Src-family kinases in the development and therapy of Philadelphia
chromosome-positive chronic myeloid leukemia and acute
The Jackson Laboratory, Bar Harbor, ME, USA
(Received 25 September 2007; accepted 27 September 2007)
The BCR-ABL kinase inhibitor imatinib has shown significant efficacy in chronic myeloid leukemia (CML) and is the standard
front-line therapy for patients in chronic phase. However, a substantial number of patients are either primarily refractory or
acquire resistance to imatinib. While a number of mechanisms are known to confer resistance to imatinib, increasing evidence
has demonstrated a role for BCR-ABL–independent pathways. The Src-family kinases (SFKs) are one such pathway and have
been implicated in imatinib resistance. Additionally, these kinases are key to the progression of CML and Philadelphia
chromosome-positive acute lymphoblastic leukemia (Phþ ALL). The dual SFK/BCR-ABL inhibitor dasatinib is now clinically
available and has markedly greater potency compared with imatinib against native BCR-ABL and the majority of imatinib-
resistant BCR-ABL mutants. Therefore, this agent, as well as other dual SFK/BCR-ABL inhibitors under development, could
provide added therapeutic advantages by overcoming both BCR-ABL–dependent (i.e., BCR-ABL mutations) and –
independent forms of imatinib resistance and delaying transition to advanced phase disease. In this review, we discuss the
preclinical and clinical evidence demonstrating the involvement of SFKs in imatinib resistance and the progression of CML and
Phþ ALL, as well as the potential role of dual SFK/BCR-ABL inhibition in the management of these diseases.
Keywords: Src, leukemia, BCR-ABL, dasatinib, imatinib resistant
The constitutively active BCR-ABL tyrosine kinase is
the defining molecular abnormality in Philadelphia
chromosome-positive (Phþ) chronic myeloid leuke-
mia (CML) and acute lymphoblastic leukemia (ALL)
[1–6]. The pathogenic role of BCR-ABL in CML
and Phþ ALL provided the rationale for therapeutic
targeting of this signaling protein. Imatinib was the
first available BCR-ABL targeted therapy and is
currently the standard front-line therapy for CML in
chronic phase (CP). However, despite the significant
efficacy of this agent, a substantial number of patients
are either primarily resistant to treatment or acquire
resistance during the course of treatment [7–14].
Additionally, imatinib does not completely eradicate
residual leukemic stem cells and progenitors [15,16],
which present a persistent risk of disease relapse.
The Src-family kinases (SFKs) have been impli-
cated in BCR-ABL signaling [17,18] and in the
progression of CML and Phþ ALL [19–27].
SFKs are involved in BCR-ABL-independent forms
of imatinib resistance [26,27]. Here we will review
the preclinical and clinical evidence demonstrating
SFK involvement in BCR-ABL signaling, the trans-
forming activity of BCR-ABL, progression of CML
and Phþ ALL, and imatinib resistance.
Oncogenic signaling pathways in CML
and PHþ ALL
BCR-ABL is a constitutively active, non-receptor
tyrosine kinase [2,3,28]. The central role of this
oncogenic kinase in the pathogenesis of CML has
been well established [3,29]. BCR-ABL initiates
Correspondence: Shaoguang Li, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA. Tel: þ207-288-6734. Fax: þ207-288-6078.
Leukemia & Lymphoma, January 2008; 49(1): 19–26
ISSN 1042-8194 print/ISSN 1029-2403 online ? 2008 Informa UK Ltd.
