BCR/ABL Genes and Leukemic Phenotype: from Molecular Mechanisms to Clinical Correlations

BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations

Fabrizio Pane*,1, Mariano Intrieri1,2, Concetta Quintarelli1, Barbara Izzo1, Giada Casadei Muccioli1 and Francesco Salvatore1

1CEINGE Biotechnologie Avanzate, and Dipartimento di Biochimica e Biotecnologie Mediche, Facolta` di Medicina, Universita` di Napoli Federico II, Italy; 2Dipartimento STAT, Facolta` di Scienze MFN, Universita` degli Studi del Molise - Isernia, Italy

The Philadelphia chromosome (Ph), a minute chromo- leukemias, the intimate role of the BCR/ABL protein some that derives from the balanced translocation in leukemic transformation has not yet been between chromosomes 9 and 22, was first described in completely elucidated. The knowledge of the role of 1960 and was for a long time the only genetic lesion BCR/ABL fusion gene in leukemia is further consistently associated with human cancer. This chro- complicated by cytogenetic and epidemiological mosomal translocation results in the fusion between the observations. This hybrid gene was originally 5’ part of BCR gene, normally located on chromosome described in a chronic type of leukemia affecting 22, and the 3’ part of the ABL gene on chromosome 9 the myeloid compartment, the chronic myelogenous giving origin to a BCR/ABL fusion gene which is leukemia (CML), and is present in almost all the transcribed and then translated into a hybrid protein. CML cases with a classical clinical picture, which is Three main variants of the BCR/ABL gene have been now considered the hallmark (Bennett et al., 1994). described, that, depending on the length of the sequence However, unlike other genetic lesions that are of the BCR gene included, encode for the p190BCR/ABL, specifically associated with particular types of leuke- P210BCR/ABL, and P230BCR/ABL proteins. These three mias (Rowley, 1998), the BCR/ABL hybrid gene is main variants are associated with distinct clinical types not restricted to CML, but it can also be found in a of human leukemias. Herein we review the data on the significant proportion of acute lymphoblastic leuke- correlations between the type of BCR/ABL gene and the mia (ALL) and less frequently in other chronic and corresponding leukemic clinical features. Lastly, drawing acute hemopoietic tumors (Melo, 1996). We described on experimental data, we provide insight into the a relatively benign and rather rare form of different transforming power of the three hybrid BCR/ myeloproliferative disease that we called ‘Neutrophi- ABL proteins. lic-chronic myeloid leukemia’ (CML-N), which is also Oncogene (2002) 21, 8652 – 8667. doi:10.1038/sj.onc. associated with a BCR/ABL gene, that shows a novel 1206094 position for the breakpoint in the BCR gene far downstream from the breakpoints already described Keywords: Philadelphia chromosome; Ph positive leu- (Pane et al., 1996b). Therefore, the identification of kemias; molecular pathogenesis; phenotype of leukemia this subset of patients carrying the novel type of BCR/ABL gene led to the hypothesis that the structure of this gene and, in particular, the location Introduction of the breakpoint on the BCR sequences correlates with the leukemic phenotype (Pane et al., 1996b). A Ph-positive leukemias have been the object of masterly account of these data and their far reaching extensive studies to define at molecular and cellular implications can be found in Melo (1996). level the complex mechanisms whereby the hybrid To minimize overlapping with the excellent reviews BCR/ABL protein may disrupt, in hemopoietic devoted to the intracellular interactions of BCR/ABL precursors, the maturation and replication processes proteins that have appeared in recent years (Faderl et and the physiological response to cytokines and al., 1999; Holyoake, 2001) or that are included in this growth factor leading, ultimately, to their neoplastic issue of the journal, we first trace the milestones that transformation. Although the expression of the have culminated in today’s state of the art, and then hybrid BCR/ABL protein is presumed to be an early discuss: (i) the molecular structure of BCR/ABL fusion pathogenic event and its elevated tyrosine kinase genes; (ii) the correlation between leukemic clinical activity to be a central event in Ph positive features and the position of the breakpoint in the BCR gene; (iii) the clinical characteristics of the CML-N cases described so far; and (iv) the hypothesis that *Correspondence: F Pane, CEINGE and Dipartimento di Biochimica could explain at molecular level the differential e Biotecnologie Mediche, Universita` di Napoli Federico II, Via S. transforming power of the various BCR/ABL fusion Pansini 5, 80131 Naples, Italy; E-mail: [email protected] proteins. BCR/ABL genes and leukemic phenotype F Pane et al 8653 Milestones neoplasias. The identification of an abnormally large (8.0 kb) v-ABL related mRNA in Ph positive cell lines The history of the hybrid BCR/ABL gene started in and in CML cells, but not in normal cells and in Ph 1960, when an abnormal, shortened chromosome 22, negative leukemias (Gale and Canaani, 1984), and the termed the Philadelphia chromosome, was described in characterization of a large (210 kDa) ABL related the leukemic cells of a patient affected by chronic phosphoprotein, the P210 (Konopka et al., 1984), myeloid leukemia (Nowell and Hungerford, 1960). reinforced the concept that CML was related to a Only 13 years later, the use of quinacrine fluorescence fusion gene. Importantly, the tyrosine kinase activity of and Giemsa banding in chromosome studying, allowed P210 resembled that of protein v-ABL (Witte et al., to clarify that Ph chromosome is the result of a 1980; Davis et al., 1985), which was phosphorylated, translocation of part of chromosome 22 to chromo- whereas the 145 kDa c-ABL protein which was not some 9 (Rowley, 1973). The molecular characterization phosphorylated in vivo, in normal cells (Konopka and of this translocation was facilitated when the c-ABL Witte, 1985). gene, the human homologous of v-ABL, an oncogene The definitive proof of the presence of a fusion gene originally identified in the Abelson murine leukemia in CML cells, came from the studies of Shtivelman et virus, was localized to the long arm of the chromosome al. (1995) that first cloned the full length cDNA 9 (Heisterkamp et al., 1982). Two ABL probes, which corresponding to the 6.0 kb cABL encoding transcript, normally map to chromosome 9, were shown to and used the 5’ part of this cDNA to clone the CML- hybridize on chromosome 22q- (the Ph chromosome) specific transcript from cDNA libraries prepared from of somatic cell hybrids originating from human CML both K562 and EM2 PH positive cell lines. The leukocytes and rodent cells (de Klein et al., 1982), sequence analysis of these transcripts isolated from while sequences of the c-sis oncogene, generally found both cell lines revealed that they contain a novel 5’ on chromosome 22, were shown to be translocated to sequence fused in frame with c-ABl exon 2, and the chromosome 9 in CML cells (Groffen et al., 1983). interestingly, a genomic fragment which hybridized to These studies indicated a reciprocal translocation for these non-ABL sequences contained the M-bcr region the Ph chromosome, and suggested that activation of previously identified by Groffen et al. (1984). Taken the transforming potential of the c-ABL proto- together, these data suggested that the 8 kb CML- oncogene was an important consequence of the specific mRNA contains the BCR joined to the c-ABL translocation. The cloning of the BCR/ABL break- sequences that was transcribed from a BCR/ABL point was the subsequent milestone. Heisterkamp and fusion gene. In addition the authors suggested that colleagues applied the ‘chromosome walking’ technique the amino terminal substitution was responsible for the to show a chromosomal breakpoint within a 14 kb increased tyrosine kinase activity of the ABL-derived sequence of chromosome 9 homologous to the v-ABL, sequences. These first but extremely important data in one of three patients with CML (Heisterkamp et al., open the way to a huge amount of studies on the 1983). However, the chromosome 9 rearrangement was structure of this hybrid gene and on molecular not evident in the remaining two patients, and this mechanisms of Ph positive leukemias. prompted Groffen et al. (1984) to direct their attention to chromosome 22 breakpoints. They used the sequences complementary to chromosome 22 Molecular structure of BCR/ABL genes previously isolated from the genomic clone of the positive CML patients as probe to screen for genomic At molecular level, the Ph translocation results in the rearrangements, and found a rearrangement of chro- juxtaposing of the 5’ part of the BCR gene to the 3’ mosome 22 in a second CML patient. Noteworthy, this part of the ABL gene, and, depending on chromosomal second rearrangement of chromosome 22 mapped very breakpoint locations, different parts of these two genes closely (1 kb) to that identified in the first positive may be included in the oncogenic fusion gene (Table CML patient. When the same probe and restriction 1). analysis was applied to a series of 17 leukemic patients, The ABL gene is a gene encoding a non receptor all but two cases (both Ph chromosome negative) tyrosine kinase, which spans a 230 kb region at band showed a chromosome 22 rearrangement clustered q34 of chromosome 9 and consists of 11 exons, with within a 5.8 kb BglII – BglII genomic fragment, which two first alternative exons i.e. exons Ia and Ib was thus named the ‘bcr’ or breakpoint cluster region (Kurzrock et al., 1988). In the vast majority of the (Figure 1) (Groffen et al., 1984). This region was later Ph positive patients, breakpoints in the ABL gene renamed ‘M-bcr’ (major breakpoint cluster region) to appeared to be distributed over a rather large 300 kb distinguish it from the ‘minor breakpoint region’ (m- fragment of chromosome 9 at band q34, which bcr) – a chromosome 22 fragment located further comprises the 5’ end of this gene, and may occur upstream, which appeared to harbor breakpoints of either upstream of the alternative first exon (exon Ib), Ph-positive acute leukemias (Hermans et al., 1987). It or downstream of the other first exon (Ia) or more was rapidly evident that the involvement of M-bcr was frequently between these two (Melo et al., 1993a). By highly specific for CML, as the rearrangement of this the effect of the Ph chromosomal translocation, ABL chromosome region were consistently found only in sequences sited downstream (telomeric) of breakpoint this type of leukemia and not in other hemopoietic move to the der(22) and are joined to the 5’ part of the

