DHODH Blockade Induces Ferroptosis in Neuroblastoma by Modulating the Mevalonate Pathway

Abstract

Neuroblastoma is the most common heterogeneous solid tumor in children, and current treatment options remain limited, especially for high-risk patients. Previous studies have identified dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine synthesis, as a potential therapeutic target in cancer. However, none of the existing FDA-approved DHODH inhibitors have shown effective inhibition of neuroblastoma cell growth. To address this challenge, we employed virtual screening to discover potential DHODH-targeting drugs, identifying Regorafenib as a promising candidate. Regorafenib significantly inhibited neuroblastoma growth in both neuroblastoma cells and patient-derived organoids. To unravel the underlying molecular mechanisms, we conducted Tandem Mass Tag (TMT)-based quantitative proteomics using LC-MS/MS. Our proteomic profiling revealed substantial regulation of lipid metabolism proteins, specifically those in the mevalonate pathway, correlating with ferroptosis induction. Further analysis showed that DHODH inhibition led to a reduction in total cholesterol, cholesterol esters, disrupted lipid droplet formation, and significantly decreased the expression of Squalene Epoxidase (SQLE), a key enzyme in lipid metabolism. Notably, we also observed an increase in nuclear SQLE expression following DHODH inhibition. In summary, our study highlights DHODH blockade as a novel approach to induce ferroptosis through lipid metabolism reprogramming, underscoring DHODH as a viable therapeutic target for neuroblastoma treatment. These insights open new avenues for metabolism-based interventions in aggressive pediatric cancers.

Keywords: DHODH; ferroptosis; lipid metabolism; mevalonate pathway; neuroblastoma; proteomics.

Graphical abstract

Fig. 1

Overview of the experimental workflow and methodologies used in this study. We validated DHODH as a potential therapeutic target for neuroblastoma using clinical data and cell-based DHODH silencing experiments. Docking simulations were then employed to identify and evaluate FDA-approved drugs capable of inhibiting DHODH activity in high-risk neuroblastoma. The efficacy of the candidate drug was confirmed through both cell-based assays and patient-derived organoid models. To further explore the molecular mechanisms underlying DHODH inhibition in neuroblastoma, we conducted Tandem Mass Tag (TMT) proteomic analysis to profile changes in the proteome following DHODH inhibition. Our results revealed a novel mechanism involving lipid metabolism and ferroptosis linked to DHODH inhibition, providing new insights into potential therapeutic strategies for treating high-risk neuroblastoma.

Fig. 2

Identification of DHODH as a potential therapeutic target in neuroblastoma. A, survival probability of neuroblastoma patients categorized by high or low DHODH expression levels, shown for event-free survival (left panel) and overall survival (right panel), based on neuroblastoma dataset analysis. B, DHODH gene expression in neuroblastoma patients, stratified by MYCN status. C, DHODH gene expression in neuroblastoma patients, categorized into low-risk and high-risk groups. D, Western blot analysis of DHODH protein expression across multiple neuroblastoma cell lines. E, quantification of DHODH protein levels normalized to actin expression. F, MTS assay results for DHODH-knockdown SK-N-BE(2)C cells at various time point. G, representative images of the wound healing assay for DHODH knockdown SK-N-BE(2)C cells. H, quantification of wound healing, with the initial wound area (0-h) normalized to 100%. I, Transwell migration assay measuring the migration capacity of DHODH-knockdown SK-N-BE(2)C cells. J, relative number of migrating cells quantified using ImageJ software. K, Western blot analysis showing levels of cleaved caspase-3 and cleaved PARP, with β-actin as the loading control. L, measurement of endogenous ATP of DHODH-silenced SK-N-BE(2)C cells using an ATP assay. M, mitochondria and nuclei in SK-N-BE(2)C cells stained with MitotrackerRed and DAPI respectively. Images were captured using confocal microscopy, and mitochondrial perimeter was measured using ImageJ. Scale bar, 10 μm. All quantified data are presented as mean ± SD from three independent experiments. Statistical significance: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Fig. 3

Workflow and experimental evaluation of potential DHODH inhibitors’ effects on DHODH structural stability and enzymatic activity, including in silico docking simulations, thermal shift assays to assess protein stability, and enzyme activity assays to measure functional inhibition. A, schematic representation of the docking simulation workflow. B, visualization of ligand-protein complex structures using Pymol. DHODH inhibitory sites are highlighted by two α-helix motifs in yellow, while the remaining DHODH structure is shown in pink. Leflunomide and Brequinar are known DHODH inhibitors, and the six other drugs are potential DHODH inhibitors identified through our docking simulation. Affinity scores for each drug are displayed above. C, Schematic diagram of DHODH protein purification process. D, Thermal shift assay results showing the structural stability of DHODH treated with four different concentrations of six candidate drugs and two known DHODH inhibitors. The melting temperature of DHODH was measured using a qPCR machine. E, schematic illustration of the DHODH activity assay principle. F, DHODH enzyme activity was quantified using a fluorescence-based DHODH activity assay. All quantified results are expressed as mean ± SD from three independent experiments. Statistical significance: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Fig. 4

Regorafenib inhibits neuroblastoma cell growth and suppresses the development of patient-derived organoids. A, SK-N-BE(2)C cell viability following 72-h treatment with Regorafenib, measured using a real-time cell analysis (RTCA). Drug concentrations of 0 μM, 0.4 μM, 4 μM, and 40 μM were used for screening. IC₅₀ values for each drug are indicated in the figures. B, morphological changes in MYCN-amplified and MYCN non-amplified patient-derived organoid after Regorafenib treatment. C, flow cytometry analysis of MYCN non-amplified organoids treated with 5 μM Regorafenib for 72 h, using Annexin V/PI staining. The dot plot shows apoptotic cells in the Q2 (late apoptosis) and Q3 (early apoptosis) quadrants, and PI-positive signals indicating cell death in the Q1 and Q2 quadrants. All quantified results are expressed as mean ± SD from three independent experiments. Statistical significance: ∗ p < 0.05, ∗∗ p < 0.01.

