The Biological Effects Of Bcrabl

Exactly how the molecular abnormalities are linked to the biological behavior of the Ph-positive cells and how they determine the leukemic phenotype, is still poorly understood. Our current knowledge suggests that the increased tyrosine phosphokinase activity of the BCR-ABL proteins activates multiple intracellular signaling pathways that regulate cellular proliferation, apop-tosis, cellular differentiation and maturation, as well as interactions with the microenvironment. Inappropriate or untimely activation of one or several of these pathways will eventually tilt the tightly knit balance between these processes. Although much evidence has been accumulated to support involvement of any of these cellular events, their relative contribution to the generation of CML in chronic phase and subsequent changes that take place in the course of transformation remain largely elusive.

One of the most characteristic features of CML is the disorderly expansion of myeloid cells. The normal lineage apportionment is unbalanced in CML in favor of increased granu-lopoiesis and thrombopoiesis. Expansion of these compartments suggests unregulated proliferation of the leukemic cells, increased self-renewal and life span of the Ph-positive progenitor cells, and a decreased fraction of apoptotic cells in the Ph-positive cell pool. However, cytokinetic studies of CML progenitors have shown that CML progenitors have lower mitotic indices and the same DNA synthesis time as the normal progenitors, resulting in a lower fraction of cells that are actively synthesizing DNA and subsequently longer generation times.106 In vitro clonogenic assays have also demonstrated that CML progenitor cells are less capable of producing large colonies compared to normal progenitor cells.107 Eaves and colleagues suggest that "long term culture initiating cells" (LTCIC) of CML progenitors are the leukemic counterpart to LTCIC of normal progenitor cells.108 They have observed that CML LTCIC differ in two important aspects: CML LTCIC have reduced self-renewal capacity and are therefore outgrown by their normal counterparts. However, by virtue of a deregulated cell cycle status, more CML progenitors are in cycle compared to the rather quiescent population of normal early progenitors. At some stage along the evolution of CML, the defects of cell cycle regulation will dominate the behavior of the CML cells and endow the leukemic progenitors with a growth advantage. Subtle morphological abnormalities of CML cells such as nuclear-cyto-plasmic asynchrony, an increase in dysplastic changes, and the presence of chimeric basophil/eosinophil granules are a morphologic correlate to the disordered balance of self-renewal, proliferation, differentiation, and maturation.109 Clarkson and Strife have suggested that although CML cells contain the capacity for complete maturation, maturation is delayed and shifted from the early stem cell compartment to a more committed progenitor cell compartment thus greatly increasing the myeloid cell mass.110 Defects in adhesion molecules or receptors (see below) facilitate release of early myeloid cells in the peripheral blood resulting in the characteristic maturational left shift of CML. Even a small increase in the probability of self-renewal in this stage may then disproportionately increase myeloid mass further.111 Dowding and colleagues112 calculated that an increase in the probability of self-renewal from 0.5 to only 0.55 will result in a doubling of the stem cell pool after eight stem cell division cycles. With a disproportionate shift of CML progenitors into later maturational compartments, the

CML cells may also become less responsive to growth regulatory signals from either cytokines or from the bone marrow microenvironment. Furthermore, factors to curtail cell production at high cell densities may be abrogated and facilitate the acquisition by the CML progenitor cells of a continuous growth advantage over their normal counterparts.111

In addition, BCR-ABL expressing leukemia cells are highly resistant to apoptosis and this mechanism has been suggested as an important aspect in the pathogenesis of CML.113,114 The abrogation of apoptosis resistance by a combination of mutations in the SH2 domain, GRB2 binding site, and an autophos-phorylation site (Y793F) of BCR-ABL lends evidence to the central role of the BCR-ABL protein.115 Hematopoietic progenitor cells or cell lines that express p210BCR-ABL can thus escape growth factor dependency and withstand the damaging effects of exposure to cytotoxic drugs and irradiation.116,117 In contrast, BCR-ABL cannot suppress apoptosis triggered by the activity of cytotoxic T cells, natural killer cells, and lymphocyte-activated killer cells.118,119 The detailed antiapoptotic pathways triggered by BCR-ABL have not been elucidated. They appear to involve RAS, the JAK/STAT pathways, and possibly NFkB and PI3-kinase. Pathways through PI3-kinase and activation of STAT may act by enhancing the expression of the antiapoptotic protein BCL-XL. Horita and colleagues99 demonstrated that interleukin-3-independent expression of BCL-XL is induced by BCR-ABL by activation of STAT5.99 Conversely, inhibition of the kinase activity of BCR-ABL in BCR-ABL-positive cell lines and CML progenitor cell samples from patients could reverse the antiapoptotic activity by suppressing the capacity of STAT5 to interact with the BCL-XL promoter. de Groot and colleagues98 have shown that activation of STAT5 in BCR-ABL transformed cells not only activates the BCL-XL gene product, but also targets the cell cycle regulator cyclin D1.98 The promoters of both cyclin D1 and BCL-XL products contain STAT binding sites to which STAT5 constitutively binds in BCR-ABL transformed cells. BCR-ABL expressing cells have also been shown to block release of mitochondrial cytochrome C and thus inhibit activation of caspase 3 independent from proteins of the BCL-2 family.120 Further inhibition of apoptotic pathways may also play a role in the evolution of CML to blast phase. After inhibition of the BCR-ABL kinase, expression of BCL-XL is downregulated more rapidly in chronic phase than blast phase CML cells. Furthermore, CML cells from patients in blast crisis are more refractory to apoptosis mediated by FAS than are CML cells from patients in chronic phase.121

