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Chronic Myeloid Leukaemia in The 21st Century
Introduction, what is chronic myeloid leukaemia.
Chronic Myeloid Leukaemia (CML) is a clonal, myeloproliferative disease that develops when a single, pluripotential, haemopoetic stem cell acquires the Philadelphia chromosome. CML was the first haematological malignancy to be associated with a specific genetic lesion. First recognised in 1845, CML exhibits a consistent chromosomal abnormality in leukaemic cells, identified in 1960 by Nowell and Hungerford, termed the Philadelphia (Ph) chromosome 1 . The cytogenetic hallmark of CML was identified in 1973 as the reciprocal translocation t(9;22)(q34:11). Furthermore, in 1984, the ABL (Abelson) proto-oncogene was identified as being involved in this translocation. Breakthroughs in cancer biology have led to extensive characterisation of CML and it is now heralded as a ‘model’ of cancer 2 .
The haemopoietic cell lines are transformed by the chimeric oncogene BCR-ABL . CML is an unusual malignancy in that a single oncogene product is central to its pathology 1 . CML is capable of expansion in both the myeloid or lymphoid lineages, and may involve myeloid, monocytic, erythroid, megakaryocytic, B-lymphoid and occasionally T-lymphocytic lineages, although expansion is predominantly in the granulocyte compartment of the myeloid lineages in the bone marrow 3 .
Epidemiology of CML
The incidence of CML is approximately 1-2 per 100,000 population per year. Consistent with this, there are 10-12 new cases of CML in Northern Ireland each year. The median age of presentation is 45 to 55 years, accounting for 20% of leukaemia affecting adults. As with all leukaemias, males are affected more than females in CML, with a 2:1 ratio. CML is more common with Caucasian ethnicity 3 .
Natural History and Clinical Course
The clinical course of the disease may be divided into three main sections 4 , ( Table I ). Signs and symptoms at presentation may include fatigue, weight loss, abdominal fullness, bleeding, purpura, splenomegaly, leukocytosis, anaemia, and thrombocytosis 3 . In approximately 50% of cases it is an incidental finding.
Clinical course of untreated CML 3 , 5 .
The Ph chromosome is present in 95% of patients with classic CML. The impetus for Ph chromosome formation and the time span required for overt disease progression are unknown. It is proposed that CML, similar to many other neoplasms, may be the result of a multistep pathogenetic process. There is very little evidence to support any additional acquired molecular aberrations prior to t(9;22) translocation 6 . It is generally accepted that the Ph+ clone is susceptible to the acquisition of additional molecular changes that may underlie disease progression. The Ph chromosome is generally the only cytogenetic abnormality present in the chronic phase of disease. Approximately 85% of patients are diagnosed in chronic phase, and this stage of disease responds to therapy 4 . As the disease progresses through the accelerated phase and into the blast crisis, additional cytogenetic abnormalities become evident (see Table I ) 7 .
MOLECULAR PATHOLOGY
Classic CML is characterised by a reciprocal translocation between chromosomes 9 and 22. This results in juxtaposition of 3′ sequences from the Abl-proto-oncogene on chromosome 9, with the 5′ sequences of the truncated Bcr (breakpoint cluster region) on chromosome 22. Fusion mRNA molecules of different lengths, are produced and subsequently transcribed into chimeric protein products, with varying molecular weights, the most common being p210 BCR-ABL ( Fig 1 ) 3 .
Molecular events leading to the expression of CML disease phenotype.
The SH1 domain of ABL encodes a non-receptor tyrosine kinase. Protein kinases are enzymes that transfer phosphate groups from ATP to substrate proteins, thereby governing cellular processes such as growth and differentiation. Tight regulation of tyrosine kinase activity is essential, and if not maintained, deregulated kinase activity can lead to transformation and malignancy 1 .
The portion of ABL responsible for governing regulation of the SH1 domain is lost during the reciprocal translocation. The addition of the BCR sequence constitutively activates the tyrosine kinase activity of the SH1 domain.
Its activity usurps the normal physiological functions of the ABL enzyme, as it interacts with a number of effector proteins 7 . Thus, the SH1 domain of BCR-ABL is the most crucial for oncogenic transformation.
Cellular Signalling
BCR-ABL has several substrates and impacts on key signalling pathways resulting in the CML phenotype 6 . The net result is deregulated cellular proliferation and development of growth factor independence, decreased adherence of the leukaemic cells to the bone marrow stroma, and a reduced apoptotic response to mutagenic stimuli ( Figs 1 and and2 2 ) 1 .
BCR-ABL signalling pathways.
CONVENTIONAL CYTOGENETICS
Cytogenetics is the genetic analysis of cells and assesses the structural integrity of chromosomes. The Ph chromosome, discovered in 1960, was identified as the smaller of the two chromosomes derived from a reciprocal translocation involving chromosomes 9 and 22. This translocation can be found in more than 95% of CML patients at diagnosis. CML was the first disease in which the cytogenetic abnormality was defined on a molecular basis and such work pioneered the combination of molecular cloning and hybridization techniques to produce fluorescence in situ hybridization (FISH) 8 , 9 . FISH uses specific fluorescently tagged DNA probes to map the chromosomal location of genes and identify other genetic anomalies. This technique can be applied in all stages of the cell cycle (interphase cytogenetics). This assay is based on the ability of single stranded DNA to hybridize to complementary DNA. FISH can be performed with substrates such as blood, bone marrow, body fluids, tissue touch preparation and paraffin embedded fixed tissue 9 .
FISH assays are relevant particularly at diagnosis and in relapse, when a large pool of affected cells are present. This is due to the inherent low levels of sensitivity with FISH; at best, sensitivities are within the range of 1 malignant cell in every 100 normal cells. Bone marrow and peripheral blood samples are used to diagnose CML by the presence of Ph chromosome. It is unacceptable to use FISH to detect minimal residual disease following therapy 8 , 9 .
Polymerase chain reaction (PCR) analysis is used at CML diagnosis. PCR is used to detect the m-RNA that encodes for the chimeric BCR-ABL protein in bone marrow and peripheral blood samples. As PCR is more sensitive than FISH it can be used at diagnosis and in monitoring response to treatment 9 , 10 .
MOLECULAR DIAGNOSTICS
Molecular techniques are used in the diagnosis and monitoring response to therapy. Response to treatment may be defined as occurring at haematologic, cytogenetic, or molecular levels 11 , 12 . This is illustrated in Figure 3 .
Defining response to treatment and minimal residual disease, for patients diagnosed with chronic phase CML, treated with imatinib.
Minimal Residual Disease
On current therapeutic regimens a complete cytogenetic response can be achieved for the majority of patients ( Fig 3 ), but a small proportion of these will relapse. Relapse arises from a persistent malignant cellular population present at a low level, below the level of detection by standard techniques. This reservoir of neoplastic cells detected only by sensitive molecular methods is referred to as minimal residual disease (MRD) 12 . Methods for detecting MRD, should ideally have sensitivity within the 10 5 to 10 6 range, be applicable for almost all patients with the disease, provide information on the target, be inexpensive, rapid, readily standardized and disease specific. Additionally, to utilise results effectively good interlaboratory reproducibility and standardisation of reporting is essential. Measuring patient response to imatinib may be achieved by conventional quantitative real-time PCR (RQ-PCR) or nested PCR. Analysis with RQ-PCR detects up to 1 in 10 4 –10 5 cells and nested PCR 1 malignant cell in 10 6 normal cells 9 , 10 . MRD may be designated as values below 10 9 to 10 10 . Clinical observation and experience implies a positive correlation between the improving levels of molecular response and better progression-free disease survival 12 .
RQ-PCR is used to monitor for MRD in patients that have achieved a complete cytogenetic response. This procedure is more amenable to interlaboratory standardisation, and has been introduced as it facilitates rapid and sensitive detection of the fusion gene transcript showing comparable results when simultaneous analysis has been performed on blood and bone marrow specimens, allowing follow up of imatinib treated CML patients 9 , 13 , 14 .
European laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based RQ-PCR, in an effort to analyze the prominent leukaemia-associated fusion genes (including BCR-ABL ) within the Europe Against Cancer (EAC) program. The EAC protocol has the potential to provide the basis for an international reference of MRD using RQ-PCR analysis of fusion gene transcripts 15 . The Department of Haematology at Queens University, Belfast, have been completing analysis of CML patient samples using these set protocols.
DISEASE MANAGEMENT
Allogenic stem cell transplants.
Allogenic stem cell transplant (allo-SCT) has been used since the 1970s in the treatment of CML 1 and is the only curative therapy for CML, however, it bears a significant mortality risk. Age, disease status, disease duration, recipient-donor gender combinations, degree of histocompatability between donor and recipient and the source of the transplant product have all been identified as significantly influencing long-term survival. Evidence in the pre imatinib era suggests that bone marrow transplant is best performed in the early phase of chronic CML 1 , 16 . Using blood or bone marrow derived stem cells from an HLA-identical sibling performed in the chronic phase of the disease offers a 60-80% probability of leukaemia-free survival at 5 years. If performed in the accelerated phase, disease survival decreases by half 17 .
Conventionally, conditioning treatments are necessary prior to allo-SCT. This involves ‘myeloablative’ doses of chemoradiotherapy, aiming to facilitate engraftment of healthy donor stem cells via permanent elimination of malignant haematopoiesis. This is a rather arduous regimen associated with toxicity and mortality. It is therefore preferably administered to those aged less than 65 years without other co-morbid conditions. Success is generally attributed to an immunologically mediated graft-versus-leukaemia effect 7 .
Bone marrow transplants have seen recent developments in research. Reduced intensity conditioning treatments (RICT) or non-myeloablative transplants have been proposed. This endeavours to produce graft-versus-leukaemia effects without exposing the patient to the potential toxicity of conditioning treatments. Here, reconstitution of the immune system and associated anti-leukaemia effect of the donor graft, compete against the growth of the malignancy. Preliminary data suggests that this approach may confer benefit, particularly in chronic phase CML 16 .
Interferon Alpha
Interferon alpha (INFα), is a glycoprotein, of biological origin. It displays antiviral and antiproliferative properties. INFα was the first effective therapy for CML. The drug entered clinical trials in the early 1980s, and remained the treatment of choice for CML patients, until a shift in therapeutic strategy after the arrival of imatinib 18 . In CML INFα prolongs survival in patients, especially of those who are cytogenetic responders. It is able to induce a cytogenetic response in 35 to 55% of patients, with a longer survival achievable in combination with chemotherapy. With this therapy the level of disease decreased with time, but CML was rarely completely eliminated 16 .
Imatinib Mesylate
The BCR-ABL protein is an ideal drug target for CML treatment. Unique to leukaemic cells, the BCR-ABL protein is expressed at high levels and its tyrosine kinase activity of the SH1 domain is essential for its ability to induce CML 7 . The SH1 domain responsible for oncogenic transformation is an extremely attractive target in combating CML.
The most successful synthetic ATP inhibitor designed was imatinib mesylate (STI 571, Gleevec (Glivec), Novartis, Switzerland), approved by the Food and Drug Administration in May 2001 in the United States, later licensed for use in the UK by the European Medicines Evaluation Agency (EMEA) in November 2001 for the treatment of CML 6 , 19 . The introduction of this drug has dramatically changed the management of CML 20 . It is currently considered as the ‘gold standard’ in treating CML, approved for the first line treatment of adult patients with Ph + CML at all disease stages 21 , 22 .
Imatinib functions as a mimic of ATP, in the ATP binding pocket in the BCR-ABL SH1 domain ( Fig 4 ). A further characteristic of imatinib is its striking degree of specificity for the ATP binding pocket, as its effect on other cellular tyrosine kinases is negligible 19 , 23 .
Comparing the mode of action of BCR-ABL and imatinib in CML pathogenesis.
In the treatment of chronic phase CML, imatinib produces a superior and sustainable response compared to INFα. The IRIS study (International Randomised Study of Interferon and STI571), a Phase III clinical trial, compared the use of imatinib and conventional drugs used in the treatment of patients with newly diagnosed CML. Conventional drugs included recombinant INFα, and low dose cytarabine having demonstrated superior rates of cytogenetic response and survival than interferon monotherapy. The results of this trial concluded that the haematologic and cytogenetic responses in terms of tolerability and likelihood of progression to accelerated or blast phase CML, provided superior results with imatinib 24 – 26 .
Imatinib has produced a sustained cytogenetic response in the majority of patients and it is clinically well tolerated. The advantages of imatinib therapy have lead to the revision of allo-SCT protocol, even in patients who may be good allo-SCT candidates. Clinicians are currently recommending that all newly diagnosed patients are treated with imatinib. Only upon failure to respond satisfactorily on imatinib will allo-SCT be considered in suitable candidates.
Imatinib Resistance
Despite its remarkable efficacy in treating CML, secondary resistance is emerging in a minority of patients. This involves the emergence of a resistant leukaemic clone after regular drug administration 27 – 29 .
Primary or intrinsic resistance differs, and is relatively less common in its incidence. It may be defined by a lack of haematologic or cytogenetic response, treatment having had negligible effects since initiation. It is uncommon in chronic phase CML, as is secondary resistance. In accelerated phase of CML primary resistance is relatively common, whilst in accelerated or indeed blast phase it is the rule, as is acquired resistance 29 – 31 .
Acquired resistance to imatinib therapy is caused most commonly by mutations in the BCR-ABL kinase domain, thus preventing imatinib binding sucessfully. A frequent mutation in this domain, conferring a particularly poor prognosis, is in the ATP phosphate binding loop (P-loop). This is a highly conserved domain involved in ATP binding 32 . Further mechanisms of secondary resistance involve over expression of BCR-ABL ; acquired additional mutations, clonal evolution, that is the addition of novel chromosomal aberrations, and pharmacological mechanisms, resulting in a reduction in the quantity of available unbound imatinib, resulting in suboptimal levels of imatinib for effect 27 , 31 .
Monitoring treatment response
The advent of imatinib therapy has added significantly to the cohort of patients in whom a complete cytogenetic response is achieved. It would therefore be logical to utilize molecular assays in monitoring treatment response. Indeed, molecular monitoring has become routine in CML management 33 . The aim of monitoring therapy is to identify sub-optimal responders to imatinib therapy and to consider alternative approaches to management in an effort to prolong progression-free disease survival 16 .
Studies using RQ-PCR have shown that an early reduction of BCR-ABL gene transcript levels can predict a subsequent cytogenetic response in CML 26 , 34 . Once patients achieve MRD status ( Fig 3 ), it is important to continue monitoring closely. The determination of the trend in the quantitative numbers of residual BCR-ABL positive cells is considered to provide important therapeutic information in the follow up of CML patients, providing key prognostic information allowing treatment optimization 15 .
Branford, et al. 35 , concluded from their research that a more than two fold rise in BCR-ABL levels by RQ-PCR identified 97% of patients with BCR-ABL domain kinase mutations. Therefore, monitoring levels of BCR-ABL could potentially serve as an early indicator or predictor of relapse and precipitant for reassessment of therapeutic management, identifying patients for whom imatinib may not be the best form of long term treatment 1 , 2 .
