REVIEW Signal Transduction, Cell Cycle Regulatory, and Anti

Leukemia (1999) 13, 1109–1166  1999 Stockton Press All rights reserved 0887-6924/ 99 $12.00 http:/ / www.stockton-press.co.uk/ leu REVIEW Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs <ul style="display: flex;"><li style="flex:1">WL Blalock1, C Weinstein-Oppenheimer1,2, F Chang1, PE Hoyle1, X-Y Wang3, PA Algate4, RA Franklin1,5, SM Oberhaus1,5 </li><li style="flex:1">,</li></ul>LS Steelman1 and JA McCubrey1,5 1Department of Microbiology and Immunology, 5Leo Jenkins Cancer Center, East Carolina University School of Medicine Greenville, NC, USA; 2Escuela de Qu´ımica y Farmacia, Facultad de Medicina, Universidad de Valparaiso, Valparaiso, Chile; 3Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN, USA; and 4Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Over the past decade, there has been an exponential increase growth factor), Flt-L (the ligand for the flt2/3 receptor), erythropoietin (EPO), and others affect the growth and differentiation in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. of these early hematopoietic precursor cells into cells of the Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified myeloid, lymphoid and erythroid lineages (Table 1).1–4 This review will concentrate on IL-3 since much of the knowledge of how cytokines affect cell growth, signal transduction, and apoptosis has been elucidated from research with IL-3-dependent cell lines. Some other cytokines (eg GM-CSF and IL-5) exert their effects through similar mechanisms. by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malig- nant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate mul- tiple receptors and be more effective in stimulating the repopu- lation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently exam- ined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the poten- tial to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated. IL-3 was initially defined by its ability to induce the enzyme 20-␣-hydroxysteroid dehydrogenase in cultures of splenic lymphocytes from nude mice.5 However, it soon became apparent that IL-3 was being studied by a number of investigators under a variety of aliases. It was called persisting cellstimulating factor (PSF),6 mast cell growth factor (MCGF),7 hematopoietic cell growth factor (HCGF),8 histamine-producing cell-stimulating factor,9 multi-colony stimulating factor (multi-CSF),10 Thy-1-inducing factor,5 and burst promoting activity (BPA).11 All of these growth stimulatory activities were subsequently identified as the same protein and renamed IL-3. IL-3 was shown to act on both myeloid and lymphoid lineages by in vitro studies. In vivo administration of pharmacological doses of recombinant IL-3 to mice resulted in the increased production of red blood cells, leukocytes and platelets.12 Moreover, over-expression of the IL-3 gene in hematopoietic progenitors via retroviral transduction of bone marrow cells resulted in a non-neoplastic, myeloproliferative syndrome in vivo.13 To further understand the role of IL-3 in vivo, transgenic mice expressing antisense IL-3 RNA were created. These mice, which exhibited decreased serum levels of IL-3, developed either a B cell lymphoproliferative syndrome or a neurological dysfunction.14 In more recent studies with IL-3- deficient mice, a role for IL-3 in contact hypersensitivity was observed. IL-3 was determined to be necessary for efficient priming of hapten-specific contact hypersensitivity.15 Keywords: cytokines; signal transduction; anti-neoplastic drugs; oncogenes; apoptosis Cytokines and hematopoiesis Cytokines stimulate cell cycle progression, proliferation, and differentiation, as well as inhibit apoptosis of hematopoietic cells.1–3 Peripheral blood cells are generated from self-renewable, pluripotential hematopoietic stem cells in the bone IL-3 transgenic mice have also been used to examine the effects of constitutive IL-3 expression on development and neoplasia. Recent evidence indicative of neurological dysfunction in IL-3-transgenic mice supports the idea that this hematopoietic cytokine performs key roles in the central nervous system as well.14–19 The pleiotropic roles of this cytokine must be considered when therapies are developed based upon alteration of IL-3 expression, down-regulation of cognate receptor expression and function, or destruction of IL-3 receptor (IL-3R)-positive cells by chimeric IL-3-toxin molecules. <ul style="display: flex;"><li style="flex:1">marrow. </li><li style="flex:1">Cytokines </li><li style="flex:1">such </li><li style="flex:1">as </li><li style="flex:1">interleukin-3 </li><li style="flex:1">(IL-3), </li></ul>granulocyte/macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF, or steel factor, c-Kit-L, macrophage Correspondence: JA McCubrey, Department of Microbiology and Immunology, East Carolina University School of Medicine, Brody Building 5N98C, Greenville, NC 27858, USA; Fax: 252-816-3104 Received 2 March 1999; accepted 14 May 1999 Review WL Blalock et al 1110 Table 1 Abbreviations of cytokines, growth factors, their receptors and cellular targets <ul style="display: flex;"><li style="flex:1">Abbreviation </li><li style="flex:1">Full name </li><li style="flex:1">Target cells or function </li></ul>EGF epo Epidermal growth factor Erythropoietin Affects the growth of many cells Erythroid cells flt-2/flt-3L G-CSF Ligand for the flt2/flt3 receptor Early hematopoietic progenitor cells Granulocyte-colony stimulating Granulocyte/monocyte progenitor cells factor <ul style="display: flex;"><li style="flex:1">GM-CSF </li><li style="flex:1">Granulocyte/macrophage- </li></ul>colony stimulating factor Many early hematopoietic precursor cells of the myeloid lineage, neutrophilic granulocytes, monocyte–macrophages, eosinophils; has the ability to activate macrophages <ul style="display: flex;"><li style="flex:1">gp130 </li><li style="flex:1">Glycoprotein 130 </li><li style="flex:1">Signal transducing receptor protein shared among IL-6, LIF, oncoM and other </li></ul>cytokines IL-3 IL-3R IL-4 IL-5 LIF Interleukin-3 Interleukin-3 receptor Interleukin 4 Interleukin 5 Leukemia inhibitory factor Many early hematopoietic precursor cells of both myeloid and lymphoid lineages Present on many early hematopoietic cells B cells, mast cells, some T cells B cells, eosinophils Homology with oncoM, inhibits proliferation of tumor cell lines, affects a broad range of cells M-CSF oncoM SCF Macrophage-colony stimulating Granulocyte–monocyte progenitor, receptor is the product of the c-fms protofactor Oncostatin M oncogene 28-kDa protein with homology to LIF, inhibits proliferation of tumor cell lines, affects a broad range of cells, expression induced by Jak/STAT pathway Stem cell factor, also known as Many early hematopoietic cells, receptor is the product of the c-kit proto-oncogene, c-kit ligand stimulates proliferation of myeloid, erythroid and lymphoid progenitors Transforming growth factor-beta Often inhibits cell proliferation, wide tissue distribution Thrombopoietin Growth and differentiation factor for megakaryocytes, receptor is the c-mpl protooncogene TGF-␤ TPO Recombinant/chimeric cytokines and therapy tration of daniplestim and G-CSF results in higher numbers of short-term and long-term clonogenic cells compared to sequential administration of daniplestim and G-CSF.29 In the rhesus myelosuppression model, daniplestim accelerated hematopoietic reconstitution in radiation-induced cytopenia. Daniplestim significantly reduced the nadir of neutropenia and the duration of thromocytopenia in animals with radiation-induced myelosuppression.27 Taken together, these data suggested that daniplestim may be clinically useful in patients with chemotherapy-induced myelosuppression and support the utility of combination therapy. Some of the chimeric cytokines developed by Searle include the myelopoietin (MPO) family (IL-3 and G-CSF receptor agonists), the promegapoietin (PMP) family (IL-3 and TPO receptor agonists) and the progenipoietin (ProGP) family (Flt3 and G-CSF receptor agonists). These chimeric molecules were developed based on the concept that a single molecule with bifunctional activities could provide synergy, that is an enhancement in activity that is greater than the addition of molecules with monofunctional activities.33,34 Additional enhancement in activity was achieved through circular permutation of the molecules such that the termini of the protein sequence were ‘linked’ and new termini were generated by ‘breaking’ the sequence at a new location. Such a process could relieve conformational constraints, add flexibility, result A variety of engineered recombinant cytokines are currently under investigation for use in hematopoietic reconstitution of <ul style="display: flex;"><li style="flex:1">patients </li><li style="flex:1">undergoing </li><li style="flex:1">myelosuppressive </li><li style="flex:1">chemotherapy </li></ul>(Table 2).20–47 A fusion molecule containing IL-3 and GM-CSF (PIXY 321) developed by Immunex activates both the IL-3 and GM-CSF receptors.22–25 GD Searle Pharmaceuticals has developed a structurally