Phosphatidylinositol-3-Phosphate Kinase Pathway Activation Protects Leukemic Large Granular Lymphocytes from Undergoing Homeostatic Apoptosis.

Abstract

T cell large granular lymphocytic leukemia (T-LGL) is characterized by chronic clonal lymphoproliferation of cytotoxic T lymphocytes (CTL). T-LGL cells exhibit phenotypic properties of antigen-activated cells, including expression of Fas/FasL and production of pro-inflammatory cytokines, and it is likely that the pathogenic CTL clone evolves in the context of an initially polyclonal autoimmune disease. A key issue regarding T-LGL pathogenesis is the question of what mechanisms are supporting the ability of the pathologic CTL to avert normal homeostatic apoptosis. Since the proinflammatory cytokines produced in T-LGL are all capable of activating the PI3K-AKT pathway, a key mediator of cellular survival, we stipulated that a defect along this pathway may be involved in pathogenesis of T-LGL.When we studied the phosphorylation status of AKT by western blot, in contrast to resting CTL from normal donors, T-LGL cells from eight patients repeatedly showed constitutive AKT phosphorylation. These studies were performed in total mononuclear cells as well as purified clonal CTL. Using the Src family kinase (SFK) inhibitor PP2, we demonstrate that phospho-AKT induction is dependent on the upstream activity of a SFK, possibly Lck or Fyn, further linking the activation of this pro-survival pathway in T-LGL to a receptor in the plasma membrane. Since the PI3K-AKT pathway can antagonize the ability of Fas to initiate apoptosis, we further hypothesized that inhibition of PI3K would lead to reacquisition of Fas sensitivity in T-LGL. When CTL from T-LGL patients are cultured with an activating anti-Fas antibody for 20 hours, there is no detectable induction of apoptosis as determined by flow cytometric detection of FITC-Annexin-V. However, addition of a PI3K inhibitor (LY294002) drastically increases apoptosis induction in these cells (8–11% Annexin-V positive with anti-Fas alone; 39–50 % Annexin-V positive with anti-Fas + LY294002). An unexpected and interesting finding in the course of these studies was that PI3K inhibition alone, in the absence of exogenous Fas engagement, led to brisk spontaneous apoptosis in clonal CTL from T-LGL patients within 20–24 hours (30–47% Annexin-V positive after LY294002 alone). As further validation of the effect of PI3K inhibition on CTL from T-LGL, we demonstrate PARP cleavage after a six hour incubation with LY294002, and distinct morphological changes after 20 hours, consistent with lymphocyte apoptosis. Furthermore, CTL from normal donors were not affected by the PI3K inhibitor, demonstrating a specific vulnerability in the T-LGL cells. Thus, the issue of defective Fas/FasL signaling in the clonal CTL may be less important than the ongoing activation of the PI3K pathway.Our results demonstrate that constitutively elevated PI3K activity in T-LGL likely plays an important role in the ability of the pathologic cells to avoid homeostatic apoptosis. More importantly, the activity of this pathway may represent a kind of “Achilles heel” for T-LGL in that PI3K inhibitors alone are quite effective at inducing spontaneous apoptosis in the clonal CTL after a short incubation. This abnormal reliance on a particular signaling pathway in T-LGL provides a unique opportunity to eradicate the clone through targeted inhibition. As novel inhibitors of PI3K and its downstream components become available for clinical applications, it will be interesting to investigate their efficacy in T-LGL.