Additionally, it has been documented that a few CML patients are beginning to exhibit clonal karyotypic abnormalities in Phnegative cells whilst completing imatinib therapy. Emergence of such events strongly elude that there is a requirement for intermittent bone marrow cytogenetic analysis 9 , 36 .
This prompts the question of how patients with CML should be monitored. Principle laboratory tests used in monitoring CML drug therapy are peripheral blood counts, cytogenetic analysis, RQ-PCR, and assessment of ABL kinase domain mutations. It is accepted that early treatment of disease relapse should translate into a greater response rate 2 , 9 , 37 , 38 . Use of such an approach will require multicentre standardisation of RQ-PCR and mutation analysis 2 . Provisional recommendations in this area have been made. These include proposals for implementing internationally standardised methodologies for measuring and recording BCR-ABL transcript levels in patients currently undergoing treatment using RQ-PCR; and reporting and detecting BCR-ABL kinase domain mutations 36 .
Molecular mutations can be used to monitor treatment response and disease progression. To date haemopoietic stem cell transplantation is the only proven cure 16 . Of the third of CML patients in whom this therapy is both feasible and appropriate, a majority achieve the status of molecular remission. The remainder of patients may have residual but stable levels of BCR-ABL transcripts. If we are comparing non transplant therapy with allotransplant, the endpoint for each must also be directly comparable, thus molecular remissions must be the goal. This further emphasises the necessity for standardisation of methodology and reporting in monitoring CML treatment response 33 .
Allo-immunity may be a factor in preventing disease relapse in allo-SCT. Imatinib confers no such benefit in its subjects treated to MRD or molecular response, and so cannot guarantee that it can maintain patients in this state indefinitely. However with the excellent response of newly diagnosed patients to imatinib, there has been a reluctance to consider allo-SCT treatment 7 . It is therefore essential that emerging resistance is recognised early, permitting timely consideration of transplant options if appropriate, before overt progression of CML 30 , 35 , 38 , 39 . It would therefore be prudent to set conservative targets for therapeutic achievements to facilitate prompt reassessment of suboptimal therapy. A modest strategy has been proposed, suggesting; complete haematologic response at 3 months, minor cytogenetic response at 6 months, major cytogenetic response at 12 months, and a complete cytogenetic response at 18 months 11 . Failure to meet these criteria would warrant a subsequent re-assessment of disease management.
Strategies to Overcome Imatinib Resistance
Imatinib resistance has been postulated to develop more rapidly and uniformly than other examples of cytotoxic drugs because of its high specificity for its target 20 . Several strategies have been proposed to overcome imatinib resistance.
Firstly, early treatment with imatinib upon diagnosis is considered crucial. Patients who are treated with imatinib within four years of initial diagnosis of CML, have a better prognosis and a significantly lower incidence of mutations than those treated outside the four year time frame. In addition to prompt administration of imatinib an adequate dose is necessary. The lowest approved dose is 400mg daily in chronic phase CML, in advanced stage 600mg daily 14 . A second strategy is imatinib dose escalation 31 , 40 .
Thirdly, combination therapy may be considered. Despite the excellent results achievable with imatinib, only 5-10% of such patients achieve a molecular remission, that is, undetected BCR-ABL transcripts. There is therefore a rationale for combining therapies effective against CML to try and improve the efficacy of therapy. Conceivably, resistance to imatinib may be caused by more than one mechanism in each cell 41 , 42 .
By targeting CML cells with combination therapies cross resistance would presumably be prevented and therapeutic performance improved as disease would be tackled by a number of different means.
The two best non transplant therapies approved for use in CML are INFα and imatinib. It would be reasonable to combine both agents to assess if response rates could be improved. One such study that considered the merits of combining imatinib with pegylated interferon was the PISCES trial (PEGIntron and Imatinib Combination Evaluation Study). In this Phase I/II study preliminary results showed that this dual therapy had improved activity over imatinib alone and was clinically well tolerated. Unfortunately, myelosupression was common. Further data would be necessary to confirm these findings, requiring a large, prospective, randomised study 7 .
The SPIRIT trial (STI571 Prospective International Randomised Trial) is currently underway. This Phase III study will compare the administration of imatinib at escalated doses of 400 mg/day, 800mg/day and imatinib at 400mg/day with interferon and low dose cytarabine, involving patients who have chronic phase CML, having been diagnosed within a three month time span 7 .
Second generation ABL kinase inhibitors
Imatinib has had unprecedented success in the treatment of CML. Despite its capability to achieve clinical remission, disease has progressed in a small minority. Progression made in IRIS is very slow and it is no longer a randomised control study. Few patients remain on the control arm of the study; IRIS follow-up may now be considered a long term imatinib follow-up study. Relapsing patients require alternative therapies, and with time the net number of such patients will increase. Whilst imatinib has proven efficacious, alternatives are now required in some patients. Figure 3 demonstrates a minority of patients will achieve a molecular response with imatinib. The remaining majority of patients still have an existing pool of approximately 10 6 -10 7 leukaemic cells, from which relapse is a possibility, even in controlled disease 43 , 44 .
Imatinib is now the keystone of disease management, and a model upon which future drug development is based, largely due to the contribution that structural biology has made in understanding imatinib resistance. This has aided the design of new kinase-inhibitors 43 , leading to two alternative types of compound.
Nilotinib (AMN107)
Strategy one involved the modification of imatinib structure. Nilotinib (developed by Novartis) is similar to its cousin imatinib as they both bind to an inactive conformation of the ABL kinase domain and function as an ATP inhibitor. There are a number of ways in which they differ. Nilotinib is capable of binding more tightly to BCR-ABL protein to enhance drug efficacy and sensitivity. Most BCR-ABL mutants are 20-fold more sensitive to nilotinib 43 – 45 . The exception to this rule is the mutant T315I 46 , 47 . Furthermore, with its superior topographical fit to the ABL protein, nilotinib proves to be more potent than imatinib.
A Phase I clinical trial with nilotinib demonstrated rates of complete haematologic response in imatinib resistant patients to be 92% in chronic phase, 75% in accelerated phase, 39% in blast phase. Cytogenetic responses were 35%, 55% and 27%, respectively 48 . Phase II studies are ongoing. With success in refractory CML recognised, further study should be focussed to evaluate if nilotinib has therapeutic potential at all stages of disease 49 .
Dasatinib (BMS-354825)
Strategy two involved preparing a compound with a completely different chemical structure to imatinib. This was based upon a drug originally synthesised as a primary Src family inhibitor. Dasatinib (developed by Bristol-Myers Squibb) was observed to inhibit wild type BCR-ABL and most resistant imatinib mutations 43 .
Src is a non-receptor tyrosine kinase that has a plethora of roles in cell signalling including cellular adhesion, motility and growth. Many substrates that Src is capable of phosphorylating with its kinase domain form part of intracellular signalling cascades ( Fig 5 ) 50 , 51 . The deregulated activity of Src has already been recognised in neoplastic cells, such as colon and breast cancer. Due to such properties and activity, Src has been considered as a target in drug development, alongside other protein kinases 50 .
Src signalling pathways.
The Src protein has three functioning molecular domains. SH2 (SRC homology 2) and SH3 are involved in protein-protein interactions. The third, SH1 is a kinase catalytic domain. Src can transfer from inactive to active state through control of its phosphorylation state, or via protein-protein interactions. FAK (focal adhesion kinase) and PDGF (platelet derived growth factor) are capable of rendering Src active by binding to its SH2 domain 50 .
GPCR: G-protein coupled receptors EGF: epidermal growth factor
Dasatinib is therefore a dual Src/ ABL kinase inhibitor. It differs from imatinib in a number of ways. Unlike imatinib, dasatinib is capable of binding to both the inactive and active forms of BCR-ABL . Thus, dasatinib can bind to a more structurally conserved area between ABL and Src kinase than is present in the inactive conformation 52 . It is also more flexible in binding to differing conformations of BCR-ABL and is able to recognise multiple states of BCR-ABL . This confers enhanced binding affinity due largely to dasatinib's less rigid conformational demands on the kinase structure 53 . Although dasatinib is the most potent ABL kinase inhibitor to date, it is not the most specific, its target profile expanding to include other Src family members 54 .
Phase I clinical trials have demonstrated that, similar to its colleague nilotinib, dasatinib too is incapable of overcoming T315I mutations. Dasatinib demonstrated complete cytogenetic responses in chronic phase, accelerated and blast phase CML of 92%, 45%, 35%; with major cytogenetic response of 45%, 27% and 35%, respectively. Clinical activity was also noted in patients who received poor or no cytogenetic benefit from imatinib. This may have implications for patients who have received a suboptimal response from imatinib although not displaying frank resistance 55 , 56 .
NOVEL THERAPIES
Hommoharringtonine.
Hommoharringtonine (HHT) is a novel plant alkaloid derived from a Chinese evergreen tree. An anticancer agent, it has recognised activity in acute myeloid leukaemia (AML), having been incorporated into the treatment regimen for AML and CML 57 , 58 . HHT is thought to conduct its anti-leukaemia effect through the inhibition of protein synthesis. HHT displays pronounced activity upon CML, in the past it has been used as salvage therapy in patients who became refractory to INFα 59 . Studies have investigated the consequences of HHT in combination with INFα or low dose cytarabine. When in dual therapy or in triple combination therapy, complete haematologic and complete cytogenetic responses equivalent to or superior to HHT single therapy have been shown, suggesting improved survival rates compared to HHT alone 58 – 60 . Shortly after such studies imatinib was introduced. In vitro HHT functions synergistically with imatinib, to decrease BCR-ABL protein expression. Research has shown imatinib and HHT to display synergistic cytotoxicity throughout different stages of disease progression. In chronic phase the duo demonstrated properties of dose dependant apoptosis and growth inhibition 7 , 16 . Additional examination of the potential therapeutic effects of HHT as a single therapy or as dual regimen with imatinib is warranted.
Arsenic Trioxide
Arsenic trioxide (As 2 O 3 ), an older therapy for CML, has been re-investigated. With the evolution of safer forms of arsenicals and efficacy of As 2 O 3 in acute promyelocytic leukaemia recently identified, interest of its potential use in CML was rekindled 59 . It is not certain how As 2 O 3 exerts its anti-CML effects. Its ability to promote apoptosis has been suggested 61 . Studies have shown dose dependant growth inhibition and a pro-apoptotic effect when CML cells were treated with clinically tolerable levels of As 2 O 3 . A significant decline in BCR-ABL protein levels was also noted, and did not coincide with reduction in any other cellular proteins, suggesting specificity of this treatment. CML cell lines studies with As 2 O 3 and imatinib have described a synergistic relationship between the two drugs, providing growth reduction and induction of apoptosis 59 , 62 .
Other Novel therapies
Proteasome inhibition has been a further area of interest in CML therapy. The ubiquitin-proteasome pathway is responsible for the degradation of cellular proteins. Proteasome have a dual role of maintenance (disposal of damaged proteins) and regulation (degradation of proteins involved in cell cycle regulation and neoplastic growth) within the cell. Due particularly to its latter property, proteasome inhibitors are being investigated as a new cancer therapy 59 . The inactivation of NF-κB is pertinent to its action. Although the mechanism has not been established by which decreased expression of BCR-ABL protein is mediated when CML cells are treated with proteasome inhibitors; caspase activation and apoptosis were recognised. The proteasome inhibitor PS-341 has shown significant effect upon growth inhibition and apoptosis of several cell lines. These have included both imatinib resistant and sensitive BCR-ABL positive cell lines 7 . Again, clinical studies in imatinib resistant patients are ongoing in this field 59 .
Further examples of a therapeutic target in CML are farnesyl transferase inhibitors. They predominantly mediate post translational modification to activate Ras G-protein. The Ras pathway is a well characterised downstream signalling cascade attributed to the tyrosine kinase activity of BCR-ABL. Thus, inhibiting Ras via farnesyl transferase inhibitors would potentially prevent expression of CML phenotype 7 . Presently, three such compounds present themselves as anti-leukaemic candidates. The most studied is SCH6636. When combined with imatinib SCH6636 is capable of suppressing the growth of CML progenitor cells in vitro, including imatinib resistant cells, with the possibility that it is capable of sensitizing imatinib resistant cells to imatinib-induced apoptosis 59 .
Other novel agents have been illustrated on Fig 6 . They include antiangiogenic agents; peptide vaccines; TNF (tumour necrosis factor) related induction of apoptosis; DNA hypomethylation; antisense oligonucleotides and RNA inhibitors; P13K effectors; destabilisation of BCR-ABL protein 7 , 59 . Many of the agents listed are in preclinical development.
Targets for CML therapy.
Imatinib is the first line agent for treatment of CML. We have examined the aims of imatinib therapy in terms of monitoring and defining disease response to treatment. Fig 7 is a suggested therapeutic algorithm for management of CML upon consideration and appraisal of the current literature. It is not however an ideal, as CML management strategies must be directed by an objective approach due to disease heterogeneity, where various subpopulations of patients may differ in their response to therapeutic regimens.
CML therapeutic algorithm.
Imatinib saw the dawn of a new era for CML management. Its success demonstrated the power and efficacy of genomic medicine and set precedents for future therapy. However, emergence of resistance remains a problem. Novel therapies appear at an impressive pace, promising to strengthen the therapeutic regimen for CML. The management of CML in the 21 st century is exciting and challenging, as it seems that cure of CML is a possibility, but still just out of reach.
Conflict of interest: none declared
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Chronic myeloid (or myelogenous) leukaemia (CML), also known as chronic granulocytic leukaemia, is a cancer of the white blood cells. It is characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood.
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Leukemia - Chronic Myeloid - CML: Latest Research
ON THIS PAGE: You will read about the scientific research being done now to learn more about CML and how to treat it. Use the menu to see other pages.
Doctors are working to learn more about CML, ways to prevent it, how to best treat it, and how to provide the best care to people diagnosed with this disease. The following areas of research may include new options for patients through clinical trials. Most cancer centers are actively involved in clinical trials aimed at increasing the number of people who are cured of CML. Always talk with your doctor about the best diagnostic and treatment options for you.
Improving current treatments. Research focused on increasing the effectiveness of CML treatments are listed below:
Combining imatinib with other drugs
Determining whether people with CML can safely stop taking TKIs after a molecular remission
Creating vaccines against BCR-ABL
Developing newer methods of bone marrow/stem cell transplantation aimed at decreasing the side effects
Evaluating other new TKIs for CML that does not respond to imatinib
Looking for unexpected long-term side effects
Treatment to target remaining CML cells. Several laboratory studies are focused on possible treatments that may help destroy the few remaining CML cells in most patients who have received TKIs so they can stop medical treatment.
Palliative care . Clinical trials are underway to find better ways of reducing symptoms and side effects of current CML treatments to improve patients’ comfort and quality of life.
Looking for More About the Latest Research?
If you would like additional information about the latest areas of research regarding CML, explore these related items that take you outside of this guide:
- To find clinical trials specific to your diagnosis, talk with your doctor or search online clinical trial databases now .