numerous signal transduction pathways that influ-
ence the growth and survival of hematopoietic cells
and collectively induce leukemic transformation,
such as STAT5, MEK1/2/ERK1/2, and NF-kB
. Several mechanisms have been implicated in
the transforming activity of BCR-ABL, including
constitutive mitogenic signaling  and reduced
dependency on external growth factors , altered
cell adhesion properties , and reduced apoptotic
potential . Additionally, evidence suggests that
[35,36], which may play a role in disease progression
by exacerbating genomic instability and promo-
ting the accumulation of additional cytogenetic
Given the central role of BCR-ABL in the
pathogenesis of CML, it is an attractive target for
BCR-ABL kinase activity alone may not be sufficient
for the management of CML, as downstream path-
ways of BCR-ABL can be activated independently of
BCR-ABL kinase activity , thereby leading to
imatinib resistance. The SFKs are an example of
such a downstream activator, and have been sug-
gested to confer BCR-ABL independence. These
non-receptor, intracellular tyrosine kinases regulate
signal-transduction pathways involved in cell growth,
differentiation, and survival [37–39] and are among
the most extensively studied oncogenes in human
cancers . There are eight known SFK members
(Src, Blk, Fgr, Fyn, Hck, Lck, Lyn, and Yes) with
each comprising a unique domain and high-sequence
homology in the four Src homology domains
(SH1-4) . SFKs exhibit a range of tissue
expression patterns and several are primarily ex-
pressed in hematopoietic cells (Table I) [39,41].
Numerous studies have indicated an association
between SFKs and myeloid and lymphoid leukemias
. Early research demonstrated the proleukemic
potential of SFKs in a variety of hematopoietic cell
lines [42–46]. Danhauser-Riedel et al. provided the
first data demonstrating that the activity of the SFKs,
Lyn and Hck, is increased in hematopoietic cells
expressing BCR-ABL . Activation of Hck or
other SFK members has been suggested to be
required for BCR-ABL–mediated transformation
[20,47]. Expression of a kinase defective mutant of
Hck blocked BCR-ABL–induced outgrowth of
cytokine dependent leukemia cell lines . Further-
more, pharmacologic inhibition of SFKs led to
growth arrest and apoptosis in CML cell lines .
Recent SFK research has centered on the pathologic
role of these signaling molecules in CML and Phþ
ALL, their involvement in disease progression and
the development of imatinib resistance.
Cooperation between BCR-ABL and SFK
SFKs are collaborative oncogenic kinases in BCR-
ABL–induced leukemias and may act to couple
BCR-ABL to certain downstream signaling pathways
involved in leukemic transformation (Figure 1)
[17,18,20,47,48]. SFKs are activated through direct
interaction with BCR-ABL [17,18,25], and likely
involves the release of intramolecular, auto-inhibitory
constraints [17,20,38]. In turn, the activity of BCR-
ABL can be enhanced through SFK-mediated
phosphorylation, which by Hck of tyrosine residues
within the activation loop of ABL was found to
increase ABL kinase activity . In another study, it
was demonstrated that Hck, Lyn, and Fyn phosphor-
ylate multiple tyrosine residues within the SH3-SH2
Table I. Expression of SFKs in hematopoietic cells .
Lineage SFK member
Blk, Fgr, Fyn, Lyn
Fgr, Hck, Lyn
SFK, Src-family kinase.
Figure 1. SFKs directly interact with BCR-ABL resulting in (1)
activation of SFKs [17,18,25] and (2) augmentation of BCR-ABL
kinase activity . Activated SFKs work cooperatively with BCR-
ABL in facilitating the growth and progression of leukemia
[48,50,51]. Several downstream effectors of SFKs have been
proposed to mediate the proleukemic effects, such as (3) STAT5
, which is known to activate genes involved in growth factor
[36,52–54] and (4) AKT , which is key in regulating cell
proliferation and survival in BCR-ABL–dependent cells .
(5) Active SFKs also phosphorylate certain tyrosine residues on
BCR-ABL to create a binding site for GRB-2. This adaptor protein
may link the BCR-ABL pathway to Ras, which is known to activate
the MEK/ERK oncogenic signaling cascade [17,48].
adhesion, and DNArepair
region of BCR-ABL, and that these phosphorylations
are required for full oncogenicity of BCR-ABL in
myeloid cell lines. This impact on BCR-ABL
function was suggested to occur through the release
of an autoregulatory function that holds the kinase
domain in an inactive state .
Role of SFKS in the progression of CML
to blast crisis
Although BCR-ABL is considered the trigger for
malignant transformation in CML, there is evidence
of an important role for SFKs in disease progression.
