High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

Introduction

Acute myeloid leukemia (AML) is a molecularly heterogeneous clonal disorder arising from malignant transformation of myeloid progenitors, characterized by uncontrolled proliferation coupled with differentiation blockade at distinct hematopoietic stages (1). Despite advances in targeted therapies (e.g., FLT3 and IDH inhibitors), the 5-year survival rate remains below 30% (2,3), necessitating novel therapeutic strategies that address both leukemogenic proliferation and drug resistance mechanisms.

Telomere protection is a fundamental biological process critical for maintaining genomic stability. At the core of this mechanism lies the shelterin complex, a specialized protein assembly, localized at chromosomal termini, which coordinates telomere protection through its multi-subunit architecture (4-6). Telomeric repeat-binding factor 2 (TERF2), a core constituent of the shelterin complex that orchestrates telomere protection mechanisms, plays a pivotal role in preserving telomere structural integrity and maintaining genomic homeostasis (7). Dysregulation of TERF2 has been implicated in chromosomal instability, cellular senescence, and oncogenesis, positioning it as a pivotal player in both telomere biology and cancer progression (8). Recent studies highlight abnormal TERF2 expression observed in cancers such as esophageal squamous cell carcinoma (ESCC) and hepatocellular carcinoma, and it correlates with poor prognosis (9-11). However, the precise role of TERF2 in AML remains incompletely elucidated.

In this study, we found that TERF2 exhibits overexpression in AML, there is a significant correlation between the levels of TERF2 and the clinicopathological characteristics of the disease, which implies that TERF2 may potentially play a role in the prognosis of AML. TERF2 knockdown significantly suppresses AML cell proliferation, induces apoptosis and downregulates key regulators of the E2F pathway, including CDK4/6 and CDKN2A. CDKN2A is a negative correlation factor for cuproptosis, and we demonstrate that TERF2 depletion enhances cuproptosis sensitivity in AML cells. This provides a new strategy for AML prognosis analysis and potential therapeutic targets. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1226/rc).

Methods

Sample collection

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Nanchang University (No. IIT-O-2024-131). A waiver of informed consent was obtained from the Ethics Committee for this clinical study. The peripheral blood mononuclear cells (PBMCs) from 50 newly diagnosed AML patients and 35 healthy donors from The Second Affiliated Hospital of Nanchang University were isolated using Ficoll density gradient centrifugation. AML Patients were diagnosed per the Chinese Guidelines for Diagnosis and Treatment of Acute Myeloid Leukemia (2022 Edition). Exclusion criteria: (I) comorbid malignancies; (II) organic lesions of vital organs or major trauma/surgery within 1 month; (III) metabolic/active infectious diseases. Healthy controls: inclusion required: (I) age 18–60 years; (II) no major diseases with normal physical/laboratory assessments; (III) no tobacco/alcohol/substance abuse; (IV) written informed consent. Exclusions: (I) chronic diseases/acute infections; (II) long-term immunosuppressants/corticosteroids; (III) pregnancy/lactation; (IV) significant blood loss (>200 mL) within 3 months.

Bioinformatic analysis

One hundred and seventy-three AML samples and 70 normal controls were obtained from The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov) and Genotype-Tissue Expression (GTEx; https://www.gtexportal.org) databases. Differential expression analysis of TERF2 was performed with the limma package. Survival correlations were assessed via Kaplan-Meier analysis using the survival. The relevance of TERF2 to hallmark sets in AML were analyzed by the “Gene set variation analysis” (GSVA) package.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot

Total RNA extraction from PBMCs and AML cell lines was performed using TRIZOL reagent (Ambion, Carlsbad, USA) in accordance with the manufacturer’s instructions. Western blot was employed to determine protein levels, with TERF2 messenger RNA (mRNA) expression levels assessed by qRT-PCR analysis following established protocols (12). The primers in Table 1 were used to amplify TERF2 mRNA. Primary antibodies targeting TERF2 (#13136, 1:500 dilution), CDK4 (#12790, 1:500), CDK6 (#1331, 1:500), and CDKN2A (#80772, 1:500) were commercially obtained from CST (Danvers, MA, USA). E2F1 (#T56580, 1:500) and GAPDH (#M20006, 1:5,000) were purchased from Abmart (Shanghai, China).

