ENO1 promotes acute myeloid leukemia progression through SCD1-mediated lipid metabolism reprogramming

Key Points

• •ENO1 regulates lipid metabolism and induces ferroptosis resistance by interacting with the SCD1 promoter to enhance its transcription in AML.
• •Combination of SCD1 inhibitor and DNR is a potential therapy strategy for ENO1 overexpression in patients with AML.

Visual Abstract

Abstract

α-Enolase (ENO1) is a potential therapeutic target in acute myeloid leukemia (AML), owing to its elevated expression in AML cells. In this study, we investigated the association between high ENO1 expression, accelerated tumor progression, and poor AML prognosis. Transcriptomic and metabolomic analyses indicated that ENO1 directly modulates lipid metabolism via regulating stearoyl–coenzyme A desaturase 1 (SCD1) expression. We further demonstrated that ENO1 functions as a DNA-binding protein, interacting with the SCD1 promoter region to enhance SCD1 transcription. This results in increased synthesis of monounsaturated fatty acids, leading to increased resistance to lipid peroxidation and ferroptosis. Based on these results, we found that SSI-4, an SCD1 inhibitor, could enhance chemosensitivity of daunorubicin (DNR), which can induce ferroptosis in tumor cells, effectively reducing the resistance to ferroptosis in AML cells exhibiting high ENO1 expression. Overall, our study elucidates the mechanism of ENO1 that promotes SCD1 transcription, driving lipid reprogramming and ferroptosis resistance within AML. In addition, it highlights the therapeutic potential of combining SCD1 inhibition with DNR for patients with AML with elevated ENO1 expression levels.

Introduction

Although continuous updates to the treatment protocols of acute myeloid leukemia (AML) have led to improvements, challenges such as high recurrence rates, toxicity, and chemoresistance continue to pose significant obstacles in AML treatment,¹ highlighting the urgent need for more effective and precise therapeutic strategies.

Enhanced glycolysis is a hallmark of tumor cells and is highly associated with poor prognosis; therefore, targeting cancer glycolysis presents a potential key strategy for oncotherapy.² The aberrant hyperactivation of rate-limiting glycolytic enzymes contributes to tumor progression and chemoresistance development in AML,3, 4, 5, 6, 7, 8, 9 indicating that glycolytic enzymes play vital roles in both AML tumor progression and chemotherapy resistance.

α-Enolase (ENO1) is a pivotal enzyme in the glycolytic pathway, facilitating the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid. ENO1 expression levels increase in various malignancies, thereby enhancing glycolytic activity in tumor cells, closely related to tumorigenesis and cancer progression.10, 11, 12, 13, 14, 15 Several oncogenic factors can enhance glycolytic activity and energy production in neoplastic cells by upregulating both the expression and modification of ENO1, ultimately contributing to tumor advancement.16, 17, 18, 19 In AML, ENO1 is aberrantly overexpressed and has been recognized as a predictor of poor prognosis as well as a potential antitumor target.20, 21, 22 Recent bioinformatics and machine learning algorithms applied to single-cell RNA sequencing (RNA-seq) data from patients with AML compared with healthy controls have demonstrated that high levels of ENO1 expression promote the self-renewal of leukemia stem cells and confer chemoresistance.²³ Collectively, these findings underscore that ENO1 actively or passively modulates multiple signaling pathways while driving metabolic reprogramming during tumorigenesis and cancer progression.

Apart from its role as a glycolytic enzyme, ENO1 is also known for its diverse nonglycolytic functions. For example, ENO1 directly participates in the regulation of tumor cell signaling pathways as an RNA-binding protein. It binds to mRNAs and modulates the stability of YAP1 and IRP1 mRNAs, thereby contributing to tumor progression.^24,25 In addition, ENO1 has been demonstrated to modulate phosphorylation with hepatocyte growth factor receptor, as well as ubiquitination with choline kinase α and proteasomal degradation of programmed death-ligand 1 through protein-protein interactions, thereby altering the stability of its downstream proteins or inducing tumor immune evasion.26, 27, 28 Finally, ENO1 also serves as a DNA-binding protein that interacts with the promoter regions of various DNAs to participate in transcriptional regulation, including CMYC, HSD3B2, and erythroblastic oncogene B homolog 2 (ERBB2/Her2).29, 30, 31 Lipid metabolic reprogramming plays a crucial role in the tumorigenesis and progression of malignant tumors.³² Stearoyl–coenzyme A desaturase 1 (SCD1) is a key enzyme responsible for the synthesis of monounsaturated fatty acids (MUFA) from the respective saturated FA (SFA),³³ which is also overexpressed in malignant cells, such as leukemia, protecting cancer cells from ferroptosis and thereby presenting itself as a potential target for cancer therapy.33, 34, 35, 36 In FLT3-mutant AML, the downregulation of SCD1 resulted in the decreased incorporation of MUFA into membrane phospholipids and the increased susceptibility of AML cells to lipid peroxidation.³⁷ This finding highlights the promising potential of SCD1 inhibitors in AML treatment.