- Visit the website of Conquer Cancer, the ASCO Foundation , to find out how to help support cancer research. Please note that this link takes you to a separate ASCO website.
The next section in this guide is Coping with Treatment . It offers some guidance in how to cope with the physical, emotional, and social changes that CML and its treatment can bring. Use the menu to choose a different section to read in this guide.
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Chronic Myeloid Leukemia in 2020
Hehlmann, Rüdiger
ELN Foundation, Weinheim; Medizinische Fakultät Mannheim, Universität Heidelberg, Mannheim, Germany.
Correspondence: Rüdiger Hehlmann (e-mail: [email protected] ).
Citation: Hehlmann R. Chronic Myeloid Leukemia in 2020. HemaSphere , 2020;4:5(e468). http://dx.doi.org/10.1097/HS9.0000000000000468
The author declares no conflicts of interest.
This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0
New insights have emerged from maturing long-term academic and commercial clinical trials regarding optimum management of chronic myeloid leukemia (CML). Velocity of response has unexpectedly proved less important than hitherto thought, does not predict survival, and is of unclear relevance for treatment-free remission (TFR). Serious and cumulative toxicity has been observed with tyrosine kinase inhibitors that had been expected to replace imatinib. Generic imatinib has become cost-effective first-line treatment in chronic phase despite chronic low-grade side-effects in many patients. Earlier recognition of end-phase by genetic assessment might improve prospects for blast crisis (BC). TFR has become an important new treatment goal of CML. To reflect this new situation ELN has recently revised and updated its recommendations for treating CML. After a brief review of 175 years of CML history this review will focus on recent developments and on current evidence for treating CML in 2020.
Introduction
Twenty-two years after the first patients with chronic myeloid leukemia (CML) were treated with the tyrosine kinase inhibitor (TKI) imatinib, outcome exceeds all expectations: most CML patients achieve a normal life expectancy, some in sustained treatment-free remissions (TFR) may operationally be cured.
Some expectations remain unmet, however. Most patients require life-long maintenance therapy. Also, progression to blast crisis still occurs in 5% to 7% of patients and remains a challenge. CML has not become the model disease for treating other leukemias or cancers. But the principle of elucidation of pathogenesis as a successful approach to treatment of cancer has been impressively shown in CML.
Success came a long way. CML was first described in 1844/5 when Virchow coined the term leukemia (Leukämie). 1–5 Bone marrow was proposed early as possible tissue of origin of CML, 6 but a definite diagnosis became possible only 82 years later when the Philadelphia (Ph)-chromosome was discovered and then the translocation t (9;22) was identified as hallmarks of the disease. 7,8 The subsequent molecular dissection of the chromosomal breakpoints with identification of the BCR-ABL fusion products laid the groundwork for molecular CML-diagnostics and for targeted therapy with BCR-ABL Tyrosine kinase inhibitors (TKI) as the current treatment principle of choice. Molecular BCR-ABL1 monitoring in CML with derivation of the International Scale (IS) has become the posterchild for molecular monitoring of other leukemias and diseases.
Early palliative treatments were arsenic (Fowler's solution, 5 to 10 drops 3× daily for several weeks) 9,10 and splenic irradiation, 11 the mainstays of treatment until 1953 when busulfan was introduced. 12 Hydroxyurea, available since 1963, 13 was easier to handle, had fewer side effects than busulfan and prolonged survival modestly. 14 Bone marrow transplantation was introduced in the late seventies 15 and provided the first cures. 16 At the same time interferon alpha (IFN) was shown to induce complete cytogenetic remissions (CCR) in a substantial minority of patients, 17 usually younger patients. Randomized studies 18–20 documented prolongation of survival with IFN which became the treatment of choice, although its exact mechanism of action is still not fully understood. 21
The benefit by IFN had just been recognized (ASH management recommendations 1999) 22 when BCR-ABL tyrosine kinase inhibition was introduced.
The detection of the ABL-oncogene was a byproduct of the search for a human leukemia virus in the 1960s and early 1970s. The first oncogenes (SRC, MYC) were isolated from chicken leukemia viruses. 23,24 ABL was isolated from the acutely transforming murine Abelson leukemia virus in 1980. 25 Numerous other oncogenes, isolated from retroviruses and from genomes of normal cells, followed.
Many oncogenes, amongst them SRC and ABL, encoded kinase activities that most notably phosphorylate tyrosine, a rarely phosphorylated amino acid. 25,26 This finding gained significance for CML when it was recognized that the human ABL oncogene homologue was located on chromosome 9 at the breakpoint of t (9;22). 27 The discovery of fusion transcripts of ABL with BCR sequences from chromosome 22 28 led to transfection experiments and the observation that BCR-ABL sequences induced leukemia in mice. 29,30 Since BCR-ABL1's oncogenic properties were mainly connected to its tyrosine kinase activity, it was the logical next step to define an inhibitor specific for bcr-abl tyrosine kinase suitable for therapeutic use in humans. 31
The first trial with imatinib, a phase I study with poor risk CML patients, started in 1998. 32 The stunning results convinced even skeptics that further studies were indicated. In 1999, a group of international investigators on CML met in Biarritz, France, to discuss the results and to convince Novartis to produce imatinib (at that time still STI571) in sufficient quantities for larger phase II and III trials. A letter sent by the group to Dr Daniel Vasella, then CEO of Novartis, recommending scale-up of the production of imatinib made the difference (The Magic Bullet 33 ).
The development of tyrosine kinase inhibitor (TKI) therapy and of molecular monitoring has been extensively reviewed by ELN 34–36 and will not be repeated here. But recent developments of current importance as discussed by ELN in its most recent recommendations, 37 will be highlighted in this review.
Epidemiology
Median age at diagnosis of CML is approximately 56 to 57 years in Western countries as estimated from the EUTOS and SIMPLICITY registries. 38,39 Patients older than 70 years make up more than 20%. In developing countries with younger populations median age is less than 50 years. 40 The incidence per year per 100,000 population varies by age and ranges between 1 and 2 depending on the age of the respective populations.
Initial diagnostic workup
The workup at baseline includes the following examinations ( Table 1 ).
The preferred risk score for CML in the TKI era is the EUTOS long-term survival (ELTS) score whose accuracy to predict death from CML is higher than the Sokal score ( Table 2 ). 41
Identification of transcript type is important for molecular monitoring, since atypical transcripts may give false negative test results – and is also of prognostic importance. The shorter e13a2 transcript is reportedly associated with shorter survival and a longer time to DMR compared with the longer e14a2 transcript. Based on evidence from a registry of transcript types in 45,503 newly diagnosed patients from 45 countries transcript type may be helpful to predict response to treatment, outcome of treatment, and TFR. 42
Several additional risk factors have been implicated, but so far none has been validated or found useful except reticulin content in a bone marrow biopsy 43–45 and high-risk additional chromosomal abnormalities (ACA; Table 3 ). High-risk ACAs predict poorer response to TKIs and a higher risk of progression. 46–48 Whereas the 2013 ELN-recommendations considered ACA a warning sign, 36 the 2020 ELN recommendations upgraded ACA to a high-risk sign for treating patients. 37
First-line treatment
With few exceptions, the current first-line treatment is a TKI. A short course of hydroxyurea may be given in symptomatic patients while a diagnosis of CML is pending. Currently, 4 TKIs are approved for first-line treatment by the FDA and EMA: imatinib (Glivec®, Novartis), dasatinib (Sprycel®, Bristol-Myers Squibb), nilotinib (Tasigna®, Novartis), and bosutinib (Bosulif®, Pfizer). Radotinib (Supect®, Dae Wong Pharma) has been approved in South-Korea only 49 and is not further considered here.
Imatinib is effective in all phases of CML, and therapy has resulted in a normal life expectancy of most patients treated in chronic phase (CP) in clinical trials 50,51 and population-based registries. 52–54 No serious toxicity has surfaced after more than 20 years of use. 37,55,56 DMR was achieved in more than 80% of patients which is stable in more than 70% 57 allowing attempts at treatment discontinuation to achieve treatment-free remissions (TFR) 58,59 alleviating chronic low-grade side-effects such as fatigue and muscle cramps.
Generic imatinib 60–62 is now available worldwide and has become cost-effective initial therapy in CP. 37,63 If a generic drug meets the national standards of a country involved in quality, bioavailability and efficacy, generic imatinib is an acceptable alternative to a branded product. The 2020 ELN recommendations 37 state generic and brand product dosing should be the same. Monitoring the response to generics should also be the same as with branded drugs. If there is a change in therapy from a brand to a generic product, enhanced vigilance for the first six months is advised. Patients should continue the same generic brand if possible, to avoid potential side-effects due to changes in drug structure, bioavailability and drug preparation.
Imatinib resistance, second generation TKI, and second-line treatment
Second generation TKIs (2G-TKI, dasatinib, nilotinib, bosutinib) were developed following recognition of imatinib kinase domain (KD) resistance mutations 64 which occur in 4.6% of 1551 CP CML patients over 10 years making it relatively rare. 51 The higher potency of 2G-TKIs resulting in more rapid responses and relief of symptoms compared to imatinib when used in second-line 65,66 led to their use also as first-line therapy. By recognizing imatinib resistant mutations, fewer patients progressed to blast crisis (BC). 67,68 These positive effects, however, were counterbalanced by drug-induced adverse effects. 5- and 10-year data of randomized trials indicate survival with 2G-TKI first-line is similar to imatinib. The high rates of adverse effects to 2G-TKI (particularly pleural effusions in more than 25% of dasatinib-treated patients and serious vascular events with linear increase to 25% by 10 years in nilotinib-treated patients) argue against the use of 2G-TKI in first-line therapy. 67–69
For second-line treatment, patients must be carefully selected considering the comorbidities and the side-effects of 2G-TKI. In the case of failure to imatinib, a change of therapy is mandatory and should be accompanied by investigating BCR-ABL1 KD mutations ( Table 3 ). In the case of intolerance, the decision to change may be subjective depending in part on the patient, the physician and options of supportive care. Response criteria are the same as for first-line treatment.
Since dasatinib has pleuro-pulmonary toxicity, previous pleuro-pulmonary disease is a strong contraindication. A dose reduction from the approved dose of 100 mg/day in CP to 50 mg/day may reduce toxicity. 70
Because of the cardiovascular toxicity of nilotinib a history of coronary heart disease, cerebrovascular accidents and/or peripheral arterial occlusive disease represent strong contraindications to nilotinib. Also, hypertension, diabetes mellitus, hypercholesterolemia and a history of pancreatitis may be contraindications to using nilotinib. A dose-increase from the approved dose of 300 mg twice daily is not recommended.
No relevant comorbidities and no strong contraindications to bosutinib have been identified. At the approved dose of 400 mg/day annoying, but typically transient diarrhea occurs. Owing to the shorter observation time compared to the other TKI, no firm statement can be made regarding long-term safety.
Selection criteria and dosing of TKI in first- and second-line have been extensively discussed in recent ELN recommendations. 37,56
Indications of 2G-TKI and of the 3 rd generation TKI (3G-TKI) ponatinib for second- and third-line treatments based on the most frequent KD resistance mutations are shown in Table 4 .
Ponatinib has been approved for patients resistant against 2 TKI and is the only approved TKI with activity against the T315I mutation. 71,72 Dosing is critical; safety and efficacy must be considered. 37
2G-TKI and ponatinib are effective against most KD resistance mutations, but cannot overcome resistance from other causes such as clonal evolution with emergence of ACA.
Table 5 summarizes the 5- and 10-year survival results of long term randomized and observational studies with imatinib or 2G-TKI. Similar survival rates are reported by population-based registries. 52–54
Current determinants of survival in CML are comorbidities, 75 major route ACA, 76 risk score, smoking 77 and treatment center, but not initial treatment selection. 51
Resistance to imatinib occurs in 10% to 15%, and to 2GTKI in <10% of patients in first-line treatment. In some patients, failure to respond may be related to poor compliance. Mutations account for resistance in about one third of resistant CP patients, and in about two thirds of resistant accelerated phase (AP) and BC patients. Alternative mechanisms of resistance include clonal evolution (emergence of high-risk ACA) and the activation of BCR-ABL1 independent pathways. A cytogenetic risk classification has been proposed to allow risk-based treatment adaptation. 47,48,78
In about two-thirds of compliant TKI resistant CP patients and in about one third of resistant AP and BC patients, a mutation is neither detected, nor is it the only cause of resistance. Analyzing the genome and expression profiles of resistant CML cells may identify somatic mutations 79–81 as early signs of progression, and lead to a genetically-based risk classification with the potential for non-BCR-ABL1 targeted therapy for resistant patients. 82
BCR-ABL1 mutations can be detected with sensitivities of about 20% by Sanger sequencing and in about 3% by NGS. NGS is the recommended technology to detect clinically relevant BCR-ABL1 resistance mutations in patients not responding adequately to TKI. 83,84
Allogeneic hematopoietic cell transplantation
Despite the superiority of drug treatment, allogeneic hematopoietic cell transplantation has retained a place in CP CML for patients with disease resistant to multiple TKIs or personal preferences. 85,86 In resource poor countries the onetime expense of a transplant may be more economical than life-long treatment with a TKI.
Transplants should be strongly considered in persons resistant to 2G-TKIs. Someone resistant to the initial 2G-TKI therapy has a low chance of achieving a durable response to an alternative TKI and should be assessed early for a transplant. Early transplantation as a rule improves outcome. 87 If the patient has also failed ponatinib, risk of progression is high. Someone progressing to AP under treatment is a candidate for an immediate transplant. For a patient presenting in BC a return to a second CP (CP2) should be attempted. Return to CP2 improves transplantation outcome. 85,88 Also, in patients with high-risk ACA and low blast counts early transplantation may improve survival. 48 Transplant mortality in CP is low, 85 but GvHD remains a problem. Transplantation in BC is a high-risk procedure and not advised. 37
Pregnancy and fertility
All TKIs are teratogenic and should be withheld during pregnancy. 89,90 Low-level secretion of TKIs in breast milk contraindicates their use during breast-feeding. 91 Sperm quality and morphology are unchanged after treatment with TKI. 92 For more in-depth information see the ELN 2020 recommendations. 37
Response monitoring and milestones
Timely recognition of suboptimal response or resistance to TKI requires regular monitoring. Hematologic and cytogenetic monitoring have been replaced in most instances by the more sensitive molecular monitoring with quantitative PCR-techniques for BCR-ABL1 transcripts. 93,94 Transcript levels are reported in a standardized fashion according to the International Scale (IS) 95–97 which underlies the response milestones guiding treatment ( Table 6 ). Complete cytogenetic remission (CCR) has been shown to be equivalent to 0.1% BCR-ABL1 on the IS. 98
DMR at the MR 4 and MR 4.5 levels is prognostic. Progression of CML is extremely rare at these levels. 57 Patients may be operationally cured and require no further treatment. To test this possibility TKI discontinuation studies have been undertaken to determine optimum duration of treatment and of deep DMR, rate of TFR after discontinuation, and markers predictive of successful discontinuation, 58,59,99 see paragraph on TFR below.
