CDKN2A deletion remodels lipid metabolism to prime glioblastoma for ferroptosis

Abstract

Malignant tumors exhibit heterogeneous metabolic reprogramming, hindering the identification of translatable vulnerabilities for metabolism-targeted therapy. How molecular alterations in tumors promote metabolic diversity and distinct targetable dependencies remains poorly defined. Here we create a resource consisting of lipidomic, transcriptomic, and genomic data from 156 molecularly diverse glioblastoma (GBM) tumors and derivative models. Through integrated analysis of the GBM lipidome with molecular datasets, we identify CDKN2A deletion remodels the GBM lipidome, notably redistributing oxidizable polyunsaturated fatty acids into distinct lipid compartments. Consequently, CDKN2A-deleted GBMs display higher lipid peroxidation, selectively priming tumors for ferroptosis. Together, this study presents a molecular and lipidomic resource of clinical and preclinical GBM specimens, which we leverage to detect a therapeutically exploitable link between a recurring molecular lesion and altered lipid metabolism in GBM.

Keywords: CDKN2A; GPX4; RNA sequencing; ferroptosis; glioblastoma; lipid droplet; lipid peroxidation; shotgun lipidomics; triacylglyceride.

Figure 1:. A molecular and lipidomic resource of glioblastoma tumors and derivative pre-clinical models.

(A) Schematic representation of data collection workflow across glioblastoma (GBM) bulk tumor tissue (n=84) and patient derived models (orthotopic xenografts (n=29) and gliomaspheres (n=43)). (B) Plot describing sample type, subtype, lipid composition (class % of total), copy number alterations and mutations in GBM, tumor recurrence, patient age, and gender. Lipid composition is plotted as a z-score across samples within a tumor microenvironment type. (C) Correlation matrix of lipid species (species % of total, n=918 lipid species) and lipid related gene expression (n=999 genes) across GBM tumors. (D) Lipid class and saturation enrichment for each lipid cluster (significance calculated by hypergeometric test against total lipidome with FDR adjustment). Dot plot summarizing gene cluster-lipid cluster associations. Significant associations derived from Kolmogorov-Smirnov testing are outlined in black (see Methods). Gene clusters are labeled with summarized interpretations of enriched Gene Ontology terms. (E) Pie charts representing the log odds ratio scores of over-enriched lipid clusters for differentially abundant lipid species in altered samples relative to WT samples. See also Fig. S1, S2, and Table S1.

Figure 2:. Comprehensive characterization of the GBM lipidome reveals the impact of CDKN2A deletion.

(A) Correlation matrix of lipid composition (species % of class, n=835) and gene expression of protein coding genes (n=2,695) in GS (n=43). (B) Scatterplot of gene- and locus-level somatic alterations (n=24 somatic events covering 109 genes) ranked by their association with the gene set composite score (see Methods). Scatterplot of the average Group 1 gene expression (log2 TPM) and shrunken log2 fold change (Group 2/Group 1) of genes at the chr9p21 locus identified. Shrunken fold changes and adjusted p-values from DESeq2 (See Methods). (C) Varimax rotated principal component analysis (PC1, PC2) of all lipids (species % of class, n=985 species) between CDKN2A WT (blue, n=12) and null (pink, n=31) GS. 50% data ellipses are shown. Quantification of difference in rotated component 1 scores between CDKN2A WT (n=12) and null (n=31) samples. Boxplots depict the median, interquartile range, and extrema (significance calculated by Student’s two-sample t-test). (D) Volcano plot of lipids (species % of class) altered by CDKN2A deletion in GS. Significantly altered lipids colored by species (Student’s t-test p-value < 0.05). Permutation p-value representing significance of differential abundance testing results displayed (see Methods) (E) Number of lipid species altered with CDKN2A deletion binned by previously defined lipid clusters (see Figure 1). (F) Acyl tail length and double bond characteristics of significantly altered lipid species identified in Figure 2D (significance calculated by Student’s two-sample t-test). (G) Acyl tail length and double bond characteristics of significantly altered lipid species in CDKN2A WT GS (GS104 and GS116) infected with: shRNA scramble control (shC) or two different shRNA targeting CDKN2A (KD-1 and KD-2). Each of these shRNAs target both p14 and p16 from the CDKN2A locus (significance calculated by Student’s two-sample t-test). For all figures: p >0.05=ns; p <0.05 = *; p<0.01 = **; p<0.001 = ***; p<0.0001 = ****. See also Fig. S3 and S4.

Figure 3:. CDKN2A deletion renders GBM susceptible to ferroptosis.

