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REVIEW ARTICLE
From the Department of Hematology/Oncology, University
of Leipzig, Germany; and the Department of Haematology, Imperial
College School of Medicine, Hammersmith Hospital, London, United
Kingdom.
Chronic myeloid leukemia (CML) is probably the most
extensively studied human malignancy. The discovery of the Philadelphia (Ph) chromosome in 19601 as the first consistent
chromosomal abnormality associated with a specific type of leukemia was
a breakthrough in cancer biology. It took 13 years before it was
appreciated that the Ph chromosome is the result of a t(9;22)
reciprocal chromosomal translocation2 and another 10 years
before the translocation was shown to involve the ABL
proto-oncogene normally on chromosome 93 and a previously
unknown gene on chromosome 22, later termed BCR for
breakpoint cluster region.4 The deregulated Abl tyrosine kinase activity was then defined as the pathogenetic
principle,5 and the first animal models were
developed.6 The end of the millennium sees all this
knowledge transferred from the bench to the bedside with the arrival of
Abl-specific tyrosine kinase inhibitors that selectively inhibit the
growth of BCR-ABL-positive cells in vitro7,8
and in vivo.9 In this review we will try to summarize what is currently known
about the molecular biology of CML. Because several aspects of CML
pathogenesis may be attributable to the altered function of the 2 genes
involved in the Ph translocation, we will also address the
physiological roles of BCR and ABL. We concede
that a review of this nature can never be totally comprehensive without losing clarity, and we therefore apologize to any authors whose work we
have not cited. The ABL gene is the human homologue of the
v-abl oncogene carried by the Abelson murine leukemia virus
(A-MuLV),10 and it encodes a nonreceptor tyrosine
kinase.11 Human Abl is a ubiquitously expressed 145-kd
protein with 2 isoforms arising from alternative splicing of the first
exon.11 Several structural domains can be defined within
the protein (Figure 1). Three SRC
homology domains (SH1-SH3) are located toward the NH2
terminus. The SH1 domain carries the tyrosine kinase function, whereas
the SH2 and SH3 domains allow for interaction with other
proteins.12 Proline-rich sequences in the center of the
molecule can, in turn, interact with SH3 domains of other proteins,
such as Crk.13 Toward the 3' end, nuclear localization
signals14 and the DNA-binding15 and
actin-binding motifs16 are found.
Several fairly diverse functions have been attributed to Abl, and the
emerging picture is complex. Thus, the normal Abl protein is involved
in the regulation of the cell cycle,17,18 in the cellular
response to genotoxic stress,19 and in the transmission of
information about the cellular environment through integrin signaling.20 (For a comprehensive review of Abl function,
see Van Etten21). Overall, it appears that the Abl protein
serves a complex role as a cellular module that integrates signals from various extracellular and intracellular sources and that influences decisions in regard to cell cycle and apoptosis. It must be stressed, however, that many of the data are based solely on in vitro studies in
fibroblasts, not hematopoietic cells, and are still controversial. Unfortunately, the generation of ABL knockout mice failed to
resolve most of the outstanding issues.22,23
The 160-kd Bcr protein, like Abl, is ubiquitously
expressed.11 Several structural motifs can be delineated
(Figure 2). The first N-terminal exon
encodes a serine-threonine kinase. The only substrates of this kinase
identified so far are Bap-1, a member of the 14-3-3 family of
proteins,24 and possibly Bcr itself.11 A
coiled-coil domain at the N-terminus of Bcr allows dimer formation in
vivo.25 The center of the molecule contains a region with dbl-like and pleckstrin-homology (PH) domains that stimulate
the exchange of guanidine triphosphate (GTP) for guanidine diphosphate (GDP) on Rho guanidine exchange factors,26 which in turn
may activate transcription factors such as NF-
The breakpoints within the ABL gene at 9q34 can occur
anywhere over a large (greater than 300 kb) area at its 5' end, either upstream of the first alternative exon Ib, downstream of the second alternative exon Ia, or, more frequently, between the
two35 (Figure 3). Regardless
of the exact location of the breakpoint, splicing of the primary hybrid
transcript yields an mRNA molecule in which BCR sequences
are fused to ABL exon a2. In contrast to ABL,
breakpoints within BCR localize to 1 of 3 so-called
breakpoint cluster regions (bcr). In most patients with CML
and in approximately one third of patients with Ph-positive acute
lymphoblastic leukemia (ALL), the break occurs within a 5.8-kb area
spanning BCR exons 12-16 (originally referred to as exons
b1-b5), defined as the major breakpoint cluster region
(M-bcr). Because of alternative splicing, fusion transcripts
with either b2a2 or b3a2 junctions can be formed. A 210-kd chimeric
protein (P210BCR-ABL) is derived from this mRNA.
