Recent developments in drug resistance mechanism in chronic myeloid leukemia: a review

Recent developments in drug resistance mechanism inchronic myeloid leukemia: a reviewShantashri Vaidya, Kanjaksha Ghosh, Babu Rao Vundinti

National Institute of Immunohaematology (ICMR), Parel, Mumbai, India

Chronic myeloid leukemia (CML) is a clonal hematopoi-

etic stem cell disorder. It is characterized by excess pro-

liferation of myeloid progenitor that retains the capacity

for differentiation during the stable or chronic phase of

the disease. Patients with CML usually present in the

chronic phase of the disease during which there is a grad-

ual expansion of mature myeloid cells in the bone mar-

row and peripheral blood. Without treatment, patients

inevitably progress through an accelerated phase of dis-

ease to a terminal acute phase known as blast crisis,

characterized by a massive increase in undifferentiated

blast that can be either myeloid or lymphoid in nature.

Hallmark of this disease is a unique chromosome, known

as the Philadelphia chromosome (Ph), which results from

the reciprocal translocation between the long arms of

chromosomes 9 and 22 [t(9;22) (q34;q11)] (Figs 1 and 2

)and leads to the fusion of the breakpoints cluster (BCR)

and human ABL1 genes (Fig. 3). The root cause of this

disease is still unknown. The majority of patients with

CML have no history of excessive exposure to ionizing

radiations or carcinogens, but the incidence does increase

progressively with exposure to increasing doses of radia-

tion. This has been noted following chronic exposure in

radiologists who practiced without adequate shielding, in

patients who received radiation treatment, and in sub-

jects exposed to a single massive dose of radiation, as in

the atomic bomb explosions in Japan in 1945 (1). CML

is not hereditary. The incidence of CML in United States

and most Western countries is about 1.5 per 100 000

population per year and accounts for about 15% of all

cases of leukemia; about 4500 new cases are diagnosed in

the United States each year. CML is slightly more fre-

quent in men than in women (incidence ratio: 1.4 to

2.2 : 1), but the course of the disease is the same. The

median age is about 50; it is rare in children, and the

incidence increases progressively with age.

Molecular biology and pathophysiology ofchronic myeloid leukemia

Chronic myeloid leukemia is probably the most exten-

sively studied human malignancy. The discovery of Phil-

adelphia chromosome (Ph) in 1960 as the first consistent

chromosomal abnormality associated with a specific type

of leukemia was a breakthrough in cancer biology. It

took 13 yrs before it was appreciated that the Ph chro-

mosome is the result of a t(9;22) reciprocal chromosomal

translocation and another 10 yrs before the translocation

Abstract

A revolution in medical science was marked with the advent of imatinib, a site-specific drug for the man-

agement of patients with chronic myeloid leukemia (CML). Imatinib mesylate (also known as Glivec, Glee-

vec, STI-571, CGP57148), an orally administered 2-phenylaminopyrimidine derivative approved by FDA in

2001 for the treatment for CML, is highly effective in treating the early stages of CML, but remission

induced in advanced phase was observed to be relatively short-lived. The primary cause of resistance in

patients with CML is the mutations in the BCR–ABL kinase domain. This review discusses the different

mechanisms leading to imatinib resistance and various treatment options to over-ride imatinib resistance.

Key words chronic myeloid leukemia; BCR–ABL fusion; drug resistance; imatinib; chronic myeloid leukemia therapy

Correspondence Babu Rao Vundinti, Department of Cytogenetics, National Institute of Immunohaematology (ICMR), 13th Floor,

New Multistoried Building, K.E.M Hospital campus, Parel, Mumbai-400012, India. Tel: +91 22 24138518, 24138519; Fax: +91 22

24138518; e-mail: [email protected]

Accepted for publication 30 July 2011 doi:10.1111/j.1600-0609.2011.01689.x

REVIEW ARTICLE

European Journal of Haematology 87 (381–393)

ª 2011 John Wiley & Sons A/S 381

shown to involve the ABL proto-oncogene normally on

chromosome 9 and a previously unknown gene on chro-

mosome 22, later termed BCR for breakpoint cluster

region. The deregulated Abl tyrosine kinase activity was

defined as the principle force behind the pathogenesis of

CML (2).

The breakpoints within the ABL (Abelson tyrosine

kinase) gene at 9q34 can occur anywhere over a large

(>300 kb) area at its 5¢end, either upstream of the first

alternative exon Ib, or downstream of the second alterna-

tive exon Ia, or more frequently, between the two.

Regardless of the exact location of the breakpoint, splic-

ing of the primary hybrid transcript yields an mRNA

molecule in which BCR sequences are fused to ABL exon

a2. In contrast to ABL, breakpoints within BCR localize

to one of three so-called breakpoint cluster regions (bcr).

In most patients with CML and in approximately one-

third of patients with Ph-positive acute lymphoblastic leu-

kemia (ALL), the break occurs within a 5.8-kb area span-

ning BCR exons 12–16 (originally referred to as exons

b1–b5), defined as the major breakpoint cluster region

(M-bcr). Because of alternative splicing, fusion transcripts

with either b2a2 or b3a2 junction can be formed. A 210-

kd chimeric protein (P210BCR–ABL) is derived from this

Figure 1 G-banded karyotype showing classical

Philadelphia pattern 46,XY,t(9;22)(q34;q11).

Figure 3 Schematic diagram showing two

types of BCR–ABL fusion transcripts.

Figure 2 FISH showing reciprocal translocation showing BCR–ABL

dual fusion using dual-color probes.

A review on drug resistance mechanisms in CML Vaidya et al.

382 ª 2011 John Wiley & Sons A/S

mRNA. In the remaining patients with ALL and rarely in

patients with CML, characterized clinically by prominent

monocytosis, the breakpoints are further upstream in the

54.4-kb region between the alternative BCR exons e2¢ ande2, termed the minor breakpoint cluster region (m-bcr).

The resultant e1a2 mRNA is translated into a 190-kd pro-

tein (P190BCR–ABL). Recently, a third breakpoint clus-

ter region (l-bcr) was identified downstream of exon 19,

giving rise to a 230-kd fusion protein (P230BCR–ABL)

associated with the rare Ph-positive chronic neutrophilic

leukemia (2).

BCR–ABL plays a central role in the pathogenesis of

CML, which was unraveled by two independent studies

(3, 4). The activity of this fusion protein is no longer

under the regulatory control mechanisms for ABL and

thus induces malignant disease by activating multiple

cytoplasmic and nuclear signal transduction pathways

that influence the growth and survival of haematopoietic

cells. The targets for Bcr–abl include members of the

Ras, phosphotidylinositol-3 kinase (PI3K) ⁄Akt, and

Jak ⁄Stat signaling pathways, which regulate cell prolifer-

ation and apoptosis. Bcr–abl abrogates cell dependence

on external growth factors by upregulating interleukin-3

production and alters the cell adhesion properties by

modulating expression and activation of focal adhesion

kinase and associated proteins. The kinase also has

diverse effects on the DNA repair response, which may

promote additional chromosomal alterations and muta-

tions involved in the progressive nature of late-stage

CML (5).

