UBE2T-Mediated HP1α Ubiquitination Enhances Nucleolar Function and Promotes the Progression of IDH1/TP53-Mutant Glioma

Abstract

Purpose:

High-grade glioma is the most aggressive form of primary brain tumor, characterized by rapid progression and a grim prognosis. The presence of mutations in IDH1 and TP53 is associated with a specific molecular phenotype in glioma, and their interaction is a potential target for therapy.

Experimental Design:

Our study utilized a combination of bioinformatics analysis, in vitro experiments, and in vivo tumor xenograft models to investigate the role of the ubiquitin-conjugating enzyme 2T (UBE2T) in the malignant progression of IDH1/TP53-mutant glioma.

Results:

We found that UBE2T is overexpressed in the context of TP53 mutations and is linked to enhanced glioma cell proliferation and stemness. Mechanistically, UBE2T was shown to degrade HP1α via the ubiquitin–proteasome pathway, leading to the release of the suppressive effects of R-2-hydroxyglutarate on nucleolar function and an increase in rDNA transcription. The therapeutic potential of targeting UBE2T is underscored by the discovery that APR-246, a mutant p53 reactivator, effectively suppresses UBE2T expression and reverses the hyperactivity of nucleolar transcription.

Conclusions:

These findings suggest that UBE2T plays a crucial role in the progression of IDH1/TP53-mutant astrocytoma and that targeting UBE2T with APR-246 could be a promising therapeutic strategy for patients with these mutations. Our study provides a foundation for further research into the role of UBE2T in glioma and the development of targeted therapies.

Translational Relevance.

This study provides critical insights into the role of ubiquitin-conjugating enzyme 2T (UBE2T) in IDH1/TP53-mutant glioma, a devastating disease with limited effective treatment options. We demonstrate that UBE2T, when overexpressed as a result of TP53 mutations, promotes glioma progression by enhancing nucleolar function and cell proliferation. The use of APR-246 to inhibit UBE2T presents a promising therapeutic strategy. This research not only advances our molecular understanding of glioma but also has direct translational implications, offering hope for more effective and personalized treatments for patients with this aggressive cancer.

Introduction

Gliomas (World Health Organization grades 1–4) are the most common primary brain tumors in adults and can be categorized into three main types: astrocytomas, oligodendrogliomas, and ependymomas. Astrocytomas, which originate from astrocytes, are further divided into low- and high-grade forms, with glioblastoma multiforme (GBM) being the most aggressive subtype (1–3). High-grade astrocytomas have a grim prognosis with median survival times of less than 2 years despite intensive treatments such as surgery, radiation, and chemotherapy (3–5). Obviously, there is an urgent demand for more effective and personalized treatments for patients with glioma. Astrocytomas commonly exhibit various genetic mutations, including IDH1/2, TP53, EGFR, ATRX, and TERT, among others (6–9). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations, particularly at the R132 residue of IDH1, are found in around 75% of grade 2/3 astrocytomas and 10% of grade 4 astrocytomas, serving as key molecular diagnostic markers (10, 11). IDH1-mutant glioma forms a distinct molecular subgroup of brain tumors with a unique metabolic profile and epigenetic landscape (12–14). The mutation in the IDH1 protein results in the production of the oncometabolite R-2-hydroxyglutarate (R-2HG), which disrupts normal cellular processes, notably affecting DNA and histone demethylation, leading to epigenetic and metabolic alterations (15–17).

Patients with mutIDH1 gliomas have a comparatively longer-term survival, but nearly all low-grade tumors progress into rapidly proliferating, lethal high-grade gliomas (18–21). Therefore, understanding the molecular mechanisms underlying malignant progression in mutIDH1 gliomas is crucial for developing targeted therapies. The relationship between TP53 and IDH1 in mutIDH1 gliomas is intricate and can have profound implications for tumor behavior (20–22). The presence of both TP53 and IDH1 mutations often indicates a poor prognosis and suggests the existence of synergistic mechanisms driving malignant progression, which are not yet fully understood (23–26). Delving deeper into the complex interactions between IDH1 and TP53 could reveal new therapeutic approaches to disrupt these collaborative processes.

Ubiquitin-conjugating enzyme 2T (UBE2T) plays a central role in the Fanconi anemia (FA) pathway (27). As an E2 ubiquitin-binding enzyme and E3 ligase, FANCL co-catalyzes the monoubiquitination of the FANCD2/FANCI heterodimer, thereby activating the DNA cross-linking damage repair mechanism and maintaining genome stability (27–29). However, in tumors, UBE2T is frequently expressed at abnormally high levels and exhibits oncogenic functions. Mechanistically, the hyperactivation of UBE2T promotes tumor progression through noncanonical ubiquitination, which suppresses tumor suppressors or activates oncogenic signals such as the Wnt/β-catenin and PI3K/AKT pathways (30–32). The regulatory relationship between p53 and UBE2T is bidirectional. Jaber and Liu’s findings illustrate a complex interplay between UBE2T and p53, wherein p53 can downregulate the FA pathway–related genes, including UBE2T, whereas UBE2T can target p53 for ubiquitination and degradation, thereby influencing cellular transformation and tumorigenesis (33–35). Our research revealed that mutTP53 upregulates the expression of UBE2T, resulting in enhanced proliferation and stemness of glioma cells. Moreover, UBE2T was identified as degrading HP1α and counteracting the inhibitory effects of R-2HG on nucleolar function. Additionally, our findings demonstrate that the mutant TP53 reactivator APR-246 successfully suppresses UBE2T expression, offering a potential therapeutic strategy for glioma.

Materials and Methods

Ethical approval and consent to participate

Tumor specimens were collected from the Second Affiliated Hospital of Zhejiang University’s School of Medicine in accordance with Institutional Review Board protocol IR2024521. Prior written informed consent was obtained from all patients or their legal guardians for research purposes. This research was carried out in accordance with the Declaration of Helsinki and observed all relevant ethical standards. Animal experiments were approved by the Animal Management Rule of the Chinese Ministry of Health and were performed in accordance with the approved guidelines and experimental protocol of Nanjing University. All animal experiments were in conformity with the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).

Reagents, patient tissue specimens, and cell lines

DMSO (Cat. # HY-Y0320), nutlin-3a (Cat. # HY-10029), APR-246 (Cat. # HY-19980), cycloheximide (Cat. # HY-12320), bafilomycin A1 (Cat. # HY-100558), 3-methyladenine (Cat. # HY-19312), Z-VAD (Cat. # HY-16658B), MG132 (Cat. # HY-13259), and TFMB-(R)-2HG (Cat. # HY-129079) were purchased from MedChemExpress. This study was approved by the Institutional Review Board and ethics committee of Nanjing University. The human GBM cell lines U251MG (RRID: CVCL_0021) and U87MG (RRID: CVCL_0022) were purchased from GeneChem and were validated by short tandem repeat DNA fingerprinting. The primary glioma cell lines SG2 (mutIDH1/mutTP53 grade 4 astrocyte), GBM22 (wtIDH1/mutTP53 GBM), and GBM30 (wtIDH1/wtTP53 GBM), derived from surgical specimens, are stored in liquid nitrogen (36, 37). For research, SG2, GBM22, and GBM30 are cultured in DMEM supplemented with 10% FBS. The details of GBM30 are shown in Supplementary Fig. S1A and S1B. The expression level of the UBE2T protein in the cell lines utilized in this experiment is illustrated in Supplementary Fig. S1C. Cell origin and mutation information are summarized in Supplementary Table S1. We used the Myco-Lumi Luminescent Mycoplasma Detection Kit (Beyotime) to detect mycoplasma in the cytoplasm. The latest test date was March 1, 2025, and the cell test results used in the experiment were negative.

