Abstract Due to the limited availability of effective treatment therapies, patients with refractory and relapsed acute myeloid leukemia (AML) often have poor prognoses. Therefore, identifying new therapeutic targets to improve the treatment landscape and enhance AML outcomes is critical. In this study, we demonstrated for the first time that alpha-enolase (ENO1) is markedly overexpressed in AML and is closely associated with poor prognosis. In vitro experiments revealed that ENO1 knockdown (shENO1) significantly inhibited cell proliferation and invasion, and concomitantly induced cell cycle arrest. In vivo, a mouse model engrafted with U937-shENO1 cells exhibited markedly prolonged overall survival compared with a model implanted with U937 cells. Mechanistically, ENO1 operates by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, as evidenced by the reversal of shENO1 function through ERK activation. Collectively, our findings highlight ENO1 as a promising therapeutic target in AML. Keywords: AML, ENO1, Invasion, MAPK/ERK signaling pathway, Proliferation 1. INTRODUCTION Acute myeloid leukemia (AML) is a heterogeneous blood stem cell cancer, characterized by the abnormal proliferation and/or impaired differentiation of myeloid lineage cells. AML is the most common type of leukemia in adults,^[40]1 and intensive chemotherapy is the main treatment modality, with a 5-year survival rate of less than 45%.^[41]2,[42]3 Advancements in allogeneic bone marrow (BM) transplantation techniques have demonstrated the potential to improve the 5-year survival rate of AML patients by more than 50% after matched-related donor transplantation.^[43]4 Recently, significant breakthroughs in molecular and cellular biology have deepened our understanding of the intricate pathophysiology of AML. These advancements have not only expanded the treatment options but have also facilitated the development of inhibitors targeting FLT3, ID, IDH2, and BCL2.^[44]5 Over recent decades, targeted therapies have markedly enhanced the survival of AML patients.^[45]6,[46]7 However, relapse and drug resistance remain barriers to better outcomes in AML.^[47]8 Therefore, identifying aberrantly expressed proteins and elucidating their functions in driving AML development may provide new targets for AML treatment. Alpha-enolase (ENO1) is a pivotal enzyme in glycolysis and catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate.^[48]9 Aberrant ENO1 expression is associated with the development of diverse cancers. ENO1 is overexpressed in more than 10 types of tumors, including lung cancer,^[49]10 gastric cancer,^[50]11 bladder cancer,^[51]12 and liver cancer.^[52]13 Its overexpression has a positive association with cancer advancement and poor prognostic outcomes for patients.^[53]14 Our previous study showed that ENO1 is overexpressed in Burkitt’s lymphoma and promotes its development.^[54]15 ENO1 localizes widely in different sites of tumor cells, which contributes to its different subcellular functions. As a plasminogen receptor on the cell membrane, ENO1 promotes extracellular matrix degradation. It enhances cell migration or invasion by activating plasminogen.^[55]16,[56]17 In the cytoplasm, in addition to functioning as a glycolytic enzyme, ENO1 also acts as an RNA-binding protein, influencing mRNA decay, thereby promoting tumor development.^[57]18,[58]19 In the cell nucleus, a truncated isoform of ENO1 (MBP-1) binds to c-Myc, thereby negatively regulating its expression and suppressing tumor growth.^[59]20 Moreover, existing in the form of exosome, ENO1 can promote the invasion of cancer cells.^[60]21 With ongoing research, the ENO1-mediated signaling pathway has garnered significant attention, and ENO1 has shown potential as a novel tumor marker. However, the clinical significance and diagnostic value of ENO1 in AML remains unclear. We aimed to investigate the expression and functions of ENO1 in AML and possibly create a new target in AML drug development. 2. MATERIALS AND METHODS 2.1. Clinical samples This study was approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University (approval number: KY2024-194-01) and informed consent was obtained from all participants. All procedures were performed according to the guidelines and rules of the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. Clinical samples and patient data were obtained from the Second Affiliated Hospital of the Dalian Medical University. BM and peripheral blood (PB) samples were obtained from 185 AML patients and 61 donors, none of whom had received chemotherapy, radiotherapy, or immunotherapy before sample collection. 