P53 Activation Research Articles

P14ARF forms meso-scale assemblies upon phase separation with NPM1.

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14ARF). Oncogenic stress induces p14ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14ARF by NPM1 are unclear because the structural features of the p14ARF-NPM1 complex were elusive. Here we show that p14ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14ARF. These hydrophobic interactions promote phase separation with NPM1, enhance p14ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

Aurora B inhibitors promote RB hypophosphorylation and senescence independent of p53-dependent CDK2/4 inhibition

Aurora B kinase (AURKB) inhibitors have been trialled in a range of different tumour types but are not approved for any indication. Expression of the human papilloma virus (HPV) oncogenes and loss of retinoblastoma (RB) protein function has been reported to increase sensitivity to AURKB inhibitors but the mechanism of their contribution to sensitivity is poorly understood. Two commonly reported outcomes of AURKB inhibition are polyploidy and senescence, although their relationship is unclear. Here we have investigated the major cellular targets of the HPV E6 and E7, p53 and RB, to determine their contribution to AURKB inhibitor induced polyploidy and senescence. We demonstrate that polyploidy is a universal feature of AURKB inhibitor treatment in all cell types including normal primary cells, but the subsequent outcomes are controlled by RB and p53. We demonstrate that p53 by regulating p21 expression is required for an initial cell cycle arrest by inhibiting both CDK2 and CDK4 activity, but this arrest is only triggered after cells have undergone two failed mitosis and cytokinesis. However, cells can enter senescence in the absence of p53. RB is essential for AURKB inhibitor-induced senescence. AURKB inhibitor induces rapid hypophosphorylation of RB independent of inhibition of CDK2 or CDK4 kinases and p53. This work demonstrates that p53 activation determines the timing of senescence onset, but RB is indispensable for senescence.

Dynamics simulations of hypoxia inducible factor-1 regulatory network in cancer using formal verification techniques

Hypoxia-inducible factor-1 (HIF-1) regulates cell growth, protein translation, metabolic pathways and therefore, has been advocated as a promising biological target for the therapeutic interventions against cancer. In general, hyperactivation of HIF-1 in cancer has been associated with increases in the expression of glucose transporter type-1 (GLUT-1) thus, enhancing glucose consumption and hyperactivating metabolic pathways. The collective behavior of GLUT-1 along with previously known key players AKT, OGT, and VEGF is not fully characterized and lacks clarity of how glucose uptake through this pathway (HIF-1) probes the cancer progression. This study uses a Rene Thomas qualitative modeling framework to comprehend the signaling dynamics of HIF-1 and its interlinked proteins, including VEGF, ERK, AKT, GLUT-1, β-catenin, C-MYC, OGT, and p53 to elucidate the regulatory mechanistic of HIF-1 in cancer. Our dynamic model reveals that continuous activation of p53, β-catenin, and AKT in cyclic conditions, leads to oscillations representing homeostasis or a stable recovery state. Any deviation from this cycle results in a cancerous or pathogenic state. The model shows that overexpression of VEGF activates ERK and GLUT-1, leads to more aggressive tumor growth in a cancerous state. Moreover, it is observed that collective modulation of VEGF, ERK, and β-catenin is required for therapeutic intervention because these genes enhance the expression of GLUT-1 and play a significant role in cancer progression and angiogenesis. Additionally, SimBiology simulation unveils dynamic molecular interactions, emphasizing the need for targeted therapeutics to effectively regulate VEGF and ERK concentrations to modulate cancer cell proliferation.

Post-transplant G-CSF impedes engraftment of gene-edited human hematopoietic stem cells by exacerbating p53-mediated DNA damage response.

Granulocyte-colony-stimulating factor (G-CSF) is commonly used to accelerate recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, its utility after exvivo gene therapy in human HSPCs remains unexplored. We show that administering G-CSF from day 1 to 14 post-transplant impedes engraftment of CRISPR-Cas9 gene-edited human HSPCs in murine xenograft models. G-CSF affects gene-edited HSPCs through a cell-intrinsic mechanism, causing proliferative stress and amplifying the early p53-mediated DNA damage response triggered by Cas9-mediated DNA double-strand breaks. This underscores a threshold mechanism where p53 activation must reach a critical level to impair cellular function. Transiently inhibiting p53 or delaying the initiation of G-CSF treatment to day 5 post-transplant attenuates its negative impact on gene-edited HSPCs. The potential for increased HSPC toxicity associated with post-transplant G-CSF administration in CRISPR-Cas9 autologous HSPC gene therapy warrants consideration in clinical trials.