Western blotting, immunofluorescence, and IHC

Western blotting, immunofluorescence (IF), and IHC were performed as described previously (37). Antibodies against UBE2T (Cat. # ab179802, RRID: AB_3677673), fibrillarin (Cat. # ab4566, RRID: AB_304523), TRIP12 (Cat. # ab86220, RRID: AB_1925533), HP1α (Cat. # ab234085, RRID: AB_3677674), linkage-specific K48 (Cat. # ab140601, RRID:AB_2783797), H3K9me3 (Cat. # ab8898, RRID: AB_306848), Goat Anti-Rabbit IgG H&L (Alexa Fluor 488, Cat. # ab150077, RRID: AB_2630356), Goat Anti-Mouse IgG H&L (Alexa Fluor 488, Cat. # ab150113, RRID: AB_2576208), Goat Anti-Rabbit IgG H&L (Alexa Fluor 594, Cat. # ab150084, RRID: AB_2734147), and Goat Anti-Mouse IgG H&L (Alexa Fluor 594, Cat. # ab150116, RRID: AB_2650601) were purchased from Abcam. Antibodies against p53 (Cat. # 9282, RRID: AB_331476), vinculin (Cat. # 13901, RRID:AB_2728768), β-tubulin (Cat. # 2146, RRID: AB_2210545), Ki-67 (Cat. # 9449, RRID:AB_2797703), Myc-Tag (Cat. # 2278, RRID: AB_490778), HA-Tag (Cat. # 3724, RRID:AB_1549585), His-Tag (Cat. # 12698, RRID: AB_2744546), Anti-rabbit IgG HRP-linked Antibody (Cat. # 7074, RRID: AB_2099233), and Anti-mouse IgG HRP-linked Antibody (Cat. # 7076, RRID: AB_330924) were purchased from Cell Signaling Technology. The antibody against Flag-tag (Cat. # F1804, RRID: AB_262044) was purchased from Sigma-Aldrich. Nucleolus Bright Red reagent (Cat. # N512) was purchased from Dojindo.

Plasmids, siRNA, short hairpin RNA, and cell transfections

Plasmids, short hairpin RNA, and corresponding lentiviral vectors were purchased from GeneChem and were performed as described previously (37). X-tremeGENE HP reagent (Cat. # XTGHP-RO), X-tremeGENE 9 reagent (Cat. # XTG9-RO), and X-tremeGENE siRNA reagent (Cat. # SITRAN-RO) were purchased from Sigma-Aldrich. Transduced cells were selected for puromycin dihydrochloride (10 μg/mL, Cat. # HY-B1743A, MedChemExpress) resistance for 1 week. The knockdown efficiency verification results of short hairpin RNA of three sequences on UBE2T expression are shown in Supplementary Fig. S1D.

5-Ethynyl-29-deoxyuridine assay, Cell Counting Kit-8-8 assay, and flow cytometric analysis

5-Ethynyl-29-deoxyuridine (EdU) assay, Cell Counting Kit-8 (CCK-8) assay, and flow cytometric analysis were performed as described previously (37). Click-iT EdU Alexa Fluor 594 Imaging Kit (Cat. # C10339) was purchased from Thermo Fisher Scientific. Annexin V-APC/7-AAD staining kit (Cat. # KGA9101) was purchased from KeyGEN BioTECH. CCK-8 assay (Cat. # CK04) was purchased from Dojindo (37).

Glioma stem cell culture and extreme limiting dilution analysis were performed as described previously

RNA extraction, real-time PCR, chromatin immunoprecipitation, and RNA sequencing

RNA extraction, real-time PCR, and chromatin immunoprecipitation (ChIP) were performed as described previously (37). Primer sequences were listed in Supplementary Tables S2 and S3. RNA sequencing transcriptome analysis was conducted by BGI Genomics. In this project, we sequenced six samples using the DNBSEQ platform, generating an average of about 1.19 Gb bases per sample. The average mapping ratio with the reference genome is 92.61%, and the average mapping ratio with genes is 89.64%; 16,634 genes were identified. The ChIP was conducted using the EZ ChIP Chromatin Immunoprecipitation Kit (Cat. # 17-371, Millipore) following the manufacturer’s instructions. Briefly, 1.5 million cells from two 10 cm plates (approximately 10 μg genomic DNA) were cross-linked with 1% formaldehyde for 10 minutes at room temperature, followed by glycine addition (0.125 mol/L). The cells were then washed with ice-cold PBS, lysed in SDS lysis buffer, and sonicated with an ultrasonic cell breaker ATPIO-650D from Nanjing ATPIO Instruments using specific parameters. After centrifugation, a control aliquot was saved, and the supernatant was diluted fivefold in ChIP dilution buffer. The diluted chromatin was incubated with the specified antibodies or IgG (ab171870, Abcam) overnight at 4°C on a rotating platform. Protein G agarose beads were then added, followed by overnight incubation at 4°C with rotation. Subsequent steps included washing the beads, de-cross-linking DNA/protein cross-links, and column purification to recover the precipitated DNA for qPCR analysis. The ChIP-PCR primers used were listed in Supplementary Table S1, and the ChIP-qPCR data were analyzed using the fold enrichment method.

Immunoprecipitation, immunoprecipitation–mass spectrometry, and isobaric tags for relative and absolute quantitation (iTRAQ)

The immunoprecipitation (IP) procedure was carried out in accordance with our previous study (37). Initially, cells were lysed using lysis buffer containing phenylmethylsulfonyl fluoride. Subsequently, an equal amount of protein was incubated with the antibody overnight at 4°C. The protein–antibody complexes were then isolated using Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). Finally, the protein underwent Western blotting or mass spectrometry analysis. The iTRAQ quantification project and IP–mass spectrometry (IP-MS) were conducted by BGI Genomics. In the iTRAQ quantification project, a total of 942,124 spectra were generated; 56,873 peptides and 7,135 proteins were identified with a 1% FDR. In the IP-MS project, the mass spectrometer Q Exactive HF-X was used. A total of 36,660 spectra were obtained from the sample group igG_101_1. After identification by the search engine, 1,392 spectra were matched, and 340 proteins and 1,052 peptides were identified. A total of 37,850 spectra were obtained from the sample group igG_102_1. After identification by the search engine, 1,293 spectra were matched, and 344 proteins and 1,005 peptides were identified. A total of 36,982 spectra were obtained from the sample group igG_1_1. After identification by the search engine, 1,171 spectra were matched, and 278 proteins and 886 peptides were identified. A total of 54,634 spectra were obtained from the sample group Flag_201_1. After identification by the search engine, 9,864 spectra were matched, and 1,405 proteins and 7,211 peptides were identified. A total of 55,387 spectra were obtained from the sample group Flag_202_1. After identification by the search engine, 11,802 spectra were matched, and 1,603 proteins and 8,644 peptides were identified. A total of 58,233 spectra were obtained from the sample group Flag_2_1. After identification by the search engine, 16,639 spectra were matched, and 1,933 proteins and 11,890 peptides were identified.

