Polo-like kinase 1 as a therapeutic target for malignant peripheral nerve sheath tumors (MPNST) and schwannomas

Abstract

Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are two dominantly inherited disorders that cause tumors in Schwann cells. NF1 patients have a high risk for malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. NF2 patients have a high risk for schwannomas. To identify potential therapeutic targets in these two tumors, we screened the NF1 MPNST cell line, ST88-14, and the NF2 schwannoma cell line, HEI-193, against ~2000 drugs of known mechanisms of action (including ~600 cancer relevant drugs), and also screened the cell lines against an siRNA library targeting most protein kinases. Both the drug screen and the siRNA screen identified Polo-like kinase 1 (PLK1) among the most potent hits in both cell lines. Since PLK1 acts on the cell cycle primarily at the G2/M transition, the same stage where aurora kinase (AURKA) acts, we explored PLK1 and its relationship to aurora kinase in MPNST. Quantitative profiling of PLK1 inhibitors against a panel of 10 neurofibromatosis cell lines found that they were potent inhibitors and, unlike AURKA inhibitors, were not more selective for NF1 over NF2 tumor cells. Furthermore, one PLK1 inhibitor, BI6727 stabilized tumor volume in MPNST xenografts. We conclude that PLK1 is a therapeutic target for MPNSTs and schwannomas, but inhibitors may have a narrow therapeutic index that limits their use as a single agent.

Keywords: HTS; heat map; high throughput screen; siRNA screen; signal transduction; synthetic lethal; von Recklinghausen disease; xenograft.

Figure 1

PLK1 inhibitors identified in a 2240 compound screen in NF1 (ST88-14) and NF2 (HEI-193) cells. (A) NF1 (left) and NF2 (right) cell line Z score distribution, Z scores higher than μ+3σ are presented in (B). μ, mean of Z scores; σ, standard deviation of Z scores. Arrows indicate where most PLK1 drugs ranked. (B) Category distribution of the top-ranked inhibitors according to a cut-off value of μ+3σ. Left, NF1; right, NF2.

Figure 2

siRNA screen of NF1 (ST88-14) and NF2 (HEI-193) cells. Cell Viability of siRNA transfected cells was measured by counting Hoechst stained nuclei 72 hours post-transfection. (A and B) Z-score plots of siRNA knockdown in ST88-14 (A) and HEI-193 (B). The relative ranking of PLK1, AURKA, and AURKB kinases are indicated. (C) Representative images of nuclei from cells transfected with NegC and PLK1 siRNA. Right side graphs demonstrate the number of nuclei counted between NegC and PLK1 siRNA transfections.

Figure 3

Comparison of NF1 and NF2 screens. Comparison of siRNA activity in independent replicates. (A) NF1 vs. NF1, (B) NF2 vs. NF2 and (C) NF1 vs. NF2. (D) Spearman’s correlation coefficients for siRNA screens.

Figure 4

PLK1 and AURK expression is increased in MPNST. Expression levels were determined from microarrays. A. Human Nerves, dermal neurofibromas (dNF), peripheral neurofibromas (pNF) and MPNST. B. Mouse nerves neurofibromas and MPNST. C. Western blot of cells used in this study compared with human peripheral Schwann cells (HPSC). A lane was deleted from the gel, due to a loading error.

Figure 5

High throughput profiling of PLK1, AURKA and MEK1/2 inhibitors. (A) Eight concentrations of each drug were tested, and the data are expressed as a heat map of the IC₅₀s. (B) IC₅₀s of selected drugs were replotted. For this analysis only human cell lines were included that were derived from NF1 and NF2 patients. (C) The cell cycle kinases profiled against neurofibromatosis cells in (A). (D) Flow cytometry analysis of the effect of PLK1 inhibitor BI6727 and the Aurora Kinase inhibitor, MLN8237 using propidium iodide DNA staining. Cells were treated for 24 hours. The histogram shows the percentage of cells in different phases of the cell cycle. (E) ST8814 cells were synchronized by thymidine (2.5 mM, 24 h) treatment followed by washed with PBS and incubated for 18 h in fresh medium supplemented with nocodazole (1 μg/mL) to arrest cells in mitosis. Cells were treated with 10 μmol/L of BI6727 and 10 μmol/L of GSK461634 for 24 h and then harvested. A western blot was probed for p-PLK1, total PLK1, and GAPDH.

Figure 6

Xenografts with the PLK1 inhibitor BI6727. A. Mice treated with BI6727 at 50 mg/kg. B. Dose escalation study of BI6727.

Figure 7

Histology of tumors from treated mice. A. Mice were treated for 5 days and stained as indicated. B. Mice were treated for 33 days and stained as indicated. Data were analyzed using multiple t-tests.