Class III receptor tyrosine kinases: role in leukaemogenesis

Haematopoiesis is a critically regulated process in which a small population of self-renewing primitive progenitors generate progeny of increasingly differentiated end cells with specific functional activities. This process is controlled by a number of growth factors and cytokines, with some of them exerting their specific functions through binding to high-affinity receptor tyrosine kinases (RTKs). The human genome, as currently sequenced, is thought to contain 90 tyrosine kinase genes, of which 58 are of the receptor type. The latter have been grouped into 20 subfamilies based on kinase domain sequence, a feature that also parallels their overall domain structure (Robinson et al, 2000). Class III RTKs (Ullrich & Schlessinger, 1990) are characterized by five immunoglobulin-like domains in the extracellular ligand-binding region, a single transmembrane domain (TM), a juxtamembrane domain (JM), two intracellular kinase domains (TK1 and TK2) divided by a kinase-insert domain (KI), and a C-terminal domain (Yarden et al, 1987) (Fig 1). Ligand binding to RTKs promotes receptor dimerization and subsequent activation of intrinsic tyrosine kinase activity that results in transphosphorylation of specific tyrosine residues (Weiss & Schlessinger, 1998). The tyrosine-phosphorylated receptor then serves as a docking site for an array of intracellular signalling molecules, including the GTPase-activating protein (GAP), the p85 subunit of phosphatidyl-inositol 3′-kinase (PI3K), phospholipase C-γ (PLC-γ), the protein tyrosine phosphatase SHP1, Grb2 and Src-like non-receptor kinases (Rosnet et al, 1996; Porter & Vaillancourt, 1998). These activated proteins then initiate serine/threonine phosphorylation cascades resulting in activation of transcription factors that determine a variety of cell responses, including cell maintenance, mitogenesis, migration and differentiation (Claesson-Welsh, 1994). A schematic of a canonical class III RTK monomer spanning the plasma membrane demonstrating five immunoglobulin-like domains (Ig1–5) in the extracellular ligand binding region, a single transmembrane domain (TM), a juxtamembrane domain (JM), two intracellular kinase domains (TK1 and TK2) divided by a kinase insert (KI). The position of reported mutations and their disease associations for FLT3 (left) and c-kit (right) are highlighted. CMPD, chronic myeloproliferative diseases; AML, acute myeloid leukaemia; GISTs, gastrointestinal stromal tumours; SNL, sinonasal lymphomas; MCD, mast cell disease; MDS, myelodysplastic syndrome; ins/del, insertion–deletion mutations. The class III RTKs, which include c-fms (Coussens et al, 1986), c-kit (Yarden et al, 1987), FLT3 (Rosnet et al, 1993a), PDGFRα (Claesson-Welsh et al, 1989) and PDGFRβ (Yarden et al, 1986), play an important role in normal haematopoiesis (with the exception of PDGFRα). c-fms, the receptor for the macrophage colony-stimulating factor (M-CSF), is crucial for the growth and differentiation of the monocyte–macrophage–osteoclast lineage (Sherr, 1990). FLT3 and c-kit are both required for the survival, proliferation and differentiation of haematopoietic progenitor cells, while c-kit is also important for the growth of mast cells, melanocytes, primordial germ cells and interstitial cells of Cajal (Drexler, 1996; Lyman & Jacobsen, 1998). The haematopoietic functions of PDGFRβ are less well defined, although the receptor and its' ligand probably play a significant role in megakaryocytopoiesis (Yang et al, 1997). The chromosomal location and genomic structure of the class III RTKs suggests a close evolutionary relationship. The c-kit and PDGFRα genes, for example, are located in tandem on chromosome subregion 4q11–q13 (Gronwald et al, 1990; Giebel et al, 1992), while the PDGFRβ gene lies approximately 350 base pairs upstream of the major transcription site of the c-fms gene on chromosome segment 5q31–q33 (Groffen et al, 1983; Roberts et al, 1988). The human FLT3 gene maps to 13q12 (Rosnet et al, 1991) and is closely linked, in a head-to-tail fashion, to FLT1, a class V RTK (characterized by seven immunoglobulin-like domains) (Rosnet et al, 1993b; Imbert et al, 1994). Recently, the FLT3 gene has been shown to comprise 24, rather than the expected 21, exons typical of other class III RTKs, with seven instead of four exons encoding the first three immunoglobulin-like repeats (Abu-Duhier et al, 2001a). In contrast, the genomic loci encoding the intracellular catalytic domains share overall conservation of exon size, number, sequence and exon/intron boundary positions, suggesting that they have arisen from a common ancestral gene by cis and trans duplication (Andre et al, 1992, 1994; Rosnet et al, 1993b; Abu-Duhier et al, 2001a). The PDGFRα and c-fms genes also contain a large intron (more than 20 kb) that disrupts