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8654

Figure 1 Breakpoint locations at the BCR and ABL loci. The sporadic breakpoints are indicated by small arrows (see also Table 1)

Table 1 Breakpoints at the BCR and ABL locus in human Philadelphia positive leukemias (see also text for details) Genomic breakpoints No of Chimeric BCR locus ABL locus cases mRNA junctions protein Reference

Minor bcr 5’ end of ABL (*300 Kb) Common e1a2 185 KDa (Fainstein et al., 1987; Clark et al., 1987; and this review) Minor bcr Intron 2 or 5’ end of ABL 4 e1a3 180 KDa (Soekarman et al., 1990; Iwata et al., 1994; Wilson et al., 2000; Mancini et al., 2001) Exon 2 5’ end of ABL (intron Ib) 1 e2-int ABL 1b-a2 187 KDa (Okamoto et al., 1997) Intron 6 5’ end of ABL (*300 Kb) 1 e6a2 195 KDa (Hochhaus et al., 1996) Intron 8 5’ end of ABL (*300 Kb) 4 Not tested ? (Saglio et al., 1988; Negrini et al., 1992) 2 e8-int-a2 197.5 KDa (How et al., 1999; Martinelli et al., 2002) Intron 10 5’ end of ABL (*300 Kb) 1 Not tested ? (Erikson et al., 1986) Major bcr 5’ end of ABL (*300 Kb) Common e13a2 and/or e14a2 210 KDa (Heisterkamp et al., 1985; Kawasaki et al., 1988 and this review) Major bcr Intron 2 or 5’ end of ABL* 4 e13a3 203 KDa (Soekarman et al., 1990; van der Plas et al., 1991; 3 e14a3 Tuszynski et al., 1993; Wilson et al., 2000) (Inukai et al., 1993; Iwata et al., 1994) Intron 15 Exon 2 (nt 78) 2 e15a2 210 KDa (Moreno Mdel et al., 2001) Micro bcr 5’ end of ABL (*300 Kb) 24 e19a2 230 KDa (Saglio et al., 1990 and this review)

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8655 BCR gene (Figure 1). Sequences of the exons Ia and/or encoding for the largest BCR/ABL chimeric protein of Ib, which may be included in the fusion gene, are predicted 230 kDa. This protein (P230BCR/ABL) was spliced out from the primary hybrid transcript. Indeed, subsequently purified from a Ph positive cell line they are never detected in the mature BCR/ABL expressing this type of BCR/ABL fusion gene (Wada mRNAs, which, with very rare exceptions (see below), et al., 1995). Afterwards, we described three additional contain only the last 10 exons, from exon 2 to exon 11, patients affected by a Ph positive leukemia carrying the of ABL gene (Morris et al., 1991; Melo et al., 1993b). e19a2 type of BCR/ABL gene (Pane et al., 1996a). Breakpoints on the BCR gene are usually clustered Interestingly, the leukemic phenotype in these three in three well defined regions (Table 1 and Figure 1). patients was very close to that of the chronic The first, now known as major breakpoint cluster neutrophilic leukemia (CNL), and, in retrospect, also region (M-bcr), is a 5.8 kb chromosomal region the clinical features of the first two cases described by spanning exons 12 – 16 (originally named b1 to b5) Saglio et al. (1990) might be considered much closer to (Heisterkamp et al., 1985; Stam et al., 1985). M-bcr CNL than the classical CML. Therefore, we proposed breakpoints are detectable in more than 95% of cases the name of mBCR (Micro-BCR) for the breakpoints (Faderl et al., 1999, however, breakpoints in this region located at the intron 19 of the BCR gene, which were can be detected in about one third of adult acute associated to a distinct form of mild Ph positive lymphoid leukemias (ALLs) with the t(9;22) transloca- myeloproliferative disease, the neutrophilic-chronic tion and in a small fraction of Ph positive ALL myeloid leukemia (CML-N) (Pane et al., 1996b). childhood cases (Arico et al., 2000; Gleissner et al., Over the past 15 years, additional types of BCR/ 2002). Depending on the position of breakpoint in this ABL genes have been described mainly as single case region, the 5’ end of the BCR gene comprising either reports (Table 1). Eight cases concerning patients with the exon 13 (formerly b2) or exon 14 (formerly b3) is a breakpoint comprised between the minor and major joined to the 3’ part of ABL gene, giving rise to a BCR regions, mainly located at, or around, the BCR BCR/ABL hybrid gene encoding the chimeric 210 kDa intron 8. The fused mRNA was detected in only three protein (P210BCR/ABL). The corresponding fusion of these patients, being the in frame e6a2 junction in mRNAs shows either the b2a2 or the b3a2 type of one case (Hochhaus et al., 1996) and an e8a2 junction junctions (Kawasaki et al., 1988). Together with which was in frame by the inclusion of an intronic Saglio’s group, we reported alternative splicing of the sequence, in the other two patients (How et al., 1999; primary BCR/ABL transcript which leads in all CML Martinelli et al., 2002). In other 11 patients, the hybrid patients to the production of also the P190-encoding transcript had an unusual junction between BCR mRNA (e1a2 junction), (Saglio et al., 1996). This sequences (exons e1, e13 or e14) and the ABL exon finding was subsequently confirmed by other groups 3, thus showing that the 174 bp sequences of the ABL (van Rhee et al., 1996; Lichty et al., 1998; Serrano et exon 2, which encode for 58 amino acids, the last 17 of al., 2000). In addition, alternative splicing allows the which are part of the SH3 domain, were not essential simultaneous expression of both b2a2 and b3a2 types for the transformation of cells (Table 1). In only one of of transcripts (Melo, 1996). these cases the Southern analysis made it possible to In 70 – 80% of Ph positive ALLs (Arico et al., 2000; detect a breakpoint at the 0.6 kb ABL intron 2, Gleissner et al., 2002), and in rare cases of CML (Melo whereas in the other two cases the restriction analysis et al., 1994), breakpoints of chromosome 22 span a excluded the presence of a breakpoint in the second 55 kb intronic sequence between the two alternative ABL intron, therefore, most likely, the unusual exons e2’ and e2 (Chissoe et al., 1995), called minor junctions derive in these latter cases, from the splicing breakpoint cluster region (m-bcr) (Clark et al., 1987; out of the ABL exon 2 from the primary hybrid Fainstein et al., 1987). In these cases only the extreme transcript (van der Plas et al., 1991; Iwata et al., 1994). 5’ end of the BCR gene is joined to the 3’ sequences of the ABL gene, and although the deriving BCR/ABL Correlation of BCR breakpoint positions with the fusion gene contains both the e1’ and the e2’ BCR leukemic phenotype exons and may contain ABL alternative first exons, all these exonic sequences are removed by splicing and the Clinical variability among the main cluster regions hybrid transcript shows a junction between the BCR exon e1 and ABL exon a2. This type of ela2 transcript Table 2 shows the different types of leukemia and the is smaller (7.4 kb) than that normally found in CML corresponding BCR/ABL breakpoints found in the Ph patients and encodes a 185 kDa chimeric protein positive patients, which shows the relationship between (P190BCR/ABL) (Clark et al., 1987; Hermans et al., the amount of BCR sequences included in the hybrid 1987; Kurzrock et al., 1987). gene and the leukemic phenotypes. More recently Saglio et al. (1990) described the In ALL, the Philadelphia chromosome and the presence of a novel type of BCR breakpoint located at corresponding BCR/ABL gene shows a different the 3’ end of the gene in two cases of chronic myeloid epidemiological distribution between adults and chil- leukemia. The breakpoints of these two patients were dren cases (see Table 2). The incidence of this genetic both comprised in the intron 19 and, hence, 19 of the abnormality seems to correlate with age and it is 23 BCR exons were included in the resulting fusion relatively common in adults where it is found in up to mRNA, which show an in frame e19a2 type junction 35% of cases (Maurer et al., 1991; Westbrook et al.,

