TOPIC REVIEW

Improving the prognosis for patients with glioblastoma:the rationale for targeting Src

John de Groot Æ Vanessa Milano

Received: 9 January 2009 / Accepted: 30 April 2009 / Published online: 13 May 2009

� Springer Science+Business Media, LLC. 2009

Abstract Glioblastoma is the most common and

aggressive form of primary brain tumor. The prognosis for

patients diagnosed with glioblastoma is poor, with a

median survival of 12–14 months and a 5-year survival rate

of \5%. The upfront standard treatment for patients with

newly diagnosed glioblastoma, consisting of surgery fol-

lowed by chemotherapy combined with radiotherapy,

provides only short-term survival benefits. Recurrent glio-

blastoma is an extremely challenging therapeutic setting

because of the aggressive and resistant nature of the tumor.

A set of key molecular targets in oncology is the Src family

of non-receptor protein kinases. Dysregulated signaling via

the Src kinases has been shown to underlie glioma-related

proliferation, angiogenesis, migration, and survival. Here

we review the biologic role of Src in malignant glioma and

discuss key preclinical studies demonstrating the potential

utility of inhibiting Src in glioma. Proof of clinical benefit

is forthcoming from the first clinical studies involving the

newest generation of small molecule Src inhibitors cur-

rently in clinical trials for recurrent glioblastoma. Blocking

Src alone will likely not translate into a significant clinical

benefit; thus, strategies for combining Src inhibitors with

potential synergistic therapeutic modalities will be dis-

cussed. This review will focus on dasatinib, the most

advanced Src inhibitor being tested in glioblastoma, which

is currently in phase I/II trials in this setting.

Keywords Glioblastoma �Src family of tyrosine kinases � Src inhibitors � Dasatinib

Introduction

Glioblastoma is designated by the World Health Organi-

zation (WHO) as a grade IV astrocytoma [1] and may arise

de novo or from a pre-existing lower-grade glioma. Glio-

blastomas are characterized pathologically by high cellu-

larity and mitotic activity, nuclear atypia, psuedopalisating

necrosis, and microvascular proliferation (angiogenesis).

Angiogenesis is driven by the secretion of vascular endo-

thelial growth factor (VEGF; originally called vascular

permeability factor or VPF), placental growth factor

(PlGF), basic fibroblast growth factor (bFGF), and other

pro-angiogenic factors which also increase vascular per-

meability. This increased vascular permeability results in

the deterioration of the blood brain barrier (BBB) and

underlies the tumor enhancement observed with T1-post

gadolinium magnetic resonance imaging (MRI) scans. In

addition to abnormal blood vessel proliferation, glioblas-

toma tumor cells are highly motile. Tumor infiltration of

the brain can be both local (confined to regions close to the

primary tumor) and more diffuse: glioblastoma cells often

eventually spread throughout the brain [2]. Those tumor

cells that migrate into healthy regions of the brain are

difficult to treat due to the inherent limitations to drug

delivery imposed by an intact BBB.

J. de Groot (&)

The University of Texas M. D. Anderson Cancer Center, 1515

Holcombe Boulevard, Box 431, Houston, TX 77030-4009, USA

e-mail: jdegroot@mdanderson.org

V. Milano

Department of Biological Sciences, The University of Notre

Dame, 107 Galvin Life Science Center, Notre Dame, IN 46556-

5645, USA

e-mail: vmilano@nd.edu

123

J Neurooncol (2009) 95:151–163

DOI 10.1007/s11060-009-9916-2

The oncogenic properties of Src signaling

Since the original discovery of viral Src almost a century

ago, the oncoprotein Src (c-Src) has become widely studied

[3]. The Src family of tyrosine kinases (SFKs), of which

there are 9 members (Src, YES, FYN, LYN, LCK, HCK,

FGR, YRK, and BLK), are all non-receptor, membrane-

associated proteins. Src is linked with the development and

progression of multiple cancer types [4]. The causal rela-

tionship between Src dysregulation and the cancer pheno-

type can be explained, in part, by downstream activation of

the phosphatidylinositol 3-kinase (PI3K) and mitogen

activated protein kinase (MAPK) pathways. These key

pathways stimulate proliferation, invasion, tumorigenesis,

angiogenesis, and impairment of apoptosis [5]. Src acti-

vation can be effected by receptor over-expression and less

commonly by mutation. High levels of activated Src have

been described in breast cancer, non-small cell lung cancer,

leukemia, colon cancer, gliomas, and other solid tumors

[4, 6–9].