the 5′-untranslated region and the signal sequence containing the initiator AUG codon. These large introns may have been inserted prior to the cis duplication of the ancestral gene, because such an insert is not present in the c-kit gene (Kawagishi et al, 1995). In view of the pivotal role of class III RTKs in normal haematopoiesis, many groups have sought evidence for their involvement in leukaemia and related disorders. The aim of this review is to provide a focused update of this work and to emphasize the importance of c-kit, FLT3 and PDGFRβ mutations in the pathogenesis of myeloid disorders. Emphasis is given to the considerable insight that such research has provided into the structure–function relationship of class III RTKs and their associated signal transduction pathways. Indeed, a better understanding of their role in leukaemogenesis may offer novel approaches for the development of more selective and less toxic antileukaemic therapy. Human c-kit (CD117), the cellular counterpart of v-kit, derived from the Hardy–Zuckerman 4-feline sarcoma virus (Besmer et al, 1986), encodes a glycoprotein receptor that binds stem cell factor (SCF, kit ligand, mast cell growth factor). Immunoprecipitation shows two proteins of approximately 140 kDa and 155 kDa, the larger presumably resulting from glycosylational processing of the smaller protein. The entire coding region has a length of 2928 bp with a deduced 976 amino acid residues (Yarden et al, 1987). c-kit and its' ligand play a pivotal role in normal haematopoiesis, as evidenced by naturally occurring murine mutations at the Sl locus, which encodes SCF, as well as in the c-kit receptor itself. These mutations result in varying degrees of macrocytic anaemia and a loss of mast cells, in addition to deficiencies in gametogenesis and melanogenesis (see Lyman & Jacobsen, 1998). Within the human haematopoietic system, c-kit is expressed by approximately 70% of CD34+ cells in bone marrow, including lineage-restricted haematopoietic progenitor cells (Ashman et al, 1991; Papayannopoulou et al, 1991) and primitive cells capable of establishing long-term in vitro haematopoiesis (Simmons et al, 1994). Importantly, c-kit is downregulated during maturation of all haematopoietic lineages except mast cells, which retain a high level of expression (Fukuda et al, 1995). A number of early observations suggested a role for c-kit in leukaemogenesis. c-kit, for example, is expressed by myeloblasts in approximately 60–80% of acute myeloid leukaemia (AML) patients, as assessed by either surface immunostaining or expression of c-kit messenger RNA (Ikeda et al, 1991). The addition of SCF results in the proliferation of some leukaemic cell lines, such as MO7E, as well as the leukaemic blasts from most cases of c-kit+ AML (Wang et al, 1989; Ikeda et al, 1991; Broudy et al, 1992), while c-kit has been reported to be activated, i.e. tyrosine phosphorylated, in the absence of exogenous SCF in a proportion of AML cases (Kanakura et al, 1993). Recently, preliminary studies demonstrating that c-kit inhibitors lead to apoptosis of AML blasts strongly support this concept (Smolich et al, 2001). The identification of SCF as the essential growth factor for human mast cells (Tsai et al, 1991) led to studies of the pathogenetic role of c-kit and its' ligand in mastocytosis. In 1993 the first c-kit gain-of-function mutations were identified in the human mast cell leukaemia cell line, HMC-1 (Furitsu et al, 1993). Sequence analysis revealed two point mutations, Val560Gly in the juxtamembrane domain and Asp816Val in the phosphotransferase domain. Transfection of murine c-kit Gly559 or Val814 mutants, corresponding to human c-kit Gly560 or Val816, into 293T human embryonic kidney-derived cells demonstrated that both mutations resulted in the constitutive activation of c-kit. Subsequently, both the P-815 mouse mastocytoma and the RBL-2H3 rat mast cell leukaemia cell line were shown to carry constitutively activating mutations of c-kit in the corresponding Asp codon of the domain et al, 1995). the murine Gly559 or Val814 were into murine cells, the c-kit proteins were not to the of the cells, also them to in et al, 1995). the strongly suggested that c-kit mutations be in human mast cell This view et reported the of c-kit Asp816Val mutations in the cells of a with and in three with associated with mastocytosis. of located at the to the has been reported in most cases of et al, et al, and in a small number of cases et al, In a of patients, et that Asp816Val mutations in cells were associated with as well as and The Asp816Val mutations and to be more common in cases with involvement et al, A kinase domain located four amino has also been associated with mast cell disease et al, 1997). In an to lineage et an Asp816Val in bone containing mast cells, not in leukaemic in a with and In contrast, et