Fig. 3: RS activated S-phase checkpoint and generated vulnerability to CHK1 inhibition in ecDNA-containing tumour cells.

a, Detection of pRPA2-S33, γH2AX, pCHK1-S345 and 53BP1/cyclin A in multiple cancer cell lines with different ecDNA amplification status. Left, representative images in COLO320DM and COLO320HSR cells. Right, mean foci number in individual cell lines. Line indicates median; every dot indicates mean foci number in each cell line. b, Comet-FISH assay in COLO320DM and COLO320HSR cells. Top, representative images. Bottom left, MYC foci number in tail. Bottom right, percentage of MYC in comet tail (two-sided Wilcoxon test, n: 47, 60, 49, 33). c, Relative cell number of Hela ecDNA⁺ and Hela ecDNA⁻ cells transduced with sgRNAs targeting CHK1 normalized to cells transduced with non-targeted (NT) sgRNA over time. d, Cell viability curves of SNU16, COLO320DM and COLO320HSR in response to CHIR-124 for three days (n = 4, mean ± s.d.). e, TUNEL assay in cells subjected to CHIR-124 for indicated time (mean ± s.d., ordinary one-way ANOVA with multiple comparison test, n = 3). f, γH2AX IF in COLO320DM and COLO320HSR cells treated with CHIR-124 with or without the combination of CDC7i (XL413), with EdU added for 30 min. Left, representative images; red lines mark EdU⁺ and white lines mark EdU⁻ nuclei. Right, mean γH2AX intensity (arbitrary units). EdU⁻, n: 3,074, 4,246, 3,291, 4,742, 3,101, 3,770, 2,608, 2,091; EdU⁺, n: 2,428, 2,859, 2,909, 2,890, 3,346, 3,491, 3,232, 2,060; two-tailed Student’s t-test. g, Schematics depicting CHK1 activation in response to RS, which sensitizes ecDNA-containing tumour cells to targeted CHK1i through unscheduled replication origin firing and accumulation of excessive DNA damage, leading to cell death. Parameters for boxplots b,f and violin plot f are the same as Fig. 2 or as otherwise specified. Scale bar, 10 µm (a,e,f), 20 µm (b). a.u., arbitrary units.