Activation Of P53 Signaling Research Articles

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.

CircUGP2 Suppresses Intrahepatic Cholangiocarcinoma Progression via p53 Signaling Through Interacting With PURB to Regulate ADGRB1 Transcription and Sponging miR-3191-5p.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and its prognosis remains poor. Although growing numbers of studies have verified the involvement of circular RNAs (circRNAs) in various cancer types, their specific functions in ICC remain elusive. Herein, a circRNA, circUGP2 is identified by circRNA sequencing, which is downregulated in ICC tissues and correlated with patients' prognosis. Moreover, circUGP2 overexpression suppresses tumor progression in vitro and in vivo. Mechanistically, circUGP2 functions as a transcriptional co-activator of PURB over the expression of ADGRB1. It can also upregulate ADGRB1 expression by sponging miR-3191-5p. As a result, ADGRB1 prevents MDM2-mediated p53 polyubiquitination and thereby activates p53 signaling to inhibit ICC progression. Based on these findings, circUGP2 plasmid is encapsulated into a lipid nanoparticle (LNP) system, which has successfully targeted tumor site and shows superior anti-tumor effects. In summary, the present study has identified the role of circUGP2 as a tumor suppressor in ICC through regulating ADGRB1/p53 axis, and the application of LNP provides a promising translational strategy for ICC treatment.

Gardenin B, A Natural Inhibitor for USP7: In vitro Evaluation and In silico Identification

Background: Ubiquitin-specific protease 7 (USP7) is one of the most widely studied deubiquitin enzymes (DUBs). The protein level of USP7 is highly expressed in a variety of malignant cancers, which suggests that it is a prognostic marker of cancers and a potential drug target for oncotherapy. Objective: The aim of this study was to identify natural and effective USP7 inhibitors, in order to understand the activation of the USP7/p53 pathway by natural inhibitors. Methods: In this study, USP7 enzyme activity screening assay system and p53 luciferase reporter assay system have been applied for the discovery of natural USP7 inhibitors targeting the catalytic active site. Molecular docking and molecular dynamics (MD) simulation revealed the combined mechanism between USP7 with gardenin B. Results: The gardenin B was screened from our home-lab natural products (160 flavonoids) and had cytotoxicity in HCT116 cells (IC50 = 46.28 ± 2.16 μM). Preliminary in vitro studies disclosed its antiproliferative activity and activated p53 signaling pathway in HCT116 cells. We found that the complex formed by gardenin B and 5vsk (Ledock score = -6.86, MM/GBSA score = -53.35) had the optimal binding conformation. Moreover, the MD simulation showed that the π-π interactions between gardenin B with His461 and Phe409, and the hydrogen bonds interaction between gardenin B with Leu406 played an important role in maintaining the close binding of the complexes. Conclusion: In conclusion, gardenin B could be used as a natural product inhibitor of USP7 for further optimized design and development potential.

HMGA2 promotes resistance against paclitaxel by targeting the p53 signaling pathway in colorectal cancer cells

Colorectal cancer is one of the most common malignancies and ranks second in terms of cancer-related mortality worldwide due to its metastasis, drug resistance, and reoccurrence. High-mobility gene group A2 (HMGA2) is overexpressed in colorectal cancer, contributing to the aggressiveness of tumor malignance, and promotes drug resistance in many types of cancer. However, the underlying molecular mechanism of HMGA2 is yet to be elucidated. In this study, we showed that HMGA2 is overexpressed in colorectal cancer tissue, and knockdown of HMGA2 significantly inhibited colorectal cancer cell growth and migratory capability. HMGA2 regulates the cancer cell response to a widely used anti-cancer drug, paclitaxel (PTX). HMGA2 knockdown increased cell death, whereas HMGA2 overexpression decreased cell death after PTX treatment. Furthermore, lower reactive oxygen species (ROS) levels and mitochondrial potential were detected in HMGA2 overexpression cells after PTX treatment. However, HMGA2 knockdown produced the opposite effect. RNA sequencing showed a p53 signaling pathway-dependent regulation in HMGA2 knockdown cells. Combined with p53 inhibitors and HMGA2 knockdown, a synergetic effect of more cell death was observed in colorectal cancer cells after PTX treatment. Thus, we showed that HMGA2 can activate p53 signaling to regulate colorectal cancer cell death after PTX treatment. Altogether, our results reveal novel insights into the molecular mechanisms underlying HMGA2-mediated cancer cell resistance against PTX and highlight the potential of targeting HMGA2 and p53 signaling for the therapeutic investigation of colorectal cancer.

