ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status

Abstract

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

Conflict of interest statement

Potential Conflicts of Interests: The other authors have no conflicts to report.

©2013 AACR.

Figures

Figure 1. ATR depletion broadly sensitizes to multiple chemotherapy agents, whereas Chk1 depletion selectively sensitizes to gemcitabine

OVCAR-8 cells were transfected with control (Luc), ATR, or Chk1 siRNAs. 48 h after transfection, cells were trypsinized and used to analyze ATR and Chk1 expression (A) or in clonogenic assays (B–F). For clonogenic assays, cells (250 per well) were plated, allowed to adhere for 4–6 h, and treated with cisplatin (B), topotecan (C), veliparib (D), or gemcitabine (E) for 8 d. In panel F, after cells were allowed to adhere for 4 h, they were treated with gemcitabine for 24 h, washed, and cultured for 8 days. A representative experiment from 3 independent experiments is shown.

Figure 2. The ATR inhibitor VE-821 and the Chk1 inhibitor MK-8776 phenocopy the effects of ATR and Chk1 depletion

OVCAR-8 cells were trypsinized, plated as single cells, allowed to adhere 4 h, treated with 0.3 µM MK-8776 or 1 µM VE-821 plus cisplatin, topotecan, veliparib, or gemcitabine for 8 d. The experiment shown is representative of 4 (SKOV3) and 5 (OVCAR-8) independent experiments.

Figure 3. MK-8776 and VE-821 disrupt chemotherapy-induced cell cycle checkpoints

OVCAR-8 cells were co-treated with vehicle, 0.1 µM MK-8776, or 1 µM VE-821 plus 10 µM veliparib, 20 nM topotecan, 0.6 µM cisplatin, or 5 nM gemcitabine for 24 h and analyzed by flow cytometry.

Figure 4. MK-8776 and VE-821 do not block Chk1 phosphorylation

(A) OVCAR-8 cells were pretreated with vehicle (−), MK-8776 (0.3 and 1.0 µM), or VE-821 (1.0 or 4.0 µM) for 15 min and then exposed to cisplatin (4 µM), topotecan (TPT, 20 nM), veliparib (10 µM), or gemcitabine (Gem, 20 nM) for 4 h in the continued presence of MK-8776 or VE-821. (B) SKOV3 cells were pretreated with vehicle (−), MK-8776 (0.3, 1.0, and 4 µM), or VE-821 (1.0, 4.0, and 6.0 µM) for 15 min and then exposed to 20 nM gemcitabine for 4 h. (C, D) MiaPaCa (C) or U937 (D) cells were pretreated with vehicle (−), MK-8776 (0.3 and 1.0 µM), or VE-821 (1.0 or 4.0 µM) for 15 min and then exposed to gemcitabine (40 nM, MiaPaCa cells; 20 nM U937 cells) for 4 h. Cell lysates were then immunoblotted for the indicated antigens.

Figure 5. ATR inhibition further sensitizes cells with defective HR to cisplatin, topotecan, and veliparib

(A) OVCAR-8 cells that have stably integrated DR-GFP HR substrate were transfected with pCβASceI plasmid plus control (Luc) or ATR siRNA and examined for GFP fluorescence 72 h after plasmid transfection. Mean +/− S.D; n = 3; *P = 0.02 by paired t-test. * indicates nonspecific band. OVCAR-8 (B–F) or SKOV3 (G) cells were transfected with control (Luc) or BRCA1 siRNA. 48 h after transfection, cells were trypsinized and used to analyze BRCA1 expression (B, OVCAR-8 cells) and for clonogenic assays (C–G). For clonogenic assays, cells were plated, allowed to adhere for 6 h, and treated 0.3 µM MK-8776 or 1 µM VE-821 plus gemcitabine (C) cisplatin (D), topotecan (E), or veliparib (F, G) for 8 d. A representative experiment from 3 independent experiments is shown.