Figure 1.

ATR-CHK1 inhibitors and homoharringtonine selectively target Ewing sarcoma cells with a partial reduction in the level of the RRM2 protein. (A) EW8 cell lines with doxycycline-inducible shRNA-mediated knockdown of RRM2, or a non-targeting shRNA (shNT), were treated with doxycycline or vehicle for 48 h. Cellular lysates were then collected for immunoblotting. (B) The shRRM2 cells, grown in the presence of doxycycline or vehicle, were treated with LY2603618 or prexasertib for 48 h. Cell viability was assessed 72 h after drug addition using the AlamarBlue assay. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (C) The shRRM2 and shNT cells were treated with doxycycline, prexasertib, or vehicle for 24 h and then cellular lysates were collected for immunoblotting. (D) Results of the small molecule drug screen, which was performed in duplicate, for the shRRM2 and shNT cells. The log2 fold difference in the growth rate between the shRRM2 and shNT cell lines is shown for each drug. A normalized growth rate (GR) inhibition approach was used to analyze the data in order to account for the effects of the different growth rates of the shRRM2 and shNT cell lines. ATR and CHK1 inhibitors, which were included in the screen as positive controls, are shown in red. (E) Ewing sarcoma cell lines were treated with DMSO, panobinostat, or romidepsin for 24 h. Cellular lysates were then collected for immunoblotting. (F) Dose response curves for shRRM2 cells treated with different concentrations of HHT in the presence or absence of doxycycline. Cell viability was assessed 72 h after drug addition using the AlamarBlue assay. A normalized growth rate (GR) inhibition approach was used to analyze the data. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (G) The shRRM2 cells, grown in the presence of DMSO or doxycycline, were treated with different concentrations of HHT for 24 h. Cellular lysates were then collected for immunoblotting. (H) The shRRM2 cells were treated with DMSO, doxycycline, HHT, or the combination of doxycycline and HHT for 24 h. Caspase-3/7 activation, relative to the cells treated with DMSO, was then quantified using a Casp-Glo 3/7 Luminescence assay. (I, J) TC71 and EW8 cells were treated with different concentrations of HHT for 24 h. Protein synthesis was then assessed using puromycin labeling. Protein loading for all the immunoblots was normalized using cell number.