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Glioblastoma Multiforme: Pathogenesis and Treatment

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DOI: 10.1016/j.pharmthera.2015.05.005

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 Pharmacology & Therapeutics 152 (2015) 63–82

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: M. Panagiotidis

Glioblastoma multiforme: Pathogenesis and treatment

Constantinos Alifieris, Dimitrios T. Trafalis ⁎
Laboratory of Pharmacology, Medical School, University of Athens, Athens, Greece

a r t i c l e i n f o a b s t r a c t

Available online 2 May 2015 Each year, about 5–6 cases out of 100,000 people are diagnosed with primary malignant brain tumors, of which
about 80% are malignant gliomas (MGs). Glioblastoma multiforme (GBM) accounts for more than half of MG
Keywords: cases. They are associated with high morbidity and mortality. Despite current multimodality treatment efforts in-
Glioblastoma cluding maximal surgical resection if feasible, followed by a combination of radiotherapy and/or chemotherapy,
Treatment the median survival is short: only about 15 months. A deeper understanding of the pathogenesis of these tumors
Review
has presented opportunities for newer therapies to evolve and an expectation of better control of this disease.
Pharmacology
Targeted therapy
Lately, efforts have been made to investigate tumor resistance, which results from complex alternate signaling
Stem cell pathways, the existence of glioma stem-cells, the influence of the blood-brain barrier as well as the expression of
06-methylguanine-DNA methyltransferase. In this paper, we review up-to-date information on MGs treatment in-
cluding current approaches, novel drug-delivering strategies, molecular targeted agents and immunomodulative
treatments, and discuss future treatment perspectives.
© 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Abbreviations: AKT, protein kinase B; BBB, blood-brain barrier; BCNU, carmustin; CCNU,
lomustin; c-MET, hepatocyte growth factor receptor; EGF(R), epidermal growth factor
(receptor); EGFRvIII, epidermal growth factor receptor variant III; ERK, extracellular
signal-regulated kinases; FTI, farnesyltransferase inhibitor; GBM, glioblastoma multiforme;
Gli1, glioma-associated oncogene-1; HDAC, histone deacetylase 1; HER2, human epidermal
growth factor receptor 2; HGF, hepatocyte growth factor; Hsp, heat-shock protein; IDH1,
NADP + -dependent isocitrate dehydrogenase; MAb, monoclonal antibody; MAPK, mito-
gen-activated protein kinases; MG, malignant glioma; MGMT, O6-methylguanine-DNA
methyltransferase; miRNA, micro ribonucleic acid; MRI, magnetic resonance imaging;
MTKI, multitargeted tyrosine kinase inhibitor; mTOR, mammalian target of rapamycin;
OS, overall survival; PARP-1, poly(ADP-ribose)polymerase-1; PCV, procarbazine, lomustine
and vincristine; PDGF(R), platelet-derived growth factor (receptor); PFS, progression-free
survival; PFS-6, six-month progression-free survival; PI3K, phosphotidylinositol 3-kinase;
PTEN, phosphatase and tensin homolog; RAF, rapidly accelerated fibrosarcoma; RAS, rat sar-
coma; RT, radiotherapy; SHH, sonic hedgehog; STAT3, signal transducer and activator of
transcription 3; TKI, tyrosine kinase inhibitor; TMZ, temozolomide; TTP, time-to-progres-
sion; VEGF(R), vascular endothelial growth factor (receptor).
⁎ Corresponding author at: Lab. Of Pharmacology, Medical School, University of Athens,
75 Mikras Asias Str., 11527-Goudi, Athens, Greece (Building 16, 1st floor). Tel.: +30 210
746 2587; fax: +30 210 746 2504.
E-mail address: dtrafal@med.uoa.gr (D.T. Trafalis).
1. Introduction
Each year, about 5–6 cases out of 100,000 people are diagnosed with
primary malignant brain tumors, of which about 80% are malignant
gliomas (MGs) (Schwartzbaum et al, 2006; Stupp et al., 2010b).
Gliomas, the most common group of primary brain tumours, include
astrocytomas, oligodendrogliomas and ependymomas. According to
World Health Organization (WHO) malignant gliomas are sub-
categorized into grade III/IV tumors such as anaplastic astrocytoma,
anaplastic oligodendroglioma, anaplastic oligoastrocytoma and ana-
plastic ependymomas, as well as grade IV/IV tumors, as glioblastoma
multiforme (GBM). The WHO grade is assigned based on certain patho-
logical features, such as nuclear atypia, mitotic activity, vascular prolif-
eration, necrosis, proliferative potential and features clinical course
and treatment outcome (Louis et al., 2007). Its incidence in the United
States is estimated around 3:100,000 while more than 10,000 cases
are diagnosed annually. It constitutes 45.2% of all malignant central ner-
vous system (CNS) tumors, 80% of all primary malignant CNS tumors

http://dx.doi.org/10.1016/j.pharmthera.2015.05.005
0163-7258/© 2015 Elsevier Inc. All rights reserved.
64 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
and approximately 54.4% of all malignant gliomas. Mean age at diagnosis
is 64 years and it is 1.5 times more common in men than women and 2
times more common in whites compared to blacks (Ostrom et al.,
2013). The incidence has increased slightly over the past 20 years mostly
due to improved radiologic diagnosis and especially in elderly (Fisher
et al., 2007). In terms of treatment, grade III tumors and GBM are
grouped together and treated similarly.
Clinically, patients with GBM may present with headaches, focal
neurologic deficits, confusion, memory loss, personality changes or
with seizures. Diagnosis and treatment response is suggested by
magnetic resonance imaging (MRI) and the use of adjunct technology
such as functional MRI, diffusion-weighted imaging, diffusion tensor
imaging, dynamic contrast-enhanced MRI, perfusion imaging, proton
magnetic resonance spectroscopy and positron-emission tomography
(Wen & Kesari, 2008).
Etiologically, there are known linked risk factors that lead to
development of GBM Environmental risk factors include primarily
exposure to therapeutic ionizing radiation and factors such as
vinyl chloride or pesticides, smoking, petroleum refining or pro-
duction work and employment in synthetic rubber manufacturing
(Wrensch et al., 2002). Additional factors such as exposure to res-
idential electromagnetic fields, formaldehyde, diagnostic irradiation
and cell phones have not been proven to lead to GBM. However, regard-
ing cell phone irradiation, a metanalysis released in 2007 did show in-
creased incidence among people who used cell phones for at least
10 years and especially those who had mostly unilateral exposure
(Hardell et al., 2007).
Currently, maximal surgical resection plus radiotherapy plus con-
comitant and adjuvant temozolomide or carmustin wafers (Gliadel) is
the standard of care in patients younger than 70 years old with newly
diagnosed GBM. However, recurrence seems to be the rule despite stan-
dard care. Lately, attention has been given to understand the initial mo-
lecular pathogenesis of these tumors including alterations in cellular
signal transduction pathways, the occurrence of resistance to therapy
and to find methods to penetrate easier the natural blood-brain barrier
(BBB). Despite these efforts to treat however, it remains an incurable
disease and the prognosis falls in a poor survival range of 12–15 months
(median 14.6 months) and a mean survival rate of only 3.3% at 2 years
and 1.2% at 3 years (Scott et al., 1998; Stupp et al., 2005).
Glioma stem cells contribute to resistance to standard radiotherapy
via preferential activation of DNA-damage-response pathways; and to
standard chemotherapy via O6-methylguanine-DNA methyltransferase
(MGMT), the inhibition of apoptosis and the up-regulation of multidrug
resistance genes (Dean et al., 2005). Thus, current efforts are directed
towards personalized treatment through blocking prime signaling
pathways in gliomagenesis, surpassing acquired resistance and by
penetration of BBB. In this article we review the current concepts as
well as emerging advances in the treatment of GBM with an emphasis
on chemotherapy and targeted agents.
Table 1
Hereditary risk factors for GBM.
Syndrome
Neurofibromatosis type 1 (NF1)
Neurofibromatosis type 2 (NF2)
Li-Fraumeni syndrome
Hereditary non-polyposis colorectal cancer (HNPCC/Lynch syndrome),
Turcot syndrome/Brain tumor-polyposis syndrome (BTPS)
Multiple endocrine neoplasia type 1 (MEN1)
Nevoid basal cell carcinoma syndrome (NBCCS), Gorlin-Gotz syndrome
Tuberous sclerosis complex (TSC)
2. Pathogenesis
The ongoing research on the pathogenesis of malignant gliomas has
given opportunities for newer therapies to evolve as well as promises
for better control of the disease. Efforts are given to understand the de-
velopment of tumor resistance (Dean et al., 2005; Furnari et al., 2007).
A small subgroup (about 5%) of patients with gliomas, is associated
with certain hereditary syndromes (Farrell & Plotkin, 2007) (Table 1).
All other patients with gliomas represent sporadic cases. An important
aspect of the pathogenesis of gliomas is that malignant transformation
results from the sequential accumulation of genetic alterations and ab-
normal regulation of growth factor signaling pathways. Aberrant prolif-
erations is thus mediated via molecules such as vascular endothelial
growth factor (VEGF), epidermal growth factor (EGF), platelet-derived
growth factor (PDGF), hepatocyte growth factor (HGF) and loss of
phosphotensin analogue (PTEN). Downstream cascades in the growth
signaling pathways (including PI3K/AKT) may be triggered (Wen &
Kesari, 2008). Also, when those tumors recur, they often show progres-
sion to a higher histologic grade and thus, acquire a different name; this
actually is a representation of progression along a classification scheme,
the natural course of the disease, rather than a new disease (Louis,
2006). Among the alterations that are most frequently seen in low-
grade astrocytomas are mutations affecting p53 and overexpression of
platelet-derived growth factor α (PDGF-α) and its receptor. The transi-
tion to a higher grade is associated to disruption of RB, p16/CDKNαA
and 19q tumor suppressor genes (Louis, 2006).
2.1. GBM clinical subtypes
GBM, is usually described in two different clinical forms, primary
and secondary GBM (Kleihues & Ohgaki, 1999). Primary GBM, is the
most common form (about 95%) and arises typically de novo, within
3–6 months, in older patients. Secondary GBM arises from prior low-
grade astrocytomas (over 10–15 years) in younger patients. While pri-
mary and secondary forms show some molecular differences, the end
result is pretty much the same since the same pathways are affected
and respond similarly to current standard treatment. Primary GBM
often has amplified, mutated epidermal-growth factor receptor
(EGFR) which encodes altered EGFR (known as EGFRvIII) whereas sec-
ondary GBM has increased signaling through PDGF-A receptor. Both
types of mutations lead to increased tyrosine kinase receptor (TKR) ac-
tivity and consequently to activation of RAS and PI3K pathways. Again,
primary GBM have commonly amplification of MDM2 gene (encodes
for an inhibitor of p53), PTEN mutations and homozygous deletions of
CDKN2A whereas secondary GBM usually has more prevalent p53
mutations, IDH1 mutations, MET amplification and overexpression of
PDGFRA. Finally, progression of low-grade glioma to high-grade is asso-
ciated with inactivation of the retinoblastoma gene (RB1) and increased
activity of human double minute 2 (HDM2) (Kleihues & Ohgaki, 1999;
Gene Associations
NF1 Neurofibromas
NF2 Schwannomas, ependymomas, meningiomas
TP53 Sarcomas, breast cancer, leukemia, adrenal cortex
carcinoma, medulloblastomas
DNA mismatch repair Colorectal adenomas, adenocarcinoma
(MSH2, MLH1, MSH6, PMS1, PMS2)
MEN1 Primary hyperparathyroidism, pancreatic
endocrine tumors, pituitary adenomas
PTC Basal cell carcinomas, medulloblastomas,
CH ovarian fibromas
TSC1, TSC2 Renal angiomyolipomas, retinal glial hamartomas,
cardiac rhabdomyomas
 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
Louis, 2006). These aberrations primarily lead to abnormal regulation of
two major cellular systems –the growth factor mediated signaling path-
ways and the cell cycle- and play a role in the increased cell prolifera-
tion, inhibition of apoptosis, invasion and angiogenesis (Louis ., 2006).
Mutation in the nicotinamide adenine dinucleotide phosphate
(NADP+)-dependent isocitrate dehydrogenase (IDH1) is conferring fa-
vorable prognostic value and prediction of response to temozolomide
in GBM. However, patients with wild type IDH1 anaplastic astrocytomas
exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1
mutation status accounts for the unfavorable prognostic effect of glio-
mas (Hartmann et al., 2010). IDH1 mutations were found in less than
5% of primary GBM and characteristically in greater than 80% of second-
ary GBM. A recent meta-analysis study associated IDH1 mutations in
GBM with improved OS (H. Cheng et al., 2013).
2.2. GBM molecular subtypes
Based on the growing understanding of molecular heterogeneity in
GBM, the Cancer Genome Atlas (TCGA) divided this tumor in molecular
subclasses termed classical, mesenchymal, proneural and neural types
based on genetic alterations and expression profiles especially of
PDGFRA, EGFR, NF1 and IDH1. Classical GBM is defined by aberrant
EGFR amplification, astrocytic cell expression pattern and loss of chro-
mosome 10 whereas IDH1, TP53 or NF1 mutations are not common.
The mesenchymal subtype is defined by NF1 and PTEN mutations, a
mesenchymal expression profile and less EGFR amplification than in
other GBM types. The proneural subtype is characterized by PDGFRA
focal amplification, TP53 and IDH1 mutations with an oligodenrocytic
cell expression profile and younger presentation age. Finally, the neural
subtype, is characterized by normal brain tissue gene expression profile
as well as astrocytic/oligodendrocytic cell markers (Verhaak et al.,
2010). Primary and secondary GBM may be indistinguishable histolog-
ically but apparently differ in genetic and epigenetic profiles. Most
GBM tumors with IDH1 mutations have the proneural gene expression
pattern but only 30% of preneural GBM has the IDH1 mutation. Thus,
IDH1 mutation is a more reliable and definitive molecular diagnostic cri-
terion of secondary GBM compared to clinical criteria. Secondary GBM
(almost always the proneural subtype) is a genetically more homoge-
nous group regarding IDH1 mutation as compared to primary GBM
(Ohgaki & Kleihues, 2013). This heterogeneity of GBM profiles leads to
different treatment efficacy among patients indicating that therapy
must be personalized to target each patient’s alterations in the molecu-
lar level.