Additional insights into the pathogenesis of CML

came from the study of transforming retroviruses, specifi-

cally the Abelson murine leukemia virus. The transform-

ing protein of this virus, v-ABL, was shown to be a

tyrosine kinase with its transforming ability dependent

on this activity. Subsequently, the Bcr–abl fusion product

proved to display similar tyrosine kinase activity. The

constitutive tyrosine kinase activity of Bcr–abl causes

activation of a variety of intracellular signaling path-

ways, leading to alterations in the proliferative, adhesive,

and survival properties of CML cells, with all of these

events dependent on the tyrosine kinase activity of the

Bcr–abl protein. Transduction of p210 bcr–abl into mur-

ine hematopoietic stem cells, followed by transplantation

into syngeneic mice, causes a CML-like syndrome; mice

transgenic for p190 bcr–abl develop acute leukemia.

These findings provide convincing evidence that Bcr–abl

is a leukemic oncogene (6).

The disease characteristically develops in three phases.

Left untreated, the initial chronic phase lasts approxi-

mately 3–6 yrs; the disease then progresses, often

through an accelerated phase, to a terminal blastic phase.

Most patients are diagnosed in chronic phase, which is

characterized by an increased number of leukocytes

and ⁄or platelets and a bone marrow blast count <10%.

The accelerated phase may be marked by one or more of

the following: increasing splenomegaly and leukocytosis,

an increase in basophils to 20% or greater, thrombocyto-

penia, and clonal evolution. In the blastic phase, for

which the median survival is 2–4 months, 30% or more

of blood and bone marrow cells are blasts, and myeloid

precursors may also form tumors in the lymph nodes,

skin, and bone. (5).

Since the description of BCR–ABL oncogene, a myr-

iad of treatment options have been explored in CML,

including arsenic trioxide, spleenic irradiation, busul-

phan, hydroxycarbamide, stem cell transplantation, and

interferon a. Among these, interferon a showed superior

activity compared with other chemotherapeutic agents. It

can promote long-term survival, but the proportion of

such patients is small. Stem cell transplantation can cure

the disease; however, as the average age of onset is

>50 yrs of age, this factor, combined with the inability

to identify suitably matched donors in all cases, limits

this option to a minority of patients (6).

Imatinib therapy in patients with CML

Imatinib was developed in the late 1990s by biochemist

Nicholas Lydon, a former researcher for Ciba Geigy

(now Novartis, Basel, Switerland), oncologist Brian

Druker of Oregon Health and Science University, and

Charles Sawyers of Memorial Sloan-Kettering Cancer

Centre, who led the clinical trials confirming its efficacy

in CML.

Imatinib was developed by rational drug design. After

the Philadelphia chromosome mutations and defective

bcr–abl protein were discovered, the investigators

screened chemical libraries to find a drug that would

inhibit that protein. With high-throughput screening,

they identified 2-phenylaminopyrimidine, which showed

low potency and poor specificity. This lead compound

was then tested and modified by the introduction of

methyl and benzamide groups to give it enhanced bind-

ing properties, resulting in imatinib (6).

Structural analyses have revealed that imatinib is sand-

wich between the N-lobe and C-lobe of the Abl kinase

domain and penetrates through the central region of the

kinase. The pyrimidine and pyridine rings of imatinib

occupy the region where an adenine ring of ATP nor-

mally binds. The rest of the molecule wedges itself into a

hydrophobic cage between the activation loop and the

C-helix of the N-lobe, specifically recognizing the inactive

and unphosphorylated conformation of Abl. Much of

the remarkable efficacy of imatinib appears to derive

from its ability to stabilize the inactive conformation

through an ‘induced fit’ mechanism. The potency of

imatinib against the activated form of bcr–abl

Vaidya et al. A review on drug resistance mechanisms in CML


presumably arises from the dynamic nature of kinase

molecules, which transiently switch between inactive and

active forms, allowing imatinib to trap the kinase in its

inactive conformation (7).

Imatinib as a single agent has shown impressive activity

in the chronic phase of CML, with >95% complete hema-

tologic response and >73% complete cytogenetic remis-

sion in patients (8). However, patients treated in the later

stages of the disease do not respond as well, with only

about 65% displaying hematologic responses (9).

Resistance to imatinib

The emergence of resistance to imatinib has dampened the

enthusiasm for this drug. The rate of relapse and resis-

tance appear to correlate with disease stage, and the inci-

dence increases as CML progresses. Resistance to imatinib

can be divided into primary resistance, in which patients

show lack of efficacy to this tyrosine kinase inhibitor

(TKI) from the start of the therapy, and secondary resis-

tance, also known as acquired resistance, which is defined

as a loss of hematologic, cytogenetic, or molecular

response, as well as progression to advanced phases of

CML. Resistance can further be divided into hematologic

(lack of normalization of peripheral blood counts), cyto-

genetic (persistence of Ph chromosome), and molecular

[persistence of BCR–ABL1 transcripts by reverse trans-

criptase polymerase chain reaction (RT-PCR)].

Patients can be classified according to their response

to treatment, which can be considered optimal, subopti-

mal, or failure. Failure indicates that primary resistance

patients in this category should be recommended a

change in therapy for second-generation TKI. Patients

with suboptimal response need close monitoring, and a

dose escalation from 400 to 800 mg is justified (10).

However, resistance to imatinib can be overcome with

various strategies at different levels (Fig. 4).

Mechanisms of imatinib resistance

Imatinib has become the standard first-line therapy for

CML. Indeed, imatinib induces complete remission in

virtually all patients with the indolent chronic phase of

CML who are treated immediately upon diagnosis (9).

When treated during the more aggressive stage of blast

crisis, most patients ultimately evolve drug resistance dis-

ease. Various mechanisms were postulated for imatinib

resistance, which can be broadly divided into BCR–

ABL-dependent and BCR–ABL-independent mechanisms

of imatinib resistance.

BCR–ABL-independent mechanisms of resistance

Quiescent CML stem cell: Quiescent CML stem cells

account for approximately 0.5% of the CD34+ popula-

tion and are characterized by intrinsic resistance to

Figure 4 Strategies to overcome imatinib resis-

tance at various levels.



imatinib. Jorgensen and Holyoake (11) noticed that

although CD34+38- HSCs carried a single copy of

BCR–ABL, they still exhibited a significant (10-fold)

increase in Bcr–abl kinase level compared with total

mature mononuclear cells. This study was in consensus

with the study carried out by Copland et al. (12).

While rationally designed TKIs have proven effective

in disease management, they do not offer a cure owing

to the persistence of insensitive HSCs. One possible

explanation for this molecular persistence is oncogene

overexpression at the message, protein and kinase activ-

ity levels that is not overcome by possible subtherapeutic

levels of TKI within HSCs (11). Nevertheless, introduc-

tion of novel agents such as the putative FTI BMS-

214662 will make significant advances towards the goal

of elimination of the diseased stem cell population, pref-

erentially targeting quiescent leukemic cells over normal.

Further elucidation of its true mechanism of action will

potentially inform drug development for use in other leu-

kemias and stem cell–derived cancers.

Pharmacokinetics: Pharmacokinetic data indicates that

patients receiving 400 mg imatinib per day have plasma

concentrations constantly higher than 1 lmol ⁄L (13). In

patients receiving higher doses (600–800 mg ⁄d), increasedplasma concentrations have been observed, with some

patients having concentrations higher than 20 lmol ⁄L. As

imatinib is metabolized largely by the cytochrome p450

isoenzymes P3A4 (CYP3A4) and P3A5 (CYP3A5), differ-

ences in the concentrations of CYP3A4 ⁄A5 or drugs that

can inhibit or induce said enzymes have the potential to

greatly affect the levels of imatinib in plasma (10).

