Seminars in Hematology
Volume 40, Issue 1 , Pages 34-49, January 2003

Management of chronic myeloid leukemia: Targets for molecular therapy☆

Department of Haematology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London, UK

Article Outline

Abstract 

Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by malignant expansion of bone marrow stem cells. Currently, the only unequivocally curative treatment for CML is allogeneic stem cell transplant. Unfortunately, a large proportion of CML patients are ineligible for such treatment and alternative forms of therapy must be employed. Conventional chemotherapy makes use of compounds, such as hydroxyurea, that are cytotoxic for actively dividing cells. Although effective, these agents are not selective for the leukemic clone and this is the cause of undesirable side effects. Moreover, as the disease progresses patients frequently become refractory to chemotherapy. In recent years, knowledge of the molecular pathology of CML has allowed the development of drugs that selectively target the malignant cells: imatinib mesylate is the most prominent example. These agents are selective because they target molecular determinants that are exclusively deregulated in the leukemic cells. In this review we consider some of the novel developments in this field. Particular emphasis is given to chemical agents that target the Bcr-Abl oncoprotein. The latter affords an excellent opportunity for therapy since CML, in contrast to many other human malignancies, is likely caused by the activity of a single oncoprotein. Furthermore, it is believed that Bcr-Abl continues to play a central role throughout the course of the disease. Semin Hematol 40:34-49. Copyright 2003, Elsevier Science (USA). All rights reserved.

 

The molecular hallmark of chronic myeloid leukemia (CML) is a reciprocal translocation involving chromosomes 9 and 22 [t(9;22)(q34;q11)]. As a result of this translocation, a small derivative chromosome 22q- is generated, known as the Philadelphia (Ph) chromosome.57 The Ph chromosome, the first consistent chromosomal abnormality to be identified in a human malignancy, bears the BCR-ABL fusion gene. This encodes a chimeric Bcr-Abl protein with a deregulated tyrosine kinase activity, the expression of which has been shown to be necessary and sufficient for the transformed phenotype of CML cells.46 CML is unusual among human cancers in that a single oncogene product has been identified as having a central role in its pathology.

In this review, we discuss methods for managing CML based on the application of molecular therapy. We describe compounds that, in contrast to traditional chemotherapeutic agents, have activity against molecular targets that are specific to leukemic cells. Special consideration is given to characteristics of the Bcr-Abl oncoprotein itself, as it is the best molecular target presented by CML cells because it is not expressed by normal cells. We describe approaches for inhibiting the activity of Bcr-Abl by targeting specific protein domains, with particular emphasis on the use of imatinib mesylate. In addition, means of inhibiting the expression of Bcr-Abl in CML cells will be reviewed. The translation of Bcr-Abl may be suppressed by targeting its mRNA through an antisense approach. Alternatively, expression of Bcr-Abl may be inhibited post-translationally by use of chemical agents that destabilize the protein and promote its proteolysis. The progress that has been made in inhibiting cell signaling pathways downstream of Bcr-Abl will be outlined, with particular attention paid to the inhibition of Ras signaling by means of farnesyl transferase inhibitors (FTI). A number of FTI are currently the subject of clinical trials. Finally, potential molecular therapies based on immunomodulation will be considered.

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Targeting the Bcr-Abl oncoprotein 

The tyrosine kinase (SH1) domain 

The Src homology 1 (SH1) domain of Bcr-Abl is an obvious molecular target since the essential leukemogenicity or transforming principle of the oncoprotein is due to its constitutive tyrosine kinase activity46 (Fig 1).

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  • Fig. 1. 

    Targets for molecular therapy. Each target is numbered and marked with a red cross. 1. The Src homology 1 (SH1) or tyrosine kinase domain of Bcr-Abl: its activity may be inhibited by signal transduction inhibitors, such as imatinib mesylate or adaphostin. 2. The oligomerization (coiled-coil) domain of Bcr-Abl: deletion/mutation or blocking of this domain with peptides that prevent oligomerization renders Bcr-Abl nontransforming. 3. The nuclear export signal (NES) receptor: inhibition of this nuclear protein with leptomycin B combined with inactivation of Bcr-Abl with imatinib entraps Bcr-Abl in the nucleus; subsequent reactivation of nuclear Bcr-Abl by removal of imatinib leads to apoptosis. 4. Heat-shock protein 90 (Hsp90): Hsp90 functions as chaperone that maintains the stability of the Bcr-Abl protein; antagonists of Hsp90, such as geldanamycin, destabilize Bcr-Abl and promote its proteasomal degradation. 5. BCR-ABL mRNA: synthesis of the Bcr-Abl oncoprotein may be suppressed by inhibiting BCR-ABL mRNA by either antisense oligonucleotides, ribozymes, or DNAzymes. 6. The SH3 domains of the adapter proteins Grb2 or CrkL: synthetic peptides that bind to these domains “uncouple” Bcr-Abl from downstream signaling pathways. 7. Farnesyl transferase: inhibitors of farnesyl transferase suppress Ras signaling by preventing the attachment of a farnesyl group to Ras; farnesyl groups are essential for the normal functioning of Ras since they tether these G-proteins to the plasma membrane. 8. Mek (MAPK or ERK Kinase): Bcr-Abl constitutively activates the Ras-Raf-Mek-Erk pathway; Mek inhibitors may be useful for inhibiting this mitogenic cascade 9. Phosphatidinylinositol-3 (PI-3) kinase: PI-3 kinase associates with Bcr-Abl and undergoes activation as a result of tyrosine phosphorylation; PI-3 kinase cell signaling may be inhibited with compounds such as wortmannin or LY294002, resulting in apoptosis by activation of Bad (pro-apoptotic) via Akt and its dissociation from Bcl-XL (anti-apoptotic). 10. Bcr-Abl junction-specific peptides used as a potential vaccine for CML patients: the aim is to stimulate an immune response mediated by T-lymphocytes.

Initial attempts at developing tyrosine kinase inhibitors involved screening natural products for compounds capable of antagonizing this catalytic activity. Candidate molecules identified by this approach included an isoflavonoid, genistein,2 and an antibiotic, herbimycin A.79 Subsequent efforts have focused on the rational design of synthetic compounds with chemical structures that are able to compete with either adenosine triphosphate (ATP) or a substrate for occupancy of the binding site in the kinase domain.44 The first synthetic tyrosine kinase inhibitors to be developed were a class of compounds known as tyrphostins.89 One of the tyrphostins, AG1112, was shown to be effective at inducing differentiation in the human CML cell line K562.5

Undoubtedly, the most successful synthetic tyrosine kinase inhibitor that has been developed to date is the 2-phenylaminopyrimidine, imatinib mesylate (formerly known as STI571) from Novartis Pharma (Basel, Switzerland). This compound was the product of a program of rational drug design conceived with the intention of synthesizing small molecule inhibitors capable of occupying the ATP-binding pocket of protein tyrosine kinases (for a recent review of the development of imatinib, see Capdeville et al14). Synthesis of appropriate chemical species was guided and informed by knowledge of the structure of the ATP-binding site of protein kinases.20 In preclinical studies, CGP 57148 (the original designation for this compound) was effective at inhibiting the autophosphorylation of Abl, the platelet-derived growth factor receptor (PDGFR),13 the Kit receptor,12 and the Arg (ABL-related gene)60 tyrosine kinases at submicromolar concentrations. The compound exhibited a remarkable degree of specificity, its effect on other tyrosine kinases being negligible. The proliferation of CML primary cells was inhibited by treatment with the inhibitor but control normal cells were unaffected.22 Selective inhibition of growth could also be demonstrated for BCR-ABL+ cell lines both in vitro17, 22 and in mice.22

The available data from the preclinical studies suggested that this compound, then renamed STI571 (STI referring to its potential as a signal transduction inhibitor), might have value as a therapeutic agent. A phase I clinical trial of STI571 was begun in 1998 in the United States,21 where the drug was administered orally to 83 chronic-phase (CP) CML patients for whom treatment with interferon alpha (IFN-α) had failed. The drug exhibited little toxicity and although a clearly defined maximal tolerated dose was not identified, a greater incidence of grade III to IV adverse effects were associated with doses above 750 mg per day. At 300 mg per day or higher, 98% of patients achieved a complete hematologic response with many of these also showing cytogenetic responses. Based on the findings of this study, it was recommended that CP patients should receive a daily dose of at least 400 mg in subsequent clinical trials of STI571.