Cell culture and transfections

AML cell lines (MOLM13 and THP-1) were obtained from the American Type Culture Collection (ATCC) and were cultured in RPMI-1640 medium (Gibco, Grand Island, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco) under standard conditions (37 ℃, 5% CO₂). For stable gene silencing, shRNA targeting TERF2 were designed and synthesized by GenePharm (Shanghai, China) in Table 1. Cells with stable TERF2 knockdown were selected using puromycin (4 µg/mL; Invitrogen, Waltham, USA) for 96 hours, followed by expansion and validation of knockdown efficiency via qRT-PCR.

Cell viability assay and flow cytometry assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; APExBIO, Houston, USA) according to the manufacturer’s protocol, with triplicate measurements for each experimental condition. Cells were fixed in 70% ethanol at 4 ℃ for 2 hours, washed with phosphate-buffered saline (PBS) and stained with propidium iodide (PI: 50 µg/mL; Biolegend, San Diego, USA) containing RNase A (100 µg/mL; Thermo Fisher, Rockford, USA) for 30 minutes in the dark. Cell cycle was analyzed using flow cytometer (BD Biosciences, New Jersey, USA). Transfected cells were washed with PBS and resuspended in 1× binding buffer, cells were incubated with fluorescein isothiocyanate-labelled annexin V (annexin V-FITC) (5 µL) and PI (2 µg/mL) for 15 minutes at room temperature in the dark, followed by immediate flow cytometry analysis apoptosis.

Animal studies

NOD-SCID IL2rg (NSG) mice (aged 5–6 weeks), housed in a specific pathogen-free (SPF) barrier facility, were injected intravenously via tail vein with luciferase-expressing MOLM13 cells (5×10⁵). The growth of AML cells in vivo was detected by LB983 system bioluminescence imager. The mice were terminated via CO₂ euthanasia at the end of the experiments. A protocol was prepared before the study without registration. All animal experiments were performed under a project license (No. NCULAE-20221031186) granted by the Animal Care Committee of Nanchang University, in compliance with the institutional guidelines for the care and use of animals, adhering to the principles of Replacement, Reduction, and Refinement (the 3Rs).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism (version 10.0) for data processing and graphical representation. To assess statistical significance, we employed the two-tailed Student’s t-test for comparisons between two groups. The data presented as the mean ± standard deviation (SD) derived from at least three independent experiments. Statistical significance was set at P<0.05.

Results

Elevated TERF2 expression in AML patients and its correlation with clinicopathological features

The mRNA levels of TERF2 in AML was analyzed using the TCGA database, which revealed significantly upregulated TERF2 mRNA levels in AML patients compared to normal controls (Figure 1A). To validate this observation, qRT-PCR was performed on PBMCs from AML patients and healthy donors. Consistently, TERF2 mRNA levels were markedly elevated in AML PBMCs compared to control group (Figure 1B), aligning with the TCGA database findings. A cohort of 150 AML patients from TCGA database was stratified into high- and low-TERF2 expression groups. Single-cell RNA sequencing data (GSE116256) demonstrate that TERF2 expression is highly enriched within malignant blasts and primitive hematopoietic subsets, including hematopoietic stem cells (HSCs), progenitor cells, and erythrocyte progenitor (EryPro) cells, while remaining low in differentiated lymphoid and erythroid lineages (Figure 1C,1D). This patterned expression suggests a role for TERF2 in proliferative capacity in AML. Comparative analysis demonstrated significant differences in key clinicopathological parameters, including white blood cell count (WBC), bone marrow (BM) blast percentage, peripheral blood (PB) blast percentage, and overall survival (OS) events (Table 2). However, no statistically significant associations were observed between TERF2 expression and recurrent AML-associated gene mutations. Kaplan-Meier survival analysis using TCGA databases revealed that AML patients with high TERF2 expression exhibited significantly shorter OS compared to the low-expression group (Figure 1E). Furthermore, receiver operating characteristic curve (ROC curve) analysis demonstrated the diagnostic potential of TERF2 in AML, with an AUC value of 0.735 (Figure 1F). Time-dependent area under the curve (AUC) analysis further validated the sensitivity and specificity of TERF2 for predicting clinical outcomes at 1-, 3-, and 5-year intervals (Figure 1G). The following findings indicate that TERF2 displays a high level of expression in patients with AML, and an upregulated expression of TERF2 is correlated with unfavorable clinicopathological traits and a dismal prognosis.

