Animal models for glioma drug discovery

1. Introduction

2. Historical account of

high-grade glioma therapy

3. Histological and molecular

characteristics of

high-grade glioma

4. Mouse models of gliomas

5. Preclinical studies using

GEMMs of glioma

6. Conclusions

7. Expert opinion

Review

Animal models for glioma drugdiscoveryTerreia S Jones* & Eric C Holland†*University of Tennessee Health Science Center, Department of Clinical Pharmacy, Memphis, TN,

USA and †Memorial Sloan-Kettering Cancer Center, Department of Neurosurgery, New York,

NY, USA

Introduction: High-grade gliomas are among the most deadly of all cancer

types and are also the most common malignant primary tumors of the CNS.

Large-scale studies that have analyzed the transcriptional and translational

expression patterns of glioma have found that the majority of these tumors

can be categorized based on specific genomic anomalies. Genetically engi-

neered mouse models (GEMMs) that represent the molecular subgroups of

the human disease harbor a variety of molecular alterations that have been

proven to drive gliomagenesis. These models provide an opportunity to assess

the effects of novel therapies in the presence of specific molecular defects.

Research using GEMMs, which are associated with these subclasses, allow

researchers to assess drug efficacy by subclass.

Areas covered: In this review, the authors discuss the histological and molec-

ular characteristics of malignant gliomas, the therapies used to treat them

and the animal models that closely recapitulate them.

Expert opinion: It is likely that GEMMs that recapitulate the molecular charac-

ter of human tumors will provide a more accurate prediction of individuals

who may be more or less likely to benefit from specific therapies. This know-

ledge can be then used to drive clinical trial design and this, in turn, could

lead to better therapeutic outcomes.

Keywords: gliomas, molecular classification, mouse models, preclinical trials

Expert Opin. Drug Discov. (2011) 6(12):1271-1283

1. Introduction

It is estimated that ~ 24,000 individuals will be diagnosed with a primary CNStumor this year in the US [1]. Diffuse gliomas (glial tumors that infiltrate into nor-mal brain tissue) are among the most difficult to treat neoplasms and mortality ratesare directly proportional to tumor grade [2]. Diffuse gliomas account for the vastmajority of primary brain tumors with grade IV glioblastoma (GBM) being themost frequent and the most malignant and deadly overall. High-grade gliomas arealmost always rapidly fatal regardless of the therapeutic management used. Mostpatients diagnosed with a GBM will die within the first 2 years from diagnosisdespite aggressive therapy. Indeed, even the low-grade tumors can undergo malig-nant transformation into a GBM over time, usually within 5 -- 10 years fromdiagnosis of the primary tumor [3].

Despite > 40 years of research, only modest improvements in survival for high-grade glioma have been made. The challenge in providing effective therapies forthe treatment of these tumors is evidenced in a 5-year survival rate of < 10% forGBM [4]. Today, the standard of care for treating the primary disease includestemozolomide and radiation and now bevacizumab is used commonly at tumorrecurrence. A number of primary papers and reviews have been published discussingthe role of signal transduction pathways in gliomagenesis and mouse models havevalidated the importance of some of these pathways [5]. There are several signal

10.1517/17460441.2011.632628 © 2011 Informa UK, Ltd. ISSN 1746-0441 1271All rights reserved: reproduction in whole or in part not permitted

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transduction inhibitors that have been developed and manyare currently being tested in clinical trials for glioma (referto Arko et al. for a review) [6,7]. Unfortunately, to date manyof these agents do not appear to significantly impact thecourse of the disease when compared to standard of care ther-apy. However, there are a few studies that have shown a mod-est benefit in primary disease when a signal transductioninhibitor is combined with temozolomide and radiation [8-10]

and a number of these agents are being tried in combinationwith other targeted therapies at tumor recurrence [11-13]. Thelack of success in developing effective novel therapies can beattributed in part to the intra- and inter-tumoral heterogene-ity. Molecular studies have revealed the complexity of thesetumors and may provide a more detailed means of character-izing them than the traditional histological classification sys-tem used by clinicians over the past several decades. In thisreview, we discuss the histologic and molecular characteristicsof malignant gliomas, the therapies used to treat them and themouse models that can be useful in testing novel therapies inpreclinical studies.

2. Historical account of high-grade gliomatherapy

Since the mid-20th century, there have been few significantadvances in the therapeutic management of glioma. Figure 1

