Mitogen-activated protein kinase (MEK/ERK) inhibition sensitizes cancer cells to centromere-associated protein E inhibition

Abstract

Inhibition of centromere-associated protein-E (CENP-E) has demonstrated preclinical anti-tumor activity in a number of tumor types including neuroblastoma. A potent small molecule inhibitor of the kinesin motor activity of CENP-E has recently been developed (GSK923295). To identify an effective drug combination strategy for GSK923295 in neuroblastoma, we performed a screen of siRNAs targeting a prioritized set of genes that function in therapeutically tractable signaling pathways. We found that siRNAs targeted to extracellular signal-related kinase 1 (ERK1) significantly sensitized neuroblastoma cells to GSK923295-induced growth inhibition (p = 0.01). Inhibition of ERK1 activity using pharmacologic inhibitors of mitogen-activated ERK kinase (MEK1/2) showed significant synergistic growth inhibitory activity when combined with GSK923295 in neuroblastoma, lung, pancreatic and colon carcinoma cell lines. Synergistic growth inhibitory activity of combined MEK/ERK and CENP-E inhibition was a result of increased mitotic arrest and apoptosis. There was a significant correlation between ERK1/2 phosphorylation status in neuroblastoma cell lines and GSK923295 growth inhibitory activity (r = 0.823, p = 0.0006). Consistent with this result we found that lung cancer cell lines harboring RAS mutations, which leads to oncogenic activation of MEK/ERK signaling, were significantly more resistant than cell lines with wild-type RAS to GSK923295-induced growth inhibition (p = 0.047). Here we have identified (MEK/ERK) activity as a potential biomarker of relative GSK923295 sensitivity and have shown the synergistic effect of combinatorial MEK/ERK pathway and CENP-E inhibition across different cancer cell types including neuroblastoma.

Figure 1

A siRNA screen identifies gene targets that sensitize neuroblastoma cells to GSK923295. (a) Relative growth inhibition of target-specific pooled siRNA (75 nM) when combined with GSK923295 (125 nM) in SK-N-FI cells. Relative growth is normalized to the cell index at 72 hr to eliminate the growth inhibitory effects of siRNA treatment alone. Bars represent mean relative viability ± (S.E.M.). (b) Real-time growth curves of SK-N-FI cells treated with pooled siRNAs (75 nM) targeted to ERK1, with intervention/read-out time points noted. Cells were significantly more sensitive to GSK923295 (125 nM) than treatment with non-targeting control (NTC) or GAPDH siRNA (p < 0.0001). (c) SK-N-FI cells treated with a dose range of GSK923295 after transfection with NTC or ERK1 siRNA (75 nM). (d) Relative ERK1 mRNA expression 48 hr after siRNA transfection normalized to β-actin expression.

Figure 2

Pharmacologic MEK/ERK inhibition sensitizes neuroblastoma cells GSK923295. (a) Combined CI-1040 (1.25 μM) and GSK923295 (125 nM) treatment for 72 hr results in significantly more growth inhibition than treatment with either agent alone (p <0.001) Bars represent mean % viability relative to DMSO control ± (S.E.M.). (b) Percentage of cells with sub-G1 (<2N) DNA content 18, 44 and 66 hr after treatment with CI-1040 (1.25 μM) and GSK923295 (125 nM).

Figure 3

Pharmacologic MEK/ERK inhibition significantly increases GSK923295-activity in colon cancer cell lines. (a) The IC₅₀ of colon cancer cell lines with differential oncogenic activation of the RAS/RAF/MEK/ERK pathway 72 hr after treatment with GSK923295 (doses of 3-fold dilutions ranging from 2.05nM–500nM). (b) Dose response curves of GSK923295, GSK1120212 and the two drug combination in RKO cells as measured by Cell-TiterGlo 96 hr after treatment. (c) Caspase-3/7 activity relative to DMSO 24 hr after treatment with indicated doses of drug in RKO cells. Caspase-3/7 bioluminescence intensity is normalized to Cell-TiterGlo bioluminescence intensity to control for cell density. Bars represent mean Caspase 3/7 activity ± (S.E.M.).

Figure 4

MEK/ERK pathway activity is predictive for the relative GSK923295 sensitivity. (a) Western blot analysis of phosphorylated ERK1/ 2 and total ERK1/2 in a panel of neuroblastoma cell lines. (b) Phospho-ERK1/2 intensity relative to total ERK1/2 intensity as measured by densitometry of bands from Western blot in (a). (c) Relative phospho-ERK1/2 intensity versus GSK923295 IC₅₀ values of neuroblastoma cell lines. X-axis shown as Log₂. (d)The GSK923295 IC₅₀ values in lung cancer cell lines with wild-type RAS are on average significantly less than those with mutant RAS (p = 0.047). Markers represent mean ± (S.E.M.).