This study demonstrated that ENO1 promotes SCD1 expression by directly binding to the promoter and regulating lipid metabolism reprogramming and ferroptosis in AML, suggesting SCD1 as a candidate therapeutic target in drug-resistant AML characterized by ENO1 overexpression.

Methods

Clinical patient data

Data of the patients with AML were derived from a retrospective study conducted at our center from October 2013 to September 2022. A total of 142 patients were included in this study.

In vitro experiments

The human AML cell lines MV4-11, Kasumi-1, HEL, HL60, U-937, MOLM-16, MOLM-13, NB4, and K562 were obtained from the National Collection of Authenticated Cell Cultures in Shanghai, China. The short hairpin RNA for ENO1 was synthesized and constructed into the pLKO.1 vector by IGE Biotechnology Ltd, Guangzhou, China. The detailed methods, reagents, short hairpin RNA sequences information and primer sequences are provided in the supplemental materials and methods insupplemental Materials and supplemental Tables 1-3.

In vivo experiments

AML cells were injected into the NOD/LtSz-SCID/IL-2Rγchain null (NSG) mice through the tail veins to establish AML cell line-derived xenografts (CDX) models. Detailed in vivo experiments are described in the supplemental materials and methods in supplemental Materials.

CUT&Tag assay

Cleavage Under Target & Tagmentation (CUT&Tag) assays were performed in MV4-11 cells using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, Nanjing, China). Detailed methods and data analysis are described in the supplemental materials and methods in supplemental Materials. The peaks identified by CUT&Tag are listed in supplemental Tables 4 and 5.

ChIP-seq data collection and analysis

We conducted a comprehensive analysis of chromatin immunoprecipitation (ChIP)–sequencing data sets derived from 11 samples from patients with AML (GSE188605³⁸), MV4-11 cell lines (GSE80779³⁹), and CD34⁺ hematopoietic progenitors (GSE71809⁴⁰). See detailed description for raw data analysis in the supplemental materials and methods in supplemental Materials.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc, La Jolla, CA). Statistical significance was set at P < .05 (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001). A detailed description on statistical analysis is provided in the supplemental Materials.

This study was approved by the Ethics Committee of the Children’s Hospital of Soochow University (number 2017047-3) and was conducted in accordance with the Declaration of Helsinki. The animal experiment in this study was approved by the Ethics Committee of Soochow University (approval number SUDA20250320A08).

Results

ENO1 represents a promising specific target for AML outcomes

To identify AML-specific therapeutic targets, we first extracted the top 1% genes with the highest expression levels in AML from the Cancer Cell Line Encyclopedia database (supplemental Table 6). Subsequently, we obtained their genetic dependency scores in both AML and control cell lines derived from CRISPR-based functional genomics screens available in DepMap. Through combined screening, we ultimately obtained 16 genes closely associated with AML (Figure 1A). Given ENO1 plays a critical role in glycolysis, the process usually enhanced in tumor cells, we focused on ENO1 as a key target for this study.

Figure 1.

ENO1 represents a promising specific target for AML outcomes. (A) Pyramid flowchart illustrated the workflow for screening AML-specific genes from the Cancer Cell Line Encyclopedia (CCLE) database. A total of 192 genes were identified as exhibiting the top 1% expression levels in AML cells based on the CCLE database. Among them, 131 genes exhibited a dependency score below −0.5 and were classified as survival-essential genes for AML cells. The genes that exhibited a dependency score below −0.5 in nontumor cells were excluded, leaving 16 candidate genes. (B) The dependence to ENO1 of 32 malignancies, with data from the CCLE database. (C) ENO1 expression level in cells from healthy donors (n = 9) and cells from patients with AML (n = 13). (D-E) Kaplan-Meier curves comparing event-free survival (D) and OS (E) in patients with AML (n = 142) from our institutional cohort based on tertiles of ENO1 expression levels. NK, natural killer. ∗∗∗P < .001.

We then investigated the expression level of ENO1 across multiple malignancies and assessed its oncogenic dependency through data obtained from CRISPR screening within the Cancer Cell Line Encyclopedia database (Figure 1B; supplemental Figure 1A). ENO1 was found to be highly expressed in AML and exhibited significant dependency in AML cell lines. Our previous studies,²² which included a cohort of 13 patients with AML and 9 healthy donors, used single-cell RNA-seq to characterize the cellular heterogeneity presented in patients with AML (supplemental Figure 1B). The expression of ENO1 was elevated in cells of the AML group compared with those from healthy donors (Figure 1C).

For potential prognostic value evaluation of ENO1, Kaplan-Meier survival analysis was performed on 142 patients with AML from our center (supplemental Table 7). The event-free survival rate for patients with high ENO1 expression was lower than that for the low ENO1 expression group (Figure 1D). Although overall survival (OS) did not reach statistical significance, a notable trend toward poorer OS was evident among patients exhibiting higher levels of ENO1 compared with those with lower levels (Figure 1E). Survival analyses in independent AML cohorts from GEPIA (supplemental Figure 1C) and The Cancer Genome Atlas (supplemental Figure 1D) databases corroborated these findings. Collectively, these results establish a robust association between ENO1 expression levels and AML outcomes, positioning ENO1 as a potential candidate biomarker for predicting clinical prognosis in this disease context.

