WO2011134023A1 - Inhibition of glioma - Google Patents

Abstract

The present invention relates to methods of identifying pharmaceutical agents useful in the treatment of glioma and methods of treatment of glioma, in particular high-grade glioma. The invention is based on the observation that nuclear factor lb (Nfib) is down- regulated in human glioma tissue samples, and the invention looks to restore Nfib activity such as through inducing expression or overexpression of Nfib which reduces the proliferation of glioma cells and induces differentiation in vitro.

Description

INHIBITION OF GLIOMA

Technical Field

The present invention relates to methods of

identifying pharmaceutical agents useful in the treatment of glioma and methods of treatment of glioma, in

particular high-grade glioma.

Background Art

Gliomas are tumours with phenotypic features of the glial elements of the central nervous system. They are commonly classified histologically as astrocytomas, oligodendrogliomas or oligoastrocytomas and are graded on a WHO consensus-derived scale of I to IV according to their degree of malignancy {Furnari et al . , 2007). Grade III and grade IV gliomas, more commonly referred to as anaplastic astrocytomas and glioblastoma multiforme (GBM) respectively, are referred to as high-grade gliomas.

These are diffusely infiltrating, anaplastic, highly proliferative tumours that are incurable by surgery.

Grade IV tumours additionally exhibit vascular

proliferation and necrosis, and, being resistant to radio/chemotherapy, are generally lethal within 12 months (Furnari et al . , 2007). High-grade gliomas are the most common primary human brain tumours (Behin et al . , 2003) . Each year, approximately the same number of Australians die from high-grade gliomas as die from melanoma. Despite this, advances in therapies for high-grade glioma

sufferers have been limited. Even with aggressive

multimodal therapy, including maximal surgical resection, fractionated radiotherapy and concurrent temozolomide chemotherapy (Aoki et al . , 2007, Wen et al . , 2008), high- grade glioma remains for the most part incurable. New therapies are therefore urgently needed. Gene expression profiling, together with DNA mutation data, have recently identified molecular

subtypes of high-grade glioma that may arise through different mechanisms iproneural, neural, classical, and mesenchymal) and can define patient groups that could benefit from particular targeted therapies (Carro et al . , 2010, Verhaak et al . ) . Although there is no general agreement on the cell of origin of high-grade glioma (Dirks efc al . , 2008), there is good evidence that at least some high-grade gliomas arise from a small

population of developmentally arrested neural progenitor or stem cells (Pardal et al . , 2003, Reya efc al . , 2001, Singh et al . , 2004). Such cells could also be responsible for tumour recurrence following treatment (Dick, 2008) and their intrinsic resistance to both chemotherapy and radiation (Bao et al . , 2006) requires new strategies to physically or functionally eradicate them. In the case of high-grade glioma, the cancer stem cell (CSC) hypothesis would suggest that the mechanism responsible for CSC differentiation into glial cells is defective.

Differentiation therapy has been shown to have positive results for some cancers (Berman efc al . , 2002, Jain efc al., 2002, Wang efc al . , 2008), including GBM (Piccirillo et al . , 2006). These studies demonstrate that targeted manipulation of factors implicated in CSC maintenance and/or differentiation can be effective in reducing the population of tumour- initiating cells and arresting tumour growth.

The ability to isolate and culture glioma CSCs under specialised conditions has underpinned much of the recent progress in this field. One such technique, the

neurosphere (NS) assay, first described for neural stem cells which grow in suspension as ¹neurospheres' , uses serum-free culture conditions with the addition of the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Deleyrolle et al . , 2009, Reynolds et al . , 1992). Significantly, GBM cell lines grown in these conditions more closely mirror the phenotype and genotype of primary resected tumours than do their serum-grown counterparts (Lee et al . , 2008). Recently this technology has been extended to a more practical adherent culture system which allows for primary glioma specimens to be grown as a de-differentiated monolayer of cells on a basement membrane of laminin. Termed glioma neural stem (GNS) cultures, primary glioma lines derived in this way exhibit gene expression patterns and differentiation behaviour that correlate with specific neural progenitor subtypes, and also permit more convenient analysis of CSC behaviour {Pollard et al . , 2009).

Nuclear Factor One DNA-binding proteins comprise a family of site- specific transcription factors, with four members in vertebrates (Nfia, Nfib, Nfic, and Nfix) (Gronostajski , 2000) . Nfi family members play key roles in specific aspects of mammalian organogenesis

(Gronostaj ski , 2000) . For example, the Nfi genes control the differentiation of glia during development by direct transcriptional activation of glial lineage genes such as glial fibrillary acidic protein (GFAP) , brain lipid binding protein (BLBP) , and astrocyte glutamate

transporter (GLAST) . Nfib is critical for glial

differentiation during development in vivo in mouse brain regions such as the hippocampus and the cerebral cortical midline (Steele-Perkins et al . , 2005). In Nfib knockout mice, progenitor cells accumulate in the ventricular zone and fail to move into the subventricular zone.

Consequently, these cells do not complete their

differentiation into mature glial cells. International Publication WO 2009/039442 describes methods and compositions for glioma therapy and treatment and, in particular, for the treatment of astrocytomas. The methods disclosed involves inhibition of expression product of nuclear factor lA{Nfia) . Inhibition can take place by inhibiting the expression of Nfia, for example, by inhibiting transcription, translation or processing of Nfia or by inhibiting a functional activity of the expression product, such as DNA binding. Still further the inhibitors include small molecule inhibitors of the protein. In some embodiments the inhibitor can be an antisense polynucleotide such as a short hairpin RNA (shRNA) targeted to Nfia. In other embodiments the inhibitor can be an antibody specific to the expressed protein.

Summary of the Invention

The present invention is based on the observation that expression of Nuclear Factor IB (Nfib) is reduced in glioma cells compared to normal brain cells. Thus the molecular pathway involving Nfib provides a new

therapeutic target for glioma.

Accordingly in a first aspect there is provided a method of identifying an agent for the treatment of glioma, comprising contacting a glioma cell with a candidate agent and establishing whether Nfib is

upregulated.

In an embodiment the glioma cell is derived from high grade glioma clinical specimens.

In an embodiment the glioma cell is derived from a glioma cell line grown under standard serum-containing culture conditions. In an embodiment the glioma cells are derived from a glioma cell line established using neurosphere culture medium.

In an embodiment the glioma cells are grown as a dedifferentiated monolayer of cells on a basement membrane of laminin.

In an embodiment the glioma cell is derived from a glioma cell line established as a xenograft.