Studies from our laboratory demonstrated that the
transition of CML to lymphoid blast crisis (LBC) in
mice requires the presence of Lyn, Hck, and Fgr
, and Donato et al. showed that overexpression
and/or activation of Hck and Lyn occur during CML
progression . Downregulation of Lyn expression
by RNAi was found to induce apoptosis in both
myeloid and lymphoid blast cells, and this effect was
more pronounced in the latter . Although the
biology of CML progression is not fully understood
and likely involves multiple factors, the studies above
clearly implicate SFKs in the development of
advanced phase CML, particularly LBC. Further-
more, the involvement of SFKs in disease progres-
sion may partially explain the aggressive nature of
advanced CML  and its relatively poor respon-
siveness to imatinib [12,57,58].
Role of SFKS in PHþ ALL
SFKs also appear to play a significant role in the
development of Phþ ALL, which may be indepen-
dent of BCR-ABL kinase activity. Data from our
laboratory with mouse knockout models demon-
strated that SFKs are required for induction of B-
ALL by BCR-ABL . We subsequently found that
although imatinib had a weak effect on the survival of
mice with B-ALL induced by BCR-ABL, coinhibi-
tion of BCR-ABL kinase activity and SFKs main-
tained long-term survival . The independent role
of SFKs in B-ALL is further supported by results
demonstrating that a SFK inhibitor reduced the
viability, and induced apoptosis, in pre-B leukemia
cells expressing the imatinib-resistant T315I BCR-
ABL mutant . Moreover, while imatinib had no
effect in mice with B-ALL induced by T315I BCR-
ABL, treatment with the dual Src/ABL inhibitor
dasatinib (which is also ineffective against the T315I
BCR-ABL mutant) significantly prolonged survival.
In addition, this improved survival correlated with
SFK inhibition . Collectively, these results
indicate that SFKs play a role in B-lymphoid
transformation that is not efficiently prevented by
inhibiting BCR-ABL kinase activity with imatinib
and that dual SFK/ABL inhibition may improve the
overall treatment outcome of patients with this
SFKS and resistance to imatinib
Resistance to imatinib develops rapidly in patients
with advanced phase CML and Phþ ALL [12,57,58].
A number of potential resistance mechanisms have
been proposed including BCR-ABL kinase domain
mutation, BCR-ABL overexpression, alterations in
drug influx and efflux, and as mentioned previously,
induction of BCR-ABL–independent pathways .
Increasing preclinical and clinical evidence implicates
SFKs in imatinib resistance. Upregulation of Lyn and
Hck was observed in blasts from patients with
imatinib-resistant CML . It was later found that
while imatinib effectively reduced activation of BCR-
ABL and downstream activation of SFKs in speci-
mens derived from patients with imatinib-sensitive
CML, this agent had no effect on SFK activation in
samples from resistant patients despite BCR-ABL
inhibition. In animal models, the antitumor activity of
imatinib was significantly reduced upon loss of
imatinib-mediated Lyn inhibition, but concomitant
inhibition of SFKs and BCR-ABL recovered this
activity . These results further indicated that SFK
activity becomes independent of BCR-ABL in pro-
gressive disease, thereby resulting in imatinib resis-
tance. Otherstudies have provided similar evidence of
SFK-mediated BCR-ABL independence. The SFKs
Lyn and Hck were found to be overexpressed in CML
cell lines with BCR-ABL–independent imatinib
resistance, and coinhibition of SFKs and BCR-ABL
in these cells resulted in an enhanced apoptotic
response [21,26]. Dai et al. showed that imatinib-
resistant cell lines demonstrated markedly increased
expression of Lyn, and treatment with a specific Src
inhibitor induced apoptosis in these cells. Further-
more, transfection of imatinib-sensitive cell lines with
a constitutively active form of Lyn conferred resis-
tance to imatinib .
