WO2009143578A1

WO2009143578A1 - Cancer drug target and methods of diagnosis and therapy

Info

Publication number: WO2009143578A1
Application number: PCT/AU2009/000672
Authority: WO
Inventors: Andrew Wallace Boyd; Bryan Day; Brett Stringer
Original assignee: The Council Of The Queensland Institute Of Medical Research; The University Of Queensland
Priority date: 2008-05-28
Filing date: 2009-05-28
Publication date: 2009-12-03

Abstract

A single nucleotide G >A polymorphism at position -126 in the promoter of the human myeloid cell leukemia-1 gene is associated with a reduced predisposition to cancer and an increased responsiveness to pro-apoptotic cancer therapeutic agents. The G>;A polymorphism is in an ELK4 transcription factor binding site, which results in reduced myeloid cell leukemia-1 gene expression. Elevated levels of ELK4 and Mcl-1 protein and nucleic acids are associated with an increased predisposition to cancer and an decreased responsiveness to pro-apoptotic agents. Reduced levels of ELK4 and Mcl-1 protein and nucleic acids are associated with less of a predisposition to cancer and an increased responsiveness to pro-apoptotic cancer therapeutic agents. Methods of cancer diagnosis and/or therapy which target ELK4 and/or Mcl-1 protein and/or nucleic acids are particularly applicable to cancers such as glioma, colon cancer, lung cancer, thyroid cancer, kidney cancer and/or melanoma. Such methods and compositions may sensitize or improve responsiveness of cancer cells to pro-apoptotic agents.

Description

TITLE CANCER DRUG TARGET AND METHODS OF DIAGNOSIS AND THERAPY

FIELD OF THE INVENTION

THIS INVENTION relates to diagnosis and/or therapy of cancer. More particularly, this invention relates to proteins and/or nucleic acids for diagnosis and/or therapy of cancers, including but not limited to glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

BACKGROUND OF THE INVENTION Cancer is generally defined as any malignant growth or tumor caused by abnormal and uncontrolled cell division. There are virtually no cells, tissues or organs that are not susceptible to, or capable of, becoming cancerous or harbouring cancer cells. Thus cancers may be found in the lung, blood, bone marrow, kidney, skin, reproductive organs, endocrine organs and brain, although without limitation thereto. Glioblastoma multiforme (GBM) (World Health Organization grade 4 astrocytoma) is the most common malignant primary brain tumour. The mean onset age for GBM is 53 years. Treatment involves surgical resection (where possible), followed by radiation and chemotherapy (Behin et al., 2003). Therapy is almost never curative due in part to the widely infiltrative nature of these tumours and the intrinsic resistance of high grade glioma to radiation and cytotoxic chemotherapy. Even with optimal treatment including post-operative radiotherapy and adjuvant temozolomide chemotherapy, the median survival is less than 15 months and only about 10% of patients survive 2 years without disease recurrence (Stupp et al., 2005).

SUMMARY OF THE INVENTION

The present invention recognizes a need for improved diagnosis and/or therapy of glioma and/or other cancers.

Accordingly, the present invention is broadly directed to a myeloid cell leukemia- 1 (McI-I) promoter polymorphism, a McI-I protein and/or an ETS transcription factor which binds said promoter, for the diagnosis and/or therapy of cancer. One preferred object of the invention is to provide a method of designing, engineering, screening or otherwise producing a cancer therapeutic agent that is preferably useful in sensitizing, or improving the responsiveness of, cancer cells to a pro-apoptotic agent. In a first aspect, the invention provides a method of designing, engineering, screening or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly reduces or inhibits the activity of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter. Preferably, the ETS transcription factor is ELK4/Sap 1 a.

In a second aspect, the invention provides a method of designing, engineering, screening or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly reduces or inhibits the activity of a myeloid cell leukemia- 1 (McI-I) protein, or modulates transcription of an mRNA encoding said protein.

In a third aspect, the invention provides a cancer therapeutic agent designed, engineered, screened or otherwise produced according to the method of the preceding aspects.

In a fourth aspect, the invention provides a pharmaceutical composition comprising the cancer therapeutic agent of the third aspect and a pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, the pharmaceutical composition further comprises one or more pro-apoptotic cancer therapeutic agents.

In a fifth aspect, the invention provides a method of treating cancer in a mammal, said method including the step of at least partly reducing or inhibiting the activity and/or expression of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter in said mammal to thereby treat, sensitize or improve the responsiveness of said mammal to cancer treatment.

