Identification of Sphingosine Kinases As Therapeutic Targets in B-Lineage Acute Lymphoblastic Leukemia

Abstract 1499

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. The role of SphK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SphK1. Little is known about the interaction of SphKs with intracellular signalling pathways. Here we assess the relevance of SphKs in B-lineage acute lymphoblastic leukemia (B-ALL).

Gene expression signatures indicative of activation of SphK1 or SphK2 were not publicly available. Therefore we treated ALL cell lines with the SphK1 or SphK2 specific inhibitors SK1-I and ABC294640 respectively, and analysed gene expression by microarray. Although a signature for SphK1 was not obtained, two independent methods of analysis generated SphK2 gene signatures that segregated control and drug treated cell lines. The signatures consisted of 11 and 35 genes and included reduced expression of the NF-κB inhibitor TRIB3 (NF-κB activity is commonly up regulated in ALL), the C/EBP family transcription factor DDIT3, the protein phosphatase 1 regulatory subunit PPP1R15A and the sphingolipid/cholesterol transporter ABCA1, all validated by quantitative RT-PCR. These gene signatures were used to interrogate a large publicly available gene expression dataset (GSE28497) obtained from pediatric ALL patients at the time of diagnosis. SphK2 activity signatures were more highly expressed in ALL samples (p=0.001 and p=0.027 for the smaller and larger signatures respectively) than normal B-cell progenitors. Although SPHK1 or SPHK2 genes were not over expressed in this dataset, SphK1 protein levels were increased in ALL cell lines and in patient samples.

The importance of SphK1 and SphK2 in the development of ALL was examined by transducing B-cell progenitors isolated from WT, SphK1^−/− or SphK2^−/− mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated wild type C57BL/6 mice. Twenty-two of 29 mice receiving cells from WT animals developed ALL, with a mean survival of 52.6 days (95% CI 42.3–62.8 days). Mice receiving cells lacking Sphk1 or Sphk2 had a significantly reduced incidence of ALL development with 14 of 30 (mean survival 71.6 days, 95% CI 60.5–82.8 days) and 16 of 29 animals (mean survival of 68.7 days, 95% CI 57.9–79.5 days) respectively (p=0.001). Lymphoblasts with a B-cell progenitor phenotype (B220⁺, CD19⁺, IgM⁻, CD11b⁻) were present in blood films and livers from leukemic mice and cells recovered from these animals produced a rapidly fatal ALL in secondary recipients. The presence of BCR/ABL and the expected deletion of Sphk1 or Sphk2 were confirmed by PCR in all murine leukemias examined.

We have previously reported that inhibitors of SphK1 and/or SphK2 inhibit proliferation and induce cell death in ALL cell lines, and that these agents can synergize with imatinib in Ph⁺ ALL cell lines. We have furthered these studies to demonstrate that patient derived ALL cells similarly respond to SphK1 and SphK2 inhibition in vitro. The SphK2 inhibitor ABC294640 (100mg/kg) reduced plasma S1P levels in mice consistent with our previous reports of reduced ALL burden following ABC294640 treatment, and using a NOD/SCID γc^−/− xenograft model of human ALL we have now shown that ABC294640 extends survival of ALL-bearing mice (p=0.0012 for Ph⁻ xenograft) and further extends the survival when combined with imatinib treatment in mice engrafted with a human Ph⁺ ALL patient sample (p=0.044).

This is the first report to suggest that sphingosine kinase 2 activity is increased in ALL, providing support for targeting SphK2 as a therapeutic strategy. Loss of SphKs reduces the incidence of ALL in a murine model of BCR/ABL-driven disease and ABC294640 reduces disease and extends survival in a human xenograft model of ALL. This compound synergizes with a number of potential therapeutic agents and further extends survival in a xenograft model of Ph⁺disease when combined with imatinib. This has potential to translate into a useful anti-leukemic strategy.