In the remaining patients with ALL and rarely in patients with CML,
characterized clinically by prominent monocytosis,36,37
the breakpoints are further upstream in the 54.4-kb region between the
alternative BCR exons e2' and e2, termed the minor
breakpoint cluster region (m-bcr). The resultant e1a2 mRNA
is translated into a 190-kd protein
(P190BCR-ABL). Recently, a third breakpoint
cluster region (µ-bcr) was identified downstream of exon
19, giving rise to a 230-kd fusion protein (P230BCR-ABL) associated with the rare
Ph-positive chronic neutrophilic leukemia,38 though not in
all cases.39 If sensitive techniques such as nested reverse transcription-polymerase chain reaction are used, transcripts with the e1a2 fusion are detectable in many patients with classical P210BCR-ABL CML.40 The low level of
expression of these P190-type transcripts compared to P210 indicates
that they are most likely the result of alternative splicing of the
primary mRNA. Occasional cases with other junctions, such as b2a3,
b3a3, e1a3, e6a2,41 or e2a2,42 have been
reported in patients with ALL and CML. These "experiments of
nature" provide important information as to the function of the
various parts of BCR and ABL in the oncogenic
fusion protein. Interestingly, ABL exon 1, even if retained
in the genomic fusion, is never part of the chimeric mRNA. Thus, it
must be spliced out during processing of the primary mRNA; the
mechanism underlying this apparent peculiarity is unknown. Based on the
observation that the Abl part in the chimeric protein is almost
invariably constant while the Bcr portion varies greatly, one may
deduce that Abl is likely to carry the transforming principle whereas the different sizes of the Bcr sequence may dictate the phenotype of
the disease. In support of this notion, rare cases of ALL express a
TEL-ABL fusion gene,43,44 indicating that the
BCR moiety can in principle be replaced by other sequences
and still cause leukemia. Interestingly, a fusion between
TEL(ETV6) and the ABL-related gene ARG
has recently been described in a patient with AML.45 Although all 3 major Bcr-Abl fusion proteins induce a CML-like disease
in mice, they differ in their ability to induce lymphoid leukemia,46 and, in contrast to P190 and P210,
transformation to growth factor independence by P230BCR-ABL
is incomplete,47 which is consistent with the relatively
benign clinical course of P230-positive chronic neutrophilic
leukemia.38
One of the most intriguing questions relates to the events responsible
for the chromosomal translocation in the first place. From
epidemiologic studies it is well known that exposure to ionizing radiation (IR) is a risk factor for CML.48,49 Moreover,
BCR-ABL fusion transcripts can be induced in hematopoietic
cells by exposure to IR in vitro50; such IR-induced
translocations may not be random events but may depend on the cellular
background and on the particular genes involved. Two recent reports
showed that the physical distance between the BCR and the
ABL genes in human lymphocytes51 and
CD34+ cells52 is shorter than might be
expected by chance; such physical proximity could favor translocation
events involving the 2 genes. However, the presence of the
BCR-ABL translocation in a hematopoietic cell is not in
itself sufficient to cause leukemia because BCR-ABL fusion
transcripts of M-bcr and m-bcr type are
detectable at low frequency in the blood of many healthy
individuals.53,54 It is unclear why Ph-positive leukemia
develops in a tiny minority of these persons. It may be that the
translocation occurs in cells committed to terminal differentiation
that are thus eliminated or that an immune response suppresses or
eliminates Bcr-Abl-expressing cells. Indirect evidence that such a
mechanism may be relevant comes from the observation that certain HLA
types protect against CML.55 Another possibility is that
BCR-ABL is not the only genetic lesion required to induce
chronic-phase CML. Indeed, a skewed pattern of G-6PD isoenzymes has
been detected in Ph-negative Epstein-Barr virus-transformed
B-cell lines derived from patients with CML, suggesting that a
Ph-negative pathologic state may precede the emergence of the Ph
chromosome.56
Essential features of the Bcr-Abl protein
Deregulation of the Abl tyrosine kinase
A host of substrates can be tyrosine phosphorylated by
Bcr-Abl (Table 1).