Figure 1 Elevated TERF2 expression in AML patients and its correlation with clinicopathological features. (A) Transcriptional expression of TERF2 in AML analyzed using TCGA database. (B) TERF2 mRNA levels quantified by qRT-PCR in PBMCs from 50 AML patients and 35 healthy donors. (C) UMAP of single cells from an AML patient (GSE116256), single-cell profiling reveals distinct expression patterns of TERF2 across cellular subpopulations in AML. (D) Heatmap depicting the average expression level of TERF2 across distinct cell types within the AML sample. (E) Univariate Cox proportional hazards regression analysis comparing survival outcomes between high- and low-TERF2 expression groups. (F) Prognostic accuracy of TERF2 evaluated using ROC curve analysis. (G) The predictive performance of the novel risk stratification model was evaluated through time-dependent ROC curve analysis, with AUC values calculated at 1-, 3-, and 5-year intervals to quantify sensitivity and specificity in AML prognosis. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; AUC, area under the curve; CI, confidence interval; FPR, false positive rate; HR, hazard ratio; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate; UMAP, uniform manifold approximation and projection.

Downregulation of TERF2 suppresses AML cells viability and proliferation

To investigate the functional role of TERF2 in AML, TERF2 was knocked down in AML cell lines (THP-1 and MOLM13) using shRNA-mediated. The knockdown efficiency was confirmed by qRT-PCR (Figure 2A,2B). AML cell viability was assessed using the CCK-8 assay, which demonstrated that TERF2 knockdown significantly inhibited cellular activity in both THP-1 and MOLM13 cells (Figure 2C,2D). To further explore the impact of TERF2 on proliferation, cell cycle was analyzed via flow cytometry. TERF2 silencing markedly reduced the S phase while increasing the G0/G1 phase in MOLM13 cells (Figure 2E,2F) and THP1 cells (Figure 2G,2H), indicating that TERF2 depletion suppresses AML cell proliferation. Collectively, these results demonstrate that downregulation of TERF2 inhibits both viability and proliferation in AML cells.

Figure 2 Downregulation of TERF2 suppresses AML cells viability and proliferation. (A) qRT-PCR assays analyzed TERF2 mRNA levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (B) Western blot assays detected TERF2 protein levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (C,D) CCK-8 assay showing the viability of NC or TERF2-knockdown MOLM13 cells (C) and THP1 cells (D). (E-H) Flow cytometry assay analyzed cell cycle in NC or TERF2-knockdown MOLM13 cells (E,F) and THP1 cells (G,H). **, P<0.01; ****, P<0.0001; ns, not significant. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

TERF2 deficiency promotes apoptosis in AML cells

To investigate the role of TERF2 in apoptosis, we analyzed its effects in AML cells using flow cytometry. Compared to control groups, TERF2 knockdown significantly induced apoptosis in MOLM13 (Figure 3A,3B) and THP1 (Figure 3C,3D) cells. Furthermore, Western blot analysis of apoptosis-related proteins revealed that cleaved caspase-3 was markedly upregulated in TERF2 deficient AML cells (Figure 3E-3H). Collectively, these findings demonstrate that TERF2 downregulation promotes apoptosis in AML cells.

Figure 3 TERF2 deficiency promotes apoptosis in AML cells. (A-D) Flow cytometry assay analyzed apoptosis in NC or TERF2-knockdown MOLM13 cells (A,B) or THP1 cells (C,D). (E-H) Western blot detected cleaved caspase 3 levels of NC or TERF2-knockdown MOLM13 cells (E,F) or THP1 cells (G,H). ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; NC, negative control.