Activation of p53 signaling and regression of breast and prostate carcinoma cells by spirooxindole-benzimidazole small molecules.

This study discusses the synthesis and use of a new library of spirooxindole-benzimidazole compounds as inhibitors of the signal transducer and activator of p53, a protein involved in regulating cell growth and cancer prevention. The text includes the scientific details of the [3 + 2] cycloaddition (32CA) reaction between azomethine ylide 7a and ethylene 3a within the framework of Molecular Electron Density Theory. The mechanism of the 32CA reaction proceeds through a two-stage one-step process, with emphasis on the highly asynchronous transition state structure. The anti-cancer properties of the synthesized compounds, particularly 6a and 6d, were evaluated. The inhibitory effects of these compounds on the growth of tumor cells (MDA-MB 231 and PC-3) were quantified using IC50 values. This study highlights activation of the p53 pathway by compounds 6a and 6d, leading to upregulation of p53 expression and downregulation of cyclin D and NF-κB in treated cells. Additionally, we explored the binding affinity of spirooxindole analogs, particularly compound 6d, to MDM2, a protein involved in regulation of p53. The binding mode and position of compound 6d were compared with those of a co-crystallized standard ligand, suggesting its potential as a lead compound for further preclinical research.

The RNA m6A writer RBM15 contributes to the progression of esophageal squamous cell carcinoma by regulating miR-3605-5p/KRT4 pathway

Cancer progression can be modulated by N6-methyladenosine (m6A) modification. RNA binding motif protein 15 (RBM15) is an essential RNA m6A writer that influences carcinogenesis, however its significance in esophageal squamous cell carcinoma (ESCC) is uncertain. This research is intended to examine how RBM15 regulates the development of ESCC. We performed qRT-PCR analysis to evaluate the expression of RBM15, microRNA (miR-3605-5p) as well as keratin 4 (KRT4) in ESCC. Target relationship between miR-3605-5p and KRT4 was validated by dual luciferase reporter assay. Western blotting analyzed the protein levels of KRT4, p53, and p21. To demonstrate that RBM15 is responsible for the m6A alteration of miR-3605-5p, RIP and Me-RIP experiments were carried out concurrently. m6A content was measured by m6A quantification assay. Cell growth and migration were assessed using the CCK-8 and transwell assays. In addition, the role of RBM15 in vivo was examined using a mouse tumor xenograft model. RBM15 and miR-3605-5p were both substantially expressed in ESCC, however KRT4 was not expressed highly. Overexpressed RBM15 triggered cell proliferation and migration in ESCC. Besides, RBM15/m6A could mediate pri-3605-5p to form the mature miR-3605-5p, and miR-3605-5p further targeted KRT4. Further investigations showed that upregulation of KRT4 overturned the promoting impact of RBM15 overexpression on cell proliferation as well as on cell migration in ESCC by activating p53 signaling pathway. This work implied the carcinogenic activity of RBM15/m6A in ESCC via miR-3605-5p/KRT4 pathway, providing a novel m6A modification pattern in the tumorigenesis of ESCC.

