Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.

Abstract Abstract 1313 Natural killer cell large granular lymphocytic leukemia (NK-LGL) is a rare lymphoproliferative disorder of cytotoxic CD3-/CD56+ natural killer cells. Patients present with a malignant clinical course and a fatal outcome with median survival time of two months from diagnosis. Aggressive NK-LGL is refractory to conventional chemotherapy and pathogenetic mechanisms remain undefined. The sphingolipid metabolic pathway or ‘sphingolipid rheostat’ has been identified as a key player in determining cell fate. We hypothesize that this sphingolipid rheostat is dysregulated in NK-LGL leukemia. Ceramide and sphingosine are pro-apoptotic members of this pathway and sphingosine-1-phosphate (S1P) is pro-survival. Sphingosine kinase 1 (SphK1) is a cytosolic enzyme that converts sphingosine into S1P and has been implicated in oncogenesis. SphK1 is overexpressed in tumors of the breast, ovary, colon, brain, liver and esophagus and various leukemias and lymphomas. Here, we show that the sphingolipid rheostat is dysregulated in leukemic NK cells. Lipids were extracted from NK cells from nine NK-LGL patients and compared to nine, age and gender matched normal controls. Ceramide and S1P levels were quantified by tandem mass spectrometry. Ceramide levels were decreased 2-fold in NK-LGL patients compared to normal NK (p < 0.05). S1P levels were increased 2-fold in NK-LGL patients compared to normal NK (p < 0.05). SphK1 mRNA levels were measured by qRT-PCR in eight NK-LGL patients and matched to eight, age and gender matched normal controls. SphK1 mRNA levels were elevated >4 times in NK-LGL patients compared to controls (p < 0.05). This same elevation in SphK1 mRNA can be seen in target recognition and activation of normal NK cells (>4 fold increase, p < 0.05). In addition, elevated SphK1 mRNA levels were detected the NK cells of Fisher 344 rats. Fischer F344 rat LGL leukemia model has been established as an important experimental model for the study of NK-LGL leukemia progression and closely resembles human aggressive NK-LGL leukemia exhibiting clonal expansion of CD3-CD8a+ lymphocytes. To investigate the role of SphK1 in survival of leukemic NK cells, pegylated cationic liposome delivery of RNAi was utilized. The human NKL cell line was established from the peripheral blood of a patient with CD3-/CD16+/CD56+ LGL leukemia and is an important model for studying the disease. RNK-16 is a spontaneous NK leukemic cell line from Fisher 344 rats. RNAi targeting SphK1 in NKL cells led to a two-fold increase of cell death by ELISA (p < 0.05). This genetic inhibition resulted in an increase in pro-apoptotic signaling proteins Bax and Bak, while leading to a decrease in anti-apoptotic proteins Bcl-2, Bcl-XLand survivin. Additionally, there was decreased phosphorylation of AKT and cleaved Poly ADP Ribose Polymerase (PARP). Treatment of NK cells with SKI-II (2.5u M), a pharmacologic inhibitor of SphK1, resulted in an increase in specific apoptosis in a dose-dependent manner by Annexin-V staining and flow cytometry in NK-LGL patients (n=4 patients, 20%, p < 0.005), NKL cells (15%, p < 0.01) and RNK-16 cells (40%, p < 0.05). This pharmacologic inhibition selectively induced apoptosis in leukemic cells and had no effect on NK cells from normal donors. Apoptosis by inhibition of SphK1 with SKI-II is caspase-dependent with significant increases in pro-caspase 3 and cleaved caspase-3. Treatment of NKL cells with z-FAD-fmk, a caspase-activity inhibitor, led to a dose-dependent increased viability of cells after treatment with SKI-II (2.5uM). The viability of NKL cells increased from 50% to 80% in a dose-dependent manner with treatment of a caspase-activity inhibitor, z-FAD-fmk (0, 10, 20, 50uM). This shows that inhibition of SphK1 results in caspase-dependent apoptosis. Through the utilization of a rat model, rat cell line, human cell line and clinical samples, the sphingolipid rheostat is shown to be dysregulated in NK-LGL leukemia. By employing both genetic and pharmacologic approaches, we have demonstrated that SphK1 is a potential therapeutic target for novel treatment of NK-LGL leukemia. Disclosures: Kester: Penn State Research Foundation: Licensed ceramide based nanotechnologies and siRNA based nanotechnologies, Keystonenano Inc. State College, PA, Co-founder and CMO Other.

Altered sphingolipid metabolism contributes to cancer progression and presents an exploitable target for the development of novel chemotherapeutics. Bioactive sphingolipid metabolites also have the potential to serve as vital biomarkers for cancer and be utilized to determine disease progression, as well as guide therapeutic regimens. Moreover, identification of these sphingolipid biomarkers is achievable based on recent technological advances in sphingolipidomics, which have aided in detection of sphingolipid metabolites through tools like mass spectrometry. Excellent reviews have previously focused on the biochemical role that sphingolipids have in cancer pathogenesis and treatment. The aim of this review is to concentrate on the critical metabolites and enzymes that contribute to the dysregulation in sphingolipid metabolism, and highlight relevant translational research that is directed towards novel therapies.

NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-beta transcripts than purified normal CD8(+) T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NKLGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy. (Blood. 2010;115:51-60)

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C₆-ceramide (C₆) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C₆-ceramide may be a promising therapeutic approach for a fatal leukemia.

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.