SFKs may also mediate imatinib resistance by
stabilizing the active form of BCR-ABL as imatinib
is unable to bind this conformation [60,61]. As
mentioned above, Meyn et al. suggested that SFK-
mediated phosphorylations in the SH3-SH2 region of
BCR-ABL promote the active conformation through
disruption of an intramolecular regulatory compo-
nent that holds the kinase domain in an inactive state
. Consistent with this, Azam et al. found that
substitution of one of these SFK-phosphorylated
tyrosine residues within the SH3 domain resulted in
imatinib resistance . Furthermore, Hck promotes
the active conformation through the phosphorylation
SFKs in Phþ CML and ALL
of specific tyrosine residues within the ABL activation
loop, which substantially decreases the sensitivity to
Dual SFK/BCR-ABL inhibition in CML
and PHþ ALL
In CML and Phþ ALL, treatment based exclusively
on the inhibition of BCR-ABL kinase activity (e.g.,
imatinib and its analogs) will clearly not improve
patient outcome if BCR-ABL–independent resis-
tance occurs, and may select for resistant clones .
Additionally, imatinib is unable to eradicate BCR-
ABL–expressing CD34þ cells , which will be
necessary to achieve curative therapy. Given the role
of SFKs in the development and progression of Phþ
ALL and CML and in BCR-ABL–independent
imatinib resistance, dual inhibition of BCR-ABL
and SFK will likely prove a more effective treatment
strategy. Additionally, this strategy may also suppress
the emergence of BCR-ABL–independent clones,
delay and possibly prevent transition of CML to blast
crisis (BC), and yield greater activity in advanced
CML and Phþ ALL .
As discussed above, preclinical studies have
demonstrated that pharmacologic or genetic inhibi-
tion of SFKs induces apoptosis and growth arrest in
BCR-ABL transformed cells [20,23–25,48] and may
overcome imatinib resistance [22,40,64]. Moreover,
dual inhibitors of BCR-ABL and SFKs may be less
susceptible to conformational resistance than imati-
nib . Although several such agents are currently
in early stage clinical development , dasatinib is
the most clinically advanced and is the only dual
SFK/BCR-ABL inhibitor approved in the United
States and Europe for the treatment of patients with
imatinib-resistant or -intolerant CML and Phþ ALL.
This novel, orally available, tyrosine kinase inhibitor
is structurally unrelated to imatinib, and capable of
binding to the ABL kinase domain in multiple
conformations [67–69]. This agent has demon-
strated 325-fold greater activity against native BCR-
ABL in vitro as compared with imatinib, and is active
against all imatinib-resistant BCR-ABL mutations
with the exception of T315I [67,68]. Additionally,
dasatinib also has activity against other oncogenic
tyrosine kinases such as c-Kit, platelet-derived
growth factor-receptor (PDGFR), and ephrin A-
Numerous preclinical studies have indicated that
dual SFK/BCR-ABL inhibition with dasatinib is
advantageous in CML and Phþ ALL. As previously
mentioned, Donato et al. showed that dasatinib was
able to recover the antitumor activity lost with
imatinib treatment as a result of BCR-ABL–inde-
pendent Lyn activation . We recently reported
that treatment with dasatinib induces complete
remission of Phþ ALL, and significantly prolongs
survival of CML in mice . It was also found that
SFKs are required for the progression of CML to
LBC, suggesting that treatment with dasatinib could
potentially delay the transition of CML from CP to
LBC. Additionally, although dasatinib does not kill
leukemic stem cells, studies in B-ALL mice suggest
that the cytostatic effects of dasatinib on this cell
population could prevent leukemic transformation
and afford long-term control of the disease .