Although the mechanisms by which Src promotes can-

cer are not completely understood, it is likely to be a

central regulator at the interface between extracellular

signals and intracellular pathway activation. Src can be

activated by integrin engagement or by the activation of

cell surface receptors including insulin-like growth factor-1

receptor (IGF-1R), epidermal growth factor receptor

(EGFR), and platelet-derived growth factor receptor

(PDGFR). In turn, Src mediates the phosphorylation of

multiple intracellular substrates (EGFR, focal adhesion

kinase [FAK], proline-rich tyrosine kinase 2 [PYK2],

paxillin, signal transducer and activator of transcription-3

[STAT3], and cyclin D) [10]. In addition to promoting

tumor growth during tumorigenesis, Src also mediates

critical functions such as cell adhesion, invasion [11],

angiogenesis [12], and the inhibition of apoptosis [13].

Furthermore, Src can cooperate with EGFR-mediated sig-

naling to enhance tumor biology [14]. Another important

pathway is the VEGF-induced signaling in endothelial cells

which is dependent on Src-mediated FAK activation. This

suggests Src plays an important role in angiogenesis

through its regulation of endothelial cell migration and any

subsequent invasion of tissues [13].

The collective evidence suggests Src plays a central role

in promoting tumorigenesis and disease progression. It is

therefore not surprising that the potential clinical benefits

of targeted therapy versus Src have garnered significant

interest in several oncology settings. There is a particularly

strong rationale for targeting Src in glioblastoma. In the

majority of these tumors, Src expression is evident and is

likely to contribute to the malignant and aggressive phe-

notype of the disease. This review provides an update on

the use of therapeutic approaches currently in development

for the treatment of glioblastoma with an emphasis on

targeting Src kinase.

Current therapeutic options for glioblastoma

Despite recent advances in the molecular classification,

technologic improvements in surgery and radiotherapy, and

the integration of novel molecular targeted therapies, the

clinical outcome of patients with malignant glioma remains

poor. The general standard of care is surgery and radio-

therapy plus temozolamide, followed by adjuvant temo-

zolamide [15]. Although malignant gliomas are diffusely

infiltrative tumors, maximal safe surgical resection of

[98%, although involving a high degree of intervention, is

associated with improved survival [16]. Following surgery,

radiotherapy is the mainstay of treatment. Adjuvant

radiotherapy of 50–60 Gy is associated with an increase in

survival by 14–36 weeks [17, 18]. Historically, chemo-

therapy was generally considered to only provide a mar-

ginal improvement in survival in this setting [19]. More

recently, however, the role of chemotherapy in the man-

agement of malignant gliomas has been further clarified. In

a landmark study, Stupp and colleagues demonstrated that

the addition of temozolomide to radiotherapy followed by

adjuvant temozolomide for 6 months improved median

overall survival by 2.5 months [20]. A greater improve-

ment was also reported in patients with a methylated

methyl guanine methyl DNA transferase (MGMT) pro-

moter [21]. Patients treated with chemoradiation and

adjuvant temozolomide had a median overall survival of

14.6 months with a 2-year survival of 26.5%, double that

of the non-temozolomide group. Ongoing trials (RTOG

0525) will determine if MGMT status is a predictive

marker of response to temozolomide and other alkylating

agents or if it is a prognostic marker of outcome.

Despite these encouraging data, almost all patients

recur. Because of the highly infiltrative nature of these

tumors, recurrence is usually within 2 cm of the original

tumor location [22]. Conventional chemotherapy has been

the mainstay therapy for recurrent malignant gliomas until

the recent development of molecularly-targeted therapy.

Nitrosoureas such as carmustine (CCNU) or lomustine

(BCNU) have been extensively evaluated in patients with

recurrent malignant gliomas prior to the introduction of

temozolomide in the last decade. Regimens showing some

activity in recurrent glioblastoma include carmustine [23],

irinotecan alone [24] or in combination with carmustine

[25] or bevacizumab [26, 27], celecoxib [28], carboplatin

[29], and procarbazine as part of the PCV regimen (pro-

carbazine, carmustine and vincristine) [30, 31]. Results

from recent clinical trials using the combination of the

angiogenesis inhibitor bevacizumab (humanized anti-VEGF

152 J Neurooncol (2009) 95:151–163

123

antibody) and irinotecan are encouraging and may herald

the approval of this regimen for the treatment of recurrent

glioblastoma [27, 32], and a phase III study of bev-

acizumab in the first-line treatment of glioblastoma is

expected to be initiated soon.

At the time of tumor recurrence, life expectancy is

severely reduced. Patients with glioblastoma have 6-month

progression-free survival (PFS) rates of 9–15%, a median

PFS of 9 weeks and overall survival of only 3–4 months.

Patients with anaplastic astrocytoma have 6-month PFS

rates of 31% and median PFS rates of 13 weeks [33].

However, the dramatic improvement in our understanding

of the molecular and genetic alterations that drive gli-

omagenesis and tumor progression heralds the develop-

ment of new targets and potentially effective therapies.

