1. Introduction
Chronic Myeloid Leukemia (CML) is a clonal disease, originated at the level of Hematopoietic Stem Cells (HSC) and characterized by the presence of the
Current treatment options in CML include tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib), compounds that inhibit the activity of the BCR-ABL protein. However some patients will develop resistance or intolerance to these drugs and resistance has been associated with different mechanism including the quiescence of leukemic stem cells and Pgp or Src kinase overexpression.
In this chapter we focus on the basic biology of hematopoietic stem and progenitor cells from CML and analyze the most relevant and current concepts in this area.
2. Chronic myeloid leukemia
Chronic myeloid leukemia (CML) is a lethal hematological malignancy characterized by the abnormal amplification of the myeloid (mainly granulocityc) compartment of the hematopoietic system. It originates from the transformation of a primitive hematopoietic cell that suffers a t(9;22) (q34; q11) balanced reciprocal translocation that results in the generation of the Philadelphia chromosome (Ph). Ph produces BCR-ABL, a constitutively active tyrosine kinase that drives a wide variety of physiological alterations [1].
CML was initially described in 1845 by John Hughes Bennett, who reported the case of a patient with “milky” blood and suggested that it was an infectious disease that caused hypertrophy of the liver and spleen, leading to the patient’s death. A few weeks later, Rudolf Virchow reported a similar case, but, in contrast to Bennett, he suggested that the disease was not infectious and implied an increase in the number of blood cells. He coined the term leukemia (from the Greek
2.1. Epidemiology and clinical characteristics
Chronic myelogenous leukemia has a worldwide incidence of 1-2 cases per 100,000 individuals [4]. The average age at diagnosis is 60 years; it occurs less frequently in young people and a tendency to increase exponentially with age has been observed. There is no geographic or genetic predisposition to acquire this condition, although some authors have associated it with exposure to high doses of ionizing radiation. The current CML prevalence of 24,000 affected patients in the United Sates is relatively low; it is expected to increase significantly over the next 20 years as a result of widespread use of BCR-ABL tyrosine kinase inhibitor therapy [5]. In Mexico, there are no official data on the incidence of such a disease, however, it has been estimated that there are about 80,000 cases of leukemia and 10% corresponds to CML [6].
The clinical presentation often includes granulocytosis, spenomegaly and marrow hypercellularity; however about 40% of patients are asymptomatic and their diagnosis is based on abnormal blood cell counts [1]. The natural course of the disease involves three sequential phases, namely chronic, accelerated and blast crises. Ninety percent of patients are diagnosed in chronic phase and they remain in it for 3 to 8 years. In this phase, the blood cells retain their ability to differentiate until the illness progresses to the accelerated phase, which is characterized by the egress of immature cells into the bloodstream. Finally, the disease progresses to the blast crisis, defined by the presence of 30 percent or more leukemic cells in peripheral blood or marrow or extramedullary infiltrates of blast. During this phase the survival of patients is reduced to months and even weeks [7].
2.2. Molecular events (Bcr-Abl oncogene)
As mentioned before, the Philadelphia chromosome, which defines CML, is a shortened chromosome 22 originated from the reciprocal translocation between the long arms of chromosomes 9 and 22 [t (9; 22)] and involves addition of 3' segments of the
The normal human ABL gene encodes for a non-receptor tyrosine kinase that is ubiquitously expressed. Such a 145 kDa protein is involved in the regulation of the cell cycle, the response to genotoxic stress, and intracellular signaling mediated by the integrin family [9]. There are three isoforms of the BCR-ABL fusion protein all of which encode the same portion of the ABL tyrosine kinase, but differ in the length of the BCR sequence at the N-terminus. p185/p190 BCR-ABL is expressed in Acute Lypmphoblastic Leukaemia (ALL), p210 BCR-ABL is characteristic of Chronic Myeloid Leukemia, and p230 BCR-ABL has been associated with a subgroup of CML patients with a more indolent disease (Figure 1) [4].
BCR-ABL fusion protein localizes in the cytoplasm and shows an increased and constitutive tyrosine kinase activity as a result of oligomerization of its coiled region and deletion of the SH domain of ABL. It activates a number of cytoplasmic and nuclear signal-transduction pathways involved in cell adherence, migration, inhibition of apoptosis, and induction of cell proliferation through activation of signaling proteins such as p21RAS, c-Myc, lipid kinasse PI3k, MAPk (mitogen-activated protein kinase family), tyrosine phosphatases, and signal transducer and activator of transcription (STATs) factors [9, 10].
2.3. Leukemic Stem Cells in chronic myeloid leukemia
There is an increasing body of evidence indicating that, similar to normal hematopoiesis, a quiescent stem cell population -within the CD34+ cell compartment- exists in the bone marrow of CML patients. Such Leukemic Stem Cells (LSC) seem to be the ones driving CML progression, following a similar pattern to the one observed in normal hematopoiesis. That is to say, LSC give rise to CML progenitor cells, which, in turn, give rise to more mature cells.
Just like normal hematopoietic stem cells (HSC), CML stem cells express high levels of CD34, and lack the cell surface markers CD38, CD45RA, or CD71, as well as lineage-specific markers. However, LSC are Ph+/BCR-ABL+, which is not present in their normal counterparts. Interestingly, it has recently been shown that a novel population of lineage-negative, CD34-negative hematopoietic stem cells from CML patients also correspond to BCR-ABL+ leukemic stem cells capable to engraft immunodeficient mice [13]. Thus, it seems that most LSC are CD34+ but a subpopulation may be CD34-. Importantly, despite the predominance of LSC in CML, a residual population of normal hematopoietic stem cells (BCR-ABL- CD34+) persists in the marrow’s patient, which seems to be responsible for hematopoietic recovery after a successful treatment using Tyrosine Kinase Inhibitors (TKIs).
As mentioned before, LSC are in a quiescent state, however, they can spontaneously exit G0 to enter a proliferating state and are capable of engrafting inmmunodeficient mice [11]. In this regard, several studies have shown that TKIs, like Imatinib, Nilotinib, Dasatinib, Bosutinib, and Lonafarnib, have antiproliferative or apoptotic effects in almost all dividing CML cells; however, the population of stem cells remains viable in a quiescent state [16-21].
In vitro studies indicate that LSCs are capable of surviving for several weeks in the absence of added growth factors due to autocrine mechanisms involving production of granulocyte colony-stimulating factor (G-CSF) and Interleukin 3 (IL-3) [12]. This, in fact, is an important difference between normal and CML HSC, since the former depends on the presence of exogenous cytokines for their growth, whereas the latter, as just mentioned, can utilize autocrine mechanisms. Although there is strong evidence that Bcr-Abl is sufficient to induce CML-like disease in transduction and transgenic murine models [14], it is still unclear whether Bcr-Abl is always the first hit in CML, since in some patients with a complete cytogenetic response after treatment, BCR-ABL transcripts are still detectable by RT-PCR, which indicates that leukemic cells persist even when the disease is reduced below detectable limits [15].