Phase II trials were begun in 1999 for CML patients in CP,36 accelerated phase (AP),74 and myeloid blast crisis (BC).67 These studies involved international, multicenter collaborations in which larger cohorts of patients were recruited: 532 for the CP, 235 for the AP, and 260 for the myeloid BC trial. Patients in CP received oral doses of 400 mg of STI571 per day, while those in AP and BC were treated with either 400 or 600 mg/d. In the CP trial, STI571 induced complete hematologic responses in 95% of patients. Major cytogenetic responses were achieved by 60% of patients, of which 41% were complete.36 For the group in AP, sustained hematologic responses (of 4 or more weeks duration) were observed in 69% of patients, with 34% of these being complete; major cytogenetic responses were achieved by 24% of patients.74 In the myeloid BC trial, STI571 induced sustained hematologic responses in 31% of patients, of which 8% were complete; major cytogenetic responses were achieved by 16% of patients, with only 7% of these being complete remissions. Median survival time was 6.9 months.67

In May 2001, STI571 was granted marketing approval by the US Food and Drug Administration (FDA) for the treatment of CML refractory to interferon. The clinical development of this drug has been remarkably rapid with less than 3 years between the start of the first phase I clinical trial and its approval by the FDA.14 Upon its introduction into clinical practice, STI571 was renamed imatinib mesylate and marketed under the proprietary names of Gleevec in the United States or Glivec in Europe.

There remain problems associated with the clinical use of imatinib mesylate for the treatment of CML patients. In advanced-phase CML the responses are often short-lived and patients invariably undergo disease progression following these brief periods of respite. Clinical resistance to the drug following its regular administration appears to be due to multiple mechanisms. Experiments in which BCR-ABL+ cell lines were systematically exposed to escalating doses of imatinib mesylate have shown that resistance can develop due to amplification of the BCR-ABL gene or through overexpression of the multidrug resistance P-glycoprotein.48 Similarly, overexpression of the BCR-ABL gene has been demonstrated in CML patients who were resistant to imatinib treatment.26 Another important mechanism responsible for the development of imatinib resistance in patients is mutation of the Bcr-Abl kinase domain. Individual point mutations within this domain have been identified that allow Bcr-Abl to remain kinase active despite the patients receiving therapeutic doses of imatinib.6, 11, 26, 29, 69, 82 Regardless of the mechanism, the mutant resistant clone will be selected for its growth advantage in the presence of the drug. It has been argued that resistance to imatinib might be expected to develop more rapidly and more uniformly than resistance to conventional cytotoxic drugs precisely because imatinib is such a highly selective agent.9, 47

There is currently great interest in treatment regimens for CML that involve combining imatinib with other therapeutic agents. Since the biological activity of imatinib is the result of binding to a well-defined molecular target, the kinase domain of Bcr-Abl, compounds that interact with other molecular targets may enhance its effects. Hence, the very specificity of imatinib mesylate in terms of its mode of action provides a rationale for multifaceted, combination therapies.58 Several prospective phase I and II studies are taking place to evaluate the efficacy of combining imatinib with other drugs that are currently used to treat CML such as IFN-α, polyethylene glycol (PEGylated) interferon, and cytosine arabinoside (Ara-C).14 In preclinical studies involving BCR-ABL+ cell lines, a number of compounds have been identified that contribute an additive cytotoxic effect when combined with imatinib. These include IFN-α and daunorubicin (DNR)76 and homoharringtonine (HHT), doxorubicin, and 4-hydroperoxycyclophosphamide.35 Importantly, a small number of compounds such as vincristine,35 mafosfamide,78 etoposide,78 and Ara-C76 have been shown to act synergistically when combined with imatinib. It should be noted, however, that etoposide and Ara-C have also been described as having merely additive effects when combined with imatinib.35 In the same study,35 the combination of IFN-α with imatinib was reported as being synergistic, while an earlier publication76 described the same combination as being additive. These discrepancies are probably due to the different methods that were chosen to analyze the data. Arsenic trioxide also has been proposed for combination with imatinib.42 Historically, arsenic compounds were among the first agents to be used for the chemotherapy of CML, with the antileukemic activity of Fowler's solution, potassium arsenite, first described in the late 19th century. The resurgence in interest in arsenic trioxide follows research from China that showed complete remission in 25 of 34 (74%) patients with CML and partial remission in seven of 34 (21%) treated patients.58 Arsenic compounds generate reactive oxygen species that promote apoptosis by causing damage to mitochondrial membranes, which leads to the release of cytochrome-C into the cytoplasm. There is evidence, however, that the combination of imatinib mesylate with certain other therapeutic compounds can result in unfavorable, antagonistic interactions. In a recent study of the effects of drug combinations on the proliferation of CML progenitors, the combination of imatinib with IFN-α was found to be less than additive,50 in contrast to data from studies using BCR-ABL+ cell lines.35, 76 Similarly, the combination of hydroxyurea with imatinib may result in antagonism.76, 78 Despite the caveat that certain therapeutic agents may reduce the efficacy of imatinib, the potential benefits to be gained from finding drugs that enhance its antileukemic effect, possibly as a result of synergism, are considerable. Moreover, where patients have acquired resistance to imatinib, the use of other compounds in conjunction with this drug may be of clinical benefit. A recent study77 demonstrated that cell lines that had been made resistant to imatinib remained sensitive to other chemotherapeutic agents. In particular, Ara-C, DNR, and HHT effectively inhibited the proliferation of imatinib-resistant cells.

Imatinib mesylate represents the prototype for an emerging class of therapeutic agents that have been refined by a process of rational drug design and which specifically target the kinase domain of Bcr-Abl. Another compound, the pyrido[2,3-d]pyrimidine derivative, PD180970 (Parke-Davis Pharmaceuticals, Ann Arbor, MI), was reported to inhibit autophosphorylation of Bcr-Abl in K562 cells with a lower IC50 than that of imatinib19 and to induce apoptosis in K562 but not in BCR-ABL− HL60 cells. Recently, the potency of PD180970 and five other pyrimidine analogs has been compared with that of imatinib in assays of Bcr-Abl–dependent cell growth.87 Of the seven compounds, another pyrimidine, PD166326, was the most potent inhibitor of growth in BCR-ABL+ cells. Whether these results are reproducible across a wider range of cell lines, or indeed whether such efficacy and apparent selectivity can also be elicited in primary CML cells, remains to be confirmed in independent studies.