Fig. 5

Proteomic profiling of neuroblastoma cells following DHODH knockdown and Regorafenib treatment. A, schematic overview of the TMT-based proteomics workflow for analyzing neuroblastoma cells subjected to DHODH knockdown and Regorafenib treatment. B, total number of identified MS/MS spectra, peptides, and proteins. The pie chart shows the quantitation and qualitative distribution of the global proteome across all groups. C, Volcano plots depicting fold change and statistical significance of quantified proteins between the Regorafenib-treated and DHODH knockdown groups (p-value <0.05; log2 fold change ≤ −0.3 or ≥0.3). Blue spots represented significantly down-regulated proteins, while red spots indicate significantly up-regulated proteins. D, Venn diagram illustrating the overlap of differentially expressed proteins (DE proteins) between Regorafenib-treated (5 μM) versus control and shDHODH-transfected (#A4 and #A5) vs. control groups. E, top GO 10 terms identified from DAVID functional annotation analysis based on biological processes for DE proteins in the DHODH knockdown group. F, Top GO 10 terms identified from DAVID functional annotation analysis based on biological processes for DE proteins in the regorafenib-treated group. G, Top 10 GO terms identified from DAVID functional annotation analysis based on biological processes for commonly differentially expressed proteins in both the DHODH knockdown and Regorafenib-treated group. H, ratios of identified proteins involved in the mevalonate pathway, with yellow boxes representing the ratios in the DHODH knockdown group and green boxes representing ratios in the regorafenib-treated group. I, validation of protein expressions in Regorafenib-treated and DHODH knockdown neuroblastoma cells using western blotting.

Fig. 6

Regorafenib induces ferroptosis by inhibiting DHODH and reduces lipid droplet production. A, confocal images of neuroblastoma cells treated with Regorafenib for 48 h and DHODH knockdown neuroblastoma cells, stained with C11-BODIPY (green: oxidized form, red: non-oxidized form) and DAPI (blue: nuclei). Scale bar = 100 μm. B, evaluation of lipid peroxidation in neuroblastoma cells using C11-BODIPY staining. The top panel shows cells treated with DMSO, 5 μM Regorafenib, 10 μM Regorafenib, or 50 μM Regorafenib for 48 h, while the bottom panel shows cells with DHODH knockdown. Images are representative of three independent experiments. C, quantified fluorescence intensity of BODIPY 581/591 C11 from three independent experiments. D, confocal imaging of neuroblastoma cells treated with Regorafenib 48 h and DHODH knockdown neuroblastoma cells, stained with BODIPY 493/503 (green: neutral lipids) and DAPI (blue: nuclei). Scale bar = 20 μm. E, evaluation of neutral lipid content in neuroblastoma cells using BODIPY 493/503 staining. The top panel shows cells treated with DMSO, 5 μM Regorafenib, or 10 μM Regorafenib for 48 h, while the bottom panel shows cells with DHODH knockdown. F, quantified fluorescence intensity of BODIPY 493/503 from three or four independent experiments. G, assessment of lipid peroxidation in MYCN non-amplified patient-derived organoids using C11-BODIPY staining and flow cytometry analysis. H, quantification of neutral lipid content in MYCN non-amplified patient-derived organoids using BODIPY 493/503 staining and flow cytometry analysis. All quantified results are presented as mean ± SD from three independent experiments. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 7

DHODH inhibition reduces cholesterol production by disrupting the mevalonate pathway, leading to decreased lipid droplet formation.A and B, cholesterol concentration in neuroblastoma cells following Regorafenib treatment (A) and in DHODH-knockdown neuroblastoma cells (B), measured using the Cholesterol/Cholesterol Ester-Glo Assay. The left panel display free cholesterol levels, while the right panel shows total cholesterol levels. C and D, spatial distribution of SQLE in relation to lipid droplets, and the ER in neuroblastoma cells treated with Regorafenib (C) or subjected to DHODH knockdown cells (D), visualized by confocal fluorescence microscopy at 100 × magnification. Cells were stained with Alexa Fluor 405 to visualize SQLE, BODIPY 493/503 to visualize lipid droplets, and ER-tracker to visualize the ER. Red arrows indicate SQLE aggregation around lipid droplets. Scale bar = 10 μm. All quantified results are presented as mean ± SD from three independent experiments. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. DHODH inhibition reduces cholesterol production by disrupting the mevalonate pathway, leading to decreased lipid droplet formation. E, Workflow for nuclear and cytoplasmic protein fractionation. F, Western blot analysis of SQLE in nuclear and cytoplasmic fractions of neuroblastoma cells following DHODH knockdown (SQLE exposure time: 4000 ms). G, enhanced detection of nuclear SQLE using extended exposure time (15,000 ms), normalized to Histone H3. H, western blot analysis of SQLE in nuclear and cytoplasmic fractions following Regorafenib treatment (SQLE exposure time: 4000 ms). I, detection of increased nuclear SQLE after Regorafenib treatment using extended exposure time (15,000 ms), normalized to Histone H3.

Fig. 8

DHODH inhibition triggers ferroptosis by downregulating key proteins in the mevalonate pathway in neuroblastoma. Our results demonstrate that DHODH inhibition disrupts the activity of the mevalonate pathway in neuroblastoma cells, leading to ferroptosis. Moreover, DHODH inhibition reduces cholesterol production as a downstream product of the mevalonate pathway, resulting in decreased lipid droplet formation and further promoting ferroptosis.