3. Functional characteristic of leukemic stem cells in CML
3.1. Proliferation
Proliferation of leukemic stem and progenitor cells is regulated by Bcr-Abl. Such a tyrosine kinase activates the Ras/Raf/MEK/ERK and JAK/STAT signal transduction pathways, and this results in an amplified proliferative state [22]. Bcr-Abl causes hyperactivity of Ras, Raf and JAK/STAT, which can occur by multiple mechanisms; i.e., by Bcr-Abl activating these pathways directly, or by the induction of autocrine cytokines, which in turn activate these pathways [23]. Bcr-Abl autophosphorylation of tyrosine 177 provides a docking site for the adapter molecule Grb-2. 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 [9, 24]. Ras activates Raf, and finally, Raf initiates a signaling cascade through the serine–threonine kinases Mek1/Mek2 and Erk, which ultimately leads to the transcription of genes involved in cell proliferation and survival (Figure 1), such as c-Myc, Cyclin D, Cyclin A, Bcl-2, cytokines, etc [22].
The JAK/STAT pathway has been demonstrated to be constitutively activated In CML. Among all the molecules participating in these pathways, STAT1 and STAT5 have been found to be the two major STATs phosphorilated by Bcr-Abl. STAT5 has pleiotropic physiologic functions, and its main effect in Bcr-Abl-transformed cells appears to be primarily anti-apoptotic, involving transcriptional activation of Bcl-xL [25]. Also, in some experimental systems there is evidence that Bcr-Abl induces an IL-3 and G-CSF autocrine loop in early progenitor cells [12].
3.2. Inhibition of apoptosis
Leukemic Stem Cells acquire the ability for long-term survival primarily by deregulation of apoptosis. In CML, blocking of apoptosis is mediated by Bcr-Abl. Bcr-Abl may block the release of cytochrome C from mitochondria and thus activation of caspases. This effect upstream of caspase activation might be mediated by the Bcl-2 family of proteins [26]. Bcr-Abl has been shown to up-regulate anti-apoptotic protein Bcl-xL in a STAT5-depend manner, as mention above [27]. Another link between Bcr-Abl and the inhibition of apoptosis might be the phosphorylation of the pro-apoptotic protein Bad through PI3k pathway. Bcr-Abl forms multimeric complexes with PI3 kinase, Cbl, and the adapter molecules Crk and Crkl, in which PI3 kinase is activated. The next substrate in this cascade appears to be the serine-threonine kinase Akt. This kinase had previously been implicated in antiapoptotic signaling and protein Bad as a key substrate of Akt (Figure 1). 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 [28].
3.3. Altered adhesion properties
In CML, progenitor cells exhibit decreased adhesion to bone marrow stroma cells and extracellular matrix. From this point of view, adhesion to stroma negatively regulates cell proliferation, and CML cells escape this regulation by virtue of their perturbed adhesion properties. Bcr-Abl directly phosphorylates Crkl, a protein involved in the regulation of cell motility and in integrin-mediated cell adhesion by association with other focal adhesion proteins such as paxillin, the focal adhesion kinase Fak, p130 Cas and Hef1 [29, 30] (Figure 1). In addition to this, it has been demonstrated that the activity of Bcr-Abl promotes expression of integrin β1, a variant not found in the normal counterpart that inhibits adhesion to stroma and cell matrix, together with the effect of expansion and premature exit of myeloid progenitors and precursors to bloodstream [31].
3.4. Self-renewal
Deregulation of self-renewal has been recognized as an important event in disease progression. In normal hematopoietic stem cells, self-renewal capacity involves several signaling pathways: Notch, Wnt, Sonic Hedgehog (Shh), FoxO and Alox5 [32-34].
Notch receptors are an evolutionarily conserved family of trans-membrane receptors that are known to be expressed and activated in normal HSC. Binding to their physiological ligands, which are part of the Delta and Serrata families, leads to separation of an intracellular portion of Notch. This fragment is capable of entering the nucleus where it binds transcriptional repressor CBF-1. Interconnection of Notch, CBF-1 and the co-factor MAML-1 (mastermind-like-1) leads to transcriptional activation of target genes [35]. Constitutively active Notch is able to mediate multilineage potential
Notch signaling may also be important in advanced stages of CML. Hes1, a key Notch target gene, was found to be highly expressed in 8 out of 20 patients with CML in blast crisis, but was not seen in the chronic phase. In mice, the combination of Hes1 and BCR-ABL expression in myeloid lineage progenitor cells resulted in an acute leukemia resembling blast crisis CML [37]. This suggests that Notch inhibitors may be useful in strategies aimed at eradicating CML LSC.
In normal hematopoiesis, Wnt pathway activity is required in the bone marrow niche to regulate HSC proliferation and to preserve self-renewal capacity [38]. Activation of the canonical Wnt/β-catenin pathway consists of binding of Wnt proteins to members of the Frizzled and low-density lipoprotein receptor related (LPR) families on the cell surface. In the absence of Wnt signals, β-catenin is associated with a large multiprotein complex that includes Axin, APC, and glycogen synthase kinase 3β (GSK3β), among others. Through a mechanism not entirely understood, when Wnt proteins bind to their target, Axin facilitates phosphorylation of β-catenin by GSK3β. Phosphorylation, in turn, results in ubiquitination, targeting β-catenin for degradation. Thus, axin serves as an inhibitor of β-catenin activity. Binding of Wnt proteins to their receptors leads to activation of Disshevled (Dsh), which inhibits phosphorylation of β-catenin by GSKβ, so it accumulates in the cytoplasm and translocates to the nucleus, where it activates transcription factors, such as LEF/TEF and allows expression of target genes [39].
This pathway has been implicated in CML. Indeed, in blast crisis CML, the LSC, which resemble granulocyte-macrophage progenitor cells (GMP), have aberrant activation of β-catenin via the canonical Wnt signaling pathway. In a proportion of these cases, the pathway is activated through abnormal missplicing of GSK3β [40].
The Hedgehog (Hh) pathway is a highly conserved developmental pathway, which regulates the proliferation, migration and differentiation of cells during development [41]. It is typically active during development, but silenced in adult tissues, except during tissue regeneration and injury repair [42]. Three distinct ligands, i.e., Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog exist in humans. Upon ligand binding to the receptor patched (Ptch), inhibition of smoothened (Smo) receptor is relieved. Smo then activates members of the Gli family of zinc-finger transcription factors, which translocate to the nucleus to regulate the transcription of Hh target genes, including Gli1, Gli2, Ptch and regulators of cell proliferation and survival [43].
Based on murine embryonic stem cell studies, it has been found that Hh signaling plays major roles during primitive hematopoiesis. Ihh is a primitive endoderm-secreted signal and is sufficient to activate embryonic hematopoiesis and vasculogenesis [44]. Furthermore, a study of zebrafish showed that the mutations of the Hh pathway members or inhibition of the Hh pathway with the Hh inhibitor cyclopamine can cause a developmental defect in adult HSC [45]. In addition, activation of Hh pathway has been observed in different human cancers. In CML patients, more than four-fold induction of the transcript levels of Gli1 and Ptch was observed in CD34+ cells in both chronic phase and blast crisis. In two studies using a CML mouse model, recipients of the Bcr-Abl transduced bone marrow cells from Smo-/- donor mice developed CML significantly slower than recipients of Bcr-Abl transduced bone marrow cells from wild-type donor mice. When the frequency and function of the LSCs were examined, Smo deletion caused a significant reduction of the percentage or LSCs [46]. By contrast, over expression of Smo led to an increased percentage of LSC and accelerated the progression of CML [47].
