Tivantinib, A c-Met Inhibitor in Clinical Trials, Is Susceptible to ABCG2-Mediated Drug Resistance

Abstract

Tivantinib, also known as ARQ-197, is a potent non-ATP competitive selective c-Met inhibitor currently under phase 3 clinical trial evaluation for liver and lung cancers. In this study, we explored factors that may lead to tivantinib resistance, especially in regards to its interaction with ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is one of the most important members of the ATP-binding cassette (ABC) transporter family, a group of membrane proteins that play a critical role in mediating multidrug resistance (MDR) in a variety of cancers, including those of the liver and lung. Tivantinib received a high score in docking analysis, indicating a strong interaction between tivantinib and ABCG2, and an ATPase assay indicated that tivantinib stimulated ABCG2 ATPase activity in a concentration-dependent manner. An MTT assay showed that ABCG2 overexpression significantly desensitized both the cancer cells and ABCG2 transfected-HEK293 cells to tivantinib and that this drug resistance can be reversed by ABCG2 inhibitors. Furthermore, tivantinib upregulated the protein expression of ABCG2 without altering the cell surface localization of ABCG2, leading to increased resistance to substrate drugs, such as mitoxantrone. Altogether, these data demonstrate that tivantinib is a substrate of ABCG2, and, therefore, ABCG2 overexpression may decrease its therapeutic effect. Our study provides evidence that the overexpression of ABCG2 should be monitored in clinical settings as an important risk factor for tivantinib drug resistance.

Keywords: ABCG2; ARQ-197; ATP-binding cassette (ABC) transporter; drug transport; multidrug resistance (MDR); tivantinib.

Figure 1

Chemical structure and cytotoxicity of tivantinib in parental and ATP-binding cassette super-family G member 2 (ABCG2)-overexpressing cells. (A) Chemical structure of tivantinib; (B) cell viability curves for NCI-H460 and NCI-H460/MX20 cells; (C) cell viability curves for S1 and S1-M1-80 cells; (D) cell viability curves for HEK293/pcDNA3.1, HEK293/ABCG2-WT, HEK293/ABCG2-R482G, and HEK293/ABCG2-R482T cells. Data are expressed as the mean ± SD from a representative of three independent experiments.

Figure 2

Effect of tivantinib on the ATPase activity of ABCG2 and accumulation of [³H]-mitoxantrone. (A) Tivantinib stimulates the ATPase activity of the ABCG2 transporter; (B) The effect of tivantinib on the intracellular accumulation of [³H]-mitoxantrone in NCI-H460 and NCI-H460/MX20 cells after 2 h treatment. Data are expressed as the mean ± SD from a representative of three independent experiments. * p < 0.05, compared with control group.

Figure 3

Tivantinib attenuated the cytotoxicity of mitoxantrone in parental and ABCG2-overexpressing cells. (A) IC₅₀ values of mitoxantrone in parental NCI-H460 and drug-selected ABCG2-overexpressing NCI-H460/MX20 cells with or without treatment of tivantinib; (B) IC₅₀ values of mitoxantrone in parental HEK293/pcDNA3.1 and transfected ABCG2-overexpressing HEK293/ABCG2-WT, HEK293/ABCG2-R482G, and HEK293/ABCG2-R482T cells with or without treatment of tivantinib. Data are expressed as the mean ± SD from a representative of three independent experiments. * p < 0.05, compared with the control group.

Figure 4

Tivantinib increases the protein expression in cells overexpressing ABCG2 without affecting the ability of ABCG2 to localize on the cell surface. (A) The effect of tivantinib on the protein expression of ABCG2 in NCI-H460/MX20 cells treated with 0.3 μM of tivantinib for 0, 24, 48, and 72 h. (B) The effect of tivantinib on the protein expression of ABCG2 in HEK/293-ABCG2-WT cells treated with 0.3 μM of tivantinib for 0, 24, 48, and 72 h. (C) Cell surface localization of ABCG2 expression in NCI-H460/MX20 cells incubated with 0.3 μM of tivantinib for 0, 24, 48, and 72 h. Data are expressed as the mean ± SD from a representative of three independent experiments. * p < 0.05, compared with the control group. scale bar: 200 μm.

Figure 5

The effect of tivantinib on the accumulation of [³H]-mitoxantrone after 72 h treatment. (A) The effect of tivantinib on the intracellular accumulation of [³H]-mitoxantrone in NCI-H460 and NCI-H460/MX20 cells after 72 h treatment. (B) The relative percentage of [³H]-mitoxantrone in the medium after 72 h tivantinib treatment in NCI-H460 and NCI-H460/MX20 cells. The percentages were calculated as the treatment groups divided by the parental control group. Data are expressed as the mean ± SD from a representative of three independent experiments. * p < 0.05, compared with the control group.

Figure 6

The binding mode of tivantinib to the human ABCG2 model, as predicted by induced-fit docking. (A) A 2D schematic diagram of the ligand–receptor interaction between tivantinib and the human ABCG2 model. Amino acids within 4 Å are indicated as colored bubbles, polar residues are depicted in blue, and hydrophobic residues are depicted in green. Purple arrows denote H-bonds and green lines denote π–π stacking aromatic interactions. (B) The docked conformation of tivantinib (ball and rod model) is shown within the ABCG2 drug-binding cavity, with the atoms colored as follows: carbon, yellow; hydrogen, white; oxygen, red; nitrogen, blue. Important amino acid residues are described (rods model) with the same color scheme as above for all atoms but with carbon atoms in gray. Dotted green lines represent hydrogen-bonding interactions, while dotted azure lines represent π–π stacking interactions. The values of the correlation distances are indicated in Å.