Synthetic lethality in CCNE1-amplified high grade serous ovarian cancer through combined inhibition of Polo-like kinase 1 and microtubule dynamics

The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.


Genomic profiling using the illumina human HT-12 expression BeadChip V4
Total RNA was isolated using the RNeasy Mini kit (Qiagen) following the manufacturer's instructions. 1 μg RNA was amplified and labeled with an Illumina TotalPrep RNA amplification kit (Life Technologies, Ambion), and hybridized according to the manufacturer's protocol (Illumina GeneExpression Direct Hyb; illumina, San Diego, USA). Arrays were read by an Illumina Bead Chip Reader, and data were normalized by quantile normalization in GenePattern (http:// genepattern. broadinstitute.org/gp/pages/index.jsf). The ratios of gene expression in OVCAR-3 cells treated for 24 h with paclitaxel versus untreated cells were determined, and the Top 30 up-and downregulated kinases were tabulated.

High-throughput siRNA-transfection
The siRNA library targeting 711 human kinases (4 siRNAs/well/gene) was purchased from Dharmacon. OVCAR-3 cells were transfected by the reverse method (simultaneous seeding and siRNA-transfection). Briefly, Lipofectamine RNAiMAX (Invitrogen) was diluted in serum-reduced medium (Opti-MEM, Invitrogen) and plated in white, clear-bottom half-area 96 well-plates (Greiner Bio-One). siRNAs were pre-diluted in siRNA-Dilution buffer (Dharmacon) and added to a final concentration of 20 nM. Plates containing the siRNAlipid mix were spun down (1,000 rpm for 10 seconds) and complexes were allowed to form for 20 min at room temperature. Meanwhile OVCAR-3 cells were trypsinized, collected in fresh medium and counted. Cell suspension (2.100 cells / 40 μL) was prepared in RPMI completed with 12.5% FCS and added to a final volume of 50 μL per well. Each plate included the following controls: Medium alone (background), untreated cells (basal level), 20 nM siGlo Red Transfection Indicator (transfection control, Dharmacon), 20 nM siPLK1 (test efficiency and consistency, Sigma) and 1 μM Campthotecin (apoptosis, cell death and consistency, Enzo Life Sciences). Each plate was analyzed in triplicate.

Phospho-histone H3 (Ser10) staining
Cells were treated with paclitaxel, BI6727 or both, trypsinized, fixed using 4% paraform-aldehyde (PFA, Sigma Aldrich) in PBS containing 0.01% Triton-X-100 and washed with PBS-T before adding a phospho-Histone H3 (Ser10) specific antibody. Staining was performed for 1 h at 37°C. Secondary staining was done for 30 min using a FITC-coupled antibody. Fractions of mitotic cells were quantified using a FACS Calibur and Cellquest Pro software (both BD Biosciences).

Western blot analysis
Protein extracts of cells were prepared by lysis in RIPA buffer (Sigma) supplemented with protease inhibitors (Complete protease inhibitor cocktail, Roche). Protein extracts (25 μg) were separated by SDS-PAGE and transferred onto PVDF membranes using the TransBlot Turbo Transfer System (BioRad). After blocking with 5% BSA in PBS with 0.1% Tween-20 for 30 min, the membrane was incubated with primary antibodies for 1 h at room temperature. HRP-linked secondary antibodies were incubated 30 min at room temperature followed by ECL detection (ECL Chemiluminescent Western Blot Substrate, Pierce).

Membrane potential
OVCAR-3 cells were trypsinized, washed in PBS and centrifuged at 700g for 5 min. The cell pellets were incubated with 10 nM DiOC 6 (3) in fresh medium at 37°C for 30 min in the dark. After incubation, cells were washed twice and resuspended in PBS and assayed using flow cytometry. The fluorescence intensity of DiOC 6 (3) was measured and calculated by BD FACS CellQuest Pro software.

Colony formation assay
Cells were treated with paclitaxel, BI6727 or both overnight followed by seeding predefined numbers in 6-well plates. Colonies were fixed using 70% EtOH and stained with Coomassie Brilliant Blue. The numbers of grown colonies were counted and images were taken using AxioObserver Z1 microscope (Zeiss) as well as the ChemiDoc MP system (BioRad).

Nerve growth factor (NGF)-induced neurite outgrowth assay.
Undifferentiated PC-12 cells were pretreated for 5 days by adding β-NGF (NGF from mouse source) (Promega) to a final concentration of 50 ng/ml, trypsinized, washed using phosphate-buffered saline (PBS), centrifuged and plated in 12-well culture chambers (Costar, Nuclepore). Optimal adherence and neurite formation required precoating the cell culture plastic using collagen I (Gibco), followed by washing with PBS. Differentiated PC-12 cells were co-incubated with paclitaxel, BI6727 or both together with 10 ng/ml β-NGF. Subsequently, cells expressing neuronal like structures were examined using an AxioObserver Z1 microscope (Zeiss). Images were taken using an AxioCam MRm camera and analyzed using ImageJ Fiji.
To discriminate specific neurotoxicity from general cytotoxicity, the analysis was performed on the basis of the fraction of neurite-forming cells instead of the absolute number of cells. The results, therefore, are given as percentage of cells expressing neurites.

Combination index
Measured effects were classified by calculating the combination index as follows: c.i.(Am) 50 / (As) 50 +(Bm) 50 /(Bs) 50 . The variables (Am) 50 /(Bm) 50 typify the concentrations of drug A/B achieving 50% growth inhibition in combinatorial treatment (IC 50 ). (As) 50 /(Bs) 50 symbolize the concentrations of drug A/B necessary to achieve 50% growth inhibition in single administration. A combination index >1 indicates an antagonistic effect, whereas an index =1 displays an additive and an index <1 a synergistic effect [2].