In an embodiment the method further comprises the step of down-regulating expression of Nfib and

ascertaining whether down-regulation impairs therapeutic activity of said candidate agent. In an embodiment of the invention there is provided a method wherein said down- regulation is induced by shRNA.

In an embodiment a reporter gene is present in the genome of the glioma cell. The reporter gene may be introduced through the use of a reporter gene construct. The reporter gene construct may comprises two reporter genes separated by an insulator or nuclear matrix

attachment sequence, the first reporter gene being preceded by a plurality of Nfi binding sites (an Nfi binding site is represented by the sequence TTGGCN₅GCCAA) fused to a minimal promoter sequence and the second reporter gene being preceded by the same minimal promoter DNA sequence. Advantageously the first reporter gene is preceded by three tandem Nfi binding sites fused to the minimal promoter DNA sequence. In an embodiment the minimal promoter sequence is the human glial fibrillary acidic protein (GFAP) genomic sequence extending from the TATA box to the translation start site sequence.

In an embodiment the two reporter genes are

separated by a nuclear scaffold/matrix attachment region (S/MAR) sequence, advantageously a human interferon beta S/MAR sequence . In an embodiment the reporter gene construct includes an antibiotic selection cassette so that

incorporation of the reporter gene(s) can be verified by antibiotic selection.

In a further aspect the present invention provides a method for the treatment of glioma comprising introducing or increasing Nfib activity in a patient in need of such treatment .

In an embodiment the method comprises introducing Nfib into the glial elements of the central nervous system in a patient in need of such treatment.

In an embodiment Nfib is modified by the addition of a cell permeable peptide sequence. Typically the cell permeable peptide sequence is a highly charged positive peptide, for example, a polyarginine sequence

(particularly an (R) ₉_₁₃ sequence, most particularly an (R) n sequence) . Other cell permeable peptides include the antennapedia peptide (RQIKIWFQNRRMKWKK) , the HIV Tat peptide (GRKKRRQRRRPPQ) , FGF signal peptide

(AAVLLFAVLLALLAP) , Arg/Trp analogue (RRWRRWWRRWWRRWRR) and further cell permeable peptides as described, for example, in EP1849798 and WO03/057725, the contents of which are incorporated herein by reference .

In an embodiment Nfib activity is introduced or increased by inducing expression or over expression of Nfib

Typically the glioma is high-grade glioma,

particularly GBM and most particularly mesenchymal GBM.

In a still further aspect the present invention provides a method of inducing differentiation of cancer stem cells comprising introducing or increasing Nfib activity in a patient in in a tissue comprising cancer stem cells. Nfib activity may be introduced or increased, for example, by inducing expression or overexpression of Nfib or administering Nfib to a tissue comprising said cancer stem cells.

In yet another aspect there is provided a method for distinguishing GBM subtypes comprising measuring Nfib mRNA expression in a patient, whereby a patient with a two- fold or greater decrease in Nfib expression is characterised as having mesenchymal GBM. Thus treatment may be adjusted as appropriate to the specific subtype.

Brief Description of the Figures

Figure 1: Nfib expression is down-regulated

in GBM (A) qPCR analysis of Nfib expression in normal brain {n=ll) vs primary high-grade glioma clinical specimens {n=31; p<0.05} and GBM cell lines (n=9;

p<0.0005). (B) Nfib expression in GBM by 1HC. (C) Nfib expression and GBM subtype. (D) Increased Nfib expression correlates strongly with improved patient survival in glioma, p=0.0168. (Data in (D) obtained from the Human Rembrandt Database) .

Figure 2: Microarray analysis data demonstrating that Nfib expression is downregulated in GBM (Data obtained from the Oncomine Database) .

Figure 3: Effect of tamoxifen- inducible expression of Nfib in GBM cell lines: (A) Lentiviral vectors for 4- hydroxytamoxifen (4HT) -inducible expression of Nfib. (B) Induction of Nfib in U87 and U251 cells with 100 nM 4HT. (C) MTS assays showing reduced proliferation in Nfib- expressing U87 and U251 cells. (D) U87 and U251 cell morphology after 7 days expression of Nfib.

Figure 4: Nfib overexpression prevents GBM

neurosphere formation. Overexpression of Nfib in (A) U87 and (B) primary, patient-derived GBM cells prevented neurosphere formation under neurosphere culture

conditions compared to WT and vector control (pCAGIG) transfected cells. Middle panel indicates relative transcription/translation efficiency of the cDNA-IRES- eGFP

cassette from the stably transfected pCAGIG plasmxd in the U87 vector control- and Nfib-transfected lines.

Figure 5: Nfib overexpression induces glioma

differentiation and reduces proliferation. Overexpression of Nfib in U87 cells increases glial (GFAP) and neuronal (βΙΙΙ tubulin) marker staining by (A) immunoblot and (B) FACS analysis. (C) Nfib overexpression reduces

proliferation in U87 cells.

Figure 6: Constitutive expression of Nfib in U87 and U251 GBM cell lines.

Figure 7: Effect of Nfib expression on glioblastoma tumorigenicity. (A) & (C) Survival curves for mice injected with Nfib-transfected or vector control

transfected U87 & U251 cells. (B) Growth of U87 Nfib transfected tumours versus vector control tumours.

Figure 8 : DNA Sequence of Nfib gene .

Figure 9: Induction of Nfib expression by

chemotherapeutic agents. Expression of Nfib measured by qPCR for U87 cells treated for 24 hours with the

indicated drugs at lOuM.

Detailed Description of the Invention

The present invention is based on the observation that nuclear factor lb (Nfib) is down-regulated in human glioma tissue samples. Further, overexpression of Nfib reduces the proliferation of glioma cells and induces differentiation in vitro. Moreover, transplantation of Nfib overexpressing cells into a mouse xenograft model was found to reduce or even prevent the growth of gliomas in vivo compared to vector control transfected cells. The sequence of human Nfib is publicly available in the Genbank database and can be found under Genbank

Accession No. NM_005596.2 (Fig. 8). The invention encompasses introducing or increasing Nfib activity by introducing Nfib itself or variants thereof with Nfib activity. Thus the present invention encompasses the use of a nucleic acid molecule that is at least 70% identical to the Nfib gene and encodes a protein with Nfib

activity, or a functional fragment thereof. Such variants will have preferably at least about 85%, and most

preferably at least about 95% sequence identity to Nfib. Sequence identity is typically calculated using the BLAST algorithm, described in Altschul et al (1997) with the BLOSHM62 default matrix.