It has been proposed that binding of a-1-acid glyco-

protein (AGP), an acute phase plasma protein, can inhi-

bit the activity of imatinib (10, 14). Therefore, imatinib

present in the plasma could be mostly bound to AGP

and thus biologically inactive. To test this hypothesis,

blood samples from two patients who were resistant to

imatinib were incubated with erythromycin (which

competes with imatinib for binding of AGP) for 1 h

before separation of mononuclear cells and lysis. The

activity of imatinib on Bcr–abl autophosphorylation was

restored, indicating that most plasma imatinib is bound

to AGP so not active (13).

Furthermore, when patients who were being treated

with imatinib received a simultaneous infusion of clinda-

mycin, another molecule known to bind AGP, large

decrease in total plasma concentrations of imatinib was

observed in minutes. This finding also shows that most

imatinib present in plasma is not in equilibrium with

tissues and that there is a gradient between plasma and

tissues (13).

Although the exact concentration of imatinib inside

cells – the final target of the drug – is not known, these

findings show that plasma concentrations of the drug are

not a reliable indicator of the concentration present

inside leukemic cells.

Drug efflux mechanism: Several other cellular mecha-

nisms of resistance to imatinib have also been identified,

but the possible importance of drug-transporter proteins

that affect drug concentration, such as permeability glyco-

protein (Pgp), human organic cationic transporter

(hOCT), and ATP-binding cassette G2, has been only

recently appreciated. The generally accepted action of

MDR1 (or adenosine triphosphate–binding cassette trans-

porter ABCB1) is to reduce intracellular drug accumula-

tion through Pgp-mediated efflux, thus hampering the

achievement of effective drug levels at the target site.

Galimberti et al. (15) and Mahon et al. (16) separately

showed that those patients who failed to attain a major

cytogenetic response or progressed exhibited MRD1 gene

overexpression. In contrast to this study, Ferrao et al. (17)

have shown that overexpression of MDR1 in K562 cells

does not confer resistance to imatinib in vitro. Association

between single-nucleotide polymorphisms in MDR1 gene

and response to imatinib has also been observed. Dulucq

et al. (18) have demonstrated the usefulness of three sin-

gle-nucleotide polymorphisms (1236C>T, 2677G>T ⁄A,

3435C>T) in the identification of CML in patients who

may or may not respond optimally to imatinib.

Inhibition of imatinib influx through the hOCT1 has

also been proposed as an important factor regulating

intracellular imatinib availability. The above-mentioned

transporters regulate in concert the active transport of

imatinib in and out of the cell and potentially play

an important role in pharmacogenetics of imatinib. Modi-

fication of imatinib regimen by increasing the standard

dose from 400 to 600–1000 mg ⁄d is suggested in some

patients showing an altered expression of transporter pro-

teins.

Cellular signaling: It is hypothesized that pathways

affected by Bcr–abl, which also contribute to the malig-

nant transformation, are involved in drug resistance

(Fig. 4). Yamada reported involvement of STAT 5, as a

critical transcription factor, confers imatinib resistance

on leukemic cells through the transcription of TERT and

MDR1 (19).

BCR–ABL-dependent mechanisms of resistance

Mutations within the ABL kinase domain (point mutations

and frame shift mutations): The mutations in the ABL

kinase domain and other domains regulating the confor-

mation of ABL kinase domain result in imatinib resis-

tance.

To date, mutations have been discovered that sterically

hinder drug occupancy of the active site, alter the

deformability of the highly conserved phosphate-binding



P-loop, and influence the conformation of the activation

loop surrounding the active site (20).

Studies carried out all over the world for screening of

mutations in BCR–ABL kinase domain have reported

many novel mutations with different frequencies occur-

ring in the population. The first mutation linked to

imatinib resistance in a cohort of relapsed patients,

in American population, discovered by Gorre et al. (21),

was T315I. Soon after the report of T315I, several other

mutations were identified in closely juxtaposed residues.

Studies carried out on German population, by von Bub-

noff et al., revealed five distinct mutations in the BCR–

ABL domain (sample size 7). All five mutations resulted

in exchange of amino acids within the ATP-binding site

(in six patients) or in the activation loop (one patient) of

Bcr–abl kinase domain. Their study strongly suggests

that a patient could be resistant to STI-571 by acquisi-

tion of different individual point mutations within the

ATP-binding pocket or activation loop of Bcr–abl (22).

Timothy Hughes, Australia, reported that among few

mutations in BCR–ABL kinase domain, there are 9 that

account for more than 85% of all mutations: M244Vg,

G250E, Y253F ⁄H, E255K ⁄V, T315I, M351T, and

F359V. The greatest degree of resistance has been associ-

ated with the T315I mutation and point mutations in the

P-loop domain, i.e., G250E, Q252H, Y253F, and

E255K ⁄V (23).

To date, 17 distinct point mutations in the ABL kinase

domain conferring resistance to imatinib have been iden-

tified (24), which mainly cluster into four main groups

(13): imatinib-binding site, nucleotide-binding site for

ATP, activation loop, and hydrophobic patch between

helices E,F, and I in the terminal lobe of the enzyme.

Despite the differences in location, several of these

mutations decrease the binding affinity of Bcr–abl for

imatinib (25) although with different indices of resistance.

Mechanisms by which mutations in the imatinib-bind-

ing site lead to reduced binding affinity are as follows:

T315: Thr315 also known as gatekeeper residue located

at the periphery of the imatinib-binding site of Abl.

Mutations of threonine 315 to isoleucine result in disrup-

tion of H-bond (10). The absence of oxygen atom nor-

mally provided by the side chain of Thr315 precludes

formation of a hydrogen bond with the secondary amino

group of STI-571. In addition, isoleucine contains an

extra hydrocarbon group in the side chain, which results

in steric clash with STI-571 and presumably inhibits

binding (25). F317L and V289A mutations cause a loss

of van der Waal’s contacts with the pyridine and piper-

azyl moiety of the inhibitor, respectively. These amino

acids are located at the two extremes of the binding site

and shield it from water; thus, the reduction in the size

of the amino acid side chain increases the accessibility of

water to the binding site, and the strength of the interac-

tion within the binding site is reduced because of the

increased competition by water (10).

In the nucleotide-binding loop, the mutation Y253H is

predominantly found. Tyrosine 253 forms a hydrogen

bond with asparagine 322, which holds in place a folded

loop between two b-strands, increasing the surface com-

plementarity with STI-571 (22). Also, a small reduction

in hydrophobic interactions is observed, because the his-

tidine side chain is smaller than the tyrosine side chain.

The loss of a hydrogen bond is probably the major con-

tributing factor to resistance, because it causes a change

in the equilibrium between the active and inactive con-

formation, forcing the balance toward the active confor-

mation (13). Mutation at G250E reduces the space

available at the ‘entrance’ of the imatinib-binding pocket;

thus, the access to the imatinb-binding pocket is hin-

dered. Mutations at Q252 and E255K probably change

the protein dynamic towards activation by modifying the

intrinsic flexibility of the nucleotide-binding loop; thus,

the ability of the inhibitor to fit in the binding pocket is

hindered (13).

The third group of mutations is located in the activa-

tion loop and close to the hinge region of the flexible

sequence. So far, a single mutation at position 396

(H396P) has been described in this region. This region of

the kinase does not interact with STI-571, except for the

anchor region, which is located at the amino-terminal of

histidine 396. However, the activation loop has been

shown to be in closed (inactive) conformation to allow

STI-571 binding to take place; stabilization of the open

(active) conformation by phosphorylation of tyrosine 393

results in diminished susceptibility of ABL to inhibition

by STI-571. Thus, H396P mutations inhibits binding of

STI-571 by stabilization of the activation loop in an

open conformation or by destabilization of the closed

conformation, both of which can result in an enzyme

that remains in the active form for longer (22).