Synthesis of 3-formyl-eudistomin U with anti-proliferation, anti-migration and apoptosis-promoting activities on melanoma cells

The discovery of new lead skeleton against melanoma are urgently needed due to its highly malignant and mortality. Herein, a new molecular entity (EU-5) derived from eudistomin U was synthesized with total yield of 46%, which displayed potent activity against malignant melanoma A375 cells (IC50 = 4.4 µM), no hemolytic toxicity and good physicochemical properties in silico. Colony formation and cell cycle arrest assays revealed that EU-5 suppressed cell proliferation by causing cell cycle arrest at G0/G1 phase. Wound healing and transwell assays suggested that EU-5 could effectively inhibit migration of A375 cells in a dose-dependent manner. Calcein-AM/PI staining, Annexin V-FITC/PI apoptosis detection, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), transcriptomics, quantitative real‑time polymerase chain reaction (qRT‑PCR), spectrometric titration and molecular docking assays indicated that EU-5 could activate p53 signaling pathway and trigger mitochondria-mediated cell apoptosis. Taken together, this study provided a promising lead structure for the design of a new generation of anti-melanoma drugs.

Knockdown of MRPL35 promotes cell apoptosis and inhibits cell proliferation in non-small-cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is a major pathological type of lung cancer. However, its pathogenesis remains largely unclear. MRPL35 is a regulatory subunit of the mitoribosome, which can regulate the assembly of cytochrome c oxidases and plays an important role in the occurrence of NSCLC.MethodsThe expression of MRPL35 in NSCLC was detected by tissue microarray and immunohistochemistry. H1299 cells were infected with lentivirus to knockdown MRPL35, and the cells were subjected to crystal violet staining to assess the results of colony formation assays. A549 cells were infected by lentiviral particles-expressing shMRPL35 or shControl, and then subcutaneously injected into nude mice. Tumorigenesis in mice was detected by in vivo imaging. The potential pathway of MRPL35 in NSCLC was assessed by Western blotting.ResultsMRPL35 was over-expressed in NSCLC tissue compared to para-cancerous and normal tissues. Knockdown of MRPL35 suppressed cell proliferation and decreased NSCLC progression both in vitro and in vivo. The possible molecular mechanisms were also clarified, which indicated that MRPL35 could be involved in cell apoptosis and proliferation by modulating the expression levels of CDK1, BIRC5, CHEK1, STMN1 and MCM2. Knockdown of MRPL35 activated p53 signaling pathway and inhibited cell cycle regulation.ConclusionsThe oncogenic role of MRPL35 in NSCLC was potentially mediated through the cell cycle regulatory genes such as BIRC5, STMN1, CDK1, CHEK1 and MCM2, as well as activation of P53 signaling pathway.

Tetraarsenic tetrasulfide triggers ROS-induced apoptosis and ferroptosis in B-cell acute lymphoblastic leukaemia by targeting HK2

PurposeAcute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children. Despite cure rates of higher than 85 %, refractory or relapsed ALL still exhibits a bleak prognosis indicative of the dearth of treatment modalities specific for relapsed or refractory ALL. Prior research has implicated metabolic alterations in leukemia pathogenesis, and literature on the therapeutic efficacy of arsenic compounds targeting metabolic pathways in B-cell acute lymphoblastic leukemia (B-ALL) cells is scarce. MethodsA compound extracted from realgar, tetraarsenic tetrasulfide (As4S4), and its antitumor effects on B-ALL were experimentally examined in vitro and in vivo. ResultsAs4S4 apparently targets B-ALL cells by inducing specific cellular responses, including apoptosis, G2/M arrest, and ferroptosis. Interestingly, these effects are attributed to reactive oxygen species (ROS) accumulation, and increased ROS levels have been linked to both the mitochondria-dependent caspase cascade and the activation of p53 signaling. The ROS scavenger N-acetylcysteine (NAC) can counteract the effects of As4S4 treatment on Nalm-6 and RS4;11 cells. Specifically, by targeting Hexokinase-2 (HK2), As4S4 induces alterations in mitochondrial membrane potential and disrupts glucose metabolism, leading to ROS accumulation, and was shown to inhibit B-ALL cell proliferation in vitro and in vivo. Intriguingly, overexpression of HK2 can partially desensitize B-ALL cells to As4S4 treatment. ConclusionTetraarsenic tetrasulfide can regulate the Warburg effect by controlling HK2 expression, a finding that provides both new mechanistic insight into metabolic alterations and pharmacological evidence for the clinical treatment of B-ALL.