Most important, because of autophosphorylation, there is a marked
increase of phosphotyrosine on Bcr-Abl itself, which creates
binding sites for the SH2 domains of other proteins. Generally,
substrates of Bcr-Abl can be grouped according to their physiologic role into adapter molecules (such as Crkl and
p62DOK), proteins associated with the organization of the
cytoskeleton and the cell membrane (such as paxillin and talin), and
proteins with catalytic function (such as the nonreceptor tyrosine
kinase Fes or the phosphatase Syp). It is important to note that the choice of substrates depends on the cellular context. For example, Crkl
is the major tyrosine-phosphorylated protein in CML
neutrophils,73 whereas phosphorylated p62DOK
is predominantly found in early progenitor cells.74
Tyrosine phosphatases counterbalance and regulate the effects of
tyrosine kinases under physiologic conditions, keeping cellular phosphotyrosine levels low. Two tyrosine phosphatases,
Syp83 and PTP1B,84 have been shown to form
complexes with Bcr-Abl, and both appear to dephosphorylate Bcr-Abl.
Interestingly, PTP1B levels increase in a kinase-dependent manner,
suggesting that the cell attempts to limit the impact of Bcr-Abl
tyrosine kinase activity. At least in fibroblasts, transformation by
Bcr-Abl is impaired by the overexpression of PTP1B.85
Interestingly, we recently observed the up-regulation of receptor
protein tyrosine phosphatase Activated signaling pathways and biologic properties of BCR-ABL-positive cells Three major mechanisms have been implicated in the malignant transformation by Bcr-Abl, namely altered adhesion to stroma cells and extracellular matrix,87 constitutively active mitogenic signaling88 and reduced apoptosis89 (Figure 5). A fourth possible mechanism is the recently described proteasome-mediated degradation of Abl inhibitory proteins.66
Altered adhesion properties CML progenitor cells exhibit decreased adhesion to bone marrow stroma cells and extracellular matrix.87,90 In this scenario, adhesion to stroma negatively regulates cell proliferation, and CML cells escape this regulation by virtue of their perturbed adhesion properties. Interferon- (IFN-), an active therapeutic agent in CML, appears to reverse the adhesion defect.91
Recent data suggest an important role for  -integrins in the
interaction between stroma and progenitor cells. CML cells express an
adhesion-inhibitory variant of 1 integrin that is not found in
normal progenitors.92 On binding to their receptors,
integrins are capable of initiating normal signal transduction from
outside to inside93; it is thus conceivable that the
transfer of signals that normally inhibit proliferation is impaired in
CML cells. Because Abl has been implicated in the intracellular
transduction of such signals, this process may be further disturbed by
the presence of a large pool of Bcr-Abl protein in the cytoplasm.
Furthermore, Crkl, one of the most prominent tyrosine-phosphorylated
proteins in Bcr-Abl-transformed cells,73 is involved in
the regulation of cellular motility94 and in integrin-mediated cell adhesion95 by association with
other focal adhesion proteins such as paxillin, the focal adhesion
kinase Fak, p130Cas,96 and Hef1.97 We
recently demonstrated that Bcr-Abl tyrosine kinase up-regulates the
expression of 6 integrin mRNA,86 which points to
transcriptional activation as yet another possible mechanism by which
Bcr-Abl may have an impact on integrin signaling. Thus, though there is
sound evidence that Bcr-Abl influences integrin function, it is more
difficult to determine the precise nature of the biologic consequences,
and, at least in certain cellular systems, integrin function appears to
be enhanced rather than reduced by Bcr-Abl.98
Activation of mitogenic signaling Ras and the MAP kinase pathways.
Several links between Bcr-Abl and Ras have been defined.