2.2. Cell culture KG-1 cells were cultured in Iscove’s Modified Dulbecco’s medium (IMDM; Gibco, Carlsbad, California ) supplemented with 20% fetal bovine serum (FBS; Gibco). U937 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS. OCI-AML3 cells were cultured in RPMI 1640 medium supplemented with 20% FBS. To modulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, we implemented a pharmacological strategy using Senkyunolide I (SENI; Selleck, Houston, Texas ) as an ERK1/2 activator and U0126-EtOH (Selleck) as an MEK1/2 inhibitor. Both compounds were dissolved in dimethyl sulfoxide (DMSO). The cells were treated with SENI (10 μM) or U0126 (5 μM), with untreated cells serving as negative controls. 2.3. Animal experiments All animal experiments were approved by the Experimental Animal Ethics Committee of Dalian Medical University (approval number: [61]AEE20061). All experiments were performed according to the guidelines and rules of the Experimental Animal Ethics Committee of Dalian Medical University. Four-to-5-week-old female non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and housed in accordance with standard procedures. To establish the tumor model, mice received tail vein injections of 2 × 10^6 U937 or U937-shENO1 cells suspended in 0.1 mL PBS. U0126, an ERK signaling pathway inhibitor, was prepared as a 10 mmol/L stock solution in DMSO and diluted with PBS to obtain a working solution containing 40% DMSO. Mice were intraperitoneally injected with U0126 at a dose of 25 μmol/kg 3 times per week. Equivalent amounts of DMSO were administered to the control group. This concentration was selected based on prior studies showing that U0126 effectively inhibits ERK activation in vivo within 24 hours at a dose that is non-toxic to mice, while downregulating ERK1/2 in tumors.^[62]22–[63]24 The mice were sacrificed by carbon dioxide asphyxiation followed by cervical dislocation on day 14, and the percentage of CD45^+ cells was measured by flow cytometry. Paraffin was used to embed the tissue samples for staining with hematoxylin and eosin (H&E). 2.4. Plasmids and establishment of stable cell lines Plasmids shENO1-1 and shENO1-2 were obtained from laboratory stocks and their sequences are listed in Table [64]1. The cells were infected with the respective lentiviruses and selected using puromycin (1 μg/mL) for 2 weeks. Table 1. Sequences of plasmids, probes, and primers used. Name Sequence shENO1-1 CCAACAUCCUGGAGAAUAA shENO1-2 GGGTACCCGGAGCACGGAGAT ENO1-Primer-F GCCGTGAACGAGAA ENO1-Primer-R TCAGCGATGAAGGTAT ENO1-Probe FAM-CTGCCTCCTGCTCAAAGTCAACCAGATTGGCTCCG-TAMRA ABL-primer-F CTAAAGGTGAAAAGCTCCG ABL-Primer-R GACTGTTGACTGGCGTGAT ABL-Probe CCATTTTTGGTTTGGGCTTCACACCATT [65]Open in a new tab AML = acute myeloid leukemia, ENO1 = alpha-enolase. 2.5. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from patient BM samples or cultured cells using the TRIzol reagent (Accurate Biology, Hunan, China) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with 1 μg of total RNA using an Evo M-MLV RT Mix Kit (Accurate Biology). qRT-PCR was conducted using Pro Taq HS Probe Premix (Accurate Biology) or SYBR Green Pro Taq HS Premix (Accurate Biology) on a Prism 7500 Real-Time PCR System (ABI, Foster City, California ). Expression levels were normalized to those of ABL or β-actin. The probe and primer sequences used are listed in Table [66]1. 2.6. RNA-seq and analysis To evaluate the expression of ENO1, we collected BM samples from 159 AML patients and 40 donors. Additionally, we established empty vector and ENO1 knockdown AML cells using the U937 cell line. After RNA extraction and library construction, RNA sequencing (RNA-seq) was performed on the NovaSeq6000 platform with a paired-end 150-bp read length at Novogene Company (Beijing, China). RNA-seq data were aligned to the reference genome (hg38) using STAR (v2.7.2a). The “cuffnorm” command from the Cufflinks package (v2.2.1) was used to measure gene expression abundances under the transcript coordinates according to the gene annotation format file from GENCODE (Release 27, GRCh38). By comparing the expression profiles between U937-shENO1 and U937 vector cells, using the “limma” package (v3.46.0), we obtained the significantly differentially expressed genes under the cutoffs of fold change (FC) >1 and p < 0.050. Volcano and bubble diagrams were generated using the “ggplot2” package. Gene set enrichment analysis (GSEA) was performed on U937-shENO1 and U937 vector cells using the JAVA program ([67]http://software.broadinstitute.org/gsea/index.jsp). Molecular signaling pathways correlated with U937-shENO1 were identified by performing 5000 permutations using the Molecular Signatures Database. Statistical over-representation and enrichment of gene sets were considered with nominal p values ≤0.050. 