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8656 Table 2 BCR/ABL junctions and leukemic phenotype BCR breakpoint Junction BCR/ABL mRNA Protein Diseases Frequency

ALL 85% of childhood Ph positive ALL 50 – 70% of adult Ph positive ALL Minor (m) e1/a2 P185 CML Very rare AML Very rare Multiple myeloma One case Non Hodgkin Lymphoma One case CML 100% of cases Major (M) b2/a2 or b3/a2 P210 ALL 15% of childhood Ph positive ALL 30 – 50% of adult Ph positive ALL AML Very rare Micro (m) c3/a2 P230 CML-N Rare

1992; Annino et al., 1994; Gleissner et al., 2002). transcript seems to predict the clinical outcome. Interestingly, among ALL cases, this genetic abnorm- Indeed, Radich et al. (1992) showed that the PCR ality is tightly associated to the B lineage and the cases analysis made it possible to detect BCR/ABL positive of true T-ALL containing the BCR/ABL gene are very cells, at least once after SCT, in bone marrow samples rare. Moreover, most of the Ph positive B-ALL cases of 23 out of 36 Ph positive ALL patients, and that the express CD34 and CD10 antigens (Kantarjian et al., PCR positivity predicted an overt clinical relapse with 1991; Westbrook et al., 1992; Gleissner et al., 2002). a relative risk of 5.7 compared to a PCR negative The relative proportion of adult ALL patients with the assay. Interestingly, these authors showed that seven P210- and P190-encoding genes is variable in the out of 10 patients with the P190 positive ALL and a various studies. The bias among the various studies is post-transplant PCR positive assay relapsed, in largely due to the selection of cases and the low contrast to only one out of eight patients with the number of patients analysed in some reports. Indeed, P210 positive ALL and a post-SCT PCR positive assay two studies, based on retrospective analysis, reported a (Radich et al., 1997). higher percentage of adult ALL cases with M-bcr In childhood ALL cases, the BCR/ABL gene is breakpoint (P210+ALL) compared to those with m- detectable in a limited proportion (up to 5 – 6%) of bcr type of breakpoint (P190+ALL) (Annino et al., newly diagnosed patients (Suryanarayan et al., 1991; 1994; Radich et al., 1994). Kantarjian et al. (1991) Pui and Evans, 1998). As for the adult patients, the Ph found the m-bcr type of breakpoint in 12 out of 24 defect is restricted to the pre-B ALL (Schrappe et al., adult ALL patients and the M-bcr in 11 patients, and 1998; Arico et al., 2000), and the cases of T-ALL with both the P210 and the P190-encoding mRNA in the the BCR/ABL gene are very rare (Arico et al., 2000). remaining patient. Most likely, this latter patient had a The P210+ALL in children is less frequent than in hybrid BCR/ABL gene with the M-bcr type of adults and represents a small minority of cases (10 – breakpoint, which gave origin to the two types of 20%) of all childhood Ph positive ALL when tested in mature mRNAs by alternative splicing of a primary large groups of patients (Maurer et al., 1991; Schrappe transcript (Dhingra et al., 1991; Saglio et al., 1996; van et al., 1998; Arico et al., 2000). Although no clinical Rhee et al., 1996). In other studies, including a large differences have been found between P210+ALL and prospective report on 175 cases of Ph positive adult P190+ALL pediatric patients, two large retrospective ALL, the percentage of adult ALL expressing the studies provided evidence that childhood Ph positive P190-encoding gene is much higher and accounts for ALL comprises patients with heterogeneous responses 70 – 75% of the total number of Ph positive cases to the intensive treatments (Schrappe et al., 1998; Arico (Maurer et al., 1991; Westbrook et al., 1992; Gleissner et al., 2000). In particular, a good response to initial et al., 2002). On the clinical ground, none of these prednisone treatment and low WBC count predict those studies provided evidence of hematological differences patients who will benefit from the treatment (Arico et between the two types of breakpoints, with the al., 2000; Schrappe et al., 1998). At the moment, exception of a trend, not statistically significant, however, the biological background of these clinical toward a longer survival probability at 3 years of the heterogeneity is not known. P190+ALL compared to the P210+ALL patients The Ph chromosome is only rarely detected in acute (0.19% vs 0.03%; P=0.7) found in the 175 Ph positive myeloid leukemia (AML), and most of the patients ALL cases published by Gleisser et al. (2002). It is to represent myeloid blast crisis of CML following a be stressed, however, that the dire prognosis of adult clinically silent chronic phase. In the few bona fide Ph ALL patients with the BCR/ABL gene might hide the positive AML cases for which molecular studies were clinical differences between the P190+ALL and the available, the P190-, differently to the P210-encoding P210+ALL cases at least as regarding the treatment gene, was always associated with the myelomonocytic outcome aspects. Noteworthy, when aggressive treat- phenotype (FAB M4 or M5), thus suggesting a pivotal ments such as allogeneic stem cell transplant (SCT), role of the BCR sequences located downstream from the were applied to the Ph positive ALL, the type of m-bcr breakpoint, in the transformation of myeloid

