Fig. 3: Elevated DDR and increased resistance of aneuploid cells to DNA damage induction.
From: Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage
a GSEA of the Reactome ‘Non-Homologous End Joining’ (NES = 2.06, q-value = 0.0026) and Hallmark ‘DNA repair’ (NES = 1.72, q-value = 0.013) gene sets in highly-aneuploid clones, SS51 and SS111, vs. pseudo-diploid clone SS48. b, c Representative IF images (b) and quantification (c) of 53BP1 and γH2AX foci in non-replicative (EdU-negative) cells across the RPE1 clones. Scale bar, 5 μm. Data obtained from 3 independent experiments, n = 137 (SS48), 146 (SS31), 157 (SS6), 157 (SS119), 156 (SS51), and 148 (SS111). ****p = 3,7 * 10−6 (pseudo-diploid/single clones) and p = 1.1 * 10−10 (pseudo-diploid/multiple clones); γH2AX: **p = 0.0015 (pseudo-diploid/single clones), ***p = 4 * 10−15 (pseudo-diploid/multiple clones); Kruskal–Wallis test, Dunn’s multiple comparison. Bars, mean ± SEM. d Top 3000 genes in genome-wide CRISPR/Cas9 screening of aneuploid clones vs. the pseudo-diploid clone SS48. Highlighted are key genes that belong to the p53 pathway (in red) or to DNA damage response (in orange). e Comparison of cellular sensitivity to drugs that directly induce DNA damage (alkylating and intercalating agents, anti-topoisomerases and PARP inhibitors) across the RPE1 clones. *p = 0.0482; ****p = 2.1 * 10−6 and p = 4.3 * 10−6 for pseudo-diploid/Single and pseudo-diploid/Multiple, respectively; Repeated-Measure One-Way ANOVA, Tukey’s multiple comparison test. n = 42 drugs. f Drug sensitivity to 72 h treatment with etoposide across the RPE1 clones. n = 12 (SS48), n = 8 (SS31), n = 5 (SS6, SS119) and n = 9 (SS51, SS111) independent experiments. *p = 0.0252 for SS119, **p = 0.0046, p = 0.0081 and p = 0.0046, for SS6, SS51 and SS111, respectively; two-sided one-sample t-test. g Western blot of p53 (top) and p21 (bottom) in pseudo-diploid clones and highly-aneuploid clones. h Protein expression of p53 and p21, relative to SS48. n = 6 independent experiments. (p53) *p = 0.0104 and **p = 0.0042, for SS51 and SS111, respectively; (p21) *p = 0.0431 and **p = 0.0061, for SS51 and SS111, respectively; two-sided one-sample t-test. i mRNA expression of the p53 transcriptional target GADD45A, in pseudo-diploid and highly-aneuploid clones. n = 4 independent experiments. **p = 0.0049 (SS51); ***p = 0.0006 (SS111); two-sided one-sample t-test. j Drug sensitivity to 72 h treatment with nutlin-3a in pseudo-diploid vs. highly-aneuploid clones. n = 4 independent experiments. *p = 0.0265 (SS51); **p = 0.0052 (SS111); two-sided One-Sample t-test. k GSEA of genes whose expression correlates with proliferation in highly-aneuploid but not in near-diploid cancer cell lines. The ranking of each DDR signature out of all signatures included in each collection is indicated. l Differential drug sensitivities between near-euploid and highly-aneuploid human cancer cell lines. Direct DNA damage inducers are highlighted in orange, green, yellow and purple. m GSEA of Hallmark ‘DNA repair’ signature in human primary tumors with high vs. low aneuploidy (NES = 1.73; q-value = 0.001). Source data are provided as a Source Data file.
