Preclinical studies of an orally-available Sphingosine Kinase inhibitor Free

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Sphingosine Kinase (SK), an oncogenic lipid kinase, is overexpressed in variety of tumor tissues. SK catalyses phosphorylation of sphingosine yielding sphingosine-1-phosphate (S1P), a potent mitogenic lipid messenger. In order to inhibit S1P production, we have identified non-lipid SK inhibitors and are characterizing their cellular and therapeutic effects. The lead compound, ABC294640, is an orally-available SK inhibitor with in vitro IC₅₀ of ~6 μM that is nontoxic in rodents up to at least 1000 mg/kg (po). When administered to a Balb/c model of mouse mammary adenocarcinoma, ABC294640 induces significant inhibition of tumor growth.
 In order to understand mechanisms of the antitumor activity of ABC294640, we performed experiments using cultured cells and intact animals. Cell cycle analyses of several cell lines revealed increased populations of cells in the G1 phase of cell cycle upon exposure to ABC294640. Early apoptosis was detected in HL60 and MV4-11 leukemia cell lines, but no subdiploid cell population was found upon exposure of either RS4;11 leukemia or A498 kidney cancer cell lines to this compound. The non-apoptotic cell death in A498 cells was confirmed by the absence of proteolytic activities of Caspases 3 and 7 in drug-treated cells. Trypan blue quantification of HL60 living and dead cells in the presence of ABC294640 indicated that, at lower concentrations, this compound has cytostatic properties. In addition, ABC294640 impaired the in vitro mobility of the highly-invasive A498 cells as measured with the Boyden chamber migration assay. This was consistent with phalloidin staining of stress fibers that revealed changes in actin structure upon exposure to ABC294640. Using the Balb/c model of mouse mammary adenocarcinoma to study combinatorial chemotherapy in vivo, the best reduction of tumor growth was observed with combination of ABC294640 with the antimetabolite Gemcitabine.
 Taken together, these data indicate that ABC294640 is cytostatic with divergent biological effects in different types of cancer cells. Of particular importance, is the identification of enhanced antitumor activity by the combination of ABC294640 and Gemcitabine. This will allow the rational design of human clinical trials with this first-in-class SK inhibitor.