Molecular targeting of glioblastoma

Like most cancers, gliomas arise through the sequential

accumulation of genetic abnormalities such as the activa-

tion of oncogenes and loss of tumor suppressor genes that

ultimately lead to increased cell proliferation, resistance to

apoptosis, increased invasion and sustained angiogenesis.

At the molecular level, these genetic changes result from

the loss of normal control of intracellular signaling path-

ways and lead to the activation of signal transduction

cascades such as the PI3K pathway. Identification of early

or critical molecular events that promote cellular trans-

formation and drive malignant cell growth is central to the

development of new molecular therapies. As our under-

standing of specific signal aberrations improves, novel

anticancer therapeutics that inhibit one or more molecular

targets can be used to block these key signals. The dramatic

clinical success seen with imatinib for the treatment of

chronic myeloid leukemia (CML) demonstrates the

potential efficacy of targeted therapy in cancer [34]. This

approach is now being enthusiastically pursued in patients

with malignant glioma.

Identifying key molecular targets in glioblastoma

The molecular profiling of glioblastoma tumors has iden-

tified numerous genes that are important for promoting

tumor proliferation and survival. Detailed analysis of

patient tumor samples has identified numerous genes in

malignant gliomas that are important for the regulation of

signaling networks responsible for sustained cellular pro-

liferation. Glioblastomas are traditionally classified into

two subgroups depending on genetic classification and the

clinical presentation of the tumor. Primary or de novo

glioblastomas are thought to arise spontaneously and

are the most common subtype of glioblastoma. They are

characterized by overexpression or amplification of EGFR,

inactivation of the tumor suppressor gene phosphatase and

tensin homolog deleted on chromosome 10 (PTEN) and

inactivation of p16INK4A or p14ARF. Secondary glioblas-

tomas are less commonly encountered. These arise from

WHO grade II tumors (low grade gliomas) and are char-

acterized by inactivating mutations or loss of TP53, loss of

heterozygosity (LOH) of chromosome 17p, and overex-

pression of PDGFR. Further progression occurs to the more

aggressive anaplastic astrocytoma (WHO grade III) tumors

which demonstrate LOH of 19q, mutation of retinoblas-

toma susceptibility locus 1 (Rb1), and overexpression or

amplification of CDK4 and HDM2. Final progression to

glioblastoma occurs with the loss of PTEN.

Two main mediators of the malignant phenotype have

been identified as potential targets for the treatment of

glioma: the cell cycle machinery that drives uncontrolled

cellular proliferation and the key growth factor receptors

can drive signaling through the MAPK and PI3K path-

ways. Dysregulation of the cell cycle leads to uncon-

trolled cellular proliferation and is typically mediated by

alterations in retinoblastoma (Rb), p53, and CDK4/6 sig-

naling [35, 36]. These molecular signals all offer potential

targets for therapy. In addition to loss of cell cycle con-

trol, extracellular growth factors can promote cell prolif-

eration, survival, and migration. The net effect of the

activation of EGFR, IGF-1R, PDGFR, and other receptor

tyrosine kinases together with loss of pathway-controlling

tumor suppressor genes is the sustained downstream

activation of multiple signal transduction networks. The

Ras-MAPK pathway, which regulates cellular prolifera-

tion [37], and the PI3K-Akt pathway, which coordinately

controls cell division, tumor growth, angiogenesis, apop-

tosis, invasion and cellular metabolism [38] are the best

characterized and most important signaling transduction

cascades in gliomas. Confirmation of decades of molec-

ular analyses of glioma tumors is supported by the pre-

liminary report recently published by the Cancer Genome

Atlas project [39]. Laboratory and clinical research efforts

have led to the development of novel, mechanism-based

strategies to identify and ‘‘target’’ specific molecular

alterations. These strategies offer exciting opportunities to

inhibit important pathways mediating cancer cell growth

and survival.

One recent study using bead-based methodology for

detecting phosphorylation of multiple tyrosine kinases

identified SRC kinase as being frequently activated in

glioblastoma cell lines and primary glioblastoma patient

samples [40]. At present, a number of targeted agents

blocking pivotal signaling pathways in glioblastoma are

under evaluation for the treatment of malignant gliomas

(Table 1). Several new agents entering into clinical trials,

J Neurooncol (2009) 95:151–163 153

123

like dasatinib and sorafenib, inhibit multiple targets, which

may allow for the simultaneous inhibition of several criti-

cal targets with the same drug. Currently there are

numerous clinical trials using combinations of agents to

inhibit multiple targets using both combinations of targeted

therapeutics as well as combining targeted agents with

chemotherapy and/or radiation (reviewed in [41, 42]).