Autophosphorylation of tyrosine 177 provides a docking site for the adapter molecule Grb-2.57 Grb-2, after binding to the Sos
protein, stabilizes Ras in its active GTP-bound form. Two other adapter molecules, Shc and Crkl, can also activate Ras. Both are substrates of
Bcr-Abl73,99 and bind Bcr-Abl through their SH2 (Shc) or SH3 (Crkl) domains. The relevance of Ras activation by Crkl is, however, questionable because it appears to be restricted to
fibroblasts.100 Moreover, direct binding of Crkl to
Bcr-Abl is not required for the transformation of myeloid
cells.101 Circumstantial evidence that Ras activation is
important for the pathogenesis of Ph-positive leukemias comes from the
observation that activating mutations are uncommon, even in the blastic
phase of the disease,102 unlike in most other tumors. This
implies that the Ras pathway is constitutively active, and no further
activating mutations are required. There is still dispute as to which
mitogen-activated protein (MAP) kinase pathway is downstream of Ras in
Ph-positive cells. Stimulation of cytokine receptors such as IL-3 leads
to the activation of Ras and the subsequent recruitment of the
serine-threonine kinase Raf to the cell membrane.103 Raf
initiates a signaling cascade through the serine-threonine kinases
Mek1/Mek2 and Erk, which ultimately leads to the activation of gene
transcription.104 Although some data indicate that this
pathway may be activated only in v-abl- but not in
BCR-ABL-transformed cells,105 this view has recently been
challenged.106 Moreover, activation of the Jnk/Sapk
pathway by Bcr-Abl has been demonstrated and is required for malignant
transformation107; thus, signaling from Ras may be
relayed through the GTP-GDP exchange factor Rac108 to
Gckr (germinal center kinase related)109 and further down
to Jnk/Sapk (Figure 6). There is also
some evidence that p38, the third pillar of the MAP kinase pathway, is
also activated in BCR-ABL-transformed cells, and there are other
pathways with mitogenic potential. In any case, the signal is
eventually transduced to the transcriptional machinery of the
cell.
It is also possible that Bcr-Abl uses growth factor pathways in a more direct way. For example, association with the c subunit of the IL-3
receptor110 and the Kit receptor111 has been
observed. Interestingly, the pattern of tyrosine-phosphorylated
proteins seen in normal progenitor cells after stimulation with Kit
ligand is similar to the pattern seen in CML progenitor
cells.112 Dok-1 (p62DOK), one of the most
prominent phosphoproteins in this setting, forms complexes with Crkl,
RasGAP, and Bcr-Abl. In fact, there may be a whole family of related
proteins with similar functions for example, the recently described
Dok-2 (p56DOK2).113 Somewhat surprisingly,
p62DOK is essential for transformation of Rat-1 fibroblasts
but not for growth-factor independence of myeloid
cells114; thus, its true role remains to be defined.
Jak-Stat pathway. The first evidence for involvement of the Jak-Stat pathway came from studies in v-abl-transformed B cells.62 Constitutive phosphorylation of Stat transcription factors (Stat1 and Stat5) has since been reported in several BCR-ABL-positive cell lines115 and in primary CML cells,116 and Stat5 activation appears to contribute to malignant transformation.117 Although Stat5 has pleiotropic physiologic functions,118 its effect in BCR-ABL-transformed cells appears to be primarily anti-apoptotic and involves transcriptional activation of Bcl-xL.119,120 In contrast to the activation of the Jak-Stat pathway by physiologic stimuli, Bcr-Abl may directly activate Stat1 and Stat5 without prior phosphorylation of Jak proteins. There seems to be specificity for Stat6 activation by P190BCR-ABL proteins as opposed to P210BCR-ABL.115 It is tempting to speculate that the predominantly lymphoblastic phenotype in these leukemias is related to this peculiarity. The role of the Ras and Jak-Stat pathways in the cellular response to growth factors could explain the observation that BCR-ABL renders a number of growth factor-dependent cell lines factor independent.105,121 In some experimental systems there is evidence for an autocrine loop dependent on the Bcr-Abl-induced secretion of growth factors,122 and it was recently reported that Bcr-Abl induces an IL-3 and G-CSF autocrine loop in early progenitor cells.123 Interestingly, Bcr-Abl tyrosine kinase activity may induce expression not only of cytokines but also of growth factor receptors such as the oncostatin M receptor.86 One should bear in mind, however, that during
the chronic phase, CML progenitor cells are still dependent on external
growth factors for their survival and proliferation,124 though less than normal progenitors.125 A recent study
sheds fresh light on this issue. FDCPmix cells transduced with a
temperature-sensitive mutant of BCR-ABL have a reduced
requirement for growth factors at the kinase permissive temperature
without differentiation block.126 This situation resembles
chronic-phase CML, in which the malignant clone has a subtle growth
advantage while retaining almost normal differentiation capacity.