and functionally distinct IL-3 receptor agonist (daniplestim) which binds to the IL-3 receptor with higher affinity than native IL- 3 but has reduced inflammatory effects.26–29 Daniplestim is a truncated form of hIL-3 but has complete biological activity. Daniplestim binds to the IL-3 receptor ␣/␤ complex with 20- fold greater affinity compared to native IL-3.26 Daniplestim induced cell proliferation with 10-fold greater potency than native IL-3 in the human acute myelogenous leukemia AML193 cell line. In colony-forming unit (CFU) assays using human bone marrow CD34+ cells, daniplestim demonstrated 22-fold greater potency than native IL-3.27 The in vivo utility of daniplestim was evident in the rhesus mobilization and myelosuppression models.28,29 In the rhesus mobilization model, the combination of daniplestim and G- CSF mobilized higher and sustained levels of stem cells in the peripheral blood. These studies demonstrated that coadminis- Table 2 Abbreviations of recombinant/chimeric cytokines and drugs and their sources <ul style="display: flex;"><li style="flex:1">Brief description </li><li style="flex:1">Name </li><li style="flex:1">Source </li></ul>Daniplestim Leridistim Modified IL-3 with higher affinity binding to the IL-3 receptor than unmodified IL-3 Monsanto/Searle <ul style="display: flex;"><li style="flex:1">IL-3/G-CSF chimera </li><li style="flex:1">Monsanto/Searle </li></ul><ul style="display: flex;"><li style="flex:1">Immunex </li><li style="flex:1">PIXY-321 </li><li style="flex:1">Fusion cytokine which binds both the IL-3 and GM-CSF receptors </li></ul><ul style="display: flex;"><li style="flex:1">Flt-3L/G-CSF chimera </li><li style="flex:1">Progenipoietin </li></ul>Promegapoietin Monsanto/Searle <ul style="display: flex;"><li style="flex:1">Monsanto/Searle </li><li style="flex:1">IL-3/TPO chimera </li></ul>Review WL Blalock et al 1111 in different conformational constraints and introduce a new structure–activity relationship.33,34 In addition, permutation of a molecule with two functional entities could impart an advantage with respect to the relative positioning of the two molecules as they bind to their respective receptors. In CFU assays, MPOs demonstrate greater potency than the coaddition of native IL-3 and G-CSF.33 In rhesus myelosuppression model, MPOs have been shown to be more effective in repopulating specific hematopoietic cell compartments than a combination of native IL-3 and G-CSF.36,37 In addition, MPOs effectively mobilize CD34+ and clonogenic cells in rhesus monkeys.36–38 In phase I/II clinical trials in breast cancer or lymphoma patients, MPOs are well-tolerated mobilizing agents.39–41 PMPs support the differentiation of progenitors along the megakaryocytic lineage and induces expansion of CD41+ cells in vitro.42–44 In rhesus myelosuppression model, PMPs significantly improve the platelet nadir and accelerates platelet recovery.45 In CFU assays, ProGPs demonstrate greater potency than coaddition of native Flt3 ligand and G-CSF suggesting a cooperativity of hematopoietic activities in a chimeric molecule.46,47 In mice and rhesus monkeys, ProGPs mobilize substantial numbers of hematopoietic stem cells into the peripheral blood.37,48 In rhesus myelosuppression model, ProGPs also support multilineage hematopoietic reconstitution.37 Interestingly, ProGPs have recently been shown to mobilize a large number of functional dendritic cells suggesting that it may also be effective in this area of immunotherapy.46,47 In summary, these chimeric cytokines represent a novel line of biotechnology, which shows significant promise in the treatment of certain leukemia patients. ases ␣ and ␤ (I-␬K␣ and I-␬K␤).55 These kinases phosphorylate I-␬B on serine residues which results in degradation of the protein and allows NF-␬B to enter the nucleus and transactivate gene expression. The I-␬ kinases are activated by NF-␬B inducing kinase (NIK) and the mitogen-activated protein kinase kinase kinase-1 (MEKK1).55 15-Deoxyspergualin (DSG), an immunosuppressive drug currently in phase I/II clinical trials may represent one approach to inhibiting NF-␬B activation. DSG inhibits the localization of heat shock protein 70 (Hsp 70) to the nucleus in response to heat stress, as well as the activation of NF- ␬B, through its interaction with Hsp70.56 Another approach to inhibiting NF-␬B activation involves introducing adenoviral vectors which overexpress I-␬B.57 This gene therapeutic approach may prove beneficial in the suppression of tumor growth. Increased levels of intracellular Ca2+ following TCR aggre- <ul