The FoxO (Forkhead-O) subfamily of transcription factors regulate cell cycle, stress resistance, differentiation, and long-term regenerative potential of HSC [48], and protect integrity of the stem cell pool. There are four members (FoxO1, FoxO3, FoxO4 and FoxO6) and are known to be effectors of the PI3k/AKT pathway, which is frequently mutated or hyperactivated in hematologic malignancies, and are abundantly expressed in the hematopoietic system. Akt directly phosporylates the FoxO members from the nucleus and promotes its degradation in the cytoplasm. FoxO members localize to the nucleus and regulate apoptosis, cell cycle progression and oxidative stress responses [49]. In a model of deficient FoxO mice it was shown a defect in the long-term expansion capacity of the HSC pool. Such a defect has been correlated with increased cell division and apoptosis of HSCs.
FoxO transcription factors have also been shown to have essential roles in the maintenance of CML LSCs [50]. FoxO3 localizes to the cell nucleus and it causes a decrease in Akt phosphorylation in the LSC population. In addition, serial CML transplantation showed that FoxO3 deficiency severely impairs the ability of LSCs to induce CML. Furthermore, transforming growth factor-β (TGF-β) is a crucial regulator of Akt activation and controls FoxO3 localization in LSCs of CML. A combination strategy of TGF-β inhibition, FoxO3 deficiency and Bcr-Abl kinase inhibition results in efficient LSCs depletion and suppression of CML development [51].
The Alox5 pathway is the only one signaling pathway not shared by LSC with normal HSC. The
4. Current therapies
The first effective treatment for CML was the solution of Fowler's, which contained arsenic as active component and was used in the early 20th century. Later between 1920 y1930 irradiation to the spleen was the main therapeutic option, since it offered patients the decrease of symptoms, although it did not prolong their lives. In 1953, busulfan was included in CML treatment. This compound provided benefit in terms of survival, although it was shown to be extremely toxic for hematopoietic progenitor cells. The next drugs effective in the treatment of CML were hydroxyurea and cytosine arabinoside, both less toxic than busulfan and able to block proliferation of cells, but unable to induce specific damage to leukemic cells; thus, patients usually progressed to the accelerated and blast crisis phases [57].
4.1. Interferon-α
Interferon-α (IFNα) was the first drug capable of extending the chronic phase of the disease and retarding the evolution to the accelerated phase. IFNα is a nonspecific stimulant of the immune system that regulates T-cell activity and produces a complete hematologic response (CHR) in 40-80% of patients, and a complete cytogenetic response (CCR) in 6-10% of patients with a median survival of 89 months [58].
In vitro studies have indicated that IFN
Because IFNα is a nonspecific immunostimulant, it produces secondary symptoms and toxicities and many patients discontinue therapy. However there are evidence that a significant proportion of IFNα-treated patients in prolonged CCR were able to discontinue treatment without disease relapse [63], and it was recently reported that in a specific group of patients treated with monotherapy there are increased numbers of NK cells and clonal γδ T cells [64].
4.2. Tyrosine kinase inhibitors
Having identified that tyrosine kinase activity of Bcr-Abl is a major factor in the pathophysiology of CML, it was clear that such a molecule was an attractive target for designing a selective kinase inhibitor. In 1996, Buchdunger et al, synthesized several compounds that inhibit the activity of platelet-derived growth factor receptor (PDGF-R) and ABL kinase. One of these was the 2- phenylaminopyrimidine, which served as a starting point for the development of other related compounds [65]. The activity of the 2-phenylaminopyrimidine series was optimized and gave rise to STI571 (also named imatinib mesylate, CGP57148B or Gleevec®, Novartis Pharmaceuticals).
Imatinib is a highly selective inhibitor of the protein tyrosine kinase family, which includes BCR-ABL protein, PDGF-R and the c-kit receptor. It competitively binds to the ATP-binding site of BCR.ABL and inhibits protein tyrosine phosphorylation
Studies in CML marrow by Holyoake and her colleagues have demonstrated the presence of a rare, highly quiescent, CD34+ cell subpopulation in which most of the cells are Ph+ with the ability to proliferate upon specific induction [11]. These cells are insensitive to the effects of STI571 and remain quiescent and viable even in the presence of growth factors [16]. This tumor resistance feature was also reported by Bathia, who mention that STI571 suppressed but does not eliminate primitive cells even after patients remain in CCR [73]. These primitive Ph+ cells could not be detected by nested PCR, when they are obtained from Imatinib-treated patients; however, when the cells are cultured in liquid cultures for a couple of weeks, the Ph+ population becomes detectable, indicating that they were able to remain even after Imatinib treatment [74].
In clinical trials, Imatinib has been shown remarkably effective as a single agent in IFNα-resistant CML chronic phase patients. It induces complete cytogenetic responses in more than 80% of newly diagnosed patients; however, the persistence of detectable leukemic cells in a quiescent state and the presence of patients with resistance or intolerance to Imatinib, lead to the development of a second generation of Tyrosine Kinase Inhibithors.
Nilotinib (Tasigna, Novartis Pharmaceutical), is an oral aminopyrimidine that is a structural derivative of Imatinib. It was designed to be more selective against the Bcr-Abl tyrosine kinase than imatinib. Like imatinib, it acts through competitive inhibition of the ATP site in the kinase domain [75]. Clinically Nilotinib showed activity in imatinib-resistant patients in all phases of the disease. In chronic phase, it induced 92% of CHR and in accelerated phase and blast crisis the hematological responses were achieved in 72% of cases [76].
In vitro, Nilotinib is 20 times more potent than imatinib against cells expressing wild type Bcr-Abl, and similar results have been observed in studies of mutants cell lines, with the exception of the T315I mutation, which is resistant to both TKIs [77]. In primary CML CD34+ cells, Imatinib-induced apoptosis is preceded by Bim accumulation; this effect was decreased when cells were cultured in a cytokine-containing medium [78]. In contrast to Imatinib, whose main effect on CML cells seems to be induction of apoptosis, the predominant effect of nilotinib seems to be antiproliferative -rather than apoptotic [17]. Indeed, it has been suggested that Nilotinib can induce a G0/G1 cell cycle blockade in cells expressing wild type Bcr-Abl, which could result in disease persistence [79].
Dasatinib (Sprycel, Bristol-Myers Squibb) is a potent, orally bioavailable thiazolecarboxamide. It is structurally unrelated to imatinib; it has the ability to bind to multiple conformations of the Abl kinase domain and it also inhibits SRC family kinases. In vitro, Dasatinib demonstrated 325-fold greater activity against native Bcr-Abl, as compared with imatinib, and it has shown efficacy against all imatinib-resistant Bcr-Abl mutants with the exception of T351I. Dasatinib is also active against PDGFR, C-Kit and ephrin A receptor [75, 76].
Dasatinib is very effective at inducing apoptosis in CML cells –either, in the presence or absence of added growth factors- and in contrast to Imatinib, that kills those cells destined to move from G0/G1 cell cycle phases, but is unable to act on those cells destined to remain quiescent in culture, Dasatinib can act on quiescent CD34+ cells. As expected, based on its structure and mode of action, it has selective cytotoxic activity for leukemic cells over normal cells [80].
Several TKIs have been developed that exhibit a target spectrum similar to the approved drugs, although they are distinct in terms of off-target effects [81].