2.3. Stem cells
The need for better understanding and managing patients with
GBM, has led to the search for a defined cell type to target on. The dis-
covery of multi-potent neural stem cells within the CNS which are capa-
ble of proliferation, self-renewal and differentiation, has led to the
suggestion that a specific transformed CNS cell exists, which has the re-
spective biological attributes of a somatic stem cell and may behave as a
tumor initiating cell (TIC) (Facchino et al., 2011). These stem cells ex-
press surface cluster designation markers such as CD133. Extensive
studies, have led to the conclusion that those glioma stem cells promote
angiogenesis by producing VEGF and can differentiate into pericytes,
thus they create an optimal microenvironment for survival (L. Cheng
et al., 2013).
Mutations leading in gliomagenesis can occur in neural stem cells
(Koso et al., 2012). Possibly neural stem cells pass on mutations to
downstream cells such as oligodendrocyte precursor cells (OPC),
which are putative glioma cells. Introducing Neurofibromin-1 and p53
mutations into OPCs produces gliomagenesis in vitro (as occurs in Li-
Fraumeni and Neurofibromatosis-1) (Liu et al., 2011). Sonic hedgehog
(SHH) signaling pathway, is presumed to be involved in the resistance
(both chemotherapy and radiotherapy) and self renewal of cancer
65
stem cells. Glioma-associated oncogene-1 (Gli1) –originally thought of
being an oncogene involved in the pathogenesis of GBM- is part of
SHH pathway. Normally, SHH acts via activation of the Gli1 transcrip-
tion factor and thus participates in maintenance and proliferation of
neural stem cells (Clement et al., 2007).
Cancer stem cells can be useful as targets by means of immunother-
apy or inhibition of important specific pathways, as with signal trans-
ducer and activator of transcription 3 (STAT 3) or the activator bone
marrow x-linked kinase (BMX) (Nduom et al., 2012).
Targeting of stem cell signaling pathways is an evolving field which
already shows some promising results. Innovative therapeutic re-
sponses as well as preventive approaches may be directed by this type
of cells. Molecular GBM phenotyping has broadened the list of targets
in tumorigenic pathways. In GBM several tumor suppressor and onco-
genic genes seem to be modulated by miRNAs thus delivery of genes
or microRNAs (miRNA) can be another form of treatment (Møller
et al., 2013).
3. Treatment
Currently, the standard approach in managing GBM includes consid-
eration of maximum surgical resection -considering that the entire
tumor cannot be removed because GBM infiltrates surrounding tissue-
radiation therapy (RT) and medical management/chemotherapy. It
also includes symptomatic treatment of seizures, cerebral edema, infec-
tions, depression, cognitive dysfunction, fatigue and venous thrombo-
embolism (Wen et al., 2006a). However, the analysis of symptom
palliation is not in the scope of this review.
The best therapeutic target is to pursuit a more individualized per-
spective. Thus, treatment depends on several factors such as the time
of diagnosis, new onset or recurrence, the performance status and the
age of the patient. Most GBM patients will follow a standard treatment
after surgical resection consisting of external beam irradiation five times
a week for six weeks, as well as oral temozolomide daily. Most patients
will recur within 6.9 months of initial diagnosis. New agents or treat-
ment administration techniques are typically initially tested first in
the recurrent setting where there are some approved treatment alterna-
tives. Table 2 depicts a summary of the current standard options for
treatment of newly diagnosed GBM. In patients with either clinical or
radiological progression, various treatment approaches have been
assessed. As part of the multidisciplinary management of high grade gli-
omas, options include further surgery in selected patients, re-irradiation
if also eligible, systemic chemotherapy, and clinical trial participa-
tion (Butowski et al., 2006; Wen & Kesari, 2008). As most patients
are ineligible for re-operation or re-irradiation, clinical trial partici-
pation is the best option. Chemotherapy options (given as single
agents) for high-grade gliomas include carboplatin, irinotecan,
carmustine (BCNU), etoposide and procarbazine,lomustine and
vincristine (PCV) combination. Exciting preliminary data from clini-
cal phase I/II trials support the development of epidermal growth
factor receptor (EGFR) antagonists, mammalian target of rapamycin
(mTOR) inhibitors, antiangiogenesis agents and other targeted inhibi-
tors (Villano et al., 2009). Even further, investigational approaches
using convection enhanced delivery, stem cell treatment, immunother-
apy and gene therapy based on recent pathogenetical discoveries, give
excitement and promise for the future.
Table 2
Current treatment of newly diagnosed GBM#.
Maximal surgical resection plus i) RT plus concomitant and andjuvant TMZ
ii)RT plus concomitant and adjuvant BCNU
#
: See text for dosages, RT: radiotherapy, TMZ: temozolomide, BCNU: carmustine wafers.
66 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82

3.1. Chemotherapy for glioblastoma multiforme
Cytotoxic therapy for GBM has evolved, more due to the approval of
temozolomide -an alkylating agent- for newly diagnosed GBM. Active
agents include also the nitrosureas: carmustine (BCNU) and lomustine
(CCNU), platinum agents, etoposide, irinotecan and PCV combination
(Table 3).
Temozolomide (TMZ) is a newer oral alkylating agent that has
excellent penetration into the central nervous system. It is an
imidazotetrazine derivative of dacarbazine. It has 96-100% bioavailability
and promotes the methylation of the O6 position on guanine (N7-guanine
and N3 adenine). It was approved in 1999 for usage against malignant
gliomas. Major toxicities include nausea and myelosuppression (often
low platelet counts). Usually, an oral 5-HT3 antagonist is given 30–60
minutes prior to each dose. Dosage is usually started at 75 mg/m2 daily
concurrently with 6 weeks of regional radiotherapy to the surgical cavity
and followed by 6 adjuvant cycles with maintenance dose at 150 mg/m2
daily p.o. for 5 days for the first cycle and if well tolerated then scaled up
to 200 mg m−2 day−1 for 5 days in 28-day cycles (Chinot et al., 2001;
Villano et al., 2009). Concurrent RT and TMZ results in a median overall
survival (OS) of 14.6 months and 2-year survival rate of 26.5%. Temozolo-
mide can be effective for recurrent GBM while its efficacy can be increased
with metronomic rather than standard schedule as well as with high
average daily dose (N100 mg/m2) (Chen et al., 2013). Patients with pro-
moter MGMT methylation treated with RT/TMZ have a median survival
of 23 months and 5- year OS of 14% versus RT alone (15 months and 5%
respectively) (Stupp et al., 2009).
The nitrosureas, carmustine (1,2 bis[2-chloreoethyl]-1-nitrosurea
BCNU) and lomustine (CCNU), are two alkylating drugs used in the
treatment of GBM and are associated with nausea, vomiting, rash, pul-
monary fibrosis (rare) and a delayed myelosuppression and typically
given in the dosage of 80 mg m−2 day−1 i.v. on days 1–3 every 6–8
weeks with radiation (Green et al., 1983). Single agent BCNU is used
most often as second line treatment in GBM that has progressed after
TMZ. Carmustine biodegradable wafers (Gliadel) are an implantable
depot form of BCNU that are placed in the cavity that is formed after re-
section of newly diagnosed or recurrent tumor. The wafers release top-
ically BCNU for about 3 weeks. Although they are considered relatively
safe they are not used by most centers because of delayed wound
healing, intracranial edema, cerebrospinal fluid leakage, intracranial in-
fection and seizures have been reported. Also, alterations in blood-
brain-barrier make the interpretation of MRI unreliable (Nagpal, 2012).
Irinotecan, etoposide and cisplatin have been used in the treatment
of GBM demonstrating modest efficacy as adjuvant chemotherapy.

Table 3
Therapeutic categories.

Chemotherapy Alkylating drugs Temozolomide

Nitrosureas (carmustine, lomustine)

Platinum compounds Irinotecan, etoposide, cisplatin
Vinca alkaloids Vincristine (part of PCV regimen)
Other chemotherapeutic agents ANG1005, Dichloroacetate, RTA 744
Targeted Therapy EGFR 1) TKIs (erlotinib, gefitinib, afatinib, dacomitinib)
2) MTKIs (lapatinib, vandetanib, Bay846)
3) MAbs (cetuximab, MAb 806, nimotuzumab, panitumumab, AMG 595)
VEGFR 1) MTKIs (vatalanib, vandetanib, sunitinib, lenvatinib, pazopanib, cediranib, cabozantinib, sorafenib)
2) MAbs (bevacizumab, ramucirumab, IMC-18 F1
3) VEGF trap (aflibercept)
PDGFR 1) MTKIs (imatinib, dasatinib, nilotinib, tandutinib, bosutinib, sunitinib)
2) sTKI (crenolanib)
3) MAbs (IMC-3G3, MEDI-575)
HGFR 1) MTKIs (cabozantinib, vandetanib, crizotinib, amuvatinib)
2) sTKI (SGX523)
3) MAbs (rilotunumab, onartuzumab)
Integrins 1) Cilengitide
2) Lenalidomide, thalidomide
Intracellular pathways RAS/RAF/MEK/MAPK 1) FTIs (Tipifarnib, lonafarnib, manumycin)
2) RAF/MEK/MAPK(sorafenib,TLN-4601)
PI3K/AKT/mTOR 1) PI3K (XL-147, buparlisib)
2) AKT (MK-2206, perifosine)
3) mTOR (temsirolimus, sirolimus, everolimus, ridaforolimus)
4) PI3K/mTOR (XL-765, PI-103, PKI-587)
PKC Tamoxifen, enzastaurin
SHH/Gli1 Cyclopamine, SEN450, vismodegib, rismodegib
NOTCH Gamma Secretase Inhibitors (RO4929097, MK-0752, DAPT)
Other molecular targets HDACs Vorinostat, panobinostat, romidepsin, trichostatin A
Hsp Tanespimycin
Proteasome Bortezomib, marizomib, MG132
TGF-beta Trabedersen, LY2109761
SRC, SFKs dasatinib
Glutamate Talampanel, riluzole
Topoisomerase inhibitors 1) Topoisomerase 1 (captothecin, irinotecan, NKTR-102)
2) Topoisomerase 2 (etoposide, teniposide)
PARP-1 Iniparib, ABT-888
Other agents C-150, aprepitant, artesunate, auranofin, disulfiram, nelfinavir, sertraline, captopril, ketoconazole,
copper gluconate
Stem cell treatment Cell surface molecule targets L1/CAM antagonism
Viral vectors Delta-24-RGD oncolytic adenovirus
Immunotherapy Vaccines 1) Peptide vaccines (Rindopepimut, ITK-1, HSPPV, Vitespen)
2) Gene-modified tumor vaccines
Dendritic cells 1) microglia as therapeutic vectors
2) viral vectors as gene therapy for microglia
JAK2/STAT3 pathway inhibitors WP1193, WP1066
 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
Since the approval of TMZ, they are mostly used as treatment after pro-
gression or recurrence. Since bevacizumab (see below) is increasingly
being used in the recurrent setting, combinations of platinum com-
pounds with bevacizumab have been used (Carrillo & Munoz, 2012).
The PCV regimen combining procarbazine, lomustine (CCNU) and
vincristine is commonly used in the treatment of anaplastic astrocyto-
mas and oligodendrogliomas. Procarbazine is also a monoamine oxidase
inhibitor and is associated with allergies and hypertensive crisis espe-
cially in response to certain foods such as those containing tyramine –
aged cheese, nuts, red wine – which patients are advised to avoid con-
suming. Vincristine is a vinca alkaloid (binds to tubulin), is associated
with a syndrome of jaw pain after the first dose and maybe peripheral
neuropathy. This combination is usually given as CCNU 110 mg/m2
p.o. on day 1, procarbazine 60 mg m−2 day−1 p.o. on days 8–21 and vin-
cristine 1.4 mg/m2 (maximum dose 2 mg) i.v. on days 8 and 29, repeat-
ed every 6 weeks (Glass et al., 1992). PCV in recurrent GBM has not
shown significant results (Schmidt et al., 2006).
Investigational cytotoxic agents: GRN1005 (ANG1005) is a novel
agent that consists of three paclitaxel molecules linked to a novel pep-
tide – angiopep – that allows it to be transported across the blood-
brain barrier through the low-density lipoprotein-related protein 1 re-
ceptor. In a phase I study, prodrug GRN1005 was tried in patients with
recurrent or progressive malignant gliomas and it was fairly well toler-
ated while showed antitumor activity. It penetrated the blood-brain
barrier (Drappatz et al., 2013). Dichloroacetate (DCA) an inhibitor of
the mitochondrial pyruvate dehydrogenase kinase induces apoptosis
in vitro and in vivo of GBM cell lines (Michelakis et al., 2010). A phase
II study evaluates DCA in patients with GBM and results are pending
(NCT00540176). RTA 744 is a close chemical analogue of doxorubicin
but is more effective in crossing the blood brain barrier and is able to
achieve high concentrations in the CNS tumor tissue in animal models.
A phase I study of RTA 744 in patients with recurrent high-grade glio-
mas has been completed but results have not been published yet
(NCT00526812).
3.2. Resistance to chemotherapy and radiotherapy
Many GBMs have intrinsic or acquired resistance to chemotherapy.
MGMT gene promoter methylation and thus silencing and downregula-
tion of the gene is an important mechanism of resistance to temozolo-
mide treatment in GBM. MGMT is a repair gene that removes alkyl
groups from the O6 position of guanine and in this way it alleviates
the effects of alkylating drugs (temozolomide or the nitrosureas)
(Esteller et al., 2000; Hegi et al., 2005). Methylation of MGMT is found
in 30–60% of GBM cases and is associated with a favorable outcome if
treated with alkylating agents (Hegi et al., 2005). A common mecha-
nism that confers this resistance is mediated by MGMT. If the promoter
of this gene is methylated, glioma cells are more sensitive to temozolo-
mide. One way of surpassing this resistance is with dose-dense (days 1–
21 every 28 days) or dose-intense regimens (Clarke et al., 2009), or by
direct inhibition of MGMT via O6-benzylguanine in combination with
TMZ (Quinn et al., 2009). A second mechanism that gives resistant prop-
erties to these tumors is that of poly(ADP-ribose)polymerase-1 (PARP-
1). PARP-1 is a critical base-excision-repair gene that when disrupted
results in persistence of potentially lethal N7- and N3-purine lesions
contributing to TMZ cytotoxicity especially when O6-methylguanine ad-
ducts are repaired or tolerated. PARP-1 small molecule inhibitors such
as BSI-201 (iniparib) and ABT-888 are promising either alone or in com-
bination with TMZ (Wen & Kesari, 2008; Zhang et al., 2012). In preclin-
ical trials, inhibition of another target important to base-excision-repair,
abasic (AP) endonocluease-1 (APE-1) shows potentiation of TMZ activ-
ity (Zhang et al., 2012).
