Drug resistance profiles of mutations in the RET kinase domain

Abstract

Background and purpose: Alterations in the tyrosine kinase enzyme RET are found in thyroid and lung cancer. While RET TK inhibitors (TKIs) are used to treat thyroid cancer and are in clinical trials for RET fusion-positive non-small cell lung cancer, the impact of mutations in the RET kinase domain on drug sensitivity is largely uncharacterized.

Experimental approach: We identified and analysed mutations in the RET kinase domain that conferred resistance to the TKIs cabozantinib, lenvatinib, vandetanib and nintedanib using RET kinase-dependent BaF3/KIF5B-RET (BaF3/KR) cells. We also examined the sensitivity of RET (M918T), a RET mutation prevalent in aggressive multiple endocrine neoplasia type 2B, to these TKIs in the context of BaF3/KR cells.

Key results: Fourteen mutations were analysed. Pan resistance to the four TKIs was found in six RET kinase domain mutations (L730I, V738A, V804L/M, Y806N, G810S). Seven RET kinase domain mutations (L730V, E732K, A807V, G810A, V871I, M918T, F998V) displayed selective resistance to one or more of these drugs. L730I/V and G810A/S had different drug resistance profiles. V871I, M918T and F998V mutations are located at distant sites away from the TKI binding pocket.

Conclusions and implications: A panel of TKI-resistant RET mutations were identified, and their drug sensitivities were cross-profiled. The results provide a reference for selecting appropriate TKIs to inhibit RET kinase domain mutants. Besides changes in the drug-interacting residues, mutations at distant sites could exert long-range effects resulting in TKI resistance. Among the four TKIs analysed here, nintedanib remained unaffected by mutations at the three distant sites.

Figure 1

Dose‐dependent inhibition of BaF3 cells expressing KIF5B‐RET and its RET kinase domain mutants. Left panels, BaF3/KR and indicated BaF3/KRmut cells were treated with (A) cabozantinib, (B) lenvatinib or (C) vandetanib for 3 days, and the relative numbers of viable cells were determined. Right panels, cells were treated with indicated concentrations of drugs for 4 h, and cell lysates were analysed by immunoblotting with the indicated antibodies. CBT, cabozantinib; LVT, lenvatinib; VDT, vandetanib; WT, wild‐type.

Figure 2

Analysis of effects of nintedanib in BaF3, BaF3/KR and BaF3/KRmut cells. (A) The IL‐3‐dependent BaF3 cells were cultured in medium containing IL‐3, while the IL‐3‐independent BaF3/KR cells were cultured in IL‐3‐free medium. These cells were treated with various concentrations of nintedanib for 3 days, and relative numbers of viable cells were determined. (B) BaF3/KR cells were treated with the indicated concentrations of nintedanib for 4 h, and cell lysates were analysed by immunoblotting with the indicated antibodies. (C) Comparison of the inhibitory activities of nintedanib on BaF3/KR, BaF3/KR (L730V) and BaF3/(L730V/V804M) cells. (D and E) BaF3/KR and the indicated BaF3/KRmut cells were treated with nintedanib for 4 h at the indicated concentrations, and cell lysates were analysed by immunoblotting with the indicated antibodies. NTD, nintedanib.

Figure 3

Structural analysis of the drug binding site in RET kinase. The crystal structure of 2iuv (Knowles et al., 2006) was used to make the images. The N‐lobe is at the top. The C‐lobe is at the bottom. (A) C‐α trace of the TK domain of RET is shown in grey. Vandetanib is shown in red as sticks. The wild‐type side chains at the mutated sites are shown as sticks. Site 810 is a glycine (Gly). Mutation sites in the vandetanib binding pocket are shown in green, whereas distant mutation sites are shown in cyan. The Gly‐rich loop is coloured magenta. The activation loop is coloured dark green. (B) Close up of the drug binding site. Vandetanib is shown as sticks and spheres with the carbon atoms coloured magenta, oxygen atoms coloured red, nitrogen atoms coloured blue, the fluorine atom coloured cyan and bromine atom coloured brown. N‐lobe (residues 713–805) is coloured grey, the C‐lobe (residues 812–1013) is coloured cyan and the intervening hinge between the two domains is coloured orange.

Figure 4

Comparison of apoptosis in BaF3/KR and BaF3/KR (E732K) induced by cabozantinib, lenvatinib, vandetanib and nintedanib. (A) Cells were treated with the indicated TKIs for 24 h, and cell lysates were analysed by immunoblotting with antibodies to cleaved PARP or β‐actin. (B) Cells were treated with vehicle, cabozantinib or lenvatinib for 48 h, stained with annexin V/propidium iodide and analysed by flow cytometry (n = 6). WT, wild‐type.