The role of Src in glioblastoma

The SFKs are a family of homologous members that are

located within the cytosol and act as intermediate intra-

cellular signal transducing proteins under physiologic

conditions [43, 44]. Dysregulation of the ratio of activated

to inactivated SFKs has been implicated in the develop-

ment of a number of primary tumors including colon

cancer, breast cancer, non-small cell lung cancer, pancre-

atic cancer, and glioma [4, 6–9, 45]. Such dysregulation

may be a consequence of a mutation in Src itself or over-

expression of Src-regulating proteins.

Transgenic mice which spontaneously express v-Src

develop glioblastoma tumors, the molecular biology of

which closely matches that of the human form [46]. This

finding points to the central role of Src in glioma devel-

opment and progression. Src is a key component of many

pathways important for the development and progression

of glioblastoma in that it is linked to proteins that are

overexpressed or exhibit constitutively active mutant forms

in glioblastoma cells (like EGFR, PDGFR, VEGFR, and

c-KIT).

Src plays an important role in tumor cell proliferation

via its activation of growth factor activated receptor tyro-

sine kinases, including EGFR, PDFGR, fibroblast growth

factor (FGF), and hepatocyte growth factor (HGF) [14, 47–

50]. Cyclic phosphorylation of EGFR, for example, acti-

vates the AKT pathway, a downstream effecter of PI3K,

and has been implicated in the stimulation of matrix

metalloproteinase-2 (MMP-2) secretion [51]. Src kinase

activity has also been proposed as necessary for tumor cells

to enter the cell cycle upon exposure to PDGF [10]. It is

thought that the activation of MAPK p42/44 by PDGF is

mediated through Src and Grb-2/PI3K function [52] and

Src-mediated activation of c-Abl by PDGF has also been

demonstrated to promote mitogenesis [53]. Src has also

been linked with STAT3 phosphorylation and extracellular

signal-regulated kinase (ERK) activation, resulting in the

inhibition of apoptosis in response to stress [54].

In addition to its role in regulating cell proliferation, Src

also affects cell adhesion, migration and invasion. Src

interaction with FAK leads to the formation of key com-

plexes with p130Cas, integrin avb3 and paxillin [55] and

activates PI3K [56]. Tumor cell migration is a key issue for

the diffuse nature (and unfavourable prognosis) of glio-

blastoma multiforme (GBM) in the clinic. Src also plays an

important role in focal adhesion disassembly since its

expression results in disruption of focal adhesions and

stress fibres leading to the loss of adhesion to the extra-

cellular matrix (ECM) [57]. This Src-mediated disruption

of focal adhesions leads to a decrease in cell-cell and cell-

ECM adhesion and is an important process central to cell

migration and invasion. In addition to its effects on

motility, Src may enhance cellular invasion by regulating

the expression of MMPs and tissue inhibitors of metallo-

proteinases (TIMPs) [58]. For example, FAK, a key sub-

strate of Src, can activate c-JUN kinase which results in

expression of MMP-2 and MMP-9 [59]. MMPs are known

to degrade the ECM, releasing angiogenic factors and

promoting cellular invasion.

Diffuse tumor infiltration into normal brain is one of the

key elements responsible for the unfavourable prognosis of

glioblastoma. Upregulated Src signaling may contribute to

this phenomenon via several different signaling pathways.

In human glioblastoma cells, LYN kinase activity is

exceptionally high and accounts for the majority of pan-Src

activity [60]. Additionally, LYN has been shown to aid the

localization of integrin avb3 to focal adhesion sites after

activation by PDGFR [61], thus promoting cell migration.

The promotion of the migration of glioblastoma cells by

phorbol 12-myristate 13-acetate (PMA)-activated PKC is

also regulated by Src via the Cas/Crk/Rak1 pathway [62].

Table 1 Selected multi-kinase inhibitors

Agent Company Target(s)

AEE-778 Novartis EGFR, VEGFR

AZD-2171 Astra Zeneca pan-VEGFR

Imatinib Novartis Abl, c-Kit, PDGFR

Lapatinib GSK EGFR, HER2

MLN-518 Millinium PDGFR, Flt-3, c-Kit

NVP-TAE226 Novartis FAK, IGF-1R

Pazopanib GSK VEGFR, PDGFR, c-Kit

Sorafenib Bayer Raf, VEGFR-2,-3, PDGFR,

Flt-3

Sunitinib Pfizer VEGFR, PDGFR, c-Kit, Flt-3

Vandetanib Astra Zeneca EGFR, VEGFR

Vatalinib Novartis VEGFR, PDGFR

XL-880 Exelixis c-met, VEGFR, PDGFR,

Tie-2

Bribvanib BMS VEGFR, FGFR

XL418 Exelixis AKT/p70S6K

XL765 Exelixis PI3K, mTOR

BEZ235 Novartis PI3K, mTOR

XL184 Exelixis c-met, VEGFR

OSI-906 OSI pharmaceuticals IGF-1R

154 J Neurooncol (2009) 95:151–163

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Finally, we recently demonstrated a significant decrease in

tumor cell invasion in vitro following the pharmacologic

blockade of Src activity with dasatinib. Treated glioma

cells changed morphologically to became spindle shaped

with a loss of focal adhesion complex formation. Predict-

ably, the loss of focal adhesions was accompanied by a

decrease in FAK autophosphorylation [63].