2.7. Clinical correlation and survival analysis To comprehensively evaluate the correlations between clinical features and ENO1 expression, we downloaded gene expression and clinical data from The Cancer Genome Atlas (TCGA) (n = 175), BeatAML (n = 200), cBioPortal ([68]http://www.cbioportal.org/), and LeuceGene (n = 436) from the website ([69]https://data.leucegene.iric.ca/). We compared ENO1 expression among different groups classified by the French–American–British (FAB) classification system, risk stratification, and remission status using in-house TCGA, BeatAML, and Leucegene datasets. Survival analyses were performed using in-house and TCGA cohort data. Using the R function surv_cutpoint in the Survminer package (v0.4.9), AML patients were categorized into high and low ENO1 expression groups based on the optimal cutoff point of ENO1 expression. The R package survival (v3.2-11) was used to calculate survival risk, and statistical significance was measured using hazard ratios (HR) and log-rank p values. 2.8. Western blot analysis Whole cell lysates were prepared using radio-immunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and a 1× cocktail. The 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were filled with equal amounts and masses of samples for electrophoresis in each well. After electrophoresis, the proteins were transferred onto methanol-activated polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies (1:1000; details are given in Table [70]2), followed by incubation with enzyme-labeled secondary antibodies (1:5000). Protein bands were visualized using chemiluminescent enhanced chemiluminescence (ECL) substrates and imaged using a ChemiDoc XRS+ Gel Imaging System. Table 2. Source and identifier of antibodies used. Antibody Source Identifier Rabbit anti-MEK1/2 Proteintech 11049-1-AP Rabbit anti-ERK1/2 Proteintech 11257-1-AP Rabbit anti-phospho-ERK1/2 Proteintech 28733-1-AP Rabbit anti-Phospho-MAP2K1-S217 Abclonal AP0209 Rabbit anti-C-RAF Affinity AF6065 Rabbit anti-Phospho-C-RAF (Ser338) Affinity AF3065 Rabbit anti-ENO1 Proteintech 11204-1-AP Rabbit anti-β-actin CST 8457S APC anti-human CD45 Biolegend 982304 [71]Open in a new tab ENO1 = alpha-enolase, ERK = extracellular signal-regulated kinase. 2.9. Cell proliferation assay The CCK8 kit (ApexBio, Houston, Texas ) was used to assess the proliferation of KG-1, U937, and OCI-AML3 cells and their stable cell lines. In 96-well plates, 2 × 10³ cells were seeded in each well with 200 μL complete medium. Each condition was tested in triplicate. At 0, 24, 48, 72, and 96 hours, 10 μL of CCK8 reagent was added to each well, followed by incubation at 37°C for 2 hours. Absorbance was measured at 450 nm to determine cell proliferation at each time point. 2.10. Colony formation assay Cells (2 × 10^3) were seeded in each well of 24-well plates, and each well contained 500 μL complete medium containing 3% methylcellulose. The cells were cultured at 37°C in a 5% CO[2] incubator for 14 days to allow colony formation. After incubation, the number and diameters of the colonies were determined in three random fields using a microscope (Leica, Wetzlar, Germany). Each experiment was performed independently in triplicate. 2.11. Cell cycle analysis Cells were fixed in ice-cold 70% ethyl alcohol and stored overnight at −20°C. After fixation, the cells were washed twice with PBS to remove residual ethanol. The cells were then treated with RNase A to digest the RNA and stained with propidium iodide (PI) reagent at 4°C for 30 minutes in the dark. This process allows for DNA labeling, enabling the analysis of cell cycle phases. The samples were then analyzed using flow cytometry (BD, Franklin Lakes, New Jersey ) to determine cell distribution across different phases of the cell cycle. 2.12. Apoptosis assay Apoptosis experiments were conducted using the Annexin V-APC Apoptosis Detection Kit (Elabscience, Wuhan, China). In 6-well plates, 2 × 10^5 cells/well were cultivated in complete medium. After 48 hours, cells were collected and washed with cold PBS. After washing, 500 μL of binding buffer was used to resuspend the cells, and 5 μL of Annexin V-APC reagent and 5 μL of PI reagent were used for staining. Staining was performed at room temperature for 20 minutes. Flow cytometry was used to evaluate the degree of apoptosis in the samples. 2.13. Transwell assay Cell migration and invasion assays were performed in 24-well transwell chambers (Corning, Corning, New York ). For invasion assays, the upper chamber membrane was pre-coated with 50 μL Matrigel (Corning), and incubated at 37°C for 1 hour. The upper chamber remained uncoated with Matrigel in migration assay. Cells (2 × 10⁵ in 100 μL serum-free medium) were seeded in its upper chamber. Subsequently, 500 μL of medium containing double FBS was added to the lower chamber. Flow cytometry was used to count the number of cells that migrated to or invaded the lower chamber after 24 and 48 hours. 2.14. Statistical analysis GraphPad Prism 6 (GraphPad Software, San Diego, California) was used for statistical analysis and expressed as mean ± standard deviation (SD). A t test was used to evaluate the statistical significance between the 2 groups, and the statistical differences in this study were expressed as *p < 0.050, **p < 0.010, and ***p < .001. All experiments were repeated 3 times. 