A molecular viewpoint of the intricate relationships among HNSCC, HPV infections, and the oral microbiota dysbiosis

HPV infection and the type of host microbiota play a role in the formation of HNCs. In contrast to other forms of OSCC, where the relationship between HPV and the cancer is less obvious, HPV-HNSCC is a particular type of oropharyngeal cancer. HPV has infected a stratified squamous epithelium, which includes the throat, mouth, anogenital tract, respiratory tract, and skin on the hands and feet. HPV DNA was found in high amounts in the saliva and gargle samples of patients with HPV-related HNSCC. It has been discovered that the specificity of oral mRNA (HPV) and HPV DNA identification varies from 23 % to 82 % in the identification of OPSCCs. The higher rate of HPV transmission through vaginal-oral compared to penile-oral sexual activity may be the reason for the difference in HPV-positive HNSCC patients between males and females. The researchers postulate that HPV-inactive tumours signify an advanced stage of HPV-positive HNSCC, which explains why there are racial disparities in gene expression that correspond to different disease progressions in Black and White patients. The increase of CD8+ T cells in the cancer microenvironment, linked to P16 activation, extends life expectancy in OSCC. tumour markers methylation caused by HPV and suggested using them as possible HNC biomarkers. Fusobacterium levels are much higher in patients with OSCC, while Actinobacteria phylum and Firmicutes are significantly lower. It also serves as a biomarker for notable variations found in Firmicutes, Actinobacteria, Fusobacteriales, Fusobacteriia, Fusobacterium, and Fusobacteriaceae. Therefore, based on this we evidence, we could investigate the role of oral microbiota as a maker for the HPV associated HNSCC.

Discovery of MD-265: A Potent MDM2 Degrader That Achieves Complete Tumor Regression and Improves Long-Term Survival of Mice with Leukemia.

MDM2 has been pursued as an attractive therapeutic target for human cancers. Herein, we describe our discovery of MD-265 as a promising PROTAC MDM2 degrader and extensive in vitro and in vivo evaluations of its therapeutic potential and mechanism of action. MD-265 effectively depleted MDM2 protein in cancer cells at concentrations as low as 1 nM, leading to strong activation of p53 in cancer cells carrying wild-type p53. It selectively inhibited the growth of wild-type p53 leukemia cell lines and showed no activity in mutated p53 lines. MD-265 achieved persistent tumor regression in a leukemia xenograft model without causing any signs of toxicity and dramatically improved survival of mice in a disseminated leukemia model even with a weekly administration. MD-265 displayed an excellent intravenous PK profile in mice, rats, and dogs. MD-265 is a promising MDM2 degrader for advanced preclinical development for the treatment of human cancers.

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with “epidrugs” is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Targeting BRIX1 via Engineered Exosomes Induces Nucleolar Stress to Suppress Cancer Progression.

Elevated ribosome biogenesis correlates with the rapid growth and progression of cancer. Targeted blockade of ribosome biogenesis induces nucleolar stress, which preferentially leads to the elimination of malignant cells. In this study, it is reported that the nucleolar protein BRIX1 is a critical regulator for the homeostasis between ribosome biogenesis and p53 activation. BRIX1 facilitated the processing of pre-rRNA by supporting the formation of the PeBoW complex. In addition, BRIX1 prevented p53 activation in response to nucleolar stress by impairing the interactions between MDM2 and the ribosomal proteins, RPL5, and RPL11, thereby triggering the resistance of cancer cells to chemotherapy. Conversely, depletion of BRIX1 induced nucleolar stress, which in turn activated p53 through RPL5 and RPL11, consequently inhibiting the growth of tumors. Moreover, engineered exosomes are developed, which are surface-decorated with iRGD, a tumor-homing peptide, and loaded with siRNAs specific to BRIX1, for the treatment of cancer. iRGD-Exo-siBRIX1 significantly suppressed the growth of colorectal cancer and enhanced the efficacy of 5-FU chemotherapy in vivo. Overall, the study uncovers that BRIX1 functions as an oncoprotein to promote rRNA synthesis and dampen p53 activity, and also implies that targeted inhibition of BRIX1 via engineered exosomes can be a potent approach for cancer therapy.