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8657 lineage cells (Kurzrock et al., 1987; Preudhomme et al., patients are different to the classical CML (Table 3): 1992; Secker-Walker et al., 1992; Alimena et al., 1995). out of the 24 CML-N patients, 18 were females, 17 The impact of the amount of BCR sequences on had no palpable splenomegaly (in one case this finding leukemic phenotype is further supported by chronic was not reported), and only three had WBC counts leukemias predominantly affecting the myeloid lineage. greater than 1006109/L. The mean platelet count of Classical CML usually shows, at least in the initial all the patients was 6616109/L and thrombocytosis chronic phase, a prominent, but often asymptomatic was present in 16 cases, with five patients presenting increase in peripheral blood of WBC, and in some with very high platelet count (410006109/L). Note- cases of platelet count associated with the presence of worthy, in 15 patients the mBCR type of BCR/ABL immature granulocytic elements. This leukemia is gene was the sole detectable genetic abnormality almost invariably derived by the P210-encoding BCR/ (Table 3, upper panel), whereas in nine patients the ABL gene, and is the prototype of a stem cell BCR/ABL gene rearrangement was associated with neoplasia, in which all the hemopoietic lineages derive other cytogenetic abnormalities beside the Ph chromo- from a staminal transformed cell and have the Ph some (Table 3, lower panel), and at least in the case chromosome (Fialkow et al., 1977; Jonas et al., 1992; reported by Wada et al. (1995), the associated Maguer-Satta et al., 1996). In the classical CML, chromosomal lesion, the isochromosome 17, was however, only the myeloid and megakaryotytic proven to precede the t(9;22) translocation. Interest- compartments show a neoplastic expansion, and the ingly, 14 out of the 15 patients belonging to the first granulocytic progenitors have a moderate degree of group showed a clinical picture typical of what we impairment of their differentiation and maturation have termed CML-N (i.e., a Ph-positive chronic capacity, whereas the erythroid, monocytic and B- neutrophilic leukemia). Indeed, all patients except and T-lymphoid lineages do not reveal any functional one did not show splenomegaly, the mean WBC count damage (Clarkson and Strife, 1993). By contrast, the in these patients was rather low (346109/L), and 12 of rare cases of CML with the P-190 encoding BCR/ABL the 15 patients are alive and well, in some instances gene are characterized by a prominent monocytosis, after a prolonged period of observation (Verstovsek et with a low neutrophil-to-monocyte ratio in both bone al., 2002). Only one patient of this group had a blastic marrow and peripheral blood. In addition, these transformation, while the other two patients died from patients show a relative higher proportion of immature unrelated causes (Saglio et al., 1990; Pane et al., cells in peripheral blood and lower neutrophil alkaline 1996b), and the median survival probability of this phosphatase (NAP) score than those affected by P210 group of patient is projected at 190 months by the CML (Melo et al., 1994; Roumier et al., 1999). actuarial analysis. By contrast, in the remaining nine Therefore, as for the P190 positive AML, when the patients who had, at presentation of disease, additional BCR/ABL gene with the m-BCR type of breakpoint is cytogenetic abnormalities, clinical and hematological expressed in the early myeloid compartment, the features were more similar to the classical CML: the monocytic precursors are consistently included in the spleen was enlarged in four of these patients, the mean neoplastic expansion, whereas the M-BCR breakpoint WBC count was significantly higher (926109/L), and a results in the restriction of the expansion to the transition to the more advanced phase of disease was granulocytic precursors. observed in four of this group of patients who showed The importance of the length of BCR sequence in a median survival probability of 37 months. Interest- the BCR/ABL fusion gene was corroborated by the ingly, all these chromosomal abnormalities have been CML-N patients with the P230 encoding BCR/ABL described in acute myeloid leukemia and among these, gene and the mBCR type of breakpoint. The CML-N both the trisomy of chromosome 8 and the isochromo- was first described by us in 1996 in five patients as a some 17, which has been found in three patients, may clinical entity characterized by primary chronic, non be the sole genetic lesion in acute myeloid leukemias progressive leukocytosis (Pane et al., 1996b). The (Schoch et al., 1997; Fioretos et al., 1999; Paulsson et original description required the following criteria: (1) al., 2001). Furthermore, the hybrid AML/ETO gene of moderate neutrophilic leukocytosis, (2) rare circulating the t(8;21) detected in patient #23, has proven to immature myeloid cells without a myelocyte peak, (3) transform myeloid precursors (Nucifora and Rowley, excess marrow mature myeloid cells, and (4) absent or 1995). We then investigated for the presence of the minimal splenomegaly. These first cases suggested that P230BCR/ABL protein and for the amount of P230- CML-N might have a more benign clinical course than encoding transcript copies in the bone marrow cells of classical CML. This was disputed after the report of all the available CML-N patients (Verstovsek et al., three new cases of Ph positive leukemia with the 2002). Interestingly, in all the patients analysed, the mBCR type of breakpoint whose clinical features were level of expression of the P230BCR/ABL protein was similar to those usually observed in the classical P210- always very low and below the detection limit of the expressing CML and of a single case of AML with this very sensitive technique used in this study. The type of BCR/ABL gene (Briz et al., 1997; Wilson et presence of the P230BCR/ABL protein was reported in al., 1997; Haskovec et al., 1998; Kojima et al., 1999). only one patient (#24 of Table 3), however, his Twenty-four patients with the P230-encoding leukemic blasts carried two copies of Ph chromosome BCR:ABL gene have thus far been reported (Verstov- (Haskovec et al., 1998). In addition, in all but two sek et al., 2002). Overall, the clinical features of these patients, we measured, by using both the real time and

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8658 Table 3 Clinical features of patients carrying the e19a2 type of bcr/abl rearrangement WBC Hb Platelets Additional CG BCR/ABL Survival No. Age/sex Splenomegaly (6109/L) (g/dL) (6109/L) abnormalities mRNA copiesa (cause of death) Reference

1 31/F – 11 13.7 418 – 2403 49 months (Rotoli et al., 2000) 2 57/F – 50 12.7 750 – 7694 415 months (Rotoli et al., 2000) 3 55/M NR 27 NR 258 – NR 45 months (Rotoli et al., 2000) 4 47/F – 50 NR 750 – NR 41 month (Rotoli et al., 2000) 5 53/F – 31 14.1 564 – 560 419 months (Verstovsek et al., 2002) 6 53/F – 24 13.2 837 – 705 45.5 years (Verstovsek et al., 2002) 7 57/F – 39 13.5 450 – 304.000 2 years (blastic phase) (Verstovsek et al., 2002) 8 57/M – 27 15.4 278 – 3243 43.3 years (Verstovsek et al., 2002) 9 47/F – 64 13.5 840 – 297.000 41.4 years (Verstovsek et al., 2002) 10 41/M – 43 15.8 191 – 1000 416 years (Pane et al., 1996b) 11 22/F – 45 12.7 1240 – NR 413 years (Pane et al., 1996b) 12 65/F – 58 8.8 160 – NR 3 years (Pane et al., 1996b) (myocardial infarction) 13 76/F – 28 NR 1020 – 28 10 years (heart failure) (Saglio et al., 1990) 14 62/F + 16 NR 870 – NR 43 years 15 45/F – 15 12.9 1370 – NR 45 years (Mittre et al., 1997)

