Recent advances in the molecular and cellular biology of chronic myeloid leukaemia: lessons to be learned from the laboratory (2001)

Chronic myeloid leukaemia (CML) was first recognized as early as 1845 (Bennett, 1845; Craigie, 1845; Virchow, 1845), when several cases of splenomegaly, anaemia and massive granulocytosis were described. In 1865, Virchow proposed the term 'leukamie' (Virchow, 1865) and, in 1878, Neumann deduced that the disease originated in the bone marrow and proposed the term 'myelogenous leukaemia' (Neumann, 1878). However, it was not until 1960 that the discovery of the Philadelphia (Ph) chromosome led to a better understanding of the pathogenesis of the disease (Nowell & Hungerford, 1960). Thirteen years later, the Ph chromosome was shown to be generated by a specific translocation involving chromosomes 9 and 22 (Rowley, 1973). In the 1980s, the BCR–ABL fusion oncogene was described and found to be transcribed and translated into a functional protein. This novel oncoprotein, p210BCR–ABL, differed, both in terms of its subcellular localization and its tyrosine kinase activity, from the endogenous c-ABL protein (Groffen et al, 1984; Shtivelman et al, 1985). Finally, in 1990, the first definitive evidence of the ability of BCR–ABL to transform primary myeloid cells and induce a CML-like disease in mice was published (Daley et al, 1990). The c-ABL gene, located on chromosome 9, is a member of the family of non-receptor protein tyrosine kinases (Fig 1). It comprises two alternative first exons, 1a and 1b, and common exons 2–11. The name derives from the Abelson virus, which contains the viral oncogene v-ABL and causes leukaemia in mice (Laneuville, 1995). The human homologue, c-ABL, is ubiquitously expressed and appears to have a major role in regulating cell cycle progression (van Etten, 1999). In quiescent cells, the ABL protein is bound in a complex involving the retinoblastoma (Rb) protein, which functions to inhibit the activity of ABL. However, as cells progress from G1 phase to S phase, Rb is phosphorylated, dissociates from ABL, which then becomes activated and, in turn, is phosphorylated by CDC2 kinases, with subsequent effects on transcription and progression to S phase. When overexpressed, c-ABL induces cell cycle arrest, confirming its probable role as a negative regulator of cell growth (Sawyers et al, 1994). Despite these apparent regulatory effects on cell proliferation, disruption of the c-ABL gene in mice does not lead to uncontrolled cell growth. Instead, mice homozygous for a null mutation in c-ABL, have lymphopenia, are runted and many die in the perinatal period (Schwartzberg et al, 1991; Tybulewicz et al, 1991). Thus, c-ABL appears indispensible for normal growth and development. The c-ABL and BCR genes. The individual boxes schematically represent the position of the exons and the space in between boxes represents the intronic sequences. The black arrows indicate the breakpoint regions. The BCR gene on chromosome 22 spans a region of 135 kb and comprises 23 exons (Fig 1). Gene expression gives rise to two mRNAs, both of which are translated into a 160-kDa protein. Although ubiquitously expressed, BCR belongs to a family of genes whose functions remain unclear. In quiescent cells, the BCR protein is localized in the cytoplasm, however, during mitosis it may be detected in a perichromosomal location, suggesting a possible role in cell cycle regulation. In BCR knock-out mice, haemopoiesis appears to be intact apart from an increased neutrophil respiratory burst resulting in an increased susceptibility to septic shock (Voncken et al, 1995). The majority of patients with CML have breakpoints in introns 1 or 2 of the ABL gene and in the MBCR region of the BCR gene, either between exons 13 and 14 (b2) or 14 and 15 (b3) (Fig 1). These breakpoints produce BCR–ABL fusion genes that transcribe either a b2a2 or b3a2 mRNA, both of which translate into a protein of 210 kDa. b3a2 is the more prevalent and, in 5–10% of cases, both b2a2 and b3a2 transcripts are formed. Although these are the most frequently identified forms of BCR–ABL, CML patients are described with atypical BCR–ABL transcripts, for example involving ABL exon a3 instead of a2 (van der Plas et al, 1991), or transcripts with an e1a2 (Hermans et al, 1987), e19a2 (Pane et al, 1996) or even e6a2 junction (Hochhaus et al, 1996) (see Fig 1). As a consequence of reciprocal recombination, an ABL–BCR gene is also created by the t(9;22) translocation and, in 60% of CML patients, an ABL–BCR mRNA is detected (Melo et al, 1993). However, to date, there is no definite evidence that the presence or absence of ABL–BCR transcripts correlate with disease behaviour and no ABL–BCR protein has, as yet, been identified. Both ABL and BCR have several functional domains that may contribute to the leukaemic phenotype (Fig 2). ABL shows structural homology to the Src family of kinases, having a tyrosine kinase (SH1), an SH2 and SH3 domain. The tyrosine kinase domain of p210BCR–ABL, which plays a crucial role in the phosphorylation of target proteins such as CRKL, is flanked on the amino-terminal end by the SH2 and SH3 domains. SH2 and SH3 domains are involved in protein–protein interactions that are required for assembly of signalling proteins. SH2 domains bind phosphorylated tyrosine moieties (Pawson, 1995), while SH3 domains specifically recognize proline-rich motifs (Feller et al, 1994). In addition, ABL has a nuclear localization domain, three acidic DNA binding regions and, at the extreme carboxyterminus, an actin-binding domain (van Etten et al, 1994). The most important functional domains of the ABL and BCR proteins. At the amino terminal part of ABL, an SH3 and SH2 domain are present. The SH1/tyrosine kinase domain lies carboxyterminal to the SH2 domain, followed by the nuclear localization signal. Further towards the carboxyterminus are the DNA binding and actin binding domains respectively. In BCR, the dimerization domain (DD) is at the extreme amino terminus. This domain is followed by an SH2 binding domain that can bind at the SH2 domain of ABL in a non-phosphotyrosine-dependent manner, a region with a unique serine/threonine kinase activity, a DBL or GAP-homology domain and a PH domain. The BCR protein has multiple functional domains, the most critical of which include the dimerization domain, which is involved in coiled-coil oligomerization of the BCR protein (McWhirter et al, 1993), an SH2 binding domain which functions in a non-phosphotyrosine-dependent manner (Pendergast et al, 1991), a unique serine/threonine kinase domain (Maru & Witte, 1991), a DBL- or GTPase-activating protein (GAP)-homology domain (Diekmann et al, 1991; Laneuville, 1995) and a pleckstrin (PH) domain. In contrast to ABL, BCR–ABL exhibits deregulated, constitutively active tyrosine kinase activity and is found exclusively in the cytoplasm of the cell, in complex with a number of cytoskeletal proteins. These two features appear to underlie the ability of BCR–ABL to induce a leukaemic phenotype. As a result of the increased tyrosine kinase activity, BCR–ABL phosphorylates several cellular substrates, activates multiple signal transduction pathways, and causes profound effects on cell growth and differentiation. Although the exact sequence of events and the relative importance of each signal transduction pathway remain unclear, an emerging theme is that BCR–ABL activates signalling cascades also used by certain cytokine-stimulated receptors to regulate normal haemopoietic cell behaviour and, as the BCR–ABL signal is constitutively 'on', cells under its control bypass normal regulatory mechanisms and become leukaemic. The majority of signal transduction studies involving BCR–ABL have been performed in artificial systems such as interleukin-3 (IL-3)-dependent cell lines immortalized with BCR–ABL constructs. These model systems have provided important clues about how BCR–ABL may exert its transforming activity through inhibition of apoptosis and activation of growth factor independence. However, these cell lines probably also harbour additional genetic aberrations that may contribute to the mechanism by which they achieve a transformed state following BCR–ABL transduction. In brief, signalling pathways that are activated by BCR–ABL include Ras and, in turn, Erk- and Jun-kinase, PI-3 kinase, a number of c-Cbl and CRKL-linked pathways, Jak-STAT and the Src pathway. Of these, the Ras, Jun-kinase and PI-3 kinase pathways have been demonstrated to play