Fig. 3: An isogenic cell line screen validates KRAS synthetic lethal network genes.

a MCF10A cells stably expressing eGFP or a mutant KRAS construct were reverse transfected with esiRNAs targeting specific genes. After 72 h, relative proliferation was compared between eGFP and KRAS mutant cells to score genetic interactions. b Proliferation of uniformly plated MCF10A cells expressing eGFP, KRAS G12V or G12D grown in the presence or absence of 20 ng/ml EGF for 72 h. n = 4 biologically independent samples. c Immunoblot of isogenic cells grown in the presence or absence of 20 ng/ml EGF for 24 h. Experiment was repeated twice with similar results. d Proliferation of eGFP or KRAS G12D cells grown in the indicated media conditions after non-targeting (NT) or KRAS knockdown for 72 h, normalized to NT control. n = 4 biologically independent samples. e Volcano plot reflecting the magnitude of change in proliferation after gene knockdown in KRAS G12D cells grown in minimal media compared to eGFP expressing control grown in full media versus the significance of this effect calculated among replicates. Dotted lines represent the indicated false discovery rate (FDR) cutoffs. KRAS indicated as control (red). f Relative proliferation of gene knockdown in eGFP or KRAS G12D cells compared to non-targeting control. Genes selected based on genetic interactions with <1% FDR. n = 4 biologically independent samples. g Signed genetic interaction scores for genes in the broader RAS pathway grouped into functional categories. The most negative scoring genes in each category are listed. h Signed genetic interaction score of retested literature curated KRAS synthetic lethal genes and their source. A subset of genes from Luo et al.⁴⁰ are indicated for clarity. i Comparison of genetic interaction scores for genes involved in the RAS or MAPK pathway (RAS/MAPK), RTK signaling, KRAS synthetic lethal genes from the literature (green), or predicted synthetic lethal network genes (purple). p values based on comparison against a median interaction score of zero (bottom) and between groups (above), both by two-tailed Student’s t test. Boxes represent the median, hinges span 25–75th percentile and whiskers span 10–90th percentile. Data are presented as mean values ± s.d.