There is evidence that glioma stem cells contribute to GBM
chemoresistance. Stem cells from highly chemotherapy resistant grade
IV gliomas show expression of multidrug resistance protein-1 (MRP1)
transporters and grade II gliomas show expression of P-glycoprotein
67
(Pgp) (De faria et al., 2008). Moreover, cancer stem cells express
MDR1 at higher levels than differentiated cancer cells, favoring resis-
tance to chemotherapy agents such as temozolomide, etoposide, pacli-
taxel and carboplatin in undifferentiated cells and thus serving as an
adequate reasoning for this high frequency of tumor recurrence with
existing treatment (Liu et al., 2005). TMZ also results in induction of
EGFRvIII and EGFR expansion, which can be blunted by the use of
EGFR inhibitors such as erlotinib (Munoz et al., 2014). Medication
such as verapamil and cyclosporine A that inhibit p-glycoprotein
in vitro, can increase brain retention of chemotherapy drugs such as vin-
cristine (Linnet & Ejsing, 2008).
3.3. Drug molecular targets in glioblastoma multiforme
The continuously evolving field of understanding the molecular
pathogenesis of GBM prompted to a more rational use of targeted mo-
lecular therapies. Inhibitors that target receptor tyrosine kinases (EGF,
PDGF, VEGF, HGF, IGF receptors) and signal transduction inhibitors
targeting mTOR, AKT/PI3K and farnesyltransferase are being investigat-
ed. Unfortunatelly, up till now, monotherapy with these agents show
disappointing results or at most mild-modest efficacy with response
that is between 0 to 15% and are unable to prolong the progression
free survival (Sathornsumetee et al., 2007). Because highly targeted
agents were not being successful, the current approach is combined in-
hibition of multiple molecular targets within the same pathway (prox-
imal and distal) or between separate-parallel pathways but with the
cost of increased toxicity. Nevertheless, no combination therapy has
shown enhanced clinical benefit over single agents (Wilson et al., 2014)
Tyrosine kinase associated receptors serve in the transmission of sig-
nals from extracellular ligands to cell nucleus and share some common
pathway mechanisms and intracellular signaling. They are composed of
a ligand-binding extracellular domain, a lipophilic transmembrane do-
main and an intracellular domain containing a catalytic site. In the ab-
sence of their associated ligand, tyrosine kinase receptors remain
unphosphorylated, monomeric and inactive. After the appropriate li-
gand binds its associated receptor, the receptors undergo dimerization
and then auto-phosphorylation in which the kinase domain of one re-
ceptor phosphorylates one or more intracellular tyrosine residues on
the second receptor. Those phosphotyrosine residues in turn, become
binding sites and recruit adaptor proteins and activate downstream ef-
fect on molecules which initiate specific signaling cascades with end re-
sult the regulation of gene transcription. Adaptor proteins include
growth factor receptor-bound-2/son-of-sevenless (Grb2)/(SOS) effect
on serine/threonine kinases such as RAS, phosphatidyloinositide-3-ki-
nase (PI3K) and phospholipase C (PLC). Negative kinase regulators in-
clude PTEN in the PI3K pathway. Important intracellular mediators in
oncogenic biochemical pathways include RAF-MEK-ERK, AKT and
mTOR.
The alterations in gene synthesis result in cell growth, proliferation,
migration, angiogenesis and apoptosis. Within tumor cells, dysregula-
tion can occur with various mechanisms mainly with overexpression
or mutations of receptors and intracellular domains, activation of
biomolecules, inactivating mutations of their negative regulators and
lead to constitutive activation of signaling pathways and uncontrolled
cellular survival, proliferation and invasion (Arora & Scholar, 2005).
C-Kit is another receptor tyrosine kinase that acts similarly to EGFR
and PDGFR and is found to be overexpressed in GBM. Binding of ligands
and specifically the stem cell factor to c-Kit, results in receptor dimeriza-
tion and activation of several transduction pathways including Akt, Src
family kinases, PI3K, Ras/MAPK and PLC-γ. (Arora & Scholar, 2005).
Angiogenic pathway inhibition: GBM is a highly vascularized malig-
nancy with marked angiogenesis and high expression of VEGF which is
also needed for stem cells to create an optimal functional environment.
Antiangiogenic agents, a group of continuously evolving targeted thera-
peutics, have demonstrated promising results in many trials (Onishi
et al., 2013). Older agents like thalidomide and newer anti-VEGF or
68 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
anti-VEGFR therapies, as well as anti-HGF and anti-avβ5 integrin mole-
cules are included. VEGF is a member of the platelet-derived growth fac-
tor family. There are numerous ligands for VEGF receptors (VEGFRs)
including VEGF-A to –E and placenta growth factor. VEGFRs are
expressed on vascular endothelial cells (Ferrara, 2005). Ligand binding
to VEGFR-1 (Fms-like tyrosine kinase 1 or Flt-1) induces endothelial
cell migration, whereas binding to VEGFR-2 (kinase insert domain-
containing receptor or KDR) stimulates endothelial cell proliferation,
increases vascular permeability (this probably accounts for the
vasogenic edema of GBM seen on MRIs), increases the expression of
proangiogenic factors (matrix metalloproteinase-1, urokinase-type
plasminogen activator and it’s receptor, plasminogen activator
inhibitor-1) and has antiapoptotic effects. Binding to VEGFR-3 (Flt-4)
leads to lymphangiogenesis. Several factors may upregulate VEGF ex-
pression including hypoxia (the most important factor in tumor cells),
HGF, PDGF, FGF, EGF, TNF, TGF-β, IL-1, c-KIT, the PI3K-Akt and Ras-
MAPK pathways, acidosis, wound healing, endometriosis and embryo-
genesis (Ferrara, 2005). Neoplasms such as GBM, require their own vas-
cular supply to maintain survival beyond the size of 2 mm. The early
small tumor extensions can receive the needed vascular supply from
the glial vasculature by “co-opting” along the capillaries. To achieve
this, angiogenesis must take place (Jain et al., 2007). However, blocking
the VEGF pathway provides means of blocking the new vessels with
high permeability (“leaky vessels”) and thus decreasing hypoxia in the
tumor and consequently they disrupt a vital mechanism of survival in
glioma stem cells, they decrease neoangiogenesis, hypoxia, vascular
permeability (and vasogenic edema) and increase chemotherapy deliv-
ery and radiosensitivity (Bao et al., 2006; Jain et al., 2007). Inhibitors of
VEGF signaling pathway include small molecule tyrosine inhibitors,
MAbs and VEGF binding agents.
EGFR is the most frequent amplified gene seen in malignant gliomas.
The EGFR is one of four tyrosine kinase receptors within the ErbB recep-
tor family, that is ErbB1 (EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3)
and ErbB4 (HER4). Activation of these receptors result in turn in activa-
tion of multiple downstream signaling pathways as fore-mentioned, in-
cluding the Ras/Raf/MEK/ERK1/2-mitogen-activated protein kinase
(MAPK) pathway, the phosphatidylinositol 3’-kinase(PI3K)/Akt/mTOR
pathway and the signal transducer and activator of transcription
pathways (STAT 3 and 5). Ligands for these receptors include EGF and
transforming growth factor α (TGF-α). These pathways affect cell
proliferation, migration, differentiation, inhibition of apoptosis and
upregulation of VEGF expression and thus increased angiogenesis
(Mendelsohn & Baselga, 2006). Activation of EGFR can be due to:
a) increased expression or activity of EGFR ligands or cofactors,
b) EGFR gene amplification or increase in translation (50–60% of GBM
cases), and c) activating mutations of EGFR, like the EGFRvIII mutant
(which mediates radioresistance and confers a more malignant
potential than the wild type receptor, found in 50–60% of GBMs)
(Ye et al., 2012). EGFR signaling alterations are associated with worse
clinical prognosis, decreased OS and gliomagenesis (Shinojima et al.,
2003). Currently, EGFR inhibition has been tried by using small
molecule tyrosine kinase inhibitors, monoclonal antibodies, peptide
vaccines, antisense oligonucleotides and immunotoxin conjugates.
Platelet-derived growth factor (PDGF) and its receptor (PDGFR) are
also commonly overactive in about 30% of GBM cases. The PDGF family
consists of four members, PDGF-A through –D which signal through the
alpha and beta PDGFR (PDGFR-α; PDGFR-β). Activation of PDGFR trig-
gers multiple intracellular signaling pathways, much like the EGFR.
They include PI3K, MAPK, Jak family kinase, Src family kinase and
phospholipace C-gamma (PLC-γ) (Joensuu et al., 2005). Thus, it pro-
motes tumor growth through autocrine stimulation, angiogenesis
through paracrine effects on reciprocal endothelial cells and affects
tumor fibroblast regulation (which in turn leads to transvascular trans-
port of drugs through alterations in intratumoral pressures) (Ostman,
2004). PDGF expression (but not PDGFR-α) is associated with the
tumor grade and proliferative activity of oligodendrogliomas
(Majumdar et al., 2009). PDGF-C has been shown to induce the
maturation of blood vessels in GBM experimental models and
attenuate the response to anti-VEGF treatment (di Tomaso et al.,
2009). Drugs that block the PDGFR include tyrosine kinase inhibi-
tors and MAbs.
Hepatocyte growth factor (HGF) or scatter factor activates the EGF,
VEGF and HGF pathways. When it binds to the c-MET receptor, it
activates intracellular signaling cascades similar to those triggered by
the EGF and PDGF receptors such as MAPK, PI3K-Akt, V-Src and STAT.
C-Met signaling has been associated with gliomagenesis, increased cell
growth, scattering and motility, invasion, resistance to apoptosis and
angiogenesis (Sierra & Tsao, 2011). It has also been postulated that
c-MET activation may bypass EGFR tyrosine kinase inhibition in sensitive
cells by either autophosphorylation of c-MET or transphosphorylation of
other ErbB receptors (‘cross talk’) thus acting as a mechanism of resis-
tance to anti-EGFR treatment (Sierra & Tsao, 2011). HGF autocrine
GBM may predict sensitivity of GBM cells to c-MET inhibitors and
serum HGF levels may be serve as biomarker for the presence of auto-
crine tumors and responsiveness to c-Met inhibition therapy (Xie et al.,
2012). Also, targeting of the c-Met pathway is found to potentiate GBM
rensposiveness to gamma irradiation (Lal et al., 2005). Preclinical evi-
dence have demonstrated that PTEN loss amplifies c-Met induced GBM
malignancy and it is suggested that trials of the combination of anti-
HGF blocking plus PTEN restoration or mTOR inhibition should be car-
ried out (Li et al., 2009). Elevated serum HGF may be associated with
poor response and a shorter PFS in patients with GBM undergoing
first-line RT (Liang et al., 2014).
Most important intacellular effector molecules in the pathogenesis
of malignant gliomas are biochemically active serine/threonine kinases.
These include PI3K, RAS, PLC etc. and either activation of them or inac-
tivation of their negative regulators lead to excessive phosphorylation
in the intracellular signal pathways and thus contribute to malignancy.
RAS-RAF-MEK-MAPK pathway: The Ras gene superfamily encodes
small GTP (guanine triphosphate)-binding proteins (GBPs) which are
found in the inner cell membrane and regulate cellular functions, in-
cluding cytoskeletal organization, protein trafficking, cellular differenti-
ation and proliferation as well as secretion of angiogenetic molecules.
Ras mutations are not common in malignant gliomas but they rather
show increased Ras activity due to amplification or mutation of upstream
growth factor receptors (Knobbe & Reifenberger, 2004). When Ras is
triggered by either EGFR or PDGFR activation or through independent
pathway alterations, the mitogenic signal through the mitogen activated
protein kinase (MAPK also known as ERK) and phosphatidylinositol 3-
kinase (PI3K) pathways are initiated. However, translocation of Ras to
the cell membrane for activation requires a post-translational alteration
which is catalyzed by farnesyltransferase. Farnesylation is the rate-
limiting step in RAS maturation although not unique for Ras (Sebti &
Adjei, 2004). Downstream of Ras, is the pathway Raf-Mek-MAPK path-
way. Activation of MAPK is associated with poor outcome in GBM
(Pelloski et al., 2006).
Phosphotidylinositol 3-kinase (PI3K) /AKT/mTOR (mammalian tar-
get of rapamycin) pathway is a group of serine/threonine kinases.
PI3K can be activated by other tyrosine kinase receptors (EGFR,
PDGFR, c-Met), active Ras or integrins. It regulates cellular mechanisms
and when altered can lead to decrease of apoptosis, increase in cell
growth and cell proliferation. It is a poor prognostic factor in patients
with malignant gliomas (Chakravanti et al., 2004). AKT (also known
as protein kinase B), is a downstream effector of PI3K -activated when
phosphorylated- and leads to the same cellular results. mTOR is
downstreamed from AKT and can be activated by RAS pathway also.
At least 2 separate mTOR complexes exist. Each complex is composed
of a common regulatory subunit, the target of rapamycin (TOR) and a
downstream-substrate-defining subunit. The latter includes raptor sub-
unit (in mTOR-complex-1, mTORC1) and rictor subunit (in mTOR-
complex-2, mTORC2). Rapamycin only inhibits mTORC1. The raptor
subunit of mTORC1 regulates protein transcription which involves 4E-
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BP1 (initiation factor 43 binding protein-1) and p70s6K (ribosomal S6
kinase). The most important negative regulator of the PI3K/Akt/mTOR
pathway seems to be PTEN (phosphate and tensin homologue)
(Sheppard et al., 2012). In GBM the PI3K/AKT/mTOR pathway is active
in about 70% of cases and it seems to regulate the proliferation and
tumorigenic potential in GBM cancer stem cells. Rapamycin seems to
inhibit this potential in vitro but not in vivo, hence it may be of limited
value as monotherapy in GBM (Mendiburu-Eliçabe et al., 2014).
