bcr-abl variants - biological and clinical aspects

Leukemia Research 26 (2002) 713–720

Millennium Review

Bcr–Abl variants: biological and clinical aspects

Anjali S. Advania, Ann Marie Pendergastb,∗a Departments of Hematology and Oncology, Duke University Medical Center, Durham, NC 27710, USA

b Department of Pharmacology and Cancer Biology, LSRC Room C233A, La Salle St. Extension,Duke University Medical Center, Durham, NC 27710, USA

Received 8 November 2001; accepted 25 November 2001

Abstract

Bcr–Abl is an oncogene that arises from fusion of theBcr gene with thec-Abl proto-oncogene. Three differentBcr–Abl variants canbe formed, depending on the amount ofBcr gene included:p185, p210, andp230. The three variants are associated with distinct types ofhuman leukemias. Examination of the signaling pathways differentially regulated by the Bcr–Abl proteins will help us gain better insightinto Bcr–Abl mediated leukemogenesis. © 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Philadelphia chromosome;Bcr–Abl; Acute lymphocytic leukemia; Chronic myelogenous leukemia; Chronic neutrophilic leukemia

1. Introduction

The Philadelphia chromosome (Ph) involves fusion of thebreakpoint cluster region (Bcr) gene on chromosome 22 atband q11 with theAbl proto-oncogene on chromosome 9 atband q34 [1]. Three different forms of theBcr–Abl oncogeneexist,p185, p210, andp230, that are associated with distincttypes of leukemia.P185 is associated with 20–30% of acutelymphocytic leukemia (ALL),p210 with 90% of chronicmyelogenous leukemia (CML), andp230 with a subset ofpatients with chronic neutrophilic leukemia (CNL) [2]. Thisreview will discuss the biological and clinical aspects of theBcr–Abl variants.

Abbreviations: Ph, Philadelphia chromosome; Bcr, breakpoint clusterregion; ALL, acute lymphocytic leukemia; CML, chronic myelogenousleukemia; CNL, chronic neutrophilic leukemia; AUL, acute undifferen-tiated leukemia; AML, acute myelogenous leukemia; Ph+, Philadelphiachromosome positive; CALGB, cancer and leukemia group B; BMT, bonemarrow transplant; CML-N, cases of chronic neutrophilic leukemia thatare Ph+; CMML, chronic myelomonocytic leukemia; RT-PCR, reversetranscriptase polymerase chain reaction; CFU-erythroid, colony formingunit-erythroid; CFU-GM, colony forming unit-granulocyte macrophage;PCR, polymerase chain reaction; STI-571, signal transduction inhibitor571; ATP, adenosine triphosphate; mRNA, messenger RNA

∗ Corresponding author. Fax:+1-919-681-7148.E-mail address: [email protected] (A.M. Pendergast).

2. Clinical correlates

CML typically has a biphasic course that is characterizedby chronic and blastic phases [3–6]. The chronic phase ischaracterized by an expansion of myeloid cells in the periph-eral blood, bone marrow, and spleen. The molecular basisfor the myeloid expansion is puzzling as Bcr–Abl transformsthe hematopoietic stem cell and is present in all hematopoi-etic elements [3–6]. Approximately 50% of patients in thechronic phase have no symptoms and are diagnosed by rou-tine testing [7,8]. Signs and symptoms, however, can includefatigue, weight loss, abdominal fullness, bleeding, sweats,splenomegaly, and hepatomegaly [7–9].

The blastic phase is thought to occur secondary to addi-tional mutations (see below). Prior to entering blast phase,75% of patients develop an intervening accelerated phasethat is characterized by increased basophilia, blasts, andpromyelocytes [7,10]. This usually progresses to blasticphase within 3–18 months [7]. The acute leukemia is ALLin one-third of cases, whereas two-thirds are acute undiffer-entiated leukemia (AUL) or acute myelogenous leukemia(AML) [7,11]. Patients with a lymphoid blastic phase mayrespond to treatment with ALL regimens; however, the me-dian duration of response is 9–12 months [7,12]. Becausethe disease is relatively treatment resistant, the median sur-vival is 3 months for myeloid blast crisis and 6 months forlymphoid blast crisis [13–16].