Preferably, the ETS transcription factor is ELK4/Sapla. In a sixth aspect, the invention provides a method of treating cancer in a mammal, said method including the step of at least partly reducing or inhibiting myeloid cell leukemia- 1 (McI-I) protein activity and/or expression, or transcription of an mRNA encoding said protein, in said mammal to thereby treat, sensitize or improve the responsiveness of said mammal to cancer treatment.

In one particular embodiment, the cancer treatment of the fifth aspects includes administration of one or more pro-apoptotic agents.

In a seventh aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence of at least a fragment of a myeloid cell leukemia- 1 (McI-I) gene promoter, which nucleotide sequence comprises a polymorphism in an ETS transcription factor binding site associated with reduced binding and/or activity of an ETS transcription factor.

Preferably, the polymorphism is a single nucleotide polymorphism (SNP).

In one embodiment, the SNP is located at position -126 relative to the major transcription start site of the myeloid cell leukemia- 1 (McI-I) promoter in an ETS transcription factor binding site. In a preferred form of this embodiment, the SNP is a G>A SNP.

Preferably, the ETS transcription factor is ELK4.

In an eighth aspect, the invention provides an isolated nucleoprotein complex comprising the isolated nucleic acid of the seventh aspect and an ETS transcription factor. In a ninth aspect, the invention provides a chimeric gene comprising the isolated nucleic acid of the first aspect operably linked or connected to an expressible nucleotide sequence.

In a tenth aspect, the invention provides a method of determining the susceptibility of a mammal to cancer, said method including the step of determining whether a myeloid cell leukemia- 1 (McI-I) gene promoter of said mammal comprises a nucleotide sequence polymorphism in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor, wherein said polymorphism indicates the susceptibility of said mammal to cancer

In a preferred embodiment, the polymorphism is a single nucleotide polymorphism (SNP) located at position -126 relative to the major transcription start site of the myeloid cell leukemia- 1 (McI-I) promoter in an ETS transcription factor binding site.

Preferably, an A at position -126 indicates that said mammal has a relatively lower or reduced susceptibility to cancer; and a G at position -126 indicates that said mammal has a relatively higher or increased susceptibility to cancer

In an eleventh aspect, the invention provides a method of determining responsiveness of a mammal to cancer therapy, said method including the step of determining whether a myeloid cell leukemia- 1 (McI-I) gene promoter of said mammal comprises a nucleotide sequence polymorphism in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor, wherein said polymorphism indicates responsiveness of said mammal to cancer therapy.

Preferably, (a) an A at position -126 indicates relatively increased or greater responsiveness of said mammal to cancer therapy; and (b) a G at position -126 indicates relatively decreased or lower responsiveness of said mammal to cancer therapy.

In a twelfth sixth aspect, the invention provides a method of determining the susceptibility of a mammal to cancer, said method including the step of determining a level of expression of a myeloid cell leukemia- 1 (McI-I) protein, or nucleic acid encoding said myeloid cell leukemia- 1 (McI-I) protein, in said mammal to thereby determine the susceptibility of said mammal to said cancer.

Preferably, (a) a relatively increased or higher level of expression of McI-I protein or encoding nucleic acid indicates a relatively higher or increased susceptibility to said cancer; and (b) a decreased or lower level of expression of McI-I protein or encoding nucleic acid indicates a relatively reduced or lower susceptibility to said cancer.

In a thirteenth aspect, the invention provides a method of determining responsiveness of a mammal to cancer therapy, said method including the step of determining a level of expression of a myeloid cell leukemia- 1 (McI-I) protein, or nucleic acid encoding said myeloid cell leukemia- 1 (McI-I) protein, in said mammal to thereby determine responsiveness of said mammal to cancer therapy.

Preferably, (a) a relatively increased or higher level of expression of McI-I protein or encoding nucleic acid indicates a relatively decreased or lower responsiveness of said mammal to cancer therapy; and (b) a relatively decreased or lower level of expression of McI-I protein or encoding nucleic acid indicates a relatively increased or greater responsiveness of said mammal to cancer therapy.

In one particular embodiment, cancer therapy includes treatment of said mammal with one or more pro-apoptotic cancer therapeutic agents. In a fourteenth aspect, the invention provides a method of determining the susceptibility of a mammal to cancer, said method including the step of determining a level of expression of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter, or a nucleic acid encoding said ETS transcription factor, in said mammal to thereby determine the susceptibility of said mammal to cancer. Preferably, (a) a relatively increased or higher level of expression of said ETS transcription factor indicates an increased susceptibility to cancer; and (b) a relatively decreased or lower level of expression of said ETS transcription factor indicates a relatively reduced or lower susceptibility to cancer.

