Cellular signalling pathways: new targets in leukaemia therapy.

Abstract

Cellular function, proliferation, differentiation and death are regulated through a number of extracellular stimuli which mediate the transcription of specific genes, either directly or indirectly via specific intracellular pathways (Alberts et al, 1994). In leukaemia the mediators at work are primarily haematopoietic growth factors and cytokines (Table I). These small polypeptides or glycoproteins play pivotal roles in normal and neoplastic cell proliferation by binding to their cell surface receptors and thus activating signal transduction pathways leading to gene transcription (Carpenter et al, 1999; Shuai, 1999). Three general families of cell-surface receptors have been characterized by their modes of action: ion channel-linked receptors, G-protein-linked receptors and enzyme-linked receptors. The first two families are involved in processes such as neurotransmitter signalling and are of little known significance in cytokine action. The enzyme-linked receptors, however, acting either directly as enzymes or in association with enzymes, activate intracellular events and play important roles in cytokine function. Five known classes of enzyme-linked receptors have been described: (1) receptor guanylyl cyclases, (2) receptor tyrosine phosphatases, (3) receptor serine/threonine kinases, (4) receptor tyrosine kinases, and (5) tyrosine kinase-associated receptors (Alberts et al, 1994). Most growth factors and cytokines bind to receptor tyrosine kinases and tyrosine kinase-associated receptors on the cell surface. Some tyrosine kinase-associated receptors have no inher- ent kinase activity but can regulate the haematopoietic system by activating a variety of non-receptor protein tyrosine kinases; these have been named the cytokine receptor superfamily (Bazan, 1990; Ihle et al, 1998). As aberrant signalling through these receptors and their downstream pathways plays a significant role in leukaemogenesis, their modulation will probably result in therapeutic benefits (Frank, 1999). Studies directed at understanding interferon (IFN) function have provided crucial information on the signalling pathways activated by IFNs and several other cytokines (Darnell et al, 1994; Ihle et al, 1998; Frank, 1999; Shuai, 1999). These pathways involve the activation of a family of non-receptor tyrosine kinases called Janus kinases (Jaks), and the recruitment by Jaks of another family of proteins called signal transducer and activator of transcription (Stat) that induce gene transcription (Decker, 1999; Shuai, 1999). As Jak-Stat pathways are utilized by a variety of growth factors, cytokines and hormones (Darnell et al, 1994; Taniguchi, 1995; Shuai, 1999), better understanding of their role in normal haematopoiesis and in leukaemogenesis will probably lead to more selective and less toxic therapies. This approach has already been proven to be effective in initial studies of the drug STI571 in chronic myelogenous leukaemia (CML) (Druker et al, 1999; Talpaz et al, 2000). The cytokine receptor superfamily is divided into four subfamilies on the basis of the characteristic structural motifs found in their extracellular binding domains (Fig 1A) (Bazan, 1990; Alberts et al, 1994; Taniguchi, 1995; Ihle et al, 1998). These extracellular domains are characterized by the presence of conserved motifs, suggesting their common evolution to utilize common signalling pathways. Many cytokine receptors consist of two or more subunits (named α, β and γ) with one subunit common to several cytokine receptors (Taniguchi, 1995). For example, in the gp130 subfamily, the receptors for interleukin 6 (IL-6), IL-11, oncostatin M (OSM), leukaemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) all signal through a common βc-chain called gp130 (Fig 1B). The sharing of receptor subunits allows the cytokine receptors to initiate biological cytokine activities that are both specific and redundant (Taniguchi, 1995; Carpenter et al, 1999). The cytokine receptor superfamily. (A) Cytokine receptors subfamilies. Receptor components shared by different subtypes are shown in the same colour. For example, members of the type 1 receptor group are characterized by the presence of multiple fibronectin