Philadelphia chromosome positive (Ph+) ALL is an acuteleukemia, which unlike CML, requires prompt, aggressive

0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.PII: S0145-2126(01)00197-7

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treatment with high dose chemotherapy [17]. Compared toBcr–Abl negative ALL, it tends to have a worse prognosis.In a Cancer and Leukemia Group B (CALGB) study, themedian remission duration for Ph+ ALL was only 8 months[17,18]. Because fewer than 5% of patients are cured bychemotherapy, allogeneic BMT remains the best chance ofcure [19,20]. The reason why p185 tends to be associatedwith acute leukemia is unknown. One hypothesis is that p185is more efficient at promoting secondary events necessaryfor the acute phase disease [21].

Although the majority of Ph+ ALL exhibit a B-lymphoidlineage phenotype, Ph+ ALL may also involve the stemcell, because some are biphenotypic [21,22]. In addition,involvement of the myeloid lineage can be seen in both p185and p210 ALL [21–29].

Finally, p230 is associated with a subset of CNL, whichhas a much more benign course. Bcr–Abl positive CNL wasidentified in patients who showed the presence of aBcr–Able19a2 transcript [30,31]. About 100 cases of Bcr–Abl posi-tive CNL have been reported [30,32,33]. Most cases of CNLare Ph chromosome negative, so these cases have been re-ferred to as CML-N [30]. CNL involves a sustained ma-ture neutrophilic expansion with mild hepatosplenomegaly.In general, there is a low proportion of immature granulo-cytes and milder anemia [34]. Progression to blast crisis isuncommon [30].

Although p185 is typically associated with ALL, p230with CNL, and p210 with CML, there is overlap. P210 oc-curs in 40% of Ph+ ALL, p185 occurs in 2–3% CML, andp230 in some cases of CML [2]. Patients with p185 CMLare thought to have a disease more reminiscent of chronicmyelomonocytic leukemia (CMML), and to have a mono-cytic defect [2,35]. Kantajaran et al. examined 32 patientswith p210 or p185 Ph+ ALL and found no difference inclinico-laboratory, karyotypic, or prognostic implications[13,36]. It is not clear whether intrinisic differences in theactivities of the three Bcr–Abl proteins account for theirassociation with different disease phenotypes or whether

Fig. 1. Structural features of the Bcr–Abl variants. The p185 Bcr–Abl protein is primarily associated with ALL, p210 Bcr–Abl with CML, and p230Bcr–Abl with CNL. The C-terminal (Abl) region of all Bcr–Abl variants contains Src-homology (SH3 and SH2) domains, tyrosine kinase (SH1) domain,proline-rich sequences that contain PXXP motifs for SH3 domain binding, DNA binding domain, actin binding domain, nuclear localization signals (NLS),and nuclear export signal (NES). The N-terminal (Bcr) portion of the protein differs among the variants. The variants include different amounts of Bcrsequence, with p230 including the largest amount of Bcr sequence. All three variants have a dimerization domain (DD) and serine/threonine kinase domain(P-S/T). P210 and p230 both have Dbl and PH domains. In addition, p230 has a calcium/phospholipid binding domain (CalB) and a Gap–Rac domain.

expression of each protein occurs in a distinict hematopoi-etic lineage.

In addition, some patients with CML co-expressp185andp210, based on detection by reverse transcriptase poly-merase chain reaction (RT-PCR) [2,37–39]. High levelsof both transcripts have been found in patients with blas-tic transformation of CML, whereas those with CML inchronic phase being treated with interferon have low orundetectable levels ofp185 [38,39]. This suggests that aquantitative variation of the two transcripts may play a rolein the clinical disease [38].

Different forms of p210 are also produced by alterna-tive splicing, namely,b2a2 andb3a2, with the latter encod-ing a longer transcript. There is some data indicating thatpatients withb3a2 have a better prognosis thanb2a2, ex-hibiting a long chronic phase and better response to inter-feron [2,40–42]. This is interesting, given that the longerBcr–Abl variants (i.e.p230) tend to have a more indolentbiological/clinical phenotype than the shorter variants (i.e.p185). However, more recent prospective studies suggest nodifference in prognosis between patients withb2a2 versusb3a2 transcripts [43–45]. Other data suggest that patientswith b3a2 transcripts have higher platelets than patients withb2a2 transcripts [46,47]. Relevant to this, 9/10 patients withPh+ essential thrombocythemia were found to have theb3a2variant [48–50].

