invasion of CRC cells in vitro. Subsequent assessments in mouse xenograft tumor models, established by injecting human HCT116 cells either through the tail vein or at the cecum termini, demonstrated a reduction in tumor metastasis to the lung and liver, respectively, upon NR3C1 knockdown. Functionally, NR3C1 (glucocorticoid receptor) suppressed SET binding protein 1 (SETBP1) transcription by binding to its promoter region. Notably, mouse double minute 4 (MDM4) was identified as an upstream regulator of NR3C1, orchestrating its downregulation via ubiquitination-dependent proteasomal degradation. Further investigations unveiled that SETBP1 knockdown suppressed migration and invasion, and epithelial to mesenchymal transition of CRC cells, consequently impeding in vivo metastasis in murine models. Conversely, upregulation of MDM4 exacerbated the metastatic phenotype of CRC cells, a propensity mitigated upon additional upregulation of NR3C1. In summary, this study elucidates a cascade wherein MDM4-mediated ubiquitination of NR3C1 enables the transcriptional activation of SETBP1, thereby propelling the dissemination of CRC cells.

Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme, a highly aggressive and lethal brain tumor, is a substantial clinical challenge and a focus of increasing concern globally. Hematological toxicity and drug resistance of first-line drugs underscore the necessity for new anti-glioma drug development. Here, 43 anthracenyl skeleton compounds as p53 activator XI-011 analogs were designed, synthesized, and evaluated for their cytotoxic effects. Five compounds (13d, 13e, 14a, 14b, and 14n) exhibited good anti-glioma activity against U87 cells, with IC50 values lower than 2 µM. Notably, 13e showed the best anti-glioma activity, with an IC50 value up to 0.53 µM, providing a promising lead compound for new anti-glioma drug development. Mechanistic analyses showed that 13e suppressed the MDM4 protein expression, upregulated the p53 protein level, and induced cell cycle arrest at G2/M phase and apoptosis based on Western blot and flow cytometry assays.

p53 biology and reactivation for improved therapy in MDS and AML.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) originate from preleukemic hematopoietic conditions, such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenia of undetermined significance (CCUS) and have variable outcomes despite the successful implementation of targeted therapies. The prognosis differs depending on the molecular subgroup. In patients with TP53 mutations, the most inferior outcomes across independent studies were observed. Myeloid malignancies with TP53 mutations have complex cytogenetics and extensive structural variants. These factors contribute to worse responses to induction therapy, demethylating agents, or venetoclax-based treatments. Survival of patients with biallelic TP53 gene mutations is often less than one year but this depends on the type of treatment applied. It is still controversial whether the allelic state of mutant TP53 impacts the outcomes in patients with AML and high-risk MDS. Further studies are needed to justify estimating TP53 LOH status for better risk assessment. Yet, TP53-mutated MDS, MDS/AML and AML are now classified separately in the International Consensus Classification (ICC). In the clinical setting, the wild-type p53 protein is reactivated pharmacologically by targeting p53/MDM2/MDM4 interactions and mutant p53 reactivation is achieved by refolding the DNA binding domain to wild-type-like conformation or via targeted degradation of the mutated protein. This review discusses our current understanding of p53 biology in MDS and AML and the promises and failures of wild-type and mutant p53 reactivation in the clinical trial setting.

MDM4 was associated with poor prognosis and tumor-immune infiltration of cancers.

MDM4 is one of the MDM protein family and is generally recognized as the key negative regulator of p53. As a cancer-promoting factor, it plays a non-negligible role in tumorigenesis and development. In this article, we analyzed the expression levels of MDM4 in pan-cancer through multiple databases. We also investigated the correlations between MDM4 expression and prognostic value, immune features, genetic mutation, and tumor-related pathways. We found that MDM4 overexpression is often accompanied by adverse clinical features, poor prognosis, oncogenic mutations, tumor-immune infiltration and aberrant activation of oncogenic signaling pathways. We also conducted transcriptomic sequencing to investigate the effect of MDM4 on transcript levels in colon cancer and performed qPCR to verify this. Finally, we carried out some in vitro experiments including colony formation assay, chemoresistance and senescence-associated ß-galactosidase activity assay to study the anti-tumor treatment effect of small molecule MDM4 inhibitor, NSC146109. Our research confirmed that MDM4 is a prognostic biomarker and potential therapeutic target for a variety of malignancies.

Comparative Clinicopathologic and Genomic Analysis of Hepatocellular Neoplasm, Not Otherwise Specified, and Hepatoblastoma.

Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathologic and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathologic characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids DNA- and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoters, were employed. We found that patients with HCN-NOS were older (P < .001) and more frequently classified as high risk (P < .01), yet they showed no significant differences in alpha fetoprotein levels or survival outcomes compared with those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs 11/24; P < .001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs 6/24; P < .05) and chromosome 11 (7/17 vs 1/24; P < .01) when compared with HB. Furthermore, the recurrent loss/LOH of chromosomes 3, 4p, 9, 15q, and Y was only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the 2 groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathologic features and greater structural complexity compared with HB. However, patients with HCN-NOS exhibit comparable alpha fetoprotein levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared with those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.

Ginsenoside Rg3 improves microcystin-induced cardiotoxicity through the miR-128-3p/MDM4 axis.

Microcystin (MC) is the byproduct of cyanobacteria metabolism that is associated with oxidative stress and heart damage. This study aimed to investigate the effect of ginsenoside Rg3 on MC-induced cardiotoxicity. A mouse model of myocardial infarction was constructed by oral MC administration. H9C2 cells were used for in vitro analysis. Cellular oxidative stress, apoptosis, and the relationship between miR-128-3p and double minute 4 protein (MDM4) were analyzed. MiR-128-3p expression was upregulated in vitro and in vivo after MC treatment, which was downregulated after Rg3 treatment. Left ventricular ejection fraction (LVEF) and left ventricular systolic pressure (LVSP) were increased and left ventricular end-diastolic pressure (LVEDP) was decreased after Rg3 treatment. Moreover, Rg3 alleviated MC-induced pathological changes and apoptosis in myocardial tissues. Meanwhile, Rg3 treatment decreased the lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels and inhabited cell apoptosis and oxidative stress in MC-treated myocardial cells. MiR-128-3p overexpression attenuated the protective effect of Rg3 on MC-induced cardiotoxicity. MiR-128-3p negatively regulated MDM4 expression. This study revealed that Rg3 alleviated MC-induced cardiotoxicity through the miR-128-3p/MDM4 axis, which emphasized the potential of Rg3 as a therapeutic agent for MC-induced cardiotoxicity, and miR-128-3p as a target for the Rg3 therapy.

Astaxanthin protects against pirarubicin-induced H9c2 cardiomyocytes by adjusting microRNA-494-3p-mediated MDM4/p53 signalling pathway.

PURPOSE: Pirarubicin (THP) is an antitumour drug widely used in clinical practice, but its cardiotoxicity limits its application. THP cardiotoxicity must be treated as soon as possible. There is an urgent need to find drugs that alleviate THP cardiotoxicity. The purpose of this study was to investigate the effects and mechanisms of Astaxanthin (AST) on THP-induced cardiomyocytes. METHODS: Rat cardiomyocytes H9c2 were induced with THP. The effects of AST on THP-induced H9c2 and its mechanism were investigated by CCK8, reactive oxygen species assay, tunnel assay, flow cytometry, RT-qPCR, and Western blot. RESULTS: AST increased cell viability, inhibited apoptosis and accelerated cell cycle progression, reduced oxidative damage and inflammatory response in THP-induced H9c2; down-regulated miR-494-3p expression, promoted MDM4 expression, inhibited p53 activation, and suppressed apoptosis-related protein expression. Overexpression of MiR-494-3p reversed the above effects of AST. CONCLUSIONS: AST can inhibit H9c2 apoptosis induced by THP and attenuate H9c2 damage by THP, which may be achieved by downregulating miR-494-3p, upregulating MDM4, and inhibiting p53.

MiR-494-3p aggravates pirarubicin-induced cardiomyocyte injury by regulating MDM4/p53 signaling pathway.

OBJECTIVE: Pirarubicin (THP) is a widely used antitumor drug in clinical practice, but its cardiotoxicity limits its use. There is an urgent need to find drugs to alleviate the cardiotoxicity of THP. This study aimed to investigate the effect and mechanism of miR-494-3p on THP-induced cardiomyocytes. METHODS: THP induced immortalized mouse cardiomyocytes HL-1, silenced or overexpressed miR-494-3p. The effects of miR-494-3p on HL-1 contained in THP were investigated by CCK8, flow cytometry, ROS detection, JC-1 mitochondrial membrane potential detection, TUNEL cell apoptosis detection, RT-qPCR, and Western blot. RESULTS: miR-494-3p could reduce cell viability, increase oxidative damage, and promote cell apoptosis; at the same time, it inhibited the expression of MDM4, promoted the activation of p53, and promoted the expression of apoptosis-related proteins. MiR-494-3p inhibitors have the opposite effect. CONCLUSION: miR-494-3p can aggravate THP damage to HL-1, which may be achieved by downregulating MDM4 and promoting p53. miR-494-3p is one of the important miRNAs in THP-induced cardiotoxicity, which provides theoretical support for its possible use as a therapeutic target for THP-induced cardiovascular disease.

