Fig. 2: Tumor-specific cytotoxicity through small molecule inhibition of Chk1 in vitro. | Oncogenesis

Fig. 2: Tumor-specific cytotoxicity through small molecule inhibition of Chk1 in vitro.

From: Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death

Fig. 2

a Dose–response curves shows relative cell viability of HNSCC cell lines (red line) and untransformed primary oral fibroblasts and primary oral keratinocytes (two individual donors each, green lines) for the Chk1 inhibitor LY2603618/Rabusertib (72 h exposure). Experiments were performed three times in triplicate and the averaged value is indicated. Note the therapeutic window between tumor and primary cells, indicating tumor-specific cytotoxicity of Chk1 inhibition. b Treatment with LY2606368/Prexasertib, a dual Chk1/Chk2 inhibitor, resulted in cytotoxic effects on HNSCC cells (in red) and primary oral keratinocytes (in green), but no therapeutic window was found. The increased viability of the keratinocytes at higher concentrations suggests an off-target effect. c Half maximal effective concentrations (EC50) of LY2603618/Rabusertib represented per tested HNSCC cell lines (red bars) and primary mucosal cell type (green bars). TP53 mutational status, and presence of hrHPV are depicted below and in Table 1. d Long-term exposure (10 days) of LY2603618/Rabusertib indicated an intrinsic difference in sensitivity for the most sensitive (UM-SCC-22A) and moderately sensitive (VU-SCC-096) HPV-negative HNSCC cell lines. After drug treatment, cells were fixed and stained with crystal violet in situ. e. Quantification of protein levels (Fig. S3a) did not reveal a correlation between either Chk1 expression levels or basal DNA damage levels measured by γH2Ax Ser139 (Fig. S3b, c). Protein levels were normalized by the loading control HSP90α/ß. Cell lines are ordered to their sensitivity to Chk1 inhibition (left to right, most to less sensitive). f EC50 values of four HPV-negative and two HPV-positive HNSCC cell lines were determined for Chk1 inhibitor LY2603618/Rabusertib and ATR inhibitor VE-821. Pearson correlation showed a significant correlation between responses to ATR inhibition and Chk1 inhibition, which was expected since Chk1 is a direct substrate of ATR. However, no therapeutic window was found for ATR inhibition with primary cells (Fig. S2c, d), which may relate either to the specificity of the inhibitors, or the apparent novel role of Chk1 in malignant cells

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