Introduction of polar residues into the hydrophobic

patch, as in the mutations M351T and F486S, decreases

the strength of the hydrophobic interactions and thus

decreases the stability of the structure, shifting the equi-

librium in favor of the active conformation resulting in a

decreased binding affinity for imatinib.

Rare cases of splicing events inducing deletions or

insertions of multiple nucleotides in the ABL kinase

domain leading to imatinib resistance have been

described. Hayette et al. (26) reported a novel frame shift

mutation acquired at the moment of imatinib resistance,

consisting an insertion of 12 nts and leading to the

conservation of open reading frame.

Mutations outside the kinase domain: It has been

shown in vitro that mutations outside the kinase domain

in the neighboring linker, SH2, SH3, and Cap domains

can confer imatinib resistance (27). In the context of



ABL, these domains have an autoinhibitory effect on

kinase activity, and mutations in this region can activate

the enzyme. In an attempt to determine the frequency

and relevance to resistance of regulatory domain muta-

tions, Sherbenou et al. screened for such mutations in a

cohort of consecutive patients with CML with various

levels of response. They found mutation T212R con-

ferred resistance to tyrosine kinase inhibitor and was

associated with relapse, whereas most other mutations

did not affect drug sensitivity (28).

BCR–ABL amplification: Overexpression of the Bcr–

abl protein owing to amplification of the BCR–ABL

gene was first observed in vitro when resistant CML cell

lines were generated by exposure to gradually increasing

doses of imatinib (29, 30). In one study, three of 11

patients with CML in blast crisis who relapsed after ini-

tially responding to imatinib were shown to have multi-

ple copies of the BCR–ABL gene by fluorescence in situ

hybridization (FISH) (25). In another study, seven of 55

patients showed a more than 10-fold increase in

BCR–ABL transcript levels, and two of the 32 patients

evaluated were found to have genomic amplification of

BCR–ABL by FISH (31). In the latter two patients, resis-

tance was primary and not acquired. 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. It is also possible that a transient over-

expression of BCR–ABL may be an early phenomenon in

the establishment of imatinib resistance, preceding the

emergence of a dominant clone with a mutant kinase

domain, as suggested by kinetic studies in cell lines (30).

Gene amplification as a mechanism of drug resistance

is also commonly observed where the drug inhibits an

enzyme within a critical biosynthetic pathway. For exam-

ple, methotrexate resistance is associated with amplifica-

tion of the dihydrofolate reductase (DHFR) gene 22.

Thus, amplification of genes encoding drug targets or

enzymes that can bypass a drug effect is a frequent

mechanism of drug resistance. However, amplification of

a tyrosine kinase in response to a tyrosine kinase inhibi-

tor has not previously been reported. The mechanisms of

gene amplification are not well understood. Genes may

be amplified in situ at intrachromosomal locations

through a mechanism that may involve repeated cycles

of breakage–fusion–bridge cycles, where chromosome

breakage is initiated at fragile sites (32).

Strategies to overcome imatinib resistance

Targeting tyrosine kinase activity using second-gener-ation and third-generation tyrosine kinase inhibitors

Second-generation TKIs: Several clinical observations

have shown impressing results against second-generation

Abl inhibitors nilotinib (AMN107, developed by Novar-

tis Pharmaceuticals, Basel, Switzerland) and dasatinib

(BMS-354825, developed by Bristol-Myers Squibb,

Princeton, USA). Nilotinib, like imatinib, requires the

Abl protein to be in the inactive conformation for opti-

mal binding. Nilotinib was found to be 10- to 25-fold

more potent as compared to imatinib in the reduction in

both autophosphorylation and proliferation (33) and sig-

nificantly active against 32 ⁄ 33 imatinib-resistant BCR–

Abl mutants (25). Dasatinib is a dual-specific SRC and

ABL inhibitor, structurally unrelated to imatinib that is

able to bind and inhibit both the active and inactive con-

formations of Abl, resulting in 100- to 300-fold higher

activity than imatinib. Shah et al. (34) recently demon-

strated that dasatinib has up to 100-fold increased activ-

ity against the Abl kinase compared to imatinib and

retains activity against 14 of 15 imatinib-resistant BCR–

ABL mutants in vitro.

Third-generation TKIs: The second-generation TKI, da-

satinib, holds great promise for the management of practi-

cally all imatinib-resistant mutations except for the T315I

mutation. T315I currently remains the most troublesome

mutant, and there are no effective treatment options for

patients harboring this mutation. Preclinical research has

demonstrated that AP24534 (ponatinib), an orally active

pan inhibitor of Bcr–abl, can inhibit the entire spectrum

of mutations that cause resistance to other Bcr–abl inhibi-

tors including the most resistant T315I (35). Ponatinib is

currently in phase II trials and showing impressing results

against T315I. Carbon–carbon triple bond linker is the

key structural feature of ponatinib that makes hydropho-

bic contact with the side chain of I315, allowing inhibition

of the T315I mutant. It also acts as an inflexible connector

that makes obligatory correct positioning of the two bind-

ing segments into their binding pockets. Its inhibitor pro-

file incorporates multiple contact points to confer very

high potency and balances the overall binding affinity.

The extensive network of optimized molecular contacts

leads to high potency and renders binding less susceptible

to disruption by single-point mutations (36). Another

third-generation TKI, bosutinib (SKI-606), is a dual Src–

abl inhibitor that is active in the low nanomolar range

against Bcr–abl (37). Bosutinib is now in phase III clinical

trials and has shown good activity in patients resistant to

imatinib or other TKIs in phase II (38). At nanomolar

concentrations, it inhibits the autophosphorylation of

both Abl and Src kinase, resulting in inhibition of cell

growth and apoptosis. Because of dual mechanism of

action, this agent shows activity in resistant CML disease,

other myeloid malignancies, and solid tumors. It seems to

cause fewer side effects because it more selectively inhibits

the faulty proteins in the leukemic cells and does not

affect similar proteins in normal cells as much as the ear-

lier drugs do.



Recently, two novel compounds were reported,

ONO12380 (39) and MK-0457 (40), to inhibit Bcr–abl

kinase activity through a distinct mechanism in cell lines

expressing the T315I mutation. Table 1 describes the

response of patients to second- and third-generation

TKIs.

Combination therapy

TKIs alone can induce remission in CML but do not elim-

inate leukemia stem cells, which remains a potential source

of relapse. Monotherapy leads to the generation of

mutants. Combination therapy involves the combination

of two or more anticancer drugs of which one is mostly a

TKI. Treatment with histone deacetylase inhibitor (HDA-

Cis) combined with imatinib effectively induced apoptosis

in quiescent CML progenitors resistant to elimination by

imatinib alone and eliminated CML stem cells capable of

engrafting immunodeficient mice. The interaction between

imatinib and HDACis inhibited genes regulating hemato-

poietic stem cell maintenance and survival (41). Combina-

tion of interferon-a and imatinib has also showed

impressing results. At 12 months, the rate of superior

molecular response was significantly higher among

patients receiving imatinib and peginterferon alfa-2a

(30%) than among those receiving 400 mg of imatinib

alone (14%) (42). Burchert et al. demonstrated that treat-

ment with IFN enables discontinuation of imatinib in

most patients after prior imatinib–IFN combination ther-

apy and may result in improved molecular response.

