Journal: bioRxiv
Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma
doi: 10.64898/2026.05.02.722429
Figure Lengend Snippet: (A) Box plots of expression-AUC Z-scores for the 13 ferroptosis resistance candidate genes across DepMap cancer cell lines. For each gene, the Pearson correlation between basal expression and ferroptosis inducer AUC was Z-scored across all compounds in the CTRP library. Individual points represent correlations for RSL3 (red circles), ML162 (blue triangles), ML210 (purple squares), and erastin (green diamonds). Dotted lines indicate the threshold of Z = ±0.5 (adjusted P < 0.05). Positive Z-scores indicate that higher gene expression correlates with ferroptosis resistance across cell lines. (B) Box plots of expression-AUC Z-scores for the six ferroptosis sensitizer candidate genes, formatted as in (A). Negative Z-scores indicate that lower gene expression correlates with ferroptosis resistance across cell lines. (C) Box plots of CRISPR-AUC Z-scores for the 13 resistance candidate genes. For each gene, Pearson correlations between CRISPR gene effect scores (Chronos) and GPX4 inhibitor AUC (RSL3, ML162, ML210) were Z-scored across all genes. Individual points represent correlations for each drug as in (A). Dotted lines indicate the threshold of Z = ±0.3 (adjusted P < 0.05). Negative Z-scores indicate that gene loss is disproportionately lethal in ferroptosis-resistant cell lines, reflecting functional dependency of the resistant state on those genes. (D) Box plots of CRISPR-AUC Z-scores for the six sensitizer candidate genes, formatted as in (B). Positive Z-scores indicate that gene loss correlates with increased ferroptosis sensitivity, consistent with a role in sustaining the ferroptosis-susceptible state. (E) Scatter plot of expression-AUC correlation (r, x-axis) versus CRISPR dependency-AUC correlation (r, y-axis) for candidate genes relative to canonical ferroptosis regulators. Points are colored by category: anchor genes (green), resistance candidates (red), and sensitizer candidates (blue). GPX4 and ACSL4 serve as positive and negative reference anchor points for expected resistance and sensitivity signals, respectively. (F) Cell viability of Caki-2 reconstituted cells following co-treatment with RSL3 (0.33 µM, 18 h) and the SCD inhibitor CAY10566 (SCDi; 0.5 µM). Viability is normalized to DMSO-treated controls within each genotype. data are mean ± SD ( n = 3 biologically independent replicates). (G) CUT&RUN genome browser tracks at the SCD locus (chr10:102,105,143–102,126,035) in Caki-2 cells. Tracks show H3K4me3, IgG, PBRM1, and PHF10 (PBAF-specific complex subunit) occupancy. Gene structure is shown below. (H) CUT&RUN genome browser tracks at the SREBF1 locus (chr17:17,714,907–17,714,724) in Caki-2 cells. Tracks are displayed as in (G). (I) Species-level heatmap of the top remodeled phosphatidylcholine (PC) lipid species across individual biological replicates for each genotype (Empty, PBRM1, VHL, PBRM1+VHL). Each row represents one lipid species annotated at sum-composition level. Rows are colored by unsaturation category: green indicates low-unsaturation species (≤1 double bond; resistance-associated) and red indicates high-unsaturation species (≥2 double bonds; sensitivity-associated). Color scale represents log2 abundance relative to Empty vector mean within each batch. (J) Species-level heatmap of the top remodeled phosphatidylethanolamine (PE) lipid species across individual biological replicates, formatted as in (G). (K) TAG low-to-high unsaturation ratio (double bonds ≤ 1 versus ≥ 2) across Empty vector, PBRM1, VHL, and PBRM1+VHL cells. Data are from lipidomics batch 2 ( n = 4 per genotype, except VHL, n = 3; see legend and Methods). Individual replicates are shown with mean ± SEM. Statistical significance was assessed by unpaired two-sided Welch’s t -test relative to Empty vector. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.
Article Snippet: Libraries were prepared using the CUTANATM CUT&RUN Library Prep Kit (EpiCypher, Durham, NC, USA; 14-1001 and 14-1002) and quality-checked by Qubit and Agilent TapeStation at Purdue Genomics Core Facility (Purdue University, West Lafayette, IN, USA).
Techniques: Expressing, Gene Expression, CRISPR, Functional Assay, Plasmid Preparation