Bosutinib (Wyeth) is a 4 anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases without effect in c-Kit or PDGFR. It has 200-fold grater potency for Bcr-Abl than imatinib and has activity against a number of mutations, but not T315I [76]. In clinical trials, Bosutinib induced 73% of complete hematological response in patients pretreated with Imatinib followed by Dasatinib [82]. In vitro, Bosutinib effectively inhibits Bcr-Abl kinase activity and Src phosphorylation, and reduces the proliferation and CFC growth in CML CD34+ cells; however, it does not seem to induce apoptosis [19].
Ponatinib, is a mulitargeted kinase inhibitor that is active against all BCR-ABL mutants, including T315I. This drug also inhibits FLT3, FGFR, VEGFR, c-Kit, and PDGFR and is able to reduce the proliferation of different cell lines and prolong survival of mice that have been injected intravenously with BCR-ABL. Ponatinib showed significant activity in a phase I study of patients with Ph+ cells who had failed to other TKIs [81, 83].
4.3. Hematopoietic cell transplant
Although molecular therapy for CML is highly effective and generally non-toxic, it is unclear whether long-term outcomes with the different therapies (IFNα or TKIs) will be equivalent to cases treated with allogeneic stem cell transplantation, which has shown the highest percentage of long-term disease-free survival of any therapy [75].
In patients younger than 50 years of age and who receive a transplant before 1 year after diagnosis, 5 years survival rates superior to 70% have been attained. However, the application of this procedure is limited by the availability of matched donors and by the toxicity of the procedure in older patients. Moreover, outcomes deteriorate with disease duration [76]. This information associated with the knowledge that quiescent leukemic stem cells remain in patients after treatment, several other agents has been reported.
4.4. Other agents
Danusertib (PHA 739358) is a small molecule with activity against BCR-ABL and aurora kinases and it is able to block the proliferation of leukemia cell lines as well as CD34+ cells from newly diagnosed CML patients including the mutation T315I. However, similarly to other tyrosine kinase inhibitors, no induction of apoptosis in quiescent hematopoietic stem cells could be achieved and resistant BCR-ABL positive clones emerged in the course of Danusertib treatment. This latter observation is related to Abcg2 proteins over-expression [84].
Lonafarnib (SCH66336) is an orally bioavailable non peptidomimetic farnesyl trransferase inhibitor with significant activity against Bcr-Abl+ cell lines and primary CML cells. It can enhance the toxicity of Imatinib in K562 cell line and can inhibit the proliferation of imatinib-resistant cells and increases imatinib-induced apoptosis. However it is unable to kill quiescent CD34+ leukemic cells [20]. In a clinical phase 1 study, it was shown that the combination of Lonafarnib and Imatinib is well tolerated in patients with CML who failed Imatinib, with some patients achieving a complete hematologic response and a complete cytogenetic response [85].
INNO 406 is a 2 phenylaminopyrimidine Bcr-Abl inhibitor with activity against PDGF, c-kit and Lyn that have shown to be 25-55 times more potent than Imatinib in Bcr-Abl+ cell lines. In contrast to other molecules INNO406 does not inhibit all SRC kinases, but it induces programmed cell death in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways [86].
MK0457 is an aurora kinase inhibitor with activity against Bcr-Abl. This agent was observed to inhibit autophosphorylation of T315I mutant and demonstrate antiproliferative effects in CML cells derived from patients with this mutation, an event that may lead to its use as a combination partner with the approved and established TKI [76].
5. TKI resistance mechanisms
The knowledge of the central role of BCR-ABL in the pathogenesis of CML has allowed the development of several drugs that inhibit the constitutive activity of such an ABL tyrosine kinase. However, although the treatment with tyrosine kinase inhibitors has proven effective in about 80% of CML patients at any stage, the remaining 20% can’t respond to it [87].
In CML, the criteria for successful response to treatment, as established by the European consortium LeukemiaNet and subsequently adopted by the National Comprehensive Cancer Network (NCCN) [88], include: complete hematologic remission (CHR), that is to say, a normal blood cell count and complete disappearance of signs and symptoms of the disease; complete cytogenetic response (CCR), which means the total absence of Ph+ metaphases; and complete molecular response, in which transcripts for BCR-ABL are no longer detectable. Using these response criteria, drug resistance is defined as the inability to achieve any of the following: a complete hematologic response (CHR) at 3 months, any cytogenetic response (CyR) at 6 months, partial cytogenetic response (PCyR) at 12 months, or a complete cytogenetic response (CCR) at 18 months of treatment with Imatinib [89].
Two types of resistance mechanisms to TKIs have been described: 1) Primary resistance, which occurs in less than 10% of cases and is defined as the failure of therapeutic effect during the chronic phase of CML without changing clones; and 2) secondary resistance, defined as the loss of the response initially obtained, and commonly occurs in accelerated phase (40-50%) and blast (80%) [90]
It is estimated that the probability of an individual to stay in CCR for 5 years after diagnosis, after treatment with Imatinib is approximately 63%; however, this percentage may represent a sub-estimation since in a significant proportion of cases there is discontinuation of treatment and this, of course, may underestimate the efficacy of the drug [91].
The molecular mechanisms of acquired drug resistance can be divided into two categories: BCR-ABL-dependent and BCR-ABL-independent.
5.1. Bcr-Abl-dependent resistance mechanisms
The inhibition of the activity of tyrosine kinase turned out to be an ideal target for molecular therapy in CML [67]. However, shortly after the introduction of Imatinib, in vitro studies demonstrated that some cell lines became refractory to the drug, suggesting a possible inherent or acquired resistance to therapy [92]. This was quickly followed by the clinical description of patients resistant to Imatinib.
The most common mechanism against TKIs therapy are point mutations within the kinase domain, which make conformational changes that decrease the affinity of the TKIs to BCR-ABL kinase domain. These point mutations in the
The first point mutation reported in TKI resistance was in the region coding for the ATP-binding site of the ABL kinase domain resulting in a threonine to isoleucine substitution at amino acid 315 (Th315→Ile315; T315I) preventing the formation of a hydrogen bond between the oxygen atom provided by the side chain of threonine 315 and the secondary amino group of Imatinib. Moreover, isoleucine contains an extra hydrocarbon group on its side chain, and this inhibits the binding of Imatinib [94]. T315I confers resistance to all currently approved BCR–ABL kinase inhibitors. Recent reports have shown that T315I mutation can be found in approximately 15% of patients after failure of imatinib therapy [85].
Other important TKI’s resistant mutations are frequently mapped to the P-loop region (residues 244 to 256) of the kinase domain, which serves as a docking site for phosphate moieties of ATP and interacts with imatinib through hydrogen and van der Waals bonds. These mutations modify the flexibility of the P-loop and destabilize the conformation required for Imatibib binding [95]. Clinical relevance of P-loop mutations is that imatinib treated patients who harbor them have been suggested to have a worse prognosis than those with non-P-loop mutations [96]. Another study identified BCR/ABL mutations in CD34+ cells from CML patients in CCR following Imatinib treatment and suggested that these mutations could lead to imatinib resistance in a small population of progenitors, which consequently could expand and cause the relapse [97].
Several additional mutations that disrupt the interaction between TKIs and BCR-ABL have been characterized, including the P-loop, C-helix, SH2 domain, substrate binding site, A-loop, and C-terminal lobe, some even prior to the initiation of therapy [98]. Most of the reported mutants are rare, however seven mutated sites constitute two thirds of all detected mutations: G250, Y253, E255 (P loop), T315I (gatekeeper), M351, F359, and H396 (activation loop or activation loop backbone) and are frequently evident in the later disease stages [99]. Recently a pan-BCR-ABL inhibitor active against the native enzyme and all tested resistant mutants, including the uniformly resistant T315I mutation has been developed [100].
