Andrzej Ptasznik

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80–95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.

A Tyrosine Kinase Signaling Pathway Accounts for the Majority of Phosphatidylinositol 3,4,5-Trisphosphate Formation in Chemoattractant-stimulated Human Neutrophils

The signaling pathway leading from G protein-coupled chemoattractant receptors to the generation of oxidants by NADPH oxidase in human neutrophils requires the formation of the lipid mediator phosphatidylinositol 3,4,5-trisphosphate (PIP3). Two mechanisms through which PIP3 can be generated have been described in human leukocytes. One pathway involves the coupling of the src-related tyrosine kinase Lyn to the “classical” p85/p110 form of phosphatidylinositol 3-kinase. The second paradigm utilizes a novel form of phosphatidylinositol 3-kinase whose activity is directly regulated by G protein βγ subunits. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block tyrosine kinase activity. These data suggest that the Lyn activation pathway plays a major role in the formation of this important lipid messenger during chemoattractant stimulation of human neutrophils.

Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells

Stromal-derived factor (SDF)-1 and its G protein–coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein–coupled receptor signaling and function of mammalian precursors.

Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131 and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation in the NPM/ALK-transfected BaF3 cells that do not express detectable Jak3 and in the NPM/ALK-positive malignant T cells with either Jak3 activity impaired by a pan-Jak or Jak3-selective inhibitor or Jak3 expression abrogated by Jak3 siRNA. The above results represent the ‘proof-of-principle’ experiments with regard to the ALK enzymatic activity as an attractive therapeutic target in T-cell lymphomas and other malignancies that express the kinase in an active form.

Signaling Mechanism of HIV-1 gp120 and Virion-Induced IL-1β Release in Primary Human Macrophages

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of proinflammatory cytokines, including IL-1β from macrophages, that are implicated in the pathogenesis of HIV-associated dementia. However, the signal transduction pathways involved have not been fully defined. Previously, our laboratory reported that soluble gp120 activates multiple protein kinases in primary human monocyte-derived macrophages, including the Src family kinase Lyn, PI3K, and the focal adhesion-related proline-rich tyrosine kinase Pyk2. In this study we showed that gp120 induces IL-1β release from macrophages in a time- and concentration-dependent manner through binding to the chemokine receptor CCR5 and coupling to Giα protein. Using pharmacological inhibitors and small interfering RNA gene knockdown, we demonstrated that concomitant activation of Lyn, Pyk2, and class IA PI3K are required for gp120-induced IL-1β production. By coimmunoprecipitation and immunofluorescence confocal microscopy, we showed that CCR5 activation by gp120 triggered the assembly of a signaling complex involving endogenous Lyn, PI3K, and Pyk2 and is associated with PI3K and Pyk2 translocation from the cytoplasm to the membrane where they colocalized with Lyn. Finally, we demonstrated that virion-associated gp120 induced similar response, as structurally intact whole virions also triggered IL-1β release and re-localization of PI3K and Pyk2. This study identifies a novel signaling mechanism for HIV-1-induced IL-1β production by primary human macrophages that may be involved in the neuropathogenesis of HIV-associated dementia.

An arrestin-dependent multi-kinase signaling complex mediates MIP-1β/CCL4 signaling and chemotaxis of primary human macrophages

MIP‐1β/CCL4 is a principal regulator of macrophage migration and signals through CCR5. Several protein kinases are linked to CCR5 in macrophages including the src kinase Lyn, PI3K, focal adhesion related kinase Pyk2, and members of the MAPK family, but whether and how these kinases regulate macrophage chemotaxis are not known. To define the role of these signaling molecules, we examined the functions and interactions of endogenous proteins in primary human macrophages. Using siRNA gene silencing and pharmacologic inhibition, we show that chemotaxis in response to CCR5 stimulation by MIP‐1β requires activation of Pyk2, PI3K p85, and Lyn, as well as MAPK ERK. MIP‐1β activation of CCR5 triggered translocation of Pyk2 and PI3K p85 from the cytoplasm to colocalize with Lyn at the plasma membrane with formation of a multimolecular complex. We show further that arrestins were recruited into the complex, and arrestin down‐regulation impaired complex formation and macrophage chemotaxis toward MIP‐1β. Together, these results identify a novel mechanism of chemokine receptor regulation of chemotaxis and suggest that arrestins may serve as scaffolding proteins linking CCR5 to multiple downstream signaling molecules in a biologically important primary human cell type.