3. RESULTS 3.1. Highly expressed ENO1 is related to poor clinical outcomes in AML To explore the correlation between clinical features and ENO1 expression in AML, we analyzed the expression and clinical data from publicly available datasets, including the BeatAML (n = 200), TCGA (n = 175), and Leucegene project (n = 436) cohorts. We discovered that high ENO1 expression was significantly associated with poor molecular features in TCGA cohort (Fig. [72]1A) and with adverse patient groups according to the 2008 European LeukemiaNet classification in the BeatAML cohort (Fig. S1A, [73]https://links.lww.com/BS/A123). Furthermore, survival analysis demonstrated that AML patients with high ENO1 expression experienced a significant decline in overall survival (OS) and disease-free survival in the TCGA cohort (Fig. [74]1B, C). Figure 1. [75]Figure 1. [76]Open in a new tab Overexpression of ENO1 is associated with poor clinical outcomes in AML. (A) ENO1 expression in AML patients with different risk levels from the TCGA database. (B–C) Survival analysis of AML patients OS and DFS based on ENO1 expression in TCGA database. (D) Boxplot of ENO1 expression in AML patients (n = 159) compared with donors (n = 40) in our in-house cohort. (E) ENO1 expression in AML patients with CR (n = 58) vs NR (n = 12) in our in-house cohort. (F) Survival analysis of AML patients OS based on ENO1 expression in our in-house cohort (n = 98). (G) Differential ENO1 expression across FAB subtypes in our in-house cohort. (H) Analysis of ENO1 expression by qRT-PCR in AML patients (n = 185) compared with donors (n = 61). (I) ENO1 expression was analyzed by qRT-PCR in AML patients with CR (n = 94) and NR (n = 34). *p < 0.050, **p < 0.010, ***p < .001. AML = acute myeloid leukemia, CI = confidence interval, CR = complete remission, DFS = disease-free survival, ENO1 = alpha-enolase, FAB = French–American–British, NR = non-remission, OS = overall survival, qRT-PCR = quantitative reverse transcription polymerase chain reaction, TCGA = The Cancer Genome Atlas. From our in-house RNA-seq data, we observed that ENO1 expression in samples from AML patients was notably higher than that in donor samples (Fig. [77]1D). We also found significantly higher ENO1 expression in the refractory patient group, based on their response to chemotherapy (Fig. [78]1E), indicating that abnormally high ENO1 expression may contribute to chemotherapy resistance in AML patients. Additionally, high ENO1 expression was significantly associated with shorter OS in our in-house cohort (Fig. [79]1F). By comparing the expression of ENO1 among different FAB subtypes, we found that ENO1 expression was higher in the M5 subtype than in other AML subtypes in our in-house cohort (Fig. [80]1G) and in the TCGA, BeatAML, and Leucegene cohorts (Fig. S1B, C, D, [81]https://links.lww.com/BS/A123). Additionally, qRT-PCR analysis of BM samples from 185 AML patients and 61 donors confirmed that ENO1 was highly expressed in AML patients (Fig. [82]1H). We also found that patients with low ENO1 expression were more likely to achieve complete remission (Fig. [83]1I). These results indicate that ENO1 expression is aberrantly high in AML and serves as a predictor of poor clinical outcomes. To validate the functional role of ENO1 in AML, we assessed the cytotoxicity of the commercial ENO1 inhibitor, ENOblock, in AML cell lines (KG-1, U937, and OCI-AML3). The results demonstrated that ENOblock effectively induced cell death in the 3 cell lines (Fig. S2A, [84]https://links.lww.com/BS/A123). 3.2. Knockdown of ENO1 inhibits the proliferation and invasion of AML cells To explore the role and mechanism of ENO1 in AML, we established stable ENO1 knockdown cell lines in KG-1, U937, and OCI-AML3. The efficiency of ENO1 knockdown was assessed by qRT-PCR (Fig. S2B, [85]https://links.lww.com/BS/A123), and western blotting (Fig. S2C, [86]https://links.lww.com/BS/A123). Three independent replicates demonstrated that the mRNA levels of ENO1 in KG-1-shENO1-1 and KG-1-shENO1-2 cells decreased by 78% and 91%, respectively, and the protein levels decreased by 50% and 70%, respectively. The mRNA level of ENO1 in U937-shENO1-1 and U937-shENO1-2 cells decreased by 78% and 82%, and the protein level decreased by 85% and 45%, respectively. ENO1 mRNA levels in OCI-AML3-shENO1-1 and OCI-AML3-shENO1-2 cells decreased by 68% and 65%, respectively, and protein levels decreased by 53% and 83%, respectively. To assess the effect of ENO1 on cell growth, CCK8 assays (Fig. [87]2A) were performed using AML cell lines (KG-1, U937, and OCI-AML3) and their stable cell lines. After 96 h, compared with KG-1, the proliferation of KG-1-shENO1-1 and KG-1-shENO1-2 cells