Medical Oncology

, 26:437

Translational research in complex etiopathogenesis and therapy of hematological malignancies: the specific role of tyrosine kinases signaling and inhibition

Authors

    • Department of Biochemistry, Medical facultyUniversity of Novi Sad
    • CEA/Institut de Génomique Centre National de Génotypage
  • Sunčica Stankov
    • Health Center Novi Sad
  • Stevan Popović
    • Hematology Clinics, Medical facultyUniversity of Novi Sad
Original Paper

DOI: 10.1007/s12032-008-9143-2

Cite this article as:
Stankov, K., Stankov, S. & Popović, S. Med Oncol (2009) 26: 437. doi:10.1007/s12032-008-9143-2
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Abstract

During the recent genomics and proteomics era, high-resolution, genome-wide approaches have revealed numerous promising new drug targets and disease biomarkers, accelerating and emphasizing the need for targeted molecular therapy compounds. Significant progress has been made in understanding the pathogenesis of hematological malignancies there by, revealing new drug targets. Introduction of multiple new technologies in cancer research have significantly improved the drug discovery process, leading to key success in targeted cancer therapeutics, including tyrosine kinase inhibitors. The studies of receptor tyrosine kinases and their role in malignant transformation are already translated from the preclinical level (cell-based and animal models) to clinical studies, enabling the more complete understanding of tumor cell biology and improvement of tumor therapy.

Keywords

LeukemiaReceptor protein–tyrosine kinasesProtein kinase inhibitors

Introduction

In the last 15 years, the introduction of molecular biology methods and techniques, for identifying mutations and recently measuring gene expression levels of mutated genes, have enabled precise molecular diagnostics, classification and assessment of prognosis and therapeutic response of malignant disease to specific therapies [1].

In the early genomics era during the late 1980s and 1990s, the characterization of the precise molecular pathology of different cancers became the central issue in drug discovery, aimed to identification of genes that initiate and support cancerogenesis. During the recent genomics and proteomics era, high-resolution, genome-wide approaches have also revealed numerous promising new drug targets, and disease biomarkers, accelerating and emphasizing the need for targeted molecular therapy compounds, including tyrosine kinase inhibitors (TKIs) [27].

The introduction of imatinib mesylate (IM), revolutionized the management of patients with chronic myeloid leukemia (CML). Furthermore, the second generation of TKIs may soon prove to be superior to IM. The use of targeted small molecule therapeutics by the development of rationally designed inhibitors targeting crucial molecular signaling effectors involved in cell proliferation, invasion and metastasis, angiogenesis, and apoptosis, shed a new light to the role of TKIs in normal cellular homeostasis, but also of their mutations and deregulated activity in the etiopathogenesis and therapy of human malignancies [812].

Tyrosine kinase signaling in leukemia

Receptor tyrosine kinases (RTKs) and growth factor receptors play a key role in the transmission of information from outside the cell into the cytoplasm and the nucleus. The signaling cascade mediates the initiation, regulation, and execution of important cellular functions and processes, such as differentiation, growth, and survival/programmed cell death. Upon the ligand binding to the extracellular (ligand-binding) domain, RTK dimerizes and increases the transphosphorylation of tyrosine residues in the cytoplasmic domains. Several adaptor molecules harboring SH2 domain or PTB (phosphotyrosine binding) domain are docked in RTK cytoplasmic domain. Adaptor molecules activate numerous downstream signaling molecules and pathways through tyrosine phosphorylations. Many of them, such as RAS/MAPK, PI3K, and STAT pathways act as the major oncogenic signaling pathways [13, 14].

More than 50 mammalian RTKs are known so far, including the Fms-like tyrosine kinase 3 (FLT3), whose mutations result in constitutive tyrosine kinase activity and aberrant signal transduction through the Ras/MAPK pathway in acute myeloid leukemia (AML) [15].

Despite differences in structure, function, and subcellular location, many of the RTK oncogenes signal through the same pathways to typically enhance proliferation and prolong viability. These pathways include activation of the Ras/Raf/MAPK (mitogen-activated protein kinase), signal transducers and activators of transcription (STATs), and phosphatidylinositol 3-kinase (PI3K). The latter signals through the protein serine/threonine kinase Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) [16].

