P50 - Impact of lenvatinib, mitotane and radiotherapy on adrenocortical carcinoma cells: an in vitro analysis

Background Adrenocortical carcinoma (ACC) is a rare, therapy-resistant malignancy with limited treatment options. Considering the role of radiotherapy in managing metastatic disease, as well as associated complications, it is desirable to investigate the impact of irradiation on ACC. Therefore, it is our aim to characterise the effects of radiotherapy, the tyrosine kinase inhibitor lenvatinib and the adrenolytic drug mitotane on ACC cell lines. Purpose The objective of this in vitro study is to identify novel therapeutic options for advanced ACC by assessing the potential additive effects of lenvatinib or mitotane in combination with radiation on ACC cell lines. Methods and preliminary Results Lenvatinib or mitotane were applied to the ACC cell lines NCI-H295R and JIL-2266 at ascending doses. After 72h, cell viability was measured using the CellTiter-Glo assay, and IC50-values were calculated. JIL-2266- cells were found to be less sensitive to mitotane, but susceptible to lenvatinib. In contrast, NCI-H295R-cells displayed increased sensitivity to mitotane and reasonable sensitivity to lenvatinib. Additionally, the cells were irradiated at 0, 2 and 5 Gy, and then fixed 30 minutes, eight hours and 24h after treatment. Afterwards cell cycle and yH2AX, a marker for DNA damage, was measured via flow cytometry. Within 24h of being exposed to the radiation, both cell lines arrested in the G2-phase. However, the NCI-H295R-cells exhibited a slower response in terms of DNA repair following irradiation, as yH2AX increased until the 24h timepoint. To detect apoptosis, the cells were treated with lenvatinib or mitotane and harvested 96h after treatment and analysed using flow cytometry after annexin-V/PI staining. Mitotane did not initiate apoptosis in JIL-2266-cells, whereas lenvatinib did. NCI-H295R-cells exhibited an inverse reaction, initiating apoptosis after mitotane treatment but not lenvatinib. Furthermore, colony formation assays were conducted on irradiated cells (0-8 Gy). Clonogenicity was measured 3-4 weeks later. The JIL-2266-cells were highly sensitive at lower and the NCI-H295R-cells at higher radiation doses. Conclusion(s) Radiation impacts clonogenicity of both cell lines in different degrees. Mitotane is effective in the NCI-H295R cell line and lenvatinib in the JIL-2266 cell line. In the future we will characterise these differences, as well as possible additive effects.