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.

IntroductionLarge granular lymphocyte (LGL) leukemia is a lymphoproliferative disease of either CD3 ϩ cytotoxic T lymphocytes (CTLs) or CD3 Ϫ natural killer cells (NK cells). The majority of LGL patients with T-cell (CD3 ϩ , CD8 ϩ /CD57 ϩ ) or NK-cell (CD3 Ϫ , CD16 ϩ / CD56 ϩ ) leukemia have a clinically indolent course. 1,2 LeukemicLGLs of T-cell phenotype reflect polarized expansion of CD8 ϩ terminal-effector memory cells. 3 Expanded NK cells have an activated phenotype with dysregulated NK receptor expression. 4,5 Fas resistance is an important biologic feature in leukemic LGLs of both T-cell and NK-cell type. 3,6 Constitutive activation of survival signaling pathways is a central pathogenetic mechanism in LGL leukemia. Previously, phosphatidylinositol-3 (PI3) kinase activation and signal transducer and activator of transcription 3 upregulation of Mcl-1 were shown to be important for survival of leukemic T-LGLs. [7][8][9] More recently, molecular profiling of T-LGL leukemia revealed a survival role for constitutive sphingolipid signaling. 10 Survival mechanisms in the NK type of LGL leukemia have been less extensively studied; however, a constitutively active retrovirus-associated DNA sequence (RAS)/mitogen-activated protein kinase (MEK)/extracellular signal-related kinase (ERK) survival pathway was identified. 6 Given the complexity and interactive nature of signaling pathways, it is difficult to determine the importance of individual pathway components when studied in isolation. Using a network modeling approach, we found that the presence of interleukin-15 (IL-15) and platelet-derived growth factor (PDGF) is sufficient to reproduce all known deregulations in T-LGL leukemia. 11 Work in this study focused on further examining the pivotal role of PDGF. We found that PDGF mediates survival of leukemic LGLs of both T-and NK-cell origin through an autocrine regulatory pathway. Methods ReagentsAll chemicals were purchased from Sigma-Aldrich unless otherwise specified. Recombinant human (rh) PDGF-BB was purchased from ProSpec-TANY TechnoGene LTD; rhIL-2, from Promega Corporation; and human T-lymphotropic virus-I (HTLV-I)-and HTLV-II-infected plasma, from Zeptometrix. Antibodies and inhibitors were obtained from the following sources and used at the dilutions recommended by the manufacturers: anti-PDGFR-␣ (951) and anti-PDGFR-␤ (958) polyclonal antibodies, anti-phospho-Tyr monoclonal antibody (PY99), goat anti-mouse immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology Inc); anti-PDGF-BB neutralizing antibody and anti-PDGF-BB antibody for immunocytochemistry/immunofluorescence (ICC/IF; Abcam Inc); anti-phospho-AKT and total AKT polyclonal antibodies, anti-MEK1/2, anti-phospho-MEK1/2, anti-ERK1/2, and phospho-ERK1/2 (Cell Signaling Technology); antiphospho-Src (Tyr419) and anti-Src antibodies (Upstate Cell Signaling Solutions); ␤-actin monoclonal antibody (Sigma-Aldrich); mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International); PI3K inhibitor LY294002 (Cell Signali...

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.

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Large granular lymphocyte (LGL) leukemia characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis.