Extended Data Fig. 5: Forward Genetic Screen Identifies CDC20 Mutants that Cause Resistance to a Cyclin A/B RxL Inhibitor.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, Dose response curve of iHCT116 cells treated with increasing doses of cyclin A/B RxL inhibitor (CIRc-004). b-c, Dose-response curves for six clones isolated from Mut-low iHCT116 cells (b) and 12 clones from Mut-high iHCT116 cells treated with increasing doses of CIRc-004 for 3 days (c). d, Dose response curve to MLN4924 of different CIRc-004 resistant iHCT116 clones. For a-d n = 2 technical replicates. Data are mean +/− SEM. e, Barcode sequences identified in 18 different CIRc-004 resistant clones, common sequences are marked in blue. f, Genes recurrently mutated (≥2 clones) among eight CIRc-004-resistant iHCT-116 clones from the forward genetic screen. g, Mutated residues (blue) in CDC20 (PDB 4GGD) present in CIRc-004 resistant clones. h, Immunoblot analysis of iHCT116 cells stably expressing vector, Flag-CDC20 WT, or Flag-CDC20 R445Q mutant. i, Dose response assay of iHCT-116 cells from h treated with increasing doses of CIRc-004. For i, n = 3 technical replicates. Data are mean +/− SEM. j, Immunoblot analysis of NCI-H1048 cells stably expressing empty vector, Flag-CDC20 WT, or Flag-CDC20 R445Q; Actin run on separate gel as a sample processing control. k-m, NCI-H1048 cells from j assessed for CIRc-004 dose response (k), cleaved PARP (l), and phospho-histone H3 (m) by flow cytometry following CIRc-004 treatment (20 nM). For k-m, n = 3 biological replicates; data represent mean ± SD. Statistical significance determined by unpaired, two-tailed Student’s t-test. Where indicated, *=p < 0,05, ****=p < 0.0001.