displays the limited number of significant advances inGBM therapy and is reflective of the modest improvementsin survival over time. In the early 1970s, the median sur-vival for patients diagnosed with a high-grade glioma wasonly 6 months [14]. Surgical removal of as much of thetumor mass as possible is directly correlated with sur-vival [15,16]; hence, surgery has remained the primary treat-ment modality for glioma. Historically, post-surgicalglioma management that provided the best opportunity atextending survival in these patients included lipophilicdrugs and radiation therapy. In the 1980s, results fromclinical studies solidified the role of radiation and chemo-therapy in improving survival in these patients and thistherapy subsequently became the standard of care [17-22].One of the biggest challenges to identifying effective drugtherapies for glial tumors was designing drugs that werecapable of penetrating the BBB to achieve adequate druglevels at the tumor site. The first drugs proven to be effec-tive against glioma were lipophilic drugs belonging to thenitrosourea drug class. These drugs function by generatinglethal DNA damage in tumor cells leading to cell death.However, the damage exerted by these drugs was not onlyrestricted to tumor cells, hence a significant amount ofadverse effects are associated with them. Drugs in this classinclude carmustine (BCNU) and lomustine (CCNU).BCNU was approved by the FDA in 1977 and becamethe most commonly used agent in this class. In a clinicalstudy that compared BCNU alone, radiation therapy aloneand radiation plus BCNU, it was found that by combiningBCNU with radiation the median survival can be extendedby as much as 12 months [19,23]. Later, in an effort todecrease the toxic side effects associated with systemicadministration of BCNU and increase drug concentrationsat the tumor site, a biodegradable wafer impregnated withBCNU was developed and approved by the FDA (1996)for implantation at post-surgical resection of recurrent dis-ease. However, this local delivery approach would onlyextend survival by ~ 2 months in GBM patients [24]. Stud-ies are being conducted that combine BCNU implants withstandard therapy [25].

After almost 10 years of practically no advances in survivalor therapeutic management for glioma, temozolomide (aderivative of the alkylating agent dacarbazine) was approvedby the FDA for refractory high-grade disease in 1999 andwas later approved for primary disease in 2005. Today, thepost-surgical standard of care for treating the primary diseaseconsists of concurrent temozolomide and radiation followedby adjuvant temozolomide. This combination has providedthe most significant improvement in survival since the1980s (median survival of 14.6 months) [4,26]. Morerecently, bevacizumab is being used more often at tumorrecurrence [27-30] and clinical studies are being performed toinvestigate bevacizumab’s utility in treating primary dis-ease [31]. However, it remains to be determined whether thereis a survival benefit with bevacizumab therapy.

Article highlights.

. The lack of success in developing more effectivetherapies against glioma can be attributed to the intra-and inter-tumoral heterogeneity and expression studieshave confirmed the molecular complexity ofthese tumors.

. Genetically engineered mouse models (GEMMs) ofglioma have been developed by overexpressingcomponents of signal transduction pathways thatpromote cell proliferation and survival.

. The Cre-Lox system allows for targeted deletion of agene flanked by loxP sites within a specific cell typewhen Cre recombinase is expressed under the control ofthe tissue-specific promoter.

. The RCAS/tv-a system allows for the delivery of anoncogene of interest into brain cells by expressing theTVA receptor under the GFAP astrocytic promoter or thenestin neural progenitor promoter.

. The GEMMs that are associated with the molecularcharacter of human glioma can be used to screen novelcompounds and therapeutic approaches for efficacy andtoxicity prior to being used in the clinical setting.

. The use of imaging modalities such as MRI andbioluminescence imaging provides the opportunity toconfirm tumor presence and monitor the tumor’sresponse to therapy in vivo over time without sacrificingthe animal.

This box summarizes key points contained in the article.

Animal models for glioma drug discovery

1272 Expert Opin. Drug Discov. (2011) 6(12)

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3. Histological and molecular characteristicsof high-grade glioma

In 1979, the WHO formalized a system to classify CNStumors using criteria based primarily on tumor histopathol-ogy. In this system, tumors are grouped based on the resem-blance of tumor cells to normal glial cells and the relativedegree of malignancy. In 2007, the classification system wasrevised to reflect changes in tumor grade, new entities and var-iants of some CNS tumors [32,33]. This system groups braintumors by grade (WHO grades I -- IV) (Table 1), with lowergrades reflecting the more benign and treatable tumors andhigher grades reflecting the more aggressive and difficult totreat tumors. Grade I gliomas are slow growing well-circumscribed tumors that are curable by complete surgicalresection. Grade II tumors are relatively slow growing tumorsexhibiting cellular differentiation but can diffusely infiltrateinto normal brain tissue and have a modest proliferativecharacter. Grade III tumors exhibit regions of anaplasia,

diffuse infiltration and are highly proliferative. And grade IVgliomas have additional characteristics including microvascu-lar proliferation and pseudopallisading necrosis [34]. Thesetumors are further divided into subcategories based on glialcell morphology (i.e., astrocytic, oligodendroglial, mixed oli-goastrocytic and ependymal). The most common high-grade (or malignant) gliomas include anaplastic astrocytomas(grade III), anaplastic oligodendrogliomas (grade III), ana-plastic oligoastrocytoma (grade III), anaplastic ependymoma(grade III) and GBM (grade IV).