Mean 1/14 33.86 13.3 666.4 53.1 months

16 66/M – 136 13.1 255 +8, iso (17) 933 410 months (Verstovsek et al., 2002) 17 13/F – 17 10.3 1442 lq+ NR 41 year (Yamagata et al., 1996) 18 50/F – 9 9.5 762 Iso (17) NR 2.5 years (blastic phase) (Wada et al., 1995) 19 29/F + 205 9.4 627 Extra Ph NR 8 months (How et al., 1998) (sepsis after BMT) 20 24/F + 203 NR 689 ND (absence of NR 4 5 years (Briz et al., 1997) mitosis) 21 70/M + 68 9.5 373 7Y NR NR (Wilson et al., 1997) 22 32/F – 65 11.8 66 Polyploidy in NR 47 years; BMT while (Polak et al., 2000) 28% of cells in accelerated phase 23 78/M – 38 NR 1650 t(8;21) upon NR 7 months (blastic phase) (Kojima et al., 1999) transformation 24 49/M + 87 NR 75 Extra Ph, +8, NR 3 months (blastic phase) (Haskovec et al., 1998) iso(17), del(7), +19

Mean 4/9 92 10.6 659 25.7 months

anumber of molecules of p230 per mg total RNA – WBC: white blood cells; Hb: hemoglobin; Plt: Platelets; F: female; M: male; BMT: bone marrow transplantation; NR: not reported

the competitive quantitative PCR, very few copies of in the cases in which additional chromosomal the P230-encoding mRNA (Verstovsek et al., 2002). alterations are absent (see also below). The mean copy number of e19a2 fusion transcripts was in those patients around two orders of magnitude Breakpoint position within M-bcr and the clinical less than the P210-encoding mRNA in classical CML heterogeneity of CML (Saglio et al., 1996; Pane et al., 1999), and more similar to those usually found in CML patients who Approximately 90% of CML patients are in chronic achieved cytogenetic remission (Hochhaus et al., 2000; phase at diagnosis. Although in many of the CML Martinelli et al., 2000). Interestingly, the first of the patients the diagnosis is made incidentally, i.e. the two patients with high copy number of P230 encoding chronic phase is often subclinical and indolent, its transcript (#7, Table 3) had a blast crisis after 2 years duration is highly variable thus supporting the of follow up and the second (#9, Table 3) failed to hypothesis of a genetic background which underlies respond to the interferon and cytosine arabinoside this clinical variability including the rate of progression treatment. Taken together, these data suggest that the to blast crisis (Lee, 2000; Goldman and Druker, 2001). BCR/ABL gene with the e19a2 type of junction It was disputed for a long time whether the structure of provides only a limited proliferative advantage to the the BCR/ABL gene, and in particular the position of Ph positive clone, and that the additional genetic the breakpoint in the M-bcr region might influence the lesions, when present, have an important role to prognosis of CML or the hematological features of the sustain the leukemic cell growth and phenotype. The CML patients. As regarding the prognosis, small low expression of the e19a2 BCR/ABL gene and of the studies have previously suggested a correlation between P230BCR/ABL protein found in the majority of the the 3’ breakpoint in the M-bcr region and the onset of CML-N patients might at least in part account for the blast crisis and indicated that breakpoints located limited expansion of the myeloid lineage in these downstream of the central HindIII restriction site (3’ patients and explain the indolent course of the disease breakpoints, see Figure 1) were significantly more

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8659 common in patients in blast crisis or accelerated phase with WBC51006109/L, Shepherd et al. (1995) found disease compared to those with chronic phase that the patients with the b3a2 type of transcript had a (Schaefer-Rego et al., 1987; Eisenberg et al., 1988). In mean platelet count significantly higher than those with addition, Mills and Birnie (1991) found that the the b2a2 type of transcript (653 vs 3816109/L. In chronic phase duration was fourfold longer in patients addition, two different studies investigated the type of with breakpoints located upstream to the central transcript in a total of 18 patients with thrombocy- HindIII restriction site (5’ breakpoint) in relation to temic onset of CML, and found the b3a2 transcript in those with 3’ breakpoints. This finding was not 13 cases (Cervantes et al., 1996; Blickstein et al., 1997). confirmed in subsequent series of patients including The inverse correlation between the WBC count with those reported in very large studies (Fioretos et al., platelet count and the length of BCR sequence 1993; Zaccaria et al., 1993a; Shepherd et al., 1995; included in the BCR/ABL fusion gene in accordance Verschraegen et al., 1995). Due to the proximity with the high frequency of thrombocytosis found in the between the HindIII restriction site of the Mbcr region CML-N patients (Table 3) (Verstovsek et al., 2002). To and the BCR exon 14 (Figure 1), the comparison of the conclude, it appears that the longer the BCR sequence data of Southern blot with those of RT – PCR included in the fusion protein, the higher the platelet indicated that a significant proportion of the patients count. with the 5’ breakpoint in the M-bcr had a b3a2 variant The importance of the genetic background on the of transcript which is a constant feature of the 3’ prognosis of CML patients, seem to be in part due to breakpoint group (Zaccaria et al., 1993b; Shepherd et the large deletions of the derivative chromosome 9 al., 1995). Therefore, other studies have focused on the which may occur at the time of the t(9;22) chromoso- prognostic role of the transcript variants (b2a2 and mal translocation in up to 30% of cases (Sinclair et al., b3a2) in CML patients with the M-bcr type of 2000). These deletions, which have proven to be the breakpoint. This prompted studies as to the prognostic most potent prognostic factor at least in retrospective role of transcript variants (b2a2 and b3a2) in CML studies, may involve a variable part of the derivative patients carrying the M-bcr type of breakpoint. chromosome 9 with losses of one of two critical genes. Although transcript type was not significantly corre- This would contribute to the genetic heterogeneity of lated with phenotype, there was a trend toward a most CML patients and to their variable clinical behaviour favorable prognosis, both in terms of cytogenetic (Huntly et al., 2001, 2002; Kolomietz et al., 2001). The conversion and in chronic phase duration for the genes potentially involved in these deletions are, patients with the b3a2 type of transcript (ICSG, 1995; however, not yet identified. Martinez-Mancilla et al., 2002). A matter of discussion is also the correlation Clinical features of patients with sporadic BCR and ABL between the breakpoint location within the M-bcr breakpoints and the platelet count. The majority of the studies in which the platelet count was stratified according to the Among the sporadic breakpoints, eight patients showed breakpoint position in the M-bcr region or to the type a breakpoint comprised between the minor and the of transcript, showed a bias, although not statistically major BCR regions that gave rise to a chimeric protein significant, toward higher platelet number in the of approximately 198 kDa, unlike the p210BCR/ABL patients with the b3a2 transcript or the 3’ breakpoint protein, this chimeric protein lacks the BCR-deriving (Table 4) (Jaubert et al., 1990; Opalka et al., 1992; pleckstrin domain. Both types of BCR/ABL fusion Zaccaria et al., 1993a; Martinez-Mancilla et al., 2002). proteins however retain the BCR gene deriving CDC24 In two studies, the platelet count increases of patients and the DBL homology domains that seems important with the 3’ breakpoint or the b3a2 transcript were to transform myeloid cells in the CML (see below). As statistically significant (Inokuchi et al., 1991; Futaki et a whole, clinical features of these patients are not al., 1992). These findings were, at least in part, different from those with the classical CML (Melo, confirmed by data on the CML patients with low 1996; How et al., 1999; Martinelli et al., 2002). WBC count at presentation. In a series of 23 patients Similarly, there were no distinctive features with