Monocyte Migration and LFA-1-Mediated Attachment to Brain Microvascular Endothelia Is Regulated by SDF-1α through Lyn Kinase

Infiltration of activated monocytes into the brain is a prerequisite for the development of various neurological disorders such as HIV-associated dementia, multiple sclerosis, and other inflammatory processes. In these pathologies, the chemokine SDF-1α (CXCL12) is over-expressed and might attract monocytes into the CNS. We demonstrate here that SDF-1α stimulates migration of monocytes through its receptor, CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for β2 integrins. SDF-1α also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) that are activated with TNF-α, IL-1β, or recombinant envelope glycoprotein from HIV-1, which increase BMVEC expression of ICAM-1. The decreased adherence is linked to down-regulation on monocytes of the activation-dependent epitope of the β2 integrin LFA-1 by SDF-1α. Knockdown of Lyn in monocytes using small interfering RNA decreases SDF-1α-mediated migration and prevents the inhibition of monocyte attachment to ICAM-1 and activated BMVEC. Thus, in SDF-1α-stimulated monocytes, Lyn acts as a positive regulator of migration and a negative regulator of adhesion to BMVEC through the LFA-1 integrin. These results provide a novel Lyn-mediated signaling mechanism for the regulation of monocyte movement at the blood-brain barrier.

Cutaneous T Cell Lymphoma Expresses Immunosuppressive CD80 (B7-1) Cell Surface Protein in a STAT5-Dependent Manner

In this article, we report that cutaneous T cell lymphoma (CTCL) cells and tissues ubiquitously express the immunosuppressive cell surface protein CD80 (B7-1). CD80 expression in CTCL cells is strictly dependent on the expression of both members of the STAT5 family, STAT5a and STAT5b, as well as their joint ability to transcriptionally activate the CD80 gene. In IL-2–dependent CTCL cells, CD80 expression is induced by the cytokine in a Jak1/3- and STAT5a/b-dependent manner, whereas in the CTCL cells with constitutive STAT5 activation, CD80 expression is also STAT5a/b dependent but is independent of Jak activity. Although depletion of CD80 expression does not affect the proliferation rate and viability of CTCL cells, induced expression of the cell-inhibitory receptor of CD80, CD152 (CTLA-4), impairs growth of the cells. Coculture of CTCL cells with normal T lymphocytes consisting of both CD4+ and CD8+ populations or the CD4+ subset alone, transfected with CD152 mRNA, inhibits proliferation of normal T cells in a CD152- and CD80-dependent manner. These data identify a new mechanism of immune evasion in CTCL and suggest that the CD80–CD152 axis may become a therapeutic target in this type of lymphoma.

Src as the link between inflammation and cancer

Although a causal link between chronic inflammation and cancer has been established, the exact molecular mechanism linking inflammation to cancer remains largely unknown. It was previously postulated that molecular switches responsible for cancer cell development, and for infiltration of inflammatory cells into cancer, were divided into a distinct set of intracellular proteins and signaling pathways. However, recent evidence suggests that both tumor cells and tumor-infiltrating immune cells utilize the same kinases, mostly that of Src family, to facilitate cancer development and progression. In the past few years several groups have found that Src activation both in cancer and inflammatory cells is mainly driven by pro-inflammatory cytokines within the tumor microenvironment. Here we evaluate the cross talks between Src kinase pathways in immune cells and cancer cells. We conclude that Src might serve as a critical mechanistic link between inflammation and cancer, mediating and propagating a cycle between immune and tissue cells that can ultimately lead to the development and progression of cancer.

Cadherin-11 Is a Cell Surface Marker Up-Regulated in Activated Pancreatic Stellate Cells and Is Involved in Pancreatic Cancer Cell Migration

Chronic pancreatitis is a prominent risk factor for the development of pancreatic ductal adenocarcinoma. In both conditions, the activation of myofibroblast-like pancreatic stellate cells (PSCs) plays a predominant role in the formation of desmoplastic reaction through the synthesis of connective tissue and extracellular matrix, inducing local pancreatic fibrosis and an inflammatory response. Yet the signaling events involved in chronic pancreatitis and pancreatic cancer progression and metastasis remain poorly defined. Cadherin-11 (Cad-11, also known as OB cadherin or CDH11) is a cell-to-cell adhesion molecule implicated in many biological functions, including tissue morphogenesis and architecture, extracellular matrix-mediated tissue remodeling, cytoskeletal organization, epithelial-to-mesenchymal transition, and cellular migration. In this study, we show that, in human chronic pancreatitis and pancreatic cancer tissues, Cad-11 expression was significantly increased in PSCs and pancreatic cancer cells. In particular, an increased expression of Cad-11 can be detected on the plasma membrane of activated PSCs isolated from chronic pancreatitis tissues and in pancreatic cancer cells metastasized to the liver. Moreover, knockdown of Cad-11 in cancer cells reduced pancreatic cancer cell migration. Taken together, our data underline the potential role of Cad-11 in PSC activation and pancreatic cancer metastasis.