Adaptive resistance in RET fusion thyroid cancer cell line CUTC48; genetic inactivation of FGFR1 abrogates adaptive resistance to RET inhibitors. (A) Western blot analysis of ERK activation and downstream signaling effectors in CUTC48 cells treated with BLU6864 alone or in combination with BGJ398 over the indicated time course. A 24 + 1 timepoint indicates that the BGJ398 was added for 1 h after an initial incubation in BLU6864 for 24 h. CUTC48 cells were obtained from the University of Colorado’s Cell Culture Services Core. (B) Sanger sequencing of individual clones isolated from lentiviral-mediated CRISPR-Cas9 gene-edited TPC1 cells shows genomic deletion of FGFR1 gene in two different FGFR1 knockout clones. (C) Long-term proliferation assay of FGFR1 KO TPC1 cells treated with XL184 or BLU6864 alone or in combination with BGJ398. Cells were treated for 12 d and stained with crystal violet. (D) Quantification of long-term viability of FGFR1 KO TPC1 cells treated with the indicated compounds was performed by calculating colony intensity; mean and SD are displayed for technical replicates from one of three biological replicates. *, P < 0.05; **, P < 0.01, Student’s t test.