Figure 4. FSEN1 is synthetic lethal with GPX4 inhibitors and sensitizes cancer cells to ferroptosis through inhibition of FSP1.
A) Dose response of RSL3 and ML162-induced cell death in H460C Cas9 cells co-treated with 1 μM FSEN1 and 2 μM Fer-1 where indicated. Data are mean ± SEM (n=3 cell biological replicates). B) Heat map represents the fraction of viable H460C Cas9 cells co-treated with increasing doses of FSEN1 and RSL3 (n=3 cell biological replicates). C) Representative images of H460C Cas9 cells co-treated with 1.6 μM RSL3, 1 μM FSEN1 and 2 μM Fer-1 as indicated. Scale bar = 200 μm. Images from one of three independent experiments are shown. D) Lethal Fraction (AUC) of 5 μM RSL3-induced cell death in H460C Cas9 cells co-treated with 1 μM FSEN1 together with the indicated inhibitors of ferroptosis (Fer-1 [2 μM], DFO [100 μM], idebenone [10 μM], tocopherol [10 μM]), apoptosis (Z-VAD [10 μM]), and necroptosis (Nec1s [1 μM]). Data are mean ± SEM (n=3 cell biological replicates). **p<0.01 by one-way ANOVA with Dunnett’s multiple comparisons test. E) Representative flow cytometry histogram (left panel) and quantification (right panel) of H460C Cas9 cells treated with 200 nM RSL3 and/or 1 μM FSEN1 and labeled with the lipid peroxidation sensor BODIPY 581/591 C11. Green fluorescence intensity was analyzed by flow cytometry with the FITC channel. Data are mean ± SD (n=3 cell biological replicates). *p<0.05, ***p=0.0003 by one-way ANOVA with Tukey’s multiple comparisons test. F) Dose response of RSL3-induced cell death in H460C FSP1KO cells co-treated with 1 μM FSEN1. Data are mean ± SEM (n=3 cell biological replicates). G) H460C spheroids were treated with vehicle, 5 μM FSEN1, and 5 μM RSL3 as indicated. Representative images from one of 20 independent experiments are shown. Scale bar represents 100 μm. The total intensity of the SYTOX green signal for each spheroid was quantified and the mean ± SEM plotted (n=20 independent spheroids).