Abstract 5502: In vitro characterization of tki resistant renal cell carcinoma after sequential application of cabozantinib

The VEGFR/MET/AXL receptor tyrosine kinase inhibitor, Cabozantinib (Cbz), showed clinical benefits in first- and second-line treatment for selective metastatic renal cell carcinoma (mRCC) patients. We tested the molecular effects of Cbz in treatment-naïve cells as well as after a chronic treatment with Sunitinib (Sun) and Pazopanib (Paz) in vitro. We used Sun-resistant (786-O/S and Caki-2/S), Paz-resistant cell lines (786-O/P, Caki-2/P), and the matched wild type (WT) cells (786-O/WT and Caki-2/WT). Treatment tolerance to Cbz (IC50 method) was tested by WST-1 and the drug’ effect on signaling pathways was evaluated by immunoblotting. The pre-treatment with Sun increased the IC50 by 1.3 fold in the 786-O/S cell lines (13µM), compared to the 786-O/WT cells (10µM, p-value 0.002) but no difference was observed between Caki-2/S and Caki-2/WT cell lines (13µM vs 14µM, singly). In 786-O/WT, Cbz caused a late down-regulation of the activated receptor P-MET (FC:-2.2), the coupled proteins Src and STAT3, and it impaired FAK. Unlike, P-MET was strongly decresed in Caki-2/WT cells (FC:-10.4). We saw a compensatory activation of FAK and mTOR marker S6K during the treatment. Of note, Cbz elicited a MAPK by 1.5 FC in the 786-O/WT and by 2.5 in Caki-2/WT. The Sun-treatment enhanced P-MET only in 786-O/S (FC: 2.6). Cbz caused late inhibition of P-MET in 786-O/S cells, but an early downregulation in Caki-2/S cells. In both Sun-pretreated cells, Cbz impaired MAPK, mainly in 786-O/S. Paz-pretreatment did not change either the cell viability of the 786-O/P cells (IC50 of 10.4µM vs 10.2µM of 786-O/WT), nor that of the Caki-2/P cell lines (IC50 of 16 µM vs 17 µM of the age-matched Caki-2/WT cells). In the Paz-resistant cells, P-MET was up-regulated by a FC of 6 and 2.4 in 786-O/P and Caki-2/P cells, respectively. A strong inhibition of P-MET by Cbz was observed independent on pretreatment. In both Sun-and Paz- resistant cell lines we observed a high activity of the protein complex Src-FAK, following Cbz treatment. While the protein level of MAPK was reduced by Cbz in the Sun- resistant cells, it was boosted in the 786-O/P and Caki-2/P (FC: 2). Only the Sun-chronic treatment sensitized the resistant cells to the inhibition of S6K by Cbz. To conclude, the P-MET-driven state in Sun and Paz resistant cell lines, and in Caki -2/WT cells did not sensitize the cells towards Cbz. The activation of Src-FAK protein axis, after P-MET inhibition, might suggest a synergy that could weaken Cb