BCR-ABL kinase domain mutations are not induced by the drug, but rather, just like antibiotic-resistance in bacteria, arise through a process whereby rare pre-existing mutant clones are self-selected due to their capacity to survive and expand in the presence of the drug thus gradually outgrowing drug-sensitive cells [101].
Overexpression of Bcr-Abl leads to resistance by increasing the amount of target protein needed to be inhibited by the therapeutic dose of the drug. Amplification of the BCR–ABL gene was first described in resistant CML cell lines generated by serial passage of the cells in Imatinib containing media and demonstrated elevated Abl kinase activity due to a genetic amplification of the Bcr–Abl sequence [102, 103].
Cells expressing high amounts of Bcr-Abl in CD34+ CML cells, as in blast crisis, are much less sensitive to Imatinib and, more significantly, take a substantially shorter time for yielding a mutant subclone resistant to the inhibitor than cells with low expression levels, as in chronic phase [104]. However overexpression and amplification of the
5.2. BCR-ABL-independent resistance mechanisms
HSC are characterized by their ability to pump-out fluorescent dyes, and this led to isolation of stem cells based on this property. In fact, such an efflux capacity has become one of the most efficient methods to purify stem cells from different sources [105]. In this regard, ATP-binding cassette (ABC) transmembrane transporters have shown to be responsible for most of the efflux of the fluorescent dyes in HSCs [106].
In cancer cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation, which is attributed to the overexpression of ABC transporter proteins [107]. The first studies on imatinib-resistance showed increased levels of the multidrug resistance protein MDR1 (ABCB1) in Imatinib resistant BCR-ABL+ cell lines [108]. Later on, it was confirmed that Imatinib is a substrate of membrane ABC transporters, such as ABCB1 (MDR1, P-gp), and that variations in the activity or expression of P-gp affects the pharmacokinetics of Imatinib, reducing or increasing its bioavailability [109]. P-gp-positive leukemic cells have low intracellular levels of Imatinib; decreased Imatinib levels, in turn, were associated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of Imatinib on cellular proliferation and apoptosis. The modulation of P-gp by Ciclosporin A readily restored imatinib cytotoxicity in these cells [110].
Another drug efflux pump, the breast cancer resistance protein BRCP encoded by ABCG2, has also been implicated in Imatinib resistance. Imatinib has been variably reported to be a substrate and/or an inhibitor for the BCRP/ABCG2 drug efflux pump, which is overexpressed in many human tumors and also found to be functionally expressed in CML stem cells [111, 112].
CML stem cells have been shown to express the ATP dependent transporter cassette protein ABCG2, which could decrease the intracellular accumulation of Imatinib in CML LSC [103]. Thus, overexpression of ABC transporters gives protection to tumor cells from TKIs [114].
Inversely to the drug efflux pump proteins, the human organic cation transporter 1 (OCT1) mediates the active transport of Imatinib into cells, and inhibition of OCT1 decreases the intracellular concentration of Imatinib [115]. OCT1 was also found to be expressed in significantly higher levels in patients who achieved a CCR to Imatinib than in those who were more than 65% Ph chromosome positive after 10 months of treatment [116]. Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial suggested that patients with lower hOCT1 levels had reduced MMR rates at 12 months when receiving the standard dose of Imatinib, compared with high-dose Imatinib [117].
Recently Engler and cols. found that the intracellular uptake and retention (IUR) of imatinib, OCT-1 activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells. However, no differences in IUR or OCT-1 activity were observed between these subsets in healthy donors. Low Imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 activity may be a critical determinant of long-term disease persistence [118].
Differential interactions between drug efflux/influx pumps and kinase inhibitors might be a possible means to tailor drug selection for individual patients, because OCT-1 expression is a key determinant of intracellular availability of Imatinib but not of Nilotinib [119]. Other TKIs, such as Dasatinib and, as just mentioned, Nilotinib, do not appear to be substrates for hOCT1, but whether this difference alone will lead to reduced resistance rates with these second-generation TKIs remains unknown [120]. An adequate balance between influx (hOCT1) and efflux (MDR1, ABCG2) transporters may be a critical determinant of intracellular drug levels and, hence, resistance to Imatinib.
One feature of CML is the presence of a population of highly quiescent primitive cells [11], which, as their normal counterparts, is capable of regenerating hematopoiesis and reconstitutes the disease in immunocompromised mice [121]. These stem cells are Ph+, express high levels of CD34 and do not express CD38, CD45RA and CD71, and may spontaneously exit the G0 phase and enter a state of constant proliferation [122]. Several reports have documented that quiescent cells from CML patients are insensitive to in vitro treatment with Imatinib and Dasatinib [16, 123].
A possible cause of insensitivity to TKIs is that BCR-ABL mRNA transcript levels are 300-fold higher in the most primitive CD34+CD38-Lin- population than in terminally differentiating CD34-Lin+ CML cells [124]. It has been reported that elevated levels of Bcr-Abl confer reduced sensitivity to Imatinib [125]. Moreover, the quiescent state of CML stem cells allows them to evade chemotherapy treatments, which are designed to eliminate metabolically active cell population as well as targeted therapies, thus contributing to relapse when treatment with tyrosine kinase inhibitors is discontinued.
BCR-ABL activates different signaling pathways that promote the growth and survival of hematopoietic cells, thus inducing cell transformation. These pathways include Ras, mitogen activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), stress-activated protein kinase (SAPK), nuclear factor kappa B(NF-kB), signal transducers and activators of transcription (STAT), phosphoinositide 3- (PI-3) kinase, and c-Myc [126]. A well characterized pathway involves the Src Family Kinases (SFKs), which are activated by BCR-ABL and the subsequent inhibition of BCR-ABL by Imatinib may not result in the complete inhibition of Src family kinases elucidating a Bcr-Abl independent mechanism of imatinib resistance [127]. Phosphorylation of the Bcr-Abl SH2 and SH3 domains by the SFK may increase the activity of the Abl kinase and may alter its susceptibility to Imatinib [128].
Activation of the Janus kinase (Jak) and subsequent phosphorylation of several Signal Transducer and Activator of Transcription (STAT) family members has been identified in both Bcr–Abl–positive cell lines and in primary CML cells and may contribute to the transforming ability of Bcr–Abl [129].
The tyrosine residue at position 177 within the BCR portion is essential for the binding of adaptor proteins, including Growth Factor Receptor-Bound Protein 2 (GRB2) GRB10, 14-3-3, and the SH2 domain of ABL1 [130]. Bcr-Abl protein is able to activate the Ras/Raf/Mek kinase pathway and the phosphatidylinositol 3′ kinase (PI3K)/Erk pathways through GRB2 [131, 132].
Autocrine loops could contribute to resistance. It has been demonstrated that IL-3 and granulocyte-colony G-CSF are produced within primitive CD34+ cells from patients with CML-CP, both of these cytokines stimulate cellular proliferation in an autocrine manner and protect cells from Imatinib-induced apoptosis [122].
