Background

TP53 mutations occur in 8–17 % of patients with acute myeloid leukemia (AML) and define a high-risk subset with median overall survival < 10 months. APR-246 (eprenetapopt) is a first-in-class mutant-p53 reactivator that induces ferroptosis via depletion of the cystine importer SLC7A11 and the lipid-repair enzyme GPX4; however, single-agent activity remains modest. ENO1 (α-enolase) is a glycolytic enzyme that we previously identified as a leukemia-promoting factor. We therefore investigated whether ENO1 inhibition sensitizes AML cells to APR-246 and examined the underlying molecular circuitry.

Methods

TCGA/GEO datasets were interrogated for ENO1 expression and prognosis. Lentiviral shRNA was used to knock down ENO1 in human AML cell lines (MOLM-13, MV4-11, Kasumi-1, THP-1) with defined TP53 or FLT3-ITD status. Synergy was quantified by the ZIP model based on 72-h CCK-8 viability curves. Ferroptosis was assessed by BODIPY-C11 lipid-ROS flow cytometry, GSH quantification, transmission electron microscopy, and rescue with ferrostatin-1. Transcriptomic profiling (RNA-seq) was integrated with the DepMap CRISPR-cancer dependency map and FerrDb 2.0 to nominate ferroptosis regulators. Mechanistic validation included qRT-PCR, immunoblotting, co-immunoprecipitation, MG132/chloroquine chase assays, and ubiquitination mapping.

Results

Clinical correlation: ENO1 mRNA was elevated 2.1-fold in AML versus normal marrow (p < 0.001). High ENO1 expression correlated with shorter OS in TP53-mutant (HR 2.14, p = 0.008) and FLT3-ITD (HR 1.87, p = 0.013) cohorts.

Functional loss-of-ENO1: shENO1 reduced clonogenicity by 48–64 % and increased APR-246 sensitivity (IC50 fold-reduction: MOLM-13 2.9, Kasumi-1 2.3; ZIP synergy 29–43).

Ferroptosis signature: Combined ENO1 inhibition plus APR-246 elevated lipid-ROS 3.7-fold, decreased GSH by 62 %, and triggered mitochondrial shrinkage/cristae loss—phenotypes reversed by ferrostatin-1.

Molecular mechanism: RNA-seq revealed selective down-regulation of the glutathione/ferroptosis axis. ENO1 interacted with the E3-ligase NEDD4L; ENO1 loss augmented NEDD4L-mediated poly-ubiquitination and proteasomal degradation of SLC7A11 and GPX4, whereas MG132 restored GPX4 levels.

Genotype independence: Synergy was observed irrespective of TP53 or FLT3 status, indicating a p53-independent metabolic vulnerability.

Conclusions

ENO1 is an adverse prognostic biomarker in AML whose enzymatic or genetic suppression cooperates with APR-246 to enforce ferroptotic cell death. The ENO1-NEDD4L-SLC7A11/GPX4 ubiquitin-proteasome circuit represents a druggable metabolic checkpoint that can potentiate APR-246 across genetically heterogeneous AML, warranting in vivo validation and clinical translation.

Disclosures: No relevant conflicts of interest to declare.

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2025
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