ORF12-06 - Inhibition of mTOR Signaling Suppresses Growth and Downregulates Oxidative Phosphorylation of Lenvatinib Resistant Thyroid Cancer

Background: Lenvatinib is a multiple receptor tyrosine kinase inhibitor frequently employed for treatment of metastatic progressive thyroid cancer (TC). The partial response to lenvatinib treatment is usually transient, resulting in subsequent progressive disease in majority of patients. The goal of this study was to identify therapeutic targets in lenvatinib resistant (LR) TC.

Methods: LR was induced in the lenvatinib-sensitive (LS) TC cell lines by long-term treatment with increasing concentration leading to resistance factor of >100. RNA sequencing was performed to evaluate differential expression of transcriptome of LR vs LS cells. Untargeted metabolomics using mass spectrometry was conducted to compare the metabolic profiles of LR and LS TC. The MIPE6.0 library containing 2800 compounds was utilized for LR growth inhibition screening. The compounds with the maximum delta AUC (area under the curve) were calculated from the difference between AUC of LS and LR cells based on the TC growth curves. Cell proliferation assay was performed to determine the percentage cell viability. Seahorse mitostress assay was done to evaluate OXPHOS in TC cells.

Results: LR cells were characterized by 2175 significantly upregulated and 2193 downregulated genes compared with LS, with ABCB1 transporter, an ATP-dependent efflux pump associated with drug resistance, amongst one of the most enriched genes (fold change 31.4, p< 0.001). Gene ontology analysis revealed a significant enrichment of genes involved in response to oxygen levels (n=46) and to oxidative stress (n=42) in LR as compared with LS clones. Metabolomic data revealed 151 upregulated and 154 downregulated metabolites in LR vs LS cells. Consistent with transcriptome data, pathway enrichment analysis showed metabolites associated with ABC transporters as the most enriched in LR vs LS cells (padj=0.002), followed by serine-glycine-threonine metabolism (padj=0.001) associated with one carbon network enrichment (padj=0.001) crucial for redox balance. In addition, OXPHOS was significantly upregulated in LR compared with LS TC, oxygen consumption rate (OCR) 3999.3 ± 357.8 vs 1774.27 ± 208.2, (p < 0.0001). The high throughput screening studies suggested AZD-8055, a strong mTORC1/2 inhibitor, as an effective agent inhibiting growth of LR clones with a delta AUC between LS and LR cells of 138.46. The functional in vitro Seahorse studies revealed that treatment with AZD-8055 (10 nM, 48 hrs) significantly reduced OXPHOS in the LR TC cells (LR-TC vs LR-TC + AZD-8055, basal OCR: 1014.6±89.5 vs 743±91.6, p=0.04; maximum OCR: 2817.6±202.7 vs 1593.8±157.8, p< 0.001, respectively).

Conclusions: LR TC is characterized by altered metabolism with significantly increased mitochondrial respiration. Targeting mTOR pathway leads to inhibition of growth and mitochondrial respiration of LR TC and can be a promising strategy to overcome resistance.