Fanqi Bai

Cooperative Regulation of Mcl-1 by Janus Kinase/STAT and Phosphatidylinositol 3-Kinase Contribute to Granulocyte-Macrophage Colony-Stimulating Factor-Delayed Apoptosis in Human Neutrophils

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR–ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr–Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr–Abl kinase. In this study, we show that blocking Bcr–Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr–Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr–Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr–Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr–Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Prevalence and clinical association of clonal T-cell expansions in Myelodysplastic Syndrome

Selected patients with Myelodysplastic Syndromes (MDS) are responsive to immunosuppressive therapy, suggesting that hematopoietic suppressive T cells have a pathogenic role in ineffective hematopoiesis. We assessed T-cell receptor (TCR) clonality through combined flow cytometry and molecular analysis of the complementarity determining region (CDR)-3 of the T-cell receptor-Vβ gene. We identified clonal T cells in 50% of MDS patients (n=52) compared to 5% of age-matched normal controls (n=20). The presence of T-cell clones was not associated with features linked previously to immunosuppression response, including WHO diagnostic category, karyotype, marrow cellularity, IPSS category, sex or age ⩽60. Using flow cytometry to identify expanded Vβ-families, we found that T cells showed greater expansion in the bone marrow compared with peripheral blood, and were characterized as CD8+/CD57+/CD28− effector T cells. Expanded effector T cell were CD62L negative and expressed the natural killer C-lectin-family receptor NKG2D and CD244 (2B4). We conclude that clonal T-cell expansion is common among all MDS prognostic subgroups.

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL)

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3−, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.

Altered naive and memory CD4+ T-cell homeostasis and immunosenescence characterize younger patients with myelodysplastic syndrome

Response to immunosuppressive therapy (IST) in younger patients with myelodysplastic syndrome (MDS) has been linked to a T-cell-dominant autoimmune process that impairs hematopoiesis. Analysis of the age-adjusted CD4:CD8 ratio in 76 MDS patients compared with 54 healthy controls showed that inadequate CD4+, rather than expansion of CD8+ T cells, was associated with a lower ratio in a group that included both lower and higher risk MDS patients defined by the International Prognostic Scoring System. In younger MDS patients, naive and memory phenotypes defined by CD45RA and CD62L display showed depletion of naive CD4+ and CD8+ T cells, suggesting a possible relationship to IST responsiveness. To determine the correlation between T-cell subset distribution, T-cell turnover and autoimmunity, a cohort of 20 patients were studied before and after IST. The CD4:CD8 ratio correlated inversely with the proliferative T-cell index before treatment in IST-responsive patients, suggesting that proliferation may be linked to accelerated CD4+ T-cell turnover and hematopoietic failure. Our data show seminal findings that both CD4+ and CD8+ T-cell subsets are dysregulated in MDS. Association between these T-cell defects and response to IST suggests that aberrant T-cell homeostasis and chronic activation are critical determinants influencing autoimmune hematopoietic suppression in younger patients.

Clinical improvement by farnesyltransferase inhibition in NK large granular lymphocyte leukemia associated with imbalanced NK receptor signaling.

Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)-LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.

A critical role for DAP10 and DAP12 in CD8+ T cell–mediated tissue damage in large granular lymphocyte leukemia

Large granular lymphocyte (LGL) leukemia, or LGLL, is characterized by increased numbers of circulating clonal LGL cells in association with neutropenia, anemia, rheumatoid arthritis, and pulmonary artery hypertension (PAH). Emerging evidence suggests that LGLL cells with a CD8(+)CD28(null) phenotype induce these clinical manifestations through direct destruction of normal tissue. Compared with CD8(+)CD28(null) T cells from healthy controls, CD8(+)CD28(null) T cells from LGLL patients have acquired the ability to directly lyse pulmonary artery endothelial cells and human synovial cells. Here, we show that LGLL cells from patients possess enhanced cytotoxic characteristics and express elevated levels of activating natural killer receptors as well as their signaling partners, DAP10 and DAP12. Moreover, downstream targets of DAP10 and DAP12 are constitutively activated in LGLL cells, and expression of dominant-negative DAP10 and DAP12 dramatically reduces their lytic capacity. These are the first results to show that activating NKR-ligand interactions play a critical role in initiating the DAP10 and DAP12 signaling events that lead to enhanced lytic potential of LGLL cells. Results shown suggest that inhibitors of DAP10 and DAP12 or other proteins involved in this signaling pathway will be attractive therapeutic targets for the treatment of LGLL and other autoimmune diseases and syndromes.

Tipifarnib-mediated suppression of T-bet-dependent signaling pathways

Large granular lymphocyte (LGL) leukemia is a chronic lymphoproliferative disease in which T-bet [T-box transcription factor 21 gene (tbx21)] overexpression may play a pathogenic role. T-bet orchestrates the differentiation of mature peripheral T-cells into interferon-γ (IFN-γ) and tumor necrosis factor-α producing CD4+ T-helper type I (Th1) and CD8+ T cytotoxic cells that are necessary for antiviral responses. When IL-12 is produced by antigen–presenting cells, T-bet expression is induced, causing direct stimulation of ifng gene transcription while simultaneously acting as a transcriptional repressor of the IL4 gene, which then leads to Th1 dominance and T-helper type 2 differentiation blockade. Additionally, T-bet has been shown to regulate histone acetylation of the ifng promoter and enhancer to loosen condensed DNA, creating greater accessibility for other transcription factor binding, which further amplifies IFNγ production. We found that treatment with a farnesyltransferase inhibitor tipifarnib reduced Th1 cytokines in LGL leukemia patient T-cells and blocked T-bet protein expression and IL-12 responsiveness in T-cells from healthy donors. The mechanism of suppression was based on modulation of histone acetylation of the ifng gene, which culminated in Th1 blockade.