Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma

Abstract

RET fusions have been found in lung adenocarcinoma, of which KIF5B-RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B-RET expression. By culturing KIF5B-RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RET^V804L mutation and vandetanib-resistant-RET^G810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RET^G810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET-associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B-RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RET^G810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521-9. ©2016 AACR.

Figure 1

Derivation and analysis of tetO-KIF5B-RET transgenic mice. A, schematic representation of tetO-KIF5B-RET transgene. B, scheme for production of C/KR bitransgenic mice and induction of the KIF5B-RET transgene expression in the mouse lung type II epithelial cells. C, RT-PCR analysis of KIF5B-RET mRNA expression in various tissues in monotransgenic tetO-KIF5B-RET mouse and in C/KR bitransgenic mouse fed with Dox diet for 1 month. D, after induction with Dox diet for 1 month, cell lysates were prepared from lung tissues of CCSP-rtTA/tetO-KIF5B-RET (C/KR) bitransgenic or wildtype (W) mice. Immunoprecipitation-immunoblotting analysis of KR expression and phosphorylation was performed.

Figure 2

Dox-induced bitransgenic C/KR mice develop lung tumors. A, H&E stained sections showing C/KR bitransgenic mice developed areas of hyperplastic lesions and early tumors after one month Dox induction. B, lung sections of a C/KR mouse induced with Dox for 4 months. Left, an H&E stained lung section showing lung with desmoplastic tumors. Right, tumor cells were stained positive of nuclear TTF1. C, lung sections of a C/KR mouse induced with Dox for 5 months. Right, H&E stained lung section with extensive tumors and desmoplasia. Right, cytokeratin stain confirmed invasive lung tumor.

Figure 3

KIF5B-RET expression is required to maintain lung tumors. A, C/KR mice developed MRI-detectable lung adenocarcinoma after Dox induction for 5 months. MRI images of representative tumor bearing mouse prior to and one month after Dox withdrawal. The graph shows comparison of MRI-detectable lung tumor burden prior to and post Dox withdrawal. Each line represents an individual mouse (n = 7). VOI, Volume of interest. B, H&E stained tissue section from lung of a mouse after Dox withdrawal (left). Lung tissue image from Genie® v1 histology pattern recognition software (Aperio) analysis (middle). Lung tumor burden was analyzed from H&E stained lung tissues (right) (n = 5 in each group). AOI, areas of interest. White, not lung tissue, area excluded; pink, background; blue, normal; purple: hyperplasia/neoplasia including some areas of normal bronchial epithelia (21). C, comparison of pRET and RET protein in the lungs of C/KR mice induced with Dox (prior to Dox withdrawal) and 1 month post Dox withdrawal. W, wildtype mouse.

Figure 4

Ponatinib is the most potent KIF5B-RET inhibitors and the vandetanib resistant RET^G810A mutation is hypersensitive to ponatinib. A, growth curve of parental, vector control (B/V) and stable BaF3 cells expressing KIF5B-RET (B/KR) without IL-3. B, cells were grown in medium without IL-3 for 6, 16, 24 hours. The cell lysates were immunoblotted with anti-cleaved PARP or anti-β actin. P, parental; V, vector control; KR, KIF5B-RET. C, vector control and KIF5B-RET cells were treated with or without 1 µM cabozantinib (CBT) for 3 hours. The cell lysates were immunoblotted with anti-RET-pY905, anti-Flag, anti-cleaved PARP or anti-beta actin. D, Ba/F3 expressing KIF5B-RET, KIF5B-RET^V804L, or KIF5B-RET^G810A were seeded on 96-well plates and treated with indicated concentration of ponatinib (PNT), cabozantinib (CBT), vandetanib (VDT) or lenvatinib (LVT) for 5 days. Cell viability was measured using the CellTiter-Glo assay and IC_50s were determined. The data were from two triplicate experiments (n = 6).

Figure 5

Ponatinib treatment results in tumor regression. A, C/KR mice with MRI-detectable lung tumors were treated with vehicle (upper panel) or ponatinib (30 mg/kg/day, 5 days/week, p.o., lower panel) for 1 month and then analyzed by MRI again (n = 4 in each group). Lung tissue sections at the end point were stained with H&E. Representative images of MRI, H&E sections, and image from Genie® v1 histology pattern recognition software analysis (right) are shown. B, comparison of MRI-detectable lung tumor burden prior to and post vehicle or ponatinib treatment. Each line represents an individual mouse. Blue, vehicle-treated; Black, ponatinib-treated. C, lung tumor burden analyzed from samples of H&E stained lung tissue sections. V, vehicle-treated; P, ponatinib-treated. D, comparison of pRET, RET and β-actin proteins in the lungs of CCSP and C/KR mice treated with vehicle or ponatinib. CCSP, negative control mouse.