Boswell Hs

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) π, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo

Internal tandem duplications (ITDs) in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knockdown of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low-density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.

The effects in vivo of purified preparations of murine macrophage colony stimulating factor-1, recombinant murine granulocyte-macrophage colony stimulating factor and natural and recombinant murine interleukin 3 without and with pretreatment of mice with purified iron-saturated human lactoferrin.

The influence of purified natural colony stimulating factor-1 (CSF-1), purified recombinant granulocyte-macrophage (GM)-CSF, purified recombinant interleukin 3 (IL3) and natural IL3 were assessed in mice that were untreated or pretreated with purified iron-saturated human lactoferrin (LF) in order to first suppress myelopoiesis in the mice. S1/S1d mice responded to recombinant GM-CSF and recombinant IL3 in a manner similar to the response of their +/+ littermates. These 4 factors increased the cycling status of hematopoietic progenitors in vivo. The effects were more noticeable if myelopoiesis was first decreased by LF. The effects do not appear to be due to endotoxin contamination. It cannot be discerned from these studies whether the effects are direct ones on the progenitor cells or indirect ones mediated through growth-factor releasing accessory cells. It is possible that effects can be both direct and indirect.

Syk kinase and Shp2 phosphatase inhibition cooperate to reduce FLT3-ITD-induced STAT5 activation and proliferation of acute myeloid leukemia

Syk kinase and Shp2 phosphatase inhibition cooperate to reduce FLT3-ITD-induced STAT5 activation and proliferation of acute myeloid leukemia

Signal transduction during myeloid cell differentiation.

&NA; The intracellular signaling mechanisms that dictate myeloid differentiation and proliferation are discussed. Independent hematopoietic signaling pathways including p21ras pathway, c‐myc pathway, and Jak‐STAT pathway are defined. Emphasis is given to the process of information integration at the nucleus, by which developmental programs may be converted from binary decisions into the complex response patterns explaining hematopoietic diversity. Coupling between signaling and transcription is emphasized.

DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony-stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated (T) factors (T-granulocyte–macrophage-CSF (T-GM-CSF)) and T-interleukin-3 (T-IL-3)) on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and -IL-3 had enhanced receptor binding, but decreased CSF activity, compared with their FL forms. Importantly, T-GM-CSF and -IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and -IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster-forming cells from patients with acute myeloid leukemia regardless of cytogenetic or molecular alterations and in vivo using animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared with their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.

Phosphatase PRL2 promotes AML1-ETO-induced acute myeloid leukemia

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Genotoxic stresses promote clonal expansion of hematopoietic stem cells expressing mutant p53

Genotoxic stresses promote clonal expansion of hematopoietic stem cells expressing mutant p53

In leukemogenesis by bcr-abl cell cycle progression via cyclin D1 involves cooperation between akt/gsk3β and jnk and is c-jun-mediated

Abstract Leukemic transformation by tyrosine kinase oncogenes (eg. Bcr/Abl) involves c-jun upregulation by c-jun-promoted transcriptional feedback. This has been closely linked to activity of c-jun N-terminal kinase (JNK), which phosphorylates c-jun serines 63, 73 to augment interaction with transcriptional coactivator p300/CBP. However, p21rac-/JNK-independent activation of PI-3-kinase and akt, both downstream of p210 Bcr-Abl, potentially bear upon c-jun and its transcription. Akt phosphorylates and inactivates glycogen synthase kinase 3β GSK3β), which then can no longer mediate inhibitory phosphorylation of the C-terminal jun DNA-binding domain. We transformed IL-3-dependent H7 cells by dual expression of activated gag-akt and RafCAAX (H7gag-akt/RafCAAX 1 & 2). We compared these cells to H7 cells transformed by p210 Bcr-Abl as well as with parental H7 cells, for enzyme activity of JNK and GSK3β. Extracts of these cells were also immunoblotted for quantity of total GSK3β vs. akt-inactivated phosphoserine 9 GSK3β, as well as for total c-jun and JNK-activated phosphoserine 73 c-jun. JNK activity for GST c-jun was 2-fold greater in Bcr-Abl.A54 cells compared with H7gag-akt/RafCAAX 1&2, and yet total c-jun expression was greater in the latter cells, which had more abundant akt-inactivated phosphoserine 9 GSK3β. However, the relative level of phosphoserine 73 c-jun was higher in Bcr-Abl.A54 vs. H7gag-akt/RafCAAX, and both cells overexpressed c-jun relative to H7 parent. Corresponding to the importance of JNK toward c-jun transactivation potential, Bcr-Abl.A54 cells overexpressed the putative c-jun transcriptional target cyclin D1, and these cells underwent more rapid cell cycle transit. Confirmation was sought for the importance of JNK in c-jun-dependent cyclin D1 expression by transfecting and overexpressing a dominant-negative mutant (serines 63,73 to alanine) c-jun not phosphorylatable by JNK (JNP). In these cells with JNP and p210 Bcr-Abl, cyclin D1 expression was blunted and proliferation was slowed. Taken together, these data point to collaboration of JNK-independent/akt-mediated and JNK-dependent pathways in leukemic transformation, acting on quantitative and qualitative levels, respectively, for c-jun regulation of cell cycle progression.

AP-1/C-jun and C-myc Regulation During Megakaryocytic Differentiation of a Human Bi-potential Growth-factor-dependent Cell Line

Terminal megakaryocytic development is characterized by nuclear poly ploidization, appearance of specific granules, and enhanced expression of membrane platelet glycoproteins. We utilized a human GM-CSF-dependent cell line, MB-02, which is capable of under going megakaryocytic differentiation, to examine the molecular events underlying this process. The responses of MB-02 to the protein kinase C (PKC) agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were examined. GM-CSF dependent proliferation of MB-02, as measured by (3)H-thymidine uptake, was greater than 95% inhibited by TPA (16 nM), but was not affected by the inactive stereoisomer, 4-α-phorbol-12,13-didecanoate (4-αPDD). Transient exposure of cells to GM-CSF after growth factor deprivation led to rapid, high-level expression of normal-sized c-myc mRN A transcripts above baseline. C-myc expression was turned off by TPA (16 nM) stimulation of cells within 2-4 h. This TPA-mediated effect likely occurred at the transcriptional level since the half life of c-myc mRN A induced by GM-CSF was less than 30 min. Treatment of cells with TPA was associated with induction of c-jun and junB mRN A within 1-4 h. The protein products of these transcription factors are known to be part of the transcription factor complex Activator protein 1 (AP-1). Indeed, our data prove a rapid induction of AP-1 protein after TPA stimulation, as shown by mobility shift assays. In addition, TPA treatment resulted in expression of platelet surface glycoprotein IIb/IIIa complex (gpIIb/IIIa). These studies suggest a link between PKC stimulation by TPA and AP-1 activation with downregulation of c-myc transcription on a molecular level. At the cellular level, PKC activation was related to the acquisition of several features of the megakaryocyte development programme associated with the switch from cell proliferation to maturation.