The nucleotide sequences can be engineered using methods accepted in the art so as to alter Nfib sequences for a variety of purposes. These include, but are not limited to, modification of the cloning, processing, and/or expression of the gene product. PCR reassembly of gene fragments and the use of synthetic oligonucleotides allow the engineering of gene nucleotide sequences. For example, oligonucleotide-mediated site-directed

mutagenesis can introduce mutations that create new restriction sites, alter glycosylation patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding the genes of the invention, some that may have minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention includes each and every possible variation of

polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of the naturally occurring gene, and all such variations are to be considered as being specifically disclosed.

The polynucleotides of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified, as will be appreciated by those skilled in the art. Such modifications include labels, methylation, intercalators , alkylators and modified linkages. In some instances it may be

advantageous to produce nucleotide sequences encoding Nfib or its derivatives possessing a substantially different codon usage than that of the naturally

occurring gene. For example, codons may be selected to increase the rate of expression of the peptide in a particular prokaryotic or eukaryotic host corresponding with the frequency that particular codons are utilized by the host. Other reasons to alter the nucleotide sequence encoding a gene or its derivatives without altering the encoded amino acid sequence include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of the nucleic acid molecules entirely by synthetic chemistry. Synthetic sequences may be inserted into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements may include regulatory sequences, promoters, 5' and 3' untranslated regions and specific initiation signals (such as an ATG initiation codon and Kozak consensus sequence) which allow more efficient ion of sequences encoding Nfib or its

derivatives. In cases where the complete coding sequence including its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, additional control signals may not be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals as described above should be provided by the vector. Such signals may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf et al., 1994) .

Nucleic acid molecules that are complements of the sequences described herein may also be prepared.

The present invention allows for the preparation of purified polypeptides or proteins. In order to do this, host cells may be transfected with a nucleic acid

molecule as described above. Typically, said host cells are transfected with an expression vector comprising a nucleic acid molecule according to the invention. A variety of expression vector/host systems may be utilized to contain and express the sequences. These include, but are not limited to, microorgan sms such as bacteria transformed with plasmid or cosmid DNA expression

vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus) ; or mouse or other animal or human tissue cell systems. Mammalian cells can also be used to express a protein using various expression vectors including plasmid, cosmid and viral systems such as a vaccinia virus expression system.

The polynucleotide sequences, or variants thereof, of the present invention can be stably expressed in cell lines to allow long term production of recombinant proteins in mammalian systems. Sequences encoding Nfib or its derivatives can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. The selectable marker confers resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the

introduced sequences. Resistant clones of stably

transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode a protein may be designed to contain signal sequences which direct secretion of the protein through a prokaryotic or eukaryotic cell

membrane .

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation,

glycosylation, phosphorylation, and acylation. Post- translational cleavage of a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells having specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO or HeLa cells), are available from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct

modification and processing of the foreign protein. When large quantities of protein are needed, vectors which direct high levels of expression may be used such as those containing the T5 or T7 inducible bacteriophage promoter. The present invention also includes the use of the expression systems described above in generating and isolating fusion proteins which contain important

functional domains of the protein. These fusion proteins are used for binding, structural and functional studies as well as for the generation of appropriate antibodies.

In order to express and purify the protein as a fusion protein, the appropriate polynucleotide sequences of the present invention are inserted into a vector which contains a nucleotide sequence encoding another peptide (for example, glutathionine succinyl transferase) . The fusion protein is expressed and recovered from

prokaryotic or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the relevant protein can subsequently be obtained by enzymatic cleavage of the fusion protein.

Fragments of polypeptides of the present invention may also be produced by direct peptide synthesis using solid-phase techniques. Automated synthesis may be achieved by using the ABI 431A Peptide Synthesizer

(Perkin-Elmer) . Various fragments of polypeptide may be synthesized separately and then combined to produce the full length molecule.

The invention also encompasses the use an isolated polypeptide having at least 70%, preferably 85%, and more preferably 95%, identity to the Nfib protein, and active fragments thereof.

Sequence identity is typically calculated using the BLAST algorithm, described in Altschul et al (1997) with the BLOSUM62 default matrix. Substantially purified protein or fragments thereof can then be used in further biochemical analyses to establish secondary and tertiary structure for example by x-ray crystallography of the protein or by nuclear magnetic resonance (NMR) . Determination of structure allows for the rational design of pharmaceuticals to interact with the protein, alter protein charge

configuration or charge interaction with other proteins, or to alter its function in the cell.

Therapies may be designed to circumvent or overcome a gene defect or inadequate gene expression, and thus moderate and possibly prevent malignancy. In considering various therapies, however, it is understood that such therapies may be targeted at other malignant tissues demonstrated to have inadequate expression of Nfib.

Treatment or prevention of glioma can be accomplished by delivering normal Nfib protein to the appropriate cells. Once the biological pathway involving the Nfib protein has been completely elucidated and understood, it may also be possible to modify the pathophysiologic pathway in which the protein participates in order to correct the physiological defect.

To replace a mutant protein with normal protein, or to add protein to cells which do not express or

underexpress a gene, it is desirable to obtain large amounts of pure protein from cultured cell systems which can express the protein. Delivery of the protein to the affected cells and tissues can then be accomplished using appropriate packaging or administration systems.

In an embodiment a method is provided for directly increasing the level of intracellular Nfib. This involves contacting cells with a modified form of Nfib designed to allow its direct uptake by the contacted cell. The modification to Nfib is the addition of a cell permeable peptide sequence (or protein transduction domain) to the amino-terminus of the mature protein. In an embodiment the cell permeable peptide sequence is a polyarginine peptide (Argn) , although a person skilled in the art will understand that other cell permeable peptide sequences may be used. In an embodiment the cell

permeable peptide-modified Nfib is produced by cloning DNA encoding Argn in-frame with the coding DNA sequence for Nfib in an expression vector such as pCAGIG, although a person skilled in the art will understand that other expression vectors may be used. The vector specifying Met-Argxj-Nfib or Nfib-Argn is introduced, using standard procedures, into a eukaryotic cell line, such as CHO cells, although a person skilled in the art will

understand that other eukaryotic or bacterial cells may be used. Expressed Met-Arg_13.-Nfib or Nfib-Argn protein is purified using standard procedures, such as antibody- mediated affinity column chromatography, although a person skilled in the art will understand that other purification procedures may be used. In an embodiment Met-Argii-Nfib or Nfib-Argn is added directly to GBM cells in culture to evaluate its efficacy in inducing

differentiation and inhibiting cell growth. In an embodiment Met-Argn-Nfib or Nfib-Argn is implanted intracerebrally as a controlled release formulation, such as a wafer preparation, although a person skilled in the art will understand that other formulations may be used, following resection of a high grade glioma tumour with the intent of inducing the differentiation, or inhibiting the growth, of residual tumour cells.

