Lapatinib potentiates cytotoxicity of YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter

Abstract

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.

Figure 1

YM155 and lapatinib show high degree of synergy in neuroblastoma regardless of the MYCN and TRKA status. (A) Overview of the drug library used in the concentration matrix-based combination screen. (B) Chemical structures of lapatinib and YM155. (C) Microarray-based gene expression profiling. Bar graphs are showing the number of significantly upregulated and downregulated genes in each condition. (D) Colony formation assay; YM155 (black border) was used at 1/16 IC50 concentration (15 nM), lapatinib (gray border) at 1/8 IC50 (1 µM). Combination treatment (blue border): 15 nM YM155 and 1 µM lapatinib. DMSO (no border) treated cells were used as a control. Data shown as mean ± s.e.m. and images are representative of three individual experiments (ANOVA, **P < 0.01). (E) Heatmap of CI values in the YM155 and lapatinib combination matrix, indicating a strong synergistic interaction. Data represent the mean of triplicates. (F,G) Synergistic effect is preserved in NTRK1 and MYCN highly expressed state. (F) Left, overexpression and activation of TRKA signaling in SH-SY5Y; right, heatmap of CI values in the combination matrix; data represent the mean of triplicates. (G) Left, overexpression of MYCN in SH-SY5Y and levels of MYCN protein in the IMR5–75 MYCN amplified cell line; right, heatmap of CI values in the combination matrix; data represent the mean of triplicates.

Figure 2

Synergistic effect is a consequence of a blocked efflux of YM155 by lapatinib in vitro. The anti-tumor effect of the combination is confirmed in an in vivo model. (A) Multiple-reaction monitoring assay in the SH-SY5Y neuroblastoma cells. Lapatinib inhibits the ABCB1 transporter and allows higher intracellular concentration of YM155. Area under the curve (AUC) corresponds to intracellular YM155 levels. Cyclosporine, MK-571 and KO143 are inhibitors of ABCB1, ABCC1 and ABCG2 transporters, (respectively). Data are the mean ± s.d. of triplicates. (B) Comparison of mRNA expression profiles of SLC35F2 and ABCB1 available in CCLE between SH-SY5Y and A673 cells. (C) Dose response curves of the effects of YM155 on the viability of SH-SY5Y (green) and A673 cells (red). (D) Heatmap of CI values in the combination matrix of YM155 and lapatinib in A673 cells. Data represent the mean of triplicates. (E) Multiple-reaction monitoring assay in A673 cells. Area under the curve (AUC) corresponds to intracellular YM155 levels. Cyclosporine and KO143 are inhibitors of ABCB1 and ABCG2 transporters, (respectively). Data are the mean ± s.d. of triplicates. (F) Efficiency of ABCB1 knock down after 24 hours. (G) Combined effect of ABCB1 knockdown and YM155 and/or lapatinib treatment on apoptosis of SH-SY5Y cells. Apoptosis was measured in SH-SY5Y cells which were transfected with control siRNA or siRNA against ABCB1 (25 nM); and treated with DMSO, 1 μM lapatinib and/or 15 nM YM155 for 48 hours. Percentages of cell death were used to calculate the fold changes in apoptosis of siRNA transfected and drug treated cells compared to untreated siRNA transfected cells. (Mean ± SEM, n = 3, **P < 0.01, ANOVA). (H) Analysis of neuroblastoma growth in Tg[dβh:MYCN-EGFP] zebrafish, expressing MYCN-EGFP under the control of dopamine-β-hydroxylase promoter. Fish were treated with lapatinib and YM155 (2 µM and 6.5 nM, respectively) for 7 days. Zebrafish were imaged before and after treatment with the same settings. The fluorescent area behind gills corresponding to tumors (white arrow) were measured by using ImageJ (representative images of three independent experiments, n = 3). A quantitation of the results is shown in the graph, where each symbol represents a fish (P = 0.0016, paired t test).