Targeting the pathways downstream of RTKs is another approach to down-regulate an over-activated signalling system, as it is the case in leukemia [17]. Recently published in vitro results show the important role of mTOR, firmly established as a major determinant for cell growth, proliferation, and survival in a wide array of human cancers [18]. PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug resistance of these cells. Both the disease-free survival, and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade represents a target for innovative therapeutic treatments of AML patients [18]. It has been shown that blockade of mTOR signalling potentiates the ability of histone deacetylase inhibitor to induce growth arrest, and differentiation of acute myelogenous leukemia cells [19].

With regard to the biological roles of PI3K/Akt in hematological malignancies, it has been shown that tyrosine 719 in c-kit protein, which is utilized for the PI3K binding, is essential for the transforming activity of the tyrosine kinase domain mutant of c-kit, by exchanging tyrosine residues in the cytoplasmic domain to phenylalanine. This mutation enables the active configuration of c-kit, responsible for the aberrant constitutive activation of RTK, associated with malignant transformation in human mast cell leukemia [20]. Constitutive activation of PI3K was found in more than 50% of AML cases, and the activation of Akt was significantly higher in spontaneously proliferating AML cells [17].

The encouraging in vitro results show that normal hematopoietic progenitor cells are less affected by inhibitors targeting PI3K signaling than AML cells, further supporting the feasibility of targeting this fundamental signal transduction network for AML therapy [17].

The important role of Ras/MAPK pathway in the growth and survival of hematopoietic cells is proved from the results showing that the inhibition of Ras/MAPK activity blocks proliferation and survival of hematopoietic cells [20]. Sorafenib, a Raf-1 kinase inhibitor, and farnesyl transferase inhibitors (such as tipifarnib), affecting Ras post-translational modification, show promising biological activity in phase I clinical trials in AML patients [17].

STATs mediate cytokine-dependent cell growth and survival by regulating the expression of cyclins, c-myc, and Bcl-Xl. STATs play a crucial role in malignant transformation caused by oncogenic, constitutively active RTK [20]. The abberant activation of STAT is the common characteristics of constitutive activity of receptor tyrosine kinases. The absence of STAT expression in human leukemia cells, due to dominant-negative STAT3 mutation, suppresses the growth and survival mediated by oncogenic c-kit [21].

Tyrosine kinases as therapy targets in hematological malignancies

Strategies aimed to effectively fight against cancer, without side effects in normal cells and tissues, are mostly limited by the fact, with few exceptions, cancer lack targets that are simultaneously specific, and vital. The tumor-specific mutations in RTKs are often used as a potential therapeutic targets, as well as main predictive factors for therapy selection and treatment response [22].

RTKs represent a large family of drug targets, emerging in recent years as a fast developing field of tyrosine kinase inhibitors (TKIs), in molecular targeted therapy of various malignancies. Starting from 1996, when the first non-selective TKI, STI-571/imatinib mesylate (IM) was reported as successful inhibitor of Bcr-Abl in CML patients, new emerging more-selective TKIs have been synthesized and approved for clinical use [23].

Sunitinib is a rationally designed small molecule, with the potential to inhibit multiple members of the tyrosine kinase family and their signaling pathways, including c-kit and FLT3. Sunitinib is therefore able to block the signal transduction involved in proliferation, and progression of hematological malignancies [23, 24]. Sunitinib demonstrates in vitro inhibition of c-kit receptor phosphorylation and cellular proliferation. However, in experimental mouse model, hair depigmentation, a pharmacodynamic marker of c-kit inhibition, was observed at high doses, comparable to those inducing adverse effects in clinical trials [24]. Mutations in FLT3 in AML are associated with enhanced proliferation, and survival of leukemic blasts. Sunitinib produces sustained inhibition of FLT3 signaling, eradication of leukemia cells, and prolonged survival [24]. Sunitinib demonstrated in vitro inhibition of phosphorylation induced by three different forms of FLT3: wild type (wt), ITD (internal tandem duplication mutant form of FLT3), and activation loop mutant FLT3-Asp835 [25].

