Cenp-E inhibitor GSK923295: Novel synthetic route and use as a tool to generate aneuploidy

Abstract

Aneuploidy is a common feature of cancer, with human solid tumour cells typically harbouring abnormal chromosome complements. The aneuploidy observed in cancer is often caused by a chromosome instability phenotype, resulting in genomic heterogeneity. However, the role aneuploidy and chromosome instability play in tumour evolution and chemotherapy response remains poorly understood. In some contexts, aneuploidy has oncogenic effects, whereas in others it is anti-proliferative and tumour-suppressive. Dissecting fully the role aneuploidy plays in tumourigenesis requires tools and facile assays that allow chromosome missegregation to be induced experimentally in cells that are otherwise diploid and chromosomally stable. Here, we describe a chemical biology approach that induces low-level aneuploidy across a large population of cells. Specifically, cells are first exposed to GSK923295, an inhibitor targeting the mitotic kinesin Cenp-E; while the majority of chromosomes align at the cell's equator, a small number cluster near the spindle poles. By then driving these cells into anaphase using AZ3146, an inhibitor targeting the spindle checkpoint kinase Mps1, the polar chromosomes are missegregated. This results in, on average, two chromosome missegregation events per division, and avoids trapping chromosomes in the spindle midzone, which could otherwise lead to DNA damage. We also describe an efficient route for the synthesis of GSK923295 that employs a novel enzymatic resolution. Together, the approaches described here open up new opportunities for studying cellular responses to aneuploidy.

Keywords: Cenp-E; Chromosome Section; Mps1; aneuploidy; chromosome instability; spindle assembly checkpoint.

Figure 1. Resolution of racemic mixture to synthesise GSK923295

The desired (S)-enantiomer (2) was obtained by the resolution of the racemic mixture of 1-(2-amino-3-pyridinyl)ethanol (rac-2), through the reaction with S-ethyl thiohexanoate at 39°C with Candida Antarctica Lipase B enzyme (50% w/w). The pure enantiomer (2) and the intermediate (1) then reacted to give the pyridyl imidazole (5), with subsequent reactions yielding GSK923295 (6).

Figure 2. GSK923295 induces chromosome misalignment

A. Immunofluorescence images of DLD-1 cells treated with 50 nM GSK923295 for four hours, fixed and stained for DNA (blue), Bub1 (green) and tubulin (red), with insets showing unaligned chromosome(s). Bar: 5 μm or 2.5 μm for increased magnification. B. A bar chart quantifying the number of cells in each mitotic phase following exposure of HeLa cells to GSK923295 at various concentrations. C. Quantification of the number of HeLa cells in each mitotic phase over a time course, following 50 nM GSK923295 treatment. D. The number of DLD-1 cells in each stage of mitosis over time after 50 nM GSK923295 treatment. E. Treatment of HeLa cells with 50 nM GSK923295, and classification of 100 mitotic cells after no washout (control), or at various times following wash-out. F. Time lapse sequences of control and 50 nM Cenp-E inhibitor treated HeLa GFP Histone-H2B cells. Time zero represents nuclear envelope break down. Bar: 5 μm.

Figure 3. GSK923295 causes cell death

A. DNA content histograms of HeLa cells treated with 50 nM GSK923295, over a time course. B. Line graph showing percent confluency and caspase 3/7 activation of RKO cells treated with varying Cenp-E inhibitor concentrations. C. Fate profiles for untreated and Cenp-E inhibitor treated RKO cells. One bar represents one cell.

Figure 4. Sequential Cenp-E and Mps1 inhibition generates aneuploid daughter cells

A. Time lapse sequences of DLD-1 histone-H2B-mCherry cells treated with the Mps1 inhibitor alone or Cenp-E inhibitor treated cells, washed into the Mps1 inhibitor. Arrows indicate mitotic defects: unaligned chromosomes/lagging chromosomes. B. Quantification of the number of anaphases in HCT-116 cells with unaligned chromosomes after treatment with Cenp-E inhibitor alone or Cenp-E inhibitor and Mps1 inhibitor combined. C. DNA content histograms of Cenp-E and Cenp-E and Mps1 inhibitors treated cells. D. FISH analysis labelling chromosomes 6 and 7 in control and Cenp-E and Mps1 inhibitor treated cells. Bar: 10 μm. E. Bar chart quantifying the number of pairs of daughter cells with 3+1 foci under control, monastrol washout (100 μM), and Cenp-E and Mps1 inhibitor treated conditions.