Alternatively, small molecule analogs may be used and administered to act as Nfib agonists and in this manner produce a desired physiological effect. In order to screen for analogues, one can design functional screens based on the sequence of Nfib. One can also fuse Nfib to heterologous DNA binding proteins to design screens for agonists. Since Nfib functions by interacting with other proteins, yeast screens can be used for small molecules that may interact by promoting or disrupting Nfib binding with other proteins.

A clone of Nfib expressed as a fusion protein can be utilized to identify small peptides that bind to Nfib. In one approach, termed phage display, random peptides (up to 20 amino acids long) are expressed with coat proteins (genelll or geneVIII) of filamentous phage such that they are expressed on the surface of the phage thus generating a library of phage that express random sequences. A library of these random sequences is then selected by incubating the library with the Nfib protein or fragments thereof and phage that bind to the protein are then eluted either by cleavage of Nfib from the support matrix or by elution using an excess concentration of soluble Nfib protein or fragments. The eluted phage are then repropagated and the selection repeated many times to enrich for higher affinity interactions. The random peptides can either by completely random or constrained at certain positions through the introduction of specific residues. After several rounds of selection, the final positive phages are sequenced to determine the sequence of the peptide .

An alternative approach uses the yeast two hybrid system to identify binding partners for Nfib. Nfib or fragments are expressed in yeast as a fusion to a DNA binding domain. This fusion protein is capable of binding to target promoter elements in genes that have been engineered into the yeast. These promoters drive

expression of specific reporter genes (typically the auxotrophic marker HIS3 and the enzyme β-galactosidase) . A library of cDNAs can then be constructed from any tissue or cell line and fused to a transcriptional activation domain. Transcription of HIS3 and β- galactosidase depends on association of the Nfib fusion protein (which contains the DNA binding domain) and the target protein (which carries the activation domain) . Yeast survival on specific growth media lacking histidine requires this interaction. This approach allows for the identification of specific proteins that interact with Nfib. The approach has also been adapted to identify small peptides. Nfib, or its fragments, are fused with the DNA binding domain and are screened with a library of random peptides or peptides which are constrained at specific positions linked to a transcriptional activation domain. Interaction is detected by growth of the

interacting peptides on media lacking histidine and by detection of β-galactosidase activity using standard techniques .

The identification of proteins or small peptides that interact with Nfib can provide the basis for the design of small peptide agonists of Nfib function.

Further, the structure of these peptides determined by standard techniques such as protein NMR or X-ray

crystallography can provide the structural basis for the design of small molecule drugs .

Gene therapy is another potential therapeutic approach in which normal copies of the Nfib gene are introduced into selected tissues to successfully code for normal protein in affected cell types. It is to be understood that gene therapy techniques can only begin once a malignant genotype/phenotype has been identified. The gene must be delivered to affected cells in a form in which it can be taken up and can code for sufficient protein to provide effective function. A vector capable of expressing Nfib or a functional fragment or derivative thereof may be administered to a subject to treat glioma. Transducing retroviral vectors are often used for somatic cell gene therapy because of their high efficiency of infection and stable integration and expression. The full length gene, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the

retroviral long terminal repeat or from a promoter specific for the target cell type of interest. Other viral vectors can be used and include, as is known in the art, adenoviruses, adeno-associated virus, vaccinia virus, papovaviruses, lentiviruses and retroviruses of avian, murine and human origin.

Gene therapy would be carried out according to established methods. A vector containing a copy of the Nfib gene linked to expression control elements and capable of replicating inside the cells is prepared.

Alternatively the vector may be replication deficient and may require helper cells or helper virus for replication and virus production and use in gene therapy.

Gene transfer using non-viral methods of infection can also be used. These methods include direct injection of DNA, uptake of naked DNA in the presence of calcium phosphate, electroporation, protoplast fusion or liposome delivery. Gene transfer can also be achieved by delivery as a part of a human artificial chromosome or receptor- mediated gene transfer. This involves linking the DNA to a targeting molecule that will bind to specific cell- surface receptors to induce endocytosis and transfer of the DNA into mammalian cells. One such technique uses poly-L-lysine to link a sialoglycoprotein to DNA. An adenovirus is also added to the complex to disrupt the lysosomes and thus allow the DNA to avoid degradation and move to the nucleus.

Transplantation of normal genes into the affected cells of a patient with a malignancy can also be useful therapy, especially if the malignancy is diffuse and cannot be excised or if the tissue affected has been substantially transformed into a malignant phenotype and due to its function in the body, cannot be surgically removed. In this procedure, normal Nfib is transferred into a cultivatable cell type, either exogenously or endogenously to a patient. These cells are then

transplanted into the targeted tissue (s).

Antisense-based strategies can be employed to explore Nfib gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence- specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary antisense species. The formation of a hybrid RNA duplex may then interfere with the

processing/transport/translation and/or stability of the target Nfib mRNA. Antisense strategies may use a variety of approaches including the use of antisense

oligonucleotides, injection of antisense RNA and

transfection of antisense RNA expression vectors.

Antisense effects can be induced by control (sense) sequences; the extent of phenotypic changes, however, are highly variable. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels .

Cell lines which underexpress Nfib may be cultured, and a test compound is added to the culture medium. After a period of incubation, the expression of Nfib mRNA and resultant protein product can be quantified to determine any changes in expression as a result of the test

compound. Alternatively some other physical parameter such the rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of defective cells. High throughput screening techniques may be employed.

Compounds that can be screened in accordance with the invention include, but are not limited to peptides (such as soluble peptides) , phosphopeptides and small organic or inorganic molecules (such as natural product or synthetic chemical libraries and peptidomimetics) . The methods of the present invention may also be used for screening compounds developed as a result of

combinatorial library technology. This provides a way to test a large number of different substances for their ability to modulate activity of a polypeptide. The use of peptide libraries is preferred (see WO 97/02048) with such libraries and their use known in the art.