Imatinib mesylate antagonizes the tyrosine kinase activity of Bcr-Abl by occupying the ATP-binding pocket of its SH1 domain. In the presence of imatinib, the SH1 domain retains the ability to associate with substrate molecules but is unable to catalyze their phosphorylation. An alternative approach for inhibiting the tyrosine kinase activity of Bcr-Abl involves the use of chemical agents that inhibit or modify the binding of substrates. Under these conditions, the ATP-binding pocket of the SH1 domain is unaffected but association of the kinase with its substrates is impaired. A class of synthetic compounds, the tyrphostins, inhibits the tyrosine kinase activity of Bcr-Abl in this fashion. The therapeutic potential of tyrphostins is more limited than that of imatinib mesylate since animal studies have revealed that these compounds have a short serum half-life in vivo.55 Attempts have been made to overcome this limitation by synthesising chemical analogs with improved pharmacokinetic properties. Of particular interest is the analog adaphostin (NSC 680410), the adamantyl ester of the tyrphostin AG957, which has a longer serum half-life than AG957 and greater in vitro potency.55 A recent study55 compared the effects of adaphostin with those of imatinib mesylate on K562 cells, BCR-ABL–transduced FDC-P1 cells, and primary progenitors obtained from CML patients. In K562 cells, levels of p210 Bcr-Abl oncoprotein were reduced following 6 hours exposure to 10 μmol/L adaphostin. Caspase activation occurred by 12 hours, and by 24 hours, 90% of the K562 cells were apoptotic. In contrast, treatment of K562 cells with 20 μmol/L imatinib led to rapid inhibition of Bcr-Abl autophosphorylation without degradation of the p210 protein. Adaphostin was selectively toxic for leukemic cells, inhibiting CML granulocyte colony-forming units (CFU-G) but not normal CFU-G. Importantly, imatinib-resistant K562 cells proved to be sensitive to adaphostin, consistent with the idea that imatinib mesylate and adaphostin exert their inhibitory effects on the tyrosine kinase activity of Bcr-Abl by separate and distinct mechanisms. Treatment of K562 cells with both imatinib mesylate and adaphostin induced greater cytotoxicity than that achieved with either agent alone. Hence, adaphostin may prove to be valuable in combination with imatinib mesylate.

The oligomerization domain 

The oligomerization domain of Bcr-Abl represents an attractive target for molecular therapy because several lines of evidence suggest that this domain is also essential for the transforming activity of the oncoprotein. Examination of the amino acid sequence of Bcr's N-terminus revealed the presence of a heptad repeat (a repeated seven–amino acid pattern) with hydrophobic residues at the first and fourth positions, between residues 28 and 68.51 This motif is commonly found within amphipathic α-helices that form coiled-coil domains associated with protein oligomerization. Mutants were generated in order to test the hypothesis that this region of Bcr-Abl is involved in oligomerization. A Bcr-Abl fusion protein containing the first 509 amino acid residues of Bcr was found to co-immunoprecipitate with a protein fragment consisting of the first 191 amino acids of Bcr. When an insertion designed to disrupt the N-terminal α-helical region was introduced into this Bcr-Abl fusion, it failed to co-immunoprecipitate with the Bcr fragment.51 An experiment in which glutaraldehyde was used to cross-link Bcr protein fragments containing amino acids 1 through 71 suggested that oligomerization of these fragments occurred spontaneously in solution and that the major species formed was a homotetramer.51 In addition, insertion mutations within the putative oligomerization domain resulted in a profound reduction in the autophosphorylation of tyrosine residues on Bcr-Abl, indicating that this region has a role in activating the tyrosine kinase activity of the oncoprotein.51 Moreover, the same insertion mutants together with a mutant lacking the first 63 amino acids of the N-terminus failed to induce transformation, as assessed by the development of growth factor independence, when expressed in Ba/F3 cells.51 The correlation between oligomerization and transformation has been interpreted as evidence that Bcr-Abl must undergo tetramerization in order to become activated. It has been postulated that the formation of Bcr-Abl homotetramers promotes their intermolecular cross-phosphorylation and activation in a manner analogous to the dimerization, cross-phosphorylation, and activation of growth factor receptor tyrosine kinases.51 According to this model, monomers of Bcr-Abl are nontransforming, whereas the oncogenic, transforming species are Bcr-Abl tetramers. In agreement with this hypothesis is the finding that expression of a peptide consisting of the first 160 amino acid residues of Bcr, including the oligomerization domain, was able to restore growth factor dependence to a growth factor–independent,BCR-ABL+ murine hematopoietic cell line.27 Furthermore, by transfecting the cells with a vector encoding the Bcr fragment and a vector encoding full-length Bcr-Abl in different stoichiometric ratios, growth factor–independent colony formation, characteristic of the transformed phenotype, could be inhibited in a dose-dependent manner. The Bcr fragment was presumed to disrupt the formation of transforming Bcr-Abl homotetramers by oligomerizing with Bcr-Abl monomers.

Recently, a recombinant peptide consisting of the N-terminal 72 amino acid residues of the Bcr-Abl oligomerization domain was crystallized and subjected to x-ray diffraction.91 Crystals consisted of an asymmetric unit containing eight equivalent protein chains composed of two identical tetramers. The complete oligomerization domain was found to incorporate a short N-terminal α-helix (α1, residues 5 through 15), a flexible loop (residues 16 through 27) and a second, longer α-helix (α2, residues 28 through 67) equivalent to the predicted amphipathic helix. These regions adopt an ‘N’-shaped conformation in which the two helices are able to assume a parallel orientation due to the flexibility of the linking loop. Dimerization of Bcr-Abl involves the formation of an antiparallel coiled-coil between the α2-helices of two monomers. Packing of the monomers is completed by domain swapping in which the α1-helices from each monomer rotate around the coiled-coil domain. In the resulting dimer, the four intertwined α-helices lie in approximately the same plane to form a surface. This surface facilitates dimer-dimer stacking leading to the formation of a tetramer.

None of the currently available therapies for CML target the oligomerization domain of Bcr-Abl. It could be argued, however, that since the oncogenicity of Bcr-Abl is apparently dependent on its oligomerization, this property could be exploited for therapeutic gain. That oligomerization is essential for malignant transformation by Bcr-Abl is underscored by a recent study28 in which mice were injected with bone marrow cells that had been retrovirally transduced with mutant BCR-ABL constructs. Mice receiving cells expressing the full-length p210 Bcr-Abl oncoprotein developed a myeloproliferative disorder resembling human CML while mice receiving cells expressing a mutant Bcr-Abl protein lacking the first 63 amino acids failed to develop this disease. A case could be made for the development of synthetic molecules capable of inhibiting or disrupting Bcr-Abl oligomerization. Although inhibitors of Bcr-Abl oligomerization might be expected also to inhibit the oligomerization of wild-type Bcr, there is evidence to suggest that disruption of the normal functioning of this protein need not necessarily be deleterious to health. BCR-knockout mice are viable and, despite having neutrophils that produce excessive reactive oxygen metabolites upon stimulation, are otherwise healthy.83 This finding suggests that Bcr does not have an essential role in cell physiology. A detailed, high-resolution (2.2 Å) crystal structure for the oligomerization domain of Bcr-Abl is now available that may serve as a template for the rational design of small molecule inhibitors or peptides.91 Although it remains to be seen whether the development of compounds that target the oligomerization domain of Bcr-Abl is feasible, the potential of this approach is highlighted by a recent report7 in which the homo- and heterodimerization of members of the ErbB family of receptor tyrosine kinases was inhibited with rationally designed peptidomimetics.