From a global standpoint, elevations in platelet derivedgrowth factor (PDGF) and epidermal growth factor receptor(EGFR) signaling collectively are the most commonly observedaberrations across all GBMs. Approximately 30% of GBM’sexhibit increased PDGF signaling and ~ 45% exhibit increasedsignaling of EGFR and these aberrations can occur together insome GBMs [35]. Elevations in PDGF signaling can occurthrough PDGFRa or PDGFb amplification or activatingmutations. PDGF binding to its receptor (PDGFR) can lead

6

4

2

0

1980

1977:FDAapproves

BCNU

1980: Walker et al.,shows ~12 month

median survival withradiation plus BCNU

Med

ian

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rviv

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BM

(ye

ars)

1990 2000

Mouse modeling of glioma

Year2010

2009: Stupp et al.,shows 14.6 month

median survival withradiation plus TMZ

1999: FDA approvesTMZ for refractoryhigh-grade glioma

2005: FDA approves TMZ forprimary glioma

Molecular sub-grouping of GBMs

1996: FDA approvesGliadel wafers for

recurrent GBM

2009: FDA approvesbevacizumab

Figure 1. Historical account of survival and standard of care therapy for glioblastoma (GBM). The first drug approved for

treating GBM was carmustine (BCNU) in 1977. Combining BCNU with radiation therapy increased the median survival from

6 to ~ 12 months [19,23]. BCNU was approved in 1996 in the form of a biodegradable wafer for local delivery to the tumor bed.

It was not until 2005 that a new systemic drug was approved for treating GBM (temozolomide, TMZ) and, in 2009,

bevacizumab was approved for recurrent disease. The median survival for primary disease remains suboptimal at

14.6 months [4]. In 1998, Uhrbom et al. developed the first molecularly and histologically relevant platelet derived growth

factor (PDGF)-driven genetically engineered mouse model (GEMM) of glioma [80]. Later, several other human disease relevant

GEMMs were developed and results from expression data from The Cancer Genome Atlas (TCGA) and others suggest that

these models may mimic the molecular character of the human disease [45-47,77,104].

Jones & Holland

Expert Opin. Drug Discov. (2011) 6(12) 1273

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to cell proliferation and survival by stimulating downstream sig-nal transduction pathways such as P13K/AKT and rat sarcomaoncogene (RAS)/MAPK (Figure 2) [36,37]. These pathways canbe activated by PIK3CA and by phophastase and tensin homo-log (PTEN) or neurofibromin 1 (NF1) loss. PTEN andNF1 function by regulating AKT and RAS activation, respec-tively, preventing inappropriate cell signaling for cell growthand proliferation. EGFR overexpression and hyperactivity canbe a consequence of gene amplification or caused by mutantvariants such as EGFRvIII, a constitutively active variant ofEGFR [38-40]. EGFR is a member of the erythroblastic leukemiaviral oncogene receptor tyrosine kinase family and, similar toPDGF and its receptor, ligand binding stimulates down-stream signaling pathways involved in tumor cell growth andsurvival (Figure 2).Loss of function of the tumor suppressors P53 and retino-

blastoma (RB) is also a common occurrence in GBM; how-ever, these lesions are more often associated with tumorsthat are believed to have progressed from a lower grade [5].P53 loss of function can be precipitated by either directgene mutation or deletion or indirectly byMDM2 copy num-ber alteration [35,41,42]. Similarly, RB function can be compro-mised through increased CDK4 copy number causing RBsuppression. And both P53 and RB are vulnerable to thecyclin-dependent kinase inhibitor 2A (CDKN2A) gene dele-tion as this gene encodes for the proteins INK4A and ARFwhich are responsible for inhibiting CDK4 and MDM2(p53 binding protein homolog), respectively (Figure 2).CDKN2A loss leads to unopposed p53 and RB protein sup-pression by CDK4 and MDM2. In a small percent ofGBMs, mutations are found in the isocitrate dehydrogenase(IDH) gene [43] and can result in a gain of function for theproduction of 2-hydroxyglutarate [44]. How this metaboliteor the characteristic DNA methylation pattern seen in thesetumors contributes to tumor formation is not yet clear.

3.1 Molecular stratification of the GBMsMore recently, there have been several papers published dis-cussing the molecular character of high-grade gliomas andthe potential clinical implications associated with the new

molecularly defined subcategories of these tumors [5,35].Large-scale studies that have analyzed the transcriptional andtranslational expression patterns of glioma have found thatthe majority of these tumors can be categorized based on spe-cific genomic anomalies [45-47]. Distinct glioma subclasseshave been defined based on these molecular data, althoughmany of the aberrations are observed across subtypes (Referto Huse et al. 2011 for a complete review) [35]. However, thereappears to be at least three subgroups that can be assigned tomost gliomas. The classical, proneural, and mesenchymal sub-groups each have a characteristic lesion that plays an impor-tant role in signal transduction pathways involved in tumorgrowth. These lesions confirm the importance of these path-ways in glioma pathology. The ‘proneural’ subtype is charac-terized by elevated PDGF signaling. Elevated expression ofthe EGFR is found predominantly in the ‘classical’ subtype.And finally, loss of the tumor suppressor NF1 is concentratedin the ‘mesenchymal’ subtype.

There are several short latency and high penetrant geneti-cally engineered mouse models (GEMMs) of glioma thatexhibit the molecular anomalies found in the human diseaseand validate the importance of signal transduction pathwaysinvolved in gliomagenesis [48-50]. These GEMMs can be usefultools for testing novel drugs against malignant glioma and arediscussed in the following section.