Quality of life
This is an important evolving field building on survival, but beyond the scope of this review.
In brief, since most patients receive TKIs for many years or even indefinitely, observation of quality of life in these patients and amelioration of chronic low-grade side-effects are important. Current research preferentially addresses tolerability of different TKIs. 100,101 Replacement of one TKI by another may improve tolerability, but frequently at the expense of other, potentially more serious toxicity. 102 Dose-reductions of TKIs are an option. 70,103 Patient-reported outcome (PRO) questionnaires are encouraged to quantify chronic quality of life issues faced by CML patients. 104
Treatment-free remission (TFR)
TFR is a new significant goal of CML management. A significant proportion of patients will achieve a DMR defined as BCR-ABL1 levels of MR 4 and MR 4.5 on the IS with current TKIs. Benchmark times for molecular response rates with imatinib are shown in Figure 1 .
Median times to MR 4 are 2.9 years, to MR 4.5 4.7 years. 5-year rates are 67% for MR 4 and 53% for MR 4.5 .
Table 7 lists benchmarks of DMR that can be expected by 5 and 10 years after treatment with imatinib, nilotinib and dasatinib. 55,57,67–69 Five-year follow-up of first-line bosutinib is not yet available. 105
An attempt at treatment discontinuation can be considered, if sustained DMR of sufficiently long duration has been achieved. An initial observation of 12 patients 94 showed that about half of them in DMR (no detectable BCR-ABL transcripts by PCR) stayed in remission after cessation of imatinib. In a follow-up study of 100 patients (STop IMatinib or STIM study) 38% stayed in TFR after an observation period of 7 years. 58,106 Most relapses occurred early within the first 6–12 months. Loss of MMR indicates failure of TFR. 107 Virtually all relapsing patients regained their prior best response level after re-treatment.
A polymyalgia-like TKI withdrawal syndrome of musculo-skeletal pain may occur in a third of patients which is usually self-limited, but may require treatment with acetaminophen, non-steroidal anti-inflammatory drugs or rarely a short course of oral steroids. 108,109 A patient study reported that the TKI withdrawal syndrome if unmanaged may cause more morbidity than hitherto thought. 110
Table 8 shows a selection of discontinuation studies after treatment with imatinib or the 2G-TKI dasatinib and nilotinib.
The largest of these studies = the Euro-SKI study of 755 mostly imatinib treated CML patients who had been in DMR at the MR 4 level for at least 1 year = showed a TFR rate of 49% after 3 years. 59 Duration of MR 4 was determined as the most important predictor of TFR. Treatment discontinuation is feasible only in CP patients. Patients in advanced phases, particularly in BC, remain a challenge.
After failure of TFR, a second stop after additional treatment can result in a TFR-rate as high as 33% at 4 years, 126 updated at EHA 2019.
Interestingly, dose reduction prior to complete discontinuation to reduce side-effects may improve successful TFR (Destiny study 103 ). Another interesting observation is the finding in the ISAV study, by comparing TFR rates in younger and older patients, of significantly lower TFR rates in patients under 45 years of age 114,115 which is in line with the observation of more aggressive disease in adolescents and young adults. 127,128
Several studies addressed the issue of changing from imatinib to a 2G-TKI to shorten the interval to DMR and TFR. A more rapid response was generally observed, but toxicity of 2G-TKI limits this approach.
In the TIDEL-II study, the dose of patients receiving imatinib 600 mg/day failing to reach time benchmarks was increased to imatinib 800 mg/day or medication was changed to nilotinib 2 × 400 mg/day. 129 This approach was considered feasible.
In the ENESTcmr study, imatinib-treated patients in CCR were randomized to remain on imatinib or to change to nilotinib. The rate of DMR by 4 years was, as expected, higher in the nilotinib group, but only 57% of nilotinib-treated patients completed 4 years of nilotinib therapy. The study provided no information whether patients in DMR subsequently achieved TFR successfully. 130,131 It should be remembered that most patients in durable DMR still harbor residual BCR-ABL1 sequences in their genomic DNA. 132
The ELN 2020 recommendations define the following requirements for TKI discontinuation for successfully achieving TFR ( Table 9 ). 37
It is recommended to consider TFR in appropriate patients after careful discussion employing the concept of shared decision making. 133 First-line TKI, or a change to a 2G-TKI, for faster DMR are not recommended because of the more serious side-effects of 2G-TKI, their increased costs and absent information about the number of patients who might actually benefit. A change to 2G-TKI to improve the depth of response can be considered in selected patients in whom DMR has not been reached such as the motivated patient with a high priority for TFR, younger patients with low or intermediate risk disease or women who wish to become pregnant.
End phase CML and blast crisis
Outcome of patients in blast crisis (BC) treated with single agents, combination chemotherapy, and TKI alone and in combination with intensive chemotherapy 134,135 remains unsatisfactory. Once BC has occurred, survival is generally less than one year with death due to infection or bleeding. New approaches are urgently needed.
Genetically-based risk assessment by ACA and somatic mutations has recently been proposed for a better recognition of patients at risk for progression to end-phase CML and BC. 47,48,77,79 Currently, diagnosis of BC rests on the percentage of blasts (20% or 30%) in blood or marrow, 34,136,137 but not all patients dying of CML reach the BC-defining blast levels. 138 Earlier recognition of end-phase might enable earlier intervention to improve prospects for BC.
End-phase CML comprises early progression with emerging high-risk ACA and late progression with failing hematopoiesis and blast cell proliferation. 48 Up to 90% of BC patients show chromosomal aberrations in addition to the Ph-chromosome (termed major or minor route by Mitelman depending on their frequency in BC 139,140 ) and as many as 80% BCR-ABL1 KD mutations. 141 Also, somatic mutations have been detected in BC and are associated with poor risk disease when detected at diagnosis. 78,79 Blast increase in blood or marrow represents the end stage of progression.
High-risk ACA defined as the major route ACA +8, +Ph, i(17q), +19, +21, +17 (the ACA most frequently observed in BC), 140 the minor route ACA -7/7q-, 3q26.2 and 11q23 rearrangements (less frequently observed, but negative impact on prognosis), and complex aberrant karyotypes 47,77 herald death by CML in the presence of low blast counts. 48
Somatic mutations observed in BC and in poor risk patients include mutations of genes associated with poor outcome in other malignancies. 142 They also might enable early identification of patients at risk of progression. Frequently mutated genes include RUNX1, ASXL1 and IKZF1 78,79 ( Table 3 ).
Patients with suboptimal responses by ELN criteria 34 and with less than DMR after 2 years (less than MR 4 ) should have a genetic evaluation. In patients with high-risk ACA more intensive treatment, for example, by hematopoietic cell transplantation (allo-HCT), may be indicated. Current treatment approaches to end-phase CML are summarized in Figure 2 . Treatment depends on disease stage. Elimination of BCR-ABL1 by effective TKI treatment is expected to prevent progression.
Cytogenetic monitoring is indicated when response to therapy is unsatisfactory. When high-risk ACA emerge, intensification of treatment should be considered. There is also evidence that earlier transplantation is more successful in patients with high-risk ACA. An appropriate time for a change of treatment may be the emergence of high-risk ACA rather than waiting for an increase of blasts. AP should be treated as high-risk CML. Transplantation is recommended if response to drug treatment is not optimal. Treatment of BC consists of intensive combination chemotherapy based on AML regimens for myeloid, and ALL regimens for lymphoid, BC with or without a TKI, for instance dasatinib at the approved dose 140 mg/day for BC or ponatinib, in preparation for a prompt transplantation if possible. Flow cytometry distinguishes between lymphoid and myeloid BC allowing appropriate selection of treatment. Lymphoid BC has more treatment options and a better outcome than myeloid BC. In patients who cannot tolerate intensive chemotherapy regimens, a more palliative approach with less intensive therapy according to immunophenotype should be considered such as vincristine and prednisone in lymphoid BC.
There is evidence that emergence of high-risk ACA is an indication for a timelier change of treatment with better outcome. 48 Comparing transplantation outcome in early and late end-phase, a clinically relevant, though not statistically significant difference of 30% in 2-year survival suggests that outcome of transplanted patients with high-risk ACA depends on disease stage similar to patients without ACA. 87
Summary and prospects
Based on the results of maturing long-term clinical trials management of CP-CML is again changing profoundly. All randomized studies that compare imatinib 400 mg once daily with 2G-TKIs, imatinib 400 mg with dose increase, or imatinib combined with IFN alpha or low-dose cytarabine have failed to improve OS. Although deeper molecular responses occurred more rapidly with 2G-TKIs, with imatinib dose increase or with imatinib in combination with peg-IFN alpha, these events did not translate into better OS than with imatinib at a standard dose of 400 mg daily. Nevertheless, these studies provided greater insights in the safety and efficacy of the drugs, as well as benchmarks for molecular response as a basis for individualized treatment and eventually treatment discontinuation. The studies showed that survival has moved close to that of the general population. Now more patients die of CML-unrelated causes than from CML. The goal of treatment in these patients is better supportive care and management of side-effects of treatments aiming at best possible quality of life.
A new important development has been recognizing that treatment can be successfully stopped in a substantial minority of patients depending upon whether duration of both treatment and DMR are long enough to make TFR a feasible option. TFR is an important new goal of CML management which should be discussed with appropriate patients.
Regarding changing therapy from imatinib to a 2G-TKI in a patient with stable CCR or MMR, but in whom the level of DMR (< MR 4 ) was insufficient to warrant consideration of discontinuation, no recommendation can be made in view of the high toxicity and costs of 2G-TKI. Also, there is no information about the rate of successful TFR from large randomized trials with different initial treatment regimens addressing this specific issue.
Regarding changing from 2G-TKI to imatinib, this can be considered when no DMR is achieved within 3 years to avoid the risk of serious cumulative toxicity of 2G-TKI.
Current challenges on the path to cure of CML are increasing the proportion of patients in whom treatment can be successfully discontinued, and the further decrease of patients who progress to BC. This can be achieved by optimizing treatment with available drugs, by developing new drugs with better efficacy and by better recognition of patients at risk for progression and of optimum conditions for treatment discontinuation (duration of DMR, duration of treatment, other factors such as risk score, age, gender), and by more intensive treatment of patients not responding well enough, respectively. Of urgency is still the management of refractory disease of those 6% who progress to BC in spite of seemingly adequate treatment. Earlier recognition of such patients seems possible.
Finally, factors causing CML remain of interest. The only established risk factor is still radiation as observed after the atomic bombs on Hiroshima and Nagasaki. Better epidemiologic studies and registries may provide an answer. 143–145
Acknowledgements
The author thanks Drs. Richard T Silver and Robert P. Gale for critically reading the manuscript, and Johannes Hehlmann for support.
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The physiologic function of the translocation partners
Molecular anatomy of the bcr-abl translocation, mechanisms of bcr-abl –mediated malignant transformation, experimental models of cml, transformation to blast crisis, molecular targets for therapy, the molecular biology of chronic myeloid leukemia.
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Michael W. N. Deininger, John M. Goldman, Junia V. Melo; The molecular biology of chronic myeloid leukemia. Blood 2000; 96 (10): 3343–3356. doi: https://doi.org/10.1182/blood.V96.10.3343
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Chronic myeloid leukemia (CML) is probably the most extensively studied human malignancy. The discovery of the Philadelphia (Ph) chromosome in 1960 1 as the first consistent chromosomal abnormality associated with a specific type of leukemia was a breakthrough in cancer biology. It took 13 years before it was appreciated that the Ph chromosome is the result of a t(9;22) reciprocal chromosomal translocation 2 and another 10 years before the translocation was shown to involve the ABL proto-oncogene normally on chromosome 9 3 and a previously unknown gene on chromosome 22, later termed BCR for breakpoint cluster region. 4 The deregulated Abl tyrosine kinase activity was then defined as the pathogenetic principle, 5 and the first animal models were developed. 6 The end of the millennium sees all this knowledge transferred from the bench to the bedside with the arrival of Abl-specific tyrosine kinase inhibitors that selectively inhibit the growth of BCR-ABL –positive cells in vitro 7 , 8 and in vivo. 9
In this review we will try to summarize what is currently known about the molecular biology of CML. Because several aspects of CML pathogenesis may be attributable to the altered function of the 2 genes involved in the Ph translocation, we will also address the physiological roles of BCR and ABL . We concede that a review of this nature can never be totally comprehensive without losing clarity, and we therefore apologize to any authors whose work we have not cited.
The ABL gene is the human homologue of the v- abl oncogene carried by the Abelson murine leukemia virus (A-MuLV), 10 and it encodes a nonreceptor tyrosine kinase. 11 Human Abl is a ubiquitously expressed 145-kd protein with 2 isoforms arising from alternative splicing of the first exon. 11 Several structural domains can be defined within the protein (Figure 1 ). Three SRC homology domains (SH1-SH3) are located toward the NH 2 terminus. The SH1 domain carries the tyrosine kinase function, whereas the SH2 and SH3 domains allow for interaction with other proteins. 12 Proline-rich sequences in the center of the molecule can, in turn, interact with SH3 domains of other proteins, such as Crk. 13 Toward the 3′ end, nuclear localization signals 14 and the DNA-binding 15 and actin-binding motifs 16 are found.
Structure of the Abl protein.
Type Ia isoform is slightly shorter than type Ib, which contains a myristoylation (myr) site for attachment to the plasma membrane. Note the 3 SRC-homology (SH) domains situated toward the NH 2 terminus. Y393 is the major site of autophosphorylation within the kinase domain, and phenylalanine 401 (F401) is highly conserved in PTKs containing SH3 domains. The middle of each protein is dominated by proline-rich regions (PxxP) capable of binding to SH3 domains, and it harbors 1 of 3 nuclear localization signals (NLS). The carboxy terminus contains DNA as well as G- and F-actin–binding domains. Phosphorylation sites by Atm, cdc2, and PKC are shown. The arrowhead indicates the position of the breakpoint in the Bcr-Abl fusion protein.