type III repeats in the extracellular domain. Type 1 receptors include the gp130 family, the IL-2 receptor, the gp140 family and the GH receptor. Type 2 receptors include interferon and IL-10 receptors. Type 3 receptors include members of tumour necrosis factor (TNF) and Fas receptors. Type 4 includes the IL-1 receptor. (B) The gp130 receptor family are compared with receptor tyrosine kinases (RTK). Note that some members of the RTK family, independently of Jaks, can mediate Stat activation. The receptors for IL-12, granulocyte colony-stimulating factor (G-CSF) and leptin have a similar extracellular binding domain and are therefore classed in the same family, although no clear association of these receptors with gp130 has been clearly demonstrated. All receptors of the gp130 family, with the exception of IL-12 receptor, activate Stat3. Dimerization of the cytoplasmic component of the cytokine receptor is the necessary next step in the initiation of cellular signalling (Carpenter et al, 1999). The dimerized units then associate with intracellular non-receptor tyrosine kinases such as members of the Src or the Jak families of kinases to further propagate the signal (Fig 1A). Different Src family members are associated with different receptors and phosphorylate distinct but overlapping sets of proteins (Stahl et al, 1993; Alberts et al, 1994). For example, Lck, Lyn and Fyn have been shown to be activated by IL-2 and to associate with the same region of the IL-2 receptor (Kobayashi et al, 1993). Src kinases can also associate with receptors having intrinsic kinase activity, thus accounting for the overlap seen between the pathways activated by the Src family of tyrosine kinase-associated receptors and by the receptor tyrosine kinases. The Jak family of kinases (Jak1, Jak2, Jak3 and Tyk2) includes relatively large proteins that are able to bind cytokine receptor β components. (Duhe & Farrer, 1998; Ihle et al, 1998; Yeh & Pellegrini, 1999; Ward et al, 2000). The highly conserved structure of the Jak family members consists of seven Jak homology (JH) domains (Taniguchi, 1995; Yeh & Pellegrini, 1999). The JH1 domain at the carboxy terminus of the protein includes a domain with tyrosine kinase activity (Fig 2A). In general, Jak proteins are divided into an amino-terminal region (N), a catalytically inactive kinase-like (KL) domain and the tyrosine kinase (TK) domain (Yeh & Pellegrini, 1999). The cytokines, growth factors and hormones that activate Jaks are listed in Table II. Jaks are able to associate non-covalently with the cytokine receptors through their cytoplasmic domain as well as with each other (Livnah et al, 1999; Remy et al, 1999). This association results in transphosphorylation and activation of the kinase activity of Jaks, and phosphorylation of other downstream targets (Fig 2B). The Jak-Stat pathway. (A) Domains of Jak protein. JH1 is the catalytic domain with tyrosine kinase (TK) activity. KL is a catalytically inactive kinase-like domain with unknown function. SOCS are suppressors of cytokine signalling which inhibit Jaks. RBR is the receptor binding region. ‘SH2’ is a SH2-like domain with unidentified function. (B) Mechanism of Stat activation by cytokine receptors and their negative regulation by SOCS. Ligand binding and receptor dimerization result in Jak activation, Stat dimerization, and translocation into the nucleus and transcription of genes including those coding for SOCS. The latter have a negative regulatory effect on the pathway. The crucial role of Jaks in cytokine signalling has been established in mouse knockout models. Newborn mice with homozygously disrupted Jak1 are small, die early, and are profoundly deficient in thymocyte production and B-lymphocyte differentiation (Rodig et al, 1998). Jak2-deficient mice embryos do not respond to various haematopoietic cytokines, respond poorly to colony-stimulating factor 1 (CSF-1) and stem cell factor (SCF), and die of severe anaemia as a result of ineffective erythropoiesis (Neubauer et al, 1998; Parganas et al, 1998). Jak3-deficient mice have profoundly defective immune systems characterized by a hypoplastic thymus, non-functional peripheral T cells and a lack of natural killer cells among other defects (Thomis et al, 1995; Eynon et al, 1996). Several