Selective Targeting of TP53-Y220C Mutant AML By PC14586 Results in TP53 Wild-Type Conformation and Synergistical Apoptosis Induction By Concomitant Inhibition of Xpo-1, MDM2, or Bcl-2

Patients with TP53-mutant acute myeloid leukemia (AML) constitute the worst outcome group with a survival of only several months. TP53 mutations frequently become dominant following exposure to chemotherapeutic agents, Bcl-2 or MDM2 inhibitors, and confer resistance to multiple other therapies. Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS (Gener-Ricoset al., ASH 2022). PC14586 (PMV Pharmaceuticals) is designed to bind to the p53-Y220C mutant and restore wild-type (WT) p53 protein conformation and function. PC14586 has been shown to convert Y220C-p53 mutant into WT-p53 protein conformation, induce the activation of p53 transcriptional pathways resulting in selective inhibition of proliferation ofTP53-Y220C mutant solid tumor cell lines and achieved partial responses and stable disease in an ongoing phase I clinical trial in solid tumors (Dumbrava, ASCO 2022, NCT04585750). However, the activity of PC14586 in TP53-Y220C mutated AML/MDS has not been investigated, either as monotherapy or in mechanism-based drug combinations. We investigated the mechanisms of action of PC14586 in AML using cells with TP53-WT, knockout (KO), and mutations (one allele with Y220C or R175H and the other truncated) generated by CRISPR (Boettcher et al., Science 2019) and found that PC14586 converts Y220C mutant p53 to the active form of p53 and selectively activates p53 signaling proteins including MDM2 and p21 in TP53-Y220C AML cells. PC14586 decreased cell viability but had limited apoptogenic activities in the TP53-Y220C AML cells . In primary AML cells with Y220C mutations, PC14586 induced cell death in bulk AML cells and, importantly, also in stem/progenitor cells in 2/3 samples. We reported previously that sustained nuclear retention of p53 by XPO-1 inhibition greatly enhances the transcriptional activity of activated p53 and that activation of p53 by inhibiting its negative regulator MDM2 effectively induces cell death in TP53-WT AML (Kojima et al., Blood 2013, 2005), and that XPO-1 inhibition vastly increased apoptosis induced by MDM2 inhibition through 20-60-fold increased p53 transcriptional activities. We also reported that combined Bcl-2 inhibition and p53 activation is synthetically lethal in TP53-WT AML (Pan et al., Cancer Cell 2017). We hypothesized that XPO-1 synergizes with PC14586 by retaining PC14586-activated p53 in the nucleus and that inhibition of MDM2 induced by PC14586-mediated p53 activation or inhibition of Bcl-2 synergizes with PC14586 in TP53-Y220C AML cells. Indeed, we found that PC14586-induced p53 target proteins were further upregulated when PC14586 was combined with XPO-1 or MDM2 inhibition. Inhibition of XPO-1, MDM2, or Bcl-2 synergistically induced massive apoptosis and decreased cell viability in PC14586 treated TP53 Y220C mutant AML cells (Fig.1), in primary AML blasts, and in CD34+CD38- stem/progenitor cells but not in normal bone marrow cells or stem/progenitor cells through conformational changes of mutant p53 to p53-WT. While PC14586 had limited activity on proliferation in TP53-Y220C AML cells, the combination with Bcl-2 inhibition largely blocked G1-S transition and enhanced apoptosis. The G1-S transition block and apoptosis induction were even more pronounced when PC14586 was combined with XPO-1 or MDM2 inhibitors as determined by flow cytometry measurement of DNA content, and PARP cleavage. Experiments in TP53-Y220C AML PDX models are ongoing and will be reported. In summary, PC14586 converts Y220C-mutant p53 into WT-p53 conformation and induces p53 target proteins. Mechanism-based combinations with XPO-1, MDM2, and Bcl-2 inhibitors induce massive apoptosis. PC14586 is presently in early clinical trials.