Nuclear entrapment of the Bcr-Abl oncoprotein 

It has recently been demonstrated that Bcr-Abl can induce apoptosis in CML cells if its nuclear import and subsequent entrapment are achieved by chemical means.81 This discovery represents a novel approach to the problem of selectively killing a leukemic clone and could form the basis of a therapy.53 Although there may appear to be a paradox associated with an oncoprotein that can be either anti- or pro-apoptotic depending on its subcellular context, this finding makes sense when the normal physiological role of the Abl component of Bcr-Abl is considered. Abl, the product of the ABL proto-oncogene, is a 145-kd nonreceptor protein tyrosine kinase that is a key component of cell signaling networks responsible for essential processes such as control of the cell cycle and the cellular response to genotoxic stress (reviewed by Van Etten80). Abl functions in both the nucleus and the cytoplasm and is exchanged between the two subcellular locations. This nuclear-cytoplasmic “shuttling” requires three nuclear localization signals (NLS)85 and a single nuclear export signal (NES)73 that are present within the C-terminus of the Abl protein. The nuclear export of Abl may be blocked by treating cells with leptomycin B, an inhibitor of the NES-receptor CRM1/exportin-1. In contrast to Abl, the Bcr-Abl oncoprotein is exclusively localized in the cytoplasm despite containing the intact C-terminus of Abl with its full complement of NLS and NES. In a series of ingenious experiments using murine Abl−/− fibroblasts transfected with BCR-ABL,81 the cytoplasmic location of Bcr-Abl was found to be due to the oncoprotein being excluded from the nucleus, rather than by an imbalance in nuclear import and export, since leptomycin B treatment failed to cause an accumulation of nuclear Bcr-Abl. Although the precise mechanism by which Bcr-Abl inhibits its own nuclear import is not known, it is evidently dependent on its kinase activity since a kinase defective mutant of Bcr-Abl was imported into the nucleus and its export could be blocked with leptomycin B. Moreover, the nuclear import of the wild-type, Bcr-Abl oncoprotein could be stimulated by incubating the cells with the tyrosine kinase inhibitor, imatinib mesylate. Treatment with imatinib alone was insufficient to cause the nuclear accumulation of Bcr-Abl, but a combination of imatinib and leptomycin B was found to be effective at trapping the oncoprotein in the nucleus. Significant apoptosis did not occur under these conditions because the tyrosine kinase activity of Bcr-Abl was inhibited by imatinib. When the cells were washed to remove imatinib, however, the tyrosine kinase activity of Bcr-Abl was restored and signs of apoptosis were observed in 70% to 80% of Bcr-Abl+ cells. Thus, the mere accumulation of the kinase inactive Bcr-Abl oncoprotein in the nucleus is not especially pro-apoptotic but the restoration of the tyrosine kinase activity of this nuclear pool of Bcr-Abl causes apoptosis. The same treatment regimen of imatinib in combination with leptomycin B followed by extensive washing to remove the tyrosine kinase inhibitor was also found to be effective at inducing apoptosis in the murine hematopoietic cell line, TonB210, and the human CML cell line, K562. Importantly, the combined treatment with imatinib and leptomycin B was found to be better at inducing apoptosis in K562 cells than treatment with imatinib alone. The use of leptomycin B in vivo is precluded by its neurotoxicity, but now that its considerable potential as an antineoplastic therapeutic agent has been demonstrated toxic inhibitors of nuclear export may be developed. Alternatively, the combination of imatinib mesylate and leptomycin B could be used ex vivo to purge bone marrow of CML cells.68 Such a purging strategy could make autologous bone marrow transplantation a viable therapeutic option for the treatment of CML.

Destabilizing the Bcr-Abl oncoprotein 

Since Bcr-Abl has a central and causative role in CML it would be therapeutically advantageous if this oncogenic fusion protein could be selectively eliminated. Molecular therapies that target particular domains of Bcr-Abl in order to inhibit its function have already been discussed. In all cases, however, the Bcr-Abl oncoprotein, despite being functionally inhibited, continues to be expressed by leukemic cells. The persistence of Bcr-Abl presents particular problems for treatment with imatinib mesylate, where the selection of mutant forms of the oncoprotein or expansion of a clone in which the oncoprotein is overexpressed lead to the development of resistance. Hence, the development of therapies that aim at inhibiting expression of Bcr-Abl is particularly desirable. The use of antisense to suppress translation of BCR-ABL mRNA is described in a later section. An alternative approach is the use of chemical agents to destabilize the Bcr-Abl oncoprotein in order to promote its proteasomal degradation. A benzoquinone ansamycin antibiotic, geldanamycin, has been shown to destabilize the p210 Bcr-Abl protein in K562 cells4, 56 and a p190 Bcr-Abl protein expressed ectopically in HL-60 cells.56 Another benzoquinone ansamycin, herbimycin A, has already been mentioned as a compound that was identified in a screening program for natural products capable of inhibiting tyrosine kinases.79 Herbimycin A was initially regarded as a tyrosine kinase inhibitor because it could restore the untransformed morphological phenotype to cells transformed with v-Src,79 and subsequently reported as an inhibitor of the kinase domain of Bcr-Abl.30 A detailed analysis of the mode of action of this compound revealed that herbimycin A actually exerted its inhibitory effect on Src via an indirect mechanism involving heat shock proteins (Hsp).86 In particular, herbimycin A and other benzoquinone ansamycins bind to Hsp90, a protein that serves to stabilize a number of important cell signaling molecules such as protein kinases and transcription factors.4 The stability of Bcr-Abl is dependent on it forming a multiprotein complex with Hsp90 and a co-chaperone protein, p23. Brief exposure to geldanamycin, a specific inhibitor of Hsp90, causes p210 Bcr-Abl to dissociate from Hsp90 and p23 and to form another complex with the chaperone proteins, Hsp70 and p60Hop.4 The p210-Hsp70-p60Hop complex is less stable than the p210-Hsp90-p23 complex since increased degradation of p210 Bcr-Abl occurs following treatment with geldanamycin.4, 56 In addition, geldanamycin and its less toxic analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) caused degradation of p190 Bcr-Abl exogenously expressed in HL-60 cells.56 Pretreatment of the BCR-ABL+ cells with proteasome inhibitors abrogated the geldanamycin-mediated degradation of the Bcr-Abl proteins, indicating that proteolysis was occurring via the ubiquitin-proteasomal pathway4, 56 Interestingly, geldanamycin and 17-AAG induced the cytosolic accumulation of cytochrome c and an increase in the activities of caspases 9 and 3 in BCR-ABL+ cells.56 These events were accompanied by an increase in the proportion of cells undergoing apoptosis. Since Hsp90 is required for the stability of proteins involved in normal cell physiology as well as for oncoproteins, it might be expected that drugs that inhibit Hsp90 would have toxic effects. Clinical trials of 17-AAG for the treatment of solid tumors are currently ongoing and a number of dose-limiting toxicities have been identified (reviewed in Blagosklonny8). It may be possible to ameliorate some side effects by shortening the duration of treatment. A recent report25 suggests that these drugs may be of particular benefit to patients who have relapsed following treatment with imatinib mesylate. Both geldanamycin and 17-AAG induced the degradation of wild-type p210Bcr-Abl and two mutant Bcr-Abl proteins (T315I and E255K) found in imatinib-resistant patients. Moreover, there was a discernible trend with both compounds exhibiting greater potency against the mutant forms of Bcr-Abl than the wild-type. Clinical studies to investigate the efficacy of 17-AAG in imatinib-resistant patients are warranted.

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Targeting Bcr-Abl mRNA 

Antisense oligonucleotides 

A variety of antisense (AS) strategies have been attempted in order to suppress translation of the Bcr-Abl oncoprotein. A typical AS approach involves the synthesis of short oligonucleotides that are complementary to sequences present within mRNA transcribed from the gene of interest. Antisense oligonucleotides (ASO) are introduced into cells, whereupon they bind to the target mRNA to form duplexes. The effect of duplex formation is twofold: it prevents the mRNA from associating with ribosomes and it renders the transcripts susceptible to degradation by RNase H. Consequently, translation is inhibited by a mechanism that involves both the obstruction of protein synthesis and destruction of the target mRNA (“killing the messenger”). Some degree of chemical modification of ASO is usually required to prevent their degradation by nucleases (recently reviewed by Braasch and Corey10). The most common modification is the substitution of sulfur for an oxygen atom in each of the phosphodiester linkages of the sugar-phosphate backbone of the oligonucleotide. The resulting phosphorothioate linkages enhance the stability of ASO by making them more resistant to nucleases. This modification also improves the pharmacokinetic properties of ASO by increasing their binding to serum proteins leading to prolonged in vivo half-lives. The majority of the ASO evaluated as possible therapeutic agents for the treatment of CML have been “engineered” to include phosphorothioate linkages.