a major role in transformation (Raitano et al, 1995; Sawyers et al, 1995; Skorski et al, 1995a, b, 1997). Inhibition of apoptosis is thought to result from activation of the PI-3 kinase and Ras pathways, with induction through AKT of c-myc and bcl-2 (Sawyers et al, 1995; Skorski et al, 1997; Warmuth et al, 1999). Signalling and phosphorylation, either directly or via c-Cbl, link p210BCR–ABL to proteins that play important roles in cellular adhesion and motility, such as talin, tensin, paxillin, actin, vinculin and FAK, thus producing the adhesion defect and cytoskeletal abnormalities characteristic of BCR–ABL-positive cells (Gordon et al, 1987; Salgia et al, 1997). Multiple pathways are thought to influence cellular proliferation, and many of these processes cannot be dissected in terms of individual effects on apoptosis, proliferation and transformation. Much research has recently focused on the activation by p210BCR–ABL of signalling pathways also triggered by haemopoietic growth factor receptor binding. For example, both p210BCR–ABL and a variety of growth factors lead to phosphorylation of Ras pathway-associated proteins (Shc, SHIP, Erk-2, Ras-GAP), paxillin, JAK/STAT pathway proteins (STAT1, STAT5, JAK1 and 2, TYK2), the phosphatases SHP1 and 2, CRKL and c-Cbl, the βc of certain growth factor receptors (e.g. IL-3), c-kit, and a number of other tyrosine kinases and substrates (Sattler & Salgia, 1997). The importance of the link between p210BCR–ABL and SHIP has been emphasized in a recent study which demonstrated that expression of BCR–ABL directly inhibits expression of SHIP and that this inhibition may be reversed by treatment with STI571 (Sattler et al, 1999). Targeted disruption of the murine SHIP gene results in a myeloproliferative disorder (Helgason et al, 1998), and BCR–ABL-transformed cell lines, Ph+ cell lines and primary CML cells all show substantially reduced SHIP expression (Sattler et al, 1999). It therefore seems probable that reduced SHIP levels caused by BCR–ABL expression contribute to the pathogenesis of CML in patients (Sattler et al, 1999). The Abelson murine leukaemia virus was first isolated from a mouse that developed a pre-B lymphoma following introduction of Moloney murine leukaemia virus (Abelson & Rabstein, 1970). The viral genome was found to contain viral gag sequences and a novel gene, later identified as the c-terminus of murine c-ABL. In the viral ABL oncogene, v-ABL, the first two exons of c-ABL are deleted and fusion of gag sequences with c-ABL provides a myristoylation site that directs localization to the plasma membrane rather than the nucleus (Wang et al, 1984). As the SH3 domain of ABL is lost during retroviral transduction, and, as mutational analysis has shown that SH3 deletion activates the protein for transformation, these findings suggest that the transforming potential of v-ABL is conferred as much by loss of SH3 as through the activity of the viral gag sequences (Franz et al, 1989; Jackson & Baltimore, 1989). This view has been stengthened by studies in which the SH3 domain was added back into v-ABL, resulting in suppression of kinase activity and, hence, transformation capacity (Muller et al, 1991). The BCR–ABL protein, in fact, retains the SH3 domain and lacks the myristoylation site. These two features presumably explain its weaker transforming ability compared with v-ABL (Resh, 1990). Once the p210 cDNA was successfully cloned, attempts were made to mimic CML in vitro as well as in vivo. Infection of primary bone marrow cells with a BCR–ABL-bearing retrovirus resulted in transformation of the cells, which were then found to have significantly reduced growth factor requirements in semisolid assays (Gishizky & Witte, 1992). Similarly, enforced expression of BCR–ABL in cytokine-dependent immortalized murine haemopoietic cell lines rendered them both growth factor-independent and tumorigenic (Daley & Baltimore, 1988; Kabarowski et al, 1994). In these various in vitro assay systems, the context and level of gene expression appears to affect the transforming ability of BCR–ABL. For example, the majority of in vitro murine models use retroviral regulatory sequences that lead to higher levels of expression of BCR–ABL than are found in blast phase CML