Expression of the c-fgr and hck protein-tyrosine kinases in acute myeloid leukemic blasts is associated with early commitment and differentiation events in the monocytic and granulocytic lineages

Department of Cell Biology, University of New Mexico School of Medicine, Albuquerque 87131.
Blood (Impact Factor: 10.45). 03/1991; 77(4):726-34.
Source: PubMed


Two members of the src proto-oncogene family of intracellular tyrosine kinases, c-fgr and hck, are selectively expressed in differentiated myeloid cells. To study the expression of these genes in acute myeloid leukemia (AML) and to determine the specific myeloid lineages and stages of myeloid differentiation at which the expression of these genes is acquired, we used a series of 79 cases of de novo AML as a differentiation model. The levels of c-fgr, hck, and c-fms (encoding the colony-stimulating factor-1 receptor) mRNA transcripts were correlated with the presence of specific cell surface antigens and the morphologic and cytochemical features in these AML blasts. Relatively undifferentiated leukemic myeloblasts with an HLA-DR, CD34, CD33, CD13+/- cell surface immunophenotype (French-American-British [FAB] M1 or M2) were characterized by a lack of c-fms and c-fgr expression, while low levels of c-fms and c-fgr could be detected in undifferentiated myeloblasts (FAB M1 or M2), which also expressed CD14 at low antigen density. The hck transcripts were either undetectable in these cells or were expressed at low levels. In contrast, only hck mRNA transcripts could be identified in blasts with progranulocytic morphology (FAB M3), while c-fms, c-fgr, and hck were all expressed at high levels in blasts with differentiated myelomonocytic or monocytic features (FAB M4 and M5). No c-fms, c-fgr, or hck transcripts were evident in leukemic cells of the erythroid lineage (FAB M6). When undifferentiated leukemic myeloblasts (HLA-DR, CD34, and CD33) were induced to differentiate in vitro to cells with monocytic characteristics, the expression of c-fms, c-fgr, and the CD14 cell surface antigen were induced to high levels, accompanied by the acquisition of hck and CD13 expression. In contrast, when HLA-DR, CD34, and CD33 blasts were induced to differentiate in vitro to cells with granulocytic characteristics, only hck and CD13 expression were induced. Our data suggest that the acquisition of c-fgr and/or hck expression is associated with early commitment and differentiation events in distinct myeloid lineages. Assessment of the expression of these kinases may provide a molecular tool to assign lineage in AML in conjunction with morphology, cytochemistry, and cell surface antigen expression.

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    • "Lyn and Fgr were found to be induced and activated in HL60 cells undergoing either monocytic or granulocytic differentiation (Katagiri et al., 1991; Katagiri et al., 1996; Miyazaki et al., 1993; Notario et al., 1989). Induced expression of Fgr and Hck was also observed during myeloid differentiation of leukemic blasts (Willman et al., 1991), while Fgr was found to be upregulated during myeloid differentiation of normal hematopoietic progenitors (Link and Zutter, 1995; Willman et al., 1987). The induction of SFK activities during myeloid differentiation is consistent with their activation by differentiation-inducing cytokines. "
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    ABSTRACT: The production of mature monocytes/macrophages and granulocytes via myeloid differentiation is a central component of the host defense mechanism against invading microorganisms. However, in myeloid leukemias genetic changes lead to blockade of myeloid differentiation. When this happens, immune responses can become severely impaired and the accumulation of proliferative blasts causes bone marrow crowding and onset of symptomatic leukemia. There is considerable hope that molecular targeting of specific signaling pathways and proteins will prove to be a viable strategy for restoring differentiation potential in myeloid leukemias. Indeed, in acute promyelocytic leukemia, treatment with ATRA can overcome the differentiation blockade and is an effective curative approach. To devise strategies and reagents that can be used to induce differentiation in other myeloid leukemias, it is important to gain an understanding of the molecular pathways that drive the normal differentiation process. Emerging evidence implicates both Src family kinases and the MEK/ERK pathway in regulating myeloid differentiation. It is interesting that Src family kinases appear to be negative regulators of myelopoiesis, while the MEK/ERK pathway is an important positive regulator of both monocytic and granulocytic differentiations. This suggests that pharmacologic inhibitors of SFKs may be of value in restoring or enhancing myeloid differentiation. In this regard, the SFK inhibitor dasatinib has recently been approved by the FDA for use in imatinib-resistant CML. Evaluation of dasatinib, alone or in combination with differentiation inducers, in the treatment of differentiation-defective AML seems warranted. The important role that the MEK/ERK pathway plays in promoting myeloid differentiation appears to conflict with observations that the MEK/ERK pathway is hyperactivated or overexpressed in a majority of primary AMLs. Moreover, pharmacologic inhibition of the MEK/ERK pathway in AML results in the induction of apoptosis, indicating that MEK/ERK activation is important for the survival of AML cells. Collectively, these data suggest that the MEK/ERK pathway may play more than one role in myeloid lineage cells, depending on the cellular context. In normal myeloid cells, activation of the MEK/ERK pathway is important for promoting differentiation. However, when differentiation becomes blocked, as is the case in most AMLs, MEK/ERK activation can no longer drive differentiation, and instead begins to support cellular survival or proliferation. Thus, therapeutic strategies aimed at provoking myeloid differentiation in AML by stimulating the MEK/ERK pathway are unlikely to be successful unless the differentiation blockade is simultaneously relieved.
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    • "There are many indirect evidences that indicate an association between Src kinases and hematologic malignancies, as summarized previously (Li, 2005). (Burnett et al., 1991;Ernould, Ferry, Barret, Genton, & Boutin, 1994;Fischer et al., 1989;Lynch et al., 1993;Myers et al., 1995;O'Connor, Torigoe, Reed, & Santoli, 1992;Tilbrook et al., 2001;Torigoe, O'Connor, Santoli, & Reed, 1992;Uckun et al., 1995;Waki et al., 1994;Willman et al., 1991;Yamaguchi et al., 1997). Direct evidence for the roles of Src kinases in leukaemogenesis is inadequate; recently, we provided convincing evidence that Src kinases are required for proliferation of leukaemic cells in mice with ALL (Hu et al., 2004;Hu et al., 2006). "
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    ABSTRACT: Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.
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    • "Since we reproducibly found that the major proteins which are tyrosine phosphorylated in this experimental system migrate with a molecular weight of '~56-60 kD, we started to address whether they might be represented by members of the src family of intracellular protein tyrosine kinases. As the bands stained by antiphosphotyrosine Abs could well represent different proteins migrating at the same molecular weight as also suggested by their strong reactivity with antiphosphotyrosine Abs, we addressed the issue of a possible 82 integrin-dependent activation of members of the src family by a direct approach based on immune precipitation of p58 f~, a member of the src family which is expressed in myelomonocytic cells (Ley et al., 1989; Notario et al., 1989: Inoue et al., 1990; Willman et al., 1991), and analysis of its enzymatic activity by in vitro kinase assays, and its extent of tyrosine phosphorylation by antiphosphotyrosine immu- noblots. Fig. 2 shows results of in vitro kinase assays (A), and antiphosphotyrosine immunoblots (B) performed on anti- p58 f~ immunoprecipitates from lysates of PMN plated on fibrinogen in the presence or the absence of TNF. "
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    ABSTRACT: Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.
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