disorders are associated with Jak-related abnormalities. These include severe combined immunodeficiency (SCID) due to Jak3 mutations (Notarangelo, 1996), leukaemia due to chromosomal translocations resulting in TEL/JAK2 constructs causing constitutive Stat5 activation, and IL-3-independent cellular proliferation (Lacronique et al, 1997; Peeters et al, 1997). Furthermore, infection with human T-lymphotropic virus 1 (HTLV-1) and Abelson murine leukaemia viruses results in enhanced tyrosine kinase activity of Jaks, which may account for their leukaemogenic potential (Danial et al, 1995; Migone et al, 1995). Jak proteins interact with a number of intracellular signalling proteins, most of which undergo tyrosine phosphorylation upon cytokine stimulation and are therefore likely to be substrates and downstream effectors of Jaks (Table III). Although the biological effects of most of these interactions are still unknown, it is hypothesized that Jaks regulate many cytokine signalling events. The Stat proteins were first identified in the late 1980s in studies aimed at understanding the molecular mechanisms of IFN-mediated effects. These studies identified a number of genes whose transcription was induced by IFN-α and IFN-γ. Specific DNA elements were found to bind proteins such as interferon-α-stimulated gene factor-3 (ISGF-3) (Fu et al, 1992; Khan et al, 1993). These factors, located in the cytoplasm, are rapidly translocated to the nucleus and bind to their cognate DNA sites upon IFN stimulation (Shuai et al, 1992). ISGF-3 consists of three proteins: p48, p91/p84 and p113. The latter two are related proteins also known as Stat1α/β and Stat 2 respectively (Schindler et al, 1992). Five additional Stat proteins (Stat3, Stat4, Stat5A, Stat5B and Stat6) have been identified and their activation by various cytokines documented (Table II) (Hou et al, 1994; Yamamoto et al, 1994; Zhong et al, 1994; Mui et al, 1995). Stats are transcription factors whose activities are modified and regulated by tyrosine phosphorylation (Shuai et al, 1993a; Darnell, 1997). This process has been shown to be necessary for Stat homo- or heterodimerization, translocation to the nucleus, DNA binding and gene activation (Fig 2B) (Shuai et al, 1994; Zhong et al, 1994). All Stat proteins share conserved domains important for their function (Shuai, 1999). The SH2 domain is necessary for Stat tyrosine phosphorylation (Shuai et al, 1993b). The binding of a cytokine to the α subunit of the cytokine receptor rapidly induces tyrosine phosphorylation of the cytoplasmic domains of the receptor β subunit by activated Jak kinases, thus providing a docking site for Stat proteins (as well as other SH2-containing proteins such as the growth factor receptor binding protein 2, or Grb2). The SH2 domain of a Stat protein is thought to bind the tyrosine-phosphorylated residue of the cytokine β receptor with specific cytokine receptors activating specific Stats (Uddin et al, 1995; Yan et al, 1996). The SH2 domains are also necessary for Stat dimer formation (Heim et al, 1995; Carpenter et al, 1999; Decker, 1999; Shuai, 1999) and the N-terminal region of the Stat molecule for DNA binding (Vinkemeier et al, 1996; Xu et al, 1996). The differential DNA binding affinities of different Stat proteins are important in achieving specificity in Stat-mediated gene activation. To induce gene transcription, Stat proteins interact with other transcription factors such as the p300/CREB binding protein (CBP) family of co-activators, which requires histone acetyltransferase activity (Bhattacharya et al, 1996; Horvai et al, 1997; Decker & Kovarik, 1999; Heim, 1999). The transcriptional activity of Stats can also bemodulated by phosphorylation of their serine and threonine residues, although the mechanistic implications of such regulation are not entirely clear (Eilers et al, 1995; Decker, 1999). While serine phosphorylation is not sufficient to induce Stat activation, it can enhance it. Kinases involved in this process include Jun NH-2 kinase (JNK), mitogen-activated protein kinase (MAPK), protein kinase C and protein kinase A (Frank, 1999). The function of various Stats has been elucidated through the creation of mutant mice with disrupted Stat genes (Levy, 1999; Ward et al, 2000). Mice