cell lines and which, therefore, must be poorly representative of chronic phase disease. More recently, attempts have been made to reduce BCR–ABL expression levels by using the endogenous human BCR promoter to direct BCR–ABL cDNA expression (Groffen & Heisterkamp, 1987; Cambier et al, 1998). Structure/function studies designed to uncover key biological functions of BCR–ABL have revealed a number of important amino acid residues or protein domains that are indispensable for its full activity. The oligodimerization domain of BCR is contained within the first 61 amino acids and, in BCR–ABL, this domain is absolutely required for kinase activity, which in turn is essential for transformation of target haemopoietic cells (McWhirter et al, 1993). A number of other proteins, each possessing oligomerization properties (e.g. Ets-like transcription factor TEL, yeast transcription factor GCN4 and yeast glutathione-S-transferase GST), may substitute for BCR in terms of homodimerization, kinase activation and cell transformation, thus demonstrating the key importance of oligodimerization in BCR–ABL-induced leukaemogenesis (Warmuth et al, 1999). A further critical region in BCR is the tyrosine residue at position 177 (177Y) that has been identified as a major Grb-2-binding site (Pendergast et al, 1993; Puil et al, 1994). BCR–ABL activates the Ras pathway by binding Grb-2, which is mediated by phosphorylation of 177Y. Mutation of 177Y abolishes Grb-2 binding and turns off BCR–ABL-driven activation of Ras (Cortez et al, 1995; Goga et al, 1995). Antisense to Ras and dominant negative constructs of Ras have been shown to inhibit the growth of Ph+ cell lines and primary CML cells (Cortez et al, 1995; Sawyers et al, 1995). Similarly, a dominant negative construct of c-JNK has been shown to reduce the transforming activity of BCR–ABL (Raitano et al, 1995). Further downstream in the Ras signalling pathway, overexpression of c-myc or cyclin D1 have enabled rescue of the in vitro and/or in vivo transformation capacity of an SH2 domain mutant of BCR–ABL, which is by itself inactive, and overexpression of a dominant negative c-myc mutant abrogates transformation (Sawyers et al, 1992; Afar et al, 1994, 1995). The SH domains of ABL are important in mediating or modulating the transforming activity of BCR–ABL, with the SH1/tyrosine kinase domain being by far the most important in this respect (Warmuth et al, 1999). Regardless of the model system employed, kinase inactive mutants of BCR–ABL have no transforming activity. Furthermore, molecules that have been developed as selective inhibitors of the ABL tyrosine kinase significantly reduce proliferation of Ph+ cells (Druker et al, 1996; Deininger et al, 1997). Alternatively, deletion of the SH3 domain activates the protein for transformation, suggesting that SH3 usually negatively regulates ABL (Franz et al, 1989; Jackson & Baltimore, 1989). This view is stengthened by work combining deletion of the oligomerization domain of BCR, which alone blocks the tyrosine kinase activity of BCR–ABL, with the addition of a deletion to the ABL SH3 domain, which then restores transforming activity (Maru et al, 1996) and, more recently, by an in vivo study of the SH3 domain, in which BCR–ABL with an SH3 deletion retained its ability to induce a myeloproliferative disease in mice (Gross et al, 1999). A role for the ABL SH2 domain in transformation remains controversial. Its key biological function appears to be to direct the tyrosine kinase activity of BCR–ABL to other proteins that are themselves relevant to transformation. Mutation of the SH2 domain affects generation of protein complexes involving BCR–ABL and, at least in fibroblasts, abrogates transformation (Mayer et al, 1992). Although a single mutation of the SH2 domain does not cause transformation of haemopoietic cells, combination with other functionally relevant mutations has revealed a role for the SH2 domain in induction of Ras signalling and a further essential role in autocrine growth factor independence in IL-3-dependent cell lines (Cortez et al, 1995; Goga et al, 1995; Anderson & Mladenovic, 1996). The first evidence directly implicating BCR–ABL in the induction of myeloid and lymphoid leukaemias came from studies of BCR–ABL