Tumor xenograft model

Murine xenograft models of human GBM were conducted according to previously established methods (37). The animal experiments were approved by the Animal Management Rule of the Chinese Ministry of Health and were carried out in compliance with the guidelines and experimental protocol of Nanjing University. All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011). In brief, six-week-old female nude mice were surgically implanted with 1 × 10⁶ luciferase-expressing GBM cells per mouse. Tumor growth was monitored weekly using a live animal bioluminescence imaging system. Mice were humanely euthanized (by cervical dislocation following CO₂ anesthesia) upon exhibiting severe signs of tumor formation such as hunching, weight loss, and rough coat. The whole brains were then excised, fixed in 4% formalin, and embedded in paraffin for further analysis.

Transmission electron microscopy analysis

Cells were fixed and dehydrated before being embedded in EMbed 812. Ultrathin sections were obtained using a Leica UC7 ultramicrotome, stained with uranyl acetate and lead citrate, and examined at a magnification of 6,000× using a HITACHI HT7800 transmission electron microscope. All procedures were conducted in accordance with established transmission electron microscopy protocols.

Molecular docking

Molecular docking was employed to evaluate the binding activity of protein–protein interactions. The HDOCK online platform (http://hdock.phys.hust.edu.cn/) served as the molecular docking program for this study. HDOCK is capable of analyzing various conformations of protein–protein docking, assessing binding activities across these conformations, and identifying amino acid residues with interaction distances within 5 Å. The structural file of the HP1a adapter protein was obtained from the AlphaFold protein database, whereas the three-dimensional structure file of the UBE2T protein was retrieved from the Protein Data Bank. LigPlus software was utilized to analyze the interactions between the two proteins from a two-dimensional perspective. Additionally, PyMOL (version 4.3.0, RRID:SCR_000305) was used to visualize the interacting amino acid residues between the two proteins.

Statistical analysis

Data were analyzed using GraphPad 10.1.2 software (RRID:SCR_002798) and reported as the mean ± SD of triplicates. Significance was determined through a two-tailed Student t test or ANOVA for multivariate analysis. Statistical significance of Kaplan–Meier survival curves was assessed using a log-rank (Mantel–Cox) analysis. A significance level of P < 0.05 was considered significant for all tests, with *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 denoting increasing levels of significance.

Data availability

The RNA sequencing data generated in this study have been deposited in the NCBI database under the accession number PRJNA1269433. The mass spectrometry proteomics data, including both the differential proteomics and IP-MS datasets, have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the identifier PXD064431. All data are publicly available as of the date of publication. Additionally, supplementary data supporting the findings are provided as supplementary materials. For further information or access to additional data, please contact the corresponding author.

Results

High expression of UBE2T is associated with TP53 mutation and promotes glioma proliferation

Given the pivotal role of p53 as a transcription factor and the potential close relationship between IDH1 mutation, our study aimed to investigate how TP53 mutation status might affect the pathogenic mechanism of IDH1 mutation in glioma. Importantly, analysis of the Chinese Glioma Genome Atlas (CGGA) dataset indicated that TP53 mutations were specifically prognostically significant in IDH1-mutant astrocytoma (Fig. 1A). In The Cancer Genome Atlas (TCGA) database, the TP53 mutation status does not predict the prognosis of patients with astrocytoma, regardless of their IDH1 mutation status (Supplementary Fig. S2A). This lack of predictive capability may be attributed to the limited number of cases exhibiting TP53 mutations. Previous research has demonstrated that mutant p53 not only disrupts the normal tumor-suppressive function of wild-type p53 (dominant negative mutation) but also may sometimes acquire new oncogenic functions (gain of function). We conducted a screening for gene sets that exhibited a significant positive correlation with TP53 mRNA expression in samples with wild-type or mutant TP53. The comparison of these gene sets showed partial overlap (Fig. 1B, Venn diagram). Further enrichment analysis revealed that the pathways associated with these gene sets were not entirely identical; notably, the cell-cycle checkpoint pathway was uniquely enriched in genes that were positively correlated with mutant TP53 [Fig. 1B (right)]. TP53 controls cell-cycle checkpoints and determines cell fate through various mechanisms, including the regulation of DNA damage repair pathways. Typically, TP53 activates multiple DNA repair pathways, but literature has reported that wild-type p53 inhibits the FA DNA repair pathway (33). FA is a hereditary disorder linked to impaired DNA damage repair, resulting in a significantly increased risk of hematologic malignancies and other cancers (38). The FA DNA repair pathway plays a crucial role in DNA damage repair and tumorigenesis (39). Interestingly, our findings revealed that the expression levels of certain FA pathway genes were notably elevated in mutTP53 glioma samples compared with wtTP53 samples [Supplementary Fig. S2B (left)], and high expression of FA-related genes was correlated with poor prognosis [Supplementary Fig. S2B (right)]. UBE2T is a crucial gene in the FA pathway. CGGA/TCGA-based data analysis revealed that the correlation between UBE2T and TP53 transcription levels was significantly stronger in mutTP53 gliomas compared with wild-type gliomas (Fig. 1C; Supplementary Fig. S2C). We found that glioma cases with high UBE2T expression in the context of mutIDH1 had poorer prognosis [Fig. 1D (left)] and UBE2T expression increased as glioma grade advanced [Fig. 1D (right)]. Moreover, this trend has been validated in both the TCGA and Gravendeel databases (Supplementary Fig. S2D). Additionally, we verified that the staining intensity of UBE2T in primary low-grade gliomas (grade 2) was significantly lower than that in relapsed gliomas (grade 4), as observed in samples from three pairs of mutIDH1 gliomas (Supplementary Fig. S2E). However, data from CGGA and TCGA indicated that the mutation status of IDH1 had a minimal impact on the correlation coefficient between IDH1 and UBE2T expression (Supplementary Fig. S2F). Furthermore, our analysis revealed that the overexpression of the IDH1 mutation did not significantly influence UBE2T protein levels (Supplementary Fig. S2G). Therefore, we speculated that the mutation status of TP53, rather than that of IDH1, has a more significant impact on the expression level of UBE2T. IHC analysis confirmed elevated UBE2T levels in mutTP53 grade 4 glioma samples compared with wild type (Fig. 1E). UV radiation led to increased levels of p53 and decreased UBE2T levels in normal human astrocytes (Fig. 1F). Knocking down p53 in wtTP53 cell lines (U87MG and GBM30) resulted in higher UBE2T levels (Fig. 1G). Treatment with the MDM2 inhibitor nutlin-3 decreased UBE2T mRNA and protein levels (Fig. 1H). However, this effect was not observed in mutTP53 cell lines (SG2 and GBM22; Fig. 1I). In conclusion, our results indicated that TP53 mutation can predict the prognosis of IDH1-mutant glioma and influence UBE2T expression. Our study also reveals a significant association between UBE2T and the malignant phenotype of glioma, particularly about proliferation and the maintenance of stemness in vitro (Supplementary Fig. S3A–S3C). Furthermore, the impact of UBE2T on proliferation capacity was evaluated using an orthotopic transplantation model with nude mice and human glioma cells. In vivo bioluminescence assays demonstrated that downregulation of UBE2T suppressed SG2 cell proliferation, whereas overexpression of UBE2T enhanced GBM30 cell proliferation in the intracranial region of nude mice (Fig. 1J; Supplementary Fig. S3D). Survival analysis revealed that nude mice with stable UBE2T knockdown in SG2 cells had prolonged survival compared with the control group, whereas nude mice with stable UBE2T overexpression in GBM30 cells had reduced survival (Fig. 1K). IHC analysis showed that the UBE2T knockdown group exhibited lower Ki67 staining intensity, whereas the UBE2T overexpressed group displayed increased Ki67 expression (Fig. 1L). Overall, our findings demonstrate that UBE2T enhances the proliferative capacity of glioma cells.