ENO1 is essential for the progression of AML cells via resistance to ferroptosis

To validate the essential role of ENO1 at the cellular level, we assessed ENO1 expression across AML cell lines. Consistent with previous findings, most of these cell lines exhibited high levels of ENO1 expression (supplemental Figure 2A-B). In addition, we observed that ENO1 expression was higher in leukemic cells isolated from 4 patients with AML (AML1, AML2, AML3, and AML4) than in bone marrow mononuclear cells (BMMCs) from four healthy donors (BMMC1, BMMC2, BMMC3, and BMMC4) (supplemental Figure 2C). These results demonstrated a higher level of ENO1 in AML cells than in normal cells. To verify the dependence of AML cells on ENO1, we established stable ENO1-knockdown (ENO1^KD) cell lines in MV4-11 and Kasumi-1 (Figure 2A). Cell Counting Kit-8 (CCK-8) assay and colony formation showed that the activity and growth of ENO1^KD AML cells were significantly suppressed in vitro (Figure 2B-C; supplemental Figure 2D-F). Furthermore, these findings were recapitulated in primary AML cells (supplemental Figure 2G-H). Notably, we observed that ENO1^KD AML cells exhibited increased ferroptosis, characterized by increased lipid peroxidation (Figure 2D-E; supplemental Figure 2I-J), elevated intracellular Fe²⁺ accumulation (Figure 2F; supplemental Figure 2K-L), and decreased glutathione (GSH) level and GSH/oxidized GSH (GSSG) ratio (Figure 2G-H). These changes could be further enhanced upon treatment with the ferroptosis inducer RSL3 (Figure 2D-F; supplemental Figure 2I-L), suggesting that ENO1-mediated tumor progression might partially rely on ferroptosis inhibition. To investigate the mechanism underlying the growth inhibition, we detected the apoptosis rate and cell cycles of ENO1^KD AML cells. It showed no significant changes in cell apoptosis rate or cell cycles profiles in ENO1^KD AML cells (supplemental Figure 3A-D). Moreover, we did not find consistent evidence for the enhancement of other cell death pathways, such as apoptosis, pyroptosis, and autophagy in ENO1^KD AML cells (supplemental Figure 3E-F).

Figure 2.

ENO1 is essential for the progression of AML cells via resistance to ferroptosis. (A) Western blot is used to verify the knockdown efficiency of ENO1 in MV4-11 and Kasumi-1 cells (left, western blot bands; right, quantification of the bands). (B) CCK-8 assay of MV4-11 and Kasumi-1 cells proliferation after knockdown of ENO1 (n = 3). (C) Colony formation assay of MV4-11 and Kasumi-1 cells proliferation after knockdown of ENO1 (left, colony formation; right, relative colony counts). (D-E) Lipid peroxidation level detected by flow cytometry with C11-BODIPY staining in shNC (negative control) and shENO1(ENO1 knockdown by short hairpin RNA plasmids) MV4-11 and Kasumi-1 cells with or without RSL3 treatment (n = 3) (top, histograms of C11-BODIPY; bottom, mean fluorescence intensity [MFI] of C11-BODIPY). (F) The intracellular level of Fe²⁺ was measured using the FerroOrange probe (n = 3) (up, measurement of the FerroOrange probe; down, relative FerroOrange fluorescence intensity). (G-H) Total GSH level and GSH/GSSG ratio in shNC and shENO1 MV4-11 and Kasumi-1 cells with or without RSL3 treatment (n = 3). (I) Bioluminescence imaging and quantification of tumor burden in NSG mice on days 11, 15, 18, and 20 after MV4-11 tail-vein injection (n = 5). (J) Bioluminescence imaging and quantification of tumor burden in NSG mice on days 7, 14, 21, and 25 after HEL cell injection (n = 5). CCK-8, Cell Counting Kit-8; cps, counts per second. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

To confirm the tumor-promoting role of ENO1 on AML in vivo, ENO1^KD MV4-11 cells were delivered into NSG mice. Compared with the negative control group mice, mice with ENO1^KD exhibited significantly reduced tumor burden and decreased infiltration in the liver, spleen, and bone marrow (Figure 2I; supplemental Figure 4A-B). In contrast, ENO1-overexpressing (ENO1^OE) HEL cells (supplemental Figure 4C) showed an accelerated growth rate in vivo compared with the control cells, as well as increased infiltration in the liver, spleen, and bone marrow (Figure 2J; supplemental Figure 4D-E). Collectively, these findings indicated that the depletion of ENO1 suppresses AML progression primarily by attenuating resistance to ferroptosis, as opposed to inducing cell apoptosis, pyroptosis, and autophagy.

