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Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer, caused by activating mutations in the RET proto-oncogene, contributing to 13% of all thyroid cancer mortalities. Patients with metastatic MTC treated with RET inhibitors (RETi) can develop resistance through mechanisms currently being investigated. We aim to understand the molecular mechanisms of acquired resistance to RETi and develop novel combination therapies to circumvent resistance to these drugs. We used TT, an MTC cell line, to develop vandetanib-resistant (VanR) lines, at two independent sites, the Medical College of Wisconsin (MCW) and the Pediatric Oncology Branch (POB) at the NCI. We applied a multiomic approach to investigate the mechanisms of resistance, including Spectral karyotyping (SKY) and multiplex interphase Fluorescence in situ Hybridization (miFISH) to study the karyotype, ploidy, and heterogeneity at a single-cell level. Whole exome and RNA-sequencing was done to identify genetic alterations such as acquired mutations, copy number variations, and gene expression changes. In parallel, we performed genome-wide CRISPR gene knockout (KO) in TT cells treated with selpercatinib or vehicle to develop potential synergistic targeted combination therapy with RETi. Additionally, we performed a cytotoxicity screening using the Mechanism Interrogation Plate (MIPE6. 0) library containing 2803 investigational drugs, novel agents, and FDA-approved drugs with known mechanisms of action. SKY and miFISH results showed that, for the parental TTPOB cell line, there were 4 copies of chromosome 10 (location of the RET gene), whereas there were 3 copies in TTPOB VanR. This was associated with an increase in the RET variant allele frequency, indicating a loss of heterozygosity of the wildtype allele as a mechanism of resistance, which was confirmed by whole exome sequencing. For TTMCW VanR, we identified a new RET solvent front (G810S), as well as an increased copy number of RET with an increase in expression. CRISPR KO demonstrated that the knockout of NF1 led to resistance and survival to selpercatinib treatment. The MIPE6. 0 screening confirmed resistance to RETi in TTMCW VanR and sensitivity to multiple MEK inhibitors. In vivo testing in a xenograft mouse model confirmed the synergistic/additive activity of combined selpercatinib with trametinib, a MEK inhibitor. In conclusion, using a multiomic approach, we show that resistance to RETi occurs by an adaptive increase in RET signaling through secondary mutations in RET, and an increase in expression or dose of the mutant RET allele. These genetic events are associated with an activation of RAS signaling, in VanR cell lines, through the ERK/MAPK pathway. Furthermore, we demonstrate that the combination of RET and ERK/MAPK inhibition is synergistic against RET-mutant MTC. Future plans include translating our findings through clinical trials at the NCI. Citation Format: Arwa Fallatah, Abdelrahman Rahmy, Sophia Khan, Hsien-chao Chou, Kerstin Heselmeyer-Haddad, Darawalee Wangsa, David Milewski, Yong Kim, Robert Hawley, Young Song, Xiaohu Zhang, Craig Thomas, Jun S. Wei, Jong-In Park, John Glod, Javed Khan. Combined MEK and RET inhibition overcomes resistance to single agent RET inhibition in medullary thyroid cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1099.
Fallatah et al. (Fri,) studied this question.