identified the in as well as in chronic cells, suggesting that both cell were derived from a common progenitor The latter view is by the of et reported c-kit Asp816Val mutations in cells as well as cells and cells in with suggesting the involvement of early progenitor mast cells, and cells not surface c-kit and may be less to the of the myelodysplastic or evidence of not c-kit mutations et al, 2001). The that and its' probably play a crucial role in the of the activating of the kinase domain of class III The for this of receptor activation et have a based on the that kinase activation in is based on the or activating from an to an in to the kinase site and to amino that phosphorylation (see et al, is suggested that the result not in a to that the activating to to an activation for kinase also in the of the the A and the activating The by which mutations is et have shown that loss of the acid to an in and the of an of c-kit while et demonstrated resulting in receptor although not in the extracellular domain. also that activation is crucial for of c-kit et al, 2001). The murine mastocytoma cell line, in contrast, a constitutively activated c-kit resulting from an in of base pairs from exon which for the juxtamembrane domain et al, is that such intracellular mutations on the kinase activity by a juxtamembrane et al, The pathogenetic c-kit activation and mast cell disease led to that a of c-kit activity et al, in disorders. the kinase activity of the has been shown to be to probably as a result of in the activation that binding et al, et al, 2000). tyrosine kinase inhibitors are currently sought and preliminary suggests that some may have activity mast cells et al, 2000). c-kit mutations have also been associated with AML either an or i.e. the binding factor et for example, reported an activating in an with and the by mutations in cases with and cases with et al, 2000). et reported novel exon mutations, with loss of in a of with and The mutations were located in the immunoglobulin-like the first evidence in support of an important role for this region of the as the amino are has been suggested that of the extracellular domain of RTKs lead to activation by the of et al, et al, 1997). The of c-kit mutations to cases of AML binding proteins as to the the two suggests that expression of transcription factor proteins is not to a leukaemia as evidenced in by the required in the murine of the and that mutations are et al, may be that the c-kit mutations provide this by proliferation while the transcription factor normal differentiation with on cellular et reported c-kit exon mutations in two with and with chronic myeloid leukaemia and suggested that this of the extracellular domain resulted in of the stem cells to SCF et al, 1997). such a results in a constitutively activated receptor to be are and that are in and are strongly associated with virus et al, 2000). Recently, et in a of patients, reported c-kit mutations in the mutations either or the juxtamembrane while seven novel The functional of the latter are is that they the and of the c-kit mutations have been to the pathogenesis of human gastrointestinal stromal and et al, et al, a that the expression of c-kit by primordial germ cells and interstitial cells of FLT3 tyrosine is expressed in haematopoietic stem cells and suggesting a role in stem cell development and differentiation et al, 1991; Rosnet et al, 1991). This view is by studies in which of the FLT3 gene results in deficiencies in primitive and myeloid haematopoietic progenitors et al, 1995). from a number of groups have suggested a role for FLT3 in acute myeloid FLT3 for example, has been in most cases of AML, as well as in the of human myeloid and cell et al, et al, 1996; Rosnet et al, In exogenous FLT3 ligand the proliferation of AML cells et al, while some leukaemia cell both FLT3 and suggesting a or (Drexler, chronic of in haematopoietic progenitors has been shown to leukaemia in a et al, 1998). et were the first to of the FLT3 gene in a small of with The mutations of tandem of the juxtamembrane domain coding sequence, exon intron and exon (see Abu-Duhier et al, for exon their location and length from to the FLT3 gene in and FLT3 with a domain. of the of all FLT3 to result in constitutive dimerization of the receptor and of specific tyrosine residues et al, et al, 2000). The FLT3 in a high proportion of AML cases of the et al, et al, Abu-Duhier et al, et al, and in myelodysplastic not in or normal haematopoietic et al, et al, with acute leukaemia and a FLT3 some with myeloid have been reported et al, et al, et al, 2000). In acute leukaemia FLT3 mutations were related to high cell and high et al, while FLT3 mutations may during of myelodysplastic or at of AML, suggesting that they leukaemic et al, et al, FLT3 mutations have been associated with a in AML et al, Abu-Duhier et al, et al, 