In theory, BCR-ABL mRNA should be an ideal molecular target for a therapy based on AS. The t(9;22) chromosomal translocation generates a unique fusion gene composed of 3' sequences from the ABL gene juxtaposed with 5' sequences from the BCR gene. Since the breakpoints on these genes occur within introns, transcription of the BCR-ABL oncogene yields fusion transcripts with sequences from intact BCR exons joined to sequences from intact ABL exons. By designing an ASO that is complementary to both the BCR and ABL sequences on either side of this junction, it should be possible to generate a species that would hybridize to BCR-ABL mRNA but not to mRNA transcribed from the wild-type BCR or ABL alleles. Hence, AS strategies can exploit the chimeric nature of the BCR-ABL mRNA to specifically inhibit translation of the oncoprotein without affecting expression of the normal, physiological Bcr and Abl proteins. Moreover, 95% of cases of CML are associated with fusion transcripts with either BCR exon 13 sequences fused to ABL exon 2 sequences (e13a2) or BCR exon 14 sequences fused to ABL exon 2 sequences (e14a2)52 so that the availability of two ASO could allow the treatment of a large cohort of patients.

In practice, however, AS has failed to fulfill its promise as a treatment for CML and BCR-ABL has not proven to be the ideal target for this approach. Early reports in which ASO were tested in murine models of CML were encouraging. In one important study,71 immunodeficient (SCID) mice were injected with the human CML blast crisis cell line, BV173, and then treated systemically with a 26-mer BCR-ABL ASO. The proliferation of the human Ph+ cells was inhibited and the development of a disease resembling leukemia was delayed. In contrast, mice that had been injected with BV173 cells but which had not received AS treatment rapidly succumbed to disease and died within 8 to 13 weeks. Treatment with the ASO reduced the levels of BCR-ABL mRNA that could be detected within tissues but did not lead to elimination of the leukemic clone. Despite receiving the oligonucleotide, all the animals in the experimental group had died of the same leukemic disease 18 to 23 weeks after injection of BV173 cells.

Formidable problems are associated with the in vivo, systemic administration of conventional BCR-ABL oligonucleotides in CML patients. In order to ensure sufficient uptake into the target CML cells, extremely large quantities of ASO are required. Not only does this high dosage makes systemic therapy prohibitively expensive, but it could also lead to potentially toxic effects as a consequence of nonspecific binding of oligonucleotides. It remains to be seen whether these problems are surmountable and whether the dream of producing an antisense nucleic acid drug for the treatment of CML can ever be realized. For these reasons, clinical trials of BCR-ABL ASO have been confined to ex vivo applications such as purging bone marrow prior to autologous transplantation. Lower doses of ASO can be used for purging because the pharmacokinetic limitations of tissue distribution and uptake that pertain in vivo no longer apply. Bone marrow cells can be washed following their in vitro incubation with ASO in order to remove excess, unbound species. Hence, toxicity to normal cells caused by nonspecific binding of ASO may be minimized. Furthermore, the effects of ASO on the clonogenicity of normal and leukemic cells may be evaluated using in vitro assays prior to purging.

A clinical trial of the efficacy of BCR-ABL ASO in purging bone marrow for autologous transplantation was conducted 4 years ago.16 This study involved eight CML patients, seven in AP and one in a second CP. Selection of patients was based on in vitro responses to the ASO. Depending on the nature of their BCR-ABL breakpoint, each patient's bone marrow was purged ex vivo with either an e13a2 (b2a2) or an e14a2 (b3a2) 26-mer ASO. Bone marrow mononuclear cells were incubated with the oligonucleotides for either 24 hours (three patients) or 72 hours (five patients). Engraftment occurred in all patients with hematologic reconstitution times comparable to those for unpurged autologous transplantation. Two patients achieved a complete cytogenetic response 90 days post-transplantation but both had relapsed by day 180 following re-emergence of the leukemic clone. The patient autografted in second CP died in BC 7 months after transplantation. Of the remaining seven patients, all transplanted in AP, three underwent disease progression to BC, one died from unrelated complications 30 months post-transplantation, and three achieved a second CP. Interestingly, the latter three patients all received an autograft that had been purged with ASO for 72 rather than 24 hours. This suggests that prolonged incubation with BCR-ABL ASO is required to produce a significant reduction in the population of leukemic cells. The comparatively long half-life of the Bcr-Abl oncoprotein, estimated to be greater than 48 hours,18 has been proposed as a possible explanation for the persistence of the malignant clone in the presence of BCR-ABL ASO.16, 72 The stability of the Bcr-Abl protein makes BCR-ABL mRNA a less than ideal candidate for a molecular therapy based on AS.

Alternative targets for AS therapy for CML have been investigated. Several potential mRNA candidates suffer from the same problem as BCR-ABL mRNA in that they encode proteins with long half-lives. The anti-apoptotic protein Bcl-2, for example, has a half-life of approximately 14 hours, and the Ras and Raf proteins, known to be important in cell signaling downstream from Bcr-Abl, both have half-lives in excess of 24 hours.45 A more promising candidate is the proto-oncogene Myb, the estimated half-life of both Myb mRNA and its encoded protein being of the order of 30 to 50 minutes. Moreover, this gene encodes a relatively hematopoietic-specific transcription factor. Results from a pilot clinical study to assess the effectiveness of Myb ASO in purging bone marrow prior to autologous transplantation have recently been published.45 CD34+ cells from 24 patients were purged with Myb ASO for either 24 hours (19 patients) or 72 hours (five patients) prior to autografting. Levels of Myb mRNA were reduced in approximately 50% of these patients. Evaluation of the 14 surviving patients at day 100, in whom engraftment of AS-treated marrow was successful, showed that five patients had achieved a major cytogenetic response as a result of the Myb ASO therapy.

Initial optimism for the AS approach has been replaced by a realization of the limitations of the technology and more realistic expectations of what can be achieved. So-called second-generation AS strategies might be applicable to the treatment of CML (phosphorothioate ASO are regarded as being “first-generation” AS molecules10). Second-generation antisense molecules include 2'-O-methoxyethyl RNA, locked nucleic acids, and peptide nucleic acids, as well as more exotic chemical species such as morpholinos (non-ionic DNA analogs in which the backbone linkages have been altered relative to the phosphodiester backbone of DNA). Other variations on the theme of AS include RNA interference (RNAi).10 At the time of writing, there are no reports of any of these novel AS strategies being evaluated as possible molecular therapies for CML.

Ribozymes and deoxyribozymes 

Ribozymes are RNA molecules that are capable of catalyzing the hydrolysis of specific phosphodiester bonds within other RNA molecules. Association of ribozymes with target RNA sequences occurs by base-pairing. As a result of the ribozyme-catalyzed hydrolysis of phosphodiester bonds, the target RNA strand is cleaved. Hammerhead ribozymes have been widely used to target specific mRNA sequences. These molecules consist of two flanking arms capable of basepairing with the substrate and a catalytic core.33 Different hammerhead ribozymes have been used to target BCR-ABL mRNA. The results from these studies have been somewhat mixed, with some groups reporting that the ribozymes had significant inhibitory effects on the levels of BCR-ABL mRNA and expressed protein and others reporting more modest effects (reviewed in James and Gibson33). In addition to hammerhead ribozymes, the catalytic RNA subunit of RNase P has been used to target BCR-ABL mRNA.15 RNase P is a ribonucleoprotein complex that catalyzes hydrolysis reactions required to remove 5' leader sequences from small RNA molecules. The catalytic RNA subunit of RNase P (M1 RNA) may be directed to specific RNA sequences by the addition of 3' terminal “guide sequences” (GS) that are complementary to the target mRNA. M1 RNA enzymes with guide sequences directed towards the junction specific sequences of the mRNA for the p190 (M1-p190-GS) and p210 (M1-p210-GS) forms of Bcr-Abl were generated. Transfection of BCR-ABL+ Ba/F3 cells with these constructs reduced the levels of the BCR-ABL target mRNA. The inhibitory effect of these enzymes was specific: M1-p190-GS only inhibited mRNA for p190 Bcr-Abl and M1-p210-GS only inhibited mRNA for p210 Bcr-Abl. More recently an artificial, allosterically controllable ribozyme known as a “maxizyme” has been described.75 BV173 cells retrovirally transduced with a vector encoding the maxizyme failed to induce leukemia in a murine model. In contrast, mice receiving control BV173 cells died of diffuse leukemia within 6 to 13 weeks.

