Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to Chemotherapy by Abrogating the TopBP1/ATR-Mediated DNA Damage Response

Abstract

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC.Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence.

Results: 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage.

Conclusions: These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: A. Ugolkov has equity interest in Actuate Therapeutics Inc. D.M. Schmitt is an employee of Actuate Therapeutics Inc. and has equity interest in Actuate Therapeutics Inc. F.J. Giles has equity interest in Actuate Therapeutics Inc. A.P. Kozikowski has equity interest in Actuate Therapeutics Inc. and is a co-inventor listed on the 9-ING-41 patent. A.P. Mazar has equity interest in Actuate Therapeutics Inc. D.D. Billadeau serves on the Scientific Advisory Board of Actuate Therapeutics Inc. and has equity interest in Actuate Therapeutics Inc. No potential conflicts of interest were disclosed by the other authors.

©2019 American Association for Cancer Research.

Figures

Figure 1. 9-ING-41 treatment synergizes with gemcitabine to abrogate PDAC cell proliferation and colony formation in vitro.

(A) The indicated PDAC cell lines were seeded in 96-well plates and treated with DMSO or increasing concentration of 9-ING-41 (nM) for 48 hours. Cell proliferation was determined by MTS assay. Data were quantified as percentage of control and expressed as mean ± SEM. n=6. (B) The 6741 PDX-derived cell line was plated and treated with 1 μM 9-ING-41 alone or with increasing concentrations of gemcitabine (nM) for 48 and 72 hours. Cell proliferation was determined by MTS assay. Data was quantified as percentage of control and expressed as mean ± SE. *P

Figure 2. 9-ING-41 and Gemcitabine abrogate tumor growth in vivo.

(A) The pancreatic PDX tumor (PCF 379419) was transplanted subcutaneously into both flanks of athymic nude mice (12 mice in total). Tumors were size matched and mice were randomized into 4 treatment groups: Vehicle, Gemcitabine, 9-ING-41 (40 mg/kg) and Gemcitabine + 9-ING-41. Gemcitabine was used as 10 mg/kg (week 1) and 5 mg/kg (week 2 and 3). Vehicle or drugs were injected i.p. Tumor volume was measured weekly and shown as mean ± SEM. (n=3/group). *P

Figure 3. GSK-3 abrogates gemcitabine-induced Chk1 activation and cell cycle arrest.

(A) PDAC cell lines were treated with gemcitabine (500 nM) over the indicated time course, harvested and lysates were prepared and immunoblotted with the indicated antibodies. (B) PDAC cell lines were pretreated with GSK-3 inhibitors Bio (5 μM) or 9-ING-41 (5 μM) for 2 hours followed by an additional 2 hour treatment with gemcitabine (500 nM). Phosphorylated Chk1 at S317 and S345 Chk1, as well as total Chk1 were examined by immunoblotting. β-actin was used as a loading control. (C) Average signal intensity of pS317 and pS345 Chk1 were analyzed and expressed as mean ± SEM. *P

Figure 4. GSK-3β regulates ATR-dependent phosphorylation of Chk1 in response to gemcitabine treatment.

(A) PDAC cell lines were depleted of GSK-3α or GSK-3β using siRNA and then treated with gemcitabine (500 nM) for 2 hours. Cell lysates were prepared and immunoblotted with the indicated antibodies. Average signal intensity of pS345 Chk1 was analyzed and expressed as mean ± SEM. *P

Figure 5. GSK-3β regulates TopBP1 protein stability.

(A) 5160 and 6741 cell lines were treated with the GSK-3 inhibitors Bio (5 μM) and 9-ING-41 (5 μM) for 4 hours. Protein lysates were prepared and immunoblotted with the indicated antibodies. The average signal intensity of TopBP1, ATR and ATRIP were analyzed and expressed as mean ± SEM. *P

Figure 6. 9-ING-41 reverses Chk1 phosphorylation induced by gemcitabine treatment.

(A) 6741 cells were grown on coverslips, treated with DMSO, 9-ING-41 (5 μM), gemcitabine (500 nM) or the combination of 9-ING-41 and gemcitabine and pulsed with EdU one hour prior to fixation. Fixed cells were subsequently stained with anti-pS317 Chk1 antibodies and detected with an Alexa 568 conjugated donkey-anti-rabbit secondary (red) and EdU-488 (green). DNA was visualized following Hoechst staining (blue). (B) The normalized MFI of nuclear pS317 Chk1, EdU-488 and the percentage of EdU positive cells were evaluated by ImageJ and expressed as mean ± SEM. *P

Figure 7. Proposed model by which GSK-3 inhibition by 9-ING-41 disrupts the TopBP1/ATR/Chk1 pathway.

In response to gemcitabine-induced DNA replication stress, TopBP1/ATR/ATRIP (not shown) complexes are recruited to stalled replication forks where ATR can fully activate Chk1 leading to cell cycle arrest and DNA repair. In the presence of 9-ING-41, TopBP1 protein levels are destabilized thus abrogating the full activation of ATR leading to reduced Chk1 phosphorylation and ultimately impaired cell cycle arrest and likely DNA repair. Circle with red P indicates phosphorylation. Dashed rectangle with red X indicates TopBP1 degradation.