A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non- peptide "small molecules" are often preferred for many in vivo pharmaceutical applications. In addition, a mimic or mimetic of the substance may be designed for

pharmaceutical use. The design of mimetics based on a known pharmaceutically active compound ("lead" compound) is a common approach to the development of novel

pharmaceuticals. This is often desirable where the original active compound is difficult or expensive to synthesise or where it provides an unsuitable method of administration. In the design of a mimetic, particular parts of the original active compound that are important in determining the target property are identified. These parts or residues constituting the active region of the compound are known as its pharmacophore. Once found, the pharmacophore structure is modelled according to its physical properties using data from a range of sources including X-ray diffraction data and NMR. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be added. The selection can be made such that the mimetic is easy to synthesxse, is likely to be pharmacologically acceptable, does not degrade in vivo and retains the biological activity of the lead compound. Further optimisation or modification can be carried out to select one or more final mimetics useful for in vivo or clinical testing.

It is also possible to isolate a target- specific antibody and then solve its crystal structure. In

principle, this approach yields a pharmacophore upon which subsequent drug design can be based as described above. It may be possible to avoid protein

crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analogue of the original binding site. The anti -id could then be used to isolate peptides from chemically or biologically produced peptide banks.

Another alternative method for drug screening relies on structure-based rational drug design. Determination of the three dimensional structure, for example, of the a protein which binds Nfib may allow for structure-based drug design to identify biologically active lead

compounds .

Three dimensional structural models can be generated by a number of applications, some of which include experimental models such as x-ray crystallography and NMR and/or from in silico studies using information from structural databases such as the Protein Databank (PDB) . In addition, three dimensional structural models can be determined using a number of known protein structure prediction techniques based on the primary sequences of the polypeptides (e.g. SYBYL - Tripos Associated, St. Louis, MO) , de novo protein structure design programs (e.g. MODELER - MSI Inc., San Diego, CA, or MOE - Chemical Computing Group, Montreal, Canada) or ab initio methods (e.g. see US Patent Numbers 5331573 and 5579250) .

Once the three dimensional structure of a

polypeptide or polypeptide complex has been determined, structure-based drug discovery techniques can be employed to design biologically-active compounds based on these three dimensional structures. Such techniques are known in the art and include examples such as DOCK (University of California, San Francisco) or AUTODOCK (Scripps

Research Institute, La Jolla, California) . A

computational docking protocol will identify the active site or sites that are deemed important for protein activity based on a predicted protein model. Molecular databases, such as the Available Chemicals Directory (ACD) are then screened for molecules that complement the protein model .

Using methods such as these, potential clinical drug candidates can be identified and computationally ranked in order to reduce the time and expense associated with typical *wet lab' drug screening methodologies.

Compounds identified from the screening methods described above form a part of the present invention, as do pharmaceutical compositions containing these and a pharmaceutically acceptable carrier.

Thus the present invention provides a cell -based screening method for identifying a pharmaceutical agent which is capable of inducing expression of or up- regulating Nfib. In embodiments of the invention up- regulation of Nfib may be detected through measurement of the protein expressed or measurement of mRNA. Techniques for measuring levels of protein expression and mRNA are well known to the person skilled in the art. In an embodiment immunoblotting or qPCR techniques are

employed. The cells employed in the screen may be GBM cells, including GBM cell lines U87 and U251, or patient derived cells. In an embodiment the glioma cells are derived from high grade glioma clinical specimens. In a further embodiment the glioma cells are grown under standard serum-containing culture conditions. In an alternative embodiment the glioma cells are established using neurosphere culture medium. In a still further embodiment the glioma cells are grown as a dedifferentiated monolayer of cells on a basement layer membrane of laminin in a culture termed "glioma neural stem culture" . In a still further embodiment the glioma cell line may be maintained as a xenograft.

In an embodiment a high- throughput assay is

provided, to more comprehensively screen for inducers of Nfib. This involves a targeting vector in which Nfib is fused to a reporter gene. In an embodiment the reporter gene is the green fluorescent protein (GFP) reporter gene, although the person skilled in the art will understand that other reporter genes such as luciferase may be used. Antibiotic-resistant colonies arising from cells

transfected with the reporter targeting vector are screened by PCR or Southern blot to identify

transfectants in which successful targeting of the Nfib locus has occurred. Confirmatory genomic sequencing is performed to confirm successful integration of the reporter gene. This Nfib reporter cell line is then able to be used to screen candidate agents by contacting a candidate agent with cells and establishing the extent of up-regulation of Nfib by measuring the degree of expression of the reporter gene. By way of example, where a Nfib - GFP fusion protein is employed, this is done using standard procedures for measuring fluorescence when the protein is exposed to blue light.

In an embodiment a second high-throughput assay is provided, to screen for chemical or other inducers of Nfib. This involves non-targeted integration into the genome of GBM or other eukaryotic cells two reporter genes separated by an insulator or nuclear matrix

attachment sequence, along with an antibiotic selection cassette. One reporter gene is preceded by three tandem Nfi binding sites (TTGGCN₅GCCAA) fused to a minimal promoter DNA sequence . In an embodiment the reporter gene is the Gaussia secreted luciferase gene, although a person skilled in the art will understand that other reporter genes may be used. In an embodiment the minimal promoter DNA sequence is the human glial fibrillary acidic protein (GFAP) genomic sequence extending from the TATA box to the translation start site sequence, although a person skilled in the art will understand that other promoter DNA sequences may be used. To discriminate Nfi binding site-mediated from non-Nfi binding site-mediated reporter gene induction, the second reporter gene is preceded by the same minimal promoter DNA sequence as the first reporter gene, minus the tandem Nfi binding sites. In an embodiment the second reporter gene is the

Cypridina secreted luciferase gene, although a person skilled in the art will understand that other reporter genes may be used. To help ensure transcription from the second reporter gene is not influenced by the presence of the distal tandem Nfi binding sites, the two reporter gene cassettes are separated by a nuclear scaffold/matrix attachment region (S/MAR) sequence. In an embodiment the S/MAR sequence is the human interferon-β S/MAR, although a person skilled in the art will understand that other S/MAR DNA sequences may be used. The antibiotic

selection cassette is incorporated to identify successful genomic integration of the reporter gene sequences. In an embodiment the antibiotic selection cassette is the puromycin N-acetyl transferase gene expressed from the SV40 promoter, although a person skilled in the art will understand that other antibiotic selection cassette sequences may be used. Cells in which successful

integration of the reporter genes has occurred are identified by their continued growth in culture medium containing puromycin. Such Nfib reporter cell lines are used to screen candidate agents by contacting a candidate agent with the cells and, after a suitable time interval, measuring the activity of the two reporter genes. By way of example, where secreted luciferase genes are employed as the two reporter genes, Gaussia and Cypridina

luciferase activity is measured sequentially using standard procedures for measuring luminescence when different substrates are added to the culture medium in which the cells are grown. Agents which induce Nfib expression are identified by an increase in the activity of the reporter gene associated with the tandem Nfi binding sites, relative to the activity of second