4. Mouse models of gliomas

As previously discussed, high-grade gliomas are heterogenoustumors both at the molecular and cellular levels. The com-plex biology of these tumors makes understanding gliomapathogenesis and the development of novel effective thera-pies extremely challenging. While using established gliomacell lines or primary glioma cultures to study glioma biologyor test novel drugs in vitro can be of some benefit [51-53],these studies lack the ability to address the more complexissues related to gliomagenesis, the role of the tumor stromaand drug pharmacodynamics (i.e., efficacy and toxicity).Over the years, a number of strategies for creating in vivomouse models of glioma have been developed that include

Table 1. Subtype, histology and distribution of common glial tumors by WHO grade. Distribution of glioma by

histology is based on the Center for Brain Tumor Registry of the United States 2011 statistical report. Grade IV

glioblastomas are the most common glial tumor overall [1].

WHO grade Common subtype(s) Histology Percent of glial tumors

I Pilocytic astrocytoma Benign, well circumscribed 5.2%II Oligodendroglioma Diffuse infiltration, low proliferation 8.1%*III Anaplastic astrocytoma Highly infiltrative, high cellular proliferation 13.7%z

IV Glioblastoma Highly infiltrative, high vascular and cellularproliferation, necrosis, hemorrhage

53.7%

All other gliomas 19.3%§

*Includes protoplasmic and fibrillary astrocytomas (1.6%).zIncludes malignant glioma not otherwise specified (7%).§Includes ependymomas (5.9%).



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tumor cell allograft and xenograft implantation models(Table 2).

4.1 Xenograft modelsClassic xenograft models are generated by subcutaneously ororthotopically implanting established human glioma cell linesinto athymic mice. Bioluminescence and green fluorescentprotein have aided the utility of these models for drug discov-ery by allowing the visualization of tumors in vivo [54-57].U87 is one human cell line that has been used extensivelybecause of the high rate of tumor take after implantationand the short survival time. A number of studies have beenconducted using U87 to assess the pharmacokinetics andtreatment efficacy of a variety of novel therapies [58,59]. Whilethis model provides a straight forward in vivo approach to pre-clinical testing, the major drawback of using cell lines is thelack of histologic similarity to the human disease [60]. Thesemodels have been of limited utility in predicting the efficacyof novel therapies in humans when used in preclinical

studies [61,62]. Other xenograft models have been developedby taking tumor cells directly from the patient and implantingthem into athymic mice resulting in a more invasivephenotype [63,64].

Genetically and histologically accurate models of humanglioma have also been developed through creating GEMMswhich appear to be the best models to date for investigatingglioma pathogenesis (Table 3). GEMMs of glioma can exhibita similar survival response to therapy as patients and thus maybe useful models in preclinical studies [65-67].

4.2 GEMMs of gliomaCarcinogenesis is a process involving serial mutagenic eventsin genes involved in cell proliferation, survival and inva-sion [68]. Human cancers primarily occur somatically andthe cell type of origin dictates the character of the resultingtumor [69]. GEMMs that possess germ-line modifications ofwell-established oncogenes are usually heterozygous at thegene locus of interest as homozygous loss is often embryonic

PTEN

PI3K

SHC GRB2 SOS RAS

RAC

RAF

RAL

NF1

CDK4 RBCell cycleregulation

P53MDM2

p16INK4a

INK4a/ARFp14ARF

MKK

MEK

JNKK JNK

ERK

p38

Promotion of cellgrowth andproliferation

Growth factor(EGF, PDGF)

Receptor tyrosinekinase (EGFR,

PDGFR)

PIP3 AKT mTORPromotion ofcell survival

Figure 2. Molecular alterations commonly observed in human glioblastoma (GBM). Alterations of components of signal

transduction pathways are commonly observed in human GBM and these anomalies define the molecular subclasses.

Overexpression of receptor tyrosine kinases or their ligands is predominately found in the classical and proneural subclasses

through alterations in the EGFR and the platelet derived growth factor (PDGF), respectively. Loss of neurofibromin 1 (NF1) is

concentrated in the mesenchymal subclass. NF1 prevents unregulated signals for cell growth by inhibiting Ras. Phophastase

and tensin homolog (PTEN) loss is responsible for inhibiting AKT and, therefore, regulates signals promoting cell survival.

Alterations in cell cycle regulation are also observed in human glioma. Disruption of cell cycle control can be caused by direct

loss of RB or P53 or through alterations in upstream regulators such as loss of INK4a/ARF or overexpression of CDK4 or MDM2.