6. Concluding remarks
The presence of a rare population of cells capable of initiating and sustaining leukemia in CML (LSC) has major implications for the biology of the disease and the development of new and more effective treatments. As recognized by several investigators, LSC are key players in the origin and progression of CML, as well as in the reappearance of the disease after treatment. Thus, it is evident that novel therapies must be directed towards the elimination of such cells. However, since their numbers within the marrow microenvironment are extremely low, as compared to the bulk of the malignant cells, and their biology is quite different from that of the rest of the CML cells, the task of finding solutions to this problem is a rather difficult one. It is a great challenge, but significant advances will surely be achieved in the years to come.
Acknowledgments
Antonieta Chàvez-González is recipient of funding from the National Council of Science and Technology CONACYT (grant CB 2008-01-105994) and from the Mexican Institute for Social Security IMSS (grant IMSS/PROT/G11/946). Dafne Moreno-Lorenzana and Socrates Avilés-Vazquez are scholarship holders from CONACYT and IMSS. Héctor Mayani is a scholar of FUNDACION IMSS (Mexico) and his research is supported by grants from the National Council of Science and Technology-CONACYT (grant SALUD-69664).
References
- 1.
Chronic myeloid leukemia. New England Journal of MedicineSawyers C. L 1999 340 1330 1340 - 2.
A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrina fluorescence and Giemsa staining. NatureRowley J 1973 243 290 293 - 3.
Leukaemia- A brief historical review from ancient times to 1950. British Journal of HaematologyPiller G 2001 112 282 292 - 4.
STI571 in Chronic Myelogenous Leukaemia. British Journal of HaematologyTsao A Kantarjian H Talpaz M et al 2002 119 24 - 5.
Chronic Myeloid Leukemia Stem Cell Biology. Current Hematologic Malignant ReportsCrews L Jamieson C 2012 7 125 132 - 6.
La leucemia Mieloide Crónica en el Siglo XXI. Biologìa y Tratamiento. Revista de Investigación ClínicaChávez-gonzález A Ayala-sanchez M Mayani H et al 2009 61 221 232 - 7.
The Biology of Chronic Myeloid Leukemia. The New England Journal MedicineFaderl S Talpaz M Estrov Z et al 1999 341 164 172 - 8.
Non random distribution of genomic features in breakpoint regions involved in chronic myeloid leukemia cases with variant t(9;22) or additional chromosomal rearrangements. Molecular Cancer.Albano F Anelli L Zagaria A et al 2010 9 120 135 - 9.
The molecular biology of chronic myeloid leukemia. BloodDeininger M Goldman J Melo J 2000 96 3343 3356 - 10.
Molecular biology of bcr-ablQuintas-cardama A Cortes J 1 positive chronic myeloid leukemia. Blood2009 - 11.
Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. BloodHolyoake T Jiang X Eaves C Eaves A 1999 2056 2064 - 12.
Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia. Proceedings of the National Academy of SciencesJiang X Lopez A Holyoake T et al 1999 96 12804 12809 - 13.
Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib. BloodLemoli R Salvestrini V Bianchi E et al 2009 114 5191 5200 - 14.
Induction of chronic myelogenous leukemia in mice by theDaley G Van Etten R Baltimore D et al 210 bcr-abl gene of the Philadelphia chromosome. Science1990 - 15.
Mayani Flores-Figueroa, Chavez-Gonzalez. In vitro biology of human myeloid leukemia. Leukemia Research.2009 33 624 637 - 16.
Primitive quiescent Philadelphia positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. BloodGraham S Jorgenssen H Allan E et al 2002 99 319 325 - 17.
Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34 CML cells. BloodJorgensen H Jordanides,Allan E et al 2007 109 4016 4019 - 18.
Dasatinib (BMS354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. BloodCopland M Hamilton A Erlick L et al 2006 107 4532 4539 - 19.
Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI 606. BloodKonig H Holyoake T Bhatia R et al 2008 111 2329 2338 - 20.
Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro. LeukemiaJorgensen H Allan E Graham S et al 2005 19 1184 1191 - 21.
Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival. BloodHamilton A Helgason V Schemionek M et al 2012 119 1401 1510 - 22.
JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. LeukemiaSteelman L. S Pohnert S. C J. G Shelton et al 2004 18 189 218 - 23.
Constitutive activation of the JAK2STAT5 signal transduction pathway correlates with growth factor independence of megakaryocytic leukemia cell lines.Liu R Fan C Garcia R et al 1999 93 2369 2379 - 24.
Oda Heaney C, Hagopian JR, et al. Crkl is the major tyrosine-phosphorylated protein in neutrophils of patients with CML. Journal of Biological Chemistry1994 269 22925 22928 - 25.
Ilarioa R Jr Van Etten R.210 and P190 (BCR/ABL) induce the tyrosine phosphorylation of SHc proteins in human tumors. Oncogene1995 - 26.
Amarante Mendes G Naekygung C, Liu L, et al. Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockade of mitochondrial release of cytochrome C and activation of caspase-3. Blood1998 91 1700 1705 - 27.
Transformation of hematopoietic cells by BCR/ABl requires activation of PI3k/Akt depend pathway. EMBO Journal.Skorski T Bellacosa A Nieborowska-skorska M et al 1997 16 6151 6161 - 28.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-xL. CellZha J Harada H Yang E et al 1996 87 619 628 - 29.
Salgia R Pisick E Sattler M et al 130CAS forms a signaling complex with the adapter protein CRKL in hematopoietic cells transformed by the BCRABL oncogene. Journal of Biological Chemistry1996 - 30.
Differential signaling after β1 integrin ligation is mediated through binding of CRKL toSattler M Salgia R Shrikhande G et al 210 CBL) and p110 (HEF1). Journal of Biological Chemistry1997 - 31.
Presence of the adhesion inhibitory β1B integrin isoform on CML but not normal progenitors is at least in part responsable for the decrease CML progenitor adhesión. Blood.Zhao R Tarone G Verfaillie C 1997 a. - 32.
Self-renewal related signaling in myeloid leukemic stem cells. International Journal of HematologyHeidel F Mar B Armstrong S 2011 94 2 109 117 - 33.
Molecular Pathways: BCR-ABL. Clinical Cancer ResearchCiloni D Saglio G 2012 18 4 1610 1613 - 34.
Insights into the stem cells of chronic myeloid leukemia. LeukemiaSloma I Jiang X Eaves A. C et al 2010 24 1823 1833 - 35.
Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nature ImmunologyDuncan A. W 2005 6 3 314 322 - 36.
Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling. Journal of Cell ScienceVarnum-finney B et al 2000 133 4313 4318 - 37.
A novel tumor-supressor function for the Notch pathway in myeloid leukaemia. NatureKlinakins A et al 2011 473 230 233 - 38.
A role for Wnt signalling in self-renewal of haematopoietic stem cells. NatureReya T Duncan A. W Ailles L et al 2003 423 409 414 - 39.
Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK- 3beta-dependent phosphorylation of beta-catenin. EMBO JournalIkeda S Kishida S Yamamoto H et al 1998 17 1371 1384 - 40.
Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer CellZhao C Blum J Chen A et al 2007 12 528 541 - 41.
Hedgehog signaling in animal development: paradigms and principles. Genes and DevelopmentIngham P. W Mcmaon A. P 2001 15 3059 3087 - 42.
In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature.Ahn S Joyner A. L 2005 437 324 331 - 43.
Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochimica et Biophysica ActaTeglund S Toftgard R 2010 1805 182 208 - 44.
Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development (Cambridge, England)Dyer M. A Farrington S. M Mohn D et al 2001 128 1717 1730 - 45.
Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos. Development CellGering M Patient R 2005 8 389 400 - 46.
Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation. Cancer CellDierks C Beigi R Guo G. R et al 2008 14 238 249 - 47.
Hedgehog signaling is essential for maintenance of cancer stem cells in myeloid leukemia. Nature.Blum J et al 2009 458 776 779 - 48.
FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem CellThotova Z Gilliland D. G 2007 1 140 152 - 49.
FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. CellTothova Z et al 2007 128 325 339 - 50.
TGF-β-FOXO signaling pathway maintains leukemia-initiating cells in chronic myeloid leukemia. NatureNaka K et al 2010 463 676 680 - 51.
TGF-beta-FOXO signaling maintains leukaemia-initiating cells in chronic myeloid leukaemia. NatureNaka K Hoshii T Muraguchi T et al 2010 463 676 680 - 52.
Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependentCatalano A Rodilossi S Caprari P et al 53 activation. EMBO Journal2005 - 53.
Henderson Jr WR. Leukotrienes. New England Journal of MedicinePeters-golden M 2007 357 1841 1854 - 54.
Altered arachidonate metabolism by leukocytes and platelets in myeloproliferative disorders. Prostaglandines Leukotrienes and MedicineTakayama H Okuma M Kanaji K et al 1983 12 261 272 - 55.
Selective inhibitors of 5-lipoxygenase reduce CML blast cell proliferation and induce limited differentiation and apoptosis. Leukemia ResearchAnderson K Seed T Plate J et al 1995 19 789 801 - 56.
Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature GeneticsChen Y Hu Y Zhang H et al 2009 41 783 792 - 57.
Targeted chronic myeloid leukemia therapy. Seeking a cure. American Journal of Health System PharmacyFausel C 2007 S9 S15. - 58.
Potential mechanisms of action of interferon-alpha in CML. Leukemia and LymphomaDowding C Gordon M Guo A et al 1993 11 185 191 - 59.
An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture. Journal of Clinical InvestigationCornelissen J Ploemacher R Wognum B et al 1998 102 976 983 - 60.
The tyrosine kinase inhibitors STI561, like interferon-alpha, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia. Experimental HematologyMarley S Deininger M Davidson J et al 2000 28 551 557 - 61.
Interferon-alfa-based treatment of chronic myeloid leukemia and implications of signal transduction inhibition. Seminars in HematologyTalpaz M 2001 38 22 27 - 62.
L-selectin expression is low on CD34+ cells from patients with chronic myeloid leucemia and interferon-a up regulates this expression. HaematologicaMartín-henao G Quiroga R Sureda A et al 2000 85 139 146 - 63.
Chronic myeloid leukemia and interferon alpha: a study of complete cytogenetic responders. BloodBonifazi F De Vivo A Rosti G et al 2001 98 3074 3081 - 64.
Chronic Myeloid Leukemia Patients in prolonged remission following interferon a monotherapy have distinct cytokine and oligoclonal lymphocyte profile. Plos OneKreutzman A Rohon P Faber E et al 2011 6 1 12 - 65.
Inhibition of the Abl protein-tyrosine kinase in vitro and in-vivo by a 2-phenylamiropyrimidine derivative. Cancer ResearchBochdunger E Zimmermann J Mett H et al 1996 56 100 104 - 66.
Structural mechanism for STI571 inhibition of Abelson tyrosine kinase. ScienceSchindler T Bornmann W Pellicena P et al 2000 289 1938 1942 - 67.
Efects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature MedicineDruker B Tamura S Buchdunger E et al 1996 2 561 566 - 68.
CGP57148 a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, REL-ABL, and TEL-PDGFR fusion proteins. BloodCarroll M Ohno-jhones S Tamura S 1997 90 4947 4952 - 69.
Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin NSC680410 on CML cells in vitro. BloodBenjamin M Chandra J Svingen P et al 2002 99 664 671 - 70.
In BCR-ABL positive cells, STAT5 tyrosine-phosphorylation integrates signal induced by imatinib mesylate and AraC. LeukemiaKindler T Breitenbuecher F Kasper S et al 2003 17 999 1009 - 71.
Sensitivity to the abl inhibitor STI571 in fresh leukaemic cells obtained from chronic myelogenous leukemia patients in different stages of disease. British Journal of HaematologyGambacorti-passerini C Barni R Marchsi E et al 2001 112 972 974 - 72.
Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation. BloodHoltz M Slovak M Zhang F et al 2002 99 3792 3800 - 73.
Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. BloodBhatia R Holtz M Niu N et al 2003 101 4701 4707 - 74.
Functional integrity in vitro of hematopoietic progenitor cells from patients with chronic myeloid leukemia that have achieved hematological remission after different therapeutic procedures. Leukemia ResearchChavez-gonzalez A Ayala-sanchez M Sanchez-valle E et al 2006 30 286 295 - 75.
Loss of response to imatinib: Mechanisms and management. HematologyShah N 2005 183 187 - 76.
Di Persio J. Therapy Options in Imatinib Failures. The OncologistRamirez P 2008 13 424 434 - 77.
E, et al. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS 354825 against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer ResearchO Hare T Walters D Stoffregen 2005 65 4500 4505 - 78.
Moreau Gaudry F, Uhalde M, et al. Imatinib and nilotinib induce apoptosis of chronic myeloid leukemia cells through a Bim-dependeant pathway modulated by cytokines. Cancer Biology and TherapyBelloc F 2007 6 912 919 - 79.
AMN 107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistan chronic myeloid leukemia. Clinical Cancer ResearchGolemovic M Verstovsek S Giles F et al 2005 11 4941 4947 - 80.
BMS 214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibithors. BloodCopland M Pellicano F Richmond L et al 2008 111 2843 2854 - 81.
Development of an effective therapy for CML. Cancer JournalWoessner D Lim C Deininger M 2011 17 477 486 - 82.
Jean Khoury H Cortes J, Dantarjian H, et al. Bosutinib is active in chronic phase chronic myeloid leukemia after imatinib and dasatinib and/or nilotinib therapy failure Blood;2012 119 4303 4312 - 83.
X, et al. AP24534 a pan BCR-ABL inhibitor for chronic myeloid leukemia potently inhibits the T315I mutant and overcomes mutation based resistance. Cancer CellO Hare T Shakespeare W Zhu 2009 16 401 412 - 84.
Abcg2 overexpression represents a novel mechanism for acquired resistance to multi-kinase inhibithor danusertib in BCR-ABL positive cells in vitro.Balabanov S Gontarewicz A Keller G et al 2011 Plos One. 6: e19146 e19164. - 85.
Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors. BloodCortes J Jabbour E Kantarjian H et al 2007 110 4005 4011 - 86.
The Bcr-Abl kinase inhibitor INNO 406 induces autophagy and different modes of cell death execution in Bcr-Abl positive leukemias. Cell Death and DifferentiationKamitsuii Y Kuroda J Kimura S et al 2008 11 1712 1722 - 87.
Choosing the best treatment strategy for chronic myeloid leukemia patients resistant to imatinib: weighing the efficacy and safety of individual drugs with BCR ABL mutations and patient history. LeukemiaJabbour E Hochhaus A Cortes J et al 2010 24 6 12 - 88.
Chronic myeloid leukemia: an update of concepts and management recommendations of European Leukemia Net. Journal of Clinical OncologyBaccarani M Cortes J Pane F et al 2009 27 6041 6051 - 89.
