Inhibition of WEE1 kinase with AZD1775 activates CDK1/2 and causes apoptosis in Ewing sarcoma cells. (A) EW8 and TC71 cells were treated with different doses of AZD1775 (0–1000 nM) for 6 h. Cellular lysates were then harvested for immunoblotting for CDK1 and P-CDK1/2. (B) EW8 and TC71 were transfected with siRNA targeting WEE1. Cellular lysates were harvested 24 h after transfection and immunoblotting was performed for CDK1 and P-CDK1/2. (C) EW8 and TC71 were treated with AZD1775 (500 nM) for 18 h. Cellular lysates were then collected for immunoblotting for cleaved and total PARP, γH2AX, CDK1, P-CDK1/2, and cleaved caspase-3. (D) EW8, TC71, and A673 cells were treated with AZD1775 (500 nM) for 18 h and then caspase-3/7 activation was quantified using Casp-Glo 3/7 Luminescence assay. (E) EW8 cells were treated with AZD1775 (500 nM), RO-3306 (10 μM), or the combination of AZD1775 and RO-3306 for 6 h. Cellular lysates were then collected for immunoblotting for γH2AX, CDK1, and P-CDK1/2. (F) EW8 and TC71 cells were treated with AZD1775 (500 nM) for 6 h. Cells were then labeled with EdU and fixed for flow cytometry for γH2AX. Results are representative of two independent experiments. (G) EW8 and TC71 cells were treated with AZD1775 (100 nM), LY2603618 (100 nM), or the combination of AZD1775 and LY2603618 for 18 h. Cellular lysates were then collected for immunoblotting for gH2AX, CDK1, P-CDK1/2, and cleaved and total PARP. (H) EW8 and TC71 cells were treated for 6 h with AZD1775 (100 nM), LY2603618 (100 nM), RO-3306 (10 μM) or the combination of AZD1775 and LY2603618 with RO-3306. Cellular lysates were then collected for immunoblotting for γH2AX. (I) A673, EW8, and TC71 cells were treated with AZD1775 (100 nM), the ATR inhibitor AZD6738 (100 nM), or the combination of AZD1775 and AZD6738 for 18 h. Cellular lysates were then collected for immunoblotting for cleaved and total PARP and γH2AX. (J, K) BJ-tert (J) and A673 (K) cells were treated for 72 h with a combination of up to 1000 nM AZD6738 and 500 nM AZD1775. Survival was assayed by Cell-Titer-Glo and each experiment was repeated 2 times. Color bars indicate % inhibition normalized to untreated cells. Loewe matrix plots for drug cooperativity are also shown. (L) A673, EW8, and BJ-tert cells were treated with AZD1775 (100 nM), AZD6738 (100 nM), and the combination of AZD1775 (100 nM) and AZD6738 (100 nM). Cellular lysates were collected for immunoblotting 24 h after the drugs were added.