Fig. 1: A pooled CRISPRi screen reveals the perturbation of GSPT1 as a sensitivity hit for WEE1 inhibitors.
From: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2
a Experimental design of pooled CRISPRi screen b CRISPRi screen results showing NormZ scores, calculated by DrugZ software, at IC95 (x-axis) and IC25 (y-axis) doses of AZD1775. Dosing information is available in Supplementary Fig. 1a. c Representative image of 200 nM AZD1775 and 62.5 nM CC-90009 compounds alone or in combination for 72 h in the RPE TP53−/− cell line in a 6 well plate format followed by quantification of the cell density (independant biological replicates n = 3). Bar charts are depicted with means ± SD, points represent each independant biological replicate. Statistical analysis was performed using one-way ANOVA with multiple comparisons: AZD1775 alone vs. Combination, p < 0.0001; CC-90009 alone vs. Combination, p < 0.0001. d Resazurin cell viability summary synergy scores (combined Loewe, Bliss and HSA synergy scores) of CC-90009 in combination with WEE1 inhibitors (AZD1775, Zn-c3, Debio0123), PKMYT1i (RP-6306), ATRi (AZD6738) or CHEK1i (LY2603618) in RPE TP53−/− cells in a 96 well plate format (independant biological replicates n = 4). Heatmaps of the drug combinations are shown in Supplementary Fig. 4. e Western Blot showing 200 nM AZD1775 and 62.5 nM CC-90009 alone or in combination treated on the RPE TP53−/− cell line for 24 h. Cycloheximide (1 μg/mL) served as a positive control for global mRNA translation shutdown. Cells were treated with puromycin (5 μg/mL, 15 min) before harvesting. The probing of puromycin was run in parallel on a separate blot. A quantification of 3 independant biological repeats of this experiment can be found in Supplementary Fig. 17d–f. f Representative image of HAP1 cells treated with 100 nM AZD1775, 4 μM CC-90009, and 500 nM ISRIB compounds alone or in combination, for 72 h in a 6 well plate format, followed by quantification of the cell density (independant biological replicates n = 3). Bar charts are depicted with means ± SD points represent each independant biological replicate. Statistical analysis was performed using an unpaired two-tailed t-test (combination [AZD1775 + CC-90009] vs. Combination + ISRIB 500 nM), p = 0.0011. Source data are provided as a Source data file.
