Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. Sphingosine 1-phosphate (S1P) is a lipid mediator of diverse cellular functions, most notably of lymphocyte trafficking, angiogenesis, cell proliferation and survival. S1P is produced intracellularly by the sphingosine kinases (SK) of which there are two isoforms, SK1 and SK2. SK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SK1. Inhibition of either SK1 or SK2 is currently under investigation as a novel anti-cancer strategy and potent anti-leukemic effects are likely. Application of the combined SK1/SK2 inhibitor SKI II and the selective SK2 inhibitor ABC294640 to ALL cells produced a reduction in cellular proliferation as measured by 3H-thymidine incorporation in all cell lines (REH, NALM6, LK63, ALL1, 2070 and TOM1) tested with IC50 values of 1μM – 7μM for SKI II and <40μM for ABC294640. Viability, measured by flow cytometry using annexin V and propidium iodide (PI) staining, was also reduced in all cell lines except the Ph+ ALL1 and 2070 cells treated with SKI II with IC50 values ranging from 2μM to >10μM for SKI II and 50–60μM for ABC294640. SKI II resulted in caspase-dependent cell death, as determined by flow cytometric assessment of intracellular caspase-3 cleavage and apoptotic morphology on light microscopy, with cell death prevented by pre-incubation with 100μM of the pan-caspase inhibitor Z-VAD-FMK. However, ABC294640 induced caspase-3 cleavage at lower than expected levels and cell death was not prevented by Z-VAD-FMK. Both agents significantly reduced intracellular S1P concentrations by 24 hours as determined by ELISA, thereby confirming the ability of these compounds to inhibit SK1 or SK2 activity. A search for agents that synergized with the SK inhibitors revealed that when Ph+ ALL cells were treated with the combination of imatinib and either ABC294640 or SKI II, a further reduction in cell death occurred than with either agent alone, thereby enhancing the therapeutic effect of ABC294640 and overcoming resistance seen with SKI II alone. Furthermore, the combination of mildly cytotoxic concentrations of ABC294640 and the novel pan histone deacetylase inhibitor AR-42 were found to significantly increase leukemic cell death at 24 and 48 hours in Ph+ and negative ALL cells. In vivo assessment of the SK inhibitors was determined by injecting NOD/SCID IL2gc−/− mice with 2–5 million human B-ALL cells and treating with 100 mg/kg/day ABC294640 or vehicle by intraperitoneal injection for 21 days after which all animals were sacrificed. Assessment of leukemia in blood, bone marrow and spleen was determined by flow cytometry using antibodies to human CD19 and murine CD45. Significant reductions in the levels of leukemia in all examined tissues were found in ABC294640-treated animals using three different human ALL xenografts, including the Ph+ positive xenograft 2070. Average absolute levels of leukemia in the bone marrow of ABC294640-treated mice for xenografts 2070, 1345 and 0398 were reduced by 40% (p = 0.00007), 55% (p = 0.004) and 72% (p = 0.000001) respectively. No overt toxicity was noted. SK inhibition, resulting in reduced intracellular S1P, is an exciting novel anti-leukemic strategy potentially adding to the repertoire of non-chemotherapeutic agents for the treatment of ALL. Combinations of SK inhibitors with newer targeted agents show promise of greater leukemia reduction.