Table 4 Mean platelet count in CML patients according to the breakpoint of the BCR/ABL gene Mean platelet count (6109/L) Number of patients 5’ Breakpoint 3’ Breakpoint b2/a2 b3/a2 Reference

57 375 841 (Inokuchi et al., 1991) 45 303 377 (Opalka et al., 1992) 133 458 448 (Fioretos et al., 1993) 244 411 453 (Zaccaria et al., 1993a) 119 530 537 395 534 (Shepherd et al., 1995) 23a 381 653 (Shepherd et al., 1995) 146 412 447 (ICSG, 1995) 70 470 545 (Martinez-Mancilla et al., 2002) aThe data concerns the 23 of the 119 patients included in the study who presented with less than 1006109/L WBC

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8660 respect to the corresponding usual breakpoints in those (Pluk et al., 2002). These data indicate that the tyrosine cases in which the BCR sequences were joined to the kinase activity of c-ABL is tightly regulated in the cells, ABL exon 3. and suggest that ABL acts as a non-receptor signal By contrast, two patients have recently been transducer role for this protein. In addition to SH described with an atypical chronic myeloproliferative motifs, ABL has other functional domains. When its disorder and a breakpoint at the BCR locus just N-terminus is codified by the first alternative exon 1b, downstream from the M-bcr region, in the intron 15 the resulting ABL protein has a myristoylated glycine (Moreno Mdel et al., 2001). In these patients, the at the N-end, which directs the molecule to the plasma hybrid BCR/ABL transcript showed a junction membrane (Jackson and Baltimore, 1989). More between sequences of the BCR exon 15 and the 3’ frequently, however, the N-terminus of the protein is part of the ABL exon 2, and hence encodes for a encoded by the other alternative first exon (1a) and the chimeric protein only three amino acids longer than the ABL protein is preferentially located in the nucleus. In classical P210BCR/ABL protein. Interestingly, the expres- addition, at the c-terminus moiety of the molecule, sion of this variant type of BCR/ABL gene was very there are three nuclear localization signal domains low and the two patients presented with a very which drive ABL protein to the nucleus and three moderate and non-progressive leukocytosis, 20 and DNA binding regions, that bind AT-rich DNA 156109/L in the two patients, without a shift to the sequences in a cooperative manner (Miao and Wang, left in the granulocytic lineage (Moreno Mdel et al., 1996). Finally, at the extreme carboxyterminus, there is 2001). an F-actin-binding domain (McWhirter and Wang, 1993), and a nuclear export signal domain, that are responsible for the shuttle of the protein between Structure and biological features of proteins ABL, BCR nucleus and cytoplasm (Taagepera et al., 1998). and of the various BCR/ABL fusion proteins Although most of the data are based on in vitro studies on fibroblasts and are still controversial, several Functional domains in the BCR and ABL proteins diverse functions have been attributed to ABL: in the The ABL protein is expressed at rather constant rate in cytoplasm, it seems to transmit signals in the frame- all human tissues and shows structural homology to work of integrin signaling through its ability to the Src family of kinases, and contains a tyrosine phosphorylate a number of different proteins (Lewis kinase (SH1), SH2 and SH3 domains (Figure 1). The and Schwartz, 1998), while in the nucleus, ABL is SH2 and SH3 domains are involved in protein – protein involved in the regulation of cell cycle (Sawyers et al., interactions, which regulate the tyrosine kinase activity. 1994; van Etten, 1999). Taken together, these observa- These domains represent the building blocks generally tions suggest that protein ABL, through found in tyrosine kinase proteins and are necessary for phosphorylation capability and the shuttling between the signal transduction function (Wang, 1993). The nucleus and cytoplasm, integrates signals from various SH2 domain seems to play a crucial role in modulating extracellular and intracellular sources and influences the activation of c-ABL protein for its capability to decisions about cell cycle and apoptosis; however, its integrate the interactions of the various SH2 binding intimate role in hemopoietic cell physiology is still proteins which, in turn, are phosphorylated in the unclear. framework of the intracellular signal transduction Like ABL, BCR protein is also ubiquitously activity. The residues located immediately c-terminal expressed and although multiple functional domains to the phosphotyrosines are responsible for the high have been recognized in its structure, the physiological affinity target interactions and confer specificity among role of this protein is still unknown. The first large different SH2-binding containing proteins (Pawson, exon of the gene encodes an oligomerization domain, 1995). The SH3 domains specifically recognize proline which is involved in coiled-coil dimerization of the rich motifs, and like SH2 domains, their binding BCR protein (McWhirter et al., 1993), a unique specificity seems to be determined by the residues serine/threonine kinase domain (Maru and Witte, immediately adjacent to the proline (Feller et al., 1994; 1991), an SH2 binding domain (Pendergast et al., Sicheri et al., 1997; Xu et al., 1997). The SH3 motif 1991). At position 177, within the amino acids appears to play an important role in the inhibition of encoded by the BCR exon 1, is a tyrosine residue, c-ABL activity because its deletion (Van Etten et al., which may be phosphorylated, thus acquiring affinity 1989) or its alteration activate the kinase activity of the to the Grb2 protein, an important adaptor protein c-ABL (Mayer and Baltimore, 1994). Furthermore, this that links this protein to the Ras pathway (Ma et al., domain is replaced by the gag sequences in the v-ABL 1997). The central part of the BCR molecule contains protein which has the highest tyrosine kinase activity of a region homologous to the dbl-like and pleckstrin all the ABL-derived transforming proteins (Danial et homology (PH) domains that stimulate the exchange al., 1995). Recent data provided evidence that the N- of guanidine triphosphate (GTP) for guanidine dipho- terminus domain of ABL, encoded by the first exon, sphate (GDP) on Rho guanidine exchange factors negatively regulates its catalytic activity by forming a (Denhardt, 1996). The carboxy-terminal part of the ‘cap’ shaped structure which blocks the SH3 domain in molecule has GTPase activity (Diekmann, 1991 #161). its proper configuration and in its intramolecular These data suggest a role for the BCR protein in the interaction for inhibition of the tyrosine kinase activity signal transduction, however, similarly to the ABL