The vitamin D receptor is essential for the replication of pseudorabies virus.

The vitamin D receptor (VDR) is a nuclear steroid receptor that regulates the expression of genes across various biological functions. However, the role of VDR in pseudorabies virus (PRV) infection has not yet been explored. We discovered that VDR positively influenced PRV proliferation because knockdown of VDR impaired PRV proliferation, whereas its overexpression promoted it. Additionally, we observed that PRV infection upregulated VDR transcription alongside 1,25-dihydroxyvitamin D3 (VD3) synthesis, contingent on p53 activation. Furthermore, VDR knockdown hindered PRV-induced lipid synthesis, implicating VDR's involvement in this process. To decipher the mechanism behind VDR's stimulation of lipid synthesis during PRV infection, we conducted RNA sequencing (RNA-seq) and found significant enrichment of genes in the Ca2+ signaling pathway. Measurements of Ca2+ indicated that VDR facilitated Ca2+ absorption. Moreover, the PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways were also enriched in our RNA-seq data. Interfering with VDR expression, or chelating Ca2+ using BAPTA-AM, markedly impacted the activation of PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways, lipid synthesis, and PRV proliferation. In summary, our study demonstrates that PRV infection promotes VDR expression, thereby enhancing Ca2+ absorption and activating PI3K/AKT/mTORC1- and AMPK/mTORC1-mediated lipid synthesis. Our findings offer new insights into strategies for PRV prevention.IMPORTANCEVitamin D, beyond its well-known benefits for bone health and immune function, also plays a pivotal role in regulating gene expression through its receptor, the vitamin D receptor (VDR). Although VDR's influence spans multiple biological processes, its relationship with viral infections, particularly pseudorabies virus (PRV), remains underexplored. Our research illustrates a complex interplay where PRV infection boosts VDR expression, which in turn enhances Ca2+ absorption, leading to the activation of critical lipid synthesis pathways, PI3K/AKT/mTORC1 and AMPK/mTORC1. These findings not only deepen our understanding of the intricate dynamics between host molecular mechanisms and viral proliferation but also open avenues for exploring new strategies aimed at preventing PRV infection. By targeting components of the VDR-related signaling pathways, we can potentially develop novel therapeutic interventions against PRV and possibly other similar viral infections.

The mitochondrial enzyme pyruvate carboxylase restricts pancreatic β-cell senescence by blocking p53 activation.

Defective glucose-stimulated insulin secretion (GSIS) and β-cell senescence are hallmarks in diabetes. The mitochondrial enzyme pyruvate carboxylase (PC) has been shown to promote GSIS and β-cell proliferation in the clonal β-cell lines, yet its physiological relevance remains unknown. Here, we provide animal and human data showing a role of PC in protecting β-cells against senescence and maintaining GSIS under different physiological and pathological conditions. β-cell-specific deletion of PC impaired GSIS and induced β-cell senescence in the mouse models under either a standard chow diet or prolonged high-fat diet feeding. Transcriptomic analysis indicated that p53-related senescence and cell cycle arrest are activated in PC-deficient islets. Overexpression of PC inhibited hyperglycemia- and aging-induced p53-related senescence in human and mouse islets as well as INS-1E β-cells, whereas knockdown of PC provoked senescence. Mechanistically, PC interacted with MDM2 to prevent its degradation via the MDM2 binding motif, which in turn restricts the p53-dependent senescent program in β-cells. On the contrary, the regulatory effects of PC on GSIS and the tricarboxylic acid (TCA) anaplerotic flux are p53-independent. We illuminate a function of PC in controlling β-cell senescence through the MDM2-p53 axis.

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B.

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4 , lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Enhanced SIRT1 Activity by Galangin Mitigates UVB-Induced Senescence in Dermal Fibroblasts via p53 Acetylation Regulation and Activation.

Human skin aging, a complex process influenced by intrinsic aging and extrinsic photoaging, is marked by the accumulation of reactive oxygen species (ROS) that cause DNA damage, impaired dermal fibroblast function, and wrinkle formation. External stressors, such as ultraviolet (UV) radiation, can trigger cellular senescence. Sirtuin-1 (SIRT1), an NAD+-dependent enzyme in the sirtuin family, plays a crucial role in deacetylating p53, thereby inhibiting its nuclear translocation and reducing skin senescence. Galangin, a flavonoid found in honey and Alpinia officinarum root, has antioxidant and anti-inflammatory properties. This study investigates the protective mechanism of galangin against UVB-induced senescence in human dermal fibroblasts (HDFs) by examining its effects on SIRT1 and its target, acetylated-p53. An in vitro model of UVB-induced senescence using HDFs and an in vivo model using nude mice were employed to assess the dermal protective effects of galangin. The results demonstrate that while UVB exposure does not decrease SIRT1 protein levels, it impairs its enzymatic function. However, galangin treatment counteracts these adverse effects. Additionally, UVB exposure significantly reduces cell viability and upregulates senescence markers like p16, p21, and p53 nuclear transactivation. An increase in senescence-associated β-galactosidase (SA-β-gal) positive cells was observed in UVB-exposed dermal fibroblasts. Galangin treatment mitigates UVB-induced cellular senescence by enhancing SIRT1-mediated p53 deacetylation, thereby inhibiting nuclear translocation and reducing dermal senescence. These findings suggest that galangin is a promising agent for alleviating UVB-induced skin aging and could be a potential component in antiaging cosmetic formulations.

The NOTCH1 and miR-34a signaling network is affected by TP53 alterations in CLL

In chronic lymphocytic leukemia (CLL), TP53 mutations or deletions on chromosome 17p lead to adverse prognosis and reduced levels of miR-34a, which targets NOTCH1. Also, hyperactivated NOTCH1 signaling is crucial for CLL progression. Here we explored the interaction between p53, miR-34a, and NOTCH1 in CLL. We investigated the effect of p53 and miR-34a on NOTCH1 signaling and expression in CLL cells with altered TP53. Our results indicate that miR-34a reduces NOTCH1 3’ UTR activity but might not be a mediator between p53 signaling and NOTCH1. p53 activation increases miR-34a expression and NOTCH1 protein levels, correlating with decreased NOTCH1 and miR-34a levels in primary CLL cells with TP53 alterations. Some samples with high NOTCH1 levels presented increased BCL-2, suggesting an anti-apoptotic mechanism of a potentially direct p53-NOTCH1 relation in CLL. This study deepens the understanding of the p53-miR-34a-NOTCH1 signaling network, providing insights that could guide future therapeutic strategies for CLL.

Loss of NAT10 Reduces the Translation of Kmt5a mRNA Through ac4C Modification in Cardiomyocytes and Induces Heart Failure.

