Abstract 5553: Acquisition of adaptive and reversible drug-tolerance state to cabozantinib in renal cell carcinoma cell lines

Abstract Drug-tolerant (DT) state has been recognized as a transient, adaptive process whereby tumour cells endure therapy with tyrosine kinase inhibitors (TKIs) through non-genetic/epigenetic mechanisms, which might be responsible for driving tumour relapse and therapy resistance. Considering that resistance to the targeted systemic therapies remains challenging in metastatic renal cell carcinoma (mRCC), here we elucidated the DT-state as response to the TKI cabozantinib (CBZ) targeting c-MET/Axl/VEGFR in RCC cell lines.After chronic exposure to CBZ (IC50) for 4 months, the 786-O/DTC, Caki-2/DTC and KTCTL-26/DTC cells were profiled for cell survival (WST-1), proliferation (BrdU), migration/invasion, apoptosis (caspase 3/7) and compared to untreated wild-type (wt) cells. Molecular analyses were carried out via immunoblotting and miRNA expression assays (hs-miRNA microarray and RT-qPCR).The DTC-cell lines were successfully induced. The DT-cell lines maintained viability after long-term therapy with a slow growing state. To investigate the mechanisms that drive DT-state, we compared the DTC and WT cells under CBZ. The DTC-cell lines showed higher proliferation (p<0.001), along with lower caspase 3/7 activity in KTCTL-26/DTC and Caki-2/DTC (p<0.001). CBZ impaired migration and invasion, except in KTCTL26/DTC, which displayed higher invasion (p<0.001). Phospho-TK c-MET was inhibited in DT-cells but not Axl. Yet, c-MET was partially restored in KTCTL-26/DTC. The downstream ERK was unchanged in Caki-2/DTC and KTCTL-26/DTC or boosted in 786-O/DTC. BCL-2 was enhanced in Caki-2/DTC or unaffected in the other two DTC- cell lines hinting at an ability to bypass cell death. The DTC-state was reversible, as the cells recovered cell growth after CBZ-withdrawal implying a dynamic regulation. Among 4 miRNAs, namely hs-miR-551b-3p, hs-miR-34a-5p, hs-miR-424-5p and hs-miR-9-3p that were commonly dysregulated in DTC-cell lines, the hs-miR-424-5p and hs-miR-9-3p (regulation of cell cycle, MAPK signalling and apoptosis) were consensually upregulated in DTC-cells by 2.5-fold change (p=0.001), but their expression was reversed after treatment discontinuation. Remarkably, their regulation matched the changes in We established CBZ-DT cell lines and characterised the related spectrum of molecular changes, while considering tumour heterogeneity crucial to drug-adaptation. We identified previously unrecognised miRNAs associated with DT to emphasise the dynamic epigenetic changes that are critical in the initiation of DT-state. Citation Format: Angela Zaccagnino, Hiresh Ayoubian, Kerstin Junker. Acquisition of adaptive and reversible drug-tolerance state to cabozantinib in renal cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5553.