Abstract

Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over-expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. Furthermore, SphK1 has been shown to correlate with the clinical outcome of certain tumors. Here we focus on SphK1 as an important oncogenic target in acute lymphoblastic leukemia (ALL).

We have previously shown SphK1 protein to be over-expressed and activated (Ser225 phosphorylation) in ALL cell lines and primary patient samples compared to normal CD34+CD19+ B-cell progenitors. Furthermore, we have reported the importance of SphK1 in the development of ALL by transducing B-cell progenitors isolated from WT or SphK1-/- mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated WT mice. The absence of SphK1 significantly reduced the incidence of ALL in recipient mice (ASH 2012).

Inhibition of SphK1 by the selective inhibitor SK1-I significantly reduced intra-cellular S1P concentrations (p=0.017 and p=0.003 at 24 and 48 hours respectively) in 3 cell lines examined, indicating that the drug targets this enzyme. SK1-I killed ALL cells as determined by annexin V/PI flow cytometric analysis with IC50 values ranging from 12 µM to 18 µM at 72 hours. Furthermore, SK1-I induced cell death in primary patient ALL cells by 16 hours. This agent resulted in virtually no caspase-3 cleavage and cell death was not prevented by the pan-caspase inhibitor Z-VAD-FMK (p=0.45, n=4). Marked cytoplasmic vacuolation was detected by light microscopy, with LC3 processing present by Western blot, consistent with the development of autophagy. However, the autophagy inhibitor 3MA failed to prevent SK1-I-mediated cell death. These results suggest that the cell death associated with inhibition of SphK1 in ALL cells is caspase-independent and cannot be attributed to autophagy. Surprisingly, conventional chemotherapeutic agents such as doxorubicin and vincristine failed to synergize with SK1-I, however, synergistic killing was observed when SK1-I was combined with 500 nM imatinib over 72 hours in Philadelphia-positive (BCR/ABL+) ALL cells.

We have developed a novel SphK1 inhibitor, MP8, that targets the enzyme via a different mechanism to SK1-I, since it blocks ATP binding. MP8 reduced intra-cellular S1P in Jurkat cells by 43% compared to untreated controls, and killed Jurkat and SUP-B15 cells over 24 to 48 hours with IC50 values of 8 µM. Additionally, MP8 induced cell death in primary patient ALL cells by 24 hours. This agent resulted in classic apoptotic cell death, which was rescued by Bcl-2 over-expression, resulting in near complete reversal of PARP cleavage.

SphK1 has indisputable tumor-promoting properties and lies downstream of a number of signalling pathways known to be dysregulated in ALL. Here we show that SphK1 is over-expressed and activated in ALL cells and targeting SphK1 has potent cytotoxic effects in a wide range of cell lines and patient samples. Furthermore, genetic deletion of Sphk1 significantly reduced the incidence of murine BCR/ABL-driven ALL. These findings suggest further examination of the role SphK1 plays in ALL will uncover novel interactions with oncogenic signalling pathways and paves the way for the inclusion of SphK1 inhibitors in future pre-clinical trials.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.