Several fairly diverse functions have been attributed to Abl, and the emerging picture is complex. Thus, the normal Abl protein is involved in the regulation of the cell cycle, 17 , 18 in the cellular response to genotoxic stress, 19 and in the transmission of information about the cellular environment through integrin signaling. 20 (For a comprehensive review of Abl function, see Van Etten 21 ). Overall, it appears that the Abl protein serves a complex role as a cellular module that integrates signals from various extracellular and intracellular sources and that influences decisions in regard to cell cycle and apoptosis. It must be stressed, however, that many of the data are based solely on in vitro studies in fibroblasts, not hematopoietic cells, and are still controversial. Unfortunately, the generation of ABL knockout mice failed to resolve most of the outstanding issues. 22 , 23
The 160-kd Bcr protein, like Abl, is ubiquitously expressed. 11 Several structural motifs can be delineated (Figure 2 ). The first N-terminal exon encodes a serine–threonine kinase. The only substrates of this kinase identified so far are Bap-1, a member of the 14-3-3 family of proteins, 24 and possibly Bcr itself. 11 A coiled–coil domain at the N-terminus of Bcr allows dimer formation in vivo. 25 The center of the molecule contains a region with dbl -like and pleckstrin-homology (PH) domains that stimulate the exchange of guanidine triphosphate (GTP) for guanidine diphosphate (GDP) on Rho guanidine exchange factors, 26 which in turn may activate transcription factors such as NF-κB. 27 The C-terminus has GTPase activity for Rac, 28 a small GTPase of the Ras superfamily that regulates actin polymerization and the activity of an NADPH oxidase in phagocytic cells. 29 In addition, Bcr can be phosphorylated on several tyrosine residues, 30 especially tyrosine 177, which binds Grb-2, an important adapter molecule involved in the activation of the Ras pathway. 31 Interestingly, Abl has been shown to phosphorylate Bcr in COS1 cells, resulting in a reduction of Bcr kinase activity. 31 , 32 Although these data argue for a role of Bcr in signal transduction, their true biologic relevance remains to be determined. The fact that BCR knockout mice are viable and the fact that an increased oxidative burst in neutrophils is thus far the only recognized defect 33 probably reflect the redundancy of signaling pathways. If there is a role for Bcr in the pathogenesis of Ph-positive leukemias, it is not clearly discernible because the incidence and biology of P190 BCR-ABL -induced leukemia are the same in BCR−/− mice as they are in wild-type mice. 34
Structure of the Bcr protein.
Note the dimerization domain (DD) and the 2 cyclic adenosine monophosphate kinase homologous domains at the N terminus. Y177 is the autophosphorylation site crucial for binding to Grb-2. The center of the molecule contains a region homologous to Rho guanidine nucleotide exchange factors (Rho-GEF) as well as dbl-like and pleckstrin homology (PH) domains. Toward the C-terminus a putative site for calcium-dependent lipid binding (CaLB) and a domain with activating function for Rac-GTPase (Rac-GAP) are found. Arrowheads indicate the position of the breakpoints in the BCR-ABL fusion proteins.
The breakpoints within the ABL gene at 9q34 can occur anywhere over a large (greater than 300 kb) area at its 5′ end, either upstream of the first alternative exon Ib, downstream of the second alternative exon Ia, or, more frequently, between the two 35 (Figure 3 ). Regardless of the exact location of the breakpoint, splicing of the primary hybrid transcript yields an mRNA molecule in which BCR sequences are fused to ABL exon a2. In contrast to ABL , breakpoints within BCR localize to 1 of 3 so-called breakpoint cluster regions ( bcr ). In most patients with CML and in approximately one third of patients with Ph-positive acute lymphoblastic leukemia (ALL), the break occurs within a 5.8-kb area spanning BCR exons 12-16 (originally referred to as exons b1-b5), defined as the major breakpoint cluster region (M- bcr ). Because of alternative splicing, fusion transcripts with either b2a2 or b3a2 junctions can be formed. A 210-kd chimeric protein (P210 BCR-ABL ) is derived from this mRNA. In the remaining patients with ALL and rarely in patients with CML, characterized clinically by prominent monocytosis, 36 , 37 the breakpoints are further upstream in the 54.4-kb region between the alternative BCR exons e2′ and e2, termed the minor breakpoint cluster region (m- bcr ). The resultant e1a2 mRNA is translated into a 190-kd protein (P190 BCR-ABL ). Recently, a third breakpoint cluster region (μ- bcr ) was identified downstream of exon 19, giving rise to a 230-kd fusion protein (P230 BCR-ABL ) associated with the rare Ph-positive chronic neutrophilic leukemia, 38 though not in all cases. 39 If sensitive techniques such as nested reverse transcription–polymerase chain reaction are used, transcripts with the e1a2 fusion are detectable in many patients with classical P210 BCR-ABL CML. 40 The low level of expression of these P190-type transcripts compared to P210 indicates that they are most likely the result of alternative splicing of the primary mRNA. Occasional cases with other junctions, such as b2a3, b3a3, e1a3, e6a2, 41 or e2a2, 42 have been reported in patients with ALL and CML. These “experiments of nature” provide important information as to the function of the various parts of BCR and ABL in the oncogenic fusion protein. Interestingly, ABL exon 1, even if retained in the genomic fusion, is never part of the chimeric mRNA. Thus, it must be spliced out during processing of the primary mRNA; the mechanism underlying this apparent peculiarity is unknown. Based on the observation that the Abl part in the chimeric protein is almost invariably constant while the Bcr portion varies greatly, one may deduce that Abl is likely to carry the transforming principle whereas the different sizes of the Bcr sequence may dictate the phenotype of the disease. In support of this notion, rare cases of ALL express a TEL-ABL fusion gene, 43 , 44 indicating that the BCR moiety can in principle be replaced by other sequences and still cause leukemia. Interestingly, a fusion between TEL(ETV6) and the ABL -related gene ARG has recently been described in a patient with AML. 45 Although all 3 major Bcr-Abl fusion proteins induce a CML-like disease in mice, they differ in their ability to induce lymphoid leukemia, 46 and, in contrast to P190 and P210, transformation to growth factor independence by P230 BCR-ABL is incomplete, 47 which is consistent with the relatively benign clinical course of P230-positive chronic neutrophilic leukemia. 38
Locations of the breakpoints in the ABL and BCR genes and structure of the chimeric mRNAs derived from the various breaks.
One of the most intriguing questions relates to the events responsible for the chromosomal translocation in the first place. From epidemiologic studies it is well known that exposure to ionizing radiation (IR) is a risk factor for CML. 48 , 49 Moreover, BCR-ABL fusion transcripts can be induced in hematopoietic cells by exposure to IR in vitro 50 ; such IR-induced translocations may not be random events but may depend on the cellular background and on the particular genes involved. Two recent reports showed that the physical distance between the BCR and the ABL genes in human lymphocytes 51 and CD34 + cells 52 is shorter than might be expected by chance; such physical proximity could favor translocation events involving the 2 genes. However, the presence of the BCR-ABL translocation in a hematopoietic cell is not in itself sufficient to cause leukemia because BCR-ABL fusion transcripts of M- bcr and m- bcr type are detectable at low frequency in the blood of many healthy individuals. 53 , 54 It is unclear why Ph-positive leukemia develops in a tiny minority of these persons. It may be that the translocation occurs in cells committed to terminal differentiation that are thus eliminated or that an immune response suppresses or eliminates Bcr-Abl–expressing cells. Indirect evidence that such a mechanism may be relevant comes from the observation that certain HLA types protect against CML. 55 Another possibility is that BCR-ABL is not the only genetic lesion required to induce chronic-phase CML. Indeed, a skewed pattern of G-6PD isoenzymes has been detected in Ph-negative Epstein-Barr virus-transformed B-cell lines derived from patients with CML, suggesting that a Ph-negative pathologic state may precede the emergence of the Ph chromosome. 56
Essential features of the Bcr-Abl protein
Mutational analysis identified several features in the chimeric protein that are essential for cellular transformation (Figure 4 ). In Abl they include the SH1, SH2, and actin-binding domains (Figure 1 ), and in Bcr they include a coiled–coil motif contained in amino acids 1-63, 25 the tyrosine at position 177, 57 and phosphoserine–threonine-rich sequences between amino acids 192-242 and 298-413 58 (Figure 2 ). It is, however, important to note that essential features depend on the experimental system. For example, SH2 deletion mutants of Bcr-Abl are defective for fibroblast transformation, 59 but they retain the capacity to transform cell lines to factor independence and are leukemogenic in animals. 60
Signaling pathways activated in
BCR-ABL –positive cells. Note that this is a simplified diagram and that many more associations between Bcr-Abl and signaling proteins have been reported.
Deregulation of the Abl tyrosine kinase
Abl tyrosine kinase activity is tightly regulated under physiologic conditions. The SH3 domain appears to play a critical role in this inhibitory process because its deletion 14 or positional alteration 61 activates the kinase; it is replaced by viral gag sequences in v- abl . 62 Both cis- and trans-acting mechanisms have been proposed to mediate the repression of the kinase. Several proteins have been identified that bind to the SH3 domain. 63-65 Abi-1 and Abi-2 (Abl interactor proteins 1 and 2) activate the inhibitory function of the SH3 domain; even more interesting, activated Abl proteins promote the proteasome-mediated degradation of Abi-1 66 and Abi-2. Another candidate inhibitor of Abl is Pag/Msp23. On exposure of cells to oxidative stress such as ionizing radiation, this small protein is oxidized and dissociates from Abl, whose kinase is in turn activated. 67 These results are in line with previous observations that highly purified Abl protein is kinase-active, 61 suggesting that its constitutive inhibition derives from a trans-acting mechanism. Alternatively, the SH3 domain may bind internally to the proline-rich region in the center of the Abl protein, causing a conformational change that inhibits interaction with substrates. 68 Furthermore, a mutation of Phe 401 to Val (within the kinase domain) leads to the transformation of rodent fibroblasts. Because this residue is highly conserved in tyrosine kinases with N-terminal SH3 domains, it may bind internally to the SH3 domain. 69 It is conceivable that the fusion of Bcr sequences 5′ of the Abl SH3 domain abrogates the physiologic suppression of the kinase. This might be the consequence of homodimer formation; indeed, the N-terminal dimerization domain is an essential feature of the Bcr-Abl protein but can be functionally replaced by other sequences that allow for dimer formation, such as the N-terminus of the TEL ( ETV-6 ) transcription factor in the TEL-ABL fusion associated with the t(9;12). 43 , 70 It is possible that deregulated tyrosine kinase activity is a unifying feature of chronic myeloproliferative disorders. Several other reciprocal translocations have been cloned from patients with chronic BCR-ABL –negative myeloproliferative disorders. Remarkably, most of these turn out to involve tyrosine kinases such as fibroblast growth factor receptor 1 71 and platelet-derived growth factor β receptor (PDGFβR). 72
A host of substrates can be tyrosine phosphorylated by Bcr-Abl (Table 1 ). Most important, because of autophosphorylation, there is a marked increase of phosphotyrosine on Bcr-Abl itself, which creates binding sites for the SH2 domains of other proteins. Generally, substrates of Bcr-Abl can be grouped according to their physiologic role into adapter molecules (such as Crkl and p62 DOK ), proteins associated with the organization of the cytoskeleton and the cell membrane (such as paxillin and talin), and proteins with catalytic function (such as the nonreceptor tyrosine kinase Fes or the phosphatase Syp). It is important to note that the choice of substrates depends on the cellular context. For example, Crkl is the major tyrosine-phosphorylated protein in CML neutrophils, 73 whereas phosphorylated p62 DOK is predominantly found in early progenitor cells. 74
Substrates of BCR-ABL
Tyrosine phosphatases counterbalance and regulate the effects of tyrosine kinases under physiologic conditions, keeping cellular phosphotyrosine levels low. Two tyrosine phosphatases, Syp 83 and PTP1B, 84 have been shown to form complexes with Bcr-Abl, and both appear to dephosphorylate Bcr-Abl. Interestingly, PTP1B levels increase in a kinase-dependent manner, suggesting that the cell attempts to limit the impact of Bcr-Abl tyrosine kinase activity. At least in fibroblasts, transformation by Bcr-Abl is impaired by the overexpression of PTP1B. 85 Interestingly, we recently observed the up-regulation of receptor protein tyrosine phosphatase κ (RPTP-κ) with the inhibition of Bcr-Abl in BV173 cells treated with the tyrosine kinase inhibitor STI571, 86 which suggests that the opposite effect may also occur. Thus, though the pivotal role of Bcr-Abl tyrosine kinase activity is clearly established, much remains to be learned about the significance of tyrosine phosphatases in the transformation process.
Activated signaling pathways and biologic properties of BCR-ABL–positive cells
Three major mechanisms have been implicated in the malignant transformation by Bcr-Abl , namely altered adhesion to stroma cells and extracellular matrix, 87 constitutively active mitogenic signaling 88 and reduced apoptosis 89 (Figure 5 ). A fourth possible mechanism is the recently described proteasome-mediated degradation of Abl inhibitory proteins. 66
Mechanisms implicated in the pathogenesis of CML.
Altered adhesion properties
CML progenitor cells exhibit decreased adhesion to bone marrow stroma cells and extracellular matrix. 87 , 90 In this scenario, adhesion to stroma negatively regulates cell proliferation, and CML cells escape this regulation by virtue of their perturbed adhesion properties. Interferon-α (IFN-α), an active therapeutic agent in CML, appears to reverse the adhesion defect. 91 Recent data suggest an important role for β-integrins in the interaction between stroma and progenitor cells. CML cells express an adhesion-inhibitory variant of β1 integrin that is not found in normal progenitors. 92 On binding to their receptors, integrins are capable of initiating normal signal transduction from outside to inside 93 ; it is thus conceivable that the transfer of signals that normally inhibit proliferation is impaired in CML cells. Because Abl has been implicated in the intracellular transduction of such signals, this process may be further disturbed by the presence of a large pool of Bcr-Abl protein in the cytoplasm. Furthermore, Crkl, one of the most prominent tyrosine-phosphorylated proteins in Bcr-Abl–transformed cells, 73 is involved in the regulation of cellular motility 94 and in integrin-mediated cell adhesion 95 by association with other focal adhesion proteins such as paxillin, the focal adhesion kinase Fak, p130Cas, 96 and Hef1. 97 We recently demonstrated that Bcr-Abl tyrosine kinase up-regulates the expression of α6 integrin mRNA, 86 which points to transcriptional activation as yet another possible mechanism by which Bcr-Abl may have an impact on integrin signaling. Thus, though there is sound evidence that Bcr-Abl influences integrin function, it is more difficult to determine the precise nature of the biologic consequences, and, at least in certain cellular systems, integrin function appears to be enhanced rather than reduced by Bcr-Abl. 98
Activation of mitogenic signaling
Ras and the map kinase pathways..
Several links between Bcr-Abl and Ras have been defined. Autophosphorylation of tyrosine 177 provides a docking site for the adapter molecule Grb-2. 57 Grb-2, after binding to the Sos protein, stabilizes Ras in its active GTP-bound form. Two other adapter molecules, Shc and Crkl, can also activate Ras. Both are substrates of Bcr-Abl 73 , 99 and bind Bcr-Abl through their SH2 (Shc) or SH3 (Crkl) domains. The relevance of Ras activation by Crkl is, however, questionable because it appears to be restricted to fibroblasts. 100 Moreover, direct binding of Crkl to Bcr-Abl is not required for the transformation of myeloid cells. 101 Circumstantial evidence that Ras activation is important for the pathogenesis of Ph-positive leukemias comes from the observation that activating mutations are uncommon, even in the blastic phase of the disease, 102 unlike in most other tumors. This implies that the Ras pathway is constitutively active, and no further activating mutations are required. There is still dispute as to which mitogen-activated protein (MAP) kinase pathway is downstream of Ras in Ph-positive cells. Stimulation of cytokine receptors such as IL-3 leads to the activation of Ras and the subsequent recruitment of the serine–threonine kinase Raf to the cell membrane. 103 Raf initiates a signaling cascade through the serine–threonine kinases Mek1/Mek2 and Erk, which ultimately leads to the activation of gene transcription. 104 Although some data indicate that this pathway may be activated only in v-abl– but not in BCR-ABL–transformed cells, 105 this view has recently been challenged. 106 Moreover, activation of the Jnk/Sapk pathway by Bcr-Abl has been demonstrated and is required for malignant transformation 107 ; thus, signaling from Ras may be relayed through the GTP–GDP exchange factor Rac 108 to Gckr (germinal center kinase related) 109 and further down to Jnk/Sapk (Figure 6 ). There is also some evidence that p38, the third pillar of the MAP kinase pathway, is also activated in BCR-ABL–transformed cells, and there are other pathways with mitogenic potential. In any case, the signal is eventually transduced to the transcriptional machinery of the cell.
