Adaptive resistance and efficacy of combined treatment with RET and FGFR inhibitors in a transgenic model of RET - rearranged thyroid cancer. (A) Schematic of tol2 transgene constructed to express CCDC6-RET fusion under control of a zebrafish thyroglobulin promoter (tg). (B) Fluorescence imaging of transgenic larval zebrafish at 7 dpf. Expression of CCDC6-RET (top) compared with wild-type thyroid (bottom). Thyroid cells are visualized in follicles and express TdTomato. ERK activation analyzed by whole-mount in situ fluorescence microscopy, pERK (green) and tERK (blue). Scale bar for bright-field images = 2 mm; scale bar for fluorescence confocal = 100 μm. Four transgenic larvae were imaged, and a representative animal is shown. (C and D) Juvenile zebrafish (28 dpf) with mass protruding from ventral jaw (C) and corresponding histopathologic section (D), H&E stained. (A) Representative tumor-bearing animal is shown from a clutch of 28 tumor-bearing animals; scale bar = 5 mm. (E) High-resolution photomicrograph from RET transgenic zebrafish tumor; scale bar = 100 μm. This image is also used in Fig. S4 H. (F) Immunohistochemical staining for thyroglobulin performed on a RET transgenic zebrafish tumor, scale bar = 100 μm. (G) Tumor-free survival curve comparing transgenic RET zebrafish (red, n = 110) to transgenic BRAF(V600E);p53(M214K); blue, n = 37) zebrafish. (A) log-rank test was used to compare tumor-free survival; **, P < 1 × 10−4. (H) GSEA using a zebrafish RET gene signature (n = 178 genes; Table S1) and examining papillary thyroid cancers (TCGA) stratified by RET-rearranged status in the TCGA cohort. Statistical tests were performed using the GSEA software. (I) Western blot analysis of phospho-RET, phospho-ERK, and phospho-AKT in lysates harvested from tumors exposed to BLU6864 or XL184. (J) Western blot analysis of ERK activation in lysates prepared from zebrafish tumors after treatment with RET inhibitors over time. A representative Western blot is shown from three independent experiments. (K) Comparison of thyroid volume from RET transgenic animals treated with BLU6864, BGJ398, or the combination. Larval zebrafish were treated from 3 to 12 dpf, and thyroid volume was measured using confocal microscopy. The mean and SD are displayed. Each transgenic animal is represented by a symbol. Statistical significance was assessed by Student’s t test; *, P < 0.01; ***, P < 0.001.