deficient in Stat1 are extremely sensitive to viral infections, consistent with Stat1's presumed importance in IFN function. (Durbin et al, 1996; Meraz et al, 1996) Mice deficient in Stat3, however, demonstrate early embryonic lethality (Takeda et al, 1997). Stat4-deficient mice have no developmental abnormalities but are completely resistant to IL-12 stimulation and fail to induce Th1 polarization of T-helper lymphocytes (Kaplan et al, 1996a; Thierfelder et al, 1996). Stat5A/B knockout mice develop normally except for defects in adult mammary gland development and lactogenesis (Liu et al, 1997; Udy et al, 1997). Stat6-deficient mice are not overtly abnormal but do exhibit defects in IL-4 function (e.g. a complete lack of IL-4-induced Th2 polarization of T-helper cells) (Shimoda et al, 1996). Stat activation and signalling are involved in diverse and sometimes opposite cellular events affecting growth, differentiation and apoptosis (Schindler, 1998; Frank, 1999; Mui, 1999). Stat activation, for example, may result in both growth arrest and cellular proliferation. Stat1 mediates the growth-inhibitory effects of IFN-γ by inducing the cyclin-dependent kinase inhibitor p21waf1, whereas Stat5 induces the proliferative effects of IL-3 and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Chin et al, 1996; Mui et al, 1996). Similarly, in myeloid cells, phosphorylation of Stat3 can lead to IL-6 and IL-10-induced growth arrest (via induction of the cyclin-dependent kinase inhibitor p19INK4d) on the one hand and to GM-CSF- and IL-3-induced proliferation on the other (Mui et al, 1996; Chaturvedi et al, 1998; O'Farrell et al, 1998). Whether these diverse effects are tissue-specific or mediated by other factors is yet to be determined. Stats also mediate transcriptional events associated with cellular differentiation (Mui, 1999). Stat3 and Stat5 have been implicated in myeloid differentiation and Stat4 and Stat6 in Th cell differentiation. Stat3 is involved in IL-6-induced differentiation of M1 myeloid leukaemia cells, but suppresses differentiation in other cell lines (Yamanaka et al, 1996). Stat5 has been implicated in cell differentiation in a variety of systems, including thrombopoietin-dependent maturation of megakaryocytes and erythropoietin-dependent maturation of erythroid cell lines (Mui, 1999). When activated by IL-4, Stat6 helps regulate the expression of cell-surface proteins such as major histocompatibility complex (MHC) class II antigens, immunoglobulin class switching and Th2 differentiation (Rothman et al, 1991; Kaplan et al, 1996b). Therefore, Stat activation may play a key role in overcoming differentiation arrest. Stats are also important in regulating programmed cell death. In fact, constitutive expression of caspases necessary for induction of apoptosis in fibroblasts may depend on Stat1 (Kumar et al, 1997). For example, reconstitution of Stat1 in Stat1-null U3A cells, which do not respond to tumour necrosis factor α (TNF-α), restores basal caspase expression and sensitivity to TNF-induced apoptosis (Kumar et al, 1997). Conversely, Stat3 and Stat5 contribute to the antiapoptotic effects of IL-6 and IL-2 respectively (Fukada et al, 1996; Zamorano & Keegan, 1998). In response to IFN-γ, Stat1 mediates the upregulated expression of Fas expression and FasL, thereby activating the caspase cascade (Xu et al, 1998). Activation of the Jak-Stat pathway is both rapid and transient. Following stimulation by IFNs, Stat1 phosphorylation peaks within 15–30 min and returns to basal levels after 1–2 h. Three possible mechanisms of Stat inactivation following stimulation have been postulated: proteasome-mediated degradation, tyrosine dephosphorylation, and inhibition by various inhibitory proteins (Shuai, 1999). The first mechanism involves the ubiquitin-proteasome pathway, which is responsible for the degradation of many proteins involved in fundamental cellular processes (Kim & Maniatis, 1996). The second mechanism, tyrosine dephosphorylation, involves cytoplasmic phosphatases such as SH-2-domain-containing protein tyrosine phosphatase-1 (SHP)-1 that are believed to inhibit cytokine-activated Jak-Stat pathways through their dephosphorylation and inactivation of Jak (Hilton, 1999; Starr & Hilton, 1999). SHP-1-deficient