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8661

Figure 2 Structural domains of the three main BCR/ABL chimeric proteins protein, the physio-logical role of this protein is still have the same transforming activity, but their expres- to be clarified. sion might be restricted to different hemopoietic compartments. There are experimental data supporting the former or the latter model. Differential transforming power of the various BCR/ABL P190BCR/ABL is the smallest chimeric protein that is chimeric proteins encoded by the hybrid BCR/ABL gene with the m-bcr Figure 2 shows schematic representations of the three type of breakpoint and is typically associated with main forms of BCR/ABL proteins. The BCR/ABL acute lymphoblastic leukemia (Table 2). Of the BCR rearrangements deriving from BCR breakpoints in M- protein, it retains only the coiled-coil dimerization bcr, m-bcr, and m-bcr, differ in the number and motif, the GRB2 binding site at tyrosine 177 residue, integrity of the BCR protein domains included in the and the phospho-serine/threonine-rich SH2 binding corresponding proteins. Over the past decade, huge domains, all encoded by the BCR exon 1 (Figure 2). efforts have been dedicated to the study of individual These domains per se can activate and deregulate the domains of the c-ABL, the BCR and the BCR/ABL kinase activity of c-ABL derived domains (Lugo et al., proteins. The fine dissection of the domains of these 1990; Ilaria and van Etten, 1996). Indeed, the analysis proteins and the evaluation of the interaction with of its tyrosine kinase activity and the measurement of intracellular proteins and signaling pathways, has its transformation potency indicated that this chimeric begun to provide some clues to the cellular and protein has a constitutive activated kinase function. molecular mechanisms of leukemogenic transformation Furthermore, Li et al. (1999) recently provided an induced by the BCR/ABL protein (reviewed in accurate measure of the tyrosine kinase activity of the Holyoake, 2001) and in other article of this issue). three types of BCR/ABL proteins by expressing their However, the molecular mechanisms by which the activity as fold of increase of phosphorylation three different forms of chimeric BCR/ABL proteins capability of the Crk substrate relative to the cABL exert different transforming activity, are much less well protein. They show that the P190BCR/ABL has the known. In principle, these three forms of BCR/ABL highest activity (sevenfold increase with respect to protein could have different intrinsic transforming cABL) followed by P210BCR/ABL (5.4-fold increase), activity, and when expressed in a hemopoietic and by P230BCR/ABL (3.7-fold increase) (Li et al., 1999). precursor might differentially drive the expansion of Interestingly, these authors also showed that although lymphoid blastic clone with blocked differentiation the P190BCR/ABL transformed both the IL-3 dependent capacity in the case of ALL, or alternatively, of 32D myeloid and Ba/F3 lymphoid cell lines similarly to myeloid cells with not severely impaired differentiation the other BCR/ABL proteins, its ability to stimulate capacity in the case of CML, or lastly, of myeloid cells proliferation of lymphoid cell lines is correlated to its with full retained differentiation capacity in the case of tyrosine kinase activity, and hence, is higher than the CML-N. On the other hand, the three proteins might P210BCR/ABL and P230BCR/ABL. Moreover, they also

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8662 showed that mice not pretreated by 5-fluorouracyl and staminal cells rather than committed progenitor cells reconstituted with P190BCR/ABL transfected bone are the target of the Ph-induced neoplastic transforma- marrow cells, developed lymphoid as well as other tion in ALL (Cobaleda et al., 2000). leukemias four times faster than mice reconstituted The most common P210BCR/ABL protein which is with P210BCR/ABL or P230BCR/ABL expressing bone encoded by the BCR/ABL gene associated with the M- marrow cells (Li et al., 1999). These findings are in bcr type of breakpoint, contains, in addition to the agreement with data from Quackenbush et al. (2000), above mentioned BCR domains, the dbl-like, the who demonstrated that, in primary mouse bone CDC24 and the plekstrin domains of the BCR protein marrow cultures, the transfection of the P190BCR/ABL, (see Figure 2). The dbl-like domain has a GDP-GTP but not of the P210BCR/ABL and P230BCR/ABL, results exchange activity for the RAS like molecule Rac and in a potent induction of lymphoid expansion even RhoA (Chuang et al., 1995; Whitehead et al., 1997), under experimental conditions that favor myeloid enhancing the activity of these latter through the expansions, and independently from cytokine supple- acceleration of the conversion of GTD into GTP mentation. These observations indicate that BCR (Boguski and McCormick, 1993). Because the domains retained in the P190BCR/ABL protein are sequences encoded by the BCR first exon play a sufficient to provide it with the highest kinase activity, pivotal role in the activation of BCR/ABL by binding and also that its transforming power seems to be to the ABL SH2 domain (Chopra et al., 1999; restricted, at least in particular experimental contexts, Deininger et al., 2000; Pluk et al., 2002), and the to lymphoid precursors. These data are consistent with P190BCR/ABL, which does not contain the dbl-like the predominant association of P190BCR/ABL with ALL domain, has the strongest biochemical activity, it has and the rarity of P190BCR/ABL positive CML, which, been suggested that the dbl-like domain is simply an however, is characterized by a marked monocytosis inhibitory spacer. However, the ability to stimulate (Melo et al., 1994). Therefore, this property might be lymphoid expansion and proliferation of the various correlated to the high kinase activity of the BCR/ABL proteins seems to correlate with their P190BCR/ABL protein, or alternatively to the prefer- tyrosine kinase activity at least in the Ba/F3 lymphoid ential deregulation of metabolic pathways of lymphoid cell lines (Li et al., 1999), and conversely, the ability to precursors, or lastly to the preferential expression in promote the myeloid expansion in vivo was recently these cells. Regarding these latter issues, there is not associated with the presence of BCR domains in the clear-cut evidence of a differential effect of hybrid proteins. Indeed, Gross et al. (2002) recently P190BCR/ABL on intracellular signaling pathways with compared the leukemogenic activity of the wild type the exception of prominent STAT6 activation by P210BCR/ABL protein with that of an activated form of P190BCR/ABL compared to the P210BCR/ABL (Ilaria ABL kinase lacking BCR sequences. The latter protein and Van Etten, 1996). Otherwise, the analysis of induced only lymphoid malignancies in mice and did P190BCR/ABL expression in sorted myeloid and not stimulate the growth of myeloid colonies in vitro. erythroid precursors of ALL patients gave discordant On the other hand, P210BCR/ABL protein proved to results. Indeed, while in CML the Ph chromosome and efficiently induce a myeloproliferative disease under the the BCR/ABL gene is detectable in hemopoietic stem same conditions (Gross et al., 1999; Gross and Ren, cell compartment as well as in multiple hemopoietic 2000). In addition, Rat1 cells transfected by a lineages (Takahashi et al., 1998; Delforge et al., 1999; P190BCR/ABL encoding construct, but not by a Holyoake et al., 1999, 2001), in most cases of Ph P210BCR/ABL encoding construct, showed an alteration positive ALL the BCR/ABL gene is restricted to the of F-actin structure leading to the disruption of lymphoid lineage (Schenk et al., 1998; Haferlach et al., cytoskeleton, which implies that the dbl-like domain 1997; Kasprzyk et al., 1999). However, other studies plays a role in the stabilization of actin filaments showed the presence of the BCR/ABL gene in multiple (McWhirter and Wang, 1997). Indeed, these small GTP lineage cells of Ph positive ALL (Pane et al., 1996a; binding proteins are involved in the activation of Saglio et al., 1997, 1999; Pajor et al., 2000). Very cytoskeleton and interestingly in the control of gene recently, Cobaleda et al. (2000) provided an elegant expression (Hill et al., 1995; Olson, 1996). In addition, demonstration that also in Ph positive ALL, the cell it has been recently reported that P210BCR/ABL, capable of initiating the disease possesses differentiative through its dbl domain, is able to interact, at least in and proliferative capacities and has the potential for vitro, with the xeroderma pigmentosum group B self-renewal expected of a leukemic stem cell. These protein (XPB), and inactivates its DNA unwinding authors used as a model the non-obese diabetic mice activity (Takeda et al., 1999). Furthermore, the positive with severe combined immunodeficiency (SCID/NOD) role of this domain has been reinforced by experiments which are highly receptive to the engraftment of human in which this region was replaced with homologous leukemic cells, even in small numbers, and showed that sequences derived from the Dbl protein (P210/dbl)or regardless of the heterogeneity in the maturation alternatively with non-functional sequences from LacZ features of leukemic blasts, the SCID leukemia (P210/LacZ) or Luciferase (P210/Luci) (Kin et al., initiating cells invariably are CD34+ and CD387, 2001). Transformation of the Rat1 fibroblasts or bone and therefore, very similar to the normal SCID marrow cells was observed only when transfected with repopulating cells both for the cellular and phenotypic the P210/WT or the P210/Dbl but not with the P210/ features. Therefore, this study suggests that normal LacZ or the P210/Luci. These data suggest a possible