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lethal. In these models, spontaneous loss of the second alleleleads to tumor formation [70,71]. Thus, many germ-line transgenic models tend to be more reflective of a humantumor predisposition syndrome where the threshold for trans-formation is lower. Germ-line models can be combined with‘conditional’ systems to allow for tumor induction in a tem-poral--spatial manner [72,73]. The use of the Cre-Lox andRCAS/tv-a systems are two examples of this approach. TheCre-Lox system allows for targeted deletion of a gene flankedby loxP sites (derived from the P1 bacteriophage) within a spe-cific cell type when Cre recombinase is expressed under thecontrol of the tissue-specific promoter. The replication com-petent avian leukosis virus splice acceptor (RCAS) viral vectorcan be used to deliver a gene of interest into targeted braincells when the expression of TVA (avian leukosis virusreceptor A; the RCAS receptor) is driven by a tissue-specific promoter [74]. Additionally, mice harboring germ-line aberrations in tumor suppressor genes such as INK4a/ARF have a decreased threshold for tumor formation anddevelop high penetrant malignant gliomas when additionaltumor inducing lesions are introduced [75]. GEMMs havebeen developed by overexpressing components of signal trans-duction pathways that promote cell proliferation and survival.As discussed below, disrupting normal function of compo-nents of the P13K/AKT/mammalian target of rapamycinand RAS/MAPK signaling pathways through aberrations inreceptor tyrosine kinases or their ligands (i.e., EGFR andPDGFb), overexpressing protein kinases (i.e., AKT, RAS) orby loss of tumor suppressors (i.e., NF1, P53, RB) can contrib-ute to glioma formation. Although overexpression of AKTand RAS is observed in the human disease, it is not a resultof a direct gene effect. Instead, gene overexpression is the

result of the activation of upstream receptor tyrosine kinasesand their ligands.

Perhaps the more relevant models of glioma are those thatmodel the newly identified molecular subclasses of human gli-oma. Generally speaking, these GEMMs can be considered asaccurate models of the signaling abnormalities that occur inhuman glioma; however, one could argue that they simplyconfirm causality of tumor formation. It should be notedthat even though glioma formation is driven by molecularanomalies that define the human subtypes (i.e., PDGF,EGFR, NF1), it is not yet known to what extent these tumorsharbor other molecular characteristics contained in the sub-classes that they theoretically represent. Recent gene expres-sion studies of gliomas isolated from GEMMs have revealedthat these tumors resemble the human disease at themolecular level [76,77].

4.2.1 Gliomas induced by ‘classical’ signalingGEMMs have been developed that overexpress EGFR andsome of these models mimic the classical subgroup of humanGBM. One of the first successful attempts in generating aEGFR-driven GEMM of glioma was accomplished withglial fibrillary acidic protein (GFAP)-EGFRvIII;GFAP-Ha-RASv12 compound transgenic mice which developedoligodendroglial tumors within 13 weeks with a 100% pene-trance [78]. When GFAP-Ha-RASv12 mice are injected withadeno-EGFRvIII viral vector, malignant gliomas form within8 weeks post injection [79]. Another model that investigatesthe role of EGFR overexpression in gliomagenesis usesCre-lox technology to somatically introduce EGFRvIII intothe brains of adult mice by stereotactic injection ofadeno-Cre [48]. This model uses INK4a/ARF null mice

Table 2. Pros and cons of commonly used models of glioma.

Model Pros Cons

Classic human tumorxenografts

Subcutaneous/orthotopicimplantation of establishedhuman glioma cell lines intoathymic rodents

Low breeding costsLarge numbers of tumor bearingmice can be generatedBioluminescence monitoring oftumor growth

Many do not recapitulatehuman histopathology ormolecular characterTumor-host immunologicalresponse is inhibited

Direct to xenografthuman tumor implants

Orthotopic implantation ofhuman glioma cells into athymicrodents

More accurately mimics theresponse to therapy of humangliomaHistopathologically andmolecularly relevant

Tumor-host immunologicalresponse is inhibited

GEMMs Mice harboring geneticalterations that allow forgliomagenesis de novo

Molecular and histologicallysimilar to the human diseaseTumor-host immunologicalresponse intactSeveral short latency highpenetrance models are availableBioluminescence monitoring oftumor growth

Extensive breeding costs

GEMM: Genetically engineered mouse model.



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harboring floxed PTEN and conditional EGFRvIII alleles thatare expressed by Cre recombinase mediated deletion of loxPsites flanking PTEN and a floxed stop cassette, respectively.These mice develop GBMs with high penetrance by 8 weekspost injection of adeno-Cre.

4.2.2 Gliomas induced by ‘proneural’ signalingA number of PDGF-driven GEMMs of glioma have beendeveloped that are representative of the proneural molecularsubtype. A recent study found that PDGF-driven mousemodels of glioma that harbor additional loss of function ofPTEN and p53 develop tumors that exhibit microvascularproliferation and pseudopalisading necrosis and have themolecular character of the proneural subtype [77]. Anothermodel of malignant glioma was developed by using the Molo-ney murine leukemia virus (MoMuLV) to deliver PDGFb bybrain injections into the frontal lobes of newborn mice [80,81].This model generated GBMs at a 40% penetrance within14 -- 29 weeks. The tumors co-expressed PDGFRa support-ing the concept that an autocrine loop is involved in glioma-genesis. One of the first studies that used an adult rodentmodel to induce PDGF-driven gliomas used a retrovirus to

deliver PDGFb to the brains of adult rats and all animalsdeveloped gliomas that were histologically similar to thehuman disease [82]. This model also suggested that autocrineand paracrine stimulations of glial progenitor cells wereresponsible for driving gliomagenesis.