As an alternative to ribozymes, deoxyribozymes (DNAzymes) have been used to target BCR-ABL mRNA.88 Like ribozymes, DNAzymes are able to bind to specific RNA sequences and can catalyze the cleavage of the target site. DNAzymes are smaller than ribozymes and are more efficient enzymatically. The expression of p210 Bcr-Abl in K562 cells was inhibited by about 40% when the cells were transfected with a DNAzyme that had been designed to target p210 Bcr-Abl mRNA. The DNAzyme inhibited the growth of these cells by more than 50% in a 6-day liquid culture assay. Moreover, the growth of BCR-ABL+ CFU-Mix colonies was inhibited by 53% to 80% when CD34+ bone marrow cells from CML patients were transfected with DNAzymes. DNAzymes are less expensive to synthesize than ribozymes and are more resistant to serum. In addition, DNAzymes may be generated to cleave the target sequence with a high degree of precision. One study88 used a DNAzyme designed to cleave BCR-ABL mRNA at a site only one nucleotide away from the BCR-ABL junction.

Many of the shortcomings associated with AS, particularly the lack of an efficient means of delivery, also apply to ribozymes and DNAzymes. Permeabilization of the membranes of target cells with streptolysin-O may enhance the uptake of these nucleotide enzymes as this method has been shown to improve the intracellular delivery of BCR-ABL-directed ASOs.72 Solutions to these problems will have to be devised in order for ribozymes or DNAzymes to become clinically useful molecular therapies. As with AS, systemic administration of these agents may not be possible and they may be confined to ex vivo applications such as purging bone marrow prior to an autologous transplant.

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Targeting cell signaling pathways downstream of the Bcr-Abl oncoprotein 

Ras signaling 

Biochemical and genetic studies show that Ras-signaling has a key role in the Bcr-Abl–mediated transformation of leukocytes in CML. The H-, K-, and N-ras genes encode low–molecular weight guanine nucleotide–binding proteins (G-proteins).39 Ras proteins function as molecular switches that cycle between inactive and active states. In its inactive state, the binding site of Ras is occupied by guanosine diphosphate (GDP). Ras is activated or “switched on” by the exchange of guanosine triphosphate (GTP) for GDP. Once activated, the GTP-bound form of Ras interacts with signaling molecules involved in a variety of cellular processes including cell cycling, apoptosis, and differentiation. A key cell-signaling pathway is the mitogen-activated protein kinase (MAPK) cascade that is linked to Ras by the serine-threonine kinase, Raf-1.49 Significantly, the binding site of Ras has intrinsic GTPase activity that catalyzes the hydrolysis of GTP to GDP and which ultimately leads to inactivation of the G-protein. Ras is “switched off” and returns to an inactive state once the hydrolysis of GTP is complete and its binding site contains only GDP. Uncontrolled Ras-signaling is oncogenic and the loss of regulation of Ras pathways underlies many human cancers. The physiological safeguard provided by the self-inactivation of Ras may fail if mutations occur that abrogate its GTPase activity or if it becomes subject to constitutive activation, as is the case in CML.

Bcr-Abl is linked to Ras by a number of protein intermediates. Autophosphorylation of the tyrosine 177 residue of Bcr-Abl generates a binding site for the adapter protein, Grb2.62 The bound Grb2 protein associates with the son of sevenless protein, SoS, to create a complex that functions as a guanine nucleotide exchange factor (GEF) for Ras. The Grb2-SoS complex stimulates activation of Ras by facilitating the exchange of GTP for GDP.65 In addition, Ras may be activated by two other Bcr-Abl substrates that function as adapter molecules, Shc61 and CrkL.59

Uncoupling Bcr-Abl from the Ras pathway: Inhibiting adapter proteins 

Since the activation of mitogenic Ras signaling by Bcr-Abl requires the involvement of adapter proteins it should be possible to uncouple the oncoprotein from this pathway by targeting these molecules. The potential of this approach has been demonstrated in studies in which novel peptides were designed with the aim of blocking the Src homology 3 (SH3) domains of CrkL38 and Grb2.37 SH3 domains mediate many protein-protein interactions by binding to proline-rich sequences that have a P-x-x-P consensus motif. Whereas the SH3 domain of CrkL is essential for its binding to Bcr-Abl, the SH3 domain of Grb2 is required for binding to SoS. By using naturally occurring sequences within the SH3 domains as templates, Kardinal et al were able to synthesize peptides that bound with high affinity to CrkL and Grb2. In order to make the resulting peptides cell permeable, a “shuttle” sequence derived from the third α-helix of the Drosophila transcription factor, Antennapedia (Antp), was attached. The shuttle sequence permitted the SH3-binding peptides, the “cargo” sequences, to enter cells efficiently by a receptor-independent mechanism. The Antp shuttle sequence was coupled to each SH3-binding peptide either directly, to generate a continuous peptide chain, or via an intermediate disulfide bond. Interestingly, the CrkL and Grb2-binding peptides that contained the disulfide bonds were found to be an order of magnitude more potent at inhibiting the proliferation of K562 cells than their single-chain counterparts. The relatively low potency of the single-chain peptides may be due to a continuous process of shuttling between intracellular and extracellular environments.37 By contrast, reduction of the disulfide bonds within cells would cause dissociation of the cargo, SH3-binding peptides from their Antp shuttle peptides. While the shuttle peptides would be free to leave cells, the SH3-binding peptides would be trapped and gradually accumulate within cells. Immunoprecipitation experiments revealed that the CrkL-binding peptide effectively blocked binding of CrkL to Bcr-Abl in K562 cells.38 Phosphorylation of MAPK in K562 cells was shown to be downregulated, indicating that the peptide was inhibiting signaling via the Ras-MAPK pathway. Furthermore, the CrkL-binding peptide significantly inhibited the proliferation of 11 of 16 cell cultures obtained from individual CML patients. In a similar series of experiments, the Grb2-binding peptide was found to disrupt the formation of Grb2-SoS complexes in K562 cells and to inhibit levels of phospho-MAPK.37 This peptide also inhibited proliferation in the same proportion of CML cultures, 11 of 16, as the CrkL-binding peptide. Of the two adapter proteins inhibited by the “designer” peptides, CrkL is the more attractive target for therapeutic intervention in CML. The SH3 domain of CrkL binds directly to Bcr-Abl, the oncoprotein responsible for CML. Hence Crkl allows Bcr-Abl to interfere with mitogenic cell signaling pathways. At present, there is no evidence that either CrkL or Crk have important roles in normal hematopoiesis.38 By contrast, the SH3 domain of Grb2 mediates complex formation with SoS, an interaction that is not unique to malignant cells and which is involved in cell signaling in normal hematopoietic cells. Significantly, the Grb2-binding peptide had no effect on the binding of Grb2 to Bcr-Abl since this interaction occurs via its SH2 domain rather than its SH3 domain.37, 62 Although these studies illustrate the potential of targeting the adapter molecules that link Bcr-Abl to the central Ras-MAPK mitogenic pathway, the authors acknowledge that considerable efforts will have to be made to refine the peptides before they can form the basis of a molecular therapy. They concede that a 20- to 50-fold improvement in the biological activity of the CrkL-binding peptide would be required to produce any significant effects in animal models at peptide concentrations of less than 100 μmol/L.38