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8663 differential role for the P210BCR/ABL protein compared associated with a mild, indolent clinical phenotype to the P190BCR/ABL on the metabolic pathways which because it has an intrinsic low transforming power. It regulate normal granulocytic differentiation. The shows the lowest tyrosine kinase activity among the importance of the Dbl-like domain of the P210BCR/ three main BCR/ABL proteins (Li et al., 1999), and at ABL protein was also supported by the observation that least in some experimental conditions shows a limited all the eight patients with a BCR breakpoint between capacity to promote cellular growth, which in addition, the minor and the major cluster region (Table 1) and seems restricted to the myeloid compartment. Indeed, express a BCR/ABL protein of 198 kDa which lacks when a high expression retroviral vector was used, both the pleckstrin but includes the dbl-like domains, were 32D myeloid and Ba/F3 lymphoid IL3-dependent cell affected by a typical CML. Therefore, the inclusion of lines were transformed by the transfection of P230- dbl-like domain in the BCR/ABL protein seems to play containing retroviral vector, similarly to the a role to drive the expansion of a myeloid clone, P190BCR/ABL and P210BCR/ABL (Li et al., 1999). However, instead of a lymphoid clone with reduced differentia- a experimental model more akin to clinical context, i.e. tion capability (Martinelli et al., 2002). the primary mouse bone marrow precursors expressing The P210BCR/ABL protein, besides CML, may also be the P230BCR/ABL protein, exogenous growth factors associated to ALL. The mechanisms by which were required to support the growth of these cells in P210BCR/ABL might gain higher transforming activity culture, whereas P190BCR/ABL and P210BCR/ABL expres- in these cases are not clear. However, we proved that sing precursors were growth factor independent the expression of P210BCR/ABL mRNA is at least 10- (Quackenbush et al., 2000). These cells transfected fold higher in ALL compared to CML cases (personal with P230BCR/ABL (or the P210BCR/ABL), were unable observation). In this context, recent data suggest a to promote the outgrowth of lymphoid precursors in critical role for the differential rate of BCR/ABL gene immunodeficient mice, and promoted a limited myeloid expression in supporting the myeloid and lymphoid expansion in this latter experimental model (Quacken- commitment of the hemopoietic precursors and the bush et al., 2000). Noteworthy, GAPrac domain might subsequent expansion of these compartments. We also play a role in the understanding of the low ability demonstrated that CML patients who had a complete to impair the myeloid differentiation of the P230BCR/ hematological response to the interferon alpha treat- ABL protein. This latter, unlike the P190BCR/ABL and ment, showed a significant reduction of the BCR/ABL the P210BCR/ABL proteins, has an intact GAPrac mRNA levels in their Ph positive cells, and that this function. Therefore, cells expressing the P230BCR/ABL effect was also observed, in the bone marrow protein retain both copies of functional GAPrac, one precursors of these patients when cultured in the encoded by the normal BCR allele, and the other on presence of the interferon. The decrease of cellular the hybrid protein. Consequently, functional partial levels of the hybrid BCR/ABL mRNA was not haploidy of the GAP domain could account for the observed in vivo nor in vitro, in the patients who did higher transforming capacity of the P190BCR/ABL and not respond to the drug (Pane et al., 1999). Huettner et the P210BCR/ABL genes (Melo, 1996). In addition, the al. (2002) generated double transgenic mice in which hybrid e19/a2 BCR/ABL gene retains the transcription the P210 encoding BCR/ABL gene was controlled by silencer elements located at the 3’ end of the BCR exon the mouse mammary tumor virus promoter and a 14 (Stewart et al., 1994), which are lacking in the P190- tetracycline transactivator element. Therefore, these encoding hybrid gene and may only be partially transgenic mice were highly responsive to the tetra- included in some of the M-BCR types of breakpoint. cycline administration for the induction of the BCR/ This observation could explain the very low expression ABL transcription (Huettner et al., 2000). These mice of the BCR/ABL gene with the e19a2 junction that we develop within 24 – 80 days a B cell ALL when the found in the CML-N patients (Verstovsek et al., 2002). tetracycline administration induced, and sustained, Therefore, the low transforming power may be due to high rate expression of the P210BCR/ABL protein. the low transcription rate of the P230 encoding gene Interestingly, shutting down the BCR/ABL expression which we found in patients with the CML-N. Low by tetracycline withdrawal, resulted in remission of the expression of the BCR/ABL gene and levels of the disease, as we observed in vivo (Pane et al., 1999), when chimeric protein below the detection limit were found the levels of the BCR/ABL mRNA were down- in a transgenic mouse in which the hybrid BCR/ABL regulated by the administration of interferon gene expression was restricted to the hemopoietic (Huettner et al., 2000). system by the tec gene promoter used as a regulatory The P230BCR/ABL protein includes sequences encoded sequence (Honda et al., 1998). A line of transgenic by 19 out of 23 BCR exons and carries 180 additional mice derived from the founder strain, developed after a amino acids compared to the P210BCR/ABL, i.e., all the long latency period an indolent form of myeloproli- BCR functional motifs with the exception of the ferative disease which closely resembles CML-N and carboxy-terminal part of the GAPrac domain (Figure recapitulates some of the molecular findings of the 2) (Ahmed et al., 1994; Pane et al., 1996b). This human disease. Indeed, these mice showed for a long domain shows a high homology to the GTPase period an increase of platelet and granulocyte counts activating protein for the RAS related proteins P21 without the presence of circulating myeloid immature Rho and P21 Rac (Diekmann et al., 1991). It is precursors. Noteworthy, during the preleukemic state possible that the P230BCR/ABL protein is mainly (first year of observation) the P210BCR/ABL cannot be

Oncogene BCR/ABL genes and leukemic phenotype F Pane et al 8664 detected at protein level in hemopoietic cells of these ABL proteins, even their levels within the Ph positive mice. However, the hybrid transcript levels were precursors may play an important role in determining detectable, although very low by RT – PCR, thus the severity of the hematological picture. These ﬁrst suggesting that low levels of chimeric protein expres- observations suggest that at least in some instances, sion may underlie the indolence of the pre-leukemic such as the CML-N patients, the quantitative data on long latency in this model (Honda et al., 1998). the BCR/ABL expression may be useful in predicting the evolution and providing clinical indications regarding therapeutic decisions. Conclusions

The molecular mechanisms accounting for the relation- Acknowledgements ship among the diﬀerent types of BCR/ABL gene and Work performed in the author’s laboratory was supported the leukemic phenotypes is one of the most fascinating by grants from AIRC (Milan), CNR - Progetto Strategico issues in molecular hematology, but still largely remain & PF Biotecnologie (Rome), PRIN - MIUR (Rome) to be clariﬁed. However, several data suggest that, Biogem, AIL and Regione Campania. We are grateful to besides the intrinsic tyrosine kinase activity of BCR/ Jean Ann Gilder for editing the text.

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