Dasatinib is an oral dual Bcr-Abl and Src family TKI, approved for use in patients with chronic myelogenous leukemia (CML) after IM treatment and in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) [23]. Dasatinib is an ATP-competitive dual Src/Abl inhibitor, showing potency to inhibit 21 out of 22 mutant forms of Bcr/Abl resistant to imatinib [23]. The lack of selectivity of dasatinib over the Src-family kinases is probably related to the fact that dasatinib, in contrast to both imatinib and nilotinib, inhibits Bcr-Abl by binding to the active form of Abl kinase [23]. In recent in vitro study it has been reported that dasatinib inhibits WT c-kit and juxtamembrane domain of mutant c-kit, preventing its autophosphorylation, and c-kit-dependent activation of downstream pathways important for cell viability and cell survival [26]. Dasatinib was approved for use in 2006, for the treatment of adults in all phases of CML with resistance or intolerance to imatinib therapy [23].

Nilotinib is rationally designed imatinib phenylamino-pyrimidine derivative, with remarkably improved affinity for and 30-fold more potent inhibitory activity against wild-type Bcr-Abl kinase, while preserving similar activity agains c-kit and PDGFR kinases [27]. Nilotinib has been approved in October 2007, for the treatment of patients with chronic and accelerated phase of CML, resistant or intolerant to prior therapy that included imatinib [27]. In primary cells of CML patients, imatinib mediates the increased cellular uptake and retention of nilotinib, which may explain the observed synergy between these two TKIs [28].

Sorafenib tosylate is a diphenylurea derivative, approved for the treatment of advanced renal carcinoma, and hepatocellular carcinoma. This multitargeted TKI inhibits c-kit, FLT3, PDGFR, and VEGFR. The in vitro study showed inhibition of blasts proliferation in six of nine samples from AML patients, by 0.1–10 μmol/l Sorafenib for 4 days [29]. Sorafenib and Sunitinib induce apoptosis in a time- and concentration-dependent manner, more efficiently than imatinib. These two multitargeted TKIs showed similar in vitro activities in cell proliferation, apoptosis and cell cycle assays, and inhibition of MAPK and STAT signaling [29].

PKC412 is a potent tyrosine kinase inhibitor, showing efficient in vitro inhibition towards FLT3, PDGF-Rβ, and c-kit in nanomolar doses. Dose-dependent cytotoxicity is observed in ALL blasts expressing FLT3. In primary AML patient cell lines, lestaurtinib, and PKC412 showed synergistic cytotoxic activity in vitro. Samples from patients with FLT3 activating mutations were significantly more sensitive to cytotoxic effects than those from wild-type cases [30].

Mechanisms of imatinib mesylate therapy and resistance in CML

The imatinib mesylate, as a first success of translational medicine, was based on the knowledge of precise molecular event of reciprocal translocation (t(9;22)(q34:q11)) leading to Bcr-Abl protein product, a constitutively active tyrosine kinase, promoting cellular survival, proliferation, and malignant transformation. The introduction of molecular targeted therapeutic IM in chronic phase CML, resulted in the follow-up of three crucial degrees of therapy response: complete hematological response (CHR, defined by normalization of white blood cell counts); complete cytogenetic remission (CCyR, the absence of Philadelphia (Ph+) chromosome within 20 metaphases on classic caryotype analysis); and complete molecular response (CMR; elimination of Bcr-Abl mRNA transcripts as detected by quantitative real-time polymerase chain reaction (qRT-PCR)) [31].

The tyrosine kinase activity of Bcr-Abl is the principal therapeutic target of imatinib, in addition to inhibitory effects on c-kit, ARG, and PDGF receptors. Imatinib acts by binding to the Bcr-Abl protein in the inactive conformation and is unable to bind to the active conformation [31].

The use of TKIs in CML is one of the greatest single advances in the targeted treatment of cancer. However, in most cases, TKI therapy suppresses but does not eliminate the primitive CML stem cells, which are refractory to TKI therapy. Dasatinib, a potent inhibitor of Bcr-Abl in vitro, has been shown as effective for patients with chronic myelogenous leukemia, resistant or intolerant to IM, in the recent multicenter, multinational phase II trial evaluating the efficacy and safety of dasatinib in chronic phase CML patients, resistant or intolerant to IM therapy [22, 32].

Recent data suggest that the treatment of myeloid neoplasms with TK inhibitors as single agents may be insufficient to control the disease for prolonged time periods. This can be documented for the use of imatinib in advanced CML [11, 33].