decreased by 52% and 77%, respectively. Compared with U937 cells, the proliferation of U937-shENO1-1 and U937-shENO1-2 cells was reduced by 52% and 77%, respectively. The proliferation of OCI-AML3-shENO1-1 and OCI-AML3-shENO1-2 cells was reduced by 41% and 63%, respectively, compared with that of OCI-AML3 cells. These results indicate that ENO1 knockdown inhibited the proliferation of KG-1, U937, and OCI-AML3 cells. Figure 2. [88]Figure 2. [89]Open in a new tab Knockdown of ENO1 inhibits the proliferation and invasion of AML cells and blocks the cell cycle. (A) The proliferation of KG-1, U937, OCI-AML3, and their stable cell lines was measured using CCK8 assays. (B) KG-1, U937, OCI-AML3, and their stable cell lines were seeded into 24-well plates. Colony number and diameter were assessed on day 14. (C–D) Migration and invasion of KG-1, U937, OCI-AML3, and their stable cell lines were analyzed by transwell assays and flow cytometry at 24 and 48 h. (E) The percentage of cells in G1, S, and G2/M phases of KG-1, U937, OCI-AML3, and their stable cell lines was determined by flow cytometry. *p < 0.050, **p < 0.010, ***p < .001. AML = acute myeloid leukemia, ENO1 = alpha-enolase, NS = no significance. To evaluate the effect of ENO1 knockdown on AML cell clonogenicity, colony formation assays were performed (Fig. [90]2B). The number and diameter of the colonies were analyzed on the 14th day using ImageJ software. After ENO1 knockdown in KG-1, U937, and OCI-AML3 cells, both the diameter and number of colonies decreased significantly. Given that high ENO1 expression is associated with AML clinical progression and poor outcomes and considering AML as a highly invasive malignant tumor, we assumed that ENO1 knockdown might inhibit the migratory and invasive behaviors of AML cell lines. We conducted transwell assays on AML cells and their stable cell lines to validate the effect of ENO1 on AML cell invasion. The results indicated that the number of migrating and invading KG-1, U937, and OCI-AML3 cells increased over time, and that ENO1 knockdown significantly inhibited the migration and invasion of AML cells (Fig. [91]2C, D). 3.3. ENO1 knockdown induces G1 phase arrest in AML cells The proportions of cells in the G1, S, and G2/M phases were determined using flow cytometry (Fig. [92]2E). The results demonstrated that ENO1 knockdown increased the proportion of cells in the G1 phase. However, the proportion of cells in the S phase decreased in KG-1, U937, and OCI-AML3 cell lines. At 48 h, the proportion of G1 phase cells in KG-1-shENO1-1 and KG-1-shENO1-2 cells was 1.3-fold and 1.7-fold that in KG-1 cells, respectively. The proportion of G1 phase cells in U937-shENO1-1 and U937-shENO1-2 was 1.3- and 2.0-fold that in U937 cells, respectively. Similarly, the proportion of G1 phase cells in OCI-AML3-shENO1-1 and OCI-AML3-shENO1-2 cells was 1.2- and 1.1-fold that in OCI-AML3 cells, respectively. These findings suggest that ENO1 knockdown induces G1 phase arrest in AML cell lines. 3.4. ENO1 knockdown inhibits the MAPK/ERK signaling pathway in AML cells To investigate the mechanism by which ENO1 regulates AML cells, we performed RNA-seq using U937 and U937-shENO1-1 cells. Genes with p values less than 0.050 between the 2 groups were considered differentially expressed. Our analysis identified 385 upregulated and 389 downregulated genes in U937-shENO1-1 cells compared with U937 cells (Fig. [93]3A). Figure 3. [94]Figure 3. [95]Open in a new tab ENO1 knockdown inhibits the MAPK/ERK signaling pathway in AML cell lines. (A) Volcano plot showing DEGs between U937 and U937-shENO1-1. (B) Bubble plot of KEGG pathway enrichment analysis. (C) GSEA plot showing the association between ENO1 expression and MAPK signaling pathway activity. (D) Western blot analysis of p-Raf, Raf, p-MEK, MEK, p-ERK, and ERK in control vector, shENO1-1, and shENO1-2 cells, all experiments were independently repeated 3 times. AML = acute myeloid leukemia, DEGs = differentially expressed genes, ENO1 = alpha-enolase, GSEA = gene set enrichment analysis, KEGG = Kyoto Encyclopedia of Genes and Genomes, MAPK/ERK = mitogen-activated protein kinase/extracellular signal-regulated kinase. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways revealed the top 10 significantly enriched signaling pathways, including epithelial cell signaling in Helicobacter pylori infection; adipokine signaling pathway; arginine and proline metabolism; viral protein, cytokine, and cytokine receptor interactions; insulin resistance; MAPK signaling pathway; iron death; JAK-STAT signaling pathway; ABC transporter; and chemotaxis factor signaling pathways (Fig. [96]3B). Among these, we focused on the MAPK signaling pathway because of its well-established association with cancer progression and potential role in AML. To further validate the correlation between ENO1 and the MAPK signaling pathway, we performed GSEA of the RNA-seq data (Fig. [97]3C). The results showed a significant association between ENO1 and MAPK activity (normalized enrichment score [NES] = −1.876, p value <.001, false discovery rate [FDR] = 0.1131). This finding suggests that ENO1 influences the MAPK signaling pathway, which is pivotal for regulating the growth and survival of AML cells. The MAPK signaling pathway encompasses 3 classical pathways: ERK, JNK, and p38. To determine the effects of ENO1 knockdown on these signaling pathways, we assessed the phosphorylation levels of key molecules in the MAPK signaling pathway in KG-1, U937, and OCI-AML3 cells and their stable cell lines. Our results showed that the phosphorylation levels of key ERK signaling pathway molecules (Raf, MEK, and ERK) were decreased following ENO1 knockdown (Fig. [98]3D). In contrast, the phosphorylation levels of p38 and JNK, which are key molecules in the p38 and JNK signaling pathways, respectively, did not show significant changes (Fig. S2D, [99]https://links.lww.com/BS/A123). Therefore, ENO1 selectively activates the MAPK/ERK signaling pathway rather than the p38 or JNK pathways in AML cells. 3.5. MAPK/ERK inhibition suppresses AML cell proliferation, invasion, and induces cell cycle arrest To investigate the impact of the MAPK/ERK signaling pathway on the adverse behaviors of AML cell lines, we treated KG-1, U937, and OCI-AML3 cells with the MEK inhibitor U0126 (5 μM). This concentration (5 μM) was selected for subsequent experiments because it was below the lowest IC25 value in KG-1, U937, and OCI-AML3 to minimize cytotoxicity (Fig. S2E, [100]https://links.lww.com/BS/A123). Cell proliferation, colony formation, migration, invasion, cell cycle progression, and apoptosis were assessed. Western blot analysis revealed that U0126 inhibited MEK and ERK phosphorylation, confirming its effect on the MAPK/ERK signaling pathway (Fig. [101]4A). CCK8 assays demonstrated that U0126 suppressed the proliferation of KG-1, U937, and OCI-AML3 cells (Fig. [102]4B). Colony formation assays revealed that treatment with U0126 significantly reduced the number and diameter of cell colonies compared with the untreated controls (Fig. [103]4C). Transwell assays demonstrated that U0126 treatment for 48 hours significantly inhibited the migratory capacity and invasiveness of AML cell lines (KG-1, U937, and OCI-AML3) compared with the untreated controls (Fig. [104]4D, E). At 48 hours, flow cytometry analysis indicated that the proportion of cells in the G1 phase was elevated and that in the S phase was decreased after U0126 treatment (Fig. [105]4F). Flow cytometry revealed an increase in the percentage of apoptotic KG-1, U937, and OCI-AML3 cells treated with U0126 (Fig. [106]4G). Collectively, these results demonstrate that inhibition of the ERK signaling pathway inhibits the proliferation, colony formation, migration, and invasion of AML cells, arrests the cell cycle in the G1 phase, and promotes apoptosis. Figure 4. [107]Figure 4. [108]Open in a new tab MAPK/ERK pathway inhibitor reduces malignant phenotypes in AML cell lines. (A) Western blot analysis of p-MEK, MEK, p-ERK, and ERK in KG-1, U937, OCI-AML3 treated with U0126 for 24 h. (B) The proliferative capacity of KG-1, U937, and OCI-AML3 cells treated with U0126 was assessed using CCK8 assays. (C) Representative images of colonies formed by KG-1, U937, and OCI-AML3 cells in 24-well plates treated with or without U0126. The number and diameter of colonies were quantified on day 14. (D–E) Migration and invasion of KG-1, U937, and OCI-AML3 cells treated with U0126 were analyzed by transwell assays and flow cytometry after 48 h of treatment. (F) Flow cytometry was employed to assess the distribution of KG-1, U937, and OCI-AML3 cells in distinct cell cycle phases following 48 h treatment with U0126. (G) Apoptosis rates of KG-1, U937, and OCI-AML3 cells before and after 48 h U0126 treatment was detected by flow cytometry. *p < 0.050, **p < 0.010, ***p < .001. AML = acute myeloid leukemia, MAPK/ERK = mitogen-activated protein kinase/extracellular signal-regulated kinase, NS = no significance. 3.6. The MAPK/ERK signaling pathway activator rescues AML cells from the inhibitory effects of ENO1 knockdown To investigate the functional role of ENO1 in AML cells mediated by the MAPK/ERK pathway, we used the ERK activator SENI (10 μM) to activate ERK in shENO1 cell lines and then assessed cell proliferation, migration, invasion, and cell cycle progression. CCK8 assays demonstrated that ENO1 knockdown significantly suppressed AML cell proliferation, whereas SENI treatment restored the proliferation of shENO1 cells (Fig. [109]5A). Transwell assays revealed that the ENO1 knockdown inhibited the migration and invasion of AML cells, both of which were rescued by SENI treatment (Fig. [110]5B, C). Flow cytometry demonstrated that