Induction of proteinase-3-specific CTL response by IFN

was supposed to contribute to this effect (43).

Targeting pathways downstream of BCR–ABL

The Ras ⁄Raf pathway is intimately linked to BCR–ABL

through the adaptor molecules Grb2 (growth factor

receptor–bound protein 2) and Crkl, inhibition of which

with a farnesyl transferase inhibitor (FTI), such as lona-

farnib, would theoretically reduce nuclear transcription

(11).

PI3K and its downstream targets, including the ser-

ine ⁄ threonine kinases AKT, mTOR, and p70S6 kinase,

play a key role in the regulation of cell survival and pro-

liferation. Recently, it was shown that activation of the

PI3K ⁄mTOR pathway by Bcr–abl contributes to

increased production of reactive oxygen species (44), thus

linking PI3K ⁄mTOR to mechanisms that have been

implicated in genomic instability and imatinib resistance.

Signal transduction through this pathway can be blocked

by inhibitors such as LY294002 (11) or wortmannin,

which specifically target PI3K. Cellular response to PI3K

inhibition includes induction of apoptosis and inhibition

of proliferation.

Modulating the levels of imatinib in Plasma

Imatinib is metabolized largely by the cytochrome p450

isoenzymes CYP3A4 and CYP3A5. Differences in the

concentrations of CYP3A4 ⁄CYP3A5 or drugs that can

inhibit or induce said enzymes have the potential to

greatly affect the levels of imatinib in plasma (10). Also,

in this regard, dose escalation of imatinib (600–

1000 mg ⁄d) can overcome resistance to standard-dose

imatinib (400 mg ⁄d) in some patients with CML. In

case of P-loop mutations, escalation of the dose of

imatinib is not recommended owing to highly resistant

nature of these mutations (38). Additionally, it is possi-

ble that this treatment will benefit only patients who

have suboptimal intracellular drug level with standard

dose of imatinib.

CML vaccines

In recent years, a series of DNA vaccines have been

developed based on different aspects to target the CML

cells. Lucansky et al. (45) demonstrated the use of a plas-

mid carrying either the complete BCR–ABLl fusion gene

or fragment thereof coding for a 25-amino-acid-

sequence-long junction zone (bcr–abl 25 amino acids)

linked with genes coding for a variety of immunostimula-

tory factors, as the DNA vaccines in mice. Peptide vac-

cines derived from amino acid sequences crossing the

b3a2 fusion breakpoint have proved their efficacy in

patients with chronic-phase CML (46). These vaccines

elicit class I restricted cytotoxic T lymphocytes and class

II responses. Investigators from Johns Hopkins Sidney

Kimmel Comprehensive Cancer Center have developed a

vaccine made from CML cells irradiated to halt their

cancerous potential and genetically altered to produce an

immune system stimulator called GM-CSF. The tumor

cells also carried antigens specific to CML cells, which

primed the immune system to recognize and kill circulat-

ing CML cells.

Destabilizing BCR–ABL protein

Stability of Bcr–abl is dependent upon its ability to form

complex with HSP90 and co-chaperone protein p23.

HSP90 antagonist such as 17-allylamino-17-demethoxy-

geldanamycin (17-AAG) interfere with Bcr–abl chaperon-

ing such that the mature protein would be unable to fold

correctly into its quaternary structure, leaving it suscepti-

ble to proteasome degradation (11).

Targeting mRNA of BCR–ABL

RNA Interference Technology: RNA interference is a

powerful tool that has been used to inhibit gene function



Tab

le1

Responses

tofirs

t-,

second-,

and

third-g

enera

tion

tyro

sin

ekin

ase

inhib

itors

(TK

Is)

Dru

gs

Mechanis

mof

action

Chem

ical

str

uctu

re

Response

Advanta

ges

Dis

advanta

ges

Chro

nic

phase

Refe

rences

Accele

rate

dphase

and

bla

st

phase

Refe

rences

HR

CR

MR

HR

CR

MR

Imatinib

Imatinib

functions

by

bin

din

gin

the

ATP

bin

din

gsite

and

sta

bili

zes

the

inactive

confo

rmation

of

BC

R-A

BL,

in

whic

hth

ew

ell

know

n‘D

FG

tria

d’

isin

the

‘out’

confo

rmation

93%

CH

R

45%

CC

yR

,

11%

MC

yR

,

9%

mC

yR

93%

MM

R,

7%

CM

R

54

43%

CH

R

14%

CC

yR

,

14%

MC

yR

,

14%

mC

yR

54

First-

line

treatm

ent

option

for

chro

nic

myelo

idle

ukem

ia

(CM

L).

First

site-

directe

danti-c

ancer

dru

gw

ith

few

er

sid

e

eff

ects

.S

afe

for

pedia

tric

patients

with

CM

L

Sto

mach

upset,

muscle

cra

mps,

poundin

gheart

beat,

extr

em

etire

dness,

unexpla

ined

weig

ht

gain

.R

are

:

bla

ck

⁄blo

ody

sto

ols

,

dark

urine,

yello

win

g

eyes

⁄skin

,fe

ver,

chill

s,

pers

iste

nt

sore

thro

at,

serious

alle

rgic

reaction,

itchin

g,

trouble

bre

ath

ing

Nilo

tinib

(firs

t-lin

e)

Mechanis

mof

action

of

nilo

tinib

issim

ilar

toim

atinb.

Only

diffe

rence

is,

incorp

ora

tion

of

altern

ative

bin

din

ggro

ups

to

N-m

eth

ylp

ipera

zine,

while

am

ide

pharm

acophore

is

pre

serv

ed

tore

tain

H-b

ond

inte

ractions

toG

lu286

and

Asp381

96%

CC

yR

85%

MM

R

55

Nilo

tinib

was

specifi

cally

desig

ned

toim

pro

ve

upon

the

mole

cula

rstr

uctu

re

of

GLIV

EC

as

aw

ay

of

pre

venting

Ph+

CM

Lre

sis

tance

to

the

imatinib

mole

cule

Unexpla

ined

ble

edin

g

or

bru

isin

g,

blo

od

in

urine

or

sto

ol,

fever,

sore

thro

at,

chill

s,

or

oth

er

sig

ns

of

infe

ction,

sw

elli

ng

of

hands,

ankle

s,

feet,

or

face

Nilo

tinib

(second-lin

e)

67%

CH

R

22%

mC

yR

,

11%

PC

yR

,

11%

CC

yR

33%

MM

R

56

100%

CH

R

0%

mC

yR

,

0%

CC

yR

,

50%

PC

yR

0% M

MR

56

Dasatinib

(firs

t-lin

e)

Dasatinib

bin

ds

inth

e

ATP

-bin

din

gsite

of

the

AB

L

kin

ase.