ENO1 modulates lipid metabolism in AML

To delineate and characterize the molecular pathways governing ENO1-mediated AML progression and ferroptosis resistance, we performed RNA-seq on ENO1^KD MV4-11 cells. Transcriptomic analysis identified 1005 upregulated and 523 downregulated genes, among which the most prominently ranked genes included SCD1, fatty acid desaturase 1 (FADS1), and FADS2, encoding key enzymes involved in unsaturated FA biosynthesis (Figure 3A; differentially expressed genes are listed in supplemental Table 8). KEGG pathway enrichment analysis further confirmed the significant association between FA metabolism and ENO1 expression levels, encompassing pathways involved in the biosynthesis of unsaturated FA, steroids, and steroid hormones (Figure 3B). We then evaluated the expression levels of SCD1, FADS1, and FADS2 using real-time polymerase chain reaction (PCR) and western blot assays in ENO1^KD AML cells (MV4-11, Kasumi-1 U-937, and MOLM-16, as well as primary AML1-4 cells) (Figure 3C-D; supplemental Figure 5A-E), which confirmed a consistent downregulation of SCD1 expression induced by ENO1 knockdown.

Figure 3.

ENO1 modulates lipid metabolism in AML. (A) Volcano plot of differentially expressed genes between ENO1^KD and control groups. The red and green colors indicate upregulated and downregulated genes, respectively (|log₂FoldChange|> 1, P < .05). (B) KEGG pathway enrichment analysis of downregulated differentially expressed genes. (C) Quantitative reverse transcription PCR assay of mRNA levels of SCD1, FADS1, and FADS2 in ENO1^KD MV4-11 and Kasumi-1 cells (n = 3). (D) Western blotting of protein expression levels of SCD1, FADS1, and FADS2 in ENO1^KD MV4-11 and Kasumi-1 cells (top, western blot bands; bottom, quantification of the bands). (E) Volcano plot of downregulated and upregulated metabolites in MV4-11 cells after ENO1 knockdown. (F) Alterations in long-chain and very-long-chain MUFA in MV4-11 after ENO1 knockdown (n = 3). (G) Alterations of PUFA/MUFA ratio in the phospholipid acyl chain of ENO1^KD MV4-11 cells (n = 3). Down, downregulated; FDR, false discovery rate; ns, not significant; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acids; Up, upregulated. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

To further validate our findings from RNA-seq data, we subsequently performed metabolomic analysis of ENO1^KD MV4-11 cells. Metabolomic analysis also revealed decreased activity in lipid metabolic pathways, including ether lipid metabolism, cholesterol metabolism, and phosphatidylinositol signaling pathways within ENO1^KD cells (supplemental Figure 5F). Cross-omics KEGG pathway analysis highlighted cholesterol metabolism in cancer as a key intersecting pathway (supplemental Figure 5G), further linking ENO1 to lipid metabolism reprogramming, particularly in unsaturated FA biosynthesis.

Interestingly, SCD1, which was found to be enriched in RNA-seq analysis, plays a crucial role in the MUFA biosynthesis in vivo. It catalyzes the desaturation of 16:0 and 18:0 SFA into 16:1 and 18:1 MUFA.³³ Concurrently, metabolomic analyses revealed a significant decrease in phosphatidylethanolamines and phosphatidylcholines containing MUFA acyl chains comparing to control group cells (Figure 3E). Phosphatidylcholines and phosphatidylethanolamines represent the predominant glycerophospholipid (GP) species within biological membranes. GPs with 16:1 and 18:1 MUFA acyl chains were significantly reduced in ENO1 depletion cells (Figure 3F), whereas GPs with SFA and polyunsaturated FA acyl chains also exhibited changes (supplemental Figure 5H-I). These findings strongly suggest that the reduction in MUFA-incorporated GPs in ENO1^KD cells is primarily attributed to the downregulation of SCD1 expression. Concomitantly, ENO1^KD MV4-11 displayed an increased polyunsaturated FA/MUFA ratio in multiple types of GPs (Figure 3G), a hallmark of enhanced susceptibility to membrane lipid peroxidation and ferroptosis, which could be manipulated via SCD1 inhibition.^37,41 However, other genes involved in ferroptosis-related pathways, such as GPX4, DHODH, and GCH1, were not consistently affected by ENO1 expression (supplemental Figure 5J), reinforcing the hypothesis that ENO1 regulates ferroptosis resistance predominantly through SCD1-mediated lipid metabolic remodeling.

ENO1 regulates SCD1 transcription by binding to its promoter

Although we have identified SCD1 as a downstream target of ENO1, the precise mechanism by which ENO1 influences SCD1 expression remains unclear. As a “moonlight protein,” ENO1 has been demonstrated to function as an RNA-binding protein and affect mRNA stability in various cellular contexts.^24,25,42,43 To determine whether ENO1 influences SCD1 expression via mRNA stabilization, we treated AML cells with actinomycin D and assessed the SCD1 mRNA decay. Markedly, the SCD1 RNA half-life remained unchanged in ENO1^KD AML cells compared with controls (supplemental Figure 6A-B), ruling out posttranscription regulation via mRNA stability. This suggests that ENO1 primarily regulates SCD1 at the transcriptional level.

