Signaling In Leukemia Cells Research Articles

CD5 does not regulate the signaling triggered through BCR in B cells from a subset of B-CLL patients

CD5 is a transmembrane protein expressed on all T lineage cells and a subset of B cells. It is known that CD5 is physically associated with the T-cell receptor and B-cell receptor (BCR), inhibiting the signaling triggered by both of them. CD5 is also characteristic of B-chronic lymphocytic leukemia (B-CLL) B cells, although its implication in the development of this lymphoproliferative disorder has not been studied. In the present study, we examined the effect of CD5 in apoptosis, cell viability and global protein tyrosine phosphorylation mediated by BCR in B cells from B-CLL patients. As opposed to tonsil B cells, we did not observe an increase in the apoptotic or viability signals induced by anti-immunoglobulin M or SAC/interleukin-2 when CD5 was dissociated from BCR in leukemic cells of the majority of patients. We also observed that CD5 did not regulate the BCR-induced phosphotyrosine pattern in B-CLL B cells. These findings suggest that CD5 does not inhibit properly the BCR-mediated signaling in leukemic cells. This defect in inhibiting the BCR might contribute to the enhanced survival of B-CLL B cells.

The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.

FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.

ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia

AbstractThe zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL. (Blood. 2005;106:2506-2512)

Mesenchymal Stem Cells Promote Survival of Leukemic Cells Via Integrin-Linked Kinase (ILK)-Dependent Akt and STAT3 Activation: Implications for Leukemia Therapy.

The β integrins play an important role in the cell-to-cell interactions, which trigger intracellular signal transduction pathways. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate Akt, which promotes cell survival. On the other hand, PI3K/Akt and JAK/STAT signaling pathways are also recognized as potent anti-apoptotic mediators activated by ligation of growth factor receptors. We have previously demonstrated that stroma cells protect acute promyelocytic leukemic (APL) cells from apoptosis (Tabe, Blood 103:1815–1822, 2004). Here, we investigate the ability of bone marrow stroma cells to activate Akt, STAT3, and ILK signaling in leukemic cells co-cultured with stroma in low-serum conditions (0.5% FCS). Human mesenchymal stem cells (MSC), co-cultivated with APL-derived NB4 cells in direct cell-to-cell contact, partially inhibited spontaneous apoptosis and enhanced viability of NB4, while separation from stromal cells by transwell insert abrogated this supporting effect of MSC. Western blot analysis using phosphospecific antibodies demonstrated that direct cell-to-cell contact with MSC caused strong activation of Akt and STAT3 signaling in NB4 cells, which have low baseline phosphorylation of these proteins. Treatment with PI3K inhibitor LY294002 or JAK/STAT3 inhibitor (AG480) decreased both, Akt and STAT3 activation in NB4 cells, however, in cells co-cultured in direct contact with MSC the Akt and STAT3 phosphorylation levels were still significantly higher than in suspension cultures and in cells separated by transwell. These observations indicate cross-talk between PI3K/Akt and JAK/STAT pathways, and that Akt is activated independent from PI3K in NB4 cells through direct interaction with MSC. Both, LY294002 and AG480 induced apoptosis and decreased viability of suspension NB4 cells, but this effect was partially abrogated by MSC co-culture. Next, we examined the effects of these signal transduction inhibitors on MSC. MSC expressed both, phospho-Akt and phospho-Stat3, which was inhibited by LY294002 and AG480. LY294002 but not AG480 induced moderate apoptosis in MSC (annexin V positivity; MSC alone19.7 %; LY294002, 30.8%; AG480, 20.1% at 72 hours). Finally, we investigated Akt and STAT3 activation associated with ILK in NB4 cells. Treatment with ILK inhibitor KP004 (QLT Inc., Vancouver, Canada) decreased phosphorylation of Akt and STAT3 only in NB4 cells co-cultured with MSC and not in suspension cultures. The specific abrogation of MSC-mediated signaling resulted in higher induction of apoptosis in stroma co-cultured cells compared to suspension cells (annexin V positivity; KP004 treated suspension cultures 47.4±4.3%; MSC co-cultures 64.9±10.3%). These results indicate that bone marrow stroma cells support survival of leukemic cells through β integrin linked ILK, which activates Akt in a PI3K-independent manner and also stimulates STAT3. We propose that abrogation of ILK/Akt and STAT3 signaling may overcome protective effects of the bone marrow microenvironment on APL cells and thereby greatly enhance anti-leukemic therapies.

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis.

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.

Blockade of Fas-dependent apoptosis by soluble Fas in LGL leukemia

Altered expression of the Fas-Fas ligand apoptotic pathway leads to lymphoproliferative and autoimmune diseases. In lpr/lpr mice and children with autoimmune lymphoproliferative syndrome, defective apoptosis is due to Fas mutations. Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disorder associated with rheumatoid arthritis. Leukemic LGLs are resistant to Fas-dependent apoptosis despite expressing high levels of Fas. Such resistance can be overcome by activating leukemic LGLs in vitro, suggesting inhibition of Fas signaling in leukemic cells. We report that sera from patients with LGL leukemia contain high levels of soluble Fas. Ten of these 33 patients with LGL leukemia also had rheumatoid arthritis. Cloning and sequencing revealed expression of multiple Fas messenger RNA variants in leukemic LGL. These Fas variants, including 3 newly described here, encode soluble Fas molecules. Supernatants from cells transfected with these Fas variants blocked Fas-dependent apoptosis of leukemic LGLs. These results suggest that blockade of Fas-signaling by soluble Fas may be a mechanism leading to apoptosis resistance in leukemic LGLs.

Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells.

This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO-dependent survival (but it can promote survival when activated).