TERF2 involved in cuproptosis through E2F pathway in AML

To investigate the molecular mechanisms of TERF2 in AML pathogenesis, GSVA was conducted using the GSVA package to systematically assess correlations between TERF2 expression and hallmark pathways. Bubble plot analysis revealed strong correlations between TERF2 and proliferation-related pathways, including E2F targets, c-Myc targets, DNA repair, and G2/M checkpoint regulation (Figure 4A). Notably, the E2F targets pathway exhibited the most significant enrichment (Figure 4B). The E2F pathway, a central regulator of cell cycle progression and the molecular switch for G1/S transition, aligns with our previous findings that TERF2 knockdown induced G0/G1 arrest and reduced S-phase entry in AML cells. Western blot analysis further demonstrated that TERF2 silencing downregulated key cell cycle regulators, including E2F1, CDK4/6, and CDKN2A (Figure 4C-4E). Intriguingly, a recent study identified CDKN2A as a negative regulator of cuproptosis (13). We thus investigated TERF2’s role in modulating cuproptosis sensitivity. CCK-8 assays revealed that TERF2 knockdown significantly reduced the half-maximal inhibitory concentration (IC₅₀) of elesclomol-copper (ES-Cu) (Figure 4F,4G), indicating enhanced cuproptosis susceptibility. Collectively, these data suggest that TERF2 may regulate cuproptosis sensitivity in AML through its interplay with the E2F pathway.

Figure 4 TERF2 involved in cuproptosis through E2F pathway in AML. (A) Bar graphs illustrate the associations between TERF2 expression levels and gene hallmark sets in AML patients. (B) GSEA for AML patients with high TERF2 expression in TCGA database. (C-E) Western blot analysis of E2F1, CDK4, CDK6, CDKN2A levels in MOLM13 cells and NB4 cells with or without TERF2 silence. (F,G) MOLM13 (F) and NB4 (G) cells with TERF2 knockdown were treated with specified concentrations of ES-Cu (1:1 ratio) for 48 hours, followed by cell viability assessment using the CCK-8 assay. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; ES-Cu, elesclomol-copper; GSEA, gene set enrichment analysis; IC₅₀, half-maximal inhibitory concentration; TCGA, The Cancer Genome Atlas.

Knockdown of TERF2 suppresses AML progression and enhances cuproptosis sensitivity in vivo

Given our findings that TERF2 downregulation inhibits AML cell proliferation and enhances cuproptosis susceptibility, we investigated its therapeutic potential in vivo. An orthotopic xenograft model was established by transplanting MOLM13 cells (stably expressing luciferase) into NSG mouse. Tumor engraftment and burden were monitored via bioluminescence imaging. At 3 weeks post-transplantation, mice bearing TERF2-depleted AML cells exhibited significantly reduced tumor burden (Figure 5A,5B) and prolonged OS in shTERF2 group compare to control (shNC) mice (Figure 5C). To evaluate cuproptosis modulation, elesclomol (10 mg/kg) was administered via intraperitoneal injection every 3 days starting 24 hours post AML cells injection. Combined TERF2 knockdown and elesclomol treatment synergistically suppressed tumor growth (Figure 5D,5E) and further extended survival (Figure 5F). These results demonstrate that TERF2 silencing attenuates AML progression and enhances cuproptosis in vivo.

Figure 5 Knockdown of TERF2 suppresses AML progression and enhances cuproptosis sensitivity in vivo. (A,B) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted NSG mice (A) and the quantification of luciferase signals for all mice per group (B). (C) Kaplan-Meier analysis of survival of MOLM13-engrafted mice, log rank test. (D,E) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted mice treated with elesclomol (D) and the quantification of luciferase signals for all mice per group (E). (F) Kaplan-Meier analysis of survival of MOLM13-engrafted mice treated with elesclomol, log rank test. **, P<0.01. AML, acute myeloid leukemia; NSG, NOD-SCID IL2rg.