The tumor suppressor PTEN controls the activity of the

SFK member FYN in glioblastoma cells, controlling

glioblastoma cell migration via PI3K [64]. However, de

novo glioblastoma neoplasms typically exhibit a high

frequency of PTEN mutation or dysregulation [65]. The

relationship between SFKs and PTEN is reciprocal, as Src

may phosphorylate PTEN and decrease its activity, lead-

ing to increased PI3K activity and cell growth [66].

Furthermore, CD95-induced recruitment of the SFK

member YES and the p85 subunit of PI3K have been

shown to signal invasion of glioblastoma via the glycogen

synthase kinase 3-b pathway and expression of MMPs in

a glioblastoma cell line [67]. Further evidence is provided

by Src-deficient transgenic mice, which develop consid-

erably less infiltrative glioblastomas compared with wild

type animals after orthotopic implantation of glioblastoma

cells in the brain [68]. These compelling data implicate a

role for Src in blood vessel growth and permeability

which has a reciprocal role in limiting tumor invasion into

normal brain.

Glioblastomas are highly vascularised and exhibit a high

degree of infiltration into surrounding brain tissue. EGFR is

well known as an activator of angiogenesis in normal

health and as a necessary component of tumor survival

[69]. Notably, a high frequency of glioblastomas are his-

tologically characterized by endothelial cell proliferation.

Endothelial cell proliferation is stimulated by multiple

pro-angiogenic growth factors, most notably VEGF.

VEGF-induced signaling in endothelial cells is dependent

on Src-mediated FAK activation, suggesting that Src plays

an important role in angiogenesis through its regulation of

endothelial cell migration and invasion [13]. Paugh et al.,

have shown that Src is involved in mediating the overex-

pression of plasminogen activator inhibitor-1 (PAI-1),

which is involved in glioblastoma angiogenesis, via an

EGFR-driven pathway [70]. Tumor growth may also be

driven at least in part by Src upregulation in conjunction

with cross-phosphorylation between both EGFR and FAK

[71]. Increased FAK signaling, which can be induced by

Src phosphorylation, promotes glioblastoma cell prolifer-

ation in animal models [72]. Also, Src-dependent phos-

phorylation of FAK is responsible for focal adhesion

formation and migration, and is also a promoter of survival

signaling via actin assembly and calpain activity [73].

Furthermore, hypoxia is an important stimulus for the

secretion of VEGF and Src mediates this process [74].

Finally, confirmation of the importance of Src in angio-

genesis comes from studies that block Src activity. Inac-

tivating Src [75] or blocking Src activity with small

molecule inhibitors [76] has been shown to inhibit angio-

genesis in vivo and in vitro.

Taken together, these factors suggest that targeted

inhibition of Src and its family members may be a rational

approach to treating glioblastoma. Src signaling pathways

and interactions are presented in Figs. 1 [3] and 2 [57].

Fig. 1 Overview of the

downstream multiple molecular

pathways activated by Src.

Reprinted by permission from

Macmillan Publishers Ltd:

Nature Reviews Molecular Cell

Biology [3], copyright 2001

J Neurooncol (2009) 95:151–163 155

123

Targeting Src in glioblastoma: current status

and future approaches

Src/SFK inhibition has emerged as a key strategy in several

oncology settings. Although Src has been known to be an

important molecular target in cancer for many years, only

recently have highly specific pharmaceutical compounds

become available. A number of Src inhibitors are currently

in development and testing in a range of solid tumors

(Table 1). Pharmaceutical approaches to Src targeting have

included blocking the catalytic site or ATP-binding site

with a reversible or irreversible inhibitor and interfering

with phosphotyrosine binding areas located within the

SH2 domain. Such interactions compromise the activated

Fig. 2 The interactions of Src

with other cellular components

in driving tumor proliferation,

invasion and vasculogenesis.