Given that ENO1 was shown to regulate gene transcriptional activity through nuclear translocation and subsequent binding to DNA promoter regions,³¹ we performed CUT&Tag in MV4-11 cells to elucidate whether ENO1 regulates SCD1 at the transcription level. Our analysis revealed distinct binding peaks for ENO1 in the promoter regions of SCD1, FADS1, and FADS2 (Figure 4A; supplemental Figure 6C-D). To integrate transcriptional and epigenetic data, we conducted a Venn diagram analysis of RNA-seq and CUT&Tag and identified 859 overlapped genes (Figure 4B), including SCD1, which ranked in the top 20 genes downstream of ENO1 (Figure 4C-D). These findings established SCD1 as a downstream target of ENO1 and suggested that ENO1 regulates SCD1 expression through interaction with its promoter.

Figure 4.

ENO1 directly regulates SCD1 transcription by binding to its promoter. (A) IGV visualization of the SCD1 whole-genome map. HSPC from healthy samples as negative control. (B) Venn diagram of RNA-seq and CUT&Tag showing that 859 genes were bound and regulated by ENO1 in MV4-11. (C) Volcano plot of genes in ENO1^KD MV4-11, with a colored plot showing 57 of the top 100 enriched downregulated genes that bind with ENO1. (D) Heat map of the top 20 enriched downregulated genes that bind with ENO1. (E) Dual-luciferase reporter assay of SCD1 promoter in ENO1^KD cells (n = 3). (F) Dual-luciferase reporter assay of SCD1 promoter in ENO1^KD cells with ENO1^WT or ENO1 catalytically dead mutants (n = 3). (G-H) ChIP-PCR assay verifying the ENO1-binding sequence on the SCD1 promoter region located −237 bp from the transcription start site. (I) Dual-luciferase reporter assay of SCD1 promoter in ENO1^OE cells with or without the deletion of the specific sequence (n = 3). DEG, differentially expressed genes; del, delete; Down, downregulated; HSPC, hematopoietic stem and progenitor cells; IgG; Immunoglobulin G; IGV, integrative genomics viewer; non-diff, non-differential; ns, not significant; Up, upregulated; WT, wild type. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

To validate the findings from CUT&Tag, we conducted a dual-luciferase reporter assay in ENO1^KD AML cells, which showed significantly decreased transcriptional activity of the SCD1 promoter region in ENO1^KD AML cells (Figure 4E), indicating that the transcriptional level of SCD1 was suppressed in AML cells with ENO1 depletion. ENO1 is known as a key enzyme in glycolysis that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate, the final step in preparing the substrate for ATP generation in glycolysis. Therefore, we constructed 2 catalytically dead mutants ENO1^D245R and ENO1^S40A, which have been validated as enzymatically inactive but structurally intact in a well-established system,²⁵ to investigate whether ENO1-mediated SCD1 transcription is dependent on its glycolytic enzyme activity. After complementation of the 2 catalytically dead mutants in ENO1^KD AML cells, the decreased transcriptional activity of the SCD1 promoter caused by ENO1 knockdown increased significantly (Figure 4F; supplemental Figure 6E), indicating that ENO1-mediated transcriptional regulation of SCD1 does not depend exclusively on its intact glycolytic catalytic activity. Immunofluorescence staining with ENO1 antibody further revealed nuclear localization of ENO1 in AML cells (supplemental Figure 6F), consistent with its role as a transcriptional regulator. Studies have reported sequence similarity in the ENO1 DNA-binding elements across the promoters of CMYC, the first intron of HSD3B2, and gene ZAT10.²⁹ To define the specific DNA sequence mediating ENO1-SCD1 promoter binding, we located a similar sequence, GGATAAAAGGGGGC, situated at −237 bp upstream of the transcription start site of the SCD1 promoter region and designed specific PCR primers targeting this sequence (Figure 4G). ChIP-PCR using primers targeting this motif validated the binding of ENO1 on the SCD1 promoter in MV4-11 and Kasumi-1 cells (Figure 4H), whereas a control sequence (GATATAAAAGATGAT located at +673 base pair (bp) downstream of the SCD1 transcription start site) showed no enrichment (supplemental Figure 6G-H). Moreover, we designed luciferase reporter constructs carrying a deletion of this potential binding sequence in the SCD1 promoter region and performed a dual-luciferase reporter assay. Although transcriptional activity from the SCD1 promoter was increased in ENO1^OE cells, deletion of the putative ENO1-binding sequence abolished this enhanced effect (Figure 4I). This suggested that ENO1 might directly bind to the promoter of SCD1 via the specific sequence and thereby mediate transcriptional regulation on SCD1.

All these evidences consistently support our hypothesis that ENO1 might regulate SCD1 transcription by directly interacting with its promoter as a nuclear DNA-binding protein, driving SCD1 transcription and subsequent lipid metabolic reprogramming in AML.