Protein kinase C (PKC) is another serine/threonine kinase that regu-
lates glioma cell proliferation, angiogenesis and invasion. It is activated
by other tyrosine kinase receptors and G-protein coupled receptors.
PKC-β mediates VEGFR2 signalling through its associated MEK/MAPK
pathway (Kreisl et al., 2010).
Apart from conferring resistance to chemo- and radio-therapy and
increase in survival of stem cells the Sonic hedgehog/Gli1 (SHH/Gli1)
pathway seems to also act in migration and glioma cell invasion
(Wang et al., 2010).
GBM is characterized by the presence of increased growth factor
signaling, hypoxia, angiogenesis and the presence of brain glioma
cancer-stem-cells, conditions in which Notch signaling could prove to
be a key. Specifically, regarding stem-cells there is evidence that by
blocking this pathway in combination with other targeted treatments,
a better control of these neoplasms could be achieved (Stockhausen
et al., 2012). Gamma secretases, mostly known for their role in the
final step of processing amyloid precursor protein in Alzheimer’s
disease through the generation of amyloid-beta peptides, also play a
key role in Notch signaling pathway(Lin et al., 2010; Stockhausen
et al., 2012).
The integrins are heterodimeric transmembrane cell adhesion mole-
cules and receptors that bind multiple extracellular ligands (e.g. matrix
proteins like collagen, laminins, vitronectins and fibronectins). This
binding activates integrins and thus regulates tumor cell invasion and
metastasis, migration, proliferation, angiogenesis and survival. The
regulation of these effects is mediated via downstream signaling path-
ways that signal in parallel to other pathways like EGFR, VEGF and
PDGFR. There are at least 24 integrin types and are widely expressed
throughout the vasculature. The integrins alphaVbeta3 (αVβ3) and
alphaVbeta5 (αVβ5) are highly expressed in malignant glioma tumor
cells and on endothelial cells at tumor peripheral vessels. Peptide-
based integrin inhibitors as well as monoclonal antibodies are being in-
vestigated as means of inhibiting this cellular mechanism. Integrin in-
hibitors decrease tumor hypoxia (Tabatabai et al., 2010). Integrin
overexpression has been found in glioma stem cell microenvironment
(Lathia et al., 2010).
3.4. Molecular pathway and kinase inhibitors
EGFR tyrosine kinase inhibitors (TKIs) are small molecules that bind
to the ATP-binding site on the tyrosine kinase domain of the receptor
and inhibit the catalytic activity of the kinase. They can also inhibit
fusion tyrosine kinases by blocking their dimerization. Furthermore,
they interact with VEGFR and other human epidermal receptors
(Yarden & Sliwkowski, 2001). Examples include the competitive antag-
onists, erlotinib and gefitinib (administered orally), as well as the newer
irreversible antagonists afatinib and dacomitinib.
Erlotinib in two separate phase II trials with patients with recurrent
GBM, showed that it is ineffective as monotherapy showing progression
free survival at 6 months (PFS-6) in 3% of patients and a median PFS of
2 months (Raizer et al., 2009) and PFS-6 in 11.4% vs 24% of control
(treated with TMZ or carmustine) (van den Bent et al., 2009). Gefitinib,
in a phase II study with recurrent GBM patients, demonstrated event-
free survival (EFS) of 13.2% at 6 months (Rich et al., 2004). Systemic
toxicity has been used as a way to predict the clinical efficacy of treat-
ment (e.g, rash, diarrhea). For example in a study, diarrhea from gefitin-
ib treatment was associated with better OS whereas EGFR expression
(either EGFRvIII or wild-type) did not correlate with prediction of OS
69
(Rich et al., 2005). On the other hand, co-expression of EGFRvIII and
PTEN in GBM seems to predict a better response to EGFR inhibition
(fifty times more likely) (Mellinhoff et al., 2005). In a phase II trial
with patients with recurrent GBM given afatinib alone, showed PFS-6
in 3% of patients but possibly was more effective in patients with
EGFRvIII mutations (Eisenstat et al., 2011). A study of afatinib and radio-
therapy with or without temozolomide is under way (NCT00977431).
Dacomitinib (PF-299804) is a new pan-HER irreversible inhibitor that
shows enhanced action against glioma-specific domain IV EGFR cyste-
ine mutations (Greenall et al., 2014) and is currently been evaluated
in two phase II studies, one with GBM patients with EGFR amplification
or EGFRvIII expression (NCT01520870) and another in seeking of effica-
cy in recurrent/relapsed GBM (NCT01112527). As a conclusion, erloti-
nib appears to be more effective against malignant gliomas than
gefitinib. Higher levels of EGFR expression can be associated with better
response to erlotinib (Haas-Kogan et al., 2005a). Although, EGFR has
clearly a great and important role in the pathogenesis of high grade
gliomas, trials up to date with EGFR inhibitors failed to show promising
results. Explanations may be the acquisition of new resistance muta-
tions and/or inability to cross effectively the blood brain barrier (BBB).
Also, proximal block of the EGFR pathway, may be overridden by over-
active distal effectors (high pAKT) (Haas-Kogan et al., 2005b).
Tyrosine kinase inhibitors have been developed to block the VEGF
pathway. However, although they are many and primarily antagonize
angiogenesis they also have mixed tyrosine kinase blocking effects. In-
hibitors that have primarily VEGFR blocking effects include vatalanib,
vandetanib, sunitinib, pazopanib, cediranib and cabozantinib. They
will be discussed below.
As in VEGFR pathway, small molecule inhibitors have been devel-
oped against PDGFR. However, they have mixed effects and are not
selective for PDGFR. Examples of inhibitors with primarily actions
against PDGFR include imatinib, dasatinib, nilotinib and tandutinib.
They will be discussed below. Crenolanib (CP-868,596), a novel oral
selective and potent PDGFR inhibitor, is currently being evaluated in a
phase II study of adult malignant gliomas (NCT01229644) and in a
phase I study of pediatric/young adult recurrent high grade gliomas
(NCT01393912).
Small molecule inhibitors targeting c-MET pathway have been
developed but not all of them show selectivity for the c-MET receptor.
SGX523 is a highly selective oral c-MET inhibitor that has been shown
to be effective in vitro and in vivo against GBM cell lines. Although
two separate phase I studies with SGX523 were prematurely terminat-
ed (NCT00606879, NCT00607399), it may still serve as a guide of inves-
tigating the role of MET in cancer until further clinical evaluation
(Buchanan et al., 2009; Guessous et al., 2010).
Farnesyltransferase inhibitors (FTIs) have been tried in the treat-
ment of malignant gliomas. Tipifarnib (R115777), in a phase II trial ex-
hibited modest efficacy as monotherapy in patients with recurrent
malignant gliomas. PFS-6 was 11.9% for GBM patients. Toxicity was tol-
erated well except the subset of patients that received enzyme inducing
antiepileptic drugs which presented an increased hematological toxicity
(Cloughesy et al., 2006). R115777 administered before RT in patients
with newly diagnosed GBM and residual enhancing disease did not
lead to any measurable benefit (Lustig et al., 2008; Nghiempu et al.,
2011). The R115777 plus RT combination with or without TMZ, in
newly diagnosed GBM seems to be well tolerated (Nghiempu et al.,
2011). Another phase I/II trial of R115777 with RT in patients with
newly diagnosed GBM produced a median OSof 60.4 weeks but time
to progression (TTP) was only 18.1 weeks where both FGFR1 and
αvβ3 integrin expressions being independent bad prognostic factors
(Ducassou et al., 2013). A phase I/II trial of the combination of sorafenib
combined with erlotinib, tipifanib or temsirolimus in recurrent GBM has
been completed and results are pending (NCT00335764). Lonafarnib
(SCH66336) against U87 GBM cell lines improved activity of TMZ/RT
(Chaponis et al., 2011). In a phase I study on Ionafarnib with TMZ in
malignant glioma patients demonstrated dose limiting hepatic,
70 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
gastrointestinal (vomiting, diarrhea, anorexia), renal, thrombotic and
constitutional toxicities (headaches) (Desjardins et al., 2011). In
progressing GBM, intensifying TMZ treatment and adding lonafarnib a
PFS-6 of 33% and partial response of 27% in previously non-responding
patients was achieved (Gilbert et al., 2006). In another phase I/Ib
study of lonafarnib and dose-dense schedule of TMZ in recurrent/TMZ
refractory GBM, toxicity was acceptable and PFS-6 was 38% and median
survival 13.7 months (Yust-Katz et al., 2013). Manumycin, a FTI, has
been found to induce reactive osxygen species (ROS) and glioma cell ap-
optosis, inhibits STAT3 and telomerase activity (Dixit et al., 2009) as wall
as targets the IL1β-Ras-HIF-1α axis (Sharma et al., 2012).
TLN-4601, a novel Ras-MAPK inhibitor, has been studied on first pro-
gression status of GBM in a phase II study. Although it was well tolerated
(except two patients who discontinued the medication due to toxic-
ities) at the proposed dosage, it was largely ineffective as monotherapy
(Mason et al., 2012). Sorafenib, is an oral MTKI that also targets B-Raf
and C-Raf (Guan & He, 2011). It is discussed under MTKI section.
Selective PI3K inhibitors include XL-147, buparlisib and BKM120.
XL147 (SAR245408) is a selective and reversible class I PI3K inhibitor
that has been used in an exploratory trial in patients with recurrent
GBM who were surgical candidates (NCT01240460). Buparlisib
(NVP-BKM120) is a selective PI3K inhibitor demonstrated antitumor
activity in U87 glioma cells (Koul et al., 2012). BKM120 is currently
under investigation in a phase II study with recurrent GBM patients
(NCT01339052), a phase I dose escalation trial along with TMZ/RT in
newly diagnosed GBM (NCT01473901) and a phase I/II study in combi-
nation with bevacizumab in patients with relapsed/recurrent GBM
(NCT01349660).
GBM patients who carry mutant PIK3R1 alleles may benefit from
targeted inhibition of AKT since signaling through PI3K promotes tu-
morigenesis (Quayle et al., 2012). MK-2206, an allosteric AKT inhibitor,
is currently undergoing phase I/II trials (Hirai et al., 2010). Perifosine
(KRX-0401) is a novel Akt inhibitor. It has been evidenced to inhibit
multiple intracellular signaling pathways in glial progenitors and coop-
erates with TMZ to arrest glioma cell proliferation in vivo (Momota
et al., 2005). It does not seem to be able to enhance the radiosensitivity
of human glioma cells (De la Pena et al., 2006). Also, perifosine in
combination with temsirolimus shows synergism in inducing apo-
ptosis and decrease proliferation in PTEN-intact and PTEN-deficient
PDGF-driven murine GBM cells (Pitter et al., 2011). Currently an
ongoing phase I/II study seeks to investigate the combination of
temsirolimus plus perifosine in recurrent or progressive malignant glio-
mas (NCT01051557) and a phase II study investigates perifosine mono-
therapy in recurrent/progressive malignant gliomas (NCT00590954).
mTOR inhibitors used in malignant gliomas are either selective (e.g.
temsirolimus, sirolimus, everolimus, ridaforolimus) or dual PI3k/mTOR
inhibitors (XL-765, PI-103, PKI-587). Temsirolimus (CCI-779) is a spe-
cific mTOR inhibitor. In a phase II trial of temsirolimus against recurrent
GBM, radiographic response in 36% of patients, PFS-6 7.8%, median
OS4.4 months, median TTP 2.3 months (responders: 5.4 months vs
non-responders: 1.9 months) were observed. It was generally well tol-
erated (Galanis et al., 2005). In a second phase II study of temsirolimus
on recurrent GBM no efficacy was demonstrated, though 50% of patients
were initially stable. Once again the toxicity profile was well tolerated
(Chang et al., 2005). However, temsirolimus in combination with
TMZ/RT showed increased risk of infectious complications (Sarkaria
et al., 2010). Various combinations have been either in ongoing trials
or in completed ones. Sirolimus (rapamycin, AY-22989, WY-090217),
is a selective mTOR inhibitor. It was found to achieve a partial radio-
graphic response in 6% and stable disease in 38% of patients when com-
bined with gefitinib, in a phase I study. Dose limiting toxicity included
mucositis, diarrhea, thrombocytopenia and hypertriglyceridemia
(Reardon et al., 2006). In a phase II trial, combination of elrotinib and
sirolimus in patients with recurrent GBM was ineffective and demon-
strated a dismal PFS-6 of 3.1% but was generally well tolerated
(Reardon et al., 2010). Everolimus (RAD001) is an mTOR inhibitor of
FKBP12. In a phase I trial of everolimus and temozolomide in combina-
tion with radiotherapy was found to be well tolerated in newly
diagnosed GBM patients (Sarkaria et al., 2011). A phase II study of con-
current TMZ/RT/bevacizumab followed by bevacizumab/everolimus
following surgical resection in newly diagnosed GBM patients, demon-
strated PFS of 11.3 months and median OS of 13.9 months. This efficient
regimen was associated with everolimus related dose limiting toxicities
like fatigue, pneumonitis and stomatitis. Bevacizumab related side-
effects were also observed. However, the PFS is favorable to standard
TMZ/RT historical reports (Hainsworth et al., 2012). Ridaforolimus
(Deferolimus, AP23573, MK-8669) is a selective mTOR inhibitor. In
a phase II study on progressive or recurrent malignant gliomas,
ridaforolimus given perisurgically was found to be able to cross the
blood-brain barrier and with histological evidence it achieved mTOR in-
hibition with an expected, well tolerated and no dose-limiting toxicity
profile (Reardon et al., 2012b). A phase I safety study of ridaforolimus
is completed in progressive/recurrent gliomas (NCT00087451). XL765
is a dual inhibitor of PI3K/mTOR that has been studied in vivo and
in vitro against GBM cells and demonstrated efficacy both alone and in
combination with TMZ (Prasad et al., 2011). Two ongoing phase I
studies use XL765, (a) in combination with TMZ with or without RT in
malignant gliomas (NCT00704080) and (b) along with XL147 in
surgical candidates with recurrent GBM (NCT01240460). PI-103 is a
potent, cell permeable, ATP-competitive class I PI3K inhibitor and
mTORC1/2 inhibitor. It was found to be efficient against malignant glio-
ma xenografts (Fan et al., 2006). PKI-587 (PF-05212384) is a dual PI3K/
mTOR inhibitor that was found to be effective against glioma U87 cell
lines (Mallon et al., 2011) and is now being investigated in a safety
phase I study. Two novel ATP-competitive mTOR inhibitors CC214-1
and CC214-2 suppress rapamycin resistant mTORC1 signalling, block
mTORC2 especially in EGFRvIII activated GBMs. It also seems that autoph-
agy helps cancer cells to overcome mTOR inhibition (Gini et al., 2013).