Farnesyl transferase inhibitors 

The subcellular localization of Ras is essential for its function. Post-translational modification of Ras is required to generate the functionally active, membrane associated form of the G-protein. A key post-translational modification, catalyzed by the enzyme farnesyl transferase, involves the covalent attachment of a 15-carbon farnesyl group to the C-terminus of Ras.63 This isoprenoid group serves to tether Ras to the plasma membrane. Importantly, the oncogenicity of mutant forms of Ras is dependent on prenylation and attachment to the plasma membrane.40 Rational drug design has the aim of developing compounds capable of inhibiting Ras prenylation with the expectation that oncogenic mutant forms of Ras could be rendered innocuous by preventing their association with the plasma membrane. These efforts have yielded a class of compounds, farnesyl transferase inhibitors (FTIs), which disrupt Ras prenylation.24 FTIs have been demonstrated to have potent antitumor activity during in vitro and in vivo studies, and a number of these drugs are currently the subject of clinical trials. The involvement of Ras in leukemogenesis suggests that FTIs may have potential as antileukemic agents. Two compounds in particular, SCH66336 (Schering-Plough, Kenilworth, NJ) and R115777 (Janssen Research Foundation, Beerse, Belgium), have emerged as promising candidates for the treatment of leukemia.

Clinical trials of the FTI, SCH66336, have begun in order to determine its efficacy as a treatment for solid tumors.1 Recent studies have demonstrated that this compound may have considerable potential as a possible therapy for CML.63 SCH66336 was effective against Bcr-Abl–induced acute leukemia in a murine model of the blast crisis of CML. Syngeneic Balb/c mice injected with Bcr-Abl-transformed Ba/F3 cells developed an aggressive neoplastic disease with splenomegaly, circulating blasts, and infiltration of hematopoietic and nonhematopoietic tissues by leukemic cells. SCH66336 was administered to 15 of 24 mice that had been injected with Bcr-Abl-transformed Ba/F3 cells. All nine animals in the control group that received only the vehicle died of the acute leukemic disease by day 28. In contrast, all but two mice that had received SCH66336 survived and remained disease-free for more than 12 months. Histological analysis of tissues from a surviving mouse revealed no evidence of residual leukemic disease.63 Similar results were obtained in another study using p190-BCR-ABL transgenic mice treated with SCH66336.66 Moreover, SCH66336 appears to selectively inhibit the growth of primary cells from CML patients but has little effect on primary cells from normal individuals.63 Treatment of bone marrow cells from healthy donors with SCH66336 resulted in only a modest inhibition of growth, as assessed by colony formation in methylcellulose, at a concentration that was 10-fold higher than the dose required to completely inhibit the growth of primary cells from CML patients. Thus, SCH66336 suppresses the growth of BCR-ABL+ leukemic cells but spares normal hematopoietic progenitors. SCH66336 also inhibited the proliferation of imatinib-resistant BCR-ABL+ cell lines and reduced the formation of colonies by hematopoietic cells obtained from CML patients who were unresponsive to imatinib treatment.31 Furthermore, SCH66336 potently sensitized imatinib-resistant cells to imatinib-induced apoptosis. Taken together, these findings suggest that a combination of SCH66336 and imatinib mesylate could be a useful treatment for CML, especially in cases of imatinib resistance.

Another FTI, R115777, was evaluated in a phase I clinical trial in patients with acute and poor-risk leukemias.39 This study involved 35 patients and included three individuals with CML in BC, one of whom was Ph-negative. R115777 was administered orally over a period of 21 days using a dose-escalation scheme ranging from 100 mg to 1,200 mg twice daily. The drug failed to produce a hematologic response in the Ph-negative CML patient but partial responses were observed in the other two CML patients, both of whom were Ph-positive and had complex cytogenetic aberrations. Reduced peripheral white blood cell counts were noted, together with normalization of platelet counts and decreased numbers of peripheral and bone marrow blasts. These patients were still alive 14 and 11 months after receiving R115777, while the Ph-negative individual died within 6 months. The presence of the Ph chromosome does not itself appear to be predictive of a favorable response to this drug since three patients with Ph-positive adult acute lymphoblastic leukemia (ALL), who were entered into the same trial, failed to achieve hematologic responses. Reproducible inhibition of farnesyl transferase activity could be demonstrated for a twice-daily dose of 600 mg of R115777. At double this level, central nervous system toxicity was considered to be dose-limiting. The nonhematologic toxicities associated with the 600-mg dose level were acceptable to patients. Based on the available data from this phase I study, phase II trials of R115777 have been proposed for patients with hematologic malignancies with the recommendation that a dose of 600 mg be used with possible dose escalation for younger patients.39

Progress has been made in understanding how FTIs are able to selectively target cancer cells. Inhibition of the farnesylation of Ras proteins, for which these compounds were designed, cannot account for all of their antineoplastic effects and additional mechanisms have been shown to contribute to their efficacy. SCH66336 was found to be more effective at inhibiting soft-agar colony formation of Bcr-Abl–transformed Ba/F3 cells than coexpression of a dominant-negative Ras.63 This result was surprising because experiments with the same cell line in which activated, GTP-bound Ras was precipitated with a GST-Raf1 Ras-binding domain fusion protein had revealed that coexpression of the dominant-negative mutant produced a more profound reduction in levels of the active G-protein than treatment with SCH66336. Taken together, these findings suggest that the antiproliferative action of SCH66336 in BCR-ABL+ cells must involve protein targets other than Ras. Cell cycle analysis indicated that treatment of Bcr-Abl–transformed Ba/F3 cells with SCH66336 caused an accumulation of cells at G2/M, consistent with an effect of the FTI on proteins that regulate this mitotic checkpoint.63 Although the identity of these other target proteins is unknown, possible candidates include the centromere associated mitotic kinesins, CENP-E and CENP-F, both of which are farnesylated. Alternatively, the antiproliferative effect of SCH66336 may be due to an effect upon STAT5, a member of the family of signal transducer and activator of transcription proteins, whose activation and subsequent DNA-binding is known to be stimulated as a result of tyrosine phosphorylation by Bcr-Abl.32 The binding of STAT5 to DNA may be inhibited by FTIs.63 Although, FTIs were developed as specific inhibitors of Ras farnesylation, there is substantial evidence to suggest that they can disrupt the prenylation of other proteins. Furthermore, it seems that their antitumor activity may, in part, be attributed to their effects on these other prenylated proteins. A recurrent theme appears to be alternative prenylation in the absence of farnesylation. Proteins that are normally farnesylated may become substrates for other prenylating enzymes, such as geranyl-geranyl protein transferase, in the presence of inhibitory concentrations of FTIs. A geranylgeranylated form of Rho-B, generated as a consequence of alternative prenylation, has been shown to have antiproliferative properties in transformed cells.43 Hence, it is becoming increasingly clear that the potent antitumor activity of FTIs derives from a more complex and subtle mode of action than originally hypothesized.

