Fig. 3. Inhibition of GPX4 is synthetic lethal with high MYCN.
a, MYCN synthetic lethal druggable genome-wide siRNA screening approach in IMR5/75 cells. b, Effects of individual siRNAs (gray dots): high MYCN versus low MYCN, including key players (median of two or three siRNAs) of the top MYCN synthetic lethal hits (single star symbol) of GSH metabolism (black) and biosynthesis (green). c, MYCN effects on lipid peroxide formation and intracellular amino acid levels (fold changes shown in red), the xc− system (Cys2 uptake), the two-step biosynthesis of GSH and GSH metabolism; the single star symbol marks the top MYCN synthetic lethal hits of GSH metabolism, with an FDR of 0.2. The action of ferroptosis inhibitors (CPX, Fer-1, Lip-1, Trolox and 10058-F4), class I (erastin, IKE, sulfasalazine), class II ferroptosis inducers (RSL3, ML-210) and the GSH biosynthesis inhibitor buthionine sulfoximine as indicated. d, siRNA GPX4 knockdown in the presence or absence of Fer-1. Data represent the mean ± s.e.m.; n = 4 samples. The experiment was replicated three times. e, Relative viability (survival of compound-treated cells divided by survival of vehicle-treated cells) of the KELLY cell line after cotreatment with iron sucrose (25 µg ml−1) and RSL3. Data represent the mean ± s.e.m.; n = 3 samples. The experiment was replicated three times. f, Relative viability of IMR5/75 cells treated with RSL3 in the presence or absence of Fer-1 or GSH. n = 4 samples. The experiment was replicated three times. g, DRIVE database27 (RSA values, Pearson’s correlation, P = 0.01; filled circle, MYCN-amplified; white circle, MYCN-non-amplified). h, Cellular responses of neuroblastoma cell lines to 72 h of RSL3 treatment: cells with MYCN amplification (black symbols), moderate MYCN expression (white circle) and lack thereof (white triangle). Data represent the mean ± s.e.m.; n = 3 samples. The experiment was replicated three times. i, Dox-inducible GPX4 CRISPR–Cas9 knockout in a 3D model with MYCN-amplified SK-N-DZ cells in the presence or absence of MYCN–MAX inhibition. Data represent the mean ± s.e.m. Right: representative western blot; n = 4 samples. The experiment was replicated three times. j, Orthotopic mouse neuroblastoma model allowing CRISPR–Cas9-mediated GPX4 deletion. Panel created with BioRender. k, Tumor weight after GPX4 knockout (+Dox) (n = 5 mice per group). A representative western blot for CRISPR–Cas9-mediated GPX4 deletion is shown. l, Elevated messenger RNA expression of the ferroptosis markers CHAC1 and TFRC. Data represent the mean ± s.e.m.; n = 4 samples from each group. Statistical analysis was performed using a one-tailed Student’s t-test for the in vivo experiments and a two-tailed Student’s t-test for the in vitro experiments. Box plots: the center line indicates the median value, the lower and upper hinges represent either the 25th and 75th percentiles or the minimum and maximum points and the whiskers denote 1.5× the interquartile range (IQR). Each dot corresponds to one sample; one-sided Student’s t-test; P values as indicated.