PURPOSE Medullary thyroid carcinoma (MTC) is often driven by activating mutations in the RET receptor tyrosine kinase. Multikinase and selective inhibitors targeting RET are highly effective for RET-mutant MTC, but acquired resistance is commonly observed, limiting clinical efficacy. MATERIALS AND METHODS We performed a comprehensive genomic and pharmacological analysis of acquired resistance in a previously described in vitro model of RET-mutant MTC generated from long-term treatment with the RET inhibitor vandetanib. Molecular studies using spectral karyotyping, multiplex interphase fluorescence in situ hybridization, whole-exome sequencing, and RNA sequencing revealed several mechanisms of acquired resistance. Whole-genome CRISPR knockout screening was performed to identify potential genes mediating intrinsic resistance. High-throughput drug screening was used to identify additional therapeutic targets. The combination of RET and MEK inhibitors was evaluated in preclinical animal models. RESULTS Genomic profiling revealed that resistant MTC cells acquired RET copy-number gain and the clinically observed secondary RET mutation p.G810S. Whole-genome CRISPR knockout screening on MTC cells treated with two different RET inhibitors highlighted that NF1 deletion and subsequent RAS/MAPK activation were sufficient to establish resistance to RET inhibition. High-throughput drug screening also indicated that MTC cells are sensitive to RAS/MAPK inhibition, particularly in combination with RET inhibitors. The combination of RET and MEK inhibitors was synergistic in both RET-inhibitor–naïve and resistant MTC in mouse xenograft models. CONCLUSION Resistance to RET inhibitors can be acquired through RET copy-number gain and secondary mutations as well as NF1 loss–mediated MAPK pathway activation. This mechanism of resistance can be overcome with dual inhibition of RET and downstream RAS/MAPK signaling, demonstrating clinical potential in RET-mutant MTC.
Milewski et al. (2026) studied this question.