ENO1-mediated AML progression depends on SCD1 and its metabolic product OA

To provide further evidence that ENO1 drives AML progression and ferroptosis resistance by regulating SCD1, we replenished SCD1 expression in ENO1^KD AML cells (Figure 5A-B). In vitro, the restoration of SCD1 effectively reversed ferroptosis following ENO1 knockdown, as shown by reduced lipid peroxidation (Figure 5C-E; supplemental Figure 7A), decreased intracellular Fe²⁺ level (Figure 5F; supplemental Figure 7B), along with the partial recovery of GSH level and GSH/GSSG ratio (Figure 5G). In vivo, compared with the ENO1^KD group mice, NSG mice injected with ENO1^KD-SCD1^OE cells exhibited significantly restored tumor burden and organ infiltration (Figure 5H; supplemental Figure 7C-H), indicating that SCD1 reintroduction partially reversed the tumor-suppressive effect caused by ENO1 depletion. These results highlight the dependency of ENO1-mediated tumor progression on SCD1 expression.

Figure 5.

Restoration of SCD1 reverses the ferroptotic phenotype in ENO1^KD AML cells. (A-B) Western blot verified the efficiency of ENO1 knockdown and SCD1 overexpression in MV4-11 and Kasumi-1 cells (left, western blot bands; right, quantification of the bands). (C-D) Lipid peroxidation level was assessed by flow cytometry after C11-BODIPY staining in SCD1-restored MV4-11 and Kasumi-1 cells with or without RSL3 treatment (n = 3) (left, histograms of C11-BODIPY; right, MFI of C11-BODIPY). (E) Lipid peroxidation level detected by confocal microscopy with C11-BODIPY staining in SCD1-restored MV4-11 and Kasumi-1 cells with RSL3 treatment (n = 3). (F) The intracellular level of Fe²⁺ in SCD1-restored MV4-11 and Kasumi-1 cells with or without RSL3 treatment (n = 3) (top, measurement of FerroOrange probe; bottom, relative FerroOrange fluorescence intensity). (G) Total GSH level and GSH/GSSG ratio in SCD1-restored MV4-11 and Kasumi-1 cells (n = 3). (H) Bioluminescence imaging and quantification of tumor burden in NSG mice on days 8, 12, 15, 18, and 21 after injection of SCD^OE and (or) ENO1^KD MV4-11 cells (n = 5). cps, counts per second. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

Oleic acid (OA; 18:1), one of the major catalytic products of SCD1, is a known oncogenic metabolite that drives malignant progression by promoting tumor cell proliferation, migration, and invasion.⁴⁴ To investigate whether the rescue effect could be reproduced via OA supplementation, we added exogenous OA into the media of ENO1^KD AML cells and observed similar restoration of ferroptosis resistance as follows: reduced level of lipid peroxidation (Figure 6A-C; supplemental Figure 8A), Fe²⁺ accumulation (Figure 6D; supplemental Figure 8B), and restored level of GSH level and GSH/GSSG ratio (Figure 6E-F). Consistent with the in vitro findings, continuous OA supplementation in ENO1^KD NSG mice also restored tumor progression (Figure 6G), confirming that ENO1 promotes AML progression through an SCD1-dependent lipid metabolism reprogramming.

Figure 6.

Supplementation of OA rescues ENO1^KD AML cells from ferroptosis. (A-B) Lipid peroxidation level was assessed by flow cytometry after C11-BODIPY staining in ENO1^KD MV4-11 and Kasumi-1 cells with or without OA supplementation (n = 3). (C) Lipid peroxidation level was assessed by confocal microscopy after C11-BODIPY staining in ENO1^KD MV4-11 and Kasumi-1 cells with or without OA supplementation (n = 3). (D) The intracellular level of Fe²⁺ in ENO1^KD MV4-11 and Kasumi-1 cells with or without OA treatment (n = 3) (left, measurement of FerroOrange probe; right, relative FerroOrange fluorescence intensity). (E-F) Total GSH level and GSH/GSSG ratio in ENO1^KD MV4-11 (E) and Kasumi-1 (F) cells with or without OA treatment (n = 3). (G) Bioluminescence imaging and quantification of tumor burden in NSG mice on days 8, 11, 14, 17, and 21 after injection of shNC and shENO1 MV4-11 cells, with or without oral OA treatment (600 mg/kg, twice a week) (n = 5). cps, counts per second; ns, not significant. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

Combination of SCD1 inhibitor and DNR is a potential therapy strategy for ENO1-elevated AML

In pediatric AML treatment, daunorubicin (DNR) is a frontline anthracycline chemotherapy drug that is routinely substituted for doxorubicin to mitigate cumulative cardiotoxicity, constituting a fundamental component of induction chemotherapy regimens. The resistance to ferroptosis acquired by malignant cells contributes to tumor resistance against anthracycline antibiotic chemotherapy agents, including doxorubicin.^45,46 To investigate whether ENO1 inhibited DNR-induced ferroptosis and promoted chemoresistance in AML, we treated ENO1^KD MV4-11 and Kasumi-1 cells with DNR and observed a more pronounced lipid peroxidation in ENO1^KD cells than in negative control groups (supplemental Figure 9A). Moreover, ENO1^KD cells exhibited increased sensitivity to DNR in both AML cell lines and primary AML cells (supplemental Figure 9B-C). Conversely, ENO1^OE HEL cells showed significantly inhibited DNR-induced ferroptosis (Figure 7A-B; supplemental Figure 9D) and increased resistance to DNR (Figure 7C-D). This finding suggested a potential therapeutic strategy that involved targeting the ENO1-SCD1 axis to counteract DNR resistance in ENO1-elevated samples. Accordingly, a specific inhibitor of SCD1 was chosen to perform the follow-up experiments. As anticipated, the combination of DNR and the specific SCD1 inhibitor, SSI-4, effectively reversed DNR resistance in ENO1^OE HEL and primary AML cells (Figure 7C-D; supplemental Figure 9E-F). These results demonstrated that ENO1 confers DNR resistance via SCD1-dependent mechanisms in AML cells.