In view of the fact that the concentration of IM needed to inhibit cellular growth or kinase activity by 50% (IC50) for Bcr-Abl is 0.1–0.3 μmol/l, the recommended daily dose of 400 mg should be more than adequate to inhibit Bcr-Abl activity. Oral availability differs greatly according to variability in CYP3A4 concentrations, whereas plasma-protein binding is another important factor determining the clinical response and resistance development. Imatinib is 89–96% bound to protein, predominantly to albumin, but also to alpha-1 acid glycoprotein (AGP), a hepatic acute-phase protein that binds cationic drugs at a 1:1 molar ratio. Only nonprotein-bound imatinib is available for cellular uptake. Drug transporters can potentially play a part in resistance to imatinib, either by transporting the drug out of the target cell or by transporting it out of cells of the gastrointestinal tract, as it is the case of overexpression of the cell-surface transmembrane ATPase ABCB1 [11, 23, 33].

The genetic causes of imatinib resistance are numerous, and they comprise Bcr-Abl mutations and expression alterations, as well as clonal evolution, i.e., the acquisition of additional chromosomal abnormalities in the Ph+ cell population. Despite the plethora of published work that exists, two main reasons can be given for why accurately defining the incidence of mutations or identifying the factors that affect the risk of developing a mutation remain difficult. First, the sensitivity of the techniques used to detect mutations has differed in different studies and second, studies have focused on different patient populations, with certain studies looking for mutations only in acquired resistance and others concentrating on patients with stable and responsive disease [11, 23, 33].

Other factors contributing to IM therapy failure are residual leukemia cells, resulting in fact that only 20–40% of patients treated by IM achieve a complete molecular response, due to limited IM activity against Bcr-Abl-positive CD34+CD38 stem cells [27].

At the molecular level, resistance to IM is usually caused by point mutations in the kinase domain of Bcr-Abl protein, resulting in functional Abl tyrosine kinase domain, and complete impairment of IM binding. Recent studies report Bcr-Abl mutations in 40–50% of imatinib-resistant CML patients, describing more than 50 different mutations, resulting in amino-acid substitutions in Abl1 kinase domain [11, 23, 33]. Imatinib binds to the Abl kinase domain in its unique inactive conformation. Point mutations in the Abl kinase domain can thwart drug binding by direct steric hindrance or by destabilizing the inactive kinase conformation that is required for imatinib binding [34, 35]. It is important to emphasize that the imatinib-resistant clones with Bcr-Abl mutations pre-exist in newly-diagnosed patients, and they become prevalent during therapy, due to selective pressure of imatinib mesylate [11, 23, 33].

Recent studies explored the consequences of increased cellular efflux of IM, mediated mainly by drug transporter P-gp, or by a decrease in cellular influx, mediated by the uptake carrier hOCT1 [36, 37]. Non-adherence to IM dosage regimen and PK–PD (pharmacokinetic–pharmacodynamic) relationships are additional important factors in IM resistance development. This observation is emphasized by the recent study in 20 CML patients (IM-resistant, point mutations not detected), showing that IM doses of 350 mg (corresponding to a through plasma concentration (TPC) of 570 μg/l), ensure an optimal hematological response. However, this dose may not be sufficient for a cytogenetic or molecular response, requiring TPC as high as 1,000 μg/l [38].

Other genetic resistance mechanisms to TKIs in CML include overexpression or amplification of Bcr-Abl, or its protein product, and overexpression of the Src family kinases [11].

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph+-positive) acute lymphocytic leukemia (ALL) are 2 fatal Bcr-Abl-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. A recent report highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph+ leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph+ALL patients [9]. This study underscores the safety of prolonged (6.3 months) daily administration of FTY720 and strongly supports the use of this PP2A activator as a novel therapeutic approach for treatment of CML-BC and Ph+ALL patients who are unresponsive to current kinase inhibitor therapy and, perhaps, for treatment of other cancers characterized by functional loss of PP2A activity [9].