In a fifteenth aspect, the invention provides a method of determining the responsiveness of a mammal to cancer therapy, said method including the step of determining a level of expression of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter, or a nucleic acid encoding said ETS transcription factor, in said mammal to thereby determine the responsiveness of said mammal to cancer therapy. Preferably, (a) a relatively increased or higher level of expression of said ETS transcription factor indicates a relatively decreased or lower responsiveness of said mammal to cancer therapy; and (b) a relatively decreased or lower level of expression of said ETS transcription factor indicates a relatively increased or greater responsiveness of said mammal to cancer therapy. In a sixteenth aspect, the invention provides a kit for cancer diagnosis, said kit comprising one or more probes, primers, antibodies or other reagents for detecting: (i) a myeloid cell leukemia- 1 (McI-I) protein or nucleic acid; (ii) an ELK4 protein or nucleic acid; and/or

(iii) at least a fragment of a myeloid cell leukemia- 1 (McI-I) promoter which may comprise a polymorphism in an ETS/ELK4 transcription factor binding site.

Preferably, according to the aforementioned aspects the mammal is a human. Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Bcl-2 family expression in high grade glioma A: Levels of mRNA encoding the anti-apoptotic proteins Bcl-2, Bcl-xL and McI-I was determined using q-PCR in 37 high grade glioma tissue samples. Mean of duplicate samples expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA levels.

B: Protein expression of the anti-apoptotic proteins McI-I, Bcl-2 and Bcl-xL was determined using western blotting in the GBM cell lines U87, Ul 18, D645 and U373. β-actin was used as a loading control.

C: Immunocytochemistry was performed to detect McI-I expression in the GBM cell lines U87, Ul 18, D645 and U373.

Figure 2. Identification of a novel, functional McI-I promoter SNP in high grade glioma

A: The McI-I promoter region depicting the insertion site of the 6bp or 18bp repeat, the -126G>A SNP site and ETS and SRE binding sites. The region cloned into the pGL2- Basic luciferase reporter vector is also indicated. The McI-I promoter region comprising -126G is designated SEQ ID NO:1. The McI-I promoter region comprising -126A is designated SEQ ID NO:2. B: High grade glioma cell line McI-I mRNA expression n=12. Mean of duplicate samples expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA levels. U373 and U251 are heterozygous for the novel - 126G>A SNP.

C: Luciferase reporter assays, WT promoter activity compared to the -126G>A SNP in the GBM cell lines U87, Ul 18, U251 and D645. Columns, mean of triplicate samples, +SD, (*=p<0.0\).

D: Electrophoretic mobility shift assay (EMSA) was conducted on four GBM nuclear extracts (Ul 18, U251, T46 and D645). Reduced binding potential of the -126G>A SNP probe to GBM nuclear extracts (arrowed). E: EMSA cold competition assay performed using Ul 18 nuclear extracts and the McI-I promoter ETS wild-type probe identified two specific complexes, competed out only in the presence of excess unlabelled wild-type probe (arrowed). The lowermost complex seen with the ETS SNP probe in (D) was not competed out by cold competition (data not shown).

Figure 3. ELK4 is the dominant ETS family member in high grade glioma and binds the McI-I promoter ETS site

A: ETS family member mRNA expression was measured in five high grade glioma tumour samples. ELK4 was identified as the dominant family member expressed in the tumour samples. Mean of duplicate samples expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA levels.

B: ETS family member mRNA expression was measured in five GBM cell lines. ELK4 was identified as the dominant family member expressed in GBM cell lines. Mean of duplicate samples expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA levels.

C: EMSA supershift assay was conducted to determine whether ELK4 bound the McI-I promoter ETS probe. GBM nuclear extracts for U87, Ul 18, U251 and D645 were investigated. Supershifted bands formed in the presence of a ELK4 polyclonal antibody (arrowed) for all four samples with corresponding reduction in the intensity of both arrowed bands in Figure 2E (lower band not shown). D: Chromatin immunoprecipitation assay (ChIP) using two ELK4-specific antibodies confirmed that ELK4 bound to the McI-I promoter in the Ul 18 GBM cell line. E: ChIP results for the Ul 18 GBM cell line were quantitated using q-PCR and show a ΔCt value of 4 between the ELK4 polyclonal antibody and the no antibody control.