3. An overview: Bcr–Abl structure and signaling

The Bcr–Abl fusion protein acts as an onco-protein byactivating several signaling paths that lead to transfor-mation. Among these are Myc, Ras, c-Raf, MAPK/ERK,SAPK/JNK, Stat, NFKB, PI-3 kinase, and c-Jun [51–61].Inhibition of Ras [62], Raf [63], PI3K [64], Akt [65],Jun [66,67], and Myc [68,69] impairs Bcr–Abl mediatedtransformation. The oncogenic ability of Bcr–Abl requiresderegulated tyrosine kinase activity which leads to recruit-

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ment of adaptor molecules, phosphorylation of signalingmolecules, and activation of downstream signaling events[70–73].

Structurally, Bcr–Abl contains multiple domains (Fig. 1).The Abl sequences encode Src-homology (SH3 and SH2)domains, tyrosine kinase domain, DNA-binding domain,actin-binding domain, nuclear localization signals, andnuclear export signal [3,74]. The Bcr region contains acoiled-coil oligomerization domain, serine/threonine kinasedomain, pleckstrin homology (PH) domain, a Dbl/cdc24guanine nucleotide exchange factor homology domain, sev-eral serine/threonine and tyrosine phosphorylation sites, andbinding sites for the Abl SH2 domain, Grb2, and 14-3-3proteins [3,75,76].

The SH2 domain of Bcr–Abl recruits signaling proteinssuch as p62dok, c-Cbl, and Rin1 [3,77–85]. Binding andphosphorylation of these molecules may be functionallyimportant as SH2 mutations in Bcr–Abl affect the courseof disease in biological models [85]. The actin bindingdomain directly links Bcr–Abl to the cytoskeleton, and fa-cilitates tyrosine phosphorylation of cytoskeletal proteins[86].

Expression of the Bcr–Abl kinases upregulates cell pro-liferation [68,69,87–91], decreases apoptosis [87,92–94],increases cytokine-independent growth [4,95,96], decreasesadhesion to the bone marrow stroma [4,87], and producescytoskeletal abnormalities [4,87].

4. Bcr–Abl variants: molecular structure

Bcr–Abl oncogenes differ in the amount of Bcr includedin the fusion protein, and are formed by joining of vari-ous amounts of theBcr gene to the sameAbl sequences.This difference in structure influences the biological andclinical phenotypes associated with theBcr–Abl variants[2]. Splicing at the m, M, or� breakpoints inBcr pro-duces three distinct protein products, namely, p185 (e1a2junction), p210 (b2a2 or b3a2 junction), and p230 (e19a2junction) (Fig. 1) [2,97]. P185 encodes the dimerizationand SH2- binding domains of Bcr [98,99]. P210 has in ad-dition to the domains present in p185, the Bcr PH and Dbldomains [43,100]. Finally, P230 has the largest amount ofBcr sequence, including the calcium/phospholipid bindingdomain and the first third of the domain associated withGTPase activity for p21 (GAP–Rac) [30]. The inclusion ofthe Dbl/PH domains in p210 and p230 may contribute to thegranulocytic differentiation associated with these variants.

5. Bcr–Abl proteins: biological/laboratory correlates

Although the three Bcr–Abl kinases (p185, p210, andp230) are similar in structure, they exhibit distinct proper-ties. When primary mouse bone marrow cells are infectedwith the different Bcr–Abl variants, and cultured in the

presence of cytokines and stroma, p185 cultures differ-entiate into a lymphoid lineage, whereas p210 and p230become myeloid [100]. In vitro, p185 has increased abil-ity to stimulate expansion of lymphoid cells, compared top210 [101]. P230 Bcr–Abl cultures require cytokines foroptimal growth, whereas p185 and p210 Bcr–Abl culturesare cytokine-independent for growth and survival [100].P185 Bcr–Abl has a greater tumorigenic potential thanp210 or p230 Bcr–Abl [100,102]. In soft agar assays, p185is 100-fold more effective than p210 at eliciting transfor-mation [73]. It was reported that SCID mice injected withp185-expressing primary bone marrow cells developedpre-B cell tumors, whereas no tumors developed in miceinjected with p210- or p230-expressing cells [100]. Millerand Pear have also shown a less aggressive leukemia asso-ciated with p230 Bcr–Abl in 5 FU-lethally irradiated mice[100]. All mice injected with the three Bcr–Abl variantsdeveloped a myeloproliferative disease, but there was anincreased latency in p230- injected mice (27 weeks) versus4–5 weeks in the p185- and p210-injected mice [100].