Chronic Myeloid Leukemia: Clinical Impact of BCR-ABL1 Mutations and Other Lesions Associated With Disease Progression. Seminars in OncologyErnst T Hochhaus A 2012 39 58 66 - 90.
La Rosée P. Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance. LeukemiaHochhaus A 2004 18 1321 31 - 91.
Imatinib for newly diagnosed patients with chronic myeloid leukemia: incidence of sustained responses in an intention-to-treat analysis. Journal of Clinical OncologyDe Lavallade H Apperley J. F Khorashad J. S et al 2008 26 3358 3363 - 92.
Molecular monitoring of BCR-ABL as a guide to clinical management in chronic myeloid leukaemia. Blood ReviewsHughes T Branford S 2006 20 29 41 - 93.
ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia. HaematologicaErnst T Hoffmann J Erben P et al 2008 93 1389 1393 - 94.
Clinical resistance to STI-571 cancer therapy caused by BCRABL gene mutation or amplification. ScienceGorre M Mohammed M Ellwood K et al 2001 293 876 880 - 95.
Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. LeukemiaHochhaus A Kreil S Corbin A. S et al 2002 16 2190 2196 - 96.
Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate- binding loop (P-loop) are associated with a poor prognosis. BloodBranford S Rudzki Z Parkinson,Walsh S et al 2003 102 276 283 - 97.
Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment. BloodChu S Xu H Shah N. P et al 2005 105 2093 2098 - 98.
kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. BloodO Hare T Eide C Deininger M Bcr-abl 2007 110 2242 2249 - 99.
La Rosee P Hochhaus A. Resistance to Imatinib in Chronic Myelogenous Leukemia: Mechanisms and Clinical Implications. Current Hematologic Malignant Reports2008 3 72 79 - 100.
Initial findings from the PACE trial: a pivotal phase 2 study of ponatinib in patients withCML and Ph + ALL resistant or intolerant to dasatinib or nilotinib, or with the T315I mutation. ASH Annual Meeting AbstractCortes J Kim D Pinilla-ibarz J 2011 - 101.
Mechanisms of resistance to BCR-ABL kinase inhibitors. Leukemia and LymphomaDiamond J Melo J 2011 S1):12-22. - 102.
Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL transformed hematopoietic cell lines. BloodWeisberg E Griffin J 2000 95 3498 3505 - 103.
Le Coutre P Tassi E, Varella-Garcia M, et al. Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. Blood2000 95 1758 1766 - 104.
Bcr-Abl expression levels determine the rate of development of resistance to imatinib mesylate in chronic myeloid leukemia. Cancer ResearchBarnes D Palaiologou D Panousopoulou E et al 2005 65 8912 8919 - 105.
Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities. BloodUchida N Combs J Chen S et al 1996 88 1297 1305 - 106.
Expression and activity of P glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. CellChaudhary P Roninson I 1991 - 107.
Multidrug resistance proteins: role of P glycoprotein, MRP1, MRP2 and BCRP(ABCG2) in tissue defense. Toxicology and Applied PharmacologyLeslie E Deeley R Cole S 2005 204 216 37 - 108.
Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. BloodMahon F. X Deininger M. W Schultheis B et al 2000 96 1070 1079 - 109.
Interaction of imatinib mesilate with human P-glycoprotein. Journal of Pharmacology and Experimental TherapeuticsHamada A Miyano H Watanabe H et al 2003 307 824 828 - 110.
P-Glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate. LeukemiaIllmer T Schaich M Platzbecker U et al 2004 18 401 408 - 111.
Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Biology and TherapyBurger H Van Tol H Brok M 2005 4 747 752 - 112.
Functional ABCG2 is over expressed on primary CML CD34+ cells and is inhibited by imatinib mesylate. BloodJordanides N. E Jorgensen H. G Holyoake T. L et al 2006 108 1370 1373 - 113.
Pharmacokinetic resistance to imatinib mesylate: role of the ABC drug pumps ABCG2 (BCRP) and ABCB1 (MDR1) in the oral bioavailability of imatinib. Cell CycleBurger H Nooter K 2004 3 1502 1505 - 114.
Tyrosine kinase inhibitors as modulators of ATP binding cassette multidrug transporters: substrates, chemo-sensitizers or inducers of acquired multidrug resistance?. Expert Opinion on Drug Metabolism and ToxicologyBrozik A Hegedus C Erdei Z Hegedus T Ozvegy-laczka C Szakacs G 2011 7 623 42 - 115.
Active transport of imatinib into and out of cells: implications for drug resistance. BloodThomas J Wang L Clark R. E et al 2004 104 3739 45 - 116.
hOCT 1 and resistance to imatinib. BloodCrossman L Druker B Deininger M 2005 106 1133 1134 - 117.
CML patients with low OCT-1 activity achieve better molecular responses on high dose imatinib than on standard dose. those with high OCT-1 activity have excellent responses on either dose: a TOPS correlative study. BloodWhite D Saunders V Dang P 2008 - 118.
Chronic Myeloid Leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 activity. LeukemiaEngler J Frede A Saunders V et al 2010 24 765 770 - 119.
OCT-1-mediated influx is a key determinant of the intracellular uptake of imatinib but not nilotinib (AMN107): reduced OCT-1 activity is the cause of low in vitro sensitivity to imatinib. BloodWhite D Saunders V Dang P et al 2006 108 697 704 - 120.
Dasatinib cellular uptake and efflux in chronic myeloid leukemia cells: therapeutic implications. Clinical Cancer ResearchHiwase D Saunders V Hewett D et al 2008 14 3881 3888 - 121.
High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase. BloodWang J Lapidot T Cashman J et al 1998 91 2406 2414 - 122.
Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3. BloodHolyoake T. L Jiang X Jorgensen H. G et al 2001 97 720 728 - 123.
Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. BloodCopland M Hamilton A Elrick L. J et al 2006 107 4532 4539 - 124.
Leukemic stem cells of chronic phase CML patients consistently display very high BCR-ABL transcript levels and reduced responsiveness to imatinib mesylate in addition to generating a rare subset that produce imatinib mesylate resistant differentiated progeny. BloodJiang X Zhao Y Chan W. Y et al 2004 a. - 125.
Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype. LeukemiaKeeshan K Mills K Cotter T et al 2001 - 126.
Signal transduction pathways involved in BCR-ABL transformation. Baillieres Clinical HaematologySawyers C. L 1997 10 223 231 - 127.
Association between imatinib-resistant BCR-ABL mutation negative leukemia and persistent activation of LYN kinase. Journal of the National Cancer InstituteWu J Meng F Kong L et al 2008 100 926 939 - 128.
rd, Wilson M, Abdi F, et al. Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity. Journal of Biological ChemistryMein M. A 2006 281 30907 30916 - 129.
STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells. BloodDe Groot R Raaijmakers J Lammers J et al 1999 94 1108 1112 - 130.
Molecular biology of bcr-abl1-positive chronic myeloid leukemia. BloodQuintas-cardama A Cortes J 2009 113 1619 30 - 131.
BCR/ABL- induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. CellPendergast A Quilliam L Cripe L et al 1993 75 175 185 - 132.
Critical role for Gab2 in transformation by BCR/ABL. Cancer CellSattler M Mohi M Pride Y et al 2002 1 479 492