Abstract Purpose Anaplastic lymphoma kinase (ALK) fusion is associated with various kinds of cancer, including anaplastic large cell lymphoma (ALCL). We investigated the efficacy of combination between ALK inhibitor and mammalian target of rapamycin (mTOR) inhibitor in ALK-positive ALCL cells. Methods & Results ALK-positive cancer cell lines were treated with ALK and/or mTOR inhibitors and assessed for cell viability. Treatment of ALK-positive ALCL cell lines with the combination resulted in a synergistic effect in Karpas299 cells but not in SU-DHL-1 cells. Viability of Karpas299 cell was reduced to 26.4% (p<0.001) by alectinib-everolimus combination treatment compared to the single treatment groups (alectinib: 59.5%, everolimus: 51.6%); the Combination Index (CI) value of 0.316 indicated a synergistic effect of the combination treatment. Moreover, changes in intracellular signaling were analyzed by western blot. Combination treatment augmented increase of ALK-mTOR de-phosphorylation, and cleavage of poly ADP ribose polymerase (PARP) molecules. Combination treatment significantly increased the numbers of Karpas299 cells in subG1 (p = 0.0096 everolimus), and G0/G1 (p = 0.0018 alectinib; 0.0013 everolimus) cell cycle arrest compared to single treatment groups. We investigated the biological differences between Karpas299 and SU-DHL-1 cell lines using RNA sequencing data obtained from Sequence Read Archive (SRA). The RNA expression pattern of the ALK isoforms ALK-201 and 203 was different between the two cell lines. These data indicated that the relatively high expression of ALK-203 in SU-DHL1 cells could explain the lack of synergism in response to the combination treatment observed. Conclusions Combination treatment with alectinib and everolimus synergistically reduced cell survival, augmented inhibition of cellular signaling, and increased cell cycle arrest in Karpas299 but not in SU-DHL1 cells. These differences might be attributable to differential expression of ALK isoforms between the two cell lines. Citation Format: Dongchan Kim, Hyejoo Park, Youngil Koh, Sung-Soo Yoon. Synergistic effect of alectinib and everolimus on anaplastic large cell lymphoma cells via ALK-mTOR pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5809.