reporter gene. This system, by virtue of its design, will detect increases in all four members of the Nfi family, each of which can bind Nfi sites as both homo- and hetero-dimers . Which members are induced in response to treatment of cells with candidate agents can be determined by using standard procedures, such as

immunoblotting, although a person skilled in the art will understand that other detection procedures may be used. Pharmaceutical agents such as those identified by the screening assay may be administered in a variety of physical forms depending on the mode of action. The routes of administration include topical, enteral and parenteral. Topical administration includes epicutaneous application {i.e. application onto the skin), inhalation, enema, eyedrops, eardrops and intranasal administration. Enteral administration is form of administration that involves any part of the gastrointestinal tract. This includes administration by mouth (orally) including administration in the form of tablets, capsules or drops. Enteral administration can also include administration through a gastric feeding tube or otherwise directly into the gastrointestinal tract, or rectally. Parenteral administration includes intravenous administration, intramuscular injection intracerebral administration, such as by direct injection into the brain, and the like. Other forms of administration of pharmaceutical agents include vaginal administration, transdermal

administration and the like.

In an embodiment anti -cancer drugs such lapatanib and ATRA are administered by positioning of one or more wafers into which the drugs have been absorbed into a cavity created by surgical removal of a glioma.

The person skilled in the art will understand that pharmaceutical agents often include chiral centres.

Where chiral centres are present one enantiomer may have greater activity than the other or than the racemic mixture. Reference to pharmaceutical agents herein includes their administration as any one enantiomer or mixture of enantiomers, including a racemic mixture.

Further, the person skilled in the art will understand that pharmaceutical agents are often administered in the form of salts or derivatives such as esters, and reference to pharmaceutical agents herein includes functionally equivalent derivatives such as salts and esters .

Generally, the terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease.

"Treating" as used herein covers any treatment of, or prevention of disease in a vertebrate, a mammal,

particularly a human, and includes: preventing the disease from occurring in a subject that may be

predisposed to the disease, but has not yet been

diagnosed as having it; inhibiting the disease, ie., arresting its development; or relieving or ameliorating the effects of the disease, ie., cause regression of the effects of the disease.

Pharmaceutical compositions are often prepared and administered in dosage units. Solid dosage units include tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.

Frequently used carriers or auxiliaries for solid formulations include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers . Preservatives include antimicrobial, anti-oxidants , chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405- 1412,1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed. ) .

Formulations for oral use may also be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension. Such excipients may be suspending agents such as sodium carboxymethyl

cellulose, methyl cellulose,

hydroxypropylmethylcellulose, sodium alginate,

polyvinylpyrrolidone, gum tragacanth and gum acacia;

dispersing or wetting agents, which may be (a) naturally occurring phosphatide such as lecithin; (b) a

condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example,

heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol

monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate .

Pharmaceutical agents may be formulated as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and

suspending agents such as those mentioned above. The sterile injectable preparation may also a sterile

injectable solution or suspension in a non- toxic

parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3 -butanediol . Among the acceptable vehicles and solvents which may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides . In addition, fatty acids such as oleic acid find use in the preparation of

injectables .

Pharmaceutical agents may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Dosage levels of the pharmaceutical agents will usually be of the order of about 0.5mg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.5mg to about lOmg per kilogram body weight per day (from about 0.5g to about 3g per patient per day) . The amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 5mg to lg of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 5mg to 500mg of active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of

administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In addition, some pharmaceutical agents may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the

invention.

Modes for Performing the Invention

The following examples illustrate embodiments of the invention. Example 1

Nfib expression is lower in high-grade glioma and GBM cell lines compared to normal brain

To investigate whether Nfib was involved in high- grade glioma we first compared the expression of Nfib in high-grade glioma versus normal human brain. Quantitative PCR (qPCR) was used to measure Nfib mRNA levels in 11 normal human brain samples and 31 high-grade glioma specimens, as well as 9 GBM cell lines - five common GBM lines grown under standard serum- containing culture conditions and four GBM lines grown under GNS culture conditions. Nfib expression was found to be reduced relative to normal brain tissue (n=ll) {Figure 1A) .

Reduction of Nfib expression in GBM tumor tissue compared to adjacent normal brain tissue was confirmed by

immunohistochemistry {Figure IB) . Interrogation of the Oncomine database revealed Nfib to be among the top 3% of under-expressed genes in GBM (n=22) compared to normal brain (Lee et al . 2006) (Figure 2).

Because the qPCR data for Nfib expression in GBM tumors appeared to be stratified into downregulated, normal and upregulated clusters, the possibility that this might be correlated with GBM subtypes was

investigated. TCGA database GBM microarray data (TCGA) was interrogated for Nfib expression in those samples previously classified as belonging to either proneural, neural, classical or mesenchymal subtypes of GBM (Verhaak et al., 2010). Remarkably, a two-fold or greater reduction in Nfib mRNA expression in GBM relative to adjacent normal brain tissue (n=9) was almost exclusively associated with mesenchymal GBM (8/9) , while a two-fold or greater increase in Nfib expression (n=28) was strongly associated with proneural GBM (23/28) (Figure 1C) . Significantly, a two-fold or greater reduction in Nfib expression did not occur for any of the proneural GBMs (n=53) , while none of the mesenchymal GBMs (n=56) exhibited a two- fold or greater increase in Nfib

expression. Thus a two-fold change in Nfib mRNA

expression in GBM, relative to normal brain,

distinguished mesenchymal and proneural GBMs with

absolute specificity.

Even more importantly, when the probability of patient survival with GBM was investigated using

mxcroarray gene expression data available for 300 glioma patients from the Rembrandt database (The National Cancer Institute's Repository of Molecular Brain Neoplasia

Database (Rembrandt) (Madhaven et al . , 2009).), a twofold increase in Nfib mRNA expression, as determined microarray, was associated with improved overall survival for all glioma (n=297) (Figure ID) . Additionally this data shows that increased expression of Nfib was

associated with significantly improved survival for patients with astrocytoma, oligodendroglioma and GBM (Fig. 2) .

Model GBM cell lines, when cultured in medium containing serum, exhibit a gene expression profile most closely resembling mesenchymal GBM (Verhaak et al . , 2010) . To investigate whether Nfib expression may affect the phenotype of GBM, two model GBM cell lines (U87 and U251) were transduced with a dual lentiviral system

(Vince et al., 2008) for tamoxifen-inducible expression of Nfib (Figure 3) . In the absence of tamoxifen

induction, polyclonal populations of the two transduced GBM cell lines were found by qPCR to express Nfib at levels approximately two- to four- fold greater than the mean level of Nfib in normal brain.