Targeted molecular therapies for AML

Dysregulation of receptor tyrosine kinase (RTK) activity has been implicated in the progression of a variety of human leukemias [20]. Most notably, mutations and chromosomal translocations affecting regulation of tyrosine kinase activity in c-kit receptor, the FLT3 receptor, and the PDGFβ/FGF1 receptors (PDGFR) have been demonstrated in mast cell leukemia, acute myeloid leukemia (AML), or chronic myelogenous leukemias (CML) [39]. Mutations can affect the extracellular part of the receptor and lead to a forced dimerisation that mimics the action of the ligand, such as in the case of chromosomal translocations that lead to a constitutive dimerisation of the receptor. RTKs get usually activated by dimerisation induced by the ligand and this process leads to the whole mechanism of tyrosine kinase activation, autophosphorylation, recruitement of docking effector proteins and activation of Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/Akt (PI3K/Akt), and Janus activated kinase/signal transducers and activators of transcription (STAT). These signaling pathways are known to control a range of fundamental cellular processes including survival, differentiation, and proliferation of myeloid blasts. Mutations have also been identified for these receptors in their kinase domain reponsible for the oncogenic activity of these receptors [40].

Studies on oncogenic alterations in human AML led to the proposal of a ‘‘two-hit’’ model of leukemogenesis [41]. In this model, an acute leukemia would arise from the cooperation between one class of mutations that interferes with differentiation and a second class of mutations that confers a proliferative advantage to cells. In human AML, combinations of dual mutations in the transcription factor AML1 and the kinase receptors FLT3, or c-kit have been reported and are a poor prognostic factor [42]. In erythroleukemia, c-kit mutations appear as the second and crucial hit that is associated with the progression of proerythroblasts toward malignancy. Therefore, this model of leukemic transformation requires the collaboration of at least one proliferative and one differentiation blocking event.

The major focus in AML has been directed towards FLT3, and c-kit as therapeutic targets. High expression of c-kit in AML (60–80%) has been reported, and point mutations of c-kit have been identified in 33.3–45% of AML with inversions and 12.8–46.8% of AML M2 with t(8;21) [43]. Activating c-kit activation loop mutations (notably, D816 V mutation) are found in association with acute myelogenous leukemia (AML) and mast cell disease (in particular systemic mastocytosis) [44, 45]. In AML blasts, the inhibition of c-kit in vitro results in caspase-3 activation and in cleavage of poly (adenosine diphosphate––ribose) polymerase (PARP) [46].

FLT3 receptor is expressed at high levels in most cases of AML patients. Constitutive activation of FLT3 in AML cells occurs through either endogenous coexpression of FLT3 ligand (FL), or through the presence of activating mutations within the juxtamembrane or tyrosine kinase domain of the receptor. The combined prevalence of activating FLT3 mutations in AML is 31%, representing the most common molecular lesion so far described [47].

The accumulating evidence that RTKs are important in the pathogenesis of chronic and acute myeloproliferative disorders has prompted a number of groups to search for inhibitors as possible therapeutic interventions. The use of tyrosine kinase inhibitors in patients have already been proved clinically useful, since patients harboring either juxta-membrane c-kit mutations (the V560G in mast cell leukemia), or translocations involving PDGFRβ (in eosinophilia-associated chronic myeloproliferative disorders) respond to imatinib [48, 49].

The mechanism of c-kit activation due to mutation at the D816 residue is thought to be mediated by disruption of the hydrogen bond between codon D816 and N819 in the activation loop of the enzymatic domain [50]. This destabilizes the inactive conformation of the kinase domain and results in ligand-independent constitutive activation and autophosphorylation of c-kit. An acquired c-kit D816V mutation confers full resistance to imatinib in vitro as studied in the mast cell leukemia cell line [50]. It prevents the binding of imatinib to c-kit, rendering resistance to this potent tyrosine kinase inhibitor. Imatinib retains its inhibitory effects on c-kit with the N822 mutation and mutations in the extracellular, transmembrane, and juxtamembrane domains [50].

Dasatinib, ATP-competitive, dual Bcr-Abl and Src inhibitor, can inhibit Bcr-Abl activation loop mutations that are found in some CML patients with acquired clinical resistance to imatinib. In a recent study, Schittenhelm et al. [26] hypothesized that dasatinib might inhibit the kinase activity of both wild-type (WT), and mutant c-kit isoforms. These authors reported that dasatinib potently inhibits WT c-kit and juxtamembrane domain mutant c-kit autophosphorylation and c-kit dependent activation of downstream pathways important for cell viability and cell survival, such as MAPK, PI3K/Akt, and STAT. Furthermore, they showed that dasatinib is a potent inhibitor of imatinib-resistant c-kit activation loop mutants and that it induces apoptosis in mast cell and leukemic cell lines expressing these mutations [26].