Figure 1.

The mutation of TP53 leads to the upregulation of UBE2T in gliomas. A, The CGGA database shows that patients with TP53 mutations in mutIDH1 glioma have a poorer prognosis, whereas the TP53 mutation status in wtIDH1 glioma is unrelated to patient prognosis. NS, P > 0.05; ***, P < 0.001 by log-rank test. B, The unique high-expressed genes associated with TP53 mutation status in mutIDH1 glioma samples from the CGGA dataset are significantly enriched in the cell checkpoint pathway (analyzed on the Metascape website). C, The scatter plot illustrates the correlation between the transcriptional levels of UBE2T and TP53 across different mutation combinations. NS, P > 0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001 by Pearson correlation analysis. D, High expression of UBE2T in mutIDH1 glioma is associated with poor prognosis (***, P < 0.001 by log-rank test), and the expression level of UBE2T increases with higher glioma grades (*, P < 0.05; **, P < 0.01; and ***, P < 0.001 by one-way ANOVA). E, Tissue specimens of World Health Organization grade 4 mutIDH1 gliomas show higher levels of UBE2T protein against the backdrop of mutTP53 compared with wtTP53. F, UVB (30 mJ/cm²) irradiation of normal human astrocytes (NHA) decreases UBE2T protein levels while increasing p53 levels. G, Knockdown of p53 leads to an elevation of UBE2T protein levels in wtTP53 cells. H, Treatment with nutlin-3a significantly inhibits mRNA and protein levels of UBE2T in wtTP53 glioma cell lines (U87MG and GBM30). n = 3 independent experiments. Data are shown as mean ± SEM, ***, P < 0.001 by unpaired t test.I, Nutlin-3a exposure has no significant effect on the expression of UBE2T in mutTP53 cell lines (SG2 and GBM22). n = 3 independent experiments. Data are shown as mean ± SEM; NS, P > 0.05 by unpaired t test.J,In vivo bioluminescence experiments were conducted using orthotopic xenograft models of human glioma cells (SG2 and GBM30) in nude mice. The results demonstrated that downregulation of UBE2T led to inhibited proliferation of SG2 cells, whereas overexpression of UBE2T promoted intracranial growth of GBM30 cells. K, Survival analysis demonstrated that the knockdown of UBE2T prolonged the survival of nude mice, whereas overexpression of UBE2T led to a shortened lifespan of nude mice. n = 5; the significance level was determined by log-rank analysis. L, IHC staining of intracranial tumors in nude mice demonstrated that reducing UBE2T led to decreased protein levels of UBE2T and Ki-67, whereas increasing UBE2T significantly elevated the protein levels of UBE2T and Ki-67. Scale bars, 50 μm. AR, androgen receptor; DEG, differentially expressed genes; GO, Gene Ontology; HAT, histone acetyltransferase; HDAC, histone deacetylase; NS, not significant; R-HSA, Reactome Human Species Annotation; sh, short hairpin; si, small interfering; UBE2T OE, UBE2T overexpression; WP, WikiPathways.

UBE2T knockdown disrupts nucleolar morphology and inhibits rDNA transcription

Recent studies have explored the dysregulation of UBE2T and its potential role as an oncogene in various cancers (30–32, 34). In this study, we delved deeper into the functions of UBE2T in glioma via transcriptomics and proteomics analyses (Fig. 2A). Transcriptomics results indicated that genes upregulated upon UBE2T knockdown were predominantly enriched in epigenetic pathways (Fig. 2B), whereas downregulated genes were mainly associated with cell cytoskeleton and extracellular matrix pathways (Supplementary Fig. S4A and S4B). Proteomics findings revealed that upregulated proteins following UBE2T knockdown were primarily linked to cell cycle–related pathways (Fig. 2C; Supplementary Fig. S4C and S4D). Increased UBE2T levels were found to potentially affect the polyubiquitination process targeting tumor suppressor proteins, such as p53 and BRCA1/2, leading to their degradation by the proteasome. Subsequently, an IP-MS experiment was conducted to identify UBE2T-interacting proteins, revealing associations with ribosome biogenesis and mRNA metabolism (Fig. 2D). Integrating these omics results, we postulated that UBE2T may play a role in the functions of the nucleolus and ribosome. To validate this hypothesis, we examined the distribution of UBE2T using confocal microscopy, uncovering its presence in the nucleoplasm and nucleolus (Fig. 2E). To delve into the impact of UBE2T on nucleolar integrity and function, we decreased UBE2T expression and observed nucleoli using electron microscopy. The results revealed a notable decrease in nucleolar size and increased density in SG2 and GBM22 cells upon UBE2T downregulation (Fig. 2F). Subsequent fluorescent staining with a fibrillarin antibody, a dense fibrillar component marker, confirmed the electron microscopy results and showed a significant reduction in nuclear size upon UBE2T knockdown (Fig. 2G). Furthermore, qPCR analysis demonstrated a marked inhibition in rDNA transcription after UBE2T downregulation (Fig. 2H). In summary, our study highlights the presence of UBE2T within the nucleolus and its crucial role in nucleolar function, with UBE2T knockdown leading to disruption in nucleolar structure and decreased rDNA transcription levels.

Figure 2.

