Abstract
BACKGROUND: Large Granular Lymphocyte (LGL) leukemia is an indolent clonal disorder arising from either CD3+ or CD3− immunophenotypes characterized as T-cell and Natural-Killer (NK)-cell malignancies, respectively. Due to a rare incidence, all therapeutic investigations in LGL leukemia are generated from single institution experiences, which consist primarily of retrospective cohort analyses. Pathogenesis may relate to an underlying autoimmune mechanism with low-dose methotrexate (10mg/m2 weekly), cyclophosphamide (50–100mg daily), and cyclosporine A (5–10mg/kg daily) used as first-line agents for the treatment of associated cytopenias. The mechanism(s) controlling survival of leukemic LGL are not fully characterized. We hypothesize that agents that target survival signaling will serve as effective therapeutics. Using pharmacologic inhibitors of the Mitogen Activated Protein Kinase (MAPK/ERK) signaling cascase, we found that ERK/Ras drives NK leukemia survival (Epling-Burnette, et al. Oncogene, 23:9220, 2004). The goal of this study was to investigate the in vitro actions of the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson and Johnson) on survival and immune response leukemic T cells.
METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with T LGL leukemia. All patients had increased numbers of CD3+ αβ T lymphocytes and evidence of clonality. Western blot analysis was used to examine the protein levels of phosphorylated (active) ERK1 and ERK2 (MAPK), Bcl-2, and total MAPK. Apoptosis assays were performed by staining with annexin-V-FITC and 7-aminoactinomycin (7-AAD) and flow cytometry analysis. Anti-CD3 plus anti-CD28 was used to stimulate T cells in the presence of brefelden A and IL-2, IFN-γ, TNF-α, IL-4, and IL-10 were determined in distinct T cell subpopulations by intracellular staining. T cell proliferation was assessed by Brdu incorporation in CD4+ and CD8+ T cells.
RESULTS: PBMCs from patients with T LGL displayed constitutively-active MAPK/ERK expression as determined by Western blot analysis (n=5). Using MEK pharmacological inhibitors, we found that blockade of the active MEK/ERK signaling pathway induced apoptosis in leukemic T LGL. Bcl-2 was expressed in 80% of patients and when MEK/ERK was inhibited there was a corresponding reduction in expression. Targeted disruption of farnesyl-transferase with two different inhibitors (FTI2153 and R115777, n=9) led to a dose-dependent increase in apoptosis with an apoptotic index that was significantly greater than normal T cells. In addition to apoptosis, we found that antigen-induced proliferation of CD4+ and CD8+ T cells was impaired by R115777 (11% ± 7 vs. 3% ±1.7 and 11% ± 9 vs. 3% ± 1.6, respectively). Furthermore, R115777 biased antigen-dependent cytokine response to a Th2 type with increased expressionof IL-4 and IL-10 after drug treatment (average increase 100% and 43%, respectively).
CONCLUSIONS: These findings suggest that FTIs regulate immune response and lead to apoptosis of leukemic T LGL cells. A pilot trial of R115777 (tipifarnib, Zarnestra®) is ongoing for the treatment of LGL leukemia.
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