Tumor-suppressive zinc finger protein 24 (ZNF24) sensitizes colorectal cancer cells to 5-fluorouracil by inhibiting the Wnt pathway and activating the p53 signaling

Carcinogenesis and colorectal cancer (CRC) development are associated with dysregulation of various pathways, including Wnt and p53. 5-fluorouracil (5-FU) is a common chemotherapeutic agent for CRC treatment, but its efficacy is restricted by drug resistance. Doxycycline is an orally active tetracycline antibiotic known for its antimicrobial and anticancer cell proliferation activities. This study intends to delineate the potential role of bioinformatically predicted ZNF24 in the 5-FU resistance of CRC cells. The expression of ZNF24 was measured in clinically collected CRC tissues and cells. Afterward, ectopic ZNF24 expression was induced by DOX to evaluate the viability, colony-forming ability and sphere-forming ability of CRC cells. It was found that ZNF24 was validated to be poorly expressed in CRC tissues, and ectopic expression of ZNF24 was revealed to restrict the malignant phenotypes of CRC cells. In addition, restored ZNF24 attenuated 5-FU resistance of CRC cells by inhibiting the Wnt pathway and activating p53 signaling. Furthermore, an inhibitor of Wnt production 2 (IWP-2) treatment was an alternative to ZNF24 up-regulation in sensitizing CRC cells to 5-FU treatment. In conclusion, our results indicate that ZNF24 inhibits 5-FU resistance of CRC cells by suppressing the Wnt pathway and activating p53 signaling, which offers a potential strategy for managing chemoresistance in CRC.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML

AbstractAcute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the “don’t-eat-me” signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

Licochalcone A induces G2/M phase arrest and apoptosis via regulating p53 pathways in esophageal cancer: In-vitro and in-vivo study

Licochalcone A (LCA) is a flavonoid isolated from Glycyrrhiza uralensis Fisch that has shown promising therapeutic effects in various cancers. This study attempted to analyze its therapeutic potential for esophageal cancer (EC). Combining multiple databases and network pharmacology, we found that the mechanism of LCA inhibiting EC may be closely related to p53 signaling pathway, cell cycle regulation and apoptosis. Molecular docking was then used to predict the affinity between LCA and key targets. Subsequently, we selected three common EC cell lines for in vitro validation. LCA treatment significantly inhibited EC cell proliferation and colony formation. Wound healing and transwell assay showed that LCA can reduce the migration and invasion of EC cells, and down-regulated the expression of matrix metalloproteinases (MMP). LCA promoted excessive ROS production, decreased mitochondrial membrane potential, and upregulated the expression of Bax, Caspase3 and Caspase-9, all of which are involved in apoptosis. LCA treatment blocked the cell cycle in G2/M phase and decreased the expression of cyclin D1, cyclin B1, and CDK1. LCA significantly up-regulated p53 protein and gene expression, thereby inducing apoptosis and cycle arrest. Finally, the xenograft tumor model was established by subcutaneous injection of Eca-109 cells. LCA administration inhibited tumor growth by activating p53 signaling pathways and apoptosis. Meanwhile, there was no significant weight loss and few major organotoxicity and hematotoxicity. In conclusion, LCA is an excellent candidate for EC treatment by regulating p53 pathway to induce G2/M phase arrest and apoptosis.

Sesamol serves as a p53 stabilizer to relieve rheumatoid arthritis progression and inhibits the growth of synovial organoids

