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[Preprint]. 2025 Jun 4:rs.3.rs-6779386.
doi: 10.21203/rs.3.rs-6779386/v1.

NOTCH3 Drives Fatty Acid Oxidation and Ferroptosis Resistance in Aggressive Meningiomas

Nishanth S Sadagopan  1 , Mateo Gomez  1 , Shashwat Tripathi  1 , Leah K Billingham  1 , Susan L DeLay  1 , Martha A Cady  2 , Harrshavasan T S Congivaram  1 , Tzu-Yi Chia  1 , Hanxiao Wan  1 , Si Wang  1 , David R Raleigh  2 , Faith C Kaluba  3 , Evan C Lien  3 , Amy B Heimberger  1 , Catalina Lee-Chang  1 , Mark W Youngblood  1 , Stephen T Magill  1 , Jason M Miska  1
Affiliations

NOTCH3 Drives Fatty Acid Oxidation and Ferroptosis Resistance in Aggressive Meningiomas

Nishanth S Sadagopan et al. Res Sq. .

Update in

Abstract

Purpose: NOTCH3 is increasingly implicated for its oncogenic role in many malignancies, including meningiomas. While prior work has linked NOTCH3 expression to higher-grade meningiomas and treatment resistance, the metabolic phenotype of NOTCH3 activation remains unexplored in meningioma.

Methods: We performed single-cell RNA sequencing on NOTCH3 + human meningioma cell lines. Using the CH157-MN meningioma cell model, we overexpressed NOTCH3 intracellular domain (ICD) and performed untargeted metabolomic, lipidomic, and bulk RNA sequencing analyses as well as functional metabolic assays.

Results: We show that NOTCH3 mediates a metabolic shift towards fatty acid oxidation (FAO), depleting lipid availability and conferring resistance to ferroptosis. Single-cell RNA sequencing revealed a correlation with CD36, a key fatty acid transporter. Furthermore, patient-derived primary meningioma lines stratified by NOTCH3 expression confirmed higher CD36 expression and increased maximal mitochondrial respiration in NOTCH3-high cells in the presence of palmitate, supporting enhanced FAO. NOTCH3 ICD overexpression (OE) exhibited depletion of fatty acid pools, alongside transcriptional upregulation of canonical FAO genes. Functional mitochondrial assays confirmed elevated oxidative respiration in the presence of palmitate compared with controls. Additionally, NOTCH3 OE cells exhibit increased resistance to RSL3-induced ferroptosis, a phenotype that was reversed with CPT1.

Conclusion: These data establish a link between NOTCH3 signaling, lipid metabolic reprogramming, and ferroptosis evasion in aggressive meningioma cells. This metabolic shift may contribute to the malignant behavior observed in NOTCH3 + meningiomas, offering new insight into the biochemical vulnerabilities of these tumors.

Keywords: Fatty Acid Oxidation; Ferroptosis; Meningioma Metabolism; NOTCH3; Tumor Microenvironment.

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Conflict of interest statement