The RCAS/tv-a system has been used to somatically trans-fer PDGFb to specific cell types in the brain. Transgenic micehave been developed to express the TVA receptor under theGFAP astrocytic promoter (Gtv-a) and the nestin neural pro-genitor promoter (Ntv-a) [83]. In transgenic Ntv-a;INK4a/ARF -/-;PTENfloxed mice, GBMs develop with 100% pene-trance within 8 weeks post injection of RCAS-PDGFb, andwhen the RCAS-PDGFb and RCAS-Cre vectors wereinjected, malignant gliomas form with an equal penetrancebut at a shorter tumor latency [84]. Additionally, the Gtv-a;INK4a/ARF -/-, Gtv-a;p53-/- and Gtv-a;ARF -/- mice alldevelop GBMs with regions of oliogastrocytoma and mixedastrocytoma histology. This system has also been used to gen-erate a pediatric model of PDGF-induced glioma by ortho-topically injecting RCAS-PDGFb into newborn Ntv-a orGtv-a mice [85,86]. In this model, oligodendroglial and mixedoligoastrocytic tumors form with a higher penetrance and

Table 3. GEMMs of high-grade glioma and the associated human molecular subtype based on tumor initiating

lesions.

Associated

molecular

subtype*

Molecular alteration Histology Latency (weeks);

incidence (%)

Model§ Ref.

Classical EGFRvIII gain; V12Ras gain High-grade gliomas 12; 55% Embryonic/neonatal [79]

EGFRvIII gain; Ink4a/Arf loss;PTEN loss

Glioblastoma 13; 100% Adult [48]

Proneural PDGFb gain Glioblastoma 14 -- 29; 40% Embryonic/neonatal [80,81]

PDGFb gain; Ink4a/Arf loss Glioblastoma ~ 6; 100% Adult [84]

PDGFb gain; Ink4a/Arf loss;PTEN loss

Glioblastoma ~ 6; 100% Adult [84]

PDGFb gain; p53 loss Glioblastoma 24; 25% Pediatric Embryonic/neonatal

[88]

Mesenchymal Nf1loss; p53z loss Glioblastoma 45; 70% Embryonic/neonatal [70]

Nf1 loss; p53 loss; PTEN loss Anaplastic astrocytoma;glioblastoma

~ 18; 100% Embryonic/neonatal [89]

Nf1 loss; p53 loss Anaplastic astrocytoma;glioblastoma

24; 100% Embryonic/neonatal/Adult

[90]

Other RB loss; PTEN loss Anaplastic astrocytoma 40; 100% Embryonic/neonatal [92,93]

KRas gain; PTEN loss Glioblastoma 12; 62% Embryonic/neonatal [98]

KRas gain; Akt gain Glioblastoma;glioblastoma variants

12; 21% Embryonic/neonatal [96]

KRas gain; Akt gain; Ink4a/Arfloss

Glioblastoma 12; 48% Embryonic/neonatal [96,97]

KRas gain; p53loss; Ink4a/Arfloss

Anaplastic astrocytoma;glioblastoma

~ 4 -- 5; 100% Adult [67]

PDGFb gain; Ink4a/Arf loss Brain stem glioma 4 -- 8; 96% Embryonic/neonatal [99]

*The molecular subclasses were derived using adult high-grade glioma expression data; hence. the lesions that drive gliomagenesis in these models may be more

closely aligned with the adult mouse models of glioma.zPenetrance depended on genetic background.§The ‘model’ is based solely on the time of tumor induction.

GEMM: Genetically engineered mouse model; PDGF: Platelet derived growth factor; PTEN: Phophastase and tensin homolog; RB: Retinoblastoma.

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shorter tumor latency as compared to PDGFb delivery byMoMuLV. It was also demonstrated that higher gene dosedirectly correlated with tumor grade.More recently, a Ctv-a transgenic mouse line was devel-

oped to express TVA under the control of the myelinating oli-godendrocyte progenitor cell promoter, 2’,3’-cyclic nucleotidephosphodiesterase promoter [87]. When newborn Ctv-a pupsare injected with RCAS-PDGFb low-grade oligodendroglialtumors form. Another PDGF-driven glioma transgenicmouse line was created recently by crossing mice that overex-press PDGFb under the control of the hGFAP promoter withP53 null mice. These mice develop oligodendrogliomas andGBMs by 6 months of age [88].

4.2.3 Gliomas induced by ‘mesenchymal’ signalingThe mesenchymal subclass is primarily defined by NF1 loss.NF1 is a tumor suppressor that when mutated is responsiblefor the heritable tumor predisposition syndrome neurofibro-matosis type 1. A mouse model of this subclass was generatedby crossing mice bearing a germ-line P53 mutation and NF1floxed alleles with GFAP-Cre mice [70]. In this model, tumorsform with a 100% penetrance and possess histological featuresconsistent with high-grade glioma. Another model demon-strated that when NF1/P53 loss is combined with PTEN,haploinsufficiency tumor latency is decreased and tumorgrade is increased [89]. A somatic cell gene transfer version ofthe mesenchymal subtype has also been generated usingadeno-Cre delivery to mice harboring conditional NF1 andP53 alleles [90]. In this model, tumors formed only when thevirus was delivered to the subventricular zone, suggestingthat at least in this case the cell of origin may be limited tostem cells located in this region.