PI3 kinase pathway. PI3 kinase activity is required for the proliferation of BCR-ABL-positive cells.127 Bcr-Abl forms multimeric complexes with PI3 kinase, Cbl, and the adapter molecules Crk and Crkl,95 in which PI3 kinase is activated. The next relevant substrate in this cascade appears to be the serine-threonine kinase Akt.128 This kinase had previously been implicated in anti-apoptotic signaling.129 A recent report placed Akt in the downstream cascade of the IL-3 receptor and identified the pro-apoptotic protein Bad as a key substrate of Akt.130 Phosphorylated Bad is inactive because it is no longer able to bind anti-apoptotic proteins such as BclXL and it is trapped by cytoplasmic 14-3-3 proteins. Altogether this indicates that Bcr-Abl might be able to mimic the physiologic IL-3 survival signal in a PI3 kinase-dependent manner (see also below). Ship131 and Ship-2,132 2 inositol phosphatases with somewhat different specificities, are activated in response to growth factor signals and by Bcr-Abl. Thus, Bcr-Abl appears to have a profound effect on phosphoinositol metabolism, which might again shift the balance to a pattern similar to physiologic growth factor stimulation. Myc pathway. Overexpression of Myc has been demonstrated in many human malignancies. It is thought to act as a transcription factor, though its target genes are largely unknown. Activation of Myc by Bcr-Abl is dependent on the SH2 domain, and the overexpression of Myc partially rescues transformation-defective SH2 deletion mutants whereas the overexpression of a dominant-negative mutant suppresses transformation.133 The pathway linking Myc to the SH2 domain of Bcr-Abl is still unknown. However, results obtained in v-abl-transformed cells suggest that the signal is transduced through Ras/Raf, cyclin-dependent kinases (cdks), and E2F transcription factors that ultimately activate the MYC promoter.134 Similar results were reported for BCR-ABL-transformed murine myeloid cells.135 How these findings relate to human Ph-positive cells is unknown. It seems likely that the effects of Myc in Ph-positive cells are probably not different from those in other tumors. Depending on the cellular context, Myc may constitute a proliferative or an apoptotic signal.136,137 It is therefore likely that the apoptotic arm of its dual function is counterbalanced in CML cells by other mechanisms, such as the PI3 kinase pathway. Inhibition of apoptosis Expression of Bcr-Abl in factor-dependent murine138 and human122 cell lines prevents apoptosis after growth-factor withdrawal, an effect that is critically dependent on tyrosine kinase activity and that correlates with the activation of Ras.88,139 Moreover, several studies showed that BCR-ABL-positive cell lines are resistant to apoptosis induced by DNA damage.89,140 The underlying biologic mechanisms are still not well understood. Bcr-Abl may block the release of cytochrome C from the mitochondria and thus the activation of caspases.141,142 This effect upstream of caspase activation might be mediated by the Bcl-2 family of proteins. Bcr-Abl has been shown to up-regulate Bcl-2 in a Ras-143 or a PI3 kinase-dependent128 manner in Baf/3 and 32D cells, respectively. Moreover, as mentioned previously, BclxL is transcriptionally activated by Stat5 in BCR-ABL-positive cells.119,120Another link between BCR-ABL and the inhibition of apoptosis
might be the phosphorylation of the pro-apoptotic protein Bad. In
addition to Akt, Raf-1, immediately downstream of Ras, phosphorylates Bad on 2 serine residues.144,145 Two recent studies
provided evidence that the survival signal provided by Bcr-Abl is at
least partially mediated by Bad and requires targeting of Raf-1 to the mitochondria.146,147 It is also possible that Bcr-Abl
inhibits apoptosis by down-regulating interferon consensus sequence
binding protein (ICSBP).148,149 These data are interesting
because ICSBP knockout mice develop a myeloproliferative
syndrome,150 and hematopoietic progenitor cells from
ICSBP It becomes clear that the multiple signals initiated by Bcr-Abl have
proliferative and anti-apoptotic qualities that are frequently difficult to separate. Thus, Bcr-Abl may shift the balance toward the
inhibition of apoptosis while simultaneously providing a proliferative stimulus. This is in line with the concept that a proliferative signal
leads to apoptosis unless it is counterbalanced by an anti-apoptotic signal,152 and Bcr-Abl fulfills both requirements at the
same time. There is, however, controversy. One report found 32D cells transfected with BCR-ABL to be more sensitive to IR than the
parental cells,153 whereas 2 other studies failed to
detect any difference between CML and normal primary progenitor cells
with regard to their sensitivity to IR and growth factor
withdrawal.124,154 Furthermore, based on results obtained
in transfected cell systems, it was suggested that Bcr-Abl inhibits
apoptosis mediated by the Fas receptor/Fas ligand
system.155 However, though there may be a role for this
system in mediating the clinical response to interferon- Degradation of inhibitory proteins. The recent discovery that Bcr-Abl induces the proteasome-mediated degradation of Abi-1 and Abi-2,66 2 proteins with inhibitory function, may be the first indication of yet another way by which Bcr-Abl induces cellular transformation. Most compelling, the degradation of Abi-1 and Abi-2 is specific for Ph-positive acute leukemias and is not seen in Ph-negative samples of comparable phenotype. The overall significance of this observation remains to be seen, and one must bear in mind that the data refer to acute leukemias and not to chronic phase CML. It is nevertheless tempting to speculate that other proteins, whose level of expression is regulated through the proteasome pathway, may also be degraded. A good candidate would be the cell cycle inhibitor p27, but to our knowledge no data are available yet.