Activation loop mutation D816 V promotes the resistance to imatinib inhibition. Recently, a concept of the rational redesign of imatinib led to the inhibition of the imatinib-resistant mutant and wild-type kinase [51]. This prototype better stabilizes the active induced-fit conformation of the activation loop, through a specific methylation of imatinib. This concept is confirmed through in vitro assays showing the dual inhibitory effect of the prototype by probing the downstream phosphorylation activity of wild-type and imatinib-resistant kinase in the presence of the parental, and prototype competitive ligands. Cell proliferation assays on lines that express wild-type, and imatinib-resistant kinase were also conducted in order to confirm the dual anticancer activity of the prototype [51].

Additional RTK receptors may be constitutively activated in AML via numerous mechanisms [43]. In support of this concept is the report of a case of AML that, despite lacking known RTK mutations, achieved a sustained complete hematological remission on imatinib therapy [45]. Recently, a number of other potentially useful RTK inhibitors have been identified. Levis et al. [52, 53] reported that lestaurtinib (formerly known as CEP-701, indolocarbazole derivative), is a sensitive and specific FLT3 inhibitor. CEP-701 inhibited one of the most frequent FLT3 oncogenic mutation, FLT3-ITD (internal tandem duplication), in primary leukemic blasts from AML patients and, in addition, was shown to prolong survival in a mouse model of FLT3-ITD leukemia. A clinical trial of CEP-701 has already been initiated in patients with AML and the results of clinical trials are awaited with interest [25, 5153]. Recently, SU11248 has been shown to inhibit split kinase domain RTKs, including PDGFRα and β, VEGFR2, c-kit, as well as wild-type FLT3, FLT3-ITD and FLT3 activation loop (FLT3 Asp835) mutants and promises to be a useful therapeutic agent [25].

Rapidly, trials of imatinib in aggressive-phase CML and Ph+ALL revealed emergence of resistance due to point mutations in the Bcr-Abl gene [51]. It has then been shown that mutant c-kit isoforms and most c-kit activation loop mutations are resistant to clinically achievable doses of IM. Imatinib only binds to the inactive conformation of c-kit; however, c-kit activation loop mutations not only activate kinase activity, but also stabilize the activation loop in a conformation that does not allow productive IM binding [54].

Acute myeloid leukemia with t(8;21)(q22;q22) is a distinct type of leukemia considered to have a favorable prognosis. However, the outcome of patients with AML associated with t(8;21) and treated with fludarabine-based regimens is variable and not predictable by conventional cytogenetic data. According to this study, the patients with mutated c-kit (D816 V/D816Y), FLT3 (ITD or D835) or RAS, as a group had a worse survival [55].

Despite the significant progress of targeted therapy, the disease relapse due to drug resistance is its major limitation and drawback. A recent mathematical modelling of drug resistance in CML predicts that multiple non-cross-resistant drugs are needed to prevent treatment failure [56]. The mortality of acute leukemia is 5,2 cases per 100.000 general population and a survival rate of 5 years of 50% for the women, 75% for children and 35% for men in Europe [57]. Alternative approaches and numerous agents are explored in CML and AML treatment, in order to develop targeted specific therapy, which will maximize the inhibition of multiple heterogeneous pathways present in myeloid blasts. Tyrosine kinase inhibitors are based on a very strong scientific rationale and they are used with considerable success in CML and AML therapy. However, many recent studies suggest that the treatment of myeloid neoplasms with TKIs as single agents, may be insufficient to control the disease for prolonged time periods. Translation of many pre-clinical studies to clinical trials, validating the application of drug combinations in order to achieve additive or synergistic antiproliferative effects, should enable in future to overcome the development of resistance.

Acknowledgments

This work was supported by the bilateral project of cooperation between the Ministry of Science, Republic of Serbia and CNRS, France, grant No. 451-03-2405/2007-02/12-1.

Copyright information

© Humana Press Inc. 2008