(A) Log₂ fold changes of significantly different (Student’s t-test p-value < 0.05) TAG species in CDKN2A null/WT GS, ordered by directionality of change, total tail length, and total double bond number. Stacked bar chart indicating double bond and total carbon composition (Student’s two-sample t-test). (B) Confocal microscopy images showing basal C11-BODIPY lipid peroxidation in CDKN2A WT and CDKN2A null GS. Scale bar = 10 μm (60x magnification). Quantification of basal C11-BODIPY lipid peroxidation across CDKN2A WT and CDKN2A null GS plotted as a bar chart. Each point represents an individual cell. Mean +/− s.d. (Student’s two-sample t-test). (C) Log₂ fold changes of significantly different TAG species with shCDKN2A (Student’s t-test p-value < 0.05) ordered by directionality of change, total tail length, and total double bond number. Stacked bar chart indicating double bond and total carbon composition (Student’s two-sample t-test). (D) Quantification of basal C11-BODIPY lipid peroxidation in GS116 (CDKN2A WT) infected with shC, KD-1, or KD-2. See (B). (E) Representative trace of cell death (%) under 2.5 μM RSL3 treatment over time (h) from the Incucyte live cell imaging system in a CDKN2A WT and null GS (two-way ANOVA followed by Tukey’s post-hoc test). (F) Heatmap of cell death (%) after 72 hours of treatment with RSL3 (2.5 μM and 5 μM) in CDKN2A WT and CDKN2A null GS (significance calculated by Student’s two-sample t-test). (G) Confocal microscopy images showing C11-BODIPY peroxidation induced after 24-hour treatment with 2.5 μM RSL3 in CDKN2A WT and CDKN2A null GS. Quantification of C11-BODIPY peroxidation plotted as fold change of RSL3 treatment/DMSO control. Each point represents an individual cell. Mean +/− s.d. (Student’s two-sample t-test). (H) Heatmap of cell death (%) after 72 hours of treatment with 2.5 μM RSL3 treatment with and without the addition of canonical ferroptosis inhibitors (100 μM DFO, 1 μM Ferrostatin-1, or 1 μM Liporoxstatin-1) in CDKN2A WT and null GS (Student’s two-sample t-test). (I) Confocal microscopy images showing C11-BODIPY peroxidation with DMSO control treatment or 2.5 μM RSL3 treatment after 24 hours in GS116 infected with shC, KD-1, or KD-2. See (G). (J) Heatmap of cell death (%) after 72 hours of treatment with 2.5 μM RSL3 treatment with and without the addition 1 μM Ferrostatin-1 in GS104 or GS116 infected with shC, KD-1, or KD-2 (Student’s two-sample t-test). For all figures: p >0.05=ns; p <0.05 = *; p<0.01 = **; p<0.001 = ***; p<0.0001 = ****. See also Fig. S5.

Figure 4:. PUFA TAGs are protective against lipid peroxidation and ferroptosis in CDKN2A WT GBM.

(A) Stacked bar chart showing PUFA distribution (% of total) within TAGs, PCs, and PEs in CDKN2A WT (n=3) and null (n=3) GS under basal (AA −, DGATi −) conditions or with 24 hours 75 μM AA treatment without (AA+, DGATi−) or with co-treatment with 20 μM DGAT1 and 10 μM DGAT2 inhibitors (AA+, DGATi+). Mean +/− s.e.m. (Student’s two-sample t-test). (B) Confocal microscopy images showing accumulation of the LipidTOX neutral lipid stain in CDKN2A null and WT GS treated with DMSO control, 75 μM AA (AA), or 75 μM AA + 20 μM DGAT1 inhibitor + 10 μM DGAT2 inhibitor (AA+, DGATi+) for 24 hours. Scale bar = 10 μm (60x magnification). Quantification of lipid droplet area per cell across CDKN2A WT (n=4) and null (n=4) GS across these same conditions relative to its respective DMSO control. Each dot represents the average quantification per cell line for the specified condition. Mean +/− s.d. (Student’s two-sample t-test). (C) Confocal images showing C11-BODIPY peroxidation induced after 24-hour treatment with 75 μM AA (AA), 75 μM AA + 2.5 μM RSL3 (AA+, RSL3+) or 75 μM AA + 2.5 μM RSL3 + 20 μM DGAT1 inhibitor + 10 μM DGAT2 inhibitor (AA+, RSL3+, DGATi+) in CDKN2A null and WT GS. Quantification of C11-BODIPY peroxidation plotted as fold-change relative to 24 hours of 75 μM AA treatment. Each dot represents the average quantification per cell line. Mean +/− s.d. (Student’s two-sample t-test). (D) Cell death (%) after 72 hours of treatment with the same conditions shown in Fig. 4C in CDKN2A WT (n=3) and null (n=3) GS. Each dot represents the average cell death per cell line for the specified condition. Mean +/− s.d. (Student’s two-sample t-test). (E) Confocal images showing accumulation of the LipidTOX neutral lipid stain (L image set) with 24 hours of the same treatments indicated in (B) and C11-BODIPY peroxidation (R image set) with 24 hours treatment of the same conditions indicated in (C) in GS116 (CDKN2A WT) infected with shC, KD-1, or KD-2. Quantification of C11-BODIPY peroxidation plotted as fold-change relative to 24 hours of 75 μM AA treatment. Each point represents the average quantification per image for the specified condition and cell line. Mean +/− s.d. (Student’s two-sample t-test). (F) Cell death (%) after 72 hours of the same treatment indicated in (D) in GS116 (CDKN2A WT) infected with shC, KD-1, or KD-2. Each point represents a biological replicate. Mean +/− s.d. (significance calculated by Student’s two-sample t-test). For all figures: p >0.05=ns; p <0.05 = *; p<0.01 = **; p<0.001 = ***; p<0.0001 = ****. See also Fig. S6.