Various experimental systems have been developed to study the pathophysiology of CML. All of them have their advantages and shortcomings, and it is probably fair to say that there is still no ideal in vitro or in vivo model that would cover all aspects of the human disease. Cell lines Fibroblasts.
Fibroblast lines have been used extensively in CML research because
they are easy to manipulate. Fibroblast transformation Hematopoietic cell lines. Until relatively recently, only a few BCR-ABL-positive lines derived from CML were available, but their number has grown considerably in the past few years.164 They include cell lines with myeloid differentiation, such as the well-known K562, and lymphoid phenotype, such as BV173. The main drawback common to all these lines is the fact that they are derived from blast crisis and, thus, contain genetic lesions in addition to BCR-ABL. Consequently, they may reflect blast crisis fairly well but are insufficient models of chronic phase CML. Until now, no cell line from chronic phase CML has been established, just as no cell line could be derived from normal human bone marrow. Even attempts to immortalize Ph-positive B-cells from patients in the chronic phase of disease were not successful because these lines have a limited life span,165 in contrast to their Ph-negative counterparts. One could therefore speculate that the very establishment of a line from a patient with CML would be diagnostic of advanced disease. In this context, it is surprising that most human CML lines remain dependent on Bcr-Abl tyrosine kinase activity for their proliferation and survival, as shown by their susceptibility to the effects of the Abl-specific tyrosine kinase inhibitor STI571.8 However, the phenotype of these cell lines is that of an acute leukemia. Therefore great caution is warranted if experimental results are to be transferred to chronic phase CML. A striking example is the fact that inhibition of apoptosis by Bcr-Abl is easily demonstrable in cell lines139 but not in primary cells.124 It should also be noted that many of the lines used have undergone hundreds, if not thousands, of rounds of replication, and different laboratories frequently house lines that have little in common but their names and BCR-ABL positivity. Transformation of factor-dependent cell lines to growth-factor independence is an important feature of Bcr-Abl, and, in fact, other oncoproteins that contain an activated tyrosine kinase.43,166 Although it is usually difficult to obtain stable expression of BCR-ABL in previously immortalized cell lines, this is relatively easily achieved in factor-dependent lines, presumably because BCR-ABL expression is an advantage to the latter but useless or even detrimental to the former. Murine cell lines such as Baf/3 and 32D and human cell lines such as MO7 were used to study the effects of BCR-ABL by direct comparison between transduced and parental cells. A particular advantage of the murine lines is the fact that they are derived from normal nonmalignant hematopoietic cells. Unfortunately, this does not rule out the development of additional mutations167 that confer a selective growth advantage. Furthermore, it is not clear how the transformation to complete factor independence relates to clinical CML in which the cells are still factor-dependent, though obviously less so than normal hematopoietic cells.123 The subject of growth factor independence and BCR-ABL transformation has been reviewed recently.168 None of the cell lines mentioned above is capable of multilineage hematopoietic differentiation. Two strategies are promising in overcoming this restraint. A recent report126 shows that murine FDCPmix cells, transduced with a temperature-sensitive mutant of BCR-ABL, become partially factor-independent at the permissive temperature, in analogy to chronic phase CML. They retain the capacity for terminal differentiation, similar to chronic phase CML cells. Another approach is the study of embryonic stem (ES) cells transduced with BCR-ABL. In one such experimental system, it was possible to reproduce one cardinal feature of the clinical disease in the model, namely the expansion of the myeloid compartment at the expense of the erythroid compartment.169 Interestingly, the increase in total cell numbers in the BCR-ABL-transduced ES cells was found to result from increased proliferation though there was little effect on apoptosis, another finding in line with observations in primary Ph-positive cells.124,154 In this system, a stromal cell layer is used on which the ES cells removed from leukemia-inhibitory factor (LIF) differentiate into hemangioblasts and then into hematopoietic cells. This may explain why these results are not strictly comparable to those of another study, in which BCR-ABL resulted in the decreased formation of embryonal bodies along with increased output of all kinds of hematopoietic progenitors.170 In yet another study, BCR-ABL-transformed ES cells transplanted into irradiated mice induced a leukemic syndrome with many features of CML.171 If developed further, these models may well be able to retain the major advantage of cell linestheir ease of
manipulationwhile at the same time moving the in vitro system closer
to the clinical disease.