In the past decade, the biological functions of various RNA modifications in mammals have been uncovered. N4-acetylcytidine (ac4C), a highly conserved RNA modification, has been implicated in human diseases. Despite this, the involvement of RNA ac4C modification in cardiac physiology and pathology remains incompletely understood. NAT10 (N-acetyltransferase 10) stands as the sole acetyltransferase known to catalyze RNA ac4C modification. This study aims to explore the role of NAT10 and ac4C modification in cardiac physiology and pathology. Cardiac-specific knockout of NAT10, leading to reduced RNA ac4C modification, during both neonatal and adult stages resulted in severe heart failure. NAT10 deficiency induced cardiomyocyte apoptosis, a crucial step in heart failure pathogenesis, supported by invitro data. Activation of the p53 signaling pathway was closely associated with enhanced apoptosis in NAT10-deficient cardiomyocytes. As ac4C modification on mRNA influences translational efficiency, we employed ribosome footprints coupled with RNA sequencing to explore genome-wide translational efficiency changes caused by NAT10 deficiency. We identified and validated that the translational efficiency of Kmt5a was suppressed in NAT10 knockout hearts due to reduced ac4C modification on its mRNA. This finding was consistent with the observation that Kmt5a protein levels were reduced in heart failure despite unchanged mRNA expression. Knockdown of Kmt5a in cardiomyocytes recapitulated the phenotype of NAT10 deficiency, including increased cardiomyocyte apoptosis and activated p53 signaling. Finally, overexpression of Kmt5a rescued cardiomyocyte apoptosis and p53 activation induced by NAT10 inhibition. Our study highlights the significance of NAT10 in cardiomyocyte physiology, demonstrating that NAT10 loss is sufficient to induce cardiomyocyte apoptosis and heart failure. NAT10 regulates the translational efficiency of Kmt5a, a key mediator, through mRNA ac4C modification during heart failure.

A structure-based virtual screening identifies a novel MDM2 antagonist in the activation of the p53 signaling and inhibition of tumor growth.

p53, a tumor suppressor protein, has a vital role in the regulation of the cell cycle, apoptosis, and DNA damage repair. The degradation of p53 is predominantly controlled by the murine double minute 2 (MDM2) protein, a ubiquitin E3 ligase. The overexpression or amplification of MDM2 is commonly observed in various human cancers bearing wild-type p53 alleles, leading to the rapid degradation of the p53 protein and the attenuation of p53 tumor suppression functions. Thus, a major effort in p53-based cancer therapy has been to research MDM2 antagonists that specifically stabilize and activate p53, leading to the suppression of tumor growth. However, despite numerous efforts to develop MDM2 antagonists, to date they have failed to reach clinical use, largely because of the cytotoxicity associated with these small molecules. This study used our newly designed structure-based virtual screening approach on a commercial compound library to identify a novel compound, CGMA-Q18, which directly binds to MDM2, leading to the activation of p53, the induction of apoptosis, and cell cycle arrest in cancer cells. Notably, CGMA-Q18 significantly inhibited tumor xenograft growth in nude mice without observable toxicity. These findings highlight our useful virtual screening protocol and CGMA-Q18 as a putative MDM2 antagonist.

Quinoline-based compounds can inhibit diverse enzymes that act on DNA.

DNA methylation, as exemplified by cytosine-C5 methylation in mammals and adenine-N6 methylation in bacteria, is a key epigenetic process. Developing non-nucleoside inhibitors to cause DNA hypomethylation is crucial for treating various conditions without the toxicities associated with existing cytidine-based hypomethylating agents. This study characterized fifteen quinoline-based analogs, particularly compounds with additions like a methylamine (9) or methylpiperazine (11), which demonstrate similar low micromolar inhibitory potency against human DNMT1 and Clostridioides difficile CamA. These compounds (9 and 11) intercalate into CamA-bound DNA via the minor groove, causing a conformational shift that moves the catalytic domain away from the DNA. This study adds to the limited examples of DNA methyltransferases being inhibited by non-nucleotide compounds through DNA intercalation. Additionally, some quinoline-based analogs inhibit other DNA-interacting enzymes, such as polymerases and base excision repair glycosylases. Finally, compound 11 elicits DNA damage response via p53 activation in cancer cells.

DNA damage–induced p53 downregulates expression of RAG1 through a negative feedback loop involving miR-34a and FOXP1

