A screening-based approach identifies cell cycle regulators AURKA, CHK1 and PLK1 as targetable regulators of chondrosarcoma cell survival

Abstract

Chondrosarcomas are malignant cartilage tumors that are relatively resistant towards conventional therapeutic approaches. Kinase inhibitors have been investigated and shown successful for several different cancer types. In this study we aimed at identifying kinase inhibitors that inhibit the survival of chondrosarcoma cells and thereby serve as new potential therapeutic strategies to treat chondrosarcoma patients. An siRNA screen targeting 779 different kinases was conducted in JJ012 chondrosarcoma cells in parallel with a compound screen consisting of 273 kinase inhibitors in JJ012, SW1353 and CH2879 chondrosarcoma cell lines. AURKA, CHK1 and PLK1 were identified as most promising targets and validated further in a more comprehensive panel of chondrosarcoma cell lines. Dose response curves were performed using tyrosine kinase inhibitors: MK-5108 (AURKA), LY2603618 (CHK1) and Volasertib (PLK1) using viability assays and cell cycle analysis. Apoptosis was measured at 24 h after treatment using a caspase 3/7 assay. Finally, chondrosarcoma patient samples (N = =34) were used to examine the correlation between AURKA, CHK1 and PLK1 RNA expression and documented patient survival. Dose dependent decreases in viability were observed in chondrosarcoma cell lines after treatment with MK-5108, LY2603618 and volasertib, with cell lines showing highest sensitivity to PLK1 inhibition. In addition increased sensitivity to conventional chemotherapy was observed after CHK1 inhibition in a subset of the cell lines. Interestingly, whereas AURKA and CHK1 were both expressed in chondrosarcoma patient samples, PLK1 expression was found to be low compared to normal cartilage. Analysis of patient samples revealed that high CHK1 RNA expression correlated with a worse overall survival. AURKA, CHK1 and PLK1 are identified as important survival genes in chondrosarcoma cell lines. Although further research is needed to validate these findings, inhibiting CHK1 seems to be the most promising potential therapeutic target for patients with chondrosarcoma.

Keywords: AURKA; CHK1; Chondrosarcoma; PLK1; Screen.

Fig. 1

siRNA screen and compound screen identify PLK1, AURKA and CHK1 as potentially important kinases for survival of chondrosarcoma cells. A. Set-up of siRNA screen. Primary screening was performed on 779 SMARTpools targeting kinases and kinase related genes. The secondary screen was performed in JJ012 and CH2879 cells and consisted of 35 SMARTpool siRNAs identified in the primary screen (decreased cell proliferation below 20% compared to mock conditions). Deconvolution consisted of 4 separate siRNAs and the SMARTpool targeting 9 different genes. B. Hoechst area as a percentage to mock for JJ012 cells. Each dot represents one SMARTpool targeting one Kinase or kinase related gene. Duplicates are shown for each gene and only when both screens showed a percentage below 20% it was considered as a hit. C. Kinases that showed cell killing in both JJ012 and CH2879 were selected for deconvolution (AURKA, CHK1, CNKSR1, COPB2, EPHA6, IRAK3, STK39, TRAT1, PLK1). D. Deconvolution results in JJ012 and CH2879 cells showing that AURKA, CHK1, COPB2, CNKSR1 and PLK1 are important for cell survival in both cell lines. E. Compound screen results in JJ012, CH2879 and SW1353 showing 35 hits in common in the top 50 compounds in each cell line. In addition, 8 compounds were found in JJ012 and CH2879, 6 in JJ012 and SW1353 and 2 in CH2879 and SW1353. F. Compounds that were identified in all three or two out of three cell lines were selected and showed that Aurora kinase, Pi3K-mTOR, mTOR, PLK, CDK and multi-target comprised the largest groups. In addition, compounds targeting c-MET, ALK, SRC, SYK, JAK, IKK and CHK were identified.

Fig.. 2

Chondrosarcoma cell lines are sensitive for compounds targeting AURKA, CHK1 and PLK1. A. Dose response curves showing viability measured after 72 h using presto blue viability reagent for 9 chondrosarcoma cell lines targeting AURKA (MK-5108), CHK1 (LY2603618) or PLK1 (volasertib). The top three panels represent the conventional chondrosarcoma cell lines and the bottom panel the rare chondrosarcoma cell lines including three dedifferentiated cell lines (L2975, L3252 and NDCS1) and one mesenchymal chondrosarcoma cell line (MCS-170). Highest sensitivity is observed after inhibition with Volasertib. Experiments were performed in triplicate at least three times. B. RNA expression in chondrosarcoma cell lines for AURKA, CHK1 and PLK1. No correlation between expression and sensitivity for the different inhibitors is observed. C. Excess over Bliss percentages of combination treatment of 100 nM LY2603618 and doxorubicin (DXR) or cisplatin (CDDP) showing that JJ012 and SW1353 can be sensitized to conventional chemotherapy after Chk1 inhibition. D. Western blot showing CHK1 and P-CHK1 (S345) expression after treatment for 2 or 24 h with IC50 concentrations of LY2603618. Gapdh expression is assessed as a loading control. Hela cells treated with Hydroxyurea have been used as a positive control for p-CHK1 expression.

Fig.. 3

Cell cycle and cell death analysis after AURKA, CHK1 or PLK1 inhibition. A. Cell cycle analysis after 24 h of treatment with MK-5108, LY2603618 or Volasertib in JJ012 or CH2879 cells. Both cell lines show a decrease in G1 and an increase in G2 phase after treatment with MK-5108. In addition, CH2879 cells show a decrease in S phase after treatment with MK-5108. JJ012 cells show a decrease in G1 after inhibition with either MK-5108, LY2603618 or Volasertib and an increase in S-phase and debris after LY2603618 treatment. B. Apoptosis induction measured using the caspase-glo 3/7 kit in JJ012, CH2879 and SW1353 after treatment for 24 h with IC₅₀ concentrations of MK5108, LY2603618 or Volasertib. Z-vad was added as a control. Only the positive control showed significant caspase induction in all cell lines. SW1353 showed significant upregulated caspase activity after treatment with MK-5108 and Volasertib compared to dmso treated controls. For both cell cycle and apoptosis experiments mean values are shown of three experiments performed in duplicate. P-values were calculated using a 2way ANOVA test, correcting for multiple comparisons using Tukeys test. C PARP cleavage assessed after 24 h of treatment using IC50 concentrations of MK-5108, LY2603618 and Volasertib. As a positive control CH2879 cells treated with the combination of ABT-737 and doxorubicin has been taken along.

Fig. 4

Gene expression analysis shows that high expression of CHK1 is correlated to a worse survival in chondrosarcoma patients. A.RNA expression of AURKA, CHK1 and PLK1 in chondrosarcoma patient samples and chondrosarcoma cell lines compared to expression in normal articular cartilage samples. Each dot represents one sample. B. Kaplan Meyer analysis of patient samples with low and high expression of AURKA and CHK1. High CHK1 expression shows a significant correlation with a worse overall survival in chondrosarcoma patients. Samples with <1 expression were considered as ‘low’ expression and samples >1 expression were considered as high expression. P values were calculated using a mantel cox log rank test.