Signaling pathways with mitogenic potential in
BCR-ABL –transformed cells. The activation of individual paths depends on the cell type, but the MAP kinase system appears to play a central role. Activation of p38 has been demonstrated only in v- abl –transformed cells, whereas data for BCR-ABL –expressing cells are missing.
It is also possible that Bcr-Abl uses growth factor pathways in a more direct way. For example, association with the βc subunit of the IL-3 receptor 110 and the Kit receptor 111 has been observed. Interestingly, the pattern of tyrosine-phosphorylated proteins seen in normal progenitor cells after stimulation with Kit ligand is similar to the pattern seen in CML progenitor cells. 112 Dok-1 (p62 DOK ), one of the most prominent phosphoproteins in this setting, forms complexes with Crkl, RasGAP, and Bcr-Abl. In fact, there may be a whole family of related proteins with similar functions—for example, the recently described Dok-2 (p56 DOK 2). 113 Somewhat surprisingly, p62 DOK is essential for transformation of Rat-1 fibroblasts but not for growth-factor independence of myeloid cells 114 ; thus, its true role remains to be defined.
Jak-Stat pathway.
The first evidence for involvement of the Jak-Stat pathway came from studies in v-abl–transformed B cells. 62 Constitutive phosphorylation of Stat transcription factors (Stat1 and Stat5) has since been reported in several BCR-ABL–positive cell lines 115 and in primary CML cells, 116 and Stat5 activation appears to contribute to malignant transformation. 117 Although Stat5 has pleiotropic physiologic functions, 118 its effect in BCR-ABL–transformed cells appears to be primarily anti-apoptotic and involves transcriptional activation of Bcl-xL. 119 , 120 In contrast to the activation of the Jak-Stat pathway by physiologic stimuli, Bcr-Abl may directly activate Stat1 and Stat5 without prior phosphorylation of Jak proteins. There seems to be specificity for Stat6 activation by P190 BCR-ABL proteins as opposed to P210 BCR-ABL . 115 It is tempting to speculate that the predominantly lymphoblastic phenotype in these leukemias is related to this peculiarity.
The role of the Ras and Jak-Stat pathways in the cellular response to growth factors could explain the observation that BCR-ABL renders a number of growth factor–dependent cell lines factor independent. 105 , 121 In some experimental systems there is evidence for an autocrine loop dependent on the Bcr-Abl–induced secretion of growth factors, 122 and it was recently reported that Bcr-Abl induces an IL-3 and G-CSF autocrine loop in early progenitor cells. 123 Interestingly, Bcr-Abl tyrosine kinase activity may induce expression not only of cytokines but also of growth factor receptors such as the oncostatin M β receptor. 86 One should bear in mind, however, that during the chronic phase, CML progenitor cells are still dependent on external growth factors for their survival and proliferation, 124 though less than normal progenitors. 125 A recent study sheds fresh light on this issue. FDCPmix cells transduced with a temperature-sensitive mutant of BCR-ABL have a reduced requirement for growth factors at the kinase permissive temperature without differentiation block. 126 This situation resembles chronic-phase CML, in which the malignant clone has a subtle growth advantage while retaining almost normal differentiation capacity.
PI3 kinase pathway.
PI3 kinase activity is required for the proliferation of BCR-ABL –positive cells. 127 Bcr-Abl forms multimeric complexes with PI3 kinase, Cbl, and the adapter molecules Crk and Crkl, 95 in which PI3 kinase is activated. The next relevant substrate in this cascade appears to be the serine–threonine kinase Akt. 128 This kinase had previously been implicated in anti-apoptotic signaling. 129 A recent report placed Akt in the downstream cascade of the IL-3 receptor and identified the pro-apoptotic protein Bad as a key substrate of Akt. 130 Phosphorylated Bad is inactive because it is no longer able to bind anti-apoptotic proteins such as Bcl XL and it is trapped by cytoplasmic 14-3-3 proteins. Altogether this indicates that Bcr-Abl might be able to mimic the physiologic IL-3 survival signal in a PI3 kinase-dependent manner (see also below). Ship 131 and Ship-2, 132 2 inositol phosphatases with somewhat different specificities, are activated in response to growth factor signals and by Bcr-Abl. Thus, Bcr-Abl appears to have a profound effect on phosphoinositol metabolism, which might again shift the balance to a pattern similar to physiologic growth factor stimulation.
Myc pathway.
Overexpression of Myc has been demonstrated in many human malignancies. It is thought to act as a transcription factor, though its target genes are largely unknown. Activation of Myc by Bcr-Abl is dependent on the SH2 domain, and the overexpression of Myc partially rescues transformation-defective SH2 deletion mutants whereas the overexpression of a dominant-negative mutant suppresses transformation. 133 The pathway linking Myc to the SH2 domain of Bcr-Abl is still unknown. However, results obtained in v-abl–transformed cells suggest that the signal is transduced through Ras/Raf, cyclin-dependent kinases (cdks), and E2F transcription factors that ultimately activate the MYC promoter. 134 Similar results were reported for BCR-ABL–transformed murine myeloid cells. 135 How these findings relate to human Ph-positive cells is unknown. It seems likely that the effects of Myc in Ph-positive cells are probably not different from those in other tumors. Depending on the cellular context, Myc may constitute a proliferative or an apoptotic signal. 136 , 137 It is therefore likely that the apoptotic arm of its dual function is counterbalanced in CML cells by other mechanisms, such as the PI3 kinase pathway.
Inhibition of apoptosis
Expression of Bcr-Abl in factor-dependent murine 138 and human 122 cell lines prevents apoptosis after growth-factor withdrawal, an effect that is critically dependent on tyrosine kinase activity and that correlates with the activation of Ras. 88 , 139 Moreover, several studies showed that BCR-ABL –positive cell lines are resistant to apoptosis induced by DNA damage. 89 , 140 The underlying biologic mechanisms are still not well understood. Bcr-Abl may block the release of cytochrome C from the mitochondria and thus the activation of caspases. 141 , 142 This effect upstream of caspase activation might be mediated by the Bcl-2 family of proteins. Bcr-Abl has been shown to up-regulate Bcl-2 in a Ras- 143 or a PI3 kinase-dependent 128 manner in Baf/3 and 32D cells, respectively. Moreover, as mentioned previously, Bclx L is transcriptionally activated by Stat5 in BCR-ABL –positive cells. 119 , 120
Another link between BCR-ABL and the inhibition of apoptosis might be the phosphorylation of the pro-apoptotic protein Bad. In addition to Akt, Raf-1, immediately downstream of Ras, phosphorylates Bad on 2 serine residues. 144 , 145 Two recent studies provided evidence that the survival signal provided by Bcr-Abl is at least partially mediated by Bad and requires targeting of Raf-1 to the mitochondria. 146 , 147 It is also possible that Bcr-Abl inhibits apoptosis by down-regulating interferon consensus sequence binding protein (ICSBP). 148 , 149 These data are interesting because ICSBP knockout mice develop a myeloproliferative syndrome, 150 and hematopoietic progenitor cells from ICSBP −/− mice show altered responses to cytokines. 151 The connection to interferon α, an active agent in the treatment of CML, is obvious.
It becomes clear that the multiple signals initiated by Bcr-Abl have proliferative and anti-apoptotic qualities that are frequently difficult to separate. Thus, Bcr-Abl may shift the balance toward the inhibition of apoptosis while simultaneously providing a proliferative stimulus. This is in line with the concept that a proliferative signal leads to apoptosis unless it is counterbalanced by an anti-apoptotic signal, 152 and Bcr-Abl fulfills both requirements at the same time. There is, however, controversy. One report found 32D cells transfected with BCR-ABL to be more sensitive to IR than the parental cells, 153 whereas 2 other studies failed to detect any difference between CML and normal primary progenitor cells with regard to their sensitivity to IR and growth factor withdrawal. 124 , 154 Furthermore, based on results obtained in transfected cell systems, it was suggested that Bcr-Abl inhibits apoptosis mediated by the Fas receptor/Fas ligand system. 155 However, though there may be a role for this system in mediating the clinical response to interferon-α, 156 there is no indication that Fas-triggered apoptosis is defective in primary CML cells or in “natural” Ph-positive cell lines. 157 Moreover, Bcr-Abl accelerates C2 ceramide-induced apoptosis, 158 and it does not protect against natural killer cell-induced apoptosis. 159 These inconsistencies may reflect genuine differences between cell lines and primary cells. On the other hand, it is debatable whether complete growth-factor withdrawal and IR constitute stimuli that have much physiologic relevance. To allow for a representative comparison, it would be crucial to define the signals that induce apoptosis in vivo.
Degradation of inhibitory proteins.
The recent discovery that Bcr-Abl induces the proteasome-mediated degradation of Abi-1 and Abi-2, 66 2 proteins with inhibitory function, may be the first indication of yet another way by which Bcr-Abl induces cellular transformation. Most compelling, the degradation of Abi-1 and Abi-2 is specific for Ph-positive acute leukemias and is not seen in Ph-negative samples of comparable phenotype. The overall significance of this observation remains to be seen, and one must bear in mind that the data refer to acute leukemias and not to chronic phase CML. It is nevertheless tempting to speculate that other proteins, whose level of expression is regulated through the proteasome pathway, may also be degraded. A good candidate would be the cell cycle inhibitor p27, but to our knowledge no data are available yet.
Various experimental systems have been developed to study the pathophysiology of CML. All of them have their advantages and shortcomings, and it is probably fair to say that there is still no ideal in vitro or in vivo model that would cover all aspects of the human disease.
Fibroblasts.
Fibroblast lines have been used extensively in CML research because they are easy to manipulate. Fibroblast transformation—that is, anchorage-independent growth in soft agar—is the standard in vitro test for tumorigenicity. 160 However, it became clear that the introduction of BCR-ABL into fibroblasts has diverse effects, depending on the type of fibroblast used. Thus, though P210 BCR-ABL transforms Rat-1 fibroblasts, 161 there is no such effect in NIH3T3. 162 Moreover, transformation to serum-independent growth occurs only in few cells (permissive cells 163 ), whereas most undergo growth arrest. These observations show that certain cellular requirements must be met if a cell is to be transformed by BCR-ABL . Interestingly, this is also the case for the various parts of the Bcr-Abl protein. Thus, a BCR-ABL mutant that lacks the SH2 domain retains the capacity to transform hematopoietic 32D cells to growth factor independence 60 but is defective for fibroblast transformation. 59 In addition, there are differences between hematopoietic cells and fibroblasts in terms of interactions with other proteins such as Crkl. The latter is functional in Ras activation and transformation in fibroblasts 100 but not in hematopoietic cells. 101 Thus, results obtained from studies in fibroblasts must be interpreted with great caution.
Hematopoietic cell lines.
Until relatively recently, only a few BCR-ABL –positive lines derived from CML were available, but their number has grown considerably in the past few years. 164 They include cell lines with myeloid differentiation, such as the well-known K562, and lymphoid phenotype, such as BV173. The main drawback common to all these lines is the fact that they are derived from blast crisis and, thus, contain genetic lesions in addition to BCR-ABL . Consequently, they may reflect blast crisis fairly well but are insufficient models of chronic phase CML. Until now, no cell line from chronic phase CML has been established, just as no cell line could be derived from normal human bone marrow. Even attempts to immortalize Ph-positive B-cells from patients in the chronic phase of disease were not successful because these lines have a limited life span, 165 in contrast to their Ph-negative counterparts. One could therefore speculate that the very establishment of a line from a patient with CML would be diagnostic of advanced disease. In this context, it is surprising that most human CML lines remain dependent on Bcr-Abl tyrosine kinase activity for their proliferation and survival, as shown by their susceptibility to the effects of the Abl-specific tyrosine kinase inhibitor STI571. 8 However, the phenotype of these cell lines is that of an acute leukemia. Therefore great caution is warranted if experimental results are to be transferred to chronic phase CML. A striking example is the fact that inhibition of apoptosis by Bcr-Abl is easily demonstrable in cell lines 139 but not in primary cells. 124 It should also be noted that many of the lines used have undergone hundreds, if not thousands, of rounds of replication, and different laboratories frequently house lines that have little in common but their names and BCR-ABL positivity.
Transformation of factor-dependent cell lines to growth-factor independence is an important feature of Bcr-Abl, and, in fact, other oncoproteins that contain an activated tyrosine kinase. 43 , 166 Although it is usually difficult to obtain stable expression of BCR-ABL in previously immortalized cell lines, this is relatively easily achieved in factor-dependent lines, presumably because BCR-ABL expression is an advantage to the latter but useless or even detrimental to the former. Murine cell lines such as Baf/3 and 32D and human cell lines such as MO7 were used to study the effects of BCR-ABL by direct comparison between transduced and parental cells. A particular advantage of the murine lines is the fact that they are derived from normal nonmalignant hematopoietic cells. Unfortunately, this does not rule out the development of additional mutations 167 that confer a selective growth advantage. Furthermore, it is not clear how the transformation to complete factor independence relates to clinical CML in which the cells are still factor-dependent, though obviously less so than normal hematopoietic cells. 123 The subject of growth factor independence and BCR-ABL transformation has been reviewed recently. 168
None of the cell lines mentioned above is capable of multilineage hematopoietic differentiation. Two strategies are promising in overcoming this restraint. A recent report 126 shows that murine FDCPmix cells, transduced with a temperature-sensitive mutant of BCR-ABL , become partially factor-independent at the permissive temperature, in analogy to chronic phase CML. They retain the capacity for terminal differentiation, similar to chronic phase CML cells. Another approach is the study of embryonic stem (ES) cells transduced with BCR-ABL . In one such experimental system, it was possible to reproduce one cardinal feature of the clinical disease in the model, namely the expansion of the myeloid compartment at the expense of the erythroid compartment. 169 Interestingly, the increase in total cell numbers in the BCR-ABL -transduced ES cells was found to result from increased proliferation though there was little effect on apoptosis, another finding in line with observations in primary Ph-positive cells. 124 , 154 In this system, a stromal cell layer is used on which the ES cells removed from leukemia-inhibitory factor (LIF) differentiate into hemangioblasts and then into hematopoietic cells. This may explain why these results are not strictly comparable to those of another study, in which BCR-ABL resulted in the decreased formation of embryonal bodies along with increased output of all kinds of hematopoietic progenitors. 170 In yet another study, BCR-ABL –transformed ES cells transplanted into irradiated mice induced a leukemic syndrome with many features of CML. 171 If developed further, these models may well be able to retain the major advantage of cell lines—their ease of manipulation—while at the same time moving the in vitro system closer to the clinical disease.