UBE2T knockdown impairs nucleolar structure and rDNA transcription. A, Workflow of the transcriptome [RNA sequencing (RNA-seq)] and proteome (iTRAQ and IP-MS) analyses after knockdown or overexpression of UBE2T in SG2 cells. B, Volcano plot of the RNA-seq results (left), showing the enrichment of significantly upregulated genes (DEG) after UBE2T knockdown in relevant pathways (analyzed by Metascape). C, Volcano plot showing differentially expressed proteins (DEP) detected by proteomics (iTRAQ). Processes that are associated with upregulated DEPs are shown on the right. D, Enrichment analysis revealed that potential UBE2T-interacting proteins identified by IP-MS are functionally associated with ribosome function. Related pathways are marked in red. E, IF confocal microscopy experiments demonstrated colocalization of UBE2T (green) with the nucleolar marker fibrillarin (red) in nucleolar regions (*). Scale bars, 5 μm. F, Electron microscopy observations revealed that the knockdown of UBE2T resulted in reduced nucleolar size in SG2 and GBM22 cells. Scale bars, 2 μm. G, Knockdown of UBE2T led to a reduced distribution of the nucleolar marker fibrillarin. Scale bars, 20 μm. H, Suppression of UBE2T expression decreased the transcription levels of pre-45s rRNA in SG2 and GBM22 cells. n = 3 independent experiments. Data are shown as mean ± SEM and were analyzed by an unpaired t test. FC, fold change; sh, short hairpin.

UBE2T degrades HP1α through the ubiquitin–proteasome pathway

UBE2T, as an E2 ubiquitin–conjugating enzyme, may regulate nucleolar function through protein ubiquitination and degradation. In this study, we identified 16 proteins at the intersection of differentially expressed proteins and IP-MS–enriched proteins, which could be potential targets of UBE2T-mediated ubiquitin degradation (Fig. 3A). Histone modifications, especially H3K9 methylation, are important regulatory mechanisms that inhibit rDNA transcriptional activity, and HP1α plays a key role in the recruitment of proteins involved in this methylation event (40–43). Therefore, our hypothesis suggests that UBE2T promotes rDNA transcription by ubiquitinating and degrading the HP1α protein near rDNA transcription initiation regions, thereby reducing its repressive methylation modifications. Importantly, confocal microscopy revealed colocalization of UBE2T and HP1α in the nucleolar region (Fig. 3B). Furthermore, IP experiments confirmed the protein–protein interaction between UBE2T and HP1α (Fig. 3C). To further verify the specific interaction region between UBE2T and HP1α, we utilized molecular docking to evaluate the binding affinity of these two proteins. Through computer-aided molecular docking analysis, we identified potential interaction sites located within the UBE2T protein, specifically between the 157th and 173rd amino acids [Fig. 3D (left)]. In this analysis, the docking score is −232.64, with a confidence level of 0.8393. A confidence level exceeding 0.7 indicates that the interaction is likely to be stable. Subsequently, we engineered a truncation mutant to confirm the essential domains that facilitate UBE2T–HP1α interactions. IP assays revealed that the truncation of UBE2T disrupted the interaction with HP1α [Fig. 3D (right)]. Next, Western blot assays showed that UBE2T knockdown increased HP1α levels in mutTP53 glioma cells, whereas UBE2T overexpression reduced HP1α levels in wtTP53 glioma cells (Fig. 3E). The effect of UBE2T on HP1α expression occurs at the posttranslational level, as evidenced by cycloheximide treatment showing an accelerated decrease in HP1α with UBE2T overexpression (Fig. 3F). There are multiple pathways for protein degradation. Using appropriate inhibitors, we found that treatment with the proteasome inhibitor MG132 significantly increased HP1α protein levels (Fig. 3G) and was time-dependent (Fig. 3H). Furthermore, IP assays revealed that HP1α ubiquitination levels increased after proteasome inhibition, as indicated by the presence of more HA-Ub in the protein precipitated by the HP1α antibody (Fig. 3I). We constructed a ubiquitin-conjugating enzyme function-deficient mutant of UBE2T (UBE2T-C86A). Overexpression of Flag-UBE2T C86A did not affect HP1α protein levels compared with the wild type (Fig. 3J). Importantly, the IP assay demonstrated that overexpression of the loss of function–mutant UBE2T led to a significant reduction in HP1α ubiquitination levels (Fig. 3K). We also found that overexpression of UBE2T increases K48-associated ubiquitination, a key feature of proteasomal degradation in HP1α (Fig. 3L). Our results reveal the mechanism by which UBE2T promotes rDNA transcription through HP1α ubiquitination and degradation.

Figure 3.

UBE2T negatively regulates HP1α protein levels via ubiquitination and subsequent proteasomal degradation. A, Venn diagram showing the intersection between differentially upregulated proteins identified by iTRAQ and potential UBE2T interacting proteins identified by IP-MS. B, The photos taken by confocal microscopy demonstrate a colocalization of UBE2T (green) and HP1α (red) in nucleolar regions (*). Scale bars, 5 μm. C, Flag-UBE2T and Myc-HP1α expression in SG2 cells was detected by Western blotting after coimmunoprecipitation (CoIP) with appropriate antibodies. D, Molecular docking studies of UBE2T and HP1α combinations revealed potential interaction regions in the UBE2T sequence, specifically between amino acids 157 and 173. Truncated mutant expression plasmids were transfected into SG2 cells, followed by an IP assay. E, The protein levels of UBE2T and HP1α are detected after UBE2T knockdown in mutTP53 cell lines SG2 and GBM22 cells (left); the expression of UBE2T and HP1α is examined after UBE2T overexpression in wtTP53 cell lines U87MG and GBM30 cells (right). F, SG2 and GBM22 cells were transfected with the indicated plasmids and then treated with cycloheximide (CHX, 50 μg/mL) for the indicated times. Then, cell lysates were examined by immunoblotting. G, A Western blot assay was performed to detect the protein levels of HP1α after treatment with inhibitors of different protein degradation pathways [autophagy inhibitors bafilomycin A1 (Baf-A1) and 3-methyladenine (3-MA), caspase inhibitor Z-VAD, and proteasome inhibitor MG132]. H, A Western blot assay reveals the changes in HP1α protein levels at different time points (2, 4, and 6 hours) after treatment with 10 µmol/L MG132 in SG2 cells. I, SG2 cells were treated with 10 µmol/L MG132 for 6 hours and harvested for ubiquitination experiments. Cell lysates were immunoprecipitated using anti-HP1α antibodies and immunoblotted (IB) using anti-HA and anti-HP1α antibodies. J, The Western blot assay shows the changes in HP1α protein levels after overexpression of wild-type (WT) or enzymatically inactive mutant (C86A) UBE2T. K, SG2 cells were transfected with the indicated plasmids to extract total protein, and HP1α was precipitated with magnetic beads coupled to anti-Myc antibodies. Ubiquitination of HP1α was detected by IP using anti-Myc antibody and IB using the anti-HA/Myc/Flag antibodies. L, SG2 cells were cotransfected with plasmids encoding Myc-HP1α and HA-Ub with or without the Flag-UBE2T plasmid. Then, cells were lysed with the anti-Myc antibody for IP. Total ubiquitination levels and K48-linked ubiquitination levels were detected by anti-HA or K48-linked specific antibodies. sh, short hairpin.