BackgroundRheumatoid arthritis (RA) is an autoimmune disease known as a leading cause of disability with considerable mortality. Developing alternative drugs and targets for RA treatment is an urgent issue. Sesamol is a phenolic compound isolated from natural food sesame (Sesamum indicum L.) with various biological activities. PurposeThe current research intended to illuminate the bioactivity and mechanisms of sesamol in RA fibroblast-like synoviocytes (FLS), and aimed to estimate the potential clinical application value of sesamol in RA treatment. MethodsCCK-8, EdU, and flow cytometry assays, as well as transwell tests were applied to observe the effects of sesamol on the abnormal functions of RA-FLS. Moreover, synovial organoids and a collagen-induced arthritis (CIA) mouse model were constructed to further explore the therapeutic capacity of sesamol on RA. Furthermore, RNA sequencing combined with quantitative real-time PCR assay, Western blot as well as co-immunoprecipitation were employed to clarify the mechanism of sesamol in regulating RA progression. ResultsSesamol suppressed the proliferation through inhibiting DNA replication, triggering cell cycle arrest and apoptosis of RA-FLS. Besides, sesamol impaired RA-FLS migration and invasion. Interestingly, sesamol inhibited the growth of constructed synovial organoids and alleviated RA symptoms in CIA mice. Moreover, RNA sequencing further implicated p53 signaling as a downstream pathway of sesamol. Furthermore, sesamol was shown to decrease p53 ubiquitination and degradation, thereby activating p53 signaling. Finally, bioinformatics analyses also highlighted the importance of sesamol-regulated networks in the progression of RA. ConclusionsOur investigation demonstrated that sesamol served as a novel p53 stabilizer to attenuate the abnormal functions of RA-FLS via facilitating the activation of p53 signaling. Moreover, our study highlighted that sesamol might be an effective lead compound or candidate drug and p53 could be a promising target for the therapy of RA.

Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

The molecular etiology of idiopathic pulmonary fibrosis (IPF) has been extensively investigated to identify new therapeutic targets. Although anti-inflammatory treatments are not effective for patients with IPF, damaged alveolar epithelial cells play a critical role in lung fibrogenesis. Here, we establish an organoid-based lung fibrosis model using mouse and human lung tissues to assess the direct communication between damaged alveolar type II (AT2)-lineage cells and lung fibroblasts by excluding immune cells. Using this in vitro model and mouse genetics, we demonstrate that bleomycin causes DNA damage and activates p53 signaling in AT2-lineage cells, leading to AT2-to-AT1 transition-like state with a senescence-associated secretory phenotype (SASP). Among SASP-related factors, TGF-β plays an exclusive role in promoting lung fibroblast-to-myofibroblast differentiation. Moreover, the autocrine TGF-β-positive feedback loop in AT2-lineage cells is a critical cellular system in non-inflammatory lung fibrogenesis. These findings provide insights into the mechanism of IPF and potential therapeutic targets.

FAAP100 is required for the resolution of transcription-replication conflicts in primordial germ cells

BackgroundThe maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored.ResultsWe find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice.ConclusionsOur findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.

Discovery of Sulanemadlin (ALRN-6924), the First Cell-Permeating, Stabilized α-Helical Peptide in Clinical Development.

We report the discovery of sulanemadlin (ALRN-6924), the first cell-permeating, stabilized α-helical peptide to enter clinical trials. ALRN-6924 is a "stapled peptide" that mimics the N-terminal domain of the p53 tumor suppressor protein. It binds with high affinity to both MDM2 and MDMX (also known as MDM4), the endogenous inhibitors of p53, to activate p53 signaling in cells having a non-mutant, or wild-type TP53 genotype (TP53-WT). Iterative structure-activity optimization endowed ALRN-6924 with favorable cell permeability, solubility, and pharmacokinetic and safety profiles. Intracellular proteolysis of ALRN-6924 forms a long-acting active metabolite with potent MDM2 and MDMX binding affinity and slow dissociation kinetics. At high doses, ALRN-6924 exhibits on-mechanism anticancer activity in TP53-WT tumor models. At lower doses, ALRN-6924 transiently arrests the cell cycle in healthy tissues to protect them from chemotherapy without protecting the TP53-mutant cancer cells. These results support the continued clinical evaluation of ALRN-6924 as an anticancer and chemoprotection agent.