Bearing all these caveats in mind, there is no doubt that the study of cell lines contributed significantly to our understanding of CML. Particularly, many of the proteins that interact with Bcr-Abl were identified in Ph-positive cell lines, where they are more abundantly expressed than in primary cells. Thus, though these lines are invaluable tools for screening, it is important to confirm the results in primary cells.
Primary cells.
The study of patient material and its comparison with normal hematopoietic progenitor cells is certainly the gold standard of CML research, particularly for the chronic phase of the disease. Much of the data refer to operationally defined cellular properties of CML versus normal cells, such as clonogenicity or adherence to bone marrow stroma; to give a comprehensive account of the cellular biology of CML would require a review in its own right. We will therefore focus on some areas in which the study of primary CML cells has been particularly instrumental to the study of the molecular biology of the disease.
One of the main problems when studying primary cells is inherent in the very nature of chronic phase cells—they tend to mature when placed in culture. Thus, the window of time for in vitro studies is narrow, and expansion of very primitive cells, the least prevalent but most interesting population, is difficult and carries the risk for introducing nonphysiologic alterations. 172 Furthermore, there is considerable variation between patients that frequently results in an overlap rather than a clear distinction between normal cells and CML cells. Last, results are unreliable unless clearly defined cell populations such as CD34 + cells are studied. To a large extent, these problems can be overcome by the introduction of retroviral BCR-ABL expression vectors to murine or human primary bone marrow cells (see “Animal models” below).
A striking example of how fruitful the comparison of primary cell populations can be is the study of tyrosine-phosphorylated proteins in CD34 + cells. 112 This study led to the subsequent identification of p62 DOK74;173 and SHIP2 132 as mediators of Bcr-Abl–induced transformation. Moreover, it produced the important notion that Bcr-Abl tyrosine kinase activity may have consequences similar to the activation of the KIT receptor. 112 Another example is the identification of CRKL as the major tyrosine-phosphorylated protein in CML neutrophils. 73
The recent possibility of turning off the Bcr-Abl tyrosine kinase activity in cell lines and primary cells with STI571 7 , 8 provided the opportunity to study the effects of the BCR-ABL gene when expressed from its natural BCR promoter at “physiological” levels. This is certainly an advantage over transduced cell systems; the drawback, however, is that effects related to inhibition of the KIT and PDGFRβ kinases, and potentially other unidentified tyrosine kinases, cannot be ruled out. Furthermore, the Bcr-Abl protein, though kinase-inactive, is still present in the cells and may interfere with other proteins.
Animal models
Thus far, no animals other than mice have been used for the study of CML in vivo. Various approaches have been taken.
Engraftment of BCR-ABL–transformed cell lines in syngeneic mice.
Murine factor-dependent cell lines such as 32D transduced with BCR-ABL give rise to an aggressive leukemia when transplanted into syngeneic recipients. 60 , 174 This is an excellent in vivo model to test the efficacy of new drugs, such as the tyrosine kinase inhibitor STI571, in vivo. Furthermore, the impact on leukemogenicity of modifications within the Bcr-Abl protein and modifications to the respective cell lines (such as the introduction of co-stimulatory molecules 174 ) can be tested. The main drawback is that the disease is a form of acute leukemia and is thus far from chronic phase CML.
Engraftment of immunodeficient mice with human BCR-ABL–positive cells.
Cell lines derived from human CML blast crisis are relatively easily propagated in severe combined immunodeficiency (SCID) mice. 175 The distribution of the leukemia cells is fairly similar to the human disease, that is, they home to bone marrow and peripheral blood before they metastasize to nonhematopoietic tissues. More recently, it was shown that SCID mice can be engrafted with chronic phase CML cells if the cell inoculum is large enough (in the range of 1 × 10 8 cells). 176 Up to 10% human cells were detectable in the recipient bone marrow and showed multilineage differentiation. Interestingly, most colonies were BCR-ABL negative and thus were derived from the patient's residual normal hematopoiesis. This is reminiscent of long-term bone marrow cultures of CML 177 and shows that host factors modify the disease to a great extent, a problem that will persist, even if higher percentages of engraftment can eventually be achieved. A step into this direction is the use of nonobese diabetes-SCID mice. In addition to the SCID defect in V(D)J recombination, these animals lack functional natural killer cells. Chronic phase CML cells and, even more so, cells from accelerated phase or blast crisis readily engraft in these mice, and there is a significant correlation between engraftment and disease state. 178 Interestingly, the cells were exclusively Ph-positive in most cases, in contrast to cells engrafted in SCID mice, as mentioned above. This may be attributed to technical reasons but may also reflect a genuine difference between the different strains of mice. We can anticipate that these murine models will be useful for studying certain aspects of CML, such as the response to novel forms of treatment. Their value in investigating the human disease will be limited because it is difficult to see how disease modification by host factors could ever be ruled out.
Transgenic mouse models.
Attempts to use BCR-ABL transgenic mice as a CML model go back to the late 1980s, when a full-length cDNA of BCR-ABL was not yet available and an artificial construct of human BCR sequences fused to v -abl was used instead. 179 Since then, a number of studies have been published that clearly prove the oncogenic potential of BCR-ABL . Several different promoters were used to direct the expression to the desired target tissues. However, some problems were encountered. First, it became clear that Bcr-Abl has a toxic effect on embryogenesis, 180 perhaps the consequence of a cytostatic effect in nonhematopoietic tissues. 181 Recently, the expression of BCR-ABL from a tetracycline-repressible promoter effectively overcame this problem. 182 Most striking, the leukemia in these transgenic mice is completely reversible on re-addition of tetracycline. The second problem with transgenic mice is that the P210 BCR-ABL variant relevant to CML is difficult to study because it is less efficient in inducing leukemia than P190, a finding that was again confirmed in a recent study. 47 Third and most important, the types of leukemia that developed in these mice were acute and of either B- or T-lymphoid phenotype, regardless of whether they arose in P190 or P210 transgenic animals. Thus, they resembled BCR-ABL –positive ALL but not chronic-phase CML 183 , 184 . In fact, myeloid leukemias developed rarely, if at all. A recent report 185 may represent a major advance in this respect. In this study, BCR-ABL was expressed from the Tec promoter, a cytoplasmic tyrosine kinase predominantly expressed in hematopoietic cells. Although the founder mice exhibited excessive proliferation of lymphoblasts, their progeny developed a CML-like disease, albeit after a relatively long latency period of approximately 10 months. Thus, it is likely that the problems of the transgenic models will eventually be resolved if the gene is targeted to the appropriate cell.
Transduction of murine bone marrow cells with BCR-ABL retroviruses.
In 1990, several groups reported that a CML-like myeloproliferative syndrome could be induced when P 210BCR-ABL –infected marrow was transplanted into syngeneic recipients. 6 , 186 , 187 Transplantation into secondary recipients frequently produced an identical disease while some mice developed acute leukemias of T- or B-cell phenotype, analogous to the development of lymphoid blast crisis in the clinical disease. Clonality was demonstrated in many cases. Roughly a quarter of the mice showed the myeloproliferative disease, whereas other recipients developed other distinct hematologic malignancies, such as macrophage tumors, B-ALL, T-ALL, and erythroleukemia. Most likely, these different diseases are the consequence of infection of different committed progenitor cells that, after transformation, give rise to the respective progeny. Not surprisingly, the infection conditions have a major impact on the disease phenotype. 188 Building on the foundations of this early work, major improvements to the transduction–transplantation system have been made in the past few years. High-titer BCR-ABL retroviral stocks can be produced rapidly by transient transfection of packaging lines; the culture conditions have been refined, and the murine stem cell virus LTR has been introduced that allows for more efficient expression of BCR-ABL in the desired target cell. Combining all 3 improvements, 2 recent studies 189 , 190 reported the induction of a transplantable CML-like disease in 100% of recipients. Pulmonary hemorrhage, a complication not found in human CML, was a frequent cause of death in both studies, demonstrating that these novel models, though a major step forward, may have their own distinct problems. Nevertheless, bone marrow transduction–transplantation most faithfully reproduces human CML, and further improvements are likely in the near future.
Clinically, chronic-phase CML does not represent a major management problem because the elevated white blood cell count is readily controlled with cytotoxic agents in most patients, and neutrophil and platelet functions are largely normal. However, the disease progresses inexorably to acceleration and blast crisis, often within 5 years of diagnosis. The mechanisms underlying this evolution remain enigmatic. Deletion or inactivation of p16, 191 p53, 192 and the retinoblastoma gene product 193 have been reported but occur relatively rarely and, similar to the overexpression of EVI-1, 194 are not specific for blast crisis CML. This probably indicates that a wide variety of lesions, possibly multiple “cooperating” lesions, are required to induce the phenotype of blast crisis. Perhaps even more intriguing is why the cells acquire these additional lesions in the first place. A recent report shows that Bcr-Abl enhances the mutation rate in the Na-K-ATPase and in the HPRT genomic loci, both commonly used markers to measure mutation frequency. Along with this goes enhanced expression of DNA polymerase β, the mammalian DNA polymerase with the least fidelity. 195 P210 BCR-ABL , but not P190 BCR-ABL , phosphorylates and potentially interacts with xeroderma pigmentosum group B protein (XPB); as a result, the catalytic function of XPB may be reduced, and DNA repair may be impaired. 196 In a recent study p210 BCR-ABL transgenic mice were cross-mated with p53 heterozygous mice. In the offspring, the remaining p53 was rapidly lost because of somatic mutation, and the mice developed a disease that resembled blast crisis. 197 Although this is still not a perfect model of human CML because the blasts are of T lineage, it strongly supports the concept of genomic instability in BCR-ABL –transformed cells. How Bcr-Abl leads to these phenomena is unclear, but they might form the basis of the presumed genomic instability of chronic phase CML. It is also possible that the alleged anti-apoptotic effect of Bcr-Abl favors inaccurate DNA repair where apoptosis would ensue in normal cells. In line with this concept, a prolonged G 2 arrest after IR has been observed in BCR-ABL –expressing cell lines exposed to DNA-damaging agents. 140 This arrest could allow for DNA (mis)repair, whereas in a normal cell the damage would induce apoptosis. Over time this could lead to the accumulation of mutations in BCR-ABL –positive cells that finally result in blastic transformation. There is no doubt that the excessive proliferation, with its high cell turnover, must be a risk factor per se for additional genetic lesions.
Attempts at designing therapeutic tools for CML based on our current knowledge of the molecular and cell biology of the disease have concentrated on 3 main areas—the inhibition of gene expression at the translational level by “antisense” strategies, the stimulation of the immune system's capacity to recognize and destroy leukemic cells, and the modulation of protein function by specific signal transduction inhibitors. The antisense oligonucleotide 198 , 199 and ribozyme 200 approaches received much attention in the last decade but have in general failed to fulfill their theoretical promises. New modifications to the system, such as the use of BCR-ABL junction-specific catalytic subunits of RNase P, 201 may revitalize the field. The issue of immunologic stimulation, be it in the form of adoptive immunotherapy by donor lymphocyte infusions 202 or of BCR-ABL junction peptide vaccination, 203 is another avenue being extensively explored for the treatment of CML.
Perhaps the most exciting of the molecularly designed therapeutic approaches was brought about by the advent of signal transduction inhibitors (STI), which block or prevent a protein from exerting its role in the oncogenic pathway. Because the main transforming property of the Bcr-Abl protein is effected through its constitutive tyrosine kinase activity, direct inhibition of such activity seems to be the most logical means of silencing the oncoprotein. To this effect, several tyrosine kinase inhibitors have been evaluated for their potential to modify the phenotype of CML cells. The first to be tested were compounds isolated from natural sources, such as the iso-flavonoid genistein and the antibiotic herbimycin A. 204 Later, synthetic compounds were developed through a rational design of chemical structures capable of competing with the adenosine triphosphate (ATP) or the protein substrate for the binding site in the catalytic center of the kinase 205 (Figure 7 ).
Mechanism of action of tyrosine kinase inhibitors.
The drug competes with ATP for its specific binding site in the kinase domain. Thus, whereas the physiologic binding of ATP to its pocket allows Bcr-Abl to phosphorylate selected tyrosine residues on its substrates (left diagram) , a synthetic ATP mimic such as STI571 fits this pocket equally well but does not provide the essential phosphate group to be transferred to the substrate (right diagram) . The downstream chain of reactions is then halted because, with its tyrosines in the unphosphorylated form, this protein does not assume the necessary conformation to ensure association with its effector.
The most promising of these compounds is the 2-phenylaminopyrimidine STI571 (formerly CGP57418B; Novartis Pharmaceutics, Basel, Switzerland), which specifically inhibits Abl tyrosine kinase at micromolar concentrations. 206 Inhibition of the Bcr-Abl kinase activity by this compound results in the transcriptional modulation of various genes involved in the control of the cell cycle, cell adhesion, and cytoskeleton organization, leading the Ph-positive cell to an apoptotic death. 86 Furthermore, STI571 selectively suppresses the growth of CML primary cells and cell lines in vitro 7 , 8 and in mice. 7 , 207 Its remarkable specificity and efficacy led to consideration of the drug for therapeutic use. Thus, in the spring of 1998, a phase 1 clinical trial was initiated in the United States in which patients with CML in chronic phase resistant to IFN-α were treated with STI571 in increasing doses. The drug showed little toxicity but proved to be highly effective. All patients treated with 300 mg/d or more entered a complete hematologic remission. Even more striking, many of the patients had cytogenetic responses. 9 This might mean that STI571 changes the natural course of the disease, though it is far too early to arrive at any definite conclusions. Altogether, the results were convincing enough to justify the initiation of phase 2 studies that included patients with acute Ph-positive leukemias (CML in blast crisis and Ph-positive ALL) and, at a later stage, a large cohort of interferon-intolerant or -resistant patients. These studies are ongoing. It turned out that STI571 is effective in many patients with acute Ph-positive leukemia, particularly of lymphoid phenotype. Although in many patients the remissions are not sustained, the advent of an effective oral medication with little toxicity represents a major step forward in this very poor risk group. Clearly, elucidation of the mechanisms underlying the resistance 208 will be of critical importance for the development of further treatment strategies, such as a combination of STI571 with conventional cytotoxic drugs or, perhaps, with other STIs (see below). In this context, the most interesting question is whether STI571 will be able to eradicate the malignant clone, at least in some patients with chronic-phase CML. From what we know about the disease, this seems unlikely—colony formation by CML progenitor cells is much reduced but not abrogated in the presence of STI571 7 , 8 —which might mean that a subset of these cells proliferates independently of Bcr-Abl tyrosine kinase activity and still relies on external growth factors. There is no doubt, however, that the clinical efficacy and low toxicity of STI571 sets a precedent for the further development of targeted forms of therapy in malignant disease.
