The Apoptotic Mechanism of Action of the Sphingosine Kinase 1 Selective Inhibitor SKI-178 in Human Acute Myeloid Leukemia Cell Lines

Abstract

We previously developed ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH 2 -terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

Targeting sphingosine kinase 1 induces MCL1-dependent cell death in acute myeloid leukemia

“…on June 7, 2019. by guest www.bloodjournal.org From MCL1 and reduced cell survival. 19,61 Interestingly, however, MP-A08 treatment was also associated with induction of the proapoptotic BH3-only proteins BIM, NOXA, and cleaved BID, which was not observed previously when AML cell lines were exposed to another SPHK1 inhibitor, SKI-178. 19 Notably, our findings demonstrate a role of S1PR2 in maintaining MCL1 expression because application of the S1PR2 antagonist JTE-013, but not antagonists of S1PR1, 3, and 4, induced a similar loss of MCL1 in AML cells to that observed with SPHK1 inhibitors.…”

“…19,61 Interestingly, however, MP-A08 treatment was also associated with induction of the proapoptotic BH3-only proteins BIM, NOXA, and cleaved BID, which was not observed previously when AML cell lines were exposed to another SPHK1 inhibitor, SKI-178. 19 Notably, our findings demonstrate a role of S1PR2 in maintaining MCL1 expression because application of the S1PR2 antagonist JTE-013, but not antagonists of S1PR1, 3, and 4, induced a similar loss of MCL1 in AML cells to that observed with SPHK1 inhibitors. These results are consistent with the emerging oncogenic role of S1PR2 signaling in solid tumors and hematological malignancies [62][63][64] providing independent validation of our findings with SPHK1 inhibitors and demonstrate that targeting the SPHK1/S1P axis has an important effect on regulating MCL1 expression and AML cell survival.…”

“…Two SPHK1 splice isoforms, SPHK1a and SPHK1c, were expressed in all samples examined ( Figure 1C; n 5 13), consistent with previous reports in AML cell lines. 19,20 Importantly, SPHK1 protein was below detection limits in MNCs obtained from the bone marrow of healthy volunteers. We have previously shown that activation of SPHK1 via phosphorylation of Ser225 by extracellular signal-regulated kinases 1/2 (ERK1/2) promotes oncogenic signaling by this enzyme.…”

“…[18][19][20][21] Indeed, MP-A08 treatment of mice engrafted with primary human AML cells significantly reduced leukemic burden and prolonged mouse survival without impacting on normal mouse hematopoiesis or blood, kidney, liver, heart, and spleen pathology. 33 Notably, MP-A08 is known to be equally effective at inhibiting human and mouse sphingosine kinases.…”

“…[6][7][8][9][10][11][12][13][14] Several studies have recently implicated a role for SPHK1 in leukemogenesis. 15 For example, SPHK1 inhibition has been shown to sensitize leukemic cells to chemotherapy, 16,17 directly induce cell death in HL-60 AML cells, 18,19 and reduce the growth of subcutaneous U937 AML cell line xenografts in mice, 20,21 but the mechanism of action and the efficacy in primary AML have not been studied.…”