Itbin

ds

toth

eactive

or

‘opened’

confo

rmation

of

the

BC

R-A

BL

kin

ase

dom

ain

and

ispre

dic

ted,

based

on

modelin

gstu

die

s,

toals

obin

d

toth

ein

active

or

‘clo

sed’

confo

rmation

98%

CC

yR

82%

MM

R,

10%

CM

R

55

77%

CC

yR

,46%

MM

R

57

Patients

with

new

ly

dia

gnosed

CM

L

show

superior

com

ple

te

cyto

genetic

response

and

majo

r

mole

cula

rre

sponse

in1

yr

when

treate

d

with

once

daily

dasatinib

com

pare

d

with

once

daily

imatinib

.A

lso,

active

again

st

muta

tions

whic

hchange

the

confo

rmation

of

kin

ase

from

clo

sed

toopen

Severe

alle

rgic

reactions,

bla

ck,

tarr

y,

or

bright

red

sto

ols

,blo

od

inth

e

vom

it,

depre

ssio

n,

unusualsw

elli

ng

or

weig

ht

gain

,unusual

tire

dness

or

weakness,

vom

it

that

looks

like

coff

ee

gro

unds

Dasatinib

(second-lin

e)

81%

CH

R

25%

mC

yR

,

31%

CC

yR

,

6%

PC

yR

13%

MM

R56

88%

CH

R

13%

mC

yR

,

25%

CC

yR

,

13%

PC

yR

13%

MM

R

56



either by increasing the destruction of mRNA corre-

sponding to the gene or, in some cases, by inhibiting the

transcription of the gene or the translation of mRNA to

the corresponding protein. The use of RNA interference,

using a breakpoint-specific short interfering RNA, has

been shown to selectively inhibit BCR–ABL-dependent

proliferation, as well as to enhance the effect of imatinib

against cells expressing an imatinib-resistant point

mutant and cells overexpressing Bcr–abl (47).

Antisense oligonucleotide

Bcr–abl is an ideal molecular target for therapy based

on antisense (AS) strategies. By designing an AS oligo-

nucleotide that is complementary to both the BCR and

ABL sequences on either side of the BCR–ABL tran-

script junction, it should be possible to generate a spe-

cies that would hybridize to BCR–ABL mRNA, thus

forming mRNA ⁄oligonucleotide heteroduplexes and thus

preventing BCR–ABL mRNA from associating with

ribosomes, leaving the transcript open for degradation

by RNase H.

Antisense strategies include antisense molecules such

as 2¢-O-methoxyethyl RNA, locked nucleic acids, and

peptide nucleic acids, as well as more exotic chemical

species such as morpholinos (48). Despite its encouraging

results with murine models, this technique has failed to

fulfill its promise as a treatment for CML. Lack of speci-

ficity of the junction AS oligos, the need to synthesize

them with special chemical backbones and to introduce

them into the cells via stringent permeabilization proce-

dures, and the extremely long life of the Bcr–abl protein

are some of the reasons for the failure (49). Another dis-

advantage of this method includes high quantities of

antisense oligonucleotides required to reach an effective

dose and thus the potential toxicity as a result of non-

specific binding (50).

Ribozymes

Ribozymes are RNA molecules that are capable of

associating with other RNA molecules by base-pairing

and catalyzing the hydrolysis of specific phosphodiester

bonds within the target RNA sequence. To date, vari-

ous types of ribozyme have been constructed to cleave

other RNAs and such trans-acting ribozymes include

hammerhead, hairpin, and HDV ribozymes. Ribozymes

can discriminate mutant sequences from their wild-type

counterparts, even when they differ by single nucleotide

base. Lance H. Leoplod demonstrated 3 log reduction

in the level of bcr–abl1 mRNA when multiunit ribo-

zyme was transfected via folate receptor–mediated

uptake into transformed 32Dcells (expressing bcr–abl

protein) (51).Tab

le1

(Continued)

Dru

gs

Mechanis

mof

action

Chem

ical

str

uctu

re

Response

Advanta

ges

Dis

advanta

ges

Chro

nic

phase

Refe

rences

Accele

rate

dphase

and

bla

st

phase

Refe

rences

HR

CR

MR

HR

CR

MR

Bosutinib

Bosutinib

isa

dualkin

ase

inhib

itor.

Itin

hib

its

the

auto

phosphory

lation

of

both

Abland

Src

kin

ases,

resultin

gin

inhib

itio

nof

cell

gro

wth

and

apopto

sis

86%

CH

R37%

MC

yR

,

22%

CC

yR

27%

MM

R,

5%

CM

R

58

61%

CH

R48%

MC

yR

,

33%

CC

yR

15%

MM

R,

4%

CM

R

58

Superior

rate

sof

majo

rm

ole

cula

r

response

(MM

R)

and

cum

ula

tive

com

ple

tem

ole

cula

r

response,

com

pare

d

with

the

sta

ndard

thera

py,

imatinib

Dia

rrhoea,

rash,

incre

ased

ala

nin

e

transam

inase,

thro

mbocyto

penia

,

neutr

openia

,

anaem

ia

Ponatinib

Carb

on-c

arb

on

trip

lebond

linker

makes

hydro

phobic

conta

ct

with

the

sid

echain

of

I315,

allo

win

g

inhib

itio

nof

the

T315I

muta

nt.

It

als

oacts

as

an

inflexib

le

connecto

rth

at

makes

oblig

ato

ry

corr

ect

positio

nin

gof

the

two

bin

din

gsegm

ents

into

their

bin

din

gpockets

.It

sin

hib

itor

pro

file

incorp

ora

tes

multip

le

conta

ct

poin

tsto

confe

rvery

hig

h

pote

ncy

and

bala

nce

the

overa

ll

bin

din

gaffi

nity

95%

CH

R66%

MC

yR

,

53%

CC

yR

No

CH

R,

35%

MH

R.

InT315I,

20%

MH

R

24%

MC

yR

,

12%

CC

yR

.

InT315I

muta

tions,

20%

MC

yR

Active

again

st

T315I

and

oth

er

TK

I

resis

tant

muta

nts

Sid

eeff

ects

:

Thro

mbocyto

penis

,

headache,

nausea,

art

hra

lgia

,fa

tigue,

anem

ia

HR

,haem

ato

logic

response;

CyR

,cyto

genetic

response;

MR

,m

ole

cula

rre

sponse;

CH

R,

com

ple

tehaem

ato

logic

response

(Norm

aliz

ation

of

blo

od

count)

;C

CyR

,com

ple

tecyto

genetic

response

(Ph+

0);

PC

yR

,part

ialcyto

genetic

response

(Ph+

1–35%

);M

CyR

,

min

or

cyto

genetic

response

(Ph+

36–65%

);m

CyR

,m

inim

alcyto

genetic

response

(66–95%

);C

MR

,com

ple

tem

ole

cula

rre

sponse

(BC

R–A

BL

transcripts

non

quantifiable

and

non

dete

cta

ble

).



Blocking the oligomerization of BCR–ABL

Monomeric Bcr–abl is non-transforming, whereas Bcr–

abl tetramers are oncogenic, transforming species. Thus,

Bcr–abl must undergo transformation to become acti-

vated. Beissert et al. (52) demonstrated that targeting the

N-terminal coiled coil oligomerization interface of bcr

interferes with the transformation potential of Bcr–abl

and increases sensitivity to STI571. Although currently

none of the available therapies for CML target the oligo-

merization domain of Bcr–abl, this approach could be

exploited for the therapeutic gain as a combined therapy

with imatinib.

Entrapment of BCR–ABL in the nucleus

BCR–ABL has a cytoplasmic localization, in contrast to

ABL, mostly nuclear. The shuttling of ABL between the

nucleus and the cytoplasm is accomplished by the presence

of import and export signal sequences in the COOH- ter-

minal region of ABL. Interestingly, these sequences are

conserved in BCR–ABL. It has been shown that the inhi-

bition of BCR–ABL export from the nucleus has been

shown to induce apoptosis in Bcr–abl-expressing cells.

This inhibition is accomplished by the use of export inhibi-

tors such as leptomycin. Vigneri et al. showed that com-

bining STI571 with leptomycin B blocks the nuclear

export of BCR–ABL, which in turn activates apoptosis.