Bearing all these caveats in mind, there is no doubt that the study of
cell lines contributed significantly to our understanding of CML.
Particularly, many of the proteins that interact with Bcr-Abl were
identified in Ph-positive cell lines, where they are more abundantly
expressed than in primary cells. Thus, though these lines are
invaluable tools for screening, it is important to confirm the results
in primary cells.
Primary cells. The study of patient material and its comparison with normal hematopoietic progenitor cells is certainly the gold standard of CML research, particularly for the chronic phase of the disease. Much of the data refer to operationally defined cellular properties of CML versus normal cells, such as clonogenicity or adherence to bone marrow stroma; to give a comprehensive account of the cellular biology of CML would require a review in its own right. We will therefore focus on some areas in which the study of primary CML cells has been particularly instrumental to the study of the molecular biology of the disease. One of the main problems when studying primary cells is inherent in the very nature of chronic phase cellsthey tend to mature when
placed in culture. Thus, the window of time for in vitro studies is
narrow, and expansion of very primitive cells, the least prevalent but
most interesting population, is difficult and carries the risk for
introducing nonphysiologic alterations.172 Furthermore,
there is considerable variation between patients that frequently
results in an overlap rather than a clear distinction between normal
cells and CML cells. Last, results are unreliable unless clearly
defined cell populations such as CD34+ cells are studied.
To a large extent, these problems can be overcome by the introduction
of retroviral BCR-ABL expression vectors to murine or human
primary bone marrow cells (see "Animal models" below).
A striking example of how fruitful the comparison of primary cell
populations can be is the study of tyrosine-phosphorylated proteins in
CD34+ cells.112 This study led to the
subsequent identification of p62DOK74;173 and
SHIP2132 as mediators of Bcr-Abl-induced transformation. Moreover, it produced the important notion that Bcr-Abl tyrosine kinase
activity may have consequences similar to the activation of the KIT
receptor.112 Another example is the identification of CRKL
as the major tyrosine-phosphorylated protein in CML
neutrophils.73
The recent possibility of turning off the Bcr-Abl tyrosine kinase
activity in cell lines and primary cells with STI5717,8 provided the opportunity to study the effects of the BCR-ABL
gene when expressed from its natural BCR promoter at
"physiological" levels. This is certainly an advantage over
transduced cell systems; the drawback, however, is that effects related
to inhibition of the KIT and PDGFR kinases, and potentially other
unidentified tyrosine kinases, cannot be ruled out. Furthermore, the
Bcr-Abl protein, though kinase-inactive, is still present in the cells and may interfere with other proteins.