2000). et for example, FLT3 mutations to be the factor for overall in the of has also been shown that the associated of (Abu-Duhier et al, 2000). results from large studies have et al, et al, et al, et al, as well as suggesting that with more than or with high of the gene have a et al, et al, 2000). The of on has also been demonstrated in AML et al, et al, et al, 2001). et identified FLT3 as a significant factor for with a reported at of for with FLT3 with for with et reported an for with FLT3 of and that with a FLT3 a In to most large et in a of AML patients, to the of FLT3 with an for this is that the of FLT3 in the are by their overall survival, or that FLT3 in may have to intrinsic in their myeloid most studies that the identification of FLT3 may from Indeed, has been that be given to the analysis of FLT3 in all AML of et al, 2000). In FLT3 may provide a for the of in have a normal et al, 2000). et reported that FLT3 in to and that FLT3 be a for antileukaemic therapy. The receptor is and in the absence of ligand et al, although the of constitutive activation is that the disrupts a domain of signal to the c-kit juxtamembrane reported in the murine mastocytoma cell line et al, the number of tyrosine residues in the duplication has been reported to with leukaemia cell et al, although a role is as cases tyrosine duplication have been et al, 1996; et al, This view has been by et FLT3 the and on receptor In to the of activated et constitutively activated FLT3 by its' domain to the domain of in to the that with in chronic leukaemia et al, 1996; et al, 1997). Transfection of cells with the gene resulted in and long-term phosphorylation of kinase signal and of transcription and binding protein suggesting a role for the in FLT3 of cells into resulted in from of the haematopoietic system, their leukaemic FLT3 mutations, in to result in activation of and kinase et al, et al, a that have (see et al, 2001). et reported that constitutive activation of FLT3 in cells to be on the and were to receptor activation in et although FLT3 to be constitutively activated in some AML not this with FLT3 mutations. suggested that other of activation are resulting from either mutations in the gene or from The view by the that an in mouse FLT3 resulted in constitutive into cells et al, 2000). The FLT3 because of its' to activating mutations and in mouse and human c-kit (Furitsu et al, et al, 1994; et al, et al, 2000). In et demonstrated that of the c-fms that are in the mouse haematopoietic progenitor cell line These led two groups to AML for FLT3 mutations (Abu-Duhier et al, et al, 2001). groups reported mutations in approximately of cases and that the novel mutations were of suggesting that FLT3 is the gene most in acute myeloid In et demonstrated that all FLT3 were constitutively activated, in that they were tyrosine and et that the of c-fms mutations are to amino acid that mutations of both c-fms and c-kit are probably to tyrosine or as are the for amino that be from an codon by a single base may be that the of reported mutations result from single base resulting in the of acid with the amino tyrosine and The of mutations on the to be a kDa single transmembrane glycoprotein is expressed on cells, cells and ligand, or is a of cell proliferation and and a role in & et al, 2000). has also been identified on a variety of haematopoietic cells, including and et al, 1990; et al, cells et al, et al, cells et al, and (Yang et al, 1997). is haematopoietic or haematopoietic through the of growth and differentiation factors from stromal cells et al, 2001). that activation of PDGFRβ lead to cell in the and early the protein of the sarcoma virus which has to the the ligand for shown to cells through activation of PDGFRβ et al, In reported to a myeloproliferative disease constitutively expressed in mouse bone et al, while the of PDGFRβ in the myeloid progenitor line reported to et al, The concept that PDGFRβ mutations be by et reported that a in chronic myeloid results in a a the of the receptor is by the first amino of the transcription factor which a a that the receptor to in the absence of In support of this et reported that receptor is by the of amino the domain. The of the gene is also on the PDGFRβ tyrosine kinase as PDGFRβ inhibitors the growth of cells et al, 1997). et the of by that expression of the gene, the of the in a chronic myeloproliferative characterized by and to In contrast, et with expression by a the of both and demonstrating that is not to the myeloid et that and which are for high with kinases and are for the development of the myeloproliferative as their resulted in the development of The of chromosomal PDGFRβ in with myeloproliferative the pathogenetic tyrosine kinases and myeloid (Fig et for example, reported that the in a with resulted in expression of the protein or protein is a protein that binds the