This combined therapy causes the irreversible and com-

plete killing of BCR–ABL-transformed cells, whereas the

effect of either drug alone is fully reversible (53). These

results indicate that nuclear entrapment of BCR–ABL can

be used as a therapeutic strategy to selectively kill chronic

myelogenous leukemia cells.

Conclusion

A remarkable breakthrough in cancer therapy came with

the introduction of imatinib to the treatment for CML.

Like other anticancer drugs, clinical resistance to imati-

nib therapy emerged. This picture gave rise to invention

of new second-generation and thereafter third-generation

TKI.

With so much advancement in CML therapy, the fact

that there is no permanent cure for CML and relapses is

a common scenario in patients with CML on treatment,

which still remains unavoidable. Bearing in mind that

CML is a pluripotent stem cell disorder, newer methods

targeting the CML stem cells should be designed.

References

1. Clarkson B. Chronic myelogenous leukemia: etiology,

incidence, and clinical features. Encycl Cancer

2002;1:505–18.

2. Deininger MWN, Goldman JM, Melo JV. The molecu-

lar biology of chronic myeloid leukemia. Blood

2000;96:3343–55.

3. Daley GQ, Van Etten RA, Baltimore D. Induction of

chronic myelogenous leukemia in mice by the P210bcr ⁄ ablgene of the Philadelphia chromosome. Science 1990;

247:824–30.

4. Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyro-

sine kinase activity and transformation potency of bcr ⁄ abloncogene products. Science 1990;247:1079–82.

5. Kantarjian HM, Talpaz M, Giles F, O’Brien S, Cortes J.

New insights into the pathophysiolody of chronic myeloid

leukemia and imatinib resistance. Ann Intern Med

2006;145:913–23.

6. Druker BJ. STI571 (Gleevec) as a paradigm for cancer

therapy. Trends Mol Med 2002;8(4 Suppl.):S14–8.

7. Azam M, Daley GQ. Anticipating clinical resistance to

target-directed agents: the BCR-ABL paradigm. Mol

Diagn Ther 2006;10:67–76.

8. Weisberg E, Griffin J. Resistance to imatinib (Glivec):

update on clinical mechanisms. Drug Resist Updat

2003;6:231–8.

9. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic

and cytogenetic responses to imatinib mesylate in chronic

myelogenous leukaemia. N Engl J Med 2002;346:645–52.

10. Quintas-Cardama A, Kantarjian H, Cortes J. Mechanisms

of primary and secondary resistance to imatinib in chronic

myeloid leukemia. Cancer Control 2009;16:122–31.

11. Jorgensen HG, Holyoake TL. Characterization of cancer

stem cells in chronic myeloid leukaemia. Biochem Soc

Trans 2007;35:1347–51.

12. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK,

Jordanides N, Barow M, Mountford JC, Holyoake TL.

Dasatinib targets an earlier progenitor population than

imatinib in primary CML but does not eliminate the

quiescent fraction. Blood 2006;107:4532–9.

13. Gambacorti-Passerini CB, Gundy RH, Piazza R, Galietta

A, Rostagno R, Scapozza L. Molecular mechanisms of

resistance to imatinib in Philadelphia-chromosome-posi-

tive leukaemias. Lancet Oncol 2003;4:75–85.

14. Larghero J, Mahon FX, Madeleine-Chambrin I, et al.

Elevated levels of the plasma protein alpha 1 acid glyco-

protein in chronic myelogenous leukemia in blast crisis

mediate pharmacological resistance to Gleevac (STI571,

imatinib) in vitro and are associated with primary resis-

tance in vivo. Presented at the 43rd annual meeting of the

American Society of Hematology 2001, Orlando.

15. Galimberti S, Cervetti G, Guerrini F, Testi R, Pacini S,

Fazzi R, Simi P, Petrini M. Quantitative molecular moni-

toring of BCR-ABL and MDR1 transcripts in patients

with chronic myeloid myeloid leukaemia during imatinib

treatment. Cancer Genet Cytogenet 2005;162:57–62.

16. Mahon FX, Belloc F, Lagarde V, Chollet C, Moreau-

Gaudry F, Reiffers J, Goldman J, Melo J. MDR1 gene

overexpression confers resistance to imatinib mesylate in

leukaemia cell line models. Blood 2003;101:2368–73.



17. Ferrao P, Frost MJ, Siah SP, Ashman L. Overexpression

of P-glycoprotein in K562 cells does not confer resistance

to the growth inhibitory effects of imatinib (STI571) in

vitro. Blood 2003;102:4499–503.

18. Dulucq S, Bouchet S, Turcq B, Lippert E, Etienne G,

Reiffers G, Molimard M, Krajinovic M, Mahon FX.

Multidrug resistance (MDR1) polymorphisms are associ-

ated with major molecular responses to standard-dose

imatinib in chronic myeloid leukemia. Blood

2008;112:2024–7.

19. Yamada O, Ozaki K, Furukawa T. Activation of STAT 5

confers imatinib resistance on leukemic cells through the

transcription of TERT and MDR1. Cell Signal

2011;23:1119–27.

20. Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL,

Kuriyan J, Sawyers CL. Multiple BCR ⁄ABL kinase

domain mutations confer polyclonal resistance to the tyro-

sine kinase inhibitor imatinib (STI571) in chronic myeloid

leukaemia. Cancer Cell 2002;2:117–25.

21. Nardi V, Azam M, Daley G. Mechanisms and implica-

tions of imatinib resistance mutations in BCR-ABL. Curr

Opin Hematol 2004;11:35–43.

22. von Bubnoff N, Schneller F, Peschel C, Duyster J. BCR-

ABL gene mutations in relation to clinical resistance of

Philadelphia-chromosome-positive leukemia to STI-571; a

prospective study. Lancet 2002;359:487–91.

23. Hughes T. Mechanisms of resistance, common BCR-ABL

mutations, and monitoring response to treatment in

CML. Medscape 2008. http://www.medscape.org/viewarticle/

575913. Accessed August 17, 2011.

24. Branford S, Rudzki Z, Walsh S, et al. Detection of

BCR ⁄ABL mutations in patients with CML treated with

imatinib is virtually always accompanied by clinical resis-

tance, and mutations in the ATP phosphate-binding loop

(P-loop) are associated with a poor prognosis. Blood

2003;102:276–83.

25. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette

R, Rao PN, Sawyers CL. Clinical resistance to STI-571

cancer therapy caused by BCR-ABL gene mutation or

amplification. Science 2001;293:876–80.

26. Hayette S, Chabane K, Tchirkov A, Berger M, Nicolini

F, Tournilhac O. Detection of twelve nucleotide insertions

in the BCR ⁄ABL kinase domain in an imatinib resistant

but dasatinib sensitive patient with bi-phenotypic acute

leukemia. Haematologica 2009;94:1324–6.

27. Azam M, Latek R, Daley G. Mechanisms of autoinhibi-

tion and STI-571 ⁄ imatinib resistance revealed by muta-

genesis of BCR-ABL. Cell 2003;112:831–43.

28. Sherbenou D, Hantschel O, Kaupe I. BCR-ABL SH3-

SH2 domain mutations in chronic myeloid leukemia

patients on imatinib. Blood 2010;116:3278–85.

29. Mahon FX, Deininger MW, Schultheis B, Chabrol J,

Reiffers J, Goldman JM, Melo JV. Selection and charac-

terization of BCR-ABL positive cell lines with differential

sensitivity to the tyrosine kinase inhibitor STI571: diverse

mechanisms of resistance. Blood 2000;96:1070–9.

