Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma.

Abstract

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.