Animal models Thus far, no animals other than mice have been used for the study of CML in vivo. Various approaches have been taken.Engraftment of BCR-ABL-transformed cell lines in syngeneic mice. Murine factor-dependent cell lines such as 32D transduced with BCR-ABL give rise to an aggressive leukemia when transplanted into syngeneic recipients.60,174 This is an excellent in vivo model to test the efficacy of new drugs, such as the tyrosine kinase inhibitor STI571, in vivo. Furthermore, the impact on leukemogenicity of modifications within the Bcr-Abl protein and modifications to the respective cell lines (such as the introduction of co-stimulatory molecules174) can be tested. The main drawback is that the disease is a form of acute leukemia and is thus far from chronic phase CML. Engraftment of immunodeficient mice with human BCR-ABL-positive cells. Cell lines derived from human CML blast crisis are relatively easily propagated in severe combined immunodeficiency (SCID) mice.175 The distribution of the leukemia cells is fairly similar to the human disease, that is, they home to bone marrow and peripheral blood before they metastasize to nonhematopoietic tissues. More recently, it was shown that SCID mice can be engrafted with chronic phase CML cells if the cell inoculum is large enough (in the range of 1 × 108 cells).176 Up to 10% human cells were detectable in the recipient bone marrow and showed multilineage differentiation. Interestingly, most colonies were BCR-ABL negative and thus were derived from the patient's residual normal hematopoiesis. This is reminiscent of long-term bone marrow cultures of CML177 and shows that host factors modify the disease to a great extent, a problem that will persist, even if higher percentages of engraftment can eventually be achieved. A step into this direction is the use of nonobese diabetes-SCID mice. In addition to the SCID defect in V(D)J recombination, these animals lack functional natural killer cells. Chronic phase CML cells and, even more so, cells from accelerated phase or blast crisis readily engraft in these mice, and there is a significant correlation between engraftment and disease state.178 Interestingly, the cells were exclusively Ph-positive in most cases, in contrast to cells engrafted in SCID mice, as mentioned above. This may be attributed to technical reasons but may also reflect a genuine difference between the different strains of mice. We can anticipate that these murine models will be useful for studying certain aspects of CML, such as the response to novel forms of treatment. Their value in investigating the human disease will be limited because it is difficult to see how disease modification by host factors could ever be ruled out. Transgenic mouse models. Attempts to use BCR-ABL transgenic mice as a CML model go back to the late 1980s, when a full-length cDNA of BCR-ABL was not yet available and an artificial construct of human BCR sequences fused to v-abl was used instead.179 Since then, a number of studies have been published that clearly prove the oncogenic potential of BCR-ABL. Several different promoters were used to direct the expression to the desired target tissues. However, some problems were encountered. First, it became clear that Bcr-Abl has a toxic effect on embryogenesis,180 perhaps the consequence of a cytostatic effect in nonhematopoietic tissues.181 Recently, the expression of BCR-ABL from a tetracycline-repressible promoter effectively overcame this problem.182 Most striking, the leukemia in these transgenic mice is completely reversible on re-addition of tetracycline. The second problem with transgenic mice is that the P210 BCR-ABL variant relevant to CML is difficult to study because it is less efficient in inducing leukemia than P190, a finding that was again confirmed in a recent study.47 Third and most important, the types of leukemia that developed in these mice were acute and of either B- or T-lymphoid phenotype, regardless of whether they arose in P190 or P210 transgenic animals. Thus, they resembled BCR-ABL-positive ALL but not chronic-phase CML183,184. In fact, myeloid leukemias developed rarely, if at all. A recent report185 may represent a major advance in this respect. In this study, BCR-ABL was expressed from the Tec promoter, a cytoplasmic tyrosine kinase predominantly expressed in hematopoietic cells. Although the founder mice exhibited excessive proliferation of lymphoblasts, their progeny developed a CML-like disease, albeit after a relatively long latency period of approximately 10 months. Thus, it is likely that the problems of the transgenic models will eventually be resolved if the gene is targeted to the appropriate cell. Transduction of murine bone marrow cells with BCR-ABL retroviruses. In 1990, several groups reported that a CML-like myeloproliferative syndrome could be induced when P210BCR-ABL-infected marrow was transplanted into syngeneic recipients.6,186,187 Transplantation into secondary recipients frequently produced an identical disease while some mice developed acute leukemias of T- or B-cell phenotype, analogous to the development of lymphoid blast crisis in the clinical disease. Clonality was demonstrated in many cases. Roughly a quarter of the mice showed the myeloproliferative disease, whereas other recipients developed other distinct hematologic malignancies, such as macrophage tumors, B-ALL, T-ALL, and erythroleukemia. Most likely, these different diseases are the consequence of infection of different committed progenitor cells that, after transformation, give rise to the respective progeny. Not surprisingly, the infection conditions have a major impact on the disease phenotype.188 Building on the foundations of this early work, major improvements to the transduction-transplantation system have been made in the past few years. High-titer BCR-ABL retroviral stocks can be produced rapidly by transient transfection of packaging lines; the culture conditions have been refined, and the murine stem cell virus LTR has been introduced that allows for more efficient expr |
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Introduction
The physiologic function of...
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