The molecular biology of chronic myeloid leukemia

Review article

The molecular biology of chronic myeloid leukemia Michael W. N. Deininger, John M. Goldman, and Junia V. Melo

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 abnormal- ity 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 theABL proto-oncogene normally on chromo- some 93 and a previously unknown gene on chromosome 22, later termedBCR 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 ofBCR-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 ofBCRandABL. 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 physiologic function of the translocation partners

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 ubiqui- tously 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 39 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 informa- tion 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 ofABL 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 withdbl-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-kB.27 The C-terminus has GTPase activity for Rac,28 a small GTPase of the Ras superfamily that regulates actin polymerization and the activity of an NADPH oxidase in phagocytic cells.29 In addition, Bcr can be phosphory- lated on several tyrosine residues,30 especially tyrosine 177, which binds Grb-2, an important adapter molecule involved in the activation of the Ras pathway.31 Interestingly, Abl has been shown to phosphorylate Bcr in COS1 cells, resulting in a reduction of Bcr kinase activity.31,32Although these data argue for a role of Bcr in signal transduction, their true biologic relevance remains to be determined. The fact thatBCRknockout mice are viable and the fact that an increased oxidative burst in neutrophils is thus far the only recognized defect33 probably reflect the redundancy of signaling pathways. If there is a role for Bcr in the pathogenesis of Ph-positive leukemias, it is not clearly discernible because the incidence and biology of P190BCR-ABL-induced leukemia are the same inBCR2/2 mice as they are in wild-type mice.34

Molecular anatomy of the BCR-ABL translocation

The breakpoints within theABL gene at 9q34 can occur anywhere over a large (greater than 300 kb) area at its 59 end, either upstream

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.

Submitted November 16, 1999; accepted July 12, 2000.

Supported by grants from Leukaemia Research Fund (UK) and the Dr Ernst und Anita Bauer Stiftung (Germany).

Reprints: Michael W. N. Deininger, Department of Hematology/Oncology,

University of Leipzig, Johannisallee 32, Leipzig 04103, Germany; e-mail:

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2000 by The American Society of Hematology

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of the first alternative exon Ib, downstream of the second alterna- tive 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 whichBCR sequences are fused toABL exon a2. In contrast toABL, break- points withinBCR 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 spanningBCRexons 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 alternativeBCRexons e29 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 (m-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-ABLCML.40

The low level of expression of these P190-type transcripts com- pared 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 ofBCR and ABL in the oncogenic fusion protein. Interestingly,ABLexon 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 underly- ing 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 theBCRmoiety can in principle be replaced by other sequences and still cause leukemia. Interestingly, a fusion betweenTEL(ETV6)and theABL-related geneARG 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-ABLfusion transcripts can be induced in hematopoietic cells by exposure to IR in vitro50; such IR-induced…