Extended Data Fig. 2: Cyclin A/B RxL Inhibitors Induce Apoptosis in Cancer Cells with High E2F Activity.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, Anti-proliferation GI50 waterfall plot of cyclin RxL A/B/E inhibitor (CIRc-001) tested against Horizon Discovery cancer cell line panel. n = 302 cell lines. b, Pathway enrichment scores for the top upregulated and down regulated MSigDb Hallmark gene sets within the differentially expressed genes identified between sensitive and resistant cell lines (n = 288 cell lines) to CIRc-001 using the data in A. Top pathways with FDR corrected <0.05 and Normalized Enrichment Score (NES) >= −/+ 2 are represented as blue (upregulated) or red (downregulated)). c, Average doubling time (hours) of sensitive (blue) and insensitive (red) cell lines using the data the DMSO control from Fig. 1f comparing cell counts at the endpoint (Day 6) compared to start (Day 0) of the dose-response assay. d, Dose response assays of the indicated human SCLC cell lines and insensitive human NSCLC cell lines treated for 6 days with increasing doses of the inactivate enantiomer of the cyclin A/B RxL inhibitor (CIRc-005). e, Representative histograms of flow cytometric analysis of propidium iodide (PI) stained NCI-H1048 cells treated with CIRc-004 (200 nM) or DMSO (vehicle) for 24 h. f, Dose response assays of NCI-H1048 cells treated for 6 days with increasing doses of the selective cyclin A RxL inhibitor (CIRc-018), the selective cyclin B RxL inhibitor (CIRc-019), or the cyclin A/B RxL inhibitor (CIRc-004). g, Quantitation of cleaved PARP positive cells analysed by flow cytometric analysis in NCI-H1048 cells treated for 3 days with the indicated doses of CIRc-018, CIRc-019, CIRc-004 or DMSO. Statistical significance was calculated using unpaired, two-tailed students t-test. h, Representative histograms of cell cycle distribution of NCI-H1048 cells treated with CIRc-018, CIRc-019, CIRc-004 or DMSO and then stained with PI. i) Representative histograms of cell cycle distribution of human hematopoietic stem and progenitor cells (HSPC) treated with CIRc-004, staurosporine (100 nM) or DMSO for 24 h. j) Quantitation of cleaved PARP positive cells analysed by flow cytometric analysis in HSPCs treated with CIRc-004, staurosporine (100 nM) or DMSO for 24 h. For i-j, n = 2 biological replicates. k, Immunoblot analysis of RPE1 cells treated with indicated concentrations of CIRc-004 for 72 h. l, Cell cycle phase progression measured over 27 h by time lapse imaging of RPE1 cells expressing FUCCI cell cycle reporter, treated with a higher concentration of CIRc-004 (2000 nM) which is required to promote cell cycle arrest in RPE1 cells (see Fig. 1f), or DMSO. Graph measures quantification of G1 (red FUCCI signal) cell cycle phase per image at progressive time points. Statistical significance calculated using 2-way ANOVA. n = 6 wells per treatment condition per replicate from 2 biological replicates, 1 representative experiment is shown. m, Representative histograms of cell cycle distribution of RPE1 cells treated with CIRc-004 (2000 nM) or DMSO for 72 h and then stained with PI. Data are mean +/− SD of 6 technical replicate wells. For c-h, k, m, n = 3 biological replicates. For c, f-g and j, data are mean +/- SD. Arrows in d, f indicates DMSO-treated sample which was used for normalization. Where indicated, ***=p < 0.001, ****=p < 0.0001.