MEK inhibitors 

Constitutive activation or mutation of Ras leads to excessive and inappropriate Ras-MAPK signaling. In addition to targeting the G-protein itself, efforts have been made to inhibit this pathway downstream of Ras. As described earlier, Ras is linked to the MAPK pathway by the serine-threonine kinase, Raf-1.49 Raf-1 activates the MAPK kinases, MEK-1/2 (MAPK or ERK Kinase), by catalyzing their phosphorylation. MEK-1/2 are dual-specificity kinases that activate ERK-1/2 (Extracellular signal-Regulated Kinase), the terminal MAP kinases in this cell signaling cascade.54 Specific inhibitors of MEK have been developed and three of these compounds, PD098059, PD184352 (Parke-Davis, Ann Arbor, MI) and U0126 (DuPont Merck, Wilmington, DE), have been shown to inhibit the proliferation of CML cell lines.54, 90 Colony formation of five CML blast crisis cell lines, EM-2, JK-1, K562, MEG-01, and LAMA84, was completely inhibited by 50 μmol/L U0126. The effect of 50 μmol/L PD098059 was less striking and more variable. The more pronounced inhibitory effect of U0126 was attributed to its higher affinity for all forms of MEK. Treatment of K562 cells with 20 μm PD098059 for 72 hours can induce apoptosis.34 A combination of PD184352 and imatinib produced a synergistic cytotoxic effect on K562 and LAMA84 cells, as well as on an imatinib-resistant K562 subline.90 At present, the available data concerning the antiproliferative activity of these agents against BCR-ABL+ cells have only been obtained from in vitro studies and it remains to be seen whether the efficacy of MEK inhibitors can be demonstrated in vivo. Moreover, notwithstanding its constitutive activation by Bcr-Abl, the Ras-MAPK pathway is required for the proliferation of normal cells. Since MEK inhibitors target both normal and constitutively active MAPK signaling alike, it is possible that these compounds may produce toxic effects due to an indiscriminate inhibition of cell proliferation. Further studies will be required to establish whether MEK inhibitors will be of therapeutic value for the treatment of CML.

PI-3 kinase inhibitors 

An important cell signaling molecule that has been shown to be activated by Bcr-Abl is phosphatidylinositol-3 (PI-3) kinase.70 PI-3 kinase catalyzes the phosphorylation of phosphoinositol lipids at the D-3 position of the inositol ring. The resulting 3'-phosphoinositides function as second messengers in intracellular signaling. PI-3 kinase is a heterodimer consisting of a catalytic subunit, p110, and a regulatory subunit, p85. Bcr-Abl coprecipitates with the α-isoform of the p85 subunit (the isoform that accounts for 90% of cellular p85) suggesting that Bcr-Abl undergoes an association with PI-3 kinase via its regulatory subunit.70 The first inhibitors of PI-3 kinase to be identified were obtained from natural sources, such as the fungal metabolite, wortmannin. Originally isolated from Penicillium wortmannii, wortmannin is a specific inhibitor of the p110 catalytic subunit of PI-3 kinase.84 Experiments in which CML and normal primary cells were treated with wortmannin revealed the potential of PI-3 kinase inhibitors as antileukemic agents.70 Treatment with 0.25 μmol/L wortmannin inhibited PI-3 kinase activity by 95% in both the normal and the CML progenitors. The growth of CML progenitors was inhibited by this dose of wortmannin but there was no effect on the growth of progenitor cells obtained from normal donors. Hence, the antiproliferative effect of wortmannin was selective for malignant cells. This property of wortmannin suggests that it may be of use as a purging agent for removing leukemic cells from bone marrow prior to autologous transplantation.70 Unfortunately, wortmannin is not suitable for in vivo clinical applications because it is highly unstable in solution. Synthetic PI-3 kinase inhibitors with improved stability have been developed by using natural products as lead compounds. One such agent is LY294002 (Lilly, Indianapolis, IN), the structure of which was modeled on the flavonoid quercetin.84 Similarly to wortmannin, LY294002 was shown to synergize with imatinib mesylate in the suppression of in vitro growth of BCR-ABL+ cell lines and progenitor cells from CML patients.41 It has yet to be established whether these newer PI-3 kinase inhibitors can be used as therapeutic agents for the treatment of CML. Although the synthetic PI-3 kinase inhibitors are more stable than wortmannin they are far less potent than this naturally occurring prototype. The IC50 for the inhibition of PI-3 kinase by LY294002, for example, is reported as being about 500-fold higher than that of wortmannin.84

Immunomodulation 

The rationale for an immunological approach for the treatment of CML comes from empirical evidence derived from allogeneic bone marrow transplantation (BMT). Donor lymphocyte infusions (DLI) are effective in inducing complete remissions in patients who have undergone relapse following BMT. The antileukemia effect of DLI is likely to be due to the action of cytotoxic T lymphocytes (CTL) that recognize molecular targets associated with CML. Efforts have been made to exploit this T-cell–mediated response by identifying the immunogenic molecular targets that are expressed by CML cells. By administering synthetic forms of these molecular determinants, an immunological antileukemia effect similar to that obtained with DLI might be achieved. The p210 Bcr-Abl protein is an attractive candidate molecule for an immunological approach because it can potentially function as a tumor-specific antigen. The junctional region of p210 Bcr-Abl contains an amino acid sequence that is not expressed in normal, untransformed cells. In addition, as a result of the codon split in the chimeric message fusion proteins derived from BCR-ABL e13a2 or e14a2 transcripts have amino acid insertions at the exact point of fusion in each protein. Hence, protein translated from an e13a2 transcript contains an inserted glutamic acid residue, whereas that translated from an e14a2 transcript contains an inserted lysine.64 A phase I clinical trial has been carried out in which CML patients in CP were vaccinated with short peptides (9 to 25 amino acids) encoded by BCR-ABL mRNA having an e14a2 junction.64 Twelve adults participated in this dose-escalation study and received mixtures of five Bcr-Abl peptides at doses of 10 to 300 μg. All vaccinations were well tolerated and no significant adverse effects were observed. Peptide-specific, T-cell proliferative responses or delayed-type hypersensitivity responses occurred in three of the six patients treated at the two highest doses of the vaccine (300 and 100 μg). These responses lasted up to 5 months after vaccination. However, there was no evidence of a cytotoxic T-cell response. It was concluded from this trial that a Bcr-Abl–derived peptide vaccine can be safely administered to patients with chronic-phase CML and that a peptide-specific immune response may be elicited.

A novel strategy for immunomodulation involving the transcription factor encoded by the Wilms tumor (WT1) gene has been tested in a preclinical setting.23 The WT1 transcription factor is normally expressed in immature CD34+ progenitor cells where differentiation is accompanied by its concomitant downregulation. The sustained overexpression of this protein in CML CD34+ progenitors is thought to contribute to leukemogenesis by blocking normal differentiation. Hence, the WT1 transcription factor is a suitable molecular target for immunomodulation because its continued overexpression is required to maintain the transformed phenotype. In this study,23 a synthetic peptide derived from human WT1 antigen was used to stimulate the in vitro production of CTLs from HLA-A0201–negative donors. The resulting HLA-A0201–restricted CTLs when cocultured with CD34+ cells from HLA-A0201–positive donors inhibited colony formation of CML cells but had no effect on the clonogenicity of normal CD34+ cells. These findings suggest that this antigen may be useful for the generation of CTLs for a human leukocyte antigen (HLA)-mismatch transplant protocol. Alternatively, it may be possible to develop an autologous therapy based on an anti-WT1 vaccine. A similar strategy using allo-restricted CTLs specific for the human CD45 antigen has recently been described.3

Although the low level of toxicity associated with an immunological approach, such as vaccination, is undoubtedly attractive it has yet to be established that molecular strategies based on immunomodulation (as opposed to DLI following BMT) will deliver any clinical benefit to CML patients.

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Conclusion 

Considerable progress has been made in developing therapeutic agents that are effective against molecular targets specifically expressed by CML cells. It is important to emphasize that Bcr-Abl is the ideal target for therapy because it is the only molecule that is unique to CML cells. Increased knowledge of the structure of this oncoprotein should aid the development of novel molecular therapies. In addition, the full potential of existing treatments may not yet have been realized. Synergy between drugs can occur so that a combination therapy may have significantly greater efficacy than that of either compound when administered in isolation. Thus, efforts to identify drug combinations are of primary importance.

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References 

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☆ Address reprint requests to Professor Junia V. Melo, Department of Haematology-ICSTM, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.

PII: S0037-1963(03)70041-9

Seminars in Hematology
Volume 40, Issue 1 , Pages 34-49, January 2003

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