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. 2023 Oct 1;37(19-20):913–928. doi: 10.1101/gad.351085.123

Figure 6.

Figure 6.

ATR inhibition can compensate for incomplete WRN degradation. (A–C) Human cancer cells expressing FKBP-WRN were treated as indicated for 6 d. When used in combination, the ATR inhibitor AZ-20 was always added 2 h after the start of dTAG-13 treatment. Viability assays showed that the combination regimen produced significantly superior cytotoxicity as compared with single-agent treatments in MSI-H RKO (A), KM12 (B), and HCT116 (C) clones but not in MSS OVCAR8 (D). Average (±SD) of three (OVCAR8 and KM12 c2) or four (RKO, HCT116, and KM12 c5) independent experiments is shown. (E) MSI-H cancer cells expressing FKBP-WRN were treated as indicated for 24 h. Immunoblotting showing that the combination regimen resulted in markedly elevated levels of pKAP1 as compared with either single agent alone. One of two independent experiments is shown. (F) RKO reporter cells were treated as indicated and subjected to live-cell imaging. Automated tracking of 53BP1 puncta showed that the combination regimen induced more 53BP1 than either agent alone. (G–I) Manual fate mapping of RKO reporter cells: vehicle (n = 25; 1094 tracked progeny), 10 nM dTAG-13 (n = 50; 992 tracked progeny), 0.25 μM AZ-20 (n = 40; 1074 tracked progeny), and dTAG-13/AZ-20 (n = 50; 497 tracked progeny). Cells treated with the combination regimen exhibited reduced cell division activities (G) accompanied by increases in both cell death (H) and cell cycle arrest (I). For F–I, one of two independent experiments is shown. (J) Proposed model of how ATR inhibition compensates for incomplete WRN degradation.