CB5468139, described here for the first time, provides an important indication that SK1-selective agents can be developed, but itself is likely to be too non-selective for development. In contrast, the data presented here support the hypothesis that selective targeting of SK2 by ABC294640 has excellent potential for use in cancer chemotherapy. Rapamycin Importantly, we now show that ABC294640 has similar or greater potency for inhibiting cancer cell proliferation and migration compared to SK1/2-dual and SK1-selective inhibitors. This indicates that the functions of SK2-generated S1P cannot be fully compensated by SK1-generated S1P, possibly due to their different subcellular localizations. Also, sphingolipid profiling demonstrated significant increases in ceramide Bortezomib species combined with depletion of S1P after ABC294640 treatment which likely intensifies its anti-proliferative activity. Interestingly, the significant increase of SK2 expression in response to exposure to ABC294640 may also contribute to its anti-cancer activity because SK2 possesses a pro-death BH3 domain. On one hand, ABC294640 treatment inhibits the mitogenic kinase function of SK2; while on the other hand, the overexpression of the BH3 domain could provide a magnified pro-death stimulus. This is consistent with studies that showed that overexpression of SK2 by transfection results in apoptosis. Further study of the promoter elements responsible for SK2 transcription would be of considerable interest to elucidate the mechanism for induction by ABC294640. The expression and phosphorylation of pro-survival signaling proteins such as STAT3, AKT, ERK and FAK were markedly impacted by the SK2-selective inhibitor ABC294640, and to a less degree by other SK inhibitory compounds. ABC29460 also disrupted the cell cycle with arrest in G1 and reduced expression of p53 and p21, which mimicked the selective knockdown of SK2 with siRNA. Flow cytometric analyses did not reveal significant increases in apoptosis after treatment with ABC294640; however elevation of the autophagy markers Beclin1 and LC3 suggest that the cells are dying by excessive autophagy. Although autophagy is recognized as a survival mechanism under most conditions, it is also capable of inducing cell death characterized by extensive digestion of intracellular organelles leading to large numbers of autophagic vacuoles. Furthermore, a number of small moleculesactivate autophagy in cancer cells both in vivo and in vitro. Among the SK inhibitory compounds tested, the SK1/2-dual inhibitor SKI-II is the only one that had the same degree of anti-proliferative and anti-migratory activity as ABC294640. DMS had less impact; whereas ABC294735 was largely inactive except for the induction of autophagy. Our previous RNA interference studies suggest that selective inhibition of SK1 should result in only mild suppression of cell growth and migration. CB5468139 had relatively strong activity in certain assays including cell proliferation, elevation of LC3 cleavage and inhibition of AKT phosphorylation, but only modest effects on sphingolipid profiles, cell cycle distribution and migration. This disparity is likely due to off-target effects of CB5468139, supporting the hypothesis that compounds targeting the sphingosine binding site rather than the ATP binding site provide more pure pharmacologic probes of SK activity and potentially less toxic therapeutic agents. Additionally, SK2-selective inhibitors may effectively deplete the nuclear S1P pool of tumor cells while having less impact on circulating S1P levels which are important for the maintenance of normal vascular and immune function.