Extended Data Fig. 5: De novo lipid synthesis is synthetically critical upon PIKfyve inhibition.
From: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer
a. Receiver operator characteristic (ROC) curves for the prediction of core essential genes using datasets from MIA PaCa-2 CRISPR screens. b. Gene enrichment rank plot based differential sgRNA representation in (low dose, 100 nM) apilimod- treated versus DMSO-treated endpoint populations of the CRISPR screen experiment. Lipid synthesis-related genes ranked at either extreme are highlighted. c. Scatter plot of gene fitness scores in (low dose, 100 nM) apilimod-treated versus DMSO-treated endpoint conditions in metabolic CRISPR screen. Top 10 hits are labeled, and 5 lipid synthesis-related genes are highlighted. d. qPCR showing changes in mRNA levels of FASN upon CRISPRi-mediated knockdown of FASN in MIA PaCa-2 cells. (One-way ANOVA with Dunnett’s.). e. Immunoblot of MIA PaCa-2 upon CRISPRi-mediated knockdown of FASN in MIA PaCa-2 cells. Vinculin was used as a loading control for the immunoblot. f. MIA PaCa-2 cell confluence assays upon FASN knockdown (sgFASN) or control (sgNC) treated with apilimod (100 nM) or DMSO. The sgNC DMSO and sgNC AP conditions are shared between the two graphs. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.). g. Confluence assays of MIA PaCa-2 cells upon FASN knockdown or control upon treatment with PIKfyve degrader PIK5-33d (100 nM) or DMSO. The sgNC DMSO and sgNC PIK5-33d conditions are shared between the two graphs. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.). h. Immunoblots of PANC-1, 7940B, and MIA PaCa-2 cells upon treatment with ND646 at indicated doses for 24 hours showing changes in labeled targets. i. Confluence assays of MIA PaCa-2 (left), PANC1 (middle), and 7940B (right) cells upon treatment with ND646 (100 nM for MIA-PaCa2, 1000 nM for PANC-1 and 7940B), apilimod (100 nM for MIA PaCa-2, 50 nM for PANC1 and 7940B), or both. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.) The data for DMSO and ND646 are also utilized in Extended Data Fig. 5j (MIA PaCa-2) or 5j-k (PANC1 and 7940B), as they were generated in the same experiment. j. Confluence assays of MIA PaCa-2, PANC-1, and 7940B cells upon treatment with ND646 (100 nM for MIA-PaCa2, 1000 nM for PANC-1 and 7940B), ESK981 (30 nM for MIA PaCa-2, 100 nM for PANC-1 and 7940B), or both. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.) The data for DMSO and ND646 are also utilized in Extended Data Fig. 5i (MIA PaCa-2) or 5i,k (PANC1 and 7940B), as they were generated in the same experiment. k. Confluence assays of MIA PaCa-2, PANC-1, and 7940B cells upon treatment with ND646 (100 nM for MIA-PaCa2, 1000 nM for PANC-1 and 7940B), PIK5-33d (100 nM for MIA PaCa-2 and PANC-1, 1000 nM for 7940B), or both. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.) The data for DMSO and ND646 are also utilized in Extended Data Fig. 5i–k (PANC1 and 7940B), as they were generated in the same experiment. l. qPCR of PANC1 (top) and MIA PaCa-2 (bottom) upon CRISPRi-mediated knockdown of SPTLC1, SPTLC2, or control showing changes in SPTLC1 (left) and SPTLC2 (right). Bars are +/-SD. (One-way ANOVA with Dunnett’s.). m. Immunoblot of PANC1 and MIA PaCa-2 upon CRISPRi-mediated knockdown of SPTLC1, SPTLC2, or control showing changes in SPTLC1 and SPTLC2. Vinculin was used as a loading control. n. Luminescence values from CellTiter-Glo analyses on PANC1 (left) or MIA PaCa-2 (right) cells upon knockdown of SPTLC1, SPTLC2, or control, and treatment with apilimod (3000 nM) or DMSO for the indicated duration. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.). o. Confluence assays of MIA PaCa-2, PANC1, and 7940B upon treatment with DMSO, apilimod (300 nM for MIA PaCa-2, 100 nM for PANC1, and 50 nM for 7940B, YM-53601 (5000 nM), or both. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.). p. Confluence assays of MIA PaCa-2, PANC1, and 7940B upon treatment with DMSO, apilimod (300 nM for MIA PaCa-2, 100 nM for PANC1, and 50 nM for 7940B), NB-598 (5000 nM), or both. Bars are +/-SEM. (Two-way ANOVA with Dunnett’s.). Statistics and reproducibility: d. Data shown are technical triplicates from one of two independent experiments. e. This experiment was performed twice. f. n = 4 biological replicates. P-values: sgFASN-1+apilimod vs sgNC+DMSO:1.1e-16; sgFASN-1+apilimod vs sgFASN-1 + DMSO:1.8e-13; sgFASN-1+apilimod vs sgNC+apilimod:1.8e-13; sgFASN-2+apilimod vs sgNC+DMSO:1.8e-13; sgFASN-2+apilimod vs sgFASN-2 + DMSO:1.8e-13; sgFASN-2+apilimod vs sgNC+apilimod:1.8e-13. This experiment was performed twice. g. n = 4 biological replicates. P-values: sgFASN-1 + PIK5-33d vs sgNC+DMSO:1.8e-13; sgFASN-1 + PIK5-33d vs sgFASN-1 + DMSO:1.8e-13; sgFASN-1 + PIK5-33d vs sgNC+PIK5-33d:1.8e-13; sgFASN-2 + PIK5-33d vs sgNC+DMSO:1.8e-13; sgFASN-2 + PIK5-33d vs sgFASN-2 + DMSO:1.8e-13; sgFASN-2 + PIK5-33d vs sgNC+PIK5-33d:1.8e-13. This data is representative of two independent experiments. h. These experiments were performed once each. i. n = 6 biological replicates for MIA PaCa-2. n = 4 biological replicates per group for PANC1 and 7940B. P-values: MIA PaCa-2: ND646+apilimod vs DMSO:2.9e-13; vs ND646:2.9e-13; vs apilimod:2.9e-13; PANC1: ND646+apilimod vs DMSO: < 1.0e-15; vs ND646: < 1.0e-15; vs apilimod:<1.0e-15; 7940B: ND646+apilimod vs DMSO:1.5e-13; vs ND646:1.5e-13; vs apilimod:1.5e-13. j. n = 6 biological replicates for MIA PaCa-2. n = 4 biological replicates for PANC1 and 7940B. P-values: MIA PaCa-2: ND646 + ESK981 vs DMSO:2.9e-13; vs ND646:2.9e-13; vs ESK981:2.9e-13; PANC1: ND646 + ESK981 vs DMSO: < 1.0e-15; vs ND646: < 1.0e-15; vs ESK981: < 1.0e-15; 7940B: ND646 + ESK981 vs DMSO:2.2e-13; vs ND646:2.2e-13; vs ESK981:2.2e-13. These experiments were performed thrice each. k. n = 4 biological replicates. P-values: MIA PaCa-2: ND646 + PIK5-33d vs DMSO:1.8e-13; vs ND646:1.8e-13; vs PIK5-33d:1.8e-13; PANC1: ND646 + PIK5-33d vs DMSO: < 1.0e-15; vs ND646: < 1.0e-15; vs PIK5-33d:<1.0e-15; 7940B: ND646 + PIK5-33d vs DMSO:2.2e-13; vs ND646:2.2e-13; vs PIK5-33d:2.2e-13. These experiments were performed thrice each. l. n = 3 technical triplicates. P-values: PANC1-SPTLC1: sgNC vs sgSPTLC1:2.4e-5; PANC1-SPTLC2: sgNC vs sgSPTLC2:1.7e-5; MIA PaCa-2-SPTLC1: sgNC vs sgSPTLC1:4.6e-7; MIA PaCa-2-SPTLC2: sgNC vs sgSPTLC2:1.8e-5. m. This experiment was performed twice. n. n = 4 biological replicates. P-values: PANC1: sgNC+apilimod vs sgNC+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC1+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC2+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC1+apilimod:<1.0e-15; sgNC+apilimod vs sgSPTLC2+apilimod:<1.0e-15. MIA PaCa-2: sgNC+apilimod vs sgNC+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC1+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC2+DMSO: < 1.0e-15; sgNC+apilimod vs sgSPTLC1+apilimod:<1.0e-15; sgNC+apilimod vs sgSPTLC2+apilimod:<1.0e-15. o. n = 3 biological replicates. P-values: MIA PaCa-2: YM-53601+apilimod vs DMSO:3.0e-14; vs YM-53601:3.0e-14; vs apilimod:3.0e-14; PANC1: YM-53601+apilimod vs DMSO: 3.0e-14; vs YM-53601:3.0e-14; vs apilimod:3.0e-14; 7940B: YM-53601+apilimod vs DMSO: 3.0e-14; vs YM-53601:3.0e-14; vs apilimod:3.0e-14. p. n = 3 biological replicates. P-values: MIA PaCa-2: NB-598+apilimod vs DMSO:3.0e-14; vs NB-598:3.0e-14; vs apilimod:3.0e-14; PANC1: NB-598+apilimod vs DMSO: < 1.0e-15; vs NB-598: < 1.0e-15; vs apilimod:<1.0e-15; 7940B: NB-598+apilimod vs DMSO:3.0e-14; vs NB-598:3.0e-14; vs apilimod:3.0e-14.