Remarkably, this level of Nfib expression was associated with significant reduction in GBM proliferation (Figure 3C) . Furthermore, induction of Nfib by tamoxifen not only resulted in additional reduction of GBM proliferation (Figure 3C) , but after three days was followed by significant changes in GBM cell morphology and cell death (Figure 3D) . These changes were accompanied by the induction of glial differentiation markers, as measured by qPCR. Even more strikingly, when the transduced GBM cell lines were cultured in serum- free neurosphere medium, Nfib

expression was associated with reduced neurosphere formation, indicating that the stem cell fraction of these cell lines was also susceptible to Nfib activity.

Example 2

Nfib expression promotes differentiation and reduces proliferation in GBM

Based on the above findings we investigated whether over-expressing Nfib in human GBM could affect cellular proliferation and differentiation. We used a well characterised GBM cell line (U87) (Clark et al . ) and a primary neurosphere GBM line derived from a patient sample (L3) , and cultured both under neurosphere culture conditions. Strikingly, following Nfib overexpression, the sphere-forming ability of both GBM cell lines was dramatically reduced (Fig 4) . This observation was accompanied by significant morphological changes in the cells and increased cellular adherence. To investigate the induction of differentiation by Nfib we examined the expression of GFAP, a glial differentiation marker and known target of Nfib in normal tissues (Brun et al . , 2009) by western blot analysis and found this to be elevated (Fig. 5A) . Moreover, we observed an overall increase in both neuronal and glial differentiation markers by immunohistochemical fluorescence-activated cell sorting (FACS) analysis (Fig. 5A&B) . Differentiation was accompanied by a significant reduction in

proliferation of U87 cells following Nfib overexpression (Fig. 5C) , compared to vector-alone control U87 cells.

Example 3

Nfib expression suppresses tumour formation in a xenograft model of GBM

To investigate whether the activity of Nfib affected GBM tumorigenicity, we stably transfected U87 and U251 cells with a pCAGIG (Matsuda & Cepko, 2004) or pCAGIG Nfib plasmid and established polyclonal

populations of U87 and U251 cells that constitutively expressed Nfib or the control plasmid, in the absence of antibiotic selection, by successive rounds of FACS sorting for GFP expression (Figure 6A) . Similar to the lentiviral transductants , the Nfib-expressing U87 and U251 transfectants proliferated slower (Figure 6B) , expressed glial differentiation markers (Figure 6C) and failed to form neurospheres in neurosphere culture conditions (Figure 6D) . A reduction was observed in the number of slow cycling cells - candidate cancer stem or propagating cells (Pece et al., 2010 & Deleyrolle et al . , 2011) - in neurosphere culture, as visualised by PKH26 staining (Figure 7E) . After three rounds of FACS sorting for cells that actively proliferated despite

constitutively expressing Nfib, the striking changes in cell morphology and cell death that occurred with

tamoxifen-inducible Nfib expression in the lentiviral clones were no longer When 2x10⁶ U87 vector control cells were injected subcutaneously into five to six week old NOD/SCID mice, tumors were palpable from seven days post -injection and grew rapidly, reaching 1 cm in

diameter at a mean of 21 and 23.5 days post-injection in independent experiments, at which point mice were euthansed (Figure 7A and B) . In contrast, mice injected with Nfib-expressing U87 cells failed to form tumors or formed 3-4 mm tumors that failed to grow further (Figure 7A and B) , even after 100 days of observation. To investigate orthotopic tumor formation, 2x10^s vector control or Nfib-expressing, U87 or U251 cells were injected into the striatum of five to six week old

NOD/SCID mice. Mice were euthanased when they exhibited neurological signs or behavioral changes and their brains were examined for evidence of tumor formation. All mice that exhibited neurological signs or behavioral changes were found to have intracranial tumors. Importantly, mice injected with either U87 or U251 Nfib-expressing GBM cells remained well significantly longer than mice injected with the corresponding vector control cells (U87 median survival 84 vs 36 days; U251 median survival 130 vs 56 days) (Figure 7C) .

Example 4

Assay to identify upstream inducers of Nfib

As an initial screen we examined whether selected chemotherapeutic agents in various stages of

investigation as potential therapies for GBM (or other cancers) induce Nfib. In this assay cells from the GBM cell line U87 were treated for 24 hours with selected candidate pharmaceutical agents at a concentration of ΙΟμΜ. The level of expression of Nfib in the treated cells was measured by immunoblot or qPCR.

As a guide to what level of up-regulation of Nfib may be therapeutically significant, Rembrandt survival data suggest this will be greater than two- fold (Fig. 2) .

It will be noted from Fig. 9 that all-trans retinoic acid (ATRA) increases Nfib expression approximately two- fold in U87 cells when they are exposed to the pharmaceutical agent for 24 hours. Furthermore the combination of ATRA and the lapatinib increases Nfib expression approximately three- fold in the same cells after 24 hours of treatment. This result demonstrates proof-of-principle for this screening assay for detecting candidate agents for the treatment of glioma.

Industrial Applicability

The present invention provides for the treatment of glioma and the induction of differentiation in cancer stem cells as well as and for identification of

pharmaceutical agents.

References :

The contents of the following documents are

incorporated herein by reference:

A1HW {Australian Institute of Health and Welfare) & AACR (Australian Association of Cancer Registries) 2008. Cancer in Australia: an overview, 2008. Cancer series no. 46. Cat. no. CAN 42. Canberra: A1HW.

Alison MR, Lim SML, Nicholson LJ. Cancer stem cells: problems for therapy? J Pathol 2011; 223: 147-161.

Altschul, SF. et al . (1997). Nucleic Acids Res. 25:

3389-3402.

Aoki, T., N. Hashimoto, and M. Matsutani, Management of glioblastoma. Expert Opin Pharmacother, 2007. 8(18): p. 3133-46.

Bao, S., et al . , Glioma stem cells promote

radioresistance by preferential activation of the DNA damage response. Nature, 2006. 444(7120): p. 756-60.

Barry, G. , et al . , Specific glial populations regulate hippocampal morphogenesis. J Neurosci, 2008. 28 (47) : p. 12328-40.

Behin, A., et al . , Primary brain tumours in adults. Lancet, 2003. 361(9354): p. 323-31.

Berman, D.M., et al . , Medulloblastoma growth

inhibition by hedgehog pathway blockade. Science, 2002. 297 (5586) : p. 1559-61.