Conflict of interest The authors declare no conflicts of interest

Figures

Figure 1
Figure 1. NOTCH3 expression controls lipid uptake and oxidation in meningioma cells.
A. Volcano plot of 14,080 NOTCH3+ human meningioma cells from 22 meningiomas showing upregulation of CD36. P-values were calculated using DESeq2. B. Relative mRNA expression of NOTCH3 and CD36 in 2 patient-derived meningioma lines PDX1 and PDX2 (PDX1, n = 3; PDX2, n = 3). Experiments were performed in triplicate; significance was calculated using an unpaired Student’s t-test. C. Seahorse flux tracing of PDX1 and PDX2 under BSA or palmitate incubation conditions. D. Basal oxygen consumption rate (OCR) in PDX1 and PDX2 cells treated with palmitate-BSA or BSA control (PDX1, n = 3 in each condition; PDX2, n = 3 in each condition). E. ATP-linked respiration in PDX1 and PDX2 cells treated with palmitate-BSA or BSA control (PDX1, n = 3 in each condition; PDX2, n = 3 in each condition). In C-En=2 independent experiments were performed. In D-E, a one-way ANOVA was performed with a Tukey’s post-hoc to calculate significance. P<0.05*; P<0.01**, P<0.001***.
Figure 2
Figure 2. NOTCH3 overexpression alters fatty acid composition in meningioma cells.
A. Bulk RNA sequencing FPKM values of NOTCH3 in CH157-MNEV and CH157-MNNOTCH3 ICD (CH157-MNEV, n = 3; CH157-MNNOTCH3 ICD, n = 3). B. Untargeted metabolomic analysis showing an increase in acyl carnitine species carnitine, propionylcarnitine, butyrylcarnitine, and hexanoylcarnitine in CH157-MNNOTCH3 ICD compared with CH157-MNEV (CH157-MNEV, n = 4; CH157-MNNOTCH3 ICD, n = 4). Significance was calculated using unpaired Student’s t-tests. P<0.05*; P<0.01**, P<0.001***; P<0.0001****. C. Histogram of targeted lipidomic analysis of CH157-MNEV and CH157-MNNOTCH3 ICD (CH157-MNEV, n = 3; CH157-MNNOTCH3 ICD, n = 3).
Figure 3
Figure 3. NOTCH3 overexpression enhances transcription of fatty acid oxidation genes in meningioma cells.
A. Gene set enrichment analysis (GSEA) of bulk RNA sequencing showing increased activation of fatty acid metabolism pathways in CH157-MNNOTCH3 ICD compared with CH157-MNEV (CH157-MNEV, n = 3; CH157-MNNOTCH3 ICD, n = 3). B. Bulk RNA sequencing FPKM values of CH157-MNEV and CH157-MNNOTCH3 ICD showing upregulation of FAO genes CPT1A, CPT2, and HADHA (CH157-MNEV, n = 3; CH157-MNNOTCH3 ICD, n = 3). C. Bulk RNA sequencing FPKM values of CH157-MNEV and CH157-MNNOTCH3 ICD showing no difference or decrease in fatty acid synthesis genes FASN, OXSM, and MCAT (CH157-MNEV, n = 3; CH157-MNNOTCH3 ICD, n = 3). In B-C, significance was calculated using unpaired Student’s t-tests. P<0.05*; P<0.01**, P<0.001***; P<0.0001****.
Figure 4
Figure 4. NOTCH3 overexpression increases maximal aerobic respiration in the presence of lipids.
A. Oxygen consumption rate (OCR) of CH157-MNEV and CH157-MNNOTCH3 ICD treated with palmitate-BSA or BSA control (CH157-MNEV, n = 4 in each condition; CH157-MNNOTCH3 ICD, n = 4 in each condition). B. Basal corrected oxygen consumption rate (OCR) in CH157-MNEV and CH157-MNNOTCH3 ICD cells treated with palmitate-BSA or BSA control (CH157-MNEV, n = 4 in each condition; CH157-MNNOTCH3 ICD, n = 4 in each condition). C. Maximal corrected oxygen consumption rate (OCR) in CH157-MNEV and CH157-MNNOTCH3 ICD cells treated with palmitate-BSA or BSA control (CH157-MNEV, n = 4 in each condition; CH157-MNNOTCH3 ICD, n = 4 in each condition). In B-C, a one-way ANOVA was performed with a Tukey’s post-hoc to calculate significance. P<0.05*; P<0.01**, P<0.001***.
Figure 5
Figure 5. NOTCH3-driven lipid depletion confers resistance to ferroptosis.
A. Cell viability of CH157-MNEV and CH157-MNNOTCH3 ICD treated with increasing concentration of RSL3 for 24 hours (CH157-MNEV, n = 5; CH157-MNNOTCH3 ICD, n = 5). To test for significance in curves, a two-way ANOVA was performed, and Sidak’s multiple comparison post hoc test was used to test for individual significance across rows. B. Cell viability of CH157-MNEV and CH157-MNNOTCH3 ICD treated with or without 5 μM of etomoxir with 32.3 nM RSL3 (CH157-MNEV, n = 3 in each condition; CH157-MNNOTCH3 ICD, n = 3 in each condition). A one-way ANOVA was performed with a Tukey’s post-hoc to calculate significance. P<0.05*; P<0.01**, P<0.001***.
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