4.2.4 Other GEMMs of gliomaAberrations within the RB pathway are commonoccurrences in human astrocytoma [91]. A truncated versionof the SV40 T antigen (T121) expressed under the GFAPpromoter can inactivate RB and induce malignant astro-cytomas in mice when combined with PTEN loss [92,93].In this model, T121 expression was induced by Cre-mediated deletion of an upstream lacZ stop signal andPTEN complete loss by somatic cell transfer of murine stemcell virus-cre-recombinase.GEMMs of glioma that utilize downstream effectors of the

EGFR and PDGF signaling pathways such as constitutiveexpression of AKT and RAS are also useful tools for investi-gating glioma biology. GFAP;Ha-RASv12 transgenic micethat express varying levels of RAS helped to prove the impor-tance of RAS in malignant glioma formation [94,95]. TheRCAS/tv-a system has been used to generate high penetrantshort latency models by somatically transferring a constitu-tively active version of RAS (KRAS) to nestin-expressing cells. INK4a/ARF -/-;Ntv-a mice develop GBMswhen RCAS-KRAS viral vector is injected into the brain [96,97].GBMs also formed in Gtv-a;INK4a/ARF -/- mice with

histological hallmarks of giant cell GBM with an incidenceof 42% [96]. Indeed, KRAS combined with AKT overexpres-sion in the presence of wild-type INK4a/ARF can generatemalignant glial tumors in mice although at a lower fre-quency [96]. When newborn Ntv-a mice are injected withRCAS-KRAS and RCAS-AKT viral vectors, malignant glio-mas develop with a 21% tumor incidence within 12 weeks.A similar model was developed that showed that PTEN losscan cooperate with KRAS activation to generate glioma. Inthis model, Ntv-a;PTENfloxed mice develop GBMs withhigh penetrance 12 weeks post RCAS-Cre and RCAS-KRASinjections into the brain [98].

Recently, a PDGF-driven pediatric brain stem glioma(BSG) model was developed using the RCAS/tv-a system [99].In this model, neonatal Ntv-a mice were injected with RCAS-PDGFb in the posterior fossa to generate a model oflow-grade BSGs and Ntv-a;INK4a/ARF-/- mice were used togenerate a high-grade model. In the low-grade BSG model,80% of the mice developed tumors by 12 weeks post injectionand 100% of the mice with high-grade gliomas developedtumors within 4 -- 8 weeks post injection.

GFAP-Cre;p53+/- mice have been used to generate gliomasafter orthotopic injection of a lentiviral vector containing Ha-RASv12 in which RAS is expressed after excision of upstreamloxP sites [66]. However, the development of short latency,high penetrant high-grade gliomas required vector injectioninto the hippocampal region of the brain. Another modelthat uses the Cre-lox system uses mice harboring conditionalp53;Ink4a/Arf;KRASv12 alleles to develop short latency glio-mas at 100% penetrance after orthotopic injection of cyto-megalovirus cre-recombinase (CMV-Cre) viral vector [67].High-grade gliomas have also been developed by using plas-mid DNA to deliver the Sleeping Beauty transposable elementwhich allows for the integration of relevant oncogenes intobrain cells of a variety of mouse strains [100]. Several genescan be co-transfected at once using this system includingreporter genes that allow for bioluminescence imaging (BLI)of luciferase expression. This model can generate histologi-cally relevant gliomas and offers the advantage of tumor imag-ing by BLI. Indeed, many of the GEMMs that have beendeveloped have not been evaluated for similarities in responseto drug therapy as observed in the human disease.

5. Preclinical studies using GEMMs of glioma

The knowledge gained through molecular studies of humanglioma has provided invaluable insight into the importanceof several genes in the pathogenesis of glioma. The GEMMsof glioma that are induced by proneural, mesenchymal orclassical signaling can be used to investigate novel com-pounds and therapeutic approaches that may be moredirected towards these signaling pathways. The use of imag-ing modalities such as MRI and BLI provides the opportu-nity to confirm the tumor presence and monitor thetumor’s response to therapy in vivo over time without



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sacrificing the animal. For example, T2 weighed MRI hasbeen used to visualize the presence of tumor in a newlydeveloped pediatric model of BSG [99]. Subsequently, thismodel was used to test the efficacy of perifosine (an AKTinhibitor) against this tumor type. When high-grade BSGswere treated with perifosine alone, radiation alone or perifo-sine plus radiation, it was found that perifosine provided nosurvival benefit over radiation therapy either alone or whencombined with radiation. When the therapeutic response ofa PDGF-driven GEMM of glioma was evaluated, temozolo-mide therapy significantly prolonged survival as compared tocontrols [65].

