Differential regulation of sphingolipid metabolism and signaling in leukemic LGLs. (A) Sphingolipid metabolism and signaling pathway and its inhibitors. The inhibitors of the pathway are underlined. The genes shown in reversed color are core enriched as analyzed by GSEA. Naive normal PBMCs (N PBMCs, 1 representative sample of 2 independent experiments showed) or leukemic LGLs (TLGLs, n = 3) were either left untreated or treated with vehicle or indicated concentrations of myriocin (B), fumonisin (C), desipramine (D), and GW4869 (E) for 24 hours. Induction of apoptosis was assessed using flow cytometry. There was no differential apoptosis of leukemic LGLs compared with normal naive PBMCs using each of these inhibitors. (F) Inhibition of acid ceramidase induced differential apoptosis in leukemic LGLs. Naive normal PBMCs (N PBMCs, white dots, n = 5), activated normal PBMCs (AC PBMCs, gray dots, n = 6), or leukemic LGLs (TLGLs, black dots, n = 6) were either left untreated or treated with vehicle (methanol) or 100 μM NOE for 6 hours. Induction of apoptosis was assessed using flow cytometry. NOE induced approximately 30-fold higher apoptosis in LGL leukemia PBMCs compared with normal PBMCs (*P < .001). (G) Inhibition of S1P-mediated signaling by FTY720-induced differential apoptosis in leukemic LGLs. Naive normal PBMCs (N PBMCs, white dots, n = 5), activated normal PBMCs (AC PBMCs, gray dots, n = 4), or leukemic LGLs (TLGLs, black dots, n = 5) were either left untreated or treated with vehicle (DMSO) or 5 μM FTY720 for 6 hours. Leukemic LGLs showed approximately 13-fold higher apoptosis compared with naive normal PBMCs (*P < .001). (Each open dot [○] represents mean percentage of apoptosis ± SEM of 3 separate experiments in an individual sample; marker (•) represents the mean of all samples in a given treatment.)