Figure 7.

The ENO1-SCD1 axis provides a potential strategy for AML treatment. (A-B) Lipid peroxidation assay of ENO1^OE HEL cells treated with DRB, with or without combined treatment of SSI-4, detected by flow cytometry (A) and confocal microscopy (B) (n = 3). (C) Sensitivity of ENO1^OE HEL cells to DNR with or without SSI-4 cotreatment (n = 3). (D) Colony formation in ENO1^OE HEL cells following exposure to single DNR or combination of DNR and SSI-4 treatment. (E-F) Bioluminescence imaging and quantification of tumor burden in NSG mice on days 7, 14, 21, and 26 after HEL tail-vein injection, with DNR or (and) SSI-4 treatment (DNR: 1 mg/kg every 3 days; SSI-4: 20 mg/kg every 3 days) (n = 5). (G) Tumor burden in NSG mice on day 26 after tail vein injection. cps, counts per second; ns, not significant. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

To assess in vivo relevance, we established AML xenograft models using HEL cells with or without ENO1 overexpression. The ENO1^OE group displayed accelerated disease progression and reduced responsiveness to DNR treatment, whereas cotreatment with SSI-4 and DNR significantly attenuated leukemia burden and tissue infiltration in ENO1^OE xenografts (Figure 7E-G; supplemental Figure 10A-G), indicating that SCD1 represents a potential target for overcoming chemoresistance to DNR induced by ENO1 overexpression in AML.

To explore the clinical relevance of the ENO1-SCD1 axis, we conducted survival analysis in 142 patients with AML from our center. Compared with patients from other groups, patients with ENO1-high–SCD1-high expression exhibited significantly shorter OS (supplemental Figure 11A-B). Validation in independent The Cancer Genome Atlas –LAML and the Gene Expression Omnibus GSE146173 cohorts confirmed that high ENO1 and SCD1 expression was associated with poor OS and disease-free survival (supplemental Figure 11C-G). Collectively, these findings establish the combined high expression of ENO1 and SCD1 as a robust biomarker for poor clinical outcomes in AML.

Discussion

Enhanced glycolysis is one of the hallmarks of cancer cells.⁴⁷ Multiple studies have consistently demonstrated the aberrant elevated expression of ENO1, a pivotal glycolytic enzyme that drives tumor metabolic reprogramming toward increased glucose utilization in diverse malignancies.12, 13, 14^,27 ENO1 is also reported to have high expression in gene expression profiling of AML,20, 21, 22 and its upregulation is intimately linked to self-renewal and chemoresistance in leukemia stem cells.²³ This study has characterized ENO1 as a novel prognostic biomarker and therapeutic candidate in AML, demonstrating that its overexpression correlates with adverse clinical outcomes.

As a vital enzyme of glycolysis, ENO1 orchestrates tumor development and progression through metabolic reprogramming, regulated by its upstream signaling axes. In colorectal cancer, ENO1 is regulated by NSUN2/YBX1/m⁵C axis and promotes tumor metabolism reprogramming.¹⁶ In lung cancer, protein arginine methyltransferase 6 (PRMT6) promotes oncogenesis by methylating ENO1 at arginine residues R9 and R372, thereby facilitating active dimer formation and 2-phosphoglycerate binding to ENO1, thereby promoting oncogenesis.¹⁷ In addition, PRMT5-mediated symmetrically demethylated at arginine 9 facilitates active ENO1 dimer formation, accelerating tumor growth in ovarian cancer.¹⁸ Concurrently, ENO1 is recognized as a moonlighting protein with multifunctional roles in biological systems.²⁸ First, the RNA-binding activity of ENO1 represents a critical mechanism through which it participates in the regulation of tumor signaling pathways. ENO1 affects mRNAs stability through direct interaction with mRNA.^24,25 ENO1 modulates arachidonic acid (AA) metabolism in liver cancer cells through regulating the YAP1/PLCB1/HPGD pathway,²⁵ indicating its involvement in nonglycolytic metabolic processes. Research conducted by Zhang et al has demonstrated a connection between ENO1’s RNA-binding protein role and ferroptosis in gallbladder cancer cells.²⁴ Second, ENO1 activates metabolism and immune evasion pathways through protein-protein interactions. It has been reported that ENO1 mediates tumor immune evasion by inducing ubiquitination of programmed death-ligand 1.^15,26 ENO1 also activates hepatocyte growth factor receptor and Wnt signaling through direct protein-protein interactions, ultimately promoting lung cancer metastasis.²⁷ In human glioblastoma, ENO1 directly binds to choline kinase α, inhibiting its polyubiquitination and degradation, resulting in altered choline metabolism and enhanced tumor growth.²⁸ Notably, some of these studies have elucidated the glycolysis-independent mechanism through which ENO1 drives metabolic reprogramming, particularly within lipid metabolic pathways.^25,28 In addition, previous studies have revealed that the ENO1 protein translocates into the cell nucleus and binds to gene promoter regions. It is known to function as an intranuclear DNA-binding protein at the CMYC promoter site, thereby promoting c-MYC expression along with HSD3B2 levels.^29,30 ENO1 negatively regulates ERBB2/Her2 expression through targeting −514 and −262 regions of the promoter of ERBB2, with this action further promoting breast cancer progression.³¹