Several transgenic lines have been developed that allow fortumor imaging by BLI [51,101,102]. In these models, photonlight emission directly correlates with tumor cell progressionand cell proliferation [102]. Ef-luc;Ntv-a mice develop oligo-dendrogliomas when injected with PDGFb and when treatedwith the PDGFR/VEGFR inhibitor vatalinib, light emissionwas reduced as compared to control treated mice [101]. Thismodel was used to test the effectiveness of perifosine in gli-oma [51]. In this study, mice were enrolled based on the pres-ence of tumors as determined by BLI and it was found thatcombining perifosine with temozolomide resulted in a greaterantitumor effect than did temozolomide alone. Anothermodel that has been engineered for BLI and potentially canbe used for preclinical drug testing uses Gli responsive pro-moter to drive the expression of luciferase in Ntv-a miceallowing the imaging of signaling through the sonic hedgehogpathway [102]. In a glioma model that used orthotopic deliveryof CMV-Cre lentivirus to brain cells of mice to achieve con-ditional loss of p53 and Ink4a/Arf and activation ofKRASv12 and luciferase, temozolomide treatment was shownto induce prolonged survival and tumor regression wassuggested by BLI analysis [67].

6. Conclusions

Xenograft models of glioma have been used for many years toinvestigate the utility of novel drugs and treatmentapproaches. More recently, direct to xenograft human tumorimplant models have been shown to be useful in predictingthe response to anticancer agents [103]. The large-scale expression studies of human glioma have provided theresearch community with an unprecedented amount of infor-mation on the molecular character of human glioma. It islikely that over time the subtypes identified through thesestudies will be more refined and others may be identified.There are now a variety of GEMMs of glioma available thatcan be used to investigate the biology of the disease as wellas be used for preclinical testing of novel therapies. Thesemodels are believed to be close recapitulations of the humandisease because they represent a combination of distinctgenetic events, some of which are introduced as somatically

acquired events, similar to the presumed evolution of gliomain humans.

7. Expert opinion

Therapeutic responses in GBMs have been very poor and havenot improved appreciably over several decades. These tumorsare now being molecularly subclassified into three or four dif-ferent groups. Although it is not completely clear how thesedata will ultimately be used to better stratify patients, it ishopeful that in the near future tumor histology will be usedalong with molecular character to better optimize therapy.Indeed, molecular data have already proven to be useful forpredicting clinical outcome [47]. There are mouse modelsthat recapitulate the genetic drivers and histology of theseGBM subsets. The mouse models that represent the molecu-lar subgroups harbor a variety of molecular alterations thathave been proven to drive gliomagenesis and these aberrationsare commonly observed in the human disease. These modelsprovide the opportunity to assess the effects of novel therapieson specific molecular defects involved in tumor growth andprogression. Using GEMMs that possess specific molecularanomalies as observed in the human disease allows for theassessment of drug efficacy based on molecular characteristicsof human glioma.

In vivo models of glioma are essential tools for addressingthe complex inter-play between the tumor and the host. Aswith other cancer types, glial tumor cells and the tumor-associated stromal cell populations function together to createa tumor permissive environment. Studying the effects of drugtherapy on stromal cell types can lead to the development ofnovel drugs that target the tumor stroma or their products,such as bevacizumab, an antibody against VEGF. It is likelythat these tools will help provide a more accurate predictionof individuals who may be more or less likely to benefitfrom specific therapies based on the molecular character oftheir tumors. GEMMs provide the opportunity to answerquestions of drug efficacy based on specific molecular charac-teristics. With the increasing focus on the development of tar-geted therapies, it is reasonable to presume that GEMMs willplay an increasing role in understanding the specificity ofthese agents and the feasibility of combining agents to targetmultiple factors. The hope is that knowledge gained from pre-clinical studies using these GEMMs can be used to moreeffectively drive clinical trial design to achieve bettertherapeutic outcomes.

Declaration of interest

The authors are supported by the Memorial Sloan-KetteringCancer Center, The University of Tennessee Health Centerand NIH grants RO1 CA100688, U54 CA126518-01,U01 CA141502-01 and U54 CA143798.

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AffiliationTerreia S Jones*1,2,3 & Eric C Holland†4,5,6

†Author for correspondence1University of Tennessee Health Science Center,

Department of Clinical Pharmacy,

19 S. Manassas, Memphis, TN, 39103, USA

Tel: +901 448 1136; Fax: +901 448 6064;

E-mail: [email protected] of Tennessee Health Science Center,

Department of Neurosurgery,

Memphis, TN, USA3University of Tennessee Health Science Center,

Center for Cancer Research,

Memphis, TN, USA4Memorial Sloan-Kettering Cancer Center,

Department of Neurosurgery,

408 East 69th (Z1304),

New York, NY 10065, USA

Tel: +646 888 2053;

E-mail: [email protected] Sloan-Kettering Cancer Center,

Department of Cancer Biology and Genetics,

New York, NY, USA6Brain Tumor Center,

Memorial Sloan-Kettering Cancer Center,

New York, NY, USA

Jones & Holland


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Documents

Animal models for glioma drug discovery