TOM40 regulates the progression of nasopharyngeal carcinoma through ROS-mediated AKT/mTOR and p53 signaling

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southern China, North Africa, and Southeast Asia. The translocase of the outer membrane (TOM) 40 is a transporter of mitochondrial proteins, and is involved in ovarian cancer cell growth. However, its role in the progression of NPC is still unclear. We found that TOM40 levels were upregulated in NPC tissues and multiple NPC cell lines. In addition, high TOM40 expression in the tumor tissues was associated with poor overall survival and disease specific survival. TOM40 knockdown in the NPC cell lines inhibited their proliferation in vitro and in vivo. Furthermore, TOM40 silencing also increased intracellular production of reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Mechanistically, the anti-tumor effects of TOM40 silencing were dependent on the inhibition of AKT/mTOR signaling and activation of p53 signaling. To summarize, TOM40 mediates NPC progression through ROS-mediated AKT/mTOR and p53 signaling. Our findings highlight the potential of TOM40 as a therapeutic target for NPC.

Streptozotocin induces renal proximal tubular injury through p53 signaling activation

Streptozotocin (STZ), an anti-cancer drug that is primarily used to treat neuroendocrine tumors (NETs) in clinical settings, is incorporated into pancreatic β-cells or proximal tubular epithelial cells through the glucose transporter, GLUT2. However, its cytotoxic effects on kidney cells have been underestimated and the underlying mechanisms remain unclear. We herein demonstrated that DNA damage and subsequent p53 signaling were responsible for the development of STZ-induced tubular epithelial injury. We detected tubular epithelial DNA damage in NET patients treated with STZ. Unbiased transcriptomics of STZ-treated tubular epithelial cells in vitro showed the activation of the p53 signaling pathway. STZ induced DNA damage and activated p53 signaling in vivo in a dose-dependent manner, resulting in reduced membrane transporters. The pharmacological inhibition of p53 and sodium-glucose transporter 2 (SGLT2) mitigated STZ-induced epithelial injury. However, the cytotoxic effects of STZ on pancreatic β-cells were preserved in SGLT2 inhibitor-treated mice. The present results demonstrate the proximal tubular-specific cytotoxicity of STZ and the underlying mechanisms in vivo. Since the cytotoxic effects of STZ against β-cells were not impaired by dapagliflozin, pretreatment with an SGLT2 inhibitor has potential as a preventative remedy for kidney injury in NET patients treated with STZ.

Abstract 45: Elephant p53 protects mice from carcinogen induced death

Abstract Elephants naturally have low rates of cancer, potentially due to evolved genetic changes in tumor suppressor elephant TP53 (EP53) and amplification of 19 TP53 retrogenes. A better understanding of the mechanisms of cancer suppression in elephants by EP53 and its retrogenes could lead to more effective human cancer therapeutics. EP53 induces a strong apoptotic response compared to human TP53, especially when combined with EP53-RETROGENE 9 (EP53-R9). EP53-R9 encodes a truncated p53 protein that induces apoptosis of human cancer cells independent of EP53 through a transcription-independent mechanism. To characterize EP53 and EP53-R9’s role in cancer suppression, transgenic mice were generated to replace mouse TRP53 with EP53. Additionally, a tetracycline inducible EP53-R9 gene was inserted into a safe harbor locus in mice. Carcinogenesis studies with 3-Methylcholanthene injection revealed that EP53 mice survived significantly longer compared to heterozygous or homozygousTRP53 mice (p <0.0001, 0.0102). Experiments to assess the protective role of EP53-R9 alone and combined with EP53 are ongoing. Several mouse embryonic fibroblast (MEF) cell lines were generated from these transgenic mice with 14 distinct genotypes with different combinations of TRP53, EP53, and EP53-R9. p53 target gene expression studies showed that MEFs with EP53 induce higher expression of MDM2 and p21 compared to TRP53-containing MEFs, suggesting that the cancer-protective effect observed in EP53 mice is due, in part, to greater activation of p53 signaling. These results support the need for future work to assess the potential of EP53-based therapeutics for human cancer. Citation Format: Lisa M. Abegglen, Jared S. Fowles, Aidan J. Preston, Aaron Rogers, Niraja Bhachech, Brayden B. Barney, Ryan Kennington, David H. Lum, Gareth Mitchell, Joshua D. Schiffman. Elephant p53 protects mice from carcinogen induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 45.