There are several possibilities to explain the different bio-logical effects of the three Bcr–Abl kinases. Li et al. haveshown that p230 exhibits lower intrinsic tyrosine activitythan p185 and p210 [102]. P210 also has been shown to havedecreased tyrosine kinase activity than p185 [73]. In addi-tion, Nishimura et al. have shown that different sets of tyro-sine phosphorylated proteins may exist in p185 versus p210leukemic cell lines [103]. A 36 kDa protein was detected inK562 cells (p210 cell line), but not MR87 (p185 cell line);and a 62 kDa protein was present in MR87 but not in K562[103]. The 62 kDa protein was a Gap-like protein, and is a cy-toskeletal target for tyrosine kinases [103]. Hef1, a memberof the�1 integrin signaling pathway, is tyrosine phosphory-lated in p185, but not p210 [104]. Tyrosine phosphorylationof these proteins, may lead to differential interaction withthe cytoskeleton or altered downstream signaling. AlthoughStat5, Stat1, and Stat3 are constitutively activated by tyro-sine phosphorylation in p185- and p210-expressing cells,p185 but not p210 promotes activation of Stat6 [59]. Addi-tionally, it was recently demonstrated that the SH2 domainis required for efficient induction of CML-like disease inmice by p210, but not p185, further suggesting a differencein signaling between the two variants [85].

It is possible that differences in the content of the Bcrregion itself may lead to distinct clinical disease [105].Additional Bcr sequences, such as Dbl/PH in p210 andGap–Rac in p230 may allow for increased differentiationalong the myeloid lineage [2,105,106]. The Dbl/PH do-mains present in p210 but not p185, may also contributeto the stabilization of actin fibers [107,108]. Dbl is knownto activate Rho, and activated Rho promotes the formationof actin stress fibers [109,110]. It has been reported that aBcr–Abl fusion containing Bcr (amino acids 1–191) can ef-ficiently transform rat 1-myc cells, but fusion of additionalBcr sequences present in p210 causes decreased disorgani-zation of the actin cytoskeleton [107]. Alternatively, it is


possible that Bcr breakpoints that yield p185 may occurpreferentially in immature B cells, and breakpoints thatproduce p210, in hematopoietic stem cells [105]. The latterpoint is supported by data from Li et al. [102]. In a murinebone marrow transplantation model, p185, p210, and p230were all equally potent in inducing a CML-like disease,when 5 FU donors were used. The proviral integrationsite was polyclonal, implicating a multipotential target cell[102]. When no 5 FU was used, a mixture of CML, ALL,and macrophage tumors was observed [102]. P185 in thesemice induced lymphoid leukemias with a shorter latencythan p210 or p230. In addition, lymphoid leukemias wereoligoclonal, suggesting a lineage restricted target cell [102].This data, however, has not been supported by analysis ofpatients with Ph+ ALL. Adult patients with Ph+ ALL havebeen reported to have a multipotential stem cell target basedon examination of metaphases of individual colony form-ing unit-erythroid (CFU-erythroid) and CFU-granulocytemacrophage (CFU-GM) [29,111,112]. This suggests theremay differences in the biology of Bcr–Abl in patients versusanimal models.

6. Molecular changes: blastic phase of CML

CML eventually progresses to a blastic phase, reminis-cent of acute leukemia that is much more treatment resistant[13,113]. The molecular changes that accompany this eventhave been of interest, but are not consistent. Progression toblast crisis has been shown to correlate with the addition ofcytogenetic abnormalities such as 22q−, loss of the Y chro-mosome, isochromosome 17, trisomies-8, -9, -19, an addi-tional Ph chromosome, translocation t(3;21), or inversion ofchromosome 16 [13,114,115]. Specific mutations have beenobserved inRas, p53, Myc, p16, andRb [116–121]. In addi-tion, increased transcription ofBcr–Abl, h-Ras, andc-Mycis detected in many clinical cases of accelerated phase CML[122–130].

In some cases, alterations in known oncogenes/tumorsuppressers correspond to the cytogenetic changes seenabove. For example,p53 is located on chromosome 17,Rbon chromosome 13,c-Myc on chromosome 8,p16 on chro-mosome 9, andAML/EV-1 on chromosomes 3 and 21 [127].In addition, AXL, a putative receptor with tyrosine kinaseactivity, located on chromosome 19, may be involved in theprogression of CML to blastic phase [21,128–130].