The E3 ubiquitin ligase TRIP12 cooperates with UBE2T to degrade HP1α

Our findings indicate that UBE2T plays a role in the degradation of HP1α via the ubiquitination pathway; however, the specific E3 ubiquitin ligase that collaborates with UBE2T remains to be identified. Analysis of our IP-MS data revealed six potential E3 ubiquitin ligases as interactors with UBE2T (Fig. 4A). Based on the expression profiles and protein localization of candidate genes in glioma cell lines, we selected TRIP12 for further investigation (Supplementary Fig. S5A–S5C). Initially, confocal microscopy was utilized to assess the subcellular localization of HP1α (red) and TRIP12 (green) in SG2 and GBM22 cells. The observed colocalization of these proteins in the nucleolar region suggests a spatial indication of their potential interaction (Fig. 4B). An IP assay further corroborated the interaction between UBE2T and TRIP12 (Fig. 4C), as well as the interaction between HP1α and TRIP12 (Fig. 4D). Knockdown of TRIP12 resulted in elevated levels of HP1α protein, implying that TRIP12 plays a role in regulating HP1α expression (Fig. 4E). This hypothesis was further substantiated by the creation of a ubiquitination-deficient mutant of TRIP12 (C1959A), which did not affect HP1α levels upon overexpression (Fig. 4F). The collaborative effect of UBE2T and TRIP12 in diminishing HP1α levels was evidenced by the simultaneous knockdown of both proteins, which resulted in the most pronounced increase in HP1α levels (Fig. 4G; Supplementary Fig. S5D). Conversely, the simultaneous overexpression of UBE2T and TRIP12 led to a significant reduction in HP1α protein expression (Fig. 4H; Supplementary Fig. S5E). To clarify whether UBE2T and TRIP12 can synergistically influence the morphology and function of nucleoli, we employed RNA staining to label intracellular RNA, which reveals the size and quantity of nucleoli. The results indicated that the simultaneous knockdown of both UBE2T and TRIP12 significantly reduced the number of nucleoli (Fig. 4I). Co-knockdown of UBE2T and TRIP12 exerted a more pronounced inhibitory effect on the expression of pre-45s rDNA compared with the knockdown of either UBE2T or TRIP12 alone (Fig. 4J). This finding suggests a synergistic interaction between the two genes in the regulation of nucleolar transcription. This conclusion was further supported by a rescue experiment, which demonstrated that overexpression of UBE2T can rescue the nucleolar transcription repression induced by HP1α overexpression (Fig. 4K). In summary, our results provide insight into the cooperative ubiquitination and degradation of HP1α by the E2 ubiquitin–conjugating enzyme UBE2T and the E3 ubiquitin ligase TRIP12.

Figure 4.

HP1α is ubiquitinated and degraded through the cooperative interaction of TRIP12 and UBE2T. A, The diagram depicts the procedure for selecting E3 ligases (top). Confocal microscopy was utilized to record the subcellular localization of TRIP12 across different glioma cell lines (lower). B, Confocal microscopy experiments demonstrate colocalization of TRIP12 (green) and HP1α (red) in the nucleolar region. Scale bars, 5 μm. C, Western blot analysis of coimmunoprecipitation (CoIP) assay of Flag-UBE2T and His-TRIP12 in SG2 cells. D, CoIP was performed using SG2 cells expressing His-TRIP12 and Myc-HP1α. E, Western blot analysis of TRIP12 and HP1α in control or TRIP12-knockdown SG2 and GBM22 cells. F, Western blot analysis was used to examine the changes in HP1α protein levels after overexpression of wild-type (WT) or enzymatically inactive mutant (C1959A) TRIP12. G, A Western blot assay was conducted to examine the trend in HP1α protein levels following the knockdown of UBE2T, TRIP12, or both. H, Western blot results demonstrated the effects of UBE2T and/or TRIP12 overexpression on HP1α protein levels. I, SG2 cells were labeled with the fluorescent dye Nucleolus Bright Red. To quantify the number of nucleoli, 60 cells were counted across 10 slices from each group. Results were analyzed using one-way ANOVA. J, PCR was used to evaluate the transcription efficiency of pre-45s rRNA in SG2 cells. K, The PCR experiment assessed the expression levels of pre-45s RNA in SG2 cells across different experimental groups. Data were normalized to endogenous β-actin expression. Results are presented as mean ± SD, with statistical significance indicated as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01, determined by one-way ANOVA and Tukey multiple comparisons test. IB, immunoblotted; ns, not significant; si, small interfering.

UBE2T mitigates the suppressive effects on nucleolar function caused by R-2HG

Our results indicate that the ubiquitin-conjugating enzyme UBE2T, in conjunction with the E3 ubiquitin ligase TRIP12, facilitates the degradation of the HP1α protein in the nucleolus, thereby promoting rDNA transcription. Elevated UBE2T expression in mutIDH1 gliomas is associated with poor patient prognosis and higher tumor grades. However, the exact underlying mechanism remains elusive. Previous studies have demonstrated that the addition of exogenous R-2HG to wtIDH1 glioma cells inhibits cellular proliferation and enhances sensitivity to radiotherapy and chemotherapy (21, 44–49). In our study, we employed the EdU assay and limited dilution assay to confirm that R-2HG treatment inhibited the proliferation (Supplementary Fig. S6A) and self-renewal capacity of wtIDH1 GBM cell lines (Supplementary Fig. S6B). We hypothesize that the elevated expression of UBE2T may significantly contribute to the persistence of the malignant phenotype in mutIDH1 gliomas. Importantly, we found that SG2 cells harboring IDH1/TP53 mutations exhibited greater sensitivity to UBE2T knockdown compared with the IDH1/TP53 wild-type glioma cell line GBM30. This sensitivity is evidenced by a reduction in both the size and number of nucleoli (Fig. 5A and B), alongside a more significant decrease in rDNA transcript levels (Fig. 5C). These findings suggest that mutIDH1 cells are more reliant on UBE2T for the maintenance of nucleolar morphology and function, potentially linked to the elevated levels of R-2HG. The effect of R-2HG treatment on nuclear morphology was studied using IF microscopy, showing a reduction in nuclear size in wtIDH1 GBM cells (Fig. 5D). To evaluate the impact of R-2HG on nucleolar function, we employed the RNA fluorescent dye Nucleolus Bright Red to label intracellular RNA and observed fluorescence intensity within the nucleus using fluorescence microscopy. The results demonstrated that R-2HG treatment reduced nuclear RNA density (Fig. 5E), reflecting a decrease in nucleolar rRNA content. Additionally, qPCR was conducted to assess the effect of R-2HG on nucleolar rDNA transcription in glioma cells, revealing a significant decrease in the levels of pre-45s rRNA following R-2HG treatment (Fig. 5F). Subsequently, we investigated whether overexpression of UBE2T influenced cell sensitivity to R-2HG. We found that overexpression of UBE2T partially mitigated the nucleolar shrinkage (Fig. 5G and H) and transcriptional repression induced by R-2HG treatment (Fig. 5I and J). Previous research has highlighted that R-2HG disrupts the distribution of H3K9me2/3, resulting in altered gene expression and compromised DNA repair (47). HP1α is an important player in regulating histone H3K9 methylation and was found to be enriched in promoter regions, leading to H3K9me3 enrichment and subsequent transcriptional silencing (50). Indeed, ChIP techniques revealed that R-2HG treatment enhanced H3K9me3 levels near the rDNA promoter (Fig. 5K and L), which could be reversed by UBE2T overexpression (Fig. 5M). Our study concluded that UBE2T degrades HP1α in the nucleolus and reduces H3K9me3 levels near the rDNA promoter, thereby improving nucleolar function. This finding partially elucidates the poorer prognosis observed in patients with IDH1/TP53 mutations. Specifically, TP53 mutations result in the upregulation of UBE2T, which counteracts the inhibitory effects of the oncometabolite R-2HG on the nucleolus.

