Abstract 3498: Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells

Abstract

Abstract Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas, although its clinical efficacy was not satisfactory, due possibly to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive anaplastic large cell lymphoma (ALCL). We treated ALK-positive ALCL cell lines, K-299 and SU-DHL-1, with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (0.5 nM, 1.0 nM, and 2.0 nM for everolimus, and 20 nM, 40 nM, and 80 nM for crizotinib, respectively). After 72 hours, the combination index (CI) values were less than 1 (0.596, 0.583, and 0.763 in K-299 cells, and 0.326, 0.616, and 0.271 in SU-DHL-1 cells, respectively) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. Western blot analysis demonstrated that everolimus up-regulated phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited mTOR Ser2448 phosphorylation, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased mTOR Ser2481 phosphorylation, a marker for mTORC2, as well. Cell-cycle analysis showed that the combination treatment induced G1 arrest. PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib. In summary, everolimus in combination with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis. Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL. Citation Format: Ji-Won Kim, Wendan Xu, Sung-Soo Yoon. Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2015-3498