Disscussion

AML is a heterogenous hematologic malignancy characterized by clonal expansion of myeloid progenitors. The genomic landscape of AML has been extensively mapped, revealing recurrent mutations in genes such as NPM1, FLT3, and IDH1/2, which drive leukemogenesis and serve as prognostic biomarkers, AML remains a therapeutic challenge despite advancements in molecular profiling and targeted therapies (14,15). Recent studies have implicated the roles of shelterin in cancer progression (16), dysregulation of shelterin components, promotes genomic instability and oncogenesis by enabling telomere fusion, breakage-fusion-bridge cycles, and chromothripsis (17-20). TERF2, a core constituent of the shelterin complex that orchestrates telomere protection mechanisms, plays a pivotal role in preserving telomere structural integrity and maintaining genomic homeostasis (7). The present study elucidates the role of TERF2 in AML, demonstrating its high expression correlating with poor prognosis. TERF2 knockdown induces apoptosis, suppresses AML cell proliferation, and downregulates key regulators of the E2F pathway, including CDK4/6 and CDKN2A, and simultaneously enhancing sensitivity to cuproptosis—a newly discovered copper-dependent cell death mechanism, which is distinct from other forms of cell death, such as apoptosis or ferroptosis (13). Emerging evidence delineates the therapeutic potential of cuproptosis modulation across malignancies. Cuproptosis induction enhances chemotherapeutic efficacy in prostate cancer (21). METTL16 lactylation facilitates m⁶A-mediated stabilization of FDX1 mRNA, thereby amplifying cuproptosis in gastric carcinogenesis (22). Oncogenic Wnt/β-catenin signaling confers cuproptosis resistance via transcriptional promotion of copper efflux transporters ATP7B, which reduces intracellular copper (23). LIPT1 triggers copper-dependent proteotoxic stress in bladder cancer models (24). Therefore, research on the role of TERF2 in AML provides insights into leukemogenesis and unveils potential therapeutic strategies.

TERF2 plays a pivotal role in maintaining genomic stability by protecting chromosome ends from DNA damage recognition and illegitimate repair (25-27). Notably, TERF2 overexpression is implicated in tumorigenesis across multiple cancers, including esophageal, hepatocellular carcinoma, and colorectal carcinomas (10,11,28,29). Our data reveal TERF2 overexpression in AML, correlating with OS, WBC, BM blast percentage, PB blast percentage, this aligns with studies showing that aberrant shelterin components expression links to leukemogenesis (30-32). Our results extend these observations and demonstrate that TERF2 as a potential biomarker for risk stratification in AML patients.

Knockdown of TERF2 significantly suppressed AML cell proliferation and triggered apoptosis, and similar findings in renal cell carcinoma demonstrated that TRF2 (TERF2) inhibition induced G1/S arrest and apoptosis via telomere dysfunction and DNA damage responses (29). A recent study demonstrates that TERF2 knockdown enhances chemotherapeutic sensitivity by suppressing autophagy (33). Our studies demonstrate that TERF2 silencing disrupts the E2F-CDK4/6 axis, leading to reduced expression of E2F1, CDK4/6 and CDKN2A. The E2F pathway is a well-established regulator of G1/S transition, and its inhibition has been shown to induce cell cycle arrest in AML (34,35). Downregulation of E2F1 inhibits autophagy through transcriptional regulation of LC3 and DRAM, thereby enhancing the sensitivity of AML cells to retinoic acid (36). The CDK4/6 inhibitor palbociclib synergizes with all-trans retinoic acid (ATRA) to induce differentiation in AML cells (37). Intriguingly, CDKN2A (p16) has recently been implicated to be a negatively correlated molecule for cuproptosis (13). Cuproptosis, a copper-dependent cell death mechanism, is influenced by mitochondrial metabolism and redox balance (13). To date, researchers have elucidated the roles and potential therapeutic implications of cuproptosis in different tumor types (22-24,38,39). Our data suggest that TERF2 knockdown suppresses CDKN2A and enhances cuproptosis sensitivity. However, the precise mechanism linking TERF2 and cuproptosis requires validation in the future.

Conclusions

To summarize, the study reveals that TERF2 is overexpressed in AML. Moreover, a notable correlation exists between the levels of TERF2 and clinicopathological characteristics, implying a potential involvement of TERF2 in the prognostic assessment of AML. Downregulation of TERF2 inhibits the AML cell proliferation, induces apoptosis, and modulates cuproptosis sensitivity possibly via the E2F-mediated pathway. Targeting TERF2 not only inhibits proliferation but also unlocks cuproptosis as a therapeutic vulnerability, offering a potential strategy for AML.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1226/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1226/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1226/prf

Funding: This study was supported by the National Natural Science Foundation of China (No. 82160405).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1226/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Nanchang University (No. IIT-O-2024-131). A waiver of informed consent was obtained from the Ethics Committee for this clinical study. All animal experiments were performed under a project license (No. NCULAE-20221031186) granted by the Animal Care Committee of Nanchang University, in compliance with the institutional guidelines for the care and use of animals, adhering to the principles of Replacement, Reduction, and Refinement (the 3Rs).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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