30. Barnes D, Schultheis B, Adedeji S, Melo J. Dose-depen-

dent effects of Bcr-Abl in cell line models of different

stages of chronic myeloid leukemia. Oncogene

2005;24:6432–40.

31. Hochhaus A, Kreil S, Corbin AS. Molecular and chromo-

somal mechanisms of resistance to imatinib (STI571)

therapy. Leukemia 2002;16:2190–6.

32. Weisberg E, Griffin J. Mechanism of resistance to theABL

tyrosine kinase inhibitor STI571 in BCR ⁄ABL-trans-

formed hematopoietic cell lines. Blood 2000;95:3498–505.

33. Weisberg E, Manley PW, Breitenstein W. Characterization

of AMN107, a selective inhibitor of native and mutant

BCR ⁄ABL. Cancer Cell 2005;7:129–41.

34. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers

CL. Overriding imatinib resistance with a novel ABL

kinase inhibitor. Science 2004;305:399–401.

35. O’Hare T, Shakespeare W, Zhu X, et al. AP24534, a

Pan-BCR-ABL inhibitor for chronic myeloid leukemia,

potently inhibits the T315I mutant and overcome

mutation-based resistance. Cancer Cell 2009;16:401–12.

36. Zhou T, Commodore L, Huang WS, et al. Structural

mechanism of the Pan-BCR-ABL inhibitor ponatinib

(AP24534): lessons for overcoming kinase inhibitor resis-

tance. Chem Biol Drug Des 2011;77:1.

37. Golas JM, Arndt K, Etienne C, Lucas J, Nardin D,

Gibbons J, Frost P, Ye F, Boschelli DH, Boschelli F.

SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor

of Src and Abl kinases, is a potent antiproliferative agent

against chronic myelogenous leukemia cells in culture and

causes regression of K562 xenografts in nude mice. Cancer

Res 2003;63:375–81.

38. Cortes-Franco J, Kantarjian H, Kim DW, Khoury HJ,

Turkina A, Shen ZX, Brummendorf TH, Chandy M,

Arkin S, Gambacorti-Passerini C. Efficacy and safety of

bosutinib (SKI-606) among patients with chronic phase

Ph+ chronic myelogenous leukemia (CML). Blood

2007;110:11.

39. Bartholomeusz G, Talpaz M, Kapuria V, Kong LY,

Wang S, Estrov Z, Priebe W, Wu J, Donato N. Activation

of a novel Bcr ⁄Abl destruction pathway by WP1130

induces apoptosis of chronic myelogenous leukemia cells.

Blood 2007;109:3470–8.

40. Giles FJ, Cortes J, Jones D, Bergstrom D, Kantarjian H,

Freedman SJ. MK-0457, a novel kinase inhibitor, is active

in patients with chronic myeloid leukemia or acute lym-

phocytic leukemia with the T315I BCR-ABL mutation.

Blood 2007;109:500–2.

41. Zhang B, Guilhot J, Nicolini FE. Effective targeting of

quiescent chronic myelogenous leukemic stem cells by his-

tone deacetylase inhibitors in combination with imatinib

mesylate cancer. Cell 2010;17:427–42.

42. Preudhomme C, Guilhot J, Nicolini FE, et al. Imatinib

plus peginterferon alfa-2a in chronic myeloid leukemia.

N Engl J Med 2010;363:2511–21.

43. Burchert A, Muller MC, Kostrewa P, Erben P, Bostel T,

Liebler S, Hehlmann R, Neubauer A, Hochhaus A, et al.



Sustained molecular response with interferon alfa mainte-

nance after induction therapy with imatinib plus inter-

feron alfa in patients with chronic myeloid leukemia.

J Clin Oncol 2010;28:1429–35.

44. Kim J Chu S, Gramlich J, Pride Y, Babendreier E,

Chauhan D, Salgia R, Podar K, Griffin J, Sattler M, et al.

Activation of the PI3K ⁄mTOR pathway by BCR ⁄ABL

contributes to increased production of reactive oxygen

species. Blood 2005;105:1717–23.

45. Lucansky V, Sobotkova E, Tachezy R, Duskova M,

Vonka V. DNA vaccination against bcr-abl positive cells

in mice. Int J Oncol 2009;35:941–51.

46. Pinilla-Ibarz J, Cathcart K, Korontsvit T, Soignet S,

Bocchia M, Caggiano J, Lai L, Jimenez J, Kolitz J,

Scheinberg DA. Vaccination of patients with chronic

myelogenous leukemia with bcr-abl oncogene breakpoint-

fusion peptides generates specific immune responses. Blood

2000;95:1781–7.

47. Wohlbold L, van der Kuip H, Miething C, Vornlocher H,

Knabbe C, Duyster J, Aulitzky W. Inhibition of

BCR ⁄ABL gene expression by small interfering RNA sen-

sitizes for imatinib mesylate (STI-571). Blood 2003;2:2236–

9.

48. Melo J, Hughes T, Apperley J. Chronic myeloid leukemia.

Am Soc Hematol 2003;1:132–52.

49. Clark RE. Antisense therapeutics in chronic myeloid

leukaemia: the promise, the progress and the problems.

Leukemia 2000;14:347–55.

50. Dhut S, Chaplin T, Young BD. BCR ⁄ABL and BCR

proteins: biochemical characterization and localization.

Leukemia 1990;4:745–50.

51. Leopold L, Shore S, Newkirk TA, Reddy R, Reddy EP.

Multi-Unit Ribozyme-Mediated Cleavage of bcr-abl

mRNA in Myeloid Leukemias. Blood 1995;85:2162–70.

52. Beissert T, Puccetti E, Bianchini A, Guller S, Boehrer S,

Hoelzer D, Ottmann OG, Nervi C, Ruthardt M. Target-

ing of the N-terminal coiled coil oligomerization interface

of BCR interferes with the transformation potential of

BCR-ABL and increases sensitivity to STI571. Blood

2003;102:2985–93.

53. Vigneri P, Wang J. Induction of apoptosis in chronic mye-

logenous leukemia cells through nuclear entrapment of

BCR–ABL tyrosine kinase. Nat Med 2001;7:228–34.

54. Parikh P, Saikia T, Tripathi A, Shah S, Sheth A, Shah P.

Role of Imatinib Mesylate in Chronic Myeloid Leukemia.

In: Agarwal MB, ed. Chronic Myeloid Leukemia, 1st edn.

Mumbai: Ranbaxy Super Specialities Ranbaxy House,

2007:89–105.

55. Rosti G, Castagnetti F, Gugliotta G, Palandri F,

Baccarani M. Second-generation BCR-ABL inhibitors

for frontline treatment of chronic myeloid leukemia in

chronic phase. Crit Rev Oncol Hematol 2011. doi: 10.1016/

j.critrevonc.2011.04.002. Epub ahead of print.

56. Garg R, Kantarjian H, O’Brien S, Quinta¢s-Cardama A,

Faderl S, Estrov Z, Cortes J. The use of nilotinib or

dasatinib after failure to 2 prior tyrosine kinase inhibitors:

long-term follow up. Blood 2009;114:4361–8.

57. DiBella N. First-line treatment of chronic myeloid leuke-

mia: imatinib versus nilotinib and dasatinib. Commun

Oncol 2011;8:65–72.

58. Nelson R. Bosutinib effective as second- and third-line

therapy in CML. ASCO 2010 Annual Meeting.



Documents

Recent developments in drug resistance mechanism in chronic myeloid leukemia: a review