transgenic mice (Hariharan et al, 1989; Heisterkamp et al, 1990). A p190 construct induced both acute myeloid and B-lymphoid leukaemias. With a p210 construct, T-cell leukaemias also emerged. However, the only model system in which BCR–ABL has induced a CML-like disease has been obtained by the transplantation of irradiated mice with syngeneic marrow cells that have been retrovirally transduced with BCR–ABL in vitro. In the first such experiments, p210 induced a fatal myeloproliferative syndrome in approximately 25% of animals. However, mice also succumbed to a variety of other BCR–ABL-induced leukaemias of B-lymphoid, monocytic, T-lymphoid and erythroid lineages (Daley et al, 1990). Therefore, the protocol was not specific for generating a CML-like disease. However, further progress in developing this model was hindered by technical difficulties and the low efficiency of disease generation. More recently, three groups have modified the original protocol and obtained close to a 100% incidence of transplantable CML reproducibly and with a latency that will enable study of diseased animals over a reasonable time-frame (Pear et al, 1998; Zhang & Ren, 1998; Li et al, 1999). Most recently, a conditional transgenic model of p210BCR–ABL, under the control of tetracycline, has been described. In the absence of tetracycline, BCR–ABL is expressed and animals immediately develop B-cell acute lymphoblastic leukaemia (ALL), but if tetracycline administration is reinitiated, the disease rapidly regresses. This confirms that continued expression of BCR–ABL is necessary to maintain the leukaemic phenotype. Although this is not a model of classical CML, it does represent a powerful new laboratory tool for studies of BCR–ABL-induced oncogenesis (Huettner et al, 2000). An alternative approach to developing an in vivo model of CML has come from the recognition that normal human haemopoietic cells will engraft relatively efficiently in highly immunodeficient mice or fetal sheep (Zanjani et al, 1992; Dick, 1996; Pflumio et al, 1996; Cashman et al, 1997). Engraftment of sublethally irradiated immunodeficient mice has recently been used to analyse the stem cells responsible for sustaining AML as well as CML (Terpstra et al, 1995; Dick, 1996; Sirard et al, 1996; Bonnet & Dick, 1997; Dazzi et al, 1998; et al, 1998; et al, 1998). of cell from chronic phase CML patients into irradiated or mice has required the use of relatively cell in to cells with Similarly, Ph+ cells can be in the immunodeficient murine a of the mice with normal cells is A recent has in the and disease behaviour correlate with disease phase, such that blast engraft early and at levels and while chronic phase engraft and over with of leukaemic cells more 2 et al, 1998). An even more immunodeficient mouse model has been described in which homozygous disruption of the gene has been the These mice appear to even higher levels of human cell to are to the of at a relatively early of as a model for of chronic phase CML et al, 1997). Despite such this in vivo model represents the system in which to the of CML growth and to novel (see Fig The transplantation groups of immunodeficient mice are with CML cells, either or following stem cell to later, bone marrow is and with in to the presence of human cell using may be to human and myeloid or stem cell and the human cells can then be using of the human cells further analysis of or cell for example to as in the shown the presence of BCR–ABL The between normal haemopoietic stem cells and the bone marrow provides the for of proliferation and differentiation. A variety of of adhesion the the the and the play a role in this with the to be most important 1997). In CML, the potential of cellular adhesion and is by the of stem cells and myeloid cells from the marrow into the and in which they in an uncontrolled CML cells have been shown to have abnormalities of (Sattler & Salgia, et al, 1995) and et al, 1992). A number of studies have that Ph+ cells bind well to bone marrow or than normal (Gordon et al, 1987; et al, 1992). The most adhesion detected in CML cells has been a in biological function of the et al, et al, et al, 1997). In function is reversed by of the cells with which also binding to bone marrow et al, 1994, 