ENO1 knockdown induced G1 phase cell cycle arrest in KG-1, U937, and OCI-AML3 cells, which was rescued by SENI treatment (Fig. [111]5D). Collectively, these findings demonstrate that MAPK/ERK pathway activation rescues ENO1 knockdown-induced functional deficiencies in AML cells. Figure 5. [112]Figure 5. [113]Open in a new tab The MAPK/ERK signaling pathway activator rescues AML cells from the inhibitory effects of ENO1 knockdown. (A) SENI restored proliferation of shENO1 cells. (B-C) SENI increased migration capacity and invasion capacity of shENO1 cells. (D) SENI promoted cell cycle progression by reducing the G1 phase population of shENO1 cells. *p < 0.050, **p < 0.010, ***p < .001. AML = acute myeloid leukemia, ENO1 = alpha-enolase, MAPK/ERK = mitogen-activated protein kinase/extracellular signal-regulated kinase, NS = no significance, SENI = senkyunolide I. 3.7. Knockdown of ENO1 inhibits the development of AML in mice in vivo To evaluate the roles of ENO1 and MAPK/ERK signaling in AML progression in vivo, we intravenously injected 2 × 10^6 U937 or U937-shENO1 cells into mice, followed by triweekly intraperitoneal administration of DMSO or U0126. After 14 days, PB, BM, liver, spleen, and lung tissues of the mice were collected for analysis. qRT-PCR analysis of BM and spleen cells demonstrated significantly reduced ENO1 mRNA levels in shENO1 cell-injected mice compared with U937 cell-injected controls (Fig. [114]6A). The spleen weight of the shENO1 group was 37% lower than that of the U937 group, whereas U0126 treatment caused a 39% decrease compared with the DMSO controls (Fig. [115]6B, C). The percentage of CD45^+ cells in the PB, BM, and spleen was reduced in the shENO1 and U0126 groups compared with that in their respective controls (Fig. [116]6D, E). Additionally, both ENO1 knockdown and U0126 treatment extended the OS of the mice (Fig. [117]6F). H&E staining was performed on the spleen, lungs, liver, and femurs. We found that the tumor cell burden in the spleen sections of the shENO1 and U0126 groups was significantly reduced compared with that in their respective control groups (Fig. [118]6G, S3A, [119]https://links.lww.com/BS/A123). Neither ENO1 knockdown nor U0126 treatment significantly altered the liver/lung weight or body weight measurements across the experimental groups (Fig. S3B–D, [120]https://links.lww.com/BS/A123). Collectively, these findings demonstrate that targeting ENO1 or MAPK/ERK signaling effectively suppresses AML progression in vivo by reducing leukemic infiltration. Figure 6. [121]Figure 6. [122]Open in a new tab Knockdown of ENO1 inhibits the development of AML in mice. (A) qRT-PCR analysis of ENO1 mRNA expression in the BM and spleen of mice from U937 and shENO1 groups. (B–C) Representative spleen images and spleen weights from mice in different groups. (D–E) Proportion of CD45^+ cells in PB, spleen, and BM of mice in different groups was quantified by flow cytometry. (F) Survival analysis of mice in different groups. (G) H&E staining of spleen sections in different groups. *p < 0.050, **p < 0.010, ***p < .001. AML = acute myeloid leukemia, BM = bone marrow, DMSO = dimethyl sulfoxide, ENO1 = alpha-enolase, H&E = hematoxylin and eosin, PB = peripheral blood, qRT-PCR = quantitative reverse transcription polymerase chain reaction. 4. DISCUSSION In recent years, significant advancements have been made in the understanding of AML pathogenesis through in-depth studies. Cytogenetic and molecular aberrations are crucial for determining the response to chemotherapy and long-term outcomes. Identifying potential therapeutic targets and developing small-molecule drugs targeting AML are essential strategies for addressing recurrence and drug resistance.^[123]25 Our research has definitively established ENO1 as a promising target for AML therapy. Using qRT-PCR, we detected ENO1 expression in the BM samples from 61 donors and 185 AML patients. The results, along with the analysis of patient genome sequencing data, revealed that ENO1 is overexpressed in AML and has a negative relationship with disease progression and clinical outcomes. ENO1 is widely localized in the cytoplasm, cell membrane, nucleus, and extracellular space around tumor cells, contributing to different subcellular functions. This study investigated the contribution of cytoplasmic changes in ENO1 to AML malignancy. Using RNA-seq analysis, we identified a positive correlation between ENO1 and the MAPK signaling pathway. Western blot analysis revealed that ENO1 selectively activated the MAPK/ERK signaling pathway rather than the p38 or JNK pathways. The Raf/MEK/ERK signaling pathway is a typical MAPK signaling pathway crucial for cell proliferation and survival. In proliferating cells, the activation of ERK1/2 is indispensable for the transition from the G1 to S phase of the cell cycle. Our results showed that the inhibition of the ERK signaling pathway blocked the cell cycle in the G1 phase. ENO1 knockdown in gastric cancer leads to cell cycle arrest at the G1 phase.