Enzastaurin (LY317615), a potent PKC inhibitor has been trialled
against malignant gliomas. A phase I/II study of enzastaurin monother-
apy in malignant gliomas demonstrated 25% radiographic response
with PFS-6 of 7% (in GBM) and of 6% (in anaplastic glioma). Glycogen
synthase kinase-3 in peripheral blood mononuclear cells was identified
as a potential marker for drug activity (Kreisl et al., 2010). However, a
later phase III study comparing enzastaurin with lomustine in recurrent
intracranial GBM did not show superior efficacy although it was less
hematologically toxic (Wick et al., 2010). A phase II study of TMZ plus
enzastaurin during and after RT in newly diagnosed GBM was well tol-
erated and exhibited a median OS of 74 weeks which was positively cor-
related with MGMT methylation status and a median PFS of 36 weeks
(Butowski et al., 2011). Enzastaurin before and concurrently with RT,
followed by enzastaurin mainentance therapy in the newly diagnosed
MGMT unmethylated GBM, demonstrated median OS of 15 months
and PFS-6 of 53.6% (less than primary planned outcome) while it was
well tolerated (Wick et al., 2013). A phase II trial of enzastaurin and
bevacizumab in recurrent GBM is underway (NCT00586508). Tamoxi-
fen, an antiestrogen, has been long described to act also as a PKC inhib-
itor (Baltuch et al., 1993). Generally, tamoxifen has not been proved to
be effective in the treatment of malignant gliomas (Spence et al.,
2004). In a phase II trial of RT plus high dose tamoxifen in GBM the me-
dian survival time was 9.7 months with a lower than expected throm-
boembolic incidence, a fact attributed to the PKC inhibitory effects of
tamoxifen (Robins et al., 2006).
Cyclopamine, is a specific SHH signaling pathway antagonist of
Smoothened (SMO). In vitro studies show that it leads to depletion of
stem-like cells in GBM (Bar et al., 2007). Also, it produces a significant
but incomplete GBM xenograft cell regression (Sarangi et al., 2009).
SEN450, a novel Smoothened receptor antagonist, seems to be cytostat-
ic on its own and it further reduces tumor volume after TMZ pretreat-
ment in vivo and in vitro (Ferruzzi et al., 2012). Vismodegib (GDC-
0449, HhAntag691), is a potent novel and specific SHH pathway inhibi-
tor. It has been granted FDA approval for basal cell carcinoma (Meiss &
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Zeiser, 2014). Vismodegib is being investigated in a phase II study of
patients with recurrent GBM that are operatable (NCT00980343).
Erismodegib (LDE225), a novel oral SHH pathway inhibitor and a selec-
tive Smoothened antagonist, suppresses epithelial-mesenchymal tran-
sition and self-renewal of GBM cells through modulation of miRNA-
200, -128 and -21 (Fu et al., 2013).
Gamma secretase inhibitors (GSIs) are investigated in malignant
gliomas to block the Notch mathway (Lin et al., 2010; Stockhausen
et al., 2012). RO4929097 is an oral GSI that sensitizes GBM cells to
TMZ in vivo and in vitro (Hiddingh et al., 2014). It is under evaluation
in recurrent/progressive GBM (phase II, NCT01122901). MK-0752 is a
moderately potent gamma secretase inhibitor. It has been used in a
phase I study of children wth refractory CNS malignancies and was
well tolerated (Fouladi et al., 2011). DAPT (GSI-IX) is a novel GSI that
suppresses GBM growth in xenografts via uncoupling of tumor vessel
density from vessel function as evidenced by poor perfusion and aggra-
vated hypoxia (Zou et al., 2013). Recently, a study showed that alpha
secretase inhibitors (ASIs) may be an alternative to GSIs in GBM since
they decrease Notch activity and tumor size and increase survival time
(compared to GSIs) in GBM cells (Floyd et al., 2012).
Cilengitide (EMD121974) is a synthetic Arg-Gly-Asp pentapeptide
(RGD) that recognizes the RGD ligand binding site on the integrin recep-
tors αVβ3 and αVβ5 and competitively blocks integrin ligand binding
(A.A. Reardon et al., 2008a). A phase II study of cilengitide in patients
with recurrent GBM, demonstrated a PFS-6 of 15% and median OSof
9.9 months. It was generally well tolerated (D.A. Reardon et al.,
2008b). A phase I/IIa study on cilengitide and TMZ/RT followed by the
administration of cilengitide and TMZ as maintenance was conducted
in newly diagnosed GBM, according to MGMT status of the patients. Re-
sults showed PFS-6 in 69% and PFS at 12 months in 33% of patients, me-
dian PFS at 8 months, OS of 68% (at one year) and 35% (at 2 years) and
median OS of 16.1 months. PFS and OS were longer in patients with
MGMT promoter methylation. The combination was well tolerated
(Stupp et al., 2010a). A similar phase II study to verify the safety of
cilengitide plus standard chemoradiation (TMZ/RT) also in newly diag-
nosed GBM was performed. The suggested dose was 2000 mg and in
this group, the median survival was 20.8 months, and regarding
MGMT methylation status median survival was 30 months in patients
with methylated MGMT and 17.4 months in the unmethylated status
subgroup. It was generally well tolerated (Nabors et al., 2012). In
NABTC 03-02 phase II trial evidence of the efficacy of cilengitide was
found to be modest as monotherapy in recurrent GBM while it was
adequately delivered into the tumor (Gilbert et al., 2012). Completed
trials with cilengitide include, (a) cilengitide, TMZ and RT in newly diag-
nosed GBM and MGMT promoter methylation (phase III – CENTRIC
trial- NCT00689221), (b) cilengitide, TMZ, RT in newly diagnosed
GBM and unmethylated MGMT promoter (phase II – CORE trial –
NCT00813943), and (c) Cilengitide in patients undergoing surgery for
recurrent or progressive GBM (NCT00112866). Results are pending.
Thalidomide has been considered as an older antiangiogenic agent
(Wen & Kesari, 2008). It has been associated specifically with a decrease
in the expression of adhesion molecules and especially integrins and
thus is postulated to inhibit cell migration with antiangiogenic and
anti-inflammatory (decrease in leukocyte migration) activity
(McCarty, 1997). It is also widely known for its teratogenic potential.
Generally, thalidomide has shown to be ineffective in malignant glio-
mas alone or in various combinations. In a phase II study of thalidomide
plus irinotecan in newly diagnosed GBM showed limited efficacy com-
pared to the standard approach while venous thromboembolism was
common (Fadul et al., 2008). In a phase II study on thalidomide com-
bined with RT in newly diagnosed GBM did not improve survival
(Alexander et al., 2013). Studies with the more potent analogue of tha-
lidomide, lenalidomide are under way including a combination of
lenalidomide along with RT in newly diagnosed GBM (NCT00165477).
In a phase I trial, lenalidomide monotherapy did not seem to be efficient
with the added risk of thromboembolic disease (Fine et al., 2007).
71
Increased thromboembolic risk was also found in a phase I study of
lenalidomide plus RT in newly-diagnosed GBM (Drappatz et al., 2009).
3.5. Multitargeted tyrosine kinase inhibitors (MTKIs)
Many tyrosine kinase inhibitors can antagonize more than one type
of kinase, thus it is somewhat arbitrary to classify them. However, most
MTKIs do tend to have a more predominant inhibitory action against a
specific type of kinase.
MTKIs with effect on EGFR TKI include two reversible inhibitors
(lapanitib and vandetanib) as well as an irreversible inhibitor
(Bay846). Lapatinib (GW572016) is a reversible MTKI with dual action
on EGFR/ERbB-1 and Her-2/ERbB2 family of receptors. In a phase I/II
trial in recurrent GBM, did not show any significant efficacy and when
given with anti-epileptic medication, its clearance increased about
1,000%. Neither EGFRvIII excpression, nor PTEN loss could predict a
favorable subtype (Thiesen et al., 2010). It has been used along with
temozolomide in a phase I trial without any considerable efficacy
(Karavasilis et al., 2013). Lapatinib has also been investigated in combi-
nation with pazopanib and is being investigated along with TMZ and ra-
diation therapy in patients with newly diagnosed GBM (NCT01591577).
Vandetanib (ZD6474), is a reversible MTKI with action against EGFR,
VEGFR-2, -3 and RET. In tumor models, it achieved higher reduction of
GBM tumor volume when combined with TMZ than as monotherapy
(Jo et al., 2012). It has already been through phase I and II trials in pa-
tients with recurrent GBM that received fractionated radiosurgery
(Fields et al., 2012; Kreisl et al., 2012) and it seems to be effective against
gliomas that express the EGFRvIII mutant, too (Yiin et al., 2010).
Autophagy protects glioblastoma cells from the proapoptotic effects of
vandetanib and thus it might contribute significantly to the resistance
against it (Shen et al., 2013). Bay846 is a novel irreversible MTKI against
EGFR and Her2 and shows efficacy in malignant glioma brain tumor
cells. Bay846 was more efficient compared to lapanitib and its sensitiv-
ity was associated with wild type PTEN in conjuction with the expres-
sion of EGFRvIII (Longo et al., 2012).
MTKIs with effevt on VEGFR include cabozanitinib, cediranib, E7080,
pazopanib, sorafenib, sunitinib, vandetanib and vatalanib. Cabozantinib
(XL184), is a MTKI that inhibits VEGF2, c-MET, AXL, TIE2, Flt-3 and RET.
In a phase II trial as monotherapy, in the cohort that received the 175 mg
daily dose it showed a response rate of 21% and in the cohort with the
better tolerated 125 mg daily dose showed PFS-6 in 25% of patients.
XL184 also appeared to have modest activity in patients with prior
antiangiogenic treatment. It was noted that only a small number of
patients developed distant or diffuse disease when the neoplasm
progressed, indicating antiangiogenic plus anti-invasive effects of
XL184 (Wen, 2010). Cabozantinib, is probably a promising agent that
suppresses angiogenesis, tumor growth and metastasis in neoplasms
with dysregulated VEGFR and MET signaling (Yakes et al., 2011). More-
over, two phase II studies on cabozantinib in recurrent (NCT00704288)
and newly diagnosed (NCT00960492) GBM are under way. Cediranib,
an oral MTKI, antagonizes all VEGFRs (especially VEGFR2), c-Kit and
PDGFR. In a phase II study, used as monotherapy in patients with recur-
rent GBM, it exhibited encouraging results including PFS-6 in 26% of pa-
tients, median OS of 7 months and median PFS of 3.5 months. More than
half of patients showed radiographic improvement. However, dose re-
duction or drug interruption was required in over half of the patients
due to grade ¾ toxicities such as hypertension (12.9%), diarrhea and fa-
tigue (Batchelor et al., 2010). Later, a randomized phase III study, com-
pared cediranib as monotherapy or in combination with lomustine to
lomustine monotherapy (with cediranib-matched placebo, as control)
was performed. Cediranib was used at lower doses relative to the previ-
ous phase II study (30 mg/day p.o. as monotherapy or 20 mg/day p.o.
when combined with lomustine) and lomustine was administrated
p.o. at 110 mg/m2. Both monotherapies and combinations were given
every 6 weeks. The PFS-6 in the ceriranib monotherapy arm was
lower than the one observed in the previous phase II study (16%). PFS-
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6 was 34.5% in the combination arm and 24.5% in the control arm
(Ahluwalia, 2011). The efficacy of cediranib in gliomas, seems to be
limited by effective efflux at the brain-blood barrier via P-glycoprotein
(P-gp) and breast cancer resistance protein (Bcrp) (Wang et al., 2012).
Lenvatinib (E7080), an MTKI active against VEGF, PDGF and FGF recep-
tors, suppresses tumor cell migration and invasion (Glen et al., 2011). It
is currently being investigated in a phase I/II study of the combination of
E7080 plus E7050 (a dual c-MET and VEGFR-2 TKI) in patients with re-
current GBM (NCT01433991). Pazopanib (GW786034) is an MTKI
against VEGFR-1 through -3, PDGFRα/β and c-Kit. In a phase II study
on patients with recurrent glioblastoma, it was given at 800 mg/daily
in 4 week cycles. It was generally ineffective with a response rate of
just 6%, PFS-6 of 3%, median PSF of 3 months and median survival of
35 weeks. However, radiographic response was observed. It was well
tolerated with a toxicity profile similar to that of other anti VEGFR
agents (Iwamoto et al., 2010b). A phase II study on pazopanib in combi-
nation with lapatinib in adult patients with relapsed malignant glioma
has demonstrated limited therapeutic results (Reardon et al., 2013). Su-
nitinib, another MTKI, is antagonizing VEGFR-1, -2, -3, c-Kit, PDGFRα/β,
Flt-3 and colony stimulating factor 1 receptor 9 (CSF-1R). It is approved
for gastrointestinal stromal tumors (GISTs) and advanced renal cancer
(Gan et al., 2009). In a phase I study on 25 patients with recurrent
high-grade gliomas treated with sunitinib plus irinotecan, limited
results were demonstrated with radiographic response was noted in
only in one patient and PFS -6 months was 25% (Reardon et al.,
2011b). In a phase II trial of patients with recurrent high grade glioma,
although radiographic response was noted, clinical reponse was
achieved in none with OS of 3.8 months (Neyns et al., 2011). In a pro-
spective phase II study, sunitinib monotherapy in patients with recurrent
malignant gliomas (GBM or anaplastic astrocytoma) was disappointing
and ineffective since it showed zero response rates, PFS-6 months of
16.7% and median OS of 12.6 months in GBM (Pan et al., 2012). Vatalanib
(PTK787/ZK222584)), is an oral VEGFR, PDGFR and c-Kit inhibitor. In a
phase I study on vatalanib plus imatinib plus hydroxyurea in recurrent
malignant glioma patients, it was found to be well tolerated at
1000 mg b.i.d. (Reardon et al., 2009b). In a 2010 phase I/II study on RT
plus concomitant/adjuvant TMZ plus vatalanib/ZK222584 in patients
with newly diagnosed GBM, the combination was found to be well toler-
ated and safe. However, the study was discontinued because the industry
that was developing vatalanib, chose not to carry on and withdrew it
(Brandes et al., 2010).