1996). Similarly, low expression of and in CML cells is by treatment 1997). The actin provides structural to the cell and plays an important role in signal transduction. activation of a number of proteins that have effects on adhesion and cell These include tensin, talin, and et al, 1995). factor of normal cells induces in cell mediated via the with downstream effects on cellular and (Sattler & Salgia, 1997). In its location, BCR–ABL of the actin with it in (McWhirter & 1991; Salgia et al, 1995). In transformed cells, BCR–ABL and a number of cytoskeletal proteins. It is thus thought to produce of CML This has recently been using to study the and of cell lines to and following transformation by BCR–ABL, and of primary CML cells, compared with normal The presence of BCR–ABL was shown to induce and cytoskeletal increased motility, membrane and of actin with and of on These were shown to be on tyrosine kinase activity and to be reversed by et al, 1997). remains the role of BCR–ABL in the inhibition of apoptosis in primary cells from chronic phase CML In the has of biological between Ph+ cell lines from blast phase cytokine-dependent murine cell lines transduced with p210BCR–ABL and primary chronic phase CML In cell lines, expression of BCR–ABL them at least in through suppression of apoptosis (Daley & Baltimore, 1988; et al, 1988; et al, 1994). this is mediated by of bcl-2 & 1995), by activation of the PI-3 kinase pathway (Sattler et al, 1996) or by increased transcription of following activation of the Ras pathway, is and several potential pathways may be relevant (Sawyers et al, 1995; et al, 1995; Laneuville, 1995). BCR–ABL expression has also been shown to both cell lines and primary CML cells from apoptosis induced by growth factor or and this may be reversed by to BCR–ABL et al, 1994, 1995). However, other groups have to these findings et al, 1993; et al, et al, 1995). More recently, the of induction of apoptosis with was in BCR–ABL-transformed cells, factor apoptosis was in the presence of BCR–ABL et al, 1998). it is to that there is evidence suppression of apoptosis as the primary mechanism of the leukaemic in In CML, a massive of the is in all patients and cells are found in the A combination of abnormalities induced by BCR–ABL may underlie these however, relative to disease behaviour is as of the most laboratory is that enforced expression of BCR–ABL in cytokine-dependent immortalized murine cell lines may induce growth factor-independent certain this has been with autocrine of and factor (Daley & Baltimore, 1988; & Witte, 1989; Kabarowski et al, 1994). Furthermore, using to induce CML in murine Zhang & and Li et have demonstrated that a of mice with CML-like induced by BCR–ABL, have levels of autocrine and/or in In the both and be detected by & Ren, 1998). However, until recently, have to these findings to primary CML, at least for patients in the chronic phase of the disease. groups have that the growth of CML are to in normal bone with normal to growth factors and no evidence of growth factor independence. on many abnormalities in proliferation, and stem cell of Ph+ and stem cells may be identified. For example, when compared in to the majority of which are of cycle at CML cells appear to be in the presence of bone marrow or as single cell et al, This active within the Ph+ stem cell appears to be with an towards and a reciprocal for et al, 1997). Therefore, in CML, of cell cycle control appears to be in of myeloid to this include the of CML cells to the negative regulator et al, 1993), and the between BCR–ABL and the receptor et al, 1996). state the abnormalities in cell cycle control are thought to a defect of CML cells, to an of Ph+ However, following normal haemopoiesis is to a growth for over Ph+ As results from both in vitro and in vivo murine models have that autocrine play a role in the and of A number of additional have further to this In primary CML cells or cell lines, many biological and are that are to which in normal cells to of et al, 1997; et al, 1999). Furthermore, transplantation of murine bone marrow cells, or retrovirally transduced to produce will induce a state to CML et al, 1989; et al, 1989; et al, 1993). to direct evidence for an autocrine mechanism in primary CML were several