Figure 4. ELK4 regulates McI-I expression in high grade glioma A: ELK4 and McI-I mRNA expression were measured by q-PCR in 38 GBM tissue samples. The expression pattern showed highly significant correlation (r=0.96) between ELK4 and McI-I expression levels. Mean of duplicate samples expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA levels.

B: ELK4 and McI-I expression was measured by q-PCR in three GBM cell lines. Two cell lines, U251 and U373, were heteroaygous for the -126G>A ETS SNP, while U87 was free of the SNP. U251 and U373, while expressing equivalent levels of ELK4, expressed significantly less McI-I compared to U87. C: ELK4 protein expression was down regulated using two specific siRNAs which map to different regions of the ELK4 gene. The U87 line was treated with the siRNAs for 24 hrs and McI-I and ELK4 protein expression determined by western blot. A significant decrease in ELK4 was observed for both sequences compared to the control. Following ELK4 down regulation ablation of the McI-I signal was observed. D: ELK4 cDNA was stably overexpressed in the Ul 18 GBM cell line. ELK4 and McI-I protein expression was determined by western blot. Increases in ELK4 expression were observed, resulting in a four fold increase in McI-I protein expression.

Figure 5. McI-I neutralisation induces apoptosis in high grade glioma A: The GBM cell lines U87, D645 and U373 were transfected with McI-I shRNA or control shRNA sequences. After 48 hours, transfected (eGFP^+ve) cells were selected by FACS and McI-I protein expression evaluated by western blotting, β-actin levels were used as a loading control and for densitometry analysis. U373 data not shown. B: Annexin V staining was conducted 48 hours post transfection in the GBM cell lines U87, D645 and U373 to determine the level of apoptosis when McI-I was down regulated compared to control transfected cells. C: ABT-737 (lμM) was applied to U87 and D645 cells 24 hours following McI-I down regulation. Annexin V staining was conducted 24 hours post ABT-737 (lμM) treatment to determine the level of apoptosis compared to control transfected cells. D: McI-I was inhibited using a NOXA peptide approach in the U87 GBM cell line (Chen et al., 2005). Annexin V staining was conducted 48 hours post treatment with NOXA peptide (lOμM) alone and in combination with ABT-737 (lμM) or cisplatin (lμM) and compared to control peptide (lOμM) treated cells. Columns, mean of triplicate samples, +SD, (*=/?<0.05).

Figure 6. ELK4 neutralisation induces increased sensitivity to ABT-737 and cisplatin treatment

A: The GBM cell lines U87, Ul 18, D645, U373 and U251 (the latter two being heterozygous for the McI-I promoter -126G>A ETS SNP) were treated with ABT-737 (lμM), cisplatin (lμM) or ABT-737/cisplatin (lμM) combined. Annexin V staining was conducted 72 hours post treatment to determine the level of apoptosis compared to control cells. Columns, mean of triplicate samples, +SD, (*=p<0.0l). B: U87 stable polyclonal ELK4 shRNA knockdown cells were treated with ABT-737 (lμM), cisplatin (lμM) or ABT-737/cisplatin (lμM) combined. Annexin V staining was conducted 48 hours post treatment to determine the level of apoptosis compared to control cells. Columns, mean of triplicate samples, +SD, (*=p<0.05).

C: Ul 18 GBM cells stably overexpressing ELK4 were treated with cisplatin (lOμM). Annexin V staining was conducted 48 hours post treatment to determine the level of apoptosis compared to control cells. Columns, mean of triplicate samples, +SD, (*=p<0.05).

Figure 7. GBM cultures exhibit lower Bcl-2 family expression when grown as neurospheres compared to serum differentiated cells

A: Neurosphere lines LIB and U87 NS were differentiated in media with serum (2%) with mitogens EGF and bFGF removed. Cells were allowed to grow for 7 days on cover slips and stained for neuronal (β-III tubulin) and glial (GFAP, MBP) lineage markers. Cells were counterstained with DAPI (blue). B-C: Levels of mRNA encoding the glial (GFAP, MBP) and neuronal (β-III tubulin) lineage markers and the anti-apoptotic proteins Bcl-2, Bcl-xL and McI-I and were determined using q-PCR. Lower relative expression was noted in the neurosphere LIB and U87 NS cells compared to cultures differentiated in serum (2%). Mean of duplicate samples was expressed relative to 10⁸ transcript copies of 18S ribosomal mRNA.