mice demonstrate multiple haematopoietic abnormalities, including hyperproliferation and abnormal activation of granulocytes and macrophages in the lungs and in the skin, resulting in the characteristic moth-eaten appearance of their coats (Jiao et al, 1997; Takahashi et al, 1998). They also produce fewer B-cell progenitors and natural killer cells and exhibit premature thymic involution leading to immune deficiency (Jiao et al, 1997). This suggests that SHP-1 is a negative regulator of both myeloidand lymphoid lineages (Van Zant & Shultz, 1989; Yoshimura et al, 1990; de la Chapelle et al, 1993; Klingmuller et al, 1995; Furukawa et al, 1997). Ofparticular interest is a report on families with polycythaemia in whom the C-terminal region of the erythropoietin (EPO) receptor (which includes the docking site for SHP-1) is deleted (de la Chapelle et al, 1993; Furukawa et al, 1997). The third mechanism of Stat inactivation involves various inhibitory proteins including the suppressors of cytokine signalling (SOCS) and the protein inhibitors of activated Stats (PIAS) (Alexander et al, 1999; Shuai, 1999; Starr & Hilton, 1999). The SOCS proteins (eight in number) are alternatively known as JAB (Jak-binding protein), SSI (Stat-induced Stat inhibitor) or CIS (cytokine-inducible SH2-containing protein) (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Starr et al, 1997). Recent studies have established the SOCS proteins as negative regulators of cytokines (Yoshimura et al, 1995; Endo et al, 1997; Naka et al, 1997; Adams et al, 1998; Song & Shuai, 1998; Nicholson et al, 1999). Several lines of evidence also suggest that Stats are important in the induction of SOCS expression, although it is likely that Stat proteins are capable of inducing the expression of several SOCS and that each SOCS protein is capable of being induced by several Stats (Hilton, 1999). The PIAS family includes five proteins with significant sequence homology and several highly conserved domains (Chung et al, 1997; Liu et al, 1998). They are specific inhibitors of the Stat signalling pathway, with PIAS1 and PIAS3 being associated with Stat1 and Stat3 respectively (Shuai, 1999). The IFN-induced phosphorylation of Stat1 on Tyr701 is necessary for the PIAS–Stat1 interaction. Thus, the interaction of PIAS-Stat is highly specific and dependent on cytokine stimulation (Shuai, 1999). As cellular signalling pathways are intimately involved in the physiological regulation of cell survival, proliferation and differentiation, their abnormal function can lead to neoplastic changes. Studies in the Drosophila fly, which expresses a Jak and a Stat homologue, have shown that gain-of-function mutations affecting Jak cause leukaemia-like haematopoietic defects (Harrison et al, 1995). Over recent years, several investigators have reported that inappropriate activation of Stats may have a significant role in the pathogenesis of human malignancies and leukaemia in particular (Table IV) (Frank, 1999). This inappropriate activation is initiated by the abnormal hyperfunction or mutation of physiological Stat activators (such as Jaks) or by mutation of kinases not usually involved in Stat phosphorylation (such as abl), thus causing increased cellular proliferation (Frank, 1999). Various activated cellular tyrosine kinases phosphorylate Stats, either directly or via Jak activation, and induce cellular transformation. Activation of Jak1 by the oncogenic tyrosine kinase v-abl induces malignant transformation of haematopoietic cells, whereas constitutive Stat activation by mutated members of the Src kinase family induces cellular transformation (Danial et al, 1998; Frank, 1999). For example, v-src, v-fps and v-sis activate Stat3 and induce fibroblast transformation (Yu et al, 1995; Chaturvedi et al, 1998). Similarly, Lck, another Src tyrosine kinase, is overexpressed in a mouse T-cell lymphoma and is associated with Stat3, Stat5, Jak1 and Jak2 activation (Yu et al, 1997). Stat activation plays a key role in the pathogenesis of human leukaemia. Stat5 and Stat1 for example are constitutively activated in acute lymphoblastic leukaemia (ALL) and Stat1, Stat3, and Stat5 in acute myelogenous leukaemia (AML) (Gouilleux-Gruart et al, 1996; Weber-Nordt et al, 1996; Xia et al, 1998). The