Figure 5.

UBE2T alleviates R-2HG–induced inhibition of nucleolar function. A, Confocal microscopy was employed to observe the distribution and expression of the nucleolar marker fibrillarin following the downregulation of UBE2T. Scale bars, 5 μm. B, Differential interference microscopy was utilized to examine the size and number of cell nucleoli after UBE2T downregulation. Scale bars, 5 μm.C, PCR was conducted to evaluate the impact of UBE2T on the transcription efficiency of pre-45s rDNA. n = 3 independent experiments. Data are shown as mean ± SEM, and *, P < 0.05 by unpaired t test.D, The distribution of fibrillarin was assessed using confocal microscopy following exposure to TFMB-(R)-2HG (500 µmol/L, 24 hours). Scale bars, 5 μm. E, Following TFMB-(R)-2HG treatment (500 µmol/L, 24 hours), cells were labeled with the fluorescent dye Nucleolus Bright Red to visualize changes in fluorescence intensity within the cell nucleus, as observed through fluorescence microscopy. Scale bars, 20 μm. F, PCR was used to assess the effect of TFMB-(R)-2HG (500 µmol/L, 24 hours) on the transcription efficiency of pre-45s rRNA. n = 3 independent experiments. Data are shown as mean ± SEM and **, P < 0.01; ***P < 0.001; and ****, P < 0.0001 by unpaired t test. G, Confocal microscopy was used to evaluate the influence of UBE2T overexpression (UBE2T OE) on the distribution and expression of fibrillarin in U251MG cells after TFMB-(R)-2HG treatment (500 µmol/L, 24 hours). H, Electron microscopy shows that overexpression of UBE2T alleviates nucleolar condensation caused by TFMB-(R)-2HG treatment (500 µmol/L, 24 hours). I, U251MG cells were labeled with the fluorescent dye Nucleolus Bright Red, and changes in fluorescence intensity within the cell nucleus following UBE2T overexpression and TFMB-(R)-2HG treatment (500 µmol/L, 24 hours) were observed using fluorescence microscopy. J, PCR was performed to assess the transcription efficiency of pre-45s rRNA in U251MG cells. All data were calculated as gene expression relative to endogenous β-actin expression. n = 3 independent experiments. Data are shown as mean ± SEM, and *, P < 0.05; **, P < 0.01 by unpaired t test.K, The schematic diagram illustrates the distribution of primers (H42.9, H42.9, and H1) in the specific gene region of human rDNA used in ChIP. L, The graph represents the impact of TFMB-(R)-2HG treatment (500 µmol/L, 24 hours) on the enrichment of H3K9me3 in the rDNA promoter region. n = 3 independent experiments. Data are shown as mean ± SEM, and **, P < 0.01; ***, P < 0.001 by unpaired t test.M, ChIP examined the effect of TFMB-(R)-2HG exposure (500 µmol/L, 24 hours) on H3K9me3 enrichment in UBE2T-overexpressing U251MG cells. n = 3 independent experiments. Data are shown as mean ± SEM. ns, P > 0.05; ***, P < 0.001 by unpaired t test. ns, not significant.

The mutant p53 reactivator APR-246 inhibits UBE2T expression and nucleolar function

Our results indicate that high UBE2T expression counteracts nucleolar functional inhibition caused by R-2HG exposure. Importantly, the abnormal expression of UBE2T is linked to TP53 mutation. We next investigated whether UBE2T is a potential therapeutic target in glioma. We extracted UBE2T expression data from brain tumor cell lines available in the Cancer Cell Line Encyclopedia. Subsequently, we obtained the sensitivity of these cell lines to a collection of small-molecule compounds, measured as the AUC, from the Genomics of Drug Sensitivity in Cancer 2. The correlation between UBE2T expression and AUC was calculated using DepMap. Interestingly, screening of drug sensitivity databases revealed effective drugs against UBE2T overexpressed cell lines, with MIRA-1 standing out as a mutant p53 reactivator (Fig. 6A). Previous findings showed that p53 suppresses UBE2T expression, whereas inhibiting p53 function boosts UBE2T expression. Due to the side effects of MIRA-1, APR-246 (Eprenetapopt), another mutant p53 reactivator undergoing clinical trials, was chosen (51–54). Using the CCK-8 assay, APR-246 was found to inhibit the proliferation of mutTP53 cell lines SG2 and GBM22 in a dose-dependent manner (Fig. 6B). APR-246 treatment notably decreased UBE2T mRNA and protein levels in SG2 and GBM22 cells, further confirming the inhibitory effect of p53 on UBE2T expression (Fig. 6C and D). Using the in vivo SG2-derived glioma model and treatment schedule, we examined the effect of APR-246 treatment (55). APR-246, dissolved in physiologic saline, was administered via i.p. injection on days 1, 3, 5, and 7 after the first imaging, at a dose of 400 mg/kg. Treatment with APR-246 inhibited tumor growth (Fig. 6E; Supplementary Fig. S7A) and prolonged the survival time of mice (Fig. 6F). IHC of tumor sections demonstrated decreased staining for UBE2T and Ki67 in APR-246–treated tumors (Fig. 6G). Next, we utilized IF experiments to investigate the impact of APR-246 on nucleolar morphology and function. The results demonstrated that APR-246 treatment led to a significant decrease in the expression of fibrillarin, causing a restricted distribution of this protein. Furthermore, APR-246 reversed the UBE2T-induced increase and aggregation of fibrillarin (Fig. 6H). We examined the effect of APR-246 treatment on rDNA transcription efficiency by qPCR and found a significant inhibitory effect on rRNA levels (Fig. 6I). Finally, APR-246 treatment also reduced the proportion of multinucleated cells caused by UBE2T overexpression (Supplementary Fig. S7B). In conclusion, our research identified APR-246 as a mutant p53 reactivator with inhibitory effects on the proliferation and nucleolar function of glioma cells. The direct reduction of UBE2T protein levels by APR-246 provides a promising foundation for targeted therapy in glioma (Fig. 6J).

Figure 6.