Figure 8. ABT-737 and cisplatin show increased efficacy in neurosphere cultures compared to serum differentiated cells

A: LlB-NS and LIB cells differentiated in serum (2%) for 72 hours, were treated with ABT-737 (0.5μM), cisplatin (0.5μM) or ABT-737/cisplatin (0.5μM) combined.

Annexin V staining was conducted 48 hours post treatment to determine the level of apoptosis compared to control cells.

B: McI-I was inhibited using a NOXA peptide approach in LlB-NS cultures (Chen et al., 2005). Annexin V staining was conducted 48 hours post treatment with NOXA peptide (lOμM) alone and in combination with ABT-737 (lμM) or cisplatin (lμM) and compared to control peptide (lOμM) treated cells. Columns, mean of triplicate samples,

+SD, (*=p<0.0\).

C-D: Sphere forming and diameter assays were performed on LlB-NS cultures following treatment with ABT-737 (0.5μM), cisplatin (0.5μM) or ABT-737/cisplatin (0.5μM) combined. Neurospheres were cultured for 7 days in 96 well plates and image capture was conducted and sphere formation and diameter (>50μM) quantitated.

Columns, mean of triplicate samples, +SD, (*= p<0.05, **= p<0.0l).

E: LIB neurospheres were serially passaged using the neurosphere assay (Reynolds and

Rietze, 2005). LlB-NS cultures were passaged every 7 days in triplicate in 6 well plates. Following cell passage, LlB-NS cultures were treated with ABT-737 (0.5μM), cisplatin (0.5μM) or ABT-737/cisplatin (0.5μM) combined. Serial passaging was conducted 7 times or until or no cells remained within the treated cultures. The total theoretical number of cells generated after each passage was calculated by multiplying the experimentally determined fold increase in the total number of viable cells after each passage by the theoretical number of cells generated from the previous passage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT In one broad form, the present invention arises, at least in part, from the discovery that McI-I is the most highly expressed Bcl-2 family member in high grade glioma and certain other cancers. Analysis of the McI-I promoter identified that McI-I expression is regulated by SRF accessory protein- Ia (SAP- Ia; also known as ELK4) in high grade glioma. ELK4 is an ETS domain transcription factor that is recruited to the c-fos serum response element (SRE) as a part of the ternary complex with serum response factor (SRF) Furthermore, the present invention arises, at least in part, from the discovery that the McI-I promoter region comprises a novel, functional G>A SNP in a consensus ETS {e.g. ELK4) transcription factor binding site, located 126 bp upstream of the major transcription start site of the McI-I gene. The wild-type (G) form of the SNP actively binds a nuclear protein complex from GBM whereas the (A) form of the SNP does not, instead correlating with significantly decreased promoter activity and with lower levels of McI-I mRNA. GBM cell lines harbouring the A form of the SNP showed increased sensitivity to apoptosis following inhibition of Bcl-2 and Bcl-xL (but not McI-I) with ABT-737 treatment and enhanced sensitivity to chemotherapy treatment. Furthermore apoptosis was observed in GBM cells lines and brain tumour stem cell 'neurosphere' lines free of the ETS SNP when McI-I was neutralized. ELK4 is the dominant ETS domain transcription factor in GBM and binds to the identified McI-I promoter ETS site. Furthermore a highly significant correlation (r=0.96) between ELK4 and McI-I mRNA expression in high grade glioma (n=38) was found. Down regulation of ELK4 by siRNA resulted in loss of McI-I expression and increased sensitivity to the BH3 mimetic ABT-737 and the chemotherapy agent cisplatin. Conversely ELK4 over- expressing GBM cells showed increased levels of McI-I and this was shown to be protective against higher concentrations of the chemotherapy agent cisplatin.

A summary of the relationship between McI-I promoter alleles, McI-I and Elk4 protein and/or nucleic acid expression levels, cancer predisposition and responsiveness to cancer therapy is provided in Tables 1 and 2. Accordingly, it is proposed that ELK4 is a critical regulator of McI-I, such that ELK4 and/or McI-I may be useful as therapeutic targets for treatment of high grade glioma and other cancers, and/or for diagnosis of high grade glioma and other cancers.

As hereinbefore described, a preferred object of the invention is to provide a method of designing, engineering, screening or otherwise producing a cancer therapeutic agent that is preferably useful in sensitizing, or improving the responsiveness of, cancer cells to a pro-apoptotic agent..

