Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines

Abstract

Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC(50) <1 micromol/L (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines, with a correlation between sensitivity and raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies, including a small number of genes in which differential expression was common to both histologies. In total, these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations, respectively.

Figure 1. In vitro sensitivity to selumetinib

(A) 31 cell lines with IC₅₀ represented in µM. Error bars indicate the standard error based on multiple experiments. The checkered bars represent cell lines with raf mutations. (B) 43 cell lines with IC₅₀ represented in µM. Error bars indicate the standard error based on multiple experiments. The black bars represent cell lines with ras mutations.

Figure 2. Effects of selumetinib on cell cycle

(A) Sensitive cell lines show marked G0/G1 arrest after incubation with 1µM selumetinib for 48 hours. (B) Resistant cell lines do not show significant G0/G1 arrest after incubation with 1µM selumetinib for 48 hours. Black bars- control; White bars- treated. Error bars represent standard error for two separate experiments.

Figure 3. Western blots evaluating PI3K and MEK pathway activation in ras mutant cell lines in response to selumetinib

A) Immunoblot evaluation for sensitive cell lines (IC₅₀ < 1µM) with ras mutations. B) Immunoblot evaluation for resistant cell lines (IC50 > 1µM) with ras mutations. Measured pERK 1/2 (Thr²⁰²/Tyr²⁰⁴), total ERK1/ERK2, pAKT (Ser⁴⁷³/Ser³⁰⁸), and total AKT at baseline (−) and after 30 minutes of treatment with 1µM of selumetinib (+) are shown.

Figure 4. Western blots evaluating PI3K and MEK pathway activation in ras wildtype cell lines in response to selumetinib

A) Immunoblot evaluation for sensitive cell lines (IC₅₀ < 1µM) with wildtype ras. B) Immunoblot evaluation for resistant cell lines (IC50 > 1µM) with wildtype ras. Measured pERK 1/2 (Thr²⁰²/Tyr²⁰⁴), total ERK1/ERK2, pAKT (Ser⁴⁷³/Ser³⁰⁸), and total AKT at baseline (−) and after 30 minutes of treatment with 1µM of selumetinib (+) are shown.

Figure 5. Heat map results from microarray analyses

(A) ANOVA analysis demonstrates differentially 206 differentially expressed genes among the breast cancer panel. (B) The region of the heat map including PIK3R3, the only gene differentially expressed when multiple test correction is applied, is shown. (C) The heat map of the NSCLC panel is shown, with the sensitive cell lines denoted by a rectangle and arrow.