chromosomal translocation t(9:12)(p24:p13) found in acute lymphoblastic leukaemia (ALL) results in the fusion of the helix-loop-helix oligomerization domain of the transcription factor Tel (a member of the Ets family of transcription factors) with the catalytic JH-1 domain of Jak2, thus activating Jak2 and inducing Stat-induced gene transcription (Schwaller et al, 1998; Ho et al, 1999). Interestingly, overexpression of the Tel–Jak2 fusion protein renders Ba/F3 cells growth factor independent, and transfection of mice with a retrovirus expressing this chimaeric protein induces a fatal mixed myeloproliferative/T-cell lymphoproliferative disorder(Ward et al, 2000). Activation of these pathways is also crucial in the pathogenesis of chronic myelogenous leukaemia (CML). Transfection of BCR-ABL gene constructs into haematopoietic cell lines induces growth factor-independent proliferation (Daley & Baltimore, 1988). Bcr-Abl-induced transformation of haematopoietic cells leads to tyrosine phosphorylation of Stat1 and Stat5 in cell lines and fresh cells from patients with CML (Frank & Varticovski, 1996; Shuai et al, 1996; Chai et al, 1997). As these cells do not demonstrate Jak activation, it is likely that the enhanced kinase activity of Bcr-Abl is directly responsible for Stat phosphorylation and cellular transformation (Fig 3). In fact, recent reports on the clinical activity of the specific Bcr-Abl kinase inhibitor STI571 and its ability to induce haematological and cytogenetic remission in CML have provided new evidence for the role of Stat signalling in the pathogenesis of this disease and suggest that inhibition of Stat activity may be therapeutically beneficial (Druker et al, 2001a,b). Influence of Bcr-Abl on signal transduction pathways implicated in leukaemogenesis. Possible mechanisms include activation of Stat1 and Stat5, activation of PI3K pathway through Crkl, and activation of Ras via adapter proteins such as growth factor receptor-bound protein-2 (Grb2) and avian sarcoma virus oncogene homologue-like (Crkl), and possibly the src homologous and collagen protein (shc). Bcr-abl may also influence the focal adhesion complex function through a Crkl/Paxillin complex. Adapted from Faderl et al (1999). Other chromosomal translocations leading to the fusion of kinases and transcription factors include Tel-Abl, NPM-ALK, ZNF198-FGFR1 and Tel-PDGFβR (Frank, 1999). In the case of Tel-PDGFβR, activation of the receptor tyrosine kinase PDGFβR is mediated by the oligomerization domain of the transcription factor Tel, leading to constitutive Stat activation and cytokine-independent growth of haematopoietic cells (Carroll et al, 1996; Jousset et al, 1997; Valgeirsdottir et al, 1998). Although cytokine-driven cellular proliferation associated with Jak-Stat pathway overactivity is seen in acute leukaemias and CML, no constitutive tyrosine phosphorylation of Stats has been seen to date in chronic lymphocytic leukaemia (CLL) (Jousset et al, 1997). However, serine phosphorylation of Stat1 and Stat3, which is known to modulate their function, has been seen in cells from untreated CLL patients but not in normal CD5+ peripheral blood lymphocytes (Jousset et al, 1997). This serine phosphorylation, which can enhance the transcriptional response mediated by tyrosine-phosphorylated Stats and lead to subtle derangements of B-cell function, may explain the indolent nature of CLL (Frank, 1999). Oncogenic viruses may also play a role in Stat activation and leukaemogenesis. In the best example, T cells infected with HTLV-1 become IL-2 independent and exhibit enhanced Stat3 and Stat5 activity (Migone et al, 1995). In patients with HTLV-1-associated adult T-cell leukaemia/lymphoma (ATLL), Stat1, Stat3 and/or Stat5, as well as Jak kinases, are constitutively activated (Migone et al, 1995; Xu et al, 1995; Takemoto et al, 1997). Although the mechanisms of Jak activation in HTLV-1-transformed cells are still unknown, it has been hypothesized that a viral protein interacts with the IL-2 receptor to activate the appropriate pathway. Interestingly, the antiproliferative effect of IFN-β on T cells is abolished in HTLV-1-infected T cells (Smith et al, 1999). Receptor tyrosine kinases (RTKs) are membrane-anchored enzymes with an extracellular ligand-binding