Fig. 4: WEE1 inhibitors activate the integrated stress response independent of WEE1 and independent of cell cycle status.
From: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2
a Western blot showing the time course of WEE1 degradation in the RPE TP53−/− cell line following treatment with 1 μM HRZ-057-1 or HRZ-1-098-1. Data are representative of n = 2 independant biological replicates. b A schematic of the experiment in (c). c Western blot of the RPE TP53−/− cell line pre-treated with DMSO or 1 μM WEE1 molecular glues (HRZ-057-1 and HRZ-1-098-1) for 1 h (total treatment duration: 7 h), followed by DMSO or 650 nM AZD1775 for an additional 6 h. Bands of similar molecular weights were run in parallel on separate blots. Total protein (except for ATF4) served as loading controls. A quantification of three independant biological repeats of this experiment can be found in Supplementary Fig. 17a–c. d A schematic of the in vitro FLAG-tagged GCN2 experiment e Western blot of an in vitro experiment probing the total and phosphorylated GCN2 in the presence of DMSO, Neratinib, WEE1i (AZD1775 and Debio0123), and PKMYT1i (RP-6306). p-GCN2 and total GCN2 were run in parallel on separate blots. A separate, independant biological experiment can be found in Supplementary Fig. 14b. f ADP-Glo assay of GCN2 mixed with a serial dilution of AZD1775 in the presence of ATP in a 384 well plate format (independant biological replicates n = 4). Graphs are depicted with means ± SD. g Thermal unfolding assays of GCN2 in the presence of 200 μM AZD1775 or 100 μM tRNA with a gradient of 0.5 °C/min from 25 °C to 90 °C. Dashed lines connect the negative and positive first derivative F350/F330 peaks to the x-axis. h Immunofluorescence analysis of nuclear ATF4 and γH2AX in the RPE TP53−/− cell line treated with 650 nM AZD1775 across multiple timepoints (independant biological replicates n = 3). Box plots show the median (centre line), the interquartile range (bounds of box), and the minimum and maximum values (whiskers). i Representative images showing ATF4 and γH2AX intensity in untreated (UT) cells and those treated with 650 nM AZD1775 alone or in combination with 1 μM GCN2iB for 24 h in the RPE TP53−/− cell line. Source data are provided as a Source data file.