Reprinted by permission from

Macmillan Publishers Ltd:

Nature Reviews Cancer [57],

copyright 2004

156 J Neurooncol (2009) 95:151–163

123

tertiary structure of Src and the protein scaffolding that

associates with the SH2 domain of Src. The development

of Src inhibitors has been most successful through a

combination of structure-based and screening-based

approaches. These agents may have anti-tumor activity

when used alone or augment the effects of chemotherapy or

radiotherapy. Currently, dasatinib (BMS-354825), sunitinib

(SU11248), PP2, and SU6656 have been evaluated to

varying degrees as SFK inhibitors vis-a-vis glioblastoma

[77, 78]. Of these agents, development of dasatinib is the

most advanced.

Dasatinib is an oral inhibitor of Src tyrosine kinase, as

well as Bcr-Abl, Kit, PDGFRb, and Eph receptors [79–82].

Dasatinib has potent in vitro anti-proliferative and anti-

metastatic activity mediated by Src kinase inhibition,

which has translated into preliminary evidence of clinical

activity in some patients with cancer. Dasatinib is FDA

approved for the treatment of CML and Philadelphia-

chromosome-positive (Ph?) acute lymphocytic leukemia

and is currently under investigation in a number of solid

tumors. Multiple phase I and II clinical trials of dasatinib

alone or in combination with cytotoxic chemotherapy are

ongoing in patients with solid tumors, including hormone-

refractory prostate cancer, breast cancer, metastatic colo-

rectal cancer, and non-small cell lung cancer. Preliminary

reports suggest that some patients with heavily pretreated,

chemotherapy-refractory disease respond to dasatinib.

Radiographic and prostate-specific antigen responses were

documented in a phase II study of patients with castration-

resistant prostate cancer [83] and confirmed responses have

also been reported in a phase I study of dasatinib in com-

bination with 5-fluoruracil, leucovorin, oxaliplatin (FOL-

FOX) and cetuximab in patients with metastatic colorectal

cancer [84]. These encouraging preliminary data support

the further investigation of dasatinib in patients with solid

tumors.

Dasatinib also appears to be well tolerated in patients

with solid tumors. In a phase I multiple ascending-dose

trial of dasatinib in patients with advanced solid tumors, of

11 patients treated with 140 mg once daily, only one

patient required a dose interruption and none required dose

reduction. No grade 3–4 hematologic toxicities were

observed [85]. In a separate phase I study in heavily pre-

treated patients with advanced solid tumors, of 67 subjects

treated with dasatinib twice daily, only four patients had

grade 3–4 hematologic toxicities at any time on treatment,

with no grade 3–4 thrombocytopenia reported [86].

Hematologic toxicity has also been uncommon in ongoing

phase II studies in patients with breast or prostate cancer.

Among 44 patients with advanced breast cancer treated

with 140–200 mg dasatinib twice daily, only three instan-

ces of grade 3–4 neutropenia were reported with no grade

3–4 anemia or thrombocytopenia observed [87]. These data

would suggest that dasatinib- associated myelosuppression

is rare in patients with solid tumors and that patients with

leukemia may develop myelotoxicity as a result of potent

inhibition of the disease process and not a specific toxicity

of dasatinib to normal hematopoiesis. Common nonhema-

tologic toxicities with dasatinib include fatigue, nausea/

vomiting or diarrhea, fluid retention, headache, and mus-

culoskeletal pain.

In the ongoing phase II trial of dasatinib in patients with

glioblastoma (RTOG0627), patients with glioblastoma

appeared to tolerate dasatinib better than patients with

other solid tumors. There are many potential reasons for

the improved tolerability of dasatinib in this patient pop-

ulation, including the use of high doses of corticosteroids

and the potential concurrent use of agents that activate the

CYP450 system, including proton pump inhibitors (fre-

quently given in conjunction with steroids). To evaluate

potential drug-drug interactions that might alter the

metabolism of dasatinib, an extension of this study is

planned that will evaluate dasatinib pharmacokinetics

during intrapatient dose escalation.

There is strong support for the use of dasatinib in glio-

blastoma from several preclinical studies. Dasatinib has

been shown to reduce levels of phosphorylated Src, AKT,

and ribosomal protein S6 in glioblastoma cell lines, par-

ticularly in cells with activated PTEN [63]. In addition,

dasatinib reduces glioblastoma cell growth and invasion

(Fig. 3) [88]. At a mechanistic level, dasatinib has been

suggested to reduce the invasive potential of glioma cells

by disrupting paxillin localization to focal adhesions,

decreasing autophosphoylation of FAK, and decreasing

phosphorylation of FAK by Src. Combining dasatinib with

cytotoxic chemotherapy results in an additive or synergistic

increase in cell growth inhibition, most notably combined

with the alkylating agent temozolomide. The combination

of dasatinib and temozolomide has been reported to effect

additive increases in cell cycle disruption and autophagic

cell death when compared with the effect of temozolomide

alone. Preliminary evidence also advocates the combina-

tion of radiotherapy with Src inhibition [51, 89].