Figure 5:. Loss of p16 is sufficient to render GBM sensitive to ferroptosis.

(A) Abundance of PUFA TAGs (n=260 lipid species, species % of class) between GS116 (CDKN2A WT) transfected with Control, CDKN2A ex2 KO, or CDKN2A ex2 KO + p16 addback. Z-scored across GS116 control, CDKN2A ex2 KO, and CDKN2A ex2 KO+p16. (B) Confocal microscopy images showing accumulation of the LipidTOX neutral lipid stain in GS116 Control, CDKN2A ex2 KO, or CDKN2A ex2 KO + p16 addback. Cells treated with DMSO control, 20 μM DGAT1 inhibitor + 10 μM DGAT2 inhibitor (DGATi), 75 μM AA (AA), or 75 μM AA + 20 μM DGAT1 inhibitor + 10 μM DGAT2 inhibitor (AA, DGATi) for 24 hours. Scale bar = 10 μm (60x magnification). Quantification of lipid droplet area/cell (FC relative to control). Each dot represents the average quantification per cell line for the specified condition. Mean +/− s.d. (Student’s two-sample t-test). (C) Confocal images showing basal MDA immunofluorescence staining. Quantification of MDA average fluorescent intensity/cell plotted. Each dot represents the average intensity per cell line. Mean +/− s.d. (significance calculated with parametric unpaired t-test). (D) Heatmap of cell death (%) induced by DMSO vehicle or 2.5μM RSL3 after 72 hours of treatment (Student’s two-sample t-test). For all figures: p >0.05=ns; p <0.05 = *; p<0.01 = **; p<0.001 = ***; p<0.0001 = ****. See also Fig. S7.

Figure 6.. CDKN2A deletion induces lipid peroxidation and susceptibility to ferroptosis in GBM orthotopic xenografts.

(A) Heatmap showing enrichment of conserved TAGs between GS and PDX. TAGs enriched in CDKN2A WT tumors have a log2FC(null/WT)<0 (blue), while TAGs enriched in CDKN2A null tumors have a log2FC(null/WT)>0 (pink). Total double bonds and total carbon length annotated within heatmap. (B) Immunofluorescence microscopy of CDKN2A WT and CDKN2A null tumors stained for malondialdehyde (MDA) (orange), GFP (GFP tumor) (green) and DAPI (blue). Scale bar represents 50 μm (20x magnification). Quantification of MDA (mean fluorescent intensity) across CDKN2A WT (n=8) and null (n=6) PDX tissue. Box-plots show mean (middle-line) +/− s.d. Whiskers represent minimum and maximum data points (significance calculated by Mann-Whitney t-test). (C) Western blot of GPX4 and Actin in CDKN2A null gliomaspheres (GS025, GS187) and WT (GS005, GS208) infected with: nothing (parental), CRISPR non-targeting control (NT), or sgRNAs targeting GPX4 (sgGPX4-1, sgGPX4-2, sgGPX4-3). Molecular weight of closest ladder marker indicated to the right. (D) Kaplan-Meier plot showing probability of survival (%) in mice harboring CDKN2A null and WT orthotopic tumors infected with NT (n=6), sgGPX4-1 (n=6), sgGPX4-2 (n=6 for PDX025, PDX005, PDX208, n=10 for PDX187), or sgGPX4-3 (n=6). X-axis indicates days post tumor implantation. Significance calculated with Log-rank (Mantel-Cox) test between the survival curves for NT vs individual guides. (E) Heatmap showing enrichment of conserved TAGs between GS and tumors from patients with GBM. TAGs enriched in CDKN2A WT tumors have a log2FC(null/WT)<0 (blue), while TAGs enriched in CDKN2A null tumors have a log2FC(null/WT)>0 (pink). (F) Proposed mechanism of ferroptotic sensitivity in CDKN2A WT and CDKN2A null GBM. For all figures: p >0.05=ns; p <0.05 = *; p<0.01 = **; p<0.001 = ***; p<0.0001 = ****. See also Fig. S8.