Pharmacological inhibition of MDM4 alleviates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology with no cure. A better understanding of the disease processes and identification of druggable targets will benefit the development of effective therapies for IPF. We previously reported that MDM4 promoted lung fibrosis through the MDM4-p53-dependent pathway. However, it remained unclear whether targeting this pathway would have any therapeutic potential. In this study, we evaluated the efficacy of XI-011, a small molecular inhibitor of MDM4, for treating lung fibrosis. We found that XI-011 significantly reduced MDM4 expression and increased the expression of total and acetylated p53 in primary human myofibroblasts and a murine fibrotic model. XI-011 treatment resulted in the resolution of lung fibrosis in mice with no notable impact on normal fibroblast death or the morphology of healthy lungs. Based on these findings, we propose that XI-011 might be a promising therapeutic drug candidate for treating pulmonary fibrosis.

Sex-Specific Associations of MDM2 and MDM4 Variants with Risk of Multiple Primary Melanomas and Melanoma Survival in Non-Hispanic Whites.

MDM2-SNP309 (rs2279744), a common genetic modifier of cancer incidence in Li-Fraumeni syndrome, modifies risk, age of onset, or prognosis in a variety of cancers. Melanoma incidence and outcomes vary by sex, and although SNP309 exerts an effect on the estrogen receptor, no consensus exists on its effect on melanoma. MDM2 and MDM4 restrain p53-mediated tumor suppression, independently or together. We investigated SNP309, an a priori MDM4-rs4245739, and two coinherited variants, in a population-based cohort of 3663 primary incident melanomas. Per-allele and per-haplotype (MDM2_SNP309-SNP285; MDM4_rs4245739-rs1563828) odds ratios (OR) for multiple-melanoma were estimated with logistic regression models. Hazard ratios (HR) for melanoma death were estimated with Cox proportional hazards models. In analyses adjusted for covariates, females carrying MDM4-rs4245739*C were more likely to develop multiple melanomas (ORper-allele = 1.25, 95% CI 1.03-1.51, and Ptrend = 0.03), while MDM2-rs2279744*G was inversely associated with melanoma-death (HRper-allele = 0.63, 95% CI 0.42-0.95, and Ptrend = 0.03). We identified 16 coinherited expression quantitative loci that control the expression of MDM2, MDM4, and other genes in the skin, brain, and lungs. Our results suggest that MDM4/MDM2 variants are associated with the development of subsequent primaries and with the death of melanoma in a sex-dependent manner. Further investigations of the complex MDM2/MDM4 motif, and its contribution to the tumor microenvironment and observed associations, are warranted.

CircPLXNA2 Affects the Proliferation and Apoptosis of Myoblast through circPLXNA2/gga-miR-12207-5P/MDM4 Axis.

CircRNAs are newly identified special endogenous RNA molecules that covalently close a loop by back-splicing with pre-mRNA. In the cytoplasm, circRNAs would act as molecular sponges to bind with specific miRNA to promote the expression of target genes. However, knowledge of circRNA functional alternation in skeletal myogenesis is still in its infancy. In this study, we identified a circRNA-miRNA-mRNA interaction network in which the axis may be implicated in the progression of chicken primary myoblasts' (CPMs) myogenesis by multi-omics (i.e., circRNA-seq and ribo-seq). In total, 314 circRNA-miRNA-mRNA regulatory axes containing 66 circRNAs, 70 miRNAs, and 24 mRNAs that may be relevant to myogenesis were collected. With these, the circPLXNA2-gga-miR-12207-5P-MDM4 axis aroused our research interest. The circPLXNA2 is highly differentially expressed during differentiation versus proliferation. It was demonstrated that circPLXNA2 inhibited the process of apoptosis while at the same time stimulating cell proliferation. Furthermore, we demonstrated that circPLXNA2 could inhibit the repression of gga-miR-12207-5p to MDM4 by directing binding to gga-miR-12207-5p, thereby restoring MDM4 expression. In conclusion, circPLXNA2 could function as a competing endogenous RNA (ceRNA) to recover the function of MDM4 by directing binding to gga-miR-12207-5p, thereby regulating the myogenesis.

FAM193A is a positive regulator of p53 activity.

Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.