The mutant p53 reactivator APR-246 inhibits UBE2T expression and nucleolar function. A, The scatter plot on the left shows the correlation between UBE2T expression levels and the drug sensitivity results (AUC) in the drug sensitivity database Genomics of Drug Sensitivity in Cancer 2. A negative correlation indicates that higher UBE2T expression in glioma cells is associated with increased sensitivity to the compound. The three-line plot lists the top five compounds that are most sensitive in glioma cells with high UBE2T expression. B, CCK-8 experiments were performed to assess the effect of APR-246 treatment at different concentrations for 48 hours on the proliferation capacity of SG2 and GBM22 cells. n = 3 independent experiments. Data are shown as mean ± SEM. C, A PCR assay was used to assess the influence of APR-246 (20 µmol/L, 24 hours) on UBE2T mRNA levels. n = 3 independent experiments. Data are shown as mean ± SEM, and *, P < 0.05 by unpaired t test. D, Western blot analysis was conducted to examine the impact of APR-246 treatment (20 µmol/L, 24 hours) on UBE2T protein levels. E,In vivo bioluminescence experiments were conducted using orthotopic xenograft models of human glioma cells (SG2) in nude mice. On the first, third, fifth, and seventh days following the initial imaging session, APR-246 was administered via i.p. injection at a dose of 400 mg/kg. The results indicated that APR-246 treatment inhibited the proliferation of SG2 cells. F, Survival analysis revealed that the APR-246 treatment prolonged the survival of nude mice. The sample size was n = 5, and the significance level was determined using log-rank analysis. G, IHC staining of intracranial tumors in nude mice demonstrated that APR-246 treatment decreased the protein levels of both UBE2T and Ki-67. H, IF was used to evaluate the effect of APR-246 treatment (20 µmol/L, 24 hours) on the distribution and expression of fibrillarin. Scale bars, 5 μm. I, PCR was performed to detect the effect of APR-246 treatment (20 µmol/L, 24 hours) on rDNA transcription. All data were calculated as gene expression relative to endogenous β-actin expression. n = 3 independent experiments. Data are shown as mean ± SEM and were analyzed by an unpaired t test.J, Graphical abstract. OE, overexpression.

Discussion

Mutant IDH1 catalyzes the production of the oncometabolite R-2HG, leading to the disruption of cell differentiation and the induction of a hypermethylation state throughout the genome, resulting in the malignant phenotype of gliomas (12). Although R-2HG initially promotes tumor transformation, it ultimately induces replicative stress and epigenetic changes that limit tumor growth (21). The transition of mutant IDH1 from a driver to a passenger in tumorigenesis occurs rapidly, indicating a narrow window for IDH1 inhibitor effectiveness. In the context of IDH1 mutations, the presence of coexisting mutations such as TP53 plays an important role in enhancing tumorigenesis by disrupting important tumor suppressor functions (51). Gliomas that harbor IDH1 mutations have a significantly higher frequency of TP53 mutations (8). Unfortunately, patients with both IDH1 and TP53 mutations have a worse prognosis. Therefore, studying the interaction between mutant IDH1 and TP53 is crucial to understanding the molecular mechanisms behind glioma progression.

Our research indicates that TP53 mutation status affects the expression of UBE2T. Using in vitro and in vivo models, we demonstrate that UBE2T promotes the proliferation and stemness of glioma cells, contributing to malignant progression and potential therapeutic resistance. UBE2T has been implicated in the progression of several cancers, such as hepatocellular carcinoma, gastric cancer, and non–small cell lung cancer (29, 32, 34). Targeting UBE2T has shown promise in increasing the sensitivity of cancer cells to treatments, highlighting its potential as a therapeutic target for a wide range of cancer types (28, 30, 56). Furthermore, our study reveals the nucleolar function of UBE2T and shows that its downregulation leads to the disruption of nucleolar structure and reduced rDNA transcription. This is crucial as nucleolar integrity is closely linked to ribosome biogenesis and cellular metabolism, both vital for cancer cell proliferation (57–59). Mechanistically, we found that UBE2T degrades HP1α through the ubiquitin–proteasome pathway, modulating nucleolar function by reducing HP1α levels and potentially counteracting the suppressive effects of R-2HG on the nucleolus. This finding is particularly important given the epigenetic changes caused by R-2HG, including disruption of histone methylation patterns and DNA repair machinery. Furthermore, our study of the therapeutic potential of the mutant p53 reactivator APR-246 showed its ability to inhibit UBE2T expression and nucleolar function, offering a promising targeted therapy approach for IDH1/TP53-mutant gliomas. Restoring wild-type p53 function through APR-246 could potentially reverse the oncogenic effects of TP53 mutation and suppress UBE2T-mediated tumor progression. In conclusion, this study offers a thorough analysis of the involvement of UBE2T in IDH1/TP53-mutant gliomas, emphasizing its impact on tumor growth, stem cell maintenance, and nucleolar function. The recognition of UBE2T as a promising therapeutic target, coupled with the investigation of APR-246, presents novel opportunities for the advancement of targeted treatments for patients with glioma.

Although our study has shed light on the role of UBE2T in IDH1/TP53-mutant gliomas, it is important to recognize the limitations. The functional analysis of UBE2T was mainly performed in cell lines, which may not fully represent the complexities of tumor microenvironments in patients. The genetic and epigenetic diversity of gliomas may lead to different responses to UBE2T regulation, which was not considered in our in vitro model. Therefore, further validation using patient-derived xenograft models or clinical trials is essential to better understand the clinical implications of our findings. Furthermore, our study focused on the effects of UBE2T on nuclear function and its regulation by TP53 mutations but did not examine its possible involvement in other cellular processes, such as autophagy, immune regulation, and angiogenesis. Future studies should further address these aspects to more fully understand the role of UBE2T in glioma biology. Finally, whether mutant p53 can directly promote the transcriptional activation of UBE2T through gain-of-function mechanisms, rather than merely alleviating the suppression of UBE2T expression caused by the inactivation of wild-type p53, remains to be elucidated (60). Taken together, our study lays the foundation for elucidating the role of UBE2T in IDH1/TP53-mutant glioma. Future directions include addressing the limitations of this study and extending these findings to develop potential treatment strategies for patients with glioma.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S1: GBM30 cell information and knock-down efficiency validation.

Supplementary Figure S2

Supplementary Figure S2: Supplementary data on glioma public databases and tissue samples.

Supplementary Figure S3

Supplementary Figure S3: UBE2T facilitates the growth and stem-like properties of glioma cells.

Supplementary Figure S4

Supplementary Figure S4: iTRAQ and RNA-seq analysis.

Supplementary Figure S5

Supplementary Figure S5: Expression and distribution of putative UBE2T interacting E3 ubiquitin ligases.

Supplementary Figure S6

Supplementary Figure S6: R-2HG inhibits the proliferation of IDH1 wild-type glioma cells.

Supplementary Figure S7

Supplementary Figure S7: Supplementary materials for in vivo and in vitro experiments of Figure 6 in main text.

Supplementary Table S1

Supplementary Table S1: Cell line summary,

Supplementary Table S2

Supplementary Table S2. Primers used for ChIP assay.

Supplementary Table S3

Supplementary Table S3. Primers used for real time PCR.

Authors’ Disclosures

No disclosures were reported.

Authors’ Contributions

F. Zhou: Data curation, formal analysis, visualization. Y. Sun: Data curation. X. Chen: Data curation. D. Wu: Data curation. Z. Tao: Data curation. L. Wu: Data curation. G. Chen: Conceptualization, supervision, funding acquisition. X. Liu: Conceptualization, formal analysis, supervision, funding acquisition. T. Yu: Conceptualization, formal analysis, supervision, funding acquisition, writing–original draft.