An alternative or a supplement to direct inhibition of Bcr-Abl is interference with proteins that are critical for Bcr-Abl–induced transformation (Figure 4 ). One of these proteins is Grb2, whose SH2 domain binds directly to Bcr-Abl through the phosphorylated tyrosine 177 within the Bcr portion of the chimera 57 and is essential for activation of the Ras pathway (Figure 6 ). 209 Another good candidate is Ras itself, whose activity depends on its attachment to the cell membrane through a prenyl (usually a farnesyl) group. Thus, farnesyl transferase inhibitors (FTI) have been studied for their effect in inhibiting the proliferation of ALL 210 and juvenile myelomonocytic leukemia cells, 211 and they may be useful for the control of CML cells. Additional targets worth considering are represented by PI-3 and Src kinases, of which the available inhibitors have been shown to suppress colony formation of primary progenitors, 127 proliferation of BCR-ABL –transfected cell lines, or both. 212 , 213 It is envisaged that the progressive unraveling of which pathways are really essential for the development of the disease, coupled to rapid advances in biotechnology, will bring us the ideal combination of rationally designed drugs that can tip the balance toward the re-establishment of normal hematopoiesis in CML.
Though this be madness, yet there is method in it. (Shakespeare W., Hamlet. Act 2, scene 2.)
There are 2 ways to conclude this review after going through the vast amount of data presented. Surely one could argue that despite all these data, there is still no clear picture emerging and each piece of additional information adds only more confusion. Alternatively, what might help us against capitulation in the face of complexity is to try to simplify without oversimplification.
Can we build a model of CML that incorporates all the scientific data available but still retains clarity? In other words, could we explain how Bcr-Abl works in a few sentences to somebody who has never heard of it? Perhaps the most promising approach might be to try to link the biologic behavior of a CML cell to the underlying molecular events (Figure 5 ). Crucially, we should be able to picture this scenario relying on BCR-ABL alone because, at least until now, there is no unequivocal evidence that additional genetic lesions are present during chronic phase. We do not know how long it takes to move from the initial genetic event to fully established chronic-phase CML, but there is good reason to believe that the proliferative advantage of CML over normal cells is limited. Together with the largely normal differentiation capacity and function of CML blood cells, one feels that Ph-positive hematopoiesis cannot be so much different from normal hematopoiesis until the disease accelerates. Thus, Bcr-Abl is likely to hijack pathways that normally increase blood cell output in response to physiologic stimuli rather than to interrupt or replace them with pathways that are not normally used in hematopoietic cells. Indeed, there is plenty of experimental evidence to support this notion. Importantly, Bcr-Abl is capable of activating survival pathways along with proliferative stimuli without the need for a second cooperating genetic lesion; in this way, the apoptotic response that would otherwise follow an isolated proliferative stimulus is avoided. The sustained dependence on growth factors is an indication that Bcr-Abl is not a complete substitute; rather, it tips the balance to provide a limited growth advantage in vivo. This growth advantage is also dependent on specific survival conditions: transient regeneration of Ph-negative hematopoiesis is often observed after autografting, even when the autograft seems to be comprised exclusively of Ph-positive stem cells, and long-term cultures initiated from patients with chronic-phase CML become dominated by BCR-ABL –negative cells after some time. 177 Thus, there appears to be a specific interaction (or noninteraction) of CML progenitor cells with their microenvironment that is crucial to maintain their proliferative advantage. Whether this interaction is stimulatory for CML over normal progenitor cells or inhibitory for normal over CML progenitor cells remains to be seen. Similarly, we can look at extramedullary hematopoiesis as a loss of function (ie, loss of the capacity to respond to negative signals) or a gain of function (ie, acquisition of a capacity to respond to positive signals that are not provided in the bone marrow) phenomenon. Much of the evidence implicates integrins in mediating these abnormal interactions, but other proteins may also play a role. Overall, it appears that the organization of cell membrane and cytoskeleton is more profoundly perturbed in CML progenitor cells than might be anticipated from the largely normal function of their progeny. Furthermore, Bcr-Abl may interfere with the “wiring” between integrin receptors on the cell surface and the nucleus and so disturb the communication of the cell with its environment. Another mechanism may also be important: Bcr-Abl appears to induce the degradation of certain inhibitory proteins. This might thwart cellular counter-reactions that would otherwise be activated, rather like cutting the telephone cable before the police can be called in.
Many questions remain unanswered. Why is there a predominantly myeloid expansion when all 3 lineages carry the translocation? What is the biologic basis for the extraordinary variability in the clinical course of a disease that appears to carry just a single genetic lesion? What is the molecular basis for the genomic instability that we see clinically as relentless progression to blast crisis?
Where do we go from here? The more we learn about the pathogenesis of CML, the more we realize its extraordinary complexity. Perhaps one should not be too surprised because it has become clear that cellular processes tend to rely on integrated networks rather than on straight unidirectional pathways. Only in this way can the cell achieve the flexibility required to respond to the various stimuli within a multicellular organism. Clearly, some components must be more important, and some less so, in the transformation network operated by Bcr-Abl. Absolutely essential features may be restricted to functional domains and to certain residues of the Bcr-Abl protein itself, and downstream effectors may be able to substitute for each other, at least to some extent. In this respect, the use of knockout mice that lack specific downstream molecules will allow one to define their precise relevance for Bcr-Abl–mediated cellular transformation. It may turn out that the combined elimination of several components abrogates transformation by Bcr-Abl, whereas each component individually is of limited significance. Chronic phase CML operates very much by exploiting physiologic pathways, perhaps by gently “coaxing” hematopoiesis toward the classical CML phenotype; nevertheless it prepares the ground for blast crisis. Thus, to understand CML, we must study its chronic phase. We must move away from artificial systems, such as transduced fibroblasts, and take on the demanding task of studying signal transduction in primary progenitor cells.
Supported by grants from Leukaemia Research Fund (UK) and the Dr Ernst und Anita Bauer Stiftung (Germany).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement ” in accordance with 18 U.S.C. section 1734.
Author notes
Michael W. N. Deininger, Department of Hematology/Oncology, University of Leipzig, Johannisallee 32, Leipzig 04103, Germany; e-mail: [email protected] .
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- Cancer information
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Research in chronic myelogenous leukemia (CML)
We are always learning more about cancer. Researchers and healthcare professionals use what they learn from research studies to develop better practices that will help prevent, find and treat chronic myelogenous leukemia (CML). They are also looking for ways to improve the quality of life of people with CML.
The following is a selection of research showing promise for CML. We've included information from PubMed, which is the research publication database of the National Library of Medicine. Each research article in PubMed has an identity number (called a PMID) that links to a brief overview (called an abstract). We have also included links to abstracts of the research presented at meetings of the American Society of Clinical Oncology (ASCO), which are held throughout the year. You can find information about ongoing clinical trials in Canada from CanadianCancerTrials.ca and ClinicalTrials.gov. Clinical trials are given an identifier called a national clinical trial (NCT) number. The NCT number links to information about the clinical trial.
Prognosis Researchers are trying to find better ways to help doctors predict a prognosis (how likely it is that the cancer can be successfully treated or that it will come back after treatment) for CML. They are also trying to determine the best treatment options based on certain characteristics of the disease, such as specific biomarkers. Biomarkers are substances, such as proteins, genes or pieces of genetic material like DNA and RNA, that are found naturally in the body. They can be measured in body fluids, including blood, lymph fluid and bone marrow, or on certain types of cells, such as cancer cells. Doctors can look for and measure these biomarkers to check if cancer is present or that it is responding to treatment. Prognostic and predictive biomarkers for CML can be used to help plan treatment. Prognostic biomarkers can be used to identify people who have a greater risk that the disease will progress or come back after treatment (recur or relapse). Predictive biomarkers are used to identify people who are more likely to have a favourable or unfavourable effect from treatment compared to people without the biomarker. Researchers are looking at the following biomarkers to see if they can help doctors diagnose, predict a prognosis for and find out which treatments will benefit a person with CML: BCR-ABL (Blood, PMID 26729897 ; American Journal of Hematology, PMID 28718956 , PMID 28466557 ; Oncology, PMID 29151104 ; Journal of Cancer Research and Clinical Oncology, PMID 28083711 ) clonal chromosomal abnormalities in Philadelphia chromosome-negative (CCA/Ph-) (Blood, PMID 28835440 ) NKG2TD (International Journal of Hematology, PMID 28795321 ) plasma biomarkers (Blood, PMID 27827824 ) NK cells (Leukemia, PMID 27890936 ) Treatment Researchers are looking for new ways to improve treatment for CML. Advances in cancer treatment and new ways to manage the side effects from treatment have improved the outlook and quality of life for many people with cancer. The following is noteworthy research into treatment for CML. Targeted therapy Targeted therapy drugs target specific molecules (usually proteins) that cause cancer cells to grow. Researchers are studying the following types of targeted therapy drugs in treating CML. Tyrosine kinase inhibitors Tyrosine kinase inhibitors are drugs that block the enzyme tyrosine kinase, which helps cells develop and grow. Imatinib (Gleevec) is the main targeted therapy drug used to treat CML. But 20% to 25% of people who take imatinib for CML develop resistance to the drug. When this happens, doctors offer these people different tyrosine kinase inhibitors. Researchers are looking at the following tyrosine kinase inhibitors to see if giving them in different doses or combining them with other therapies will make them effective treatments for CML. Asciminib (ABL001) is a tyrosine kinase inhibitor that is effective in treating CML and that researchers are studying in clinical trials (ClinicalTrials.gov, NCT03106779 ; ASCO, Abstract TPS7081 ). Pioglitazone is a drug approved to treat diabetes. Giving pioglitazone with tyrosine kinase inhibitors may be an effective treatment for people with CML. Although tyrosine kinase inhibitors are effective against CML, there are sometimes cancer cells called leukemia stem cells that can hide from tyrosine kinase inhibitors. Pioglitazone increased CML stem cell death when given with tyrosine kinase inhibitors in laboratory studies. Clinical trials are currently looking at pioglitazone along with tyrosine kinase inhibitors to treat CML (Cancer, PMID 28026860 ; ClinicalTrials.gov, NCT02730195 ). Nilotinib (Tasigna) seems to be more effective than imatinib at treating newly diagnosed CML that is in the chronic phase. Clinical trials also show that treatment with nilotinib made it more likely that people with CML could stay in remission after stopping treatment (called treatment-free remission) (Leukemia, PMID 28218239 ; ASCP, Abstract 7003 ). Ponatinib (Iclusig) is another tyrosine kinase inhibitor that researchers are studying as a treatment for CML in the chronic phase (Lancet Haematology, PMID 26436130 ). Switching to nilotinib after imatinib treatment could improve molecular response in people with CML and is more effective than increasing the dose of imatinib. Few people with CML stay in remission after they stop treatment, so many people need to continue treatments for the rest of their lives. If switching to nilotinib improves molecular response, some people may be able to have treatment-free remission (Lancet Haematology, PMID 27890073 ). Optimizing the dose of tyrosine kinase inhibitors for each person based on response to treatment is being studied in clinical trials. Certain people may benefit from a lower dose of tyrosine kinase inhibitor (British Journal of Haematology, PMID 28699641 ; Lancet Haematology, PMID 28566209 ). Long-term follow-up after treatment with imatinib shows favourable results. Almost 11 years of follow-up studies show that long-term treatment with imatinib was not associated with any unacceptable long-term or late effects (New England Journal of Medicine, PMID 28273028 ; Leukemia, PMID 28804124 ). Stem cell transplant Stem cell transplant replaces a person's blood-forming (hematopoietic) stem cells. It is used when stem cells or the bone marrow has been damaged by chemotherapy drugs, radiation therapy or disease (such as cancer). The new stem cells make healthy blood cells. Stem cell transplants may be used to treat CML. Researchers are studying the following types of stem cell transplant to see if they could be safer, easier and more effective for people with CML. Reduced-intensity allogeneic transplant uses lower doses of chemotherapy or radiation therapy before the transplant. The lower doses don't completely destroy the recipient's bone marrow, so blood cell counts don't drop as low as they do in standard stem cell transplants. For this reason, there is a lower risk for complications. This may be of great importance for older adults who can't tolerate the higher doses of chemotherapy and radiation normally used with stem cell transplants (Annals of Hematology, PMID 28624905 ). Tyrosine kinase inhibitor treatment given after stem cell treatment (called prophylactic targeted therapy) may help prevent a relapse of CML (Clinical Lymphoma, Myeloma and Leukemia, PMID 27297665 ; Leukemia, PMID 28218239 ; Oncotarget, PMID 27880933 ). Predicting who will benefit from a stem cell transplant can help ensure people with CML get the right treatment. Response to prior treatment with tyrosine kinase inhibitors may predict prognosis of stem cell transplant (American Journal of Hematology, PMID 28543934 ). Lowering the risk for graft-versus-host disease (GVHD) in people with CML who receive stem cell transplants is another area of research. Possible ways of reducing GVHD include studies of: antithymocyte globulin (ATG) (Cancer, PMID 28301690 ) vorinostat (Zolinza) plus tacrolimus or methotrexate (Blood, PMID 28784598 ) Learn more about cancer research Researchers continue to try to find out more about CML. Clinical trials are research studies that test new ways to prevent, detect, treat or manage CML. Clinical trials provide information about the safety and effectiveness of new approaches to see if they should become widely available. Most of the standard treatments for CML were first shown to be effective through clinical trials. Find out more about clinical trials . Expert review and references
- Versha Banerji, MD, FRCPC
IMAGES
VIDEO
COMMENTS
In 2007, a French CML study showed that 50% of patients who had been negative for BCR-ABL1 transcript for approximately 2.5 years, in whom TKI
In the treatment of chronic phase CML, imatinib produces a superior and sustainable response compared to INFα. The IRIS study (International Randomised Study of
Chronic myeloid leukaemia articles from across Nature Portfolio. Definition ... Leukemia 37, 505-517. Research 17 December 2022 | Open Access
Leukemia - Chronic Myeloid - CML: Latest Research · Combining imatinib with other drugs · Determining whether people with CML can safely stop taking TKIs after a
Twenty-two years after the first patients with chronic myeloid leukemia (CML) were treated with the tyrosine kinase inhibitor (TKI) imatinib, outcome exceeds
Prognosis of long-term survival considering disease-specific death in patients with chronic myeloid leukemia. Leukemia. 2016; 30: 48-56. View in Article. Scopus
In a phase III randomized study, patients with newly diagnosed CML-CP were assigned to one of four treatment arms (imatinib 400 mg once daily
Fibroblast lines have been used extensively in CML research because they are easy to manipulate. Fibroblast transformation—that is, anchorage-independent growth
More than two-thirds of patients with chronic phase CML achieve long-term control of the disease with TKIs. Although TKIs have not been proven
We are always learning more about cancer. Researchers and healthcare professionals use what they learn from research studies to develop better practices