style="display: flex;"><li style="flex:1">gation allows calmodulin to activate calcineurin, </li><li style="flex:1">a</li></ul>serine/threonine phosphatase.53 Activated calcineurin dephosphorylates the cytoplasmic (c) form of the transcription factor, NF-ATc (nuclear factor of activated T cells) enabling NF-AT to translocate to the nucleus (n). This results in the transactivation of cytokine gene expression, including IL-3 and GM- CSF whose promoters contain NF-AT binding sites.59–74 The immunosuppressive drugs cyclosporin A (CsA) and FK506 mediate their activity by inhibiting calcineurin activation, thereby preventing the dephosphorylation of NF-ATc58,74 (Table 4). An illustration of the regulation of IL-3 gene expression is presented in Figure 1. Similar mechanisms mediate the expression of IL-2, GM-CSF and other T cellderived cytokines. In addition to stimulating proliferation and differentiation of hematopoietic cells, cytokines such as IL-3 also promote cell survival. IL-3-dependent cells undergo apoptosis after withdrawal of IL-3 for a prolonged period of time (12 to 48 h depending upon the cell type and species of origin).48,49 However, addition of IL-3 to IL-3-deprived cells can prevent apoptosis in a significant proportion of these cells.48,49 These anti-apoptotic functions of IL-3 and other important cytokines are critical concerns in therapeutic cytokine intervention, especially with patients having certain leukemias and minimal residual disease. There are additional pathways by which activated PKC can stimulate cytokine gene expression. PKC can activate the Ras pathway by inactivating the GTPase activating protein (GAP), a negative regulator of Ras.61,68–70 Ras is a member of a large multi-gene family, which encodes small GTP-binding proteins that serve as molecular switches. Inactivation of GAP stimulates Ras activity, which results in an enhancement of activator-protein-1 (AP-1) binding activity.61,68–70 AP-1 can then stimulate cytokine gene expression, including IL-3 (Figure 2a). Interestingly, the neurofibromatosis-1 (NF1) gene, a tumor suppressor frequently lost in juvenile chronic myelogenous leukemia (CML), is functionally related to GAP.77,78 NF1 likely serves to block Ras activation, thus its loss leads to constitutive Ras activation and contributes to the generation of CML. Ras is frequently targeted by anti-neoplastic drugs including farnesyl transferase (FT) inhibitors (see below). Addition of a farnesyl group is necessary for Ras localization to the cytoplasmic membrane. Drugs, which block Ras farnesylation, are currently being developed by pharmaceutical companies for therapeutic use (eg Janssen, Merck).79 Regulation of IL-3 expression Most hematopoietic cells do not usually synthesize the 26- kDa IL-3 protein. In those cells that do express the IL-3 gene, the gene is normally under stringent controls.50–76 In peripheral blood, activated T cells, natural killer cells, mast and some megakaryocytic cells can synthesize IL-3.50–53 For optimal IL-3 expression, T cells must be activated via the T cell receptor (TCR)/CD3 pathway, or by agents that mimic this pathway, eg the combination of PMA and calcium ionophores.50–53 When a T cell is activated, aggregation of the TCR/CD3 complex occurs (Table 3). Receptor aggregation is followed by a complex series of biochemical events leading to the activation of protein kinase C (PKC) and a rise in the concentration of intracellular Ca2+.50–68 Activated PKC can phosphorylate and inactivate the repressor protein, inhibitor ␬B (I-␬B), thus permitting I␬B to disassociate from nuclear factor-␬B (NF-␬B).54 This allows NF-␬B to assume an active form (Figure 1) which subsequently enters the nucleus and transactivates cytokine gene expression. In addition, there is a complex of proteins which phosphorylates I-␬B, the I-␬B kin- Transcriptional regulation of IL-3 expression The cis-acting elements of the human IL-3 promoter include two activation regions separated by an inhibitory region.59–74 These genetic elements lie within a region that extends to ෂ300 bp upstream of the transcription start site (Figure 2a). Inhibitory elements in the IL-3 promoter which suppress IL-3 transcription include a NIP (nuclear inhibitory protein) binding sequence located between bp −271 to −250.59 Figure 2 depicts some of the transcription factor binding sites involved in the regulation of IL-3 expression. Sequence motifs common to many cytokine promoters,

REVIEW Signal Transduction, Cell Cycle Regulatory, and Anti

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