Such cancer therapeutic agents may be particularly useful in combination therapy together with pro-apoptotic agents that target members of the Bcl-2 family. As used herein, the term "cancer" includes any malignancy listed by the US

National Cancer Institute, which listing may be found at http://www.cancer.gov/cancertopics/alphalist

Preferred examples of cancers include solid tumors such as sarcomas and carcinomas. Preferably, for diagnosis and therapy relating to McI-I proteins and/or nucleic acids, the cancer is selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

Preferably, for diagnosis and therapy using ELK4 proteins and/or nucleic acids, the cancer is not melanoma. Such cancers may be selected from the group consisting of glioma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

In one preferred embodiment, the cancer is glioma.

As used herein, the term "glioma" is any benign or malignant central nervous system neoplasm derived from glial cells (e.g. astrocytes, oligodendrocytes, and ependymocytes) and includes "glioblastoma multiforme" or "GBM\ the most malignant type of astrocytoma, which is composed of spongioblasts, astroblasts, and astrocytes.

GBM usually occurs in the brain but may occur in the brain stem or spinal cord.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation.

Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

By "protein" is meant an amino acid polymer comprising D- or L-amino acids and/or natural or non-natural amino acids as are well understood in the art. A "peptide" is a protein having no more than fifty (50) amino acids.

A "polypeptide" is a protein having more than fifty (50) amino acids.

As used herein, "nucleic acid" includes and encompasses DNA, RNA and DNA- RNA hybrids. DNA includes single-stranded and double-stranded genomic DNA and cDNA as are well understood in the art. RNA includes single-stranded and double- stranded unprocessed RNA, mRNA, siRNA, shRNA, miRNA, RNAi and tRNA.

As used herein, a "gene" is a discrete structural unit of a genome which may comprise one or more elements such as an amino acid coding region typically present in one or more cistrons, an operator, a promoter, a terminator and/or any other regulatory nucleotide sequence(s). As used herein, an "oligonucleotide" is a single- or double-stranded nucleic acid having no more than one hundred (100) nucleotides (bases) or nucleotide pairs (base pairs). A "polynucleotide" has more than one hundred (100) nucleotides or nucleotide pairs.

In the particular context of nucleic acid sequence amplification, an oligonucleotide of the invention may be in the form of a primer.

As used herein, a "primer" is a single-stranded oligonucleotide which is capable of hybridizing to a nucleic acid "template" and being extended in a template-dependent fashion by the action of a suitable DNA polymerase such as Taq polymerase, RNA- dependent DNA polymerase or Sequenase™. Typically, a primer may have at least twelve, fifteen, twenty, twenty-five, thirty, thirty five or forty but no more than fifty contiguous nucleotide bases.

In particular aspects, the invention relates to methods and compositions for treatment of cancer in a mammal.

To facilitate treatment of cancer, the invention provides methods of designing, engineering, screening or otherwise producing a cancer therapeutic agent which at least partly inhibits, suppresses or reduces the binding of ELK4 to the McI-I gene promoter, transcription from the McI-I promoter and/or McI-I mRNA and protein expression.

In a particularly preferred embodiment, the cancer therapeutic agent sensitizes and/or improves the responsiveness of cancer cells to pro-apoptotic agents that target Bcl-2 family members.

Accordingly, the invention contemplates "combination therapy" that includes administration of:

(a) one or more cancer therapeutic agents designed, engineered, screened or otherwise produced according to the invention; and (b) one or more pro-apoptotic agents.

A non-limiting example of the prop-apoptotic agent is ABT-377.

Generally, cancer therapeutic agents may be identified by way of screening libraries of molecules such as synthetic chemical libraries, including combinatorial libraries, by methods such as described in Nestler & Liu, 1998, Comb. Chem. High Throughput Screen. 1 113 and Kirkpatrick et al, 1999, Comb. Chem. High Throughput

Screen 2 211.

It is also contemplated that libraries of naturally-occurring molecules may be screened by methodology such as reviewed in KoIb, 1998, Prog. Drug. Res. 51 185.

More rational approaches to designing cancer therapeutic agents may employ X- ray crystallography, NMR spectroscopy, computer assisted screening of structural databases, computer-assisted modelling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art.

A review of structural bioinformatics approaches to drug discovery is provided in Fauman et al, 2003, Meth. Biochem. Anal. 44: 477. Computer-assisted structural database searching and bioinformatic approaches are becoming increasingly utilized as a procedure for identifying and/or engineering agonists and antagonist molecules. Examples of database searching methods may be found in United States Patent No. 5,752,019 and International Publication WO 97/41526 (directed to identifying EPO mimetics) and United States Patents 7,158,891