During the maturation of pre-B cells, the recombination activating gene 1 and 2 (RAG1/2) endonuclease complex plays a crucial role in coordinating V(D)J recombination by introducing DNA breaks in immunoglobulin (Ig) loci. Dysregulation of RAG1/2 has been linked to the onset of B-cell malignancies, yet the mechanisms controlling RAG1/2 in pre-B cells exposed to excessive DNA damage are not fully understood. In this study, we show that DNA damage-induced activation of p53 initiates a negative-feedback loop which rapidly downregulates RAG1 levels. This feedback loop involves ataxia telangiectasia mutated (ATM) activation, subsequent stabilization of p53, and modulation of microRNA-34a (miR-34a) levels, which is one of the p53 targets. Notably, this loop incorporates transcription factor forkhead box P1 (FOXP1) as a downstream effector. The absence of p53 resulted in an increased proportion of IgM+ cells prompted to upregulate RAG1/2 and to undergo Ig light chain (Igl) recombination. Similar results were obtained in primary pre-B cells with depleted levels of miR-34a. We propose that in pre-B cells undergoing Ig gene recombination, the DNA breaks activate a p53/miR-34a/FOXP1-mediated negative-feedback loop that contributes to the rapid downregulation of RAG. This regulation limits the RAG-dependent DNA damage, thereby protecting the stability of the genome during V(D)J rearrangement in developing B cells.

Colchicine Prevents Cardiac Rupture in Mice with Myocardial Infarction by Inhibiting P53-Dependent Apoptosis.

Cardiac rupture is a fatal complication following myocardial infarction (MI) and there are currently no effective pharmacological strategies for preventing this condition. In this study, we investigated the effect of colchicine on post-infarct cardiac rupture in mice and its underlying mechanisms.We induced MI in mice by permanently ligating the left anterior descending artery. Oral colchicine or vehicle was administered at a dose of 0.1 mg/kg/day from day 1 to day 7 after MI. Cultured neonatal cardiomyocytes and fibroblasts were exposed to normoxia or anoxia and treated with colchicine.Colchicine significantly improved the survival rate (colchicine, n = 46: 82.6% versus vehicle, n = 42: 61.9%, P < 0.05) at 1 week after MI. Histological analysis revealed colchicine significantly reduced the infarct size and the number of macrophages around the infarct area. Colchicine decreased apoptosis in the myocardium of the border zone and cultured cardiomyocytes and fibroblasts as assessed by TUNEL assay. Colchicine also attenuated the activation of p53 and decreased the expression of cleaved-caspase 3 and bax, as assessed by Western blotting.Colchicine prevents cardiac rupture via inhibition of apoptosis, which is attributable to the downregulation of p53 activity. Our findings suggest that colchicine may be a prospective preventive medicine for cardiac rupture, however, large clinical trials are required.

An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

CBR-470-1 protects against cardiomyocyte death in ischaemia/reperfusion injury by activating the Nrf2–GPX4 cascade

Cardiac ischaemia/reperfusion (I/R) impairs mitochondrial function, resulting in excessive oxidative stress and cardiomyocyte ferroptosis and death. Nuclear factor E2-related factor 2 (Nrf2) is a key regulator of redox homeostasis and has cardioprotective effects against various stresses. Here, we tested whether CBR-470-1, a noncovalent Nrf2 activator, can protect against cardiomyocyte death caused by I/R stress. Compared with vehicle treatment, the administration of CBR-470-1 (2 mg/kg) to mice significantly increased Nrf2 protein levels and ameliorated the infarct size, the I/R-induced decrease in cardiac contractile performance, and the I/R-induced increases in cell apoptosis, ROS levels, and inflammation. Consistently, the beneficial effects of CBR-470-1 on cardiomyocytes were verified in a hypoxia/reoxygenation (H/R) model in vitro, but this cardioprotection was dramatically attenuated by the GPX4 inhibitor RSL3. Mechanistically, CBR-470-1 upregulated Nrf2 expression, which increased the expression levels of antioxidant enzymes (NQO1, SOD1, Prdx1, and Gclc) and antiferroptotic proteins (SLC7A11 and GPX4) and downregulated the protein expression of p53 and Nlrp3, leading to the inhibition of ROS production and inflammation and subsequent cardiomyocyte death (apoptosis, ferroptosis and pyroptosis). In summary, CBR-470-1 prevented I/R-mediated cardiac injury possibly through inhibiting cardiomyocyte apoptosis, ferroptosis and pyroptosis via Nrf2-mediated inhibition of p53 and Nlrp3 and activation of the SLC7A11/GPX4 pathway. Our data also highlight that CBR-470-1 may serve as a valuable agent for treating ischaemic heart disease.