7. Current treatment implications

Traditionally, CML in the chronic phase has been treatedwith a combination of hydroxyurea and interferon, with a73% hematologic response rate and 58% rate of cytogeneticremissions [13,131,132]. This has been slightly improvedwith the addition of cytarabine [13]. Despite this, the 5year survival rates are 57% [131,132], so allogeneic bone

marrow transplant (BMT) has been the treatment of choicefor younger patients with an HLA matched identical sib-ling, showing a 70% cure rate [13,133]. However, only aminority of patients are transplanted because of age, or lackof an HLA-matched identical sibling [13]. After transplant,PCR forBcr–Abl has been used to monitor patient’s diseasestatus. Repeated PCR positivity at greater than 6 monthspost-transplant or increasing values based on quantitativePCR tend to correlate with relapse [13,134,135]. Donorlymphocyte infusions have been found to be the most ef-fective form of adoptive salvage immunotherapy in patientswho relapse post-transplant [7,136].

Other treatments for CML have been investigated includ-ing antisense oligonucleotides, immunotherapy, farnesyltransferase inhibitors, and tyrosine kinase inhibitors [87].The tyrosine kinase inhibitors have been of particular interestbecause numerous studies have demonstrated that tyrosinekinase activity is required for the transforming capacity ofBcr–Abl [73,87,137]. In the mid-1990s, signal transductioninhibitor 571 (STI-571) (Gleevec), a phenylaminopyrimidederivative with potent tyrosine kinase inhibition and selec-tivity for Abl, c-Kit, and platelet-derived growth factor wasdeveloped [87,138]. STI-571 binds to a pocket of the cat-alytic domain of the Abl tyrosine kinase and competitivelyinhibits binding of adenosine triphosphate (ATP), therebyresulting in inhibition of autophosphorylation and inhibitionof substrate phosphorylation [139].

Pre-clinically, it was shown that STI-571 caused adose-dependent inhibition of 32D 210 cells injected intomice [140]. Subsequently, a phase 1 trial was carried outwith 54 patients with CML refractory to interferon-�. Ofthese patients, 23/24 patients receiving a dose of 300 mg orhigher underwent a complete hematologic remission, and8/24 had a complete cytogenetic remission [141]. Currently,a phase 3 study is underway comparing STI-571 versusinterferon-� +/− cytarabine as front-line therapy for CML[87].

STI-571 was also used in 33 patients with Ph+ acuteleukemia. Both blast crisis and Ph+ ALL are difficultto treat. Patients with a myeloid blast crisis treated withSTI-571 had a 55% response rate and 22% complete remis-sion rate. Patients with lymphoid blast crisis had an 82%response rate and 55% complete remission rate [142]. Theseresults are surprisingly encouraging, and suggest that eitherthe Bcr–Abl tyrosine kinase activity is necessary for blastictransformation of that other signaling pathways that areactivated during blast phase are inhibited by STI-571 [143].

Despite enthusiasm based on the above results, the emer-gence of STI-571 resistance in patients with acute leukemia,and the isolation of Ph+ leukemia cell lines that are resis-tant to STI-571 highlights the need for combination therapy[144,145]. The molecular mechanism of resistance includesincreasedBcr–Abl messenger RNA (mRNA), increased pro-tein, amplification of the transgene, and point mutationswithin the Abl kinase domain ofBcr–Abl [144–146]. Cur-rently, trials with ST-571 combined with chemotherapy for


Ph+ leukemias are underway. In addition, other moleculartargeted therapies are being examined. Second generationtyrosine kinase inhibitors are being developed for testing[87]. An alternative approach to inhibition of the kinase hasbeen to develop an Abl-targeted tyrosine phosphatase [147].

Because Ras activation is thought to be important inBcr–Abl mediated transformation, farnesyl transferase in-hibitors have also been of particular interest [148]. Thefarnesyl transferase inhibitor, SCHH66336 has been de-signed to block both mutant and wild-type Ras signaling intransformed cells [148]. In a murine ALL model, it has beenshown to revert early signs of leukemia and significantlyprolong survival [148]. Currently, it is being investigated inclinical trials.

The above therapies are illustrative of how understandingthe molecular biology of a disease can contribute to elegantand successful molecular targeted therapies. It is likely thisparadigm will be applied to other diseases.

Acknowledgements

A.S. Advani is supported by National Institutes of HealthTraining Grant 2-T32-HC07057-26. A.M. Pendergast is sup-ported by National Institutes of Health grants CA61033 andCA70940.

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bcr-abl variants - biological and clinical aspects

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