^[124]11 In the non–small-cell lung cancer cell line A549, ENO1 overexpression increased the levels of the oncogenic cell cycle regulators cyclin D1, cyclin E1, and c-Myc, whereas the cell cycle inhibitors p21 and p27 were negatively regulated by ENO1.^[125]26 Cyclin D1 plays an essential role in the mechanism by which ERK regulates the G1/S phase transition of the cell cycle.^[126]27,[127]28 Our results demonstrated that ENO1 knockdown inhibited cyclin D1 expression in AML cells by inhibiting the ERK signaling pathway (Fig. S4A, B, [128]https://links.lww.com/BS/A123). Therefore, ENO1 modulates cyclin D1 and other cell cycle regulators by regulating the MAPK/ERK signaling pathway, thereby affecting cell cycle progression. In addition to regulating the cell cycle, ERK influences cell growth and apoptosis by modulating biosynthetic and metabolic signaling pathways, such as mRNA translation, protein synthesis, and glycolysis.^[129]28,[130]29 ENO1, a key enzyme in glycolysis, promotes tumor cell growth and regulates apoptosis.^[131]30–[132]32 Our results demonstrated that ENO1 knockdown or inhibition of the ERK signaling pathway could inhibit proliferation and promote the apoptosis of AML cells. Furthermore, the activation of the ERK signaling pathway reversed the inhibitory effects of ENO1 knockdown on AML cell proliferation and apoptosis. Therefore, ENO1 promotes the growth of AML cells and inhibits apoptosis by activating the ERK signaling pathway. As one of the crucial steps in tumorigenesis, invasion and metastasis are regulated by multiple signaling pathways, among which the MAPK/ERK signaling pathway promotes cell metastasis and invasion through the transcriptional regulation of key regulators such as matrix metalloproteinases and epithelial-mesenchymal transition.^[133]33,[134]34 Active ERK regulates cell adhesion and migration by targeting integrin binding and activating v-Src.^[135]35 In Burkitt lymphoma, we have previously shown that downregulating ENO1 inhibits the phosphoinositide 3-kinase–protein kinase B (PI3K-AKT) signaling pathway by decreasing plasminogen recruitment and plasmin production, thereby inhibiting tumor cell invasion.^[136]15 In our cell experiments, ENO1 knockdown or inhibition of the ERK signaling pathway inhibited the migration and invasion of AML cells. Additionally, activation of the ERK signaling pathway rescued the effect of ENO1 knockdown on the migration and invasion of AML cells. Therefore, ENO1 promoted AML cell invasion via the MAPK/ERK pathway. In mice, both ENO1 knockdown and U0126 treatment reduced AML cell invasion and prolonged the OS of mice (log-rank p = 0.003 and p = 0.039, respectively). Although the latter p value approached the significance threshold, it still met the statistical standard of p < 0.050. Considering that the sample size may have limited statistical power, we will expand the animal cohort in follow-up studies to further validate this trend. However, the mechanism through which ENO1 regulates the MAPK pathway remains unclear. Metabolic enzymes regulate cell signaling pathway mainly through the following two mechanisms: First, glycolytic enzymes can directly regulate signaling pathways through “moonlighting functions.” For example, PCK1 phosphorylates INSIG to activate SREBP-driven lipogenic gene expression.^[137]36 Second, metabolic intermediates mediate signaling effects. Lactate can activate SIRT1 by increasing the NAD^+/NADH ratio, thereby promoting the MAPK phosphorylation cascade.^[138]37 KEGG analysis of the RNA-seq data after ENO1 knockdown in this study showed that lipid metabolism and amino acid metabolism-related pathways. These findings suggest that ENO1 may have dual biological functions in AML, functioning as a metabolic enzyme that regulates basal metabolic processes and activates the MAPK/ERK signaling pathway through its moonlighting functions, thereby driving AML progression. In this study, we investigated for the first time the function of ENO1 in AML occurrence and development. Additionally, we aimed to identify ENO1 as a potential target for the diagnosis and treatment of AML. In the future, we will continue to investigate the specific molecular mechanisms and downstream effector factors by which ENO1 regulates phosphorylation of the MAPK/ERK signaling pathway. 5. CONCLUSIONS ENO1 is markedly overexpressed in AML patients, as evidenced by both database analyses and clinical sample evaluations. Furthermore, ENO1 levels negatively correlated with patient remission and survival. ENO1 regulates the proliferation, migration, and invasion of AML cells via the MAPK/ERK signaling pathway. Therefore, ENO1 is a promising tumor marker and therapeutic target for AML. ACKNOWLEDGMENTS