Penetration of the BBB is a key issue when treating

primary and metastatic brain tumors. There may be less of

an impediment to drug delivery to the primary, enhancing

glioblastoma as the tumor may disrupt the BBB and allow

greater access for anticancer agents. However, the more

diffuse GBM becomes within the central nervous system

(CNS), the more of an issue the intact BBB becomes. For

example, in a highly infiltrative glioblastoma, distant sites

of the tumor within the normal brain may remain unaf-

fected by drugs. As a result, the disease is likely to progress

in distant areas of the brain. Insight into the ability of Src

inhibitors to penetrate into the CNS comes from the

treatment of metastases to the brain. For example, imatinib

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(STI-571), a close analog of dasatinib, is used to treat acute

Ph? leukemia, but this agent does not appear to prevent

CNS relapses. This has been attributed to poor drug pen-

etration of the BBB [90] as imatinib appears to be a sub-

strate of the p-glycoprotein (p-gp) efflux pump responsible

for elimination of drug from the brain [91, 92] and dasat-

inib is not [93].

Dasatinib demonstrated substantial activity in eleven

adult and pediatric patients with CNS metastases from Ph?

leukemia. All patients had clinically significant, long-last-

ing responses, which were complete in seven patients

(64%; [94]). The action of imatinib and dasatinib was also

compared in a preclinical mouse model of intracranial Ph?

leukemia. Dasatinib increased survival and stabilization,

and regression of CNS disease was achieved with contin-

ued administration. In contrast, imatinib failed to inhibit

intracranial tumor growth [94]. Although these data sug-

gest that dasatinib has the ability to cross the BBB and

eliminate tumor cells from within the neuraxis, the con-

centrations of drug delivered beyond the intact BBB are not

known. To address this question, one strategy would be to

perform a phase I study in patients who are planning to

undergo surgical resection for recurrent glioblastoma.

Using the treat–biopsy–treat paradigm previously descri-

bed [95], patients could be treated with dasatinib prior to

surgical resection of their tumor. At the time of tumor

resection, intratumoral concentrations of dasatinib could be

measured and the impact of dasatinib on target inhibition

(SRC, EGFR, Eph activation) assessed. This approach

would evaluate drug penetration across the BBB, and

whether dasatinib can reach concentrations that would

inhibit its anticipated targets. The tissue obtained at the

time of surgery could also be used to develop biomarkers

of response and to assess negative feedback signals that

might mediate treatment resistance.

Some initial clinical activity has been reported in

patients with glioblastoma treated with dasatinib. Dasatinib

is currently under evaluation in patients with recurrent

glioblastoma (NCT00423735). In this ongoing phase II

study, patients received oral dasatinib twice daily until

disease progression. The primary endpoint was PFS at

6 months, with immunohistochemical analysis of baseline

Fig. 3 Effects of dasatinib on glioblastoma cell growth in vitro. Reprinted with permission from [88]

158 J Neurooncol (2009) 95:151–163

123

tumor tissue to determine if a molecular signature of da-

satinib targets (e.g. the presence of Src, PDGFR, EPHA2,

and c-KIT) predicts sensitivity to or clinical outcome to

dasatinib treatment. An interim analysis of this study was

recently presented at the Society for Neuro-Oncology

annual meeting. A total of 78% of patients had 2 or more of

the presumptive molecular targets of dasatinib described

above. Although efficacy data are not available, dasatinib

at a dose of 100 mg/day was reasonably well tolerated with

no patients having grade 4 or 5 toxicities [96]. Although

other SFK member (such as the highly expressed LYN and

FYN) protein expression or phosphorylation status may

need to be evaluated in this study, the expression of other

SFK-related proteins may prove more predictive of

response. Among a larger panel of genes identified, Huang

et al., identified a six gene expression profile that corre-

lated with breast cancer cell line response to dasatinib. The

six genes that correlated with response included EPHA2,

CAV1, CAV2, ANXA1, PTRF, and IGFBP2 [97], all of

which are either targets of dasatinib, substrates for SFK or

are part of downstream signaling pathways mediated by

SFK. Although Src expression itself did not correlate with

response, the overall activation status of the Src pathway

was important. Ultimately, the use of a biomarker panel to

aid in the selection of patients most likely to respond to

dasatinib has enormous clinical utility and is the first step

in individualizing cancer treatment.