MTKIs with effect on PDGFR include bosutinib, cediranib, dasatinib,
imatinib, nilotinib, pazopanib, sorafenib, sunitinib, tandutinib. Bosutinib
(SKI-606), inhibits PDGFR, Src/Srk and BCR/ABL tyrosine kinase used in
the treatment of chronic myeloid leukemia (Rassi & Khoury, 2013) and
is currently being evaluated in a phase II study of patients with recur-
rent GBM (NCT01331291). Dasatinib, is an oral MTKI that affects BCR-
ABL, the Src family, c-Kit, EPHA2 and PDGFR-β. It binds to both active
and inactive ABL kinase domains. It induces apoptosis, autophagic cell
death of GBM cells, inhibits their invasion, alter cell adhesion and
motility-migration of GBM cells in vitro. In vitro findings are being
followed by ongoing preclinical trials in GBM including, (a) dasatinib
plus RT/TMZ in newly diagnosed GBM, and (b) dasatinib monotherapy
in recurrent GBM (Manmeet et al., 2010). Dasatinib plus lomustine in
recurrent GBM was marked by significant hematological toxicity and
suboptimal efficacy (Franceschi et al., 2012). A phase I trial of dasatinib
plus erlotinib in patients with recurrent malignant gliomas, showed that
it is a safe combination on a daily continuous dosing schedule. However
no radiographic response was observed and the PFS-6 was 2%. No grade
3–4 toxicities were observed and most commonly, dose limiting toxic-
ities were diarrhea and fatigue (Reardon et al., 2012a). Dasatinib, does
not appear to have any effect in combination with bevacizumab after
bevacizumab failure in recurrent heavily pretreated GBM (Lu-
Emerson et al., 2011). P-gp and Bcrp seem to cause active efflux of
dasatinib from the brain hence decreasing efficacy (Agarwal et al.,
2012). Imatinib is an oral MTKI that is effective against BCR-ABL, stem
cell factor (SCF)/c-Kit, and PDGFR-α/β. Its toxicity profile includes
myelosuppression, aplastic anemia, GI effects (nausea, vomiting, diar-
rhea, GI hemorrhage), liver toxicity (elevated transaminases/ bilirubin),
respiratory effects (cough, dyspnea, upper respiratory tract infections,
chest pain, pulmonary fibrosis), skin manifestations (erythema
multiforme/ Stevens-Johnson syndrome, pruritus, angioedema), ner-
vous system effects (headache, migraines, central nervous system hem-
orrhage, peripheral neuropathy, syncopy), fatigue, pyrexia, blurred
vision, conjunctivitis, ascites, fluid retention-edema, arthralgias/myal-
gias and electrolyte disturbances. It is metabolized through CYP 3A4
and subjective to drug interactions when this enzyme is induced (Gan
et al., 2009). In a 2005 phase II study, imanitib plus hydroxyurea in
adults with recurrent GBM showed 9% radiographic response, PFS-6 in
27% of patients and median PFS 14.4 weeks, in a median follow up of
58 weeks. It was generally well tolerated and effective in some patients
(Reardon et al., 2005). However, in a later phase I/II study, imatinib
mesylate when used in recurrent malignant gliomas as monotherapy
was largely ineffective with a PFS-6 of 3% for GBM patients. CYP3A4 in-
ducers substantially decreased its effectiveness (Wen et al., 2006b). In a
phase II study the efficacy of imatinib monotherapy in patients with re-
current malignant gliomas was tested. Imatinib was generally well tol-
erated in the doses that it was administrated and demonstrated limited
antitumor effects with a PFS-6 of 16% in GBM (Raymond et al., 2008). In
another phase II study imatinib was given as neoadjuvant imatinib be-
fore either definitive surgery or re-biopsy in GBM cases. Median surviv-
al was found to be 6.2 months. Imatinib was found in the histological
specimens but it was not associated with reduction in either prolifera-
tion or other survival mechanisms (according to biochemical evidence
of Ki67, AKT, MAPK or p27 level changes) (Razis et al., 2009). During
the same year, a multicentre phase II study of the combination of ima-
tinib plus hydroxyurea in patients with progressive GBM was not
found to be associated with clinically important antitumor activity but
was well tolerated. PFS-6 was 10.6%, median OS was 26 weeks and ra-
diographic response was noted in 3.4% of cases. 7% of patients had either
grade 3 fatigue, neutropenia, thrombocytopenia (Reardon et al., 2009a).
Since the combination of hydroxyurea and imatinib had shown mixed
results, in 2010, a randomized multicenter open label phase III study
was carried out comparing the combination of hydroxyurea plus ima-
tinib versus hydroxyurea alone in patients with recurrent progressive
GBM resistant to TMZ. No clinically important differences were found
in comparing the two arms thus implying the ineffectiveness of imatin-
ib in recurrent GBM. The median PFS was similar in both arms
(6 weeks), and PFS-6 was 5% for the combination arm and 7% for the
monotherapy arm. In the arm with imatinib, toxicities were well tolerat-
ed (Dresemann et al., 2010). Nilotinib, another PDGFR, BCR-ABL and c-
Kit inhibitor, is used in chronic myeloid leukemia (Jabbour &
Kantarjian, 2012) and is currently being investigated in a phase II study
of patients with recurrent malignant gliomas (NCT01140568).
Tandutinib (MLN518), a PDGFR, Flt-3 and c-Kit inhibitor (Lehky et al.,
2011), has been evaluated in a phase II trial of the combination
tandutinib plus bevacizumab in recurrent malignant gliomas. The trial
is completed and the results are pending (NCT00667394). A second
study on tandutinib combination in recurrent or progressive GBM is
also completed (NCT00379080).
MTKIs with action against HGF/SF include cabozantinib, crizotinib and
MP470. Crizotinib (PF02341066), a dual c-MET, ALK inhibitor, has been
tried in patients with both c-MET and ALK positive histology. Recently,
there was a report of rapid radiographic and clinical improvement after
treatment with crizotinib in MET-amplified recurrent GBM (Chi et al.,
2012). Amuvatinib (MP470), a novel oral potent c-Met, c-Kit and PDGFR in-
hibitor has been shown to radiosensitize GBM cells both in vivo and in vitro
(Welsh et al., 2009). Three different safety phase I trials have already been
completed in healthy volunteers (Choy et al., 2012).
Sorafenib is an oral MTKI with targets such as VEGFR-2, -3, Flt-3,
PDGFR-β, c-Kit and it also inhibits B-Raf and C-Raf. These inhibitory ef-
fects decrease tumor cells and angiogenesis. This drug is commonly
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used in hepatocellular carcinoma (Guan & He, 2011). In vitro results of
sorafenib plus nutritional factors such as vitamin K1, has demonstrated
enhancement of growth inhibition and apoptosis of malignant glioma
cells by blocking the Raf/MEK/ERK pathway (Du et al., 2012). A phase
I trial on concurrent RT and TMZ followed by TMZ and sorafenib in
newly diagnosed GBM did not appear to improve efficacy compared
to historical standard therapy results (Hainsworth et al., 2010). In a
Phase II study, patients with recurrent GBM were treated with sorafenib
plus daily TMZ. However, PFS-6 was only 9.4% (only one patient
achieved partial response) and this was attributed to drug interactions
with CYP3A inducing antiepileptic drugs that lowered sorafenib expo-
sure in these patients (Reardon et al., 2011c). In a phase I/II trial with pa-
tients with recurrent GBM, the combination of temsirolimus plus
sunitinib was discontinued due to zero PFS-6 (Wen et al., 2009). A
phase II trial of erlotinib along with sorafenib in patients with recurrent
or progressive GBM did not have acceptable results as there were phar-
macokinetic interactions between the two drugs (Peereboom et al.,
2013). A phase I/II trial of sorafenib combined with erlotinib, tipifanib
or temsirolimus in recurrent GBM has been completed (NCT00335764).
3.6. Other molecular targets and drugs
The importance of epigenetic alterations in cancer is well appreciat-
ed. Histone deacetylases (HDAC) are involved in chromatin structure
that organize DNA and regulate transcription of genes and are altered
in malignant gliomas. HDAC inhibitors have been used in an attempt
to halt growth arrest and to promote cancer cell apoptosis. Histone
acetylases (HATs) and histone hyperacetylation can also degrade
HIF-1a and decrease VEGF and thus can lead to antiangiogenic effects
(Galanis et al., 2009). Treatment with HDACs reduces proliferation of
glioblastoma-derived stem cells and induces their differentiation
(Alvarez et al., 2015). Vorinostat (SAHA) is an oral quinolone-based
HDAC inhibitor that in a phase II study as monotherapy in recurrent
GBM demonstrated modest activity with median OS of 5.7 months
and PFS-6 was achieved in 9 of the first 57 patients (total of 66). Toxic-
ities included hematologic grade 3 or worse (mainly thrombocytope-
nia) as well as grade 3 or worse nonhematologic toxicities (fatigue,
dehydration, hypernatremia) (Galanis et al., 2009). Vorinostat in combi-
nation with TMZ in high grade gliomas seems to be well tolerated (Lee
et al., 2012). Many trials with vorinostat are ongoing including a phase
I/II trial of vorinostat plus TMZ/RT in newly diagnosed GBM
(NCT00731731) and in various combinations such as with isotretinoin
plus TMZ or bevacizumab plus TMZ/RT. However, it seems that inhibition
of HDAC potentiates the evolution of TMZ resistance by MGMT overex-
pression in vitro (Kitange et al., 2012). Panobinostat (LBH589) another
HDAC inhibitor has been tested in patients with recurrent high-grade gli-
omas in combination with bevacizumab in a phase I trial, while a phase II
study is ongoing (Drappatz et al., 2012). Romidepsin (FR901228,
Depsipeptide), another quinolone-based HDAC inhibitor has been evalu-
ated in a phase I/II trial of patients with recurrent malignant glioma. It
was largely ineffective with a median PFS was 8 weeks, PFS-6 was 3%
and median survival duration was 34 weeks (Iwamoto et al., 2011).
Trichostatin A (TSA) is an HDAC inhibitor that promotes apoptosis on
GBM cells and potentiates innate immune response against cancer cells
in vitro and in vivo (Höring et al., 2013).
Heat shock proteins (Hsp) and specifically Hsp90 are molecular
chaperons that assist in stabilizing client proteins such as protein
kinases and several transcription factors. Hsp90 seem to be vital in main-
taining malignant transformation and in increasing the growth, survival
and invasiveness of cancer cells. It stabilizes surface expression of
EGFRvIII and EGFRvIV mutants and probably maintains invasiveness of
GBM (Pines et al., 2010). Tanespimycin (17-AAG) is a potent Hsp90 in-
hibitor. It exhibits in vitro inhibition of GBM cells and synergistic effects
with radiation. It can also attenuate glioma stem cell radioresistance but
not with TMZ (Sauvageot et al., 2009). Acquired resistance is found
during prolonged exposure of tanespimycin (Gaspar et al., 2009).
73
The 26S proteasome is a protein complex that degrades ubiquinated
proteins. The role of the ubiquitin-proteasome pathway is to regulate
intracellular concentrations of specific proteins and maintain cellular
homeostasis as it is related to regulation of the cell cycle and transcrip-
tion, cell signaling and apoptosis by degrading regulatory proteins such
as p53 and cyclin dependent kinases or CDKs (Mani & Gelmann, 2005).
Bortezomib (PS-341) is a selective reversible inhibitor of the 26S protea-
some. In a phase I of recurrent malignant glioma patients, some clinical
efficacy was observed. The overall response rate was 3%, PFS-6 was 21%
for patients without exposure to enzyme inducing antiepileptics-EIAED
and 9% for those exposed, and median PFS was 1.9 months (exposed)
versus 2.6 months (for non-exposed). Median OS was 5.8 months (in
EIAED exposed patients) versus 6.1 months (in EIAED non-exposed pa-
tients) (Phurphanich et al., 2010). A phase II trial of the combination of
vorinostat plus bortezomib in recurrent GBM was disappointing and
demonstrated an OS of 3.2 months and median TTP of 1.5 months
(Friday et al., 2012). Bortezomib has shown to overcome MGMT-
related resistance of GBM cells to TMZ in vitro by inhibiting MGMT
with scheduling of administration being critical (bortezomib should
be given prior to TMZ) (Vlachostergios et al., 2013). Bortezomib acts
synergistically along with HDAC inhibitors to eliminate GBM stem-
like cells (Asklund et al., 2012). Marizomib (NPI-0052) is a new pro-
teasome inhibitor that has been tested in models for glioma (Potts
et al., 2011). The proteasome inhibitor MG132 produces apoptosis
of GBM cells and acts as a chemosensitizer in vitro (Zanotto-Filho
et al., 2012).
Tranforming-growth factor beta (TGF-β) and in particular the β2
isoform, is considered a key factor in malignant glioma progression
(TGF-β2 was originally described as “glioblastoma-derived T-cell sup-
pressor factor”) and is associated to the depressed immune status of pa-
tients with GBM. High plasma and tissue levels of TGF-β2 are associated
with poor prognosis and advanced disease (Hau et al., 2011). It seems to
also have an essential role in regulation of glioma-stem cells through
Smad-dependent induction of LIF and the subsequent activation of
JAK-STAT pathway (Penuelas et al., 2009). Trabedersen (AP 12009)
an antisense oligonucleotide that specifically inhibits TGF-β2
mRNA was tried as monotherapy (administered intratumorally by
convection enhanced delivery) versus standard chemotherapy
(TMZ or PCV) in a recently randomized phase IIb study, of patients
with recurrent or refractory malignant gliomas. At 10 μM, it achieved
a median survival of 39.1 months compared with 21.7 months for che-
motherapy (Bogdahn et al., 2011). LY2109761 is a TGF-beta receptor I
kinase inhibitor that seems to act as a radiosensitizer and prolongs sur-
vival in vitro and in vivo (Zhang et al., 2011) and may be used as an ad-
junct to RT/TMZ in GBM (Zhang, Herion, et al., 2011).