Blow, N. , Cell migration; our protruding knowledge. Nature Methods, 2007. 4(7): p. 589-594.

Brun, M. , et al . , Nuclear factor I regulates brain fatty acid-binding protein and glial fibrillary acidic protein gene expression in malignant glioma cell lines. J Mol Biol, 2009. 391(2): p. 282-300.

Carro, M.S., et al . , The transcriptional network for mesenchymal transformation of brain tumours. Nature, 2010. 463(7279): p. 318-25. Clark, M.J., et al . , U87MG decoded: the genomic sequence of a cytogenetically aberrant

human cancer cell line. PLoS Genet. 6(1): p. el000832.

Cole, Sp. Et al. (1984). Mol . Cell Biol. 62: 10-120.

Culver, K. (1996) . Gene Therapy : A Primer for

Physicians. Second Edition. (Mary Ann Liebert)

Deleyrolle, L.P. and B.A. Reynolds, Isolation, expansion, and differentiation of adult mammalian neural stem and progenitor cells using the neurosphere assay. Methods Mol Biol, 2009. 549: p. 91-101.

Deneen, B., et al . , The transcription factor NFIA controls the onset of gliogenesis in the developing spinal cord. Neuron, 2006. 52(6): p. 953-68.

Dick, J.E., Stem cell concepts renew cancer

research. Blood, 2008. 112(13): p. 4793-807.

Dxrks , P . B . , JBrain tumour stem cells: the

undercurrents of human brain cancer and their

relationship to neural stem cells. Philos Trans R Soc Lond B Biol Sci, 2008. 363(1489): p.139-52.

Furnari, F.B., et al., Malignant astrocytic glioma: genetics , biology, and paths to treatment.

Genes Dev, 2007. 21{21): p. 2683-710.

Galli, R. , et al . , Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res, 2004. 64(19): p. 7011-21.

Ginestier C, Wicinski J, Cervera N, et al . Retinoid signaling regulates breast cancer stem cell

differentiation. Cell Cycle 2009; 8: 3297-3302

Gronostajski, R.M., Roles of the NFI/CTF gene family in transcription and development. Gene, 2000. 249(1-2): p. 31-45.

Jain, M. , et al . , Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science, 2002. 297 (5578) : p. 102-4. Kaufman, W.L., et al . , Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation. Nucleic Acids Res, 2008. 36(17): p. elll.

Lee, H.K., et al . , GRP78 is overexpressed in

glioblastomas and regulates glioma cell growth and apoptosis. Neuro Oncol, 2008. 10(3): p. 236-43.

Madhavan, S., et al., Rembrandt : helping

personalized medicine become a reality through

integrative translational research. Mol Cancer Res, 2009. 7 (2) : p. 157-67.

Marumoto, T . , et al., Development of a novel mouse glioma model using lentiviral vectors. Nat Med, 2009. 15 (1) : p. 110-6.

Matsuda, T. and C.L. Cepko, Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A, 2004. 101(1): p. 16-22.

Namihira, M. , et al . , Committed neuronal precursors confer astrocytic potential on residual neural precursor cells. Dev Cell, 2009. 16(2): p. 245-55.

Pardal, R. , M.F. Clarke, and S.J. Morrison, Applying the principles of stem-cell biology to cancer. Nat Rev Cancer, 2003. 3(12): p. 895-902.

Pece, S., et al . , Biological and molecular

heterogeneity of breast cancers correlates with their cancer stem cell content. Cell. 140(1): p. 62-73.

Piccirillo, S.G., et al . , Bone morphogenetic

proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature, 2006. 444(7120): p. 761- 5.

Pollard, S.M., et al . , Glioma stem cell lines expanded in adherent culture have tumorspecific

phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell, 2009. 4(6): p. 568-80. Reya, T., et al.. Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p. 105-11.

Reynolds, B.A. and S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992. 255(5052): p.

1707-10.

Reynolds, B.A. and R.L. Rietze, Neural stem cells and neurospheres~~re~evaluating the relationship. Nat Methods, 2005. 2(5): p. 333-6.

Scharf, D. et al. (1994). Results Probl . Cell Differ. 20: 125-162.

Singh, S.K., et al . , Identification of human brain tumour initiating cells. Nature, 2004.432(7015): p. 396- 401.

Steele-Perkins, G., et al . , The transcription factor gene Nfib is essential for both lung maturation and brain development. Mol Cell Biol, 2005. 25(2): p. 685-98.

Verhaak, R.G., et al., .Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 17(1): p. 98-110.

Wang, Z.Y. and Z. Chen, Acute promyelocytic

leukemia: from highly fatal to highly curable. Blood, 2008. Ill (5) : p. 2505-15.

Wen, P.Y. and S. Kesari, Malignant gliomas in adults. N Engl J Med, 2008. 359(5): p. 492-507.

Claims

Claims:

1. A method of identifying an agent for the treatment of glioma, comprising contacting a glioma cell with a candidate agent and establishing whether Nfib is upregulated.

2. A method as claimed in claim 1 wherein the glioma cell is derived from high grade glioma clinical specimens .

3. A method as claimed in claim 1 wherein the glioma cell is derived from a glioma cell line grown under standard serum-containing culture

conditions .

4. A method as claimed in claim 1 wherein the glioma cell is derived from a glioma cell line

established using neurosphere culture medium.

5. A method as claimed in claim 1 wherein the glioma cells are grown as a de-differentiated monolayer of cells on a basement membrane of laminin.

6. A method as claimed in claim 1 wherein the glioma cell is derived from a glioma cell line

established as a xenograft.

7. A method for the treatment of glioma comprising introducing or increasing Nfib activity in a patient in need of such treatment.

8. A method as claimed in claim 7 comprising

introducing an Nfib peptide.

9. A method as claimed in claim 8 where said Nfib peptide comprises a cell permeable peptide sequence .

10. A method as claimed in claim 9 wherein the cell permeable peptide sequence is a polyarginine sequence .

11. A method as claimed in claim 7 comprising

inducing expression or overexpression of Nfib.

12. A method of inducing differentiation of cancer stem cells comprising introducing or increasing Nfib activity in a tissue comprising said cancer stem cells.

13. A method as claimed in claim 12 comprising

introducing an Nfib peptide.

14. A method as claimed in claim 13 where said Nfib peptide comprises a cell permeable peptide sequence .

15. A method as claimed in claim 14 wherein the cell permeable peptide sequence is a polyarginine sequence .

16. A method as claimed in claim 7 comprising

inducing expression or overexpression of Nfib.