Like most targets in glioblastoma, however, single-agent

activity is likely to be limited and SFK inhibition will be

most effective in combination with other therapies. Src

inhibitors combined with radiation or cytotoxic chemo-

therapy or other novel agents could effectively inhibit

tumor growth and overcome intrinsic resistance to single-

agent therapy [98]. An alternate approach is to use agents

like dasatinib which have promiscuous inhibitory activity

against multiple tyrosine kinases (dasatinib also inhibits

PDGFR, c-KIT, and EPHA2). Currently, dasatinib is being

investigated in combination with erlotinib (an EGFR

inhibitor) in patients with malignant glioblastoma in a

phase I study (NCT00609999). Src is thought to potentiate

EGFR signaling [99] and thus targeting both EGFR and Src

may have synergistic benefit to patients with glioblastoma.

There has recently been an interest in developing combi-

nation strategies that may block the pro-invasive phenotype

driven by the use of anti-VEGF therapies in glioblastoma

[26, 100]. Given its ability to block in vitro tumor migra-

tion [63], dasatinib may be effective in inhibiting tumor

invasion when used in combination with anti-angiogenic

agents. Dasatinib, characterized previously as an anti-

invasive but cytostatic agent in glioblastoma, may also be

successfully combined with radiation therapy or anti-

angiogenic therapy, which has been recently shown to

increase tumor invasion [26]. We are opening a phase I/II

clinical of dasatinib in combination with radiation and

temozolomide followed by adjuvant dasatinib and tem-

ozolomide for patients with newly diagnosed glioblastoma.

In addition to evaluating the impact of Src inhibition on

progression free survival and outcome, we will be inter-

rogating tumor tissue for potential biomarkers of response.

These studies will improve our understanding of the role of

Src in glioma development and the impact of inhibiting Src

on patient outcome.

Summary and conclusions

Malignant gliomas are diffusely infiltrative tumors that

migrate and invade extensive areas of the brain; this

invasive phenotype often mediates tumor recurrence.

Glioblastoma is a disease with a poor prognosis; most

patients will progress within six to nine months despite

treatment with surgery, radiation and adjuvant chemo-

therapy. Clearly, new therapeutic options are needed.

Agents that prevent the invasion of tumor cells throughout

the brain are likely to provide meaningful improvements in

survival for patients with glioblastoma. The prominent role

of Src in activating downstream signaling through the Ras/

MAPK and PI3K pathways in promoting tumor prolifera-

tion and invasion makes it an attractive molecular target in

glioblastoma. Pharmacologic inhibition of Src/SFK repre-

sents a promising strategy for improving glioblastoma

treatment by limiting tumor invasion into normal brain, a

rationale supported by preclinical data.

Although Src inhibitors are poised to deliver promising

results, the optimal therapeutic strategy will involve

rationale combination of Src inhibitors with agents that

exert synergistic anti-glioma activity. Because the biology

of malignant gliomas involves a complex network of inter-

connected signaling pathways, careful preclinical interro-

gation is necessary to determine the optimal treatment

combinations. Evaluation of tumor tissue before and after

treatment with Src inhibitors will be necessary to fully

realize the impact of this therapy on the tumor and to

inform the next generation of clinical trials involving

treatments that block mechanisms of escape from Src

inhibition. As more clinical information about Src/SFK

inhibitors emerges, a clearer picture of the molecular

determinants of response will define who will benefit from

this strategy and how it integrates into current glioblastoma

therapy. In the future, molecular profiles will be used to

determine which molecular targets predict response

allowing for individualization of cancer therapy.

Integrating SFK inhibitors into the treatment of primary

brain tumors provides an invaluable opportunity to advance

the field of neuro-oncology. Realistically, single-agent

studies might be expected to lead to clinical responses in

J Neurooncol (2009) 95:151–163 159

123

recurrent glioblastoma but have little impact on progres-

sion free survival due to the activation of multiple redun-

dant signaling pathways in these tumors [101]. However,

when combined in synergistic combinations with chemo-

therapy and/or radiation, these agents may significantly

improve 6-month progression free and overall survival

rates compared with historical controls. Given the highly

invasive phenotype of malignant gliomas, Src inhibitors

may improve overall survival by inhibiting tumor invasion

into normal brain hidden from the effects of cytotoxic

therapies. Novel endpoints could be used to measure a

decrease in tumor cell invasion using noninvasive MR

imaging biomarker endpoints in these studies. There is also

a potential role for Src/SFK inhibitors in the treatment of

brain metastases from other primary tumors. Also, given

the elevated incidence of glioblastoma in children, the

inclusion of pediatric patients should be strongly encour-

aged in clinical trials. A first step toward this may be to

lower the inclusion age for recruitment to trials to include

younger adults/older children.

In summary, Src inhibitors hold great promise for the

treatment of glioblastoma. In the near future, the treatment

of glioblastoma will include combining the old SRC

(Surgery, Radiotherapy, Chemotherapy) with the new Src

(sarcoma tyrosine kinase inhibitors).

Acknowledgments Editorial and writing support was provided by

Gardiner-Caldwell US, funded by Bristol-Myers Squibb.

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