Stimulation by cytokines and expression of distinct cytokine profiles provide CML progenitor cells with a distinct growth advantage over normal bone marrow cells. Aberrant growth factor production is especially evident in the more advanced stages of CML.122 CML cells from patients in myeloid or undifferenti-ated blast crisis that are cultured on a marrow-adherent layer proliferate continuously and exhibit constitutive growth factor expression [interleukin (IL)-1P, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF)]. Furthermore, experiments showing that progenitor cells from patients with inter-feron-resistant disease have a reduced response to exogenous growth factors and a relatively autonomous growth pattern in colony assays, support the significance of autocrine/paracrine pathways in CML.123 Estrov and colleagues124 were the first to recognize the role of IL-1P as a growth-promoting cytokine in CML. IL-ip synergizes with growth factors either through upregulation of growth factor receptors or production and release of various other cytokines by the hematopoietic stroma or accessory cells. Marrow cells from patients with transformed CML produce high levels of IL-ip. Inhibition of IL-ip by either IL-1 receptor antagonist or soluble IL-1 receptors suppresses CML colony formation. These results suggest that autocrine/ paracrine production of growth factors such as IL-1P alters the relation of the leukemic cells to the adjacent microenvironment and thus influences the proliferative and differentiation state of the leukemic cells. The BCR-ABL proteins also appear to bind directly to growth factor receptors and to activate them by tyrosine phosphorylation. The activated receptors such as IL-3R and c-KIT then recruit further downstream signaling pathways contributing to the deregulation of cellular pathways in CML.125,126

Adhesion defects of hematopoietic progenitor cells to bone marrow stromal elements have been implicated in the pathogenesis of the manifestations of CML.127 It may be responsible for inappropriate release of immature precursors into the circulation, evasion of growth-regulatory mechanisms, and subsequently disinhibition of hematopoietic progenitor cell proliferation. Normal hematopoietic progenitor cells adhere to adhesive ligands of extracellular matrix components such as fibronectin receptors (a4, a5, P1 integrins), heparin sulfate or thrombospondin, or immobilized and secreted growth-regulating cytokines. The interactions are mediated by cell surface receptors on hematopoietic progenitors including the cell surface proteoglycan CD44, or the selectins, phosphatidylinositol-linked lymphocyte-associated antigen 3 (LFA 3). Especially P1-integrin-dependent interactions are crucial for the localization, regulation of adhesion, and migration of hematopoietic progenitors. Integrins are cell surface glycoproteins that consist of an a- and b-subunit. Whereas the a-chain determines ligand specificity, the b-chain initiates signal transduction pathways on binding to the ligand.127 This process generates the recruitment of cytoskeletal adhesion proteins such as actin, talin, paxillin, and FAK. The next step is the activation of adapter proteins and finally of the RAS/MAPK pathway.128 In a series of in vivo experiments, antibodies against a4P1-integrins inhibited homing and engraftment, and caused premature release of myeloid precursors and stem cells (reviewed in Ref. 129). In contrast to normal stem cells, Ph-positive precursors from the marrow of patients with chronic phase CML adhered significantly less to the feeder layer of marrow stromal cells. Ph-positive progenitors may fail to adhere to the bone marrow stroma through a functional defect of fibronectin receptors and subsequent signaling pathways without changes in receptor expression.130 Verfaillie and colleagues131 studied P1-integrin-cytoskeletal interactions in CML and normal CD34-positive primary hematopoietic progenitors, and in BCR-ABL-transfected and mock-transfected MO7e cells. Whereas in normal cells antibody-mediated cross-linking of P1-integrins redistributes them into caps through a process requiring receptor-cytoskeletal interactions, BCR-ABL-expressing cells show significantly impaired P1-integrin capping. Defective P1-integrin capping, increased actin polymerization, and altered actin cytoskeletal organization eventually result in integrin-defective adhesion and disinhibition of proliferation of CML cells. Defective cytoadhesion of CML cells has been restored by preincubation of Ph-positive cells with antisense oligonucleotides against p210BCR-ABL, tyrosine kinase inhibitors targeted against the BCR-ABL tyrosine kinase, and by incubation with interferon-alpha.129

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