SRC and SRC-family kinases (SFKs) mediate downstream signaling
pathways from several growth factor receptors and they are frequently
activated in glioblastoma (Wick et al., 2011). It is supported that in-
creased invasiveness associated with antiangiogenic treatment with
VEGF inhibitors is mediated through increased SFK signaling and thus
justifying the combination of dasatinib with bevacizumab (Huveldt
et al., 2013), however without much success [See dasatinib).
Glutamate was until recently unrecognized as significant in the
pathogenesis of the gliomas. Glutamate is produced as a byproduct of
glutathione synthesis and is released by the x(c) (−) cystine glutamate
receptors in glioma cells. It is involved by either binding on peritumoral
Glu receptors or by paracrine/autocrine effects in: a) in seizure induc-
tion, b) excitotoxicity, c) Akt/MAPK activation and cell invasion through
AMPA receptors, d) inducing focal adhesion kinases, critical for growth
factor regulation and integrin-stimulated cell motility/invasion (de
Groot & Sontheimer, 2011). Talampanel, a well tolerated oral alpha-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor
blocker was tested in a phase II trial in patients with newly diagnosed
GBM in addition to standard RT/TMZ. When compared with historical
results with standard treatment, the median survival was 20.3 versus
14.6 months and 2 month-survival was 41.7% versus 26.5% favoring
74 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
the addition of talampanel (Grossman et al., 2009). In a phase II trial of
talampanel monotherapy against recurrent malignant gliomas was
disappointing but well tolerated with most common toxicities were
fatigue, dizziness, ataxia and respectively results for GBM/anaplastic
glioma were PFS-6: 4.6%/0%, median PFS: 5.9 weeks/8.9 weeks and
OS: 13 weeks/14 months (Iwamoto et al., 2010a). Riluzole, a glutamate
release inhibitor, in combination with LY341495, a group II mGluR
inhibitor, seems to act synergistically to inhibit the growth of GBM
in vitro (Yelskaya et al., 2013).
Topoisomerase I inhibitors camptothecin, irinotecan and novel
NKTR-102 as well as topoisomerase II inhibitors etoposide and teniposide
show in vitro efficacy against glioblastoma cell lines (Tamura et al.,
2012). Irinotecan and topotecan have been tested on GBM in various
combinations including TMZ and bevacizumab with irinotecan and
have shown some promising results (Sasine et al., 2010). NKTR-102 is
a topoisomerase I inhibitor polymer conjugate that releases irinotecan
following administration and is further metabolized to SN-38, is cur-
rently being tested in a phase II study of bevacizumab resistant GBM
(NCT01663012). Etoposide in a recent meta-analysis was found to im-
prove OS more than irinotecan in patients with high grade gliomas
(Leonard & Wolff, 2013).
JAK2/STAT3 pathway, signal transducers and activators of transcrip-
tion 3, inhibitors have recently risen as approaches for cancer immuno-
therapy. STAT3 is constitutively active in some GBM cells, helping tumor
cells resist apoptosis and enhance local immunosuppression. The non-
receptor tyrosine kinase BMX activates STAT3 signaling to maintain
self renewal and tumorigenic potential of glioma stem cells and it can
serve also as a therapeutic target (Guryanova et al., 2011). WP1193
in vitro (Sai et al., 2012) and WP1066 in GBM patients (Hussain et al.,
2007), are both effective by reducing glioma cell growth as well as re-
ducing glioma stem cell lines. See also manumycin, a FTI.
Double stranded RNA (dsRNA) is an example of pattern recognition
agonists. In a phase II study of the immune modulator polyinosinic-
polycytifylic (poly-ICLC) acid in patients with recurrent anaplastic
glioma, produced discouraging results: 11% radiographic response,
PFS-6 24% and median survival of 43 weeks (Butowski et al., 2009). A
phase II study of the poly-ICLC plus standard TMZ/RT in newly diag-
nosed GBM patients, demonstrated improved results relative to histor-
ical data of standard regimens (Rosenfeld et al., 2010).
3.7. Other experimental therapeutics
C-150, a novel curry spice curcumin nanoformulative derivative, is a
multitargeted agent that reduces the transcription activation of NFkB,
inhibits PKC-alpha, VEGF, Cyclin D1, BCL-XL and promotes differentia-
tion of glioma-initiating cells by inducing autophagy in vitro and
shows efficacy in vivo (Langone et al., 2013; Zhuang et al., 2012).
When curcumin is coupled to a glioblastoma-directed antibody, it po-
tentiates its antitumor activity (Langone et al., 2013). Curcumin in-
creases GBM cell sensitivity to TMZ by generating reactive oxygen
species and by disrupting the AKT/mTOR pathway (Yin et al., 2014).
Niclosamide is an anthelminthic medication that has demonstrated
anti-tumor effects in vitro, probably mediated through inhibition
of WNT/CTNNB1-, Notch-, mTOR-, and NF-kB signaling pathways
(Wieland et al., 2013). Aprepitant is an oral neurokinin-1 receptor
(NK-1R) antagonist used in treating nausea and vomiting. Substance P
is a natural ligand of NK-1R and thus blockage of this pathway seems
to inhibit proliferation, induce apoptosis, antiangiogenic and anti-
magration effects in preclinical trials (Muñoz & Coveñas, 2012).
Artesunate, an antimalarial drug, induces oxidative DNA damage, DNA
double-strand breakage and the ATM/ATR damage response in cancer
cells (Berdelle et al., 2011) and potentiates erlotinib’s EGFR mediated cy-
totoxicity (Efferth et al., 2004). Auranofin, a medication used to treat
rheumatoid arthritis, inhibits cytoprotective thioredoxin reductase and
cathepsin B. Thioredoxin reductase is upregulated by TNF-alpha-
induced apoptosis (ATIA) protein in glioblastoma and protects from
oxidative damage whereas cathepsin B promotes GBM growth and inva-
sion, thus proposing auranofin as a GBM treatment adjunct (Kast, 2010).
Disulfiram is an oral aldehyde dehydrogenase (ALD) inhibitor and has
strong anti-GBM effects. In vitro effects include P-glycoprotein inactiva-
tion, MGMT repair function inhibition, superoxide dismutase inhibition
which secondarily inhibits proteosome, induction of oxidative stress, in-
hibition of DNA methyltransferase, reduction of DNA replication and re-
duction of NF-kB pathway (Kast & Halatsch, 2012). Nelfinavir, a
protease inhibitor used in the treatment of HIV infection, exhibits anti-
GBM potential by binding to and limiting the chaperone function of
HSP90 (Kast & Halatsch, 2012). Sertraline, an oral selective serotonine re-
uptake inhibitor (SSRI), demonstrates downregulation of AKT together
with TMZ and imatinib in vitro (Tzadok et al., 2010). Captopril, an angio-
tensin conversion enzyme inhibitor (ACEI) inhibits glioblastoma cell syn-
thesis of MMP-2 and MMP-9 thus strongly inhibiting GBM growth and
invasion (Kast & Halatsch, 2012). Furthermore, GBM cells express
AT-1R (67%) and AT-2R (53%) and this high expression is associated
with a poor prognosis (Arrieta et al., 2008). Coordinated undermining
of survival paths (CUSP9) project is investigating aprepitant, artesunate,
auranofin, captopril, disulfiram, nelfinavir, sertraline, ketoconazole and
copper gluconate as adjuvant to low dose TMZ in patients with recur-
rent GBM (Kast et al., 2013).
3.8. Monoclonal antibodies
Monoclonal antibodies (MAbs), are targeting immunoglobulins that
specifically recognize cell surface proteins/receptors as antigens, espe-
cially targeted on the surface of tumor cells. They are mainly classified
on whether they are unconjugated or conjugated (to protein toxins/
cytotoxic agents), or radioimmunoconjugates (to radioisotopes). Mabs
may be of murine, human, primate or chimeric (murine variable region
plus human constant region) origin (Harris, 2004). Examples of anti
EGFR chimeric MAbs are cetuximab and MAb 806, whereas examples
of humanized MAb are nimotuzumab and panitumumab.
Cetuximab, is an unconjugated chimeric murine-human IgG1 MAb
which binds to the extracellular domain of the EGFR, thus inhibiting
the binding of EGF to its receptor. It has a stronger affinity to EGFR
(plus the EGFRvIII) than EGF and TGF-α. It also downregulates EGFR,
inhibits cell growth and metastasis, induces apoptosis, inhibits VEGF
production and causes antibody dependent cell-mediated cytotoxicity
(Fukai et al., 2008). In recurrent high grade glioma, cetuximab as mono-
therapy has demonstrated PFS-6 in 9.2% of patients, median OS(OS) of
5 months and average time to progression (TTP) of 2 months. Although
patients where stratified according to EGFR amplification, no correla-
tion to response was found (Neyns et al., 2009). Again in the recurrent
GBM setting, the combination of bevacizumab, irinotecan and
cetuximab showed PFS-6 in 30% of patients, median OS 7.2 months
and radiographic response of 34% (Hasselbalch et al., 2010). Amplifica-
tion of EGFR and the type of EGFR mutation (EGFRvIII or EGFRvIV) may
determine the outcome in GBM patients treated with cetuximab.
Patients with EGFR amplification and lack of EGFRvIII had better OS
(p = 0.12) than patients with EGFRvIII expression (p = 0.08) (Lv
et al., 2012). No trials have been publiced with cetuximab as treatment
in newly diagnosed high grade gliomas up to this date. MAb 806, seems
to enhance the efficacy of ionizing radiation in glioma xenografts ex-
pressing the EGFRvIII mutation and can inhibit the growth of U87MG
(Johns et al., 2012). Nimotuzumab, a humanized MAb with preferential
binding to high-EGFR-density tissues (e.g tumors, sparing normal
tissue) has been tested in pediatric recurrent high grade gliomas
(MackDonald et al., 2011). Radiotherapy plus nimotuzumab or placebo
in anaplastic astrocytoma and GBM demonstrated excellent safety pro-
file and significant survival bebefit with mean survival of 31.06 months
in nimotuzumab treated group versus 21.07 for the control group
(Solomón et al., 2013). A Chinese Phase II study of nimotuzumab in
combination with TMZ/RT demonstrated good safety and tolerability
without prolonging survival compared to standard therapy (Wang
 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
et al., 2014). Panitumumab (ABX-EGF), a human monoclonal antibody
specific for HER-1, has been used in combination with irinotecan
for GBM but results have not been published (NCT01017653)
(Berezowska & Schlegel, 2011). AMG 595, an anti-EGFRvIII-DM1
immunoconjugate human monoclonal antibody is currently being eval-
uated in a phase I study patients with recurrent GBM expressing mutant
EGFRvIII (NCT01475006).
Anti-VEGFR MAbs include bevacizumab, ramucirumab (IMC-1121B)
and icrucumab (IMC 18 F1). Bevacizumab, is an unconjugated recombi-
nant humanized murine MAb which binds to and neutralizes VEGF-A,
preventing the activation of VEGF receptor tyrosine kinases VEGFR-1
and -2. Given i.v., it has an important toxicity profile. Common
manifestations of toxicity include hypertension, headache, gastrointes-
tinal effects (abdominal pain, nausea, vomiting, diarrhea, anorexia, con-
stipation), proteinuria, leucopenia, weakness, stomatitis, epistaxis,
dyspnea, upper respiratory tract infection and impaired wound healing.
Some more rare but severe side-effects are central nervous system hem-
orrhage, venous and arterial thromboembolic events, bowel perfora-
tion, left ventricular dysfunction, infusion related reactions and
nephrotic syndrome (De Fazio et al., 2012). In the newly diagnosed set-
ting of GBM, since the concern of serious toxicity is great enough, assess-
ment of risk versus benefit is important. When patients with MGMT
methylation is treated with RT and TMZ without bevacizumab, about
50% survive 2 years (Stupp et al., 2009). So it is of concern whether
the addition of bevacizumab will prolong this OS or shorten it due to
toxicity. Also, there is concern that addition of bevacizumab promotes
a more invasive and aggressive tumor hence shortening survival com-
pared to RT/TMZ alone. In a way to avoid the risk, a stratification system
has been employed to design GBM trials. The recursive partitioning
technique (RPA) can be used. Preliminary data demonstrated that
there is a trend towards longer survival (when compared to standard
RT/TMZ treatment) for patients with low RPA class (e.g. poor perfor-
mance status and older age) but not for patients with a high RPA class
when bevacizumab is added. However, a longer PFS-6 was achieved
with no association to RPA class (Curran et al., 1993; Lai et al., 2009).
In a phase II study which compared the addition of bevacizumab to
standard RT/TMZ versus standard treatment alone, showed PFS-6 in
77.5% versus 51.6% of patients respectively and median PFS 17 months
versus 7 months respectively (Gruber et al., 2009). A randomized
phase III trial of the addition of bevacizumab or placebo in newly diag-
nosed GBM, did not improve the OS (median 15.7 vs 16.1 months re-
spectively) though it improved the PFS (10.7 vs 7.3 months) but
increased symptom burden and the rate of adverse effects led to an at-
tenuation in quality of life (Gilbert et al., 2014).
In the recurrent setting, bevacizumab has been evaluated either
alone or in combination with other agents such as irinotecan, etoposide,
carboplatin and TMZ. The phase II BRAIN study, evaluated the role of
bevacizumab alone or in combination with irinotecan in patients with
recurrent GBM. Monotherapy versus combination showed respectively
PFS-6 in 42.6% versus 50.3% of patients, objective response 28.2% versus
37.8% and median OS of 9.2 versus 8.7 months. Both groups of patients
had superior radiographic response rates, median PFS and PFS-6 in rela-
tion to historical controls (Friedman et al., 2009). However, this study
was a non-comparative study, so it wasn’t designed to compare the
two arms. A second study, tested patients with recurrent GBM treated
with bevacizumab and it showed a response rate of 35%, PFS-6 in 29%
of patients and a median OS of 31 weeks, decreases in cerebral edema,
lower needs for corticosteroids and improved neurologic function.
Patients that experienced later progression, did not show any improve-
ment when irinotecan was added (Kreisl et al., 2009). After the BRAIN
study, there was still dispute on whether monotherapy of bevacizumab
was of any real benefit but after the second forementioned study,
FDA rushed into an accelerated approval in 2009 and since then,
bevacizumab is the approved monotherapy for recurrent GBM. Clinical
benefit was also demonstrated in Japanese patients with PFS-6 in
33.9% of patients, median PFS of 3.3 months and median overall survival
75
of 10.5 months (Nagane et al., 2012). However, most patients progress
and die from the disease within 6–9 months (Kang et al., 2008).