Results from phase I and II trials showed that
dasatinib induces rapid and deep responses in
imatinib-resistant patients across all phases of CML
and Phþ ALL [74–79]. While it is clear that the
more potent activity of dasatinib against native and
mutant variants of BCR-ABL is, in part, responsible
for its clinical efficacy in imatinib-resistant CML,
BCR-ABL-independent effects appear to play a role
as well. Dasatinib has shown activity in imatinib-
resistant patients with no detectable mutations at
baseline . Furthermore, in a randomized clinical
trial, dasatinib demonstrated superior efficacy com-
pared to high-dose (HD) imatinib in patients with
imatinib-resistant CP CML, and major cytogenetic
responses (CyRs) were achieved in 55% (28/51) of
patients without a BCR-ABL mutation at baseline
compared with 34% (12/35) observed with HD
imatinib . A separate study showed that the
clinical activity of dasatinib in patients with BCR-
ABL–independent imatinib resistance correlated
with inhibition of both BCR-ABL and SFKs in
primary cell samples taken from the same patients
. Dasatinib was also found to be active in patients
with CML after failure of imatinib and its analog
nilotinib . Notably, hematologic and CyRs were
achieved with dasatinib in a substantial number of
patients with advanced stage CML and Phþ ALL
[74,76–78]. This latter result is in marked contrast
to the limited activity observed with imatinib and
nilotinib in LBC CML and Phþ ALL [66,82],
further suggesting a potential role of SFKs in
advanced disease. Collectively, the clinical data
support the preclinical findings implicating SFKs in
CML progression and imatinib resistance, and
suggest that dual SFK/BCR-ABL inhibition may be
more effective than inhibition of BCR-ABL alone.
Most appropriate use of Src/ABL inhibitors
Although dasatinib has demonstrated promising
results in advanced phase patients, the outcome of
treatment in this population is clearly inferior to that
observed in CP CML. Additionally, it remains to be
determined whether the responses generated in
patients with BC CML and Phþ ALL will be
durable. In the phase I trial of dasatinib responses in
these patients were described as short-lived ;
however, improved response durations were reported
in phase II studies [77,78]. Moreover, many of these
patients do not respond to treatment of any kind.
This refractory nature is likely due to the accumula-
tion of additional genetic abnormalities that occur
with disease progression, conferring multiple and
complex mechanisms of resistance which are not
This raises the question of when it is most appro-
priate to begin treatment with dasatinib. The results
in our mouse CML model demonstrating that SFKs
are required for progression to BC and that dasatinib
significantly prolonged survival compared to imati-
nib, suggest that early and continuous treatment with
dasatinib in patients with CP CML may provide the
greatest therapeutic benefit . This strategy could
more effectively prevent disease progression as well as
the emergence of drug resistance, through suppres-
sion of leukemic stem cell transformation. Thus,
while imatinib is currently the standard first-line
therapy for newly diagnosed patients in CP, dasatinib
should also be considered in these patients.
Earlier treatment with dasatinib may also prove
beneficial given its greater potency, which could allow
for more rapid achievement of the treatment goals,
that is, complete CyR and major molecular response
(MMR) . Several studies have shown that both
overall survival and progression-free survival is
improved in patients who achieve a CyR at 3 or 6
months [8,84–88]. Similarly, early molecular re-
sponses are also associated with better outcome .
Indeed, dasatinib has shown impressive activity as
front-line therapy in CP CML. Complete CyR rates
at 3, 6, and 12 months were 77%, 92%, and 95%,
respectively, and MMRs were achieved in 19% of
patients at 6 months which increased to 32% at 12
months . Long-term follow-up from this study
and head-to-head studies evaluating dasatinib versus
imatinib as first-line treatment are necessary to
determine whether earlier treatment with dasatinib
will improve the outcome of patients with CP CML.
Overall, preclinical and clinical evidence has demon-
strated an important role for SFKs in the progression
of CML and Phþ ALL and the development of
imatinib resistance. Compared with other BCR-ABL
inhibitors, such as imatinib and nilotinib, the anti-
SFK activity of dasatinib and other dual SFK/BCR-
ABL inhibitors could provide added therapeutic
dependent and – independent imatinib resistance.
Furthermore, long-term suppression of leukemic
stem cells with dasatinib may reduce the emergence
of resistant clones, translating to more durable
follow-up of dasatinib in imatinib-pretreated as well
as early-CP patients will further elucidate the clinical
benefit of inhibiting SFKs in CML and Phþ ALL.
Writing and editorial support was provided by
Johnathan C. Maher and Josh Collis. This work
was funded by Bristol-Myers Squibb.
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