The association between SCD1 and ferroptosis has garnered significant attention in cancer research. A study focused on ovarian cancer elucidated a potential mechanism whereby the depletion of SCD1 induces the reorganization of membrane phospholipid composition by decreasing the content of monounsaturated fatty acyl chains while increasing polyunsaturated fatty acyl chains within membrane phospholipids.³⁶ Findings from various tumor studies have corroborated this theory. By producing unsaturated FA, SCD1 modulates the ratio of SFA to unsaturated FA, thereby enhancing resistance to ferroptosis in colorectal cancer cells, whereas SCD1 knockdown sensitizes colorectal cancer cells to ferroptosis.³⁵ In this study, we confirmed the essential role of ENO1 for AML both in vitro and in vivo, demonstrating that the combined expression levels of ENO1 and SCD1 serve as predictive biomarkers for the prognosis of patients with AML. Our findings indicated that ENO1 promotes tumor progression through lipid reprogramming in an SCD1-dependent manner, revealing a novel ENO1-mediated lipid metabolic pathway in AML. Although previous reports have linked ENO1 with lipid metabolism in tumors,⁴⁸ there has been no evidence establishing direct control by ENO1 over lipid metabolic pathways until now. To our knowledge, this study provided the first demonstration that ENO1 regulates lipid metabolism through interaction with the promoter region of SCD1 in AML. Furthermore, this regulatory effect is mediated by the binding between ENO1 and the promoter region of SCD1. These results suggested that ENO1’s nonglycolytic effects on cancers extend beyond RNA regulation to include its active participation in transcriptional processes.

All the evidence presented earlier indicated that ENO1 is a promising candidate for cancer therapy. A series of achievements have been made in ENO1-targeted therapies, including PhAH (phosphonoacetohydroxamate), AP-III-a4 (also known as ENOblock), and HEX. However, severe adverse effects, inconsistent efficacy, or a lack of substrate specificity toward ENO1 have limited the use of these inhibitors in tumor subtypes driven by ENO1. Specifically, PhAH is a potent enolase inhibitor for both ENO1 and ENO2, but it has been associated with erythrocyte reduction as a side effect.⁴⁹ Although AP-III-a4 serves as a pan-inhibitor of enolases, it exhibits variable efficacy against ENO1 depending on the specific tumor context.⁵⁰ HEX and its prodrug POMHEX act more effectively as substrate-competitive inhibitors against ENO2 than against ENO1.⁵¹ Meanwhile, several SCD1 inhibitors have progressed to clinical availability or are currently undergoing clinical-stage testing. We aim to focus on SCD1 inhibition in AML cells with high levels of ENO1 expression as an alternative therapeutic strategy. Studies on SCD1 inhibitors for therapeutic applications have surged over the past decades, particularly regarding repurposed drug combinations in AML and lymphoma.⁵² Concurrently, combining chemotherapy agents with SCD1 inhibitors has emerged as a promising antitumor therapeutic approach.^53,54 Previous studies have confirmed the safety and efficacy of SSI-4 when used as an adjunctive therapy in AML.⁵⁵ Our observations indicate that the efficacy of DNR improves in cases of high-level ENO1-expressing AML when combined with SSI-4, providing theoretical support for screening downstream therapeutic targets relevant to AML characterized by elevated levels of ENO1 expression.

In this study, we investigated the effects of supplementing OA, a MUFA product of SCD1, in ENO1^KD AML cells and AML cell line-derived xenografts (CDX) models. Tumor suppression induced by ENO1 knockdown was effectively rescued by OA supplementation both in vitro and in vivo. This finding highlights the theoretical feasibility of personalized dietary therapy during tumor treatment. Although MUFA are an important component of daily diet, excessive MUFA intake may serve as a detrimental factor for patients with AML exhibiting low ENO1 expression.

In conclusion, this study confirms the functional diversity of ENO1 as a moonlighting protein and elucidates its intrinsic connection to lipid metabolism. These findings provide a theoretical foundation for addressing chemotherapy-resistant AML characterized by specific genetic phenotypes.

Conflict-of-interest disclosure: The authors declare no competing financial interests.