A Novel Salt Inducible Kinase 2 Inhibitor, ARN-3261, Sensitizes Ovarian Cancer Cell Lines and Xenografts to Carboplatin

Abstract

Salt-induced kinase 2 (SIK2) is a serine-threonine kinase that regulates centrosome splitting, activation of PI3 kinase and phosphorylation of class IIa HDACs, affecting gene expression. Previously, we found that inhibition of SIK2 enhanced sensitivity of ovarian cancer cells to paclitaxel. Carboplatin and paclitaxel constitute first-line therapy for most patients with ovarian carcinoma, producing a 70% clinical response rate, but curing <20% of patients with advanced disease. We have asked whether inhibition of SIK2 with ARN-3261 enhances sensitivity to carboplatin in ovarian cancer cell lines and xenograft models. ARN-3261-induced DNA damage and apoptosis were measured with γ-H2AX accumulation, comet assays, and annexin V. ARN-3261 inhibited growth of eight ovarian cancer cell lines at an IC50 of 0.8 to 3.5 µM. ARN-3261 significantly enhanced sensitivity to carboplatin in seven of eight ovarian cancer cell lines and a carboplatin-resistant cell line tested. Furthermore, ARN-3261 in combination with carboplatin produced greater inhibition of tumor growth than carboplatin alone in SKOv3 and OVCAR8 ovarian cancer xenograft models. ARN-3261 enhanced DNA damage and apoptosis by downregulating expression of survivin. Thus, a SIK2 kinase inhibitor enhanced carboplatin-induced therapy in preclinical models of ovarian cancer and deserves further evaluation in clinical trials.

Keywords: PARP inhibitor; apoptosis; carboplatin sensitivity; comet assay; salt inducible kinase 2 (SIK2); γ-H2AX.

Figure 1

ARN-3261 synergistically enhances carboplatin-induced inhibition of ovarian cancer cell short term and clonogenic growth in cell culture. (A) Sensitivity to ARN-3261. A2780, ES2, IGROV1, MDA2774, OC316, OVCAR3, OVCAR8 and SKOv3 ovarian cancer cell lines were plated at a density of 2000 cells/well in 96-well plates, then treated with different concentrations of ARN-3261 for 96 h. Cell viability was measured with a bioluminescence assay as described in Methods and IC50 values were calculated. (B) Sensitivity to carboplatin. Eight ovarian cancer cell lines were treated as above with different concentrations of carboplatin as indicated. (C) Effect of a single concentration of ARN-3261 on the carboplatin dose response curve. Eight ovarian cancer cell lines were treated with different concentrations of carboplatin as indicated with or without a single concentration of ARN-3261 (A2780 0.75 µM, ES2 1.25 µM, IGROV1 1.25 µM, MD2774 1.15 µM, OC316 0.75 µM, OVCAR3 0.75 µM, OVCAR8 1 µM and SKOv3 1 µM). IC50s of carboplatin with or without ARN-3261 were calculated by GraphPad Prism 8 (** p < 0.01 by student t test). (D) Synergistic interaction of carboplatin and ARN-3261. IGROV1, OC316, OVCAR8 and SKOv3 were treated concomitantly with a serial dilution of ARN-3261 and carboplatin at a fixed ratio indicated in the figure. The drug concentration ratio is indicated in each plot. The combination index at 50% growth inhibition was calculated using CalcuSyn software. (E) Effect of SIK2 knockout on the carboplatin dose response curve. Cells were treated with different concentrations of carboplatin as indicated. IC50 values for (A–C,E) were calculated by GraphPad Prism 8. (F) ARN-3261 enhances carboplatin-induced inhibition of clonogenic growth. Four hundred OVCAR8 or SKOv3 ovarian cancer cells were seeded in 6-well plates in culture medium for 24 h. Cells were then treated with diluent, ARN-3261 (ES2 2.2 µM, OC316 2.5 µM, OVCAR8 2.3 µM, SKOv3 3.5 µM and MDA2774 2.5 µM), carboplatin (ES2 3.3 µM, OC316 3.0 µM, OVCAR8 4.0 µM, SKOv3 2.0 µM and MDA2774 3.0 µM) or both in triplicate for another 12–14 days. The graphs indicate the mean colony formation numbers with standard deviations. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way ANOVA analysis.