domain, a transmembrane domain, and a highly conserved intracellular domain that mediates phosphorylation of tyrosine residues on substrates in a manner similar to the Jak-Stat pathway (Drexler, 1996; Porter & Vaillancourt, 1998). These receptors are not only activated by ligand binding but also by ligand-independent mechanisms, such as cell–cell interactions via cell adhesion molecules (CAMs) in response to cellular stress and by stimulation of G-protein-coupled receptors (GPCRs) (Weiss et al, 1997). RTKs have been subdivided into four classes on the basis of their structural characteristics: class I includes the receptors for epidermal growth factor (EGF-R); class II, those for insulin-like growth factor-1 (IGF-1R); class III, those for platelet-derived growth factor (PDGF-R), macrophage colony-stimulating factor (FMS-R or CSF-1R), SCF receptor (KIT) and FMS-like tyrosine kinase-3 receptor (FLT3-R); and class IV, those for fibroblast growth factor (FGF-R) (Fig 1B) (Matthews et al, 1991; Sherr, 1990; Drexler, 1996). Human FLT3-R, KIT and FMS-R/CSF-1R show significant homology (63%) in their tyrosine kinase domains but much less homology (19%) in their extracellular domains (Drexler, 1996). All bone marrow-derived CD34+ cells uniformly express low levels of FLT3-R and most express KIT (Gabbianelli et al, 1995; McKenna et al, 1995; Drexler, 1996). Several studies have demonstrated the role of class III receptors and their ligands in the regulation of survival, proliferation and maturation of early haematopoietic progenitors, usually in synergy with other cytokines (Sherr, 1990; Matthews et al, 1991; Hirayama et al, 1995; Hudak et al, 1995; Rasko et al, 1995). Several groups have investigated the role of these growth factors in the pathogenesis of leukaemia and myelodysplastic syndrome (Ratajczak et al, 1992; Kanakura et al, 1993; Meierhoff et al, 1995; Dehmel et al, 1996; Drexler, 1996; Kimura et al, 1997; Cortelezzi et al, 1999; Sawada et al, 1999). FLT3-R is expressed at high levels by AML, ALL and lymphoid or mixed blast crisis CML cells, but at much lower levels by T-ALL cells (Drexler, 1996). Plasma SCF levels have been shown to correlate with in vitro leukaemia cell growth in myelodysplastic syndrome (Cortelezzi et al, 1999; Sawada et al, 1999). Point mutation of KIT has been reported in patients with myeloproliferative disorders (Kimura et al, 1997), and activation of KIT by its ligand has been shown to increase the proliferation of some human leukaemia cell lines (Kanakura et al, 1993). In addition, KIT expression has been reported in all French–American–British (FAB) subtypes of AML and in myeloid blast crisis CML but not in ALL (Stacchini et al, 1996; Muroi et al, 1998; Schwartz et al, 1999). A large number of intracellular signalling proteins bind the phosphotyrosine on the activated RTKs, including the GTPase-activating protein (GAP), phospholipase C-γ (PLC-γ), phosphatidylinositol 3′-kinase (PI3K), Grb2 and Src-like non-receptor tyrosine-kinases (Alberts et al, 1994; Porter & Vaillancourt, 1998; McCubrey et al, 2000). The activation of these proteins then initiates serine/threonine phosphorylation cascades, resulting in activation of transcription factors and modulation of cellular processes by gene transcription (McCubrey et al, 2000). Many major cellular signalling pathways involve tyrosine phosphorylation of Src and Jak kinases (Schwartzberg, 1998). However, several other major routes, such as the Ras signalling cascade, involve activation of serine/threonine kinases (McCubrey et al, 2000). The Ras proteins belong to the large Ras superfamily of monomeric GTPases, which also contains several other subfamilies including the Rho and Rac proteins involved in relaying signals from cell surface receptors to the actin cytoskeleton and the Rab family involved in the trafficking of intracellular vesicles (Alberts et al, 1994; Beaupre & Kurzrock, 1999). The three human Ras genes (H-ras, N-ras and K-ras) encode four isoforms (p21) that localize to the inner surface of the plasma membrane and function as GDP/GTP-regulated switches (Campbell et al, 1998). Ras proteins are central to a number of signalling pathways including those mediated by RTKs, non-receptor tyrosine kinase-associated receptors and GPCRs. Li