protein of the gene in et al, 1997). the amino of are to the transmembrane and tyrosine kinase domains of the PDGFRβ in this novel protein. Recently, two groups reported that the in cases of myeloproliferative resulted in the of to PDGFRβ et al, et al, 2001). encodes a expressed protein of which has been characterized as a of the receptor tyrosine kinase et al, and ligand is the cell factor et al, the is of a number of reported associated with and human et al, The gene is to a protein of amino that the from and the entire transmembrane and tyrosine kinase domains of et reported a with and an that resulted in a novel has been shown to be an essential and of early through with the and The protein of the as in the to the transmembrane and intracellular kinase domains of from a pathogenetic the protein three of the four domains that are in the of the In to the the is in that as a at in a with AML and a the of associated with the development of a and of myeloproliferative et al, 1997). suggests that PDGFRβ may be in at of which five have been characterized et al, 2001). the the and proteins have been to constitutive tyrosine kinase activity and although is that this be the for the and In to other class III RTKs, of PDGFRβ resulting from point mutations have not been although studies of gene analysis are & a constitutively activated murine PDGFRβ containing a located in a in the domain. This suggests that the juxtamembrane domain a role in the of receptor activity and by in of the normal PDGFRβ extracellular transmembrane tyrosine kinase tyrosine kinase and the proteins identified to to The of the intracellular and transmembrane domains of PDGFRβ are present in all The domains of the thought to be in are the and The PDGFRβ gene with a number of genes, resulting in the of In the pathogenetic in that the gene a dimerization domain that the constitutive activation of the PDGFRβ kinase domain. of proteins have not been and are to be et al, et al, 2001). with a or have been reported to have that are to in the as they of both and all cases are are in a feature et al, et al, et al, 1991; et al, et al, et al, 1996; et al, although PDGFRβ is not to be in be to determine the is present in in the cases and the of suggests an associated et al, et al, 2000). The of with PDGFRβ with from to et al, et al, be important to cases to a better understanding of the in view of the of a of kinase activity et al, et al, 2000). The human located on chromosome at encodes a amino acid transmembrane glycoprotein which functions as the receptor of the colony-stimulating factor or et al, The c-fms gene is the cellular of the gene of the of the sarcoma virus et al, In to the human gene, the gene constitutive kinase activity in the absence of ligand and cells in vitro et al, et al, 1988). and studies of c-fms and revealed the of the for the of cell include a number of point mutations and the of amino at the with amino These lead to the of a tyrosine in human which the of the gene to ligand as well as at and et al, 1988). of c-fms, have the importance of mutations at and for activity et al, et al, 1988). that the of codon on c-fms functional activity may be of tyrosine although to of cells, transduction through the receptor et al, 1987). mutations may the to in an or In contrast, mutations in the human c-fms at codon of cells which be by the of the et al, et al, 1988). In have identified other for activity in both the extracellular and intracellular domain c-fms et al, et al, studies suggested a role for of c-fms in for example, to the development of leukaemia et al, 1987). In studies to identified point mutations in approximately of AML and of et al, 1990; et al, with codon more than codon were more common in characterized by acute leukaemia and et to codon and mutations in either AML or MDS, a that has been by subsequent studies et al, et al, 1997). The role of c-fms mutations in leukaemogenesis and the for the may in the that the early studies the c-fms, may be in the pathogenesis of leukaemia as a result of an et that the protein c-fms expression by on the an that may the high c-fms expression in with The class III RTKs have been to the pathogenesis of an number of c-kit mutations, for example, have been shown to be in as well as associated with acute myeloid leukaemia and sinonasal Indeed, et have a of mast cell disease based on the of the c-kit an which may be FLT3 is the gene most in AML and the of to be the factor in the has been that analysis of FLT3 in all AML the of be The development of specific FLT3 inhibitors is currently by the of in The role of genes in chronic myeloproliferative is at genes may be to from a in may be the of the for the pathogenetic role of RTKs and a for activation of other tyrosine kinases in other of is the of RTKs and leukaemia is and is that many more are to be The and for their review of the and