Patients with progression after bevacizumab failure, have no standard op-
tions for salvage treatment. Given the lack of effective salvage regimens,
possible options include treatment, with bevacizumab again until a sec-
ond or further recurrence in patients with high performance status and
small non-rapidly enlarging tumors or combinations, with targeted ther-
apies that inhibit possible resistance mechanisms such as other growth
factors, or circulating progenitor cells (Krashraw & Lassman, 2010).
Other combinations of bevacizumab have been tested such as
plus etoposide or TMZ (Reardon et al., 2011a), plus erlotinib
(Sathornsumetee et al., 2010) and bevacizumab with dose-dense TMZ
(Gilbert et al., 2010). However, response rate, PFS-6 and OS where
similar to the bevacizumab monotherapy rates seen in BRAIN study.
Ramucirumab (IMC-1121B) (NCT00895180) and icrucumab (IMC-
18 F1) are newer monoclonal antibodies that target the VEGF receptors
VEGFR-2 and VEGFR-1 respectively. Trials on GBM are under way but
not published up to this date (Hsu & Wakelee, 2009).
Aflibercept (Hsu & Wakelee, 2009) (also known as VEGF trap), is a
recombinantly produced soluble peptide-antibody fusion that scav-
enges VEGF ligand and placental growth factor. In patients with GBM,
two separate phase II studies have been published (Gomez-Manzano
et al., 2008; de Groot et al., 2011). Generally it showed minimal activity
and specifically demonstrated PFS-6 in 7.7% of patients, response rate of
18% and median PFS of 3 months (Gomez-Manzano et al., 2008). Toxic-
ity led to discontinuation of the treatment in a quarter of patients which
included hypertension, lymphopenia, fatigue and central nervous
system toxicity (cerebral ischemia).
MAbs against PDGFR-α, include IMC-3G3 and MEDI-575. MEDI-575
is investigated in ongoing phase I/II studies in recurrent GBM patients
(NCT01268566) and results are pending. IMC-3G3, has shown inhibi-
tion of growth (69%) in U118 GBM cell lines and has been tried in
patients with ovarian cancer (see NCT00895180) (Shah et al., 2010).
Rilotumumab (AMG102), is a fully human MAb that effectively
blocks c-Met phosphorylation, thus inhibiting c-Met activated signaling
pathways (Giordano, 2009). It has been shown to radiosensitize GBM
cells in vivo and in vitro (Buchanan et al., 2011). In a phase II study, pa-
tients with recurrent GBM treated with rilotunumab. Two cohorts were
designed depending on the rilotunumab dose (10 and 20 mg/kg) and
respectively median OS was 6.5 months versus 5.4 months and PFS
was 4.1 weeks versus 4.3 weeks. No significant statistical difference
was attained regarding the pretreatment status with bevacizumab.
The results were disappointing and the most common adverse effects
were fatigue, headache and peripheral edema (Wen et al., 2011). Two
more phase II studies are currently underway evaluating the combina-
tion of AMG102 and bevacizumab in patients with recurrent malignant
glioma (NCT01113398) as well as AMG102 monotherapy in advanced
malignant gliomas (NCT00427440). Onartuzumab (MetMAb or OA-
5D5), is a novel human, monovalent (one-armed) 5D5 (OA-5D5) anti-
c-Met antibody that was found to be effective in reducing proliferation
and microvessel density, as well as in increasing apoptosis in U87
GBM cells (c-Met and HGF/scatter factor positive cell lines) (Martens
et al., 2006). A phase II study of onartuzumab in combination with
bevacizumab versus bevacizumab alone or onartuzumab alone in pa-
tients with recurrent GBM is in progress (NCT01632228).
3.9. Stem cells
It has been noted earlier that glioma stem cells have proliferative, in-
vasive capabilities and promote angiogenesis. EGFR inhibitors (that re-
duce the CD133+ population of stem cells), PI3K/Akt/mTOR pathway
inhibitors, GSIs, anti-angiogenetic agents (e.g bevacizumab or cediranib
to target the vascular niche of glioma stem cells), Notch pathway inhib-
itors, Hsp90 inhibitors, Sonic hedgehog pathway inhibitors have all
been used to block glioma stem cell population and perhaps enhance ra-
diosensitivity. Cell surface molecules on glioma stem cells may be used
76 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
as pharmacological targets. L1CAM specifically is a glycoprotein that
contains a cytoplasmic tail, a transmembrane domain and its extracellu-
lar domain that is preferentially found in glioma stem cells compared to
non-stem cells of GBM tumors. It can either bind with extracellular ma-
trix proteins, EGFR, FGFR, α5β1, avb3, neurophilin-1 or with another
L1CAM molecule. L1CAM acts as a mediator of molecular signals and reg-
ulates survival, growth, adhesion, invasion and migration glioma stem
cells. Some of its effects may be due to upregulation of Olig2 that sup-
presses p21WAF/CIP1 (Schmidt & Maness, 2008). L1CAM expression is in-
creased with mutations of TP53 or expression of TGF-beta (Tsuzuki
et al., 1998). L1CAM also contributes in the chemoresistance of GBM
cells and stem cells (Held-Feindt et al., 2012). Blocking L1CAM, seems
to be able to suppress tumor growth and prolong survival of tumor
cells in vivo (Bao et al., 2008).
Since malignant gliomas contain many genetic alterations, various
viral vectors have been employed to transmit therapeutic genes into
their neoplasmatic cells and target those alterations. The replication
oncolytic adenovirus Delta-24-RGD is capable of engaging the complete
cellular system within the glioma cells for its own replication and sur-
vival. Even resistant glioma stem cells seem to be vulnerable, suggesting
autophagy (upregulation of Atg5 in the tumor cells) followed by lysis of
the malignant cell (Jiang et al., 2007). A phase I/II trial in patients with
recurrent GBM (NCT01582516) and a phase I trial for patients with
recurrent malignant gliomas (NCT00805376) are ongoing.
3.10. Immunotherapy
Immunotherapy is a promising approach that has demonstrated an
ability to specifically eliminate cancer cells without damaging the sur-
rounding healthy tissue. It includes passive and active immunotherapy
strategies. Active immunotherapy creates a long-term immune re-
sponse against the tumor (propably effective against recurrences) and
passive immunotherapy involves the transfer of immediately and
short-acting effective molecules. Tumor-specific antigens are processed
by antigen presenting cells (APCs) and recognized by lymphocytes.
GBM has the ability to modulate the activity of APCs and this is hypoth-
esized from vaccination studies using tumor lysates that have been
unsuccesful (Jackson et al., 2013).
Passive immunotherapy utilizes monoclonal antibodies, adoptive
cell transfer and cytokine-mediated therapies. Targeted monoclonal an-
tibodies that were discussed in the above sections can be lethal to tumor
cells by immune-mediated mechanisms as well as by their specific-
targeted effects. MAbs bind on cancer cell surfaces in a major histocom-
patibility complex (MHC) unrestricted manner and induce GBM cell
death by both immune and non-immune mechanisms. Nivolumab, a
fully human MAb against programmed death receptor-1 (PD-1), a neg-
ative checkpoint regulator with immunosuppressive capabilities has
been approved for treatment in unresectable malignant melanoma in
Japan (Deeks, 2014). Ipilimumab is a MAb that binds to cytotoxic
T-lymphocyte (CTL)-associated antigen-4 (CTLA-4/CD152) and en-
hances T-cell activation thus allowing CTLs to destroy the cancer cells.
It has been approved for use in malignant melanoma (Karimkhari
et al., 2014). Nivolumab efficacy in recurrent GBM is being investigated
in a phase IIb trial as monotherapy or in combination with ipilimumab
versus bevacizumab (NCT02017717).
Adoptive cell transfer uses effector cells that are activated ex vivo
and then tranfered to a patient to attack GBM cells. Effector cells can
be lymphocyte-activated killer (LAK) cells, cytotoxic T lymphocytes
(CTLs), natural-killer (NK) cells and γδ-T-cells. Results generally
have been mixed with good tolerability but the OS was not significantly
improved (Dillman et al., 2009). Newer CTLs that are more specific
to tumor-associated antigens like CD133 which show activity
against GBM stem cells (Hua et al., 2011) or virus-specific CTLs (e.g.
against CMV proteins pp65 and IE1-72 or nucleic acids) are intensively
being investigated against GBM (Nair et al., 2014) (NCT00693095,
NCT01109095). Genetically modified T-cells with novel artificial
chimeric antigen receptors (CAR), can recognize antigens with HLA-
independent mechanisms. CARs are fusion molecules of MAb specific
for a tumor-antigen and the ζ-chain of the T-cell-receptor (TCR). CAR-
modified T-cells have been manufactured against IL-13 receptor alpha
2 (IL13Ra2) (Krebs et al., 2014) and EGFRvIII (Choi et al., 2014). A
phase I/II trial of autologous CARs against EGFRvIII in glioblastoma is
in development (NCT01454596). A phase I trial of postsurgical tumoral
cavity injection of IL13Ra2 in GBM has been completed (NCT01082926).
Active Immunotherapy – Vaccine strategies: Although we have a
vast arsenal of agents to attack the EGFRs in GBM, a large amount of
EGFRs still cannot be blocked due to the receptor’s ubiquitous and
high level expression. A peptide vaccine, rindopepimut (CDX-110), is
directed against the novel exon 1–8 junction produced by the EGFRvIII
mutation. Recent phase II studies have shown EGFRvIII specific immune
response and significantly prolonged TTP and OS in patients with newly
diagnosed GBM compared to standard care. It represents a promising
field of therapy in patients with GBM (Del Vecchio et al., 2012). A
phase III study in patients with newly diagnosed GBM is under way
(NCT01480479). ITK-1 is a personalized peptide vaccine given in HLA-
A24 positive recurrent or progressive GBM patients (Terasaki et al.,
2011).
Heat shock protein peptide vaccine (HSPPV) uses peptides com-
plexed with Hsp proteins that are effectively used by APCs (See et al.,
2011). Vitespen a heat shock protein (gp96)-peptide complex purified
from autologous tumors has been used in phase I/II trials with minimal
side-effects and clinical responses in early-stage disease (Wood &
Mulders, 2009).
Other peptide vaccines for malignant gliomas include peptides
against melanoma/testis antigens, viral antigens, cytokine receptors
and differentiation antigens (Mitchell & Sampson, 2009).
Gene-modified tumor vaccines work in a different way by inducing
GBM cells to be more immunogenic, express new antigens and thus
give more targets for the immune system to fight against. Using autolo-
gous glioma cells modified to secrete either granulocyte/macrophage
colony factor (GM-CSF) or TGFβ2 antisense vectors (Fakhrai et al.,
2006) or IL-4 (Okada et al., 2007), response has been seen in small trials
of progressive or recurrent GBM patients.
Evidence shows that microglia is controlled by glioma cells resulting
in their support for growth and thus, instead of destroying them they
help by contributing to tumor progression. This may be mediated
through colony-stimulating factors, interleukin-6, TGF-β2 and mono-
cyte chemotactic protein-1(MCP-1) that are being produced by glioma
cells (Charles et al., 2011). In turn, microglial cells support the survival,
proliferation and invasion of glioma cells via secretion of factors such as
EGF, VEGF and stress-inducible-protein-1 (STI1) (Alves et al., 2011).
Dendritic cell (antigen-presenting) vaccines are made from dendrit-
ic cells extracted from the patient, then exposed to the antigens of GBM
cells and the same (now “primed”) cells are infused back into the pa-
tient. In turn, they activate the CD8+ (cytotoxic) T cells and present
GBM antigen which is now being recognized by the host immune sys-
tem. Tumor antigens that are being used are HER2, TRP2, Epha 2,
GP100 and KI3ra (Lesniak, 2011). Glioma stem cells contribute to resis-
tance to chemoradiotherapy, thus introducing dendritic cells against
these stem cells (using specific antigens such as CD133 and nestin)
would be an important therapeutic option. In a non-randomized
phase II study, anti-EGFRvIII dendritic cell tumor vaccine addition to
standard first line treatment in GBM patients with good performance
status, when compared to a matched control group from MD Anderson,
it resulted in an increase of median OS from 15 to 26 months and an in-
crease in median PFS from 6.3 months to 14.2 months (hazard ration
2.4; P = o.o13 in favor of the vaccination group). The PFS-6 rate after
vaccination was 67% (95% CI, 40–83%) and after diagnosis was 94%
(95%, 67–99%, n = 18). The development of antibodies or delayed
type hypersensitivity had a significant effect on OS. When the tumor re-
lapsed, EGFRvIII expression was lost in 82% (95% CI, 48–97%). In subcat-
egory analysis, outcomes were improved in both O-MGMT methylated
 C. Alifieris, D.T. Trafalis / Pharmacology & Therapeutics 152 (2015) 63–82
and unmethylated patients. These results demonstrate that in EGFRvIII-
positive GBM, outcomes are improved (Sampson et al., 2010).
Other treatment strategies include microglia as gene or factor thera-
peutic vectors into the central nervous system and glioma tumors.
Examples include microglia carrying the LacZ gene (Sawada et al.,
1998), inducible thymidine kinase gene or MRI contrast agents (Ribot
et al., 2007) or biodegradable nano-vehicles carrying DNA or RNA
silencer fragmants into the glioma tumor (Carbon nanotubes) (Zhao
et al., 2011). Microglial cell gene therapy through viral vectors of either
adenovirus (Bhat & Fan, 2002) or lentivirus (the latter using an
enhanced GFP gene) (Balcaitis et al., 2005) have also been tried.
Intratumoral administration of oncolytic myxoma virus with rapamycin
was another approach (Lun et al., 2012). Although phase III data are
lacking, preclinical and phaseI/II trial results, stongly state that immuno-
therapy is an area with promising results and offer a new weapon
against this lethal tumor.
4. Conclusion
Research in GBM treatment is ongoing, vast, and rapidly evolving.
However, even all this data and progression of molecular science
it has not been able to battle effectively this tumor. No matter how
many different targets are discovered and molecules to aim them are
enginered, the end result is that we have made only a little progress for-
ward in improving overall survival. However, as is seen in this review,
every step in the way, new lessons drive us forward and small details
help us bypass the presenting obstacles. New targets, novel antagonists,
more effective drug penetration through the BBB and new methods of
manipulating the immune response are all puzzles to be solved in the
near future.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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