Figure 2

Inhibition of SIK2 activity with ARN-3261 or knockout of SIK2 protein enhances carboplatin-induced apoptosis. (A) Effect of ARN-3261 on carboplatin-induced apoptosis. OC316, OVCAR8 and SKOv3 cell lines were plated at a density of 8000 cells/well in 12-well plate in triplicate, and then treated with ARN-3261 (OC316 3 µM, OVCAR8 5 µM and SKOv3 4.5 µM) and/or carboplatin (OC316 15 µM, OVCAR8 70 µM and SKOv3 60 µM) for 72 h. Cells were dislodged and stained with Annexin V antibody and PI dye for flow cytometry. Representative images are shown on the left and the analysis of apoptotic population under different treatment conditions are on the right. (B) Effect of SIK2 knockout on carboplatin-induced apoptosis. SIK2 knockout (KO) and control cell lines were treated as in (A) and analyzed for apoptosis. The bars indicate the mean percentage of apoptotic cells with standard deviations. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. ns: not significant by one-way ANOVA analysis.

Figure 3

Treatment with ARN-3261 enhances the carboplatin-induced decrease in survivin expression. OC316, OVCAR8, and OVCAR8 SIK2 KO ovarian cancer cells were treated with diluent, ARN-3261 (OC316 3 µM and OVCAR8 5.0 µM), carboplatin (OC316 15 µM and OVCAR8 60 µM) and the combination for 48 h. Cell lysates were collected and survivin expression was measured by western blot analysis. The experiments were performed three times individually. Densitometry values were determined by Image J shareware (NIH) and normalized to the GAPDH loading control. The values relative to the untreated group were plotted at the bottom. Different treatments were compared by one-way ANOVA analysis. * p < 0.05 and ** p < 0.01 compared to untreated control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the combination treatment of ARN-3261 and carboplatin.

Figure 4

ARN-3261 enhances carboplatin-induced DNA damage. (A) OC316, OVCAR8 and SKOv3 ovarian cancer cells were treated with diluent, ARN-3261 (OC316 3 µM, OVCAR8 3.0 µM and SKOv3 3.5 µM), carboplatin (OC316 15 µM, OVCAR8 35 µM and SKOv3 35 µM) or the combination for 8 h and stained for γ-H2AX in green and for DNA with DAPI in blue. Each plot depicts the mean number of punctae (the bars indicate the standard deviation). (B) Cells were treated as described in (A) for 24 h. Then cells were dislodged, immobilized in agarose gel onto glass slide, and lysed. DNA was eletrophoresed in alkaline buffer and stained by Vista Green. Olive tail moment (OTL) was measured as described in the Methods. Each plot depicts the mean of OTL (the bars indicate the standard deviation). Statistical significance by one-way ANOVA is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 5

Treatment with ARN-3261 enhances carboplatin toxicity in cisplatin-sensitive and cisplatin-resistant sublines. (A) A cisplatin-resistant ovarian cancer cell subline is also resistant to carboplatin. Cisplatin-resistant A2780-CP20 and cisplatin-sensitive A27801 PAR sublines were plated at a density of 2000/well in 96-well plates, then treated with different concentrations of carboplatin for 96 h as indicated. Cell viability was measured with a bioluminescence assay and the IC50 was calculated. (B) Growth of both cisplatin-resistant and cisplatin-sensitive sublines are inhibited by ARN-3261 as indicated. Cells were similarly cultured and treated for 96 h with different concentrations of ARN-3261, before measuring cell viability and calculating IC50. (C,D). The interaction of ARN-3261 and carboplatin in cisplatin sensitive (C) and cisplatin resistant cell lines was evaluated by the combination index at 50% growth inhibition using CalcuSyn software.

Figure 6

ARN-3261 enhances the activity of carboplatin in human ovarian cancer cell xenografts. (A) Design of xenograft experiments (n = 10/group); (B) the combination of ARN-3261 and carboplatin inhibits tumor growth in an OVCAR8 i.p. model. After treatment as indicated in (A) for three weeks, mice were weighed, and intraperitoneal nodules were excised and weighed. (C) Design of xenograft experiments (n = 10/groups); (D) The combination of ARN-3261 and primary chemotherapeutic drugs of carboplatin and paclitaxel inhibits tumor growth in an SKOv3 subcutaneous xenograft model. Mice were treated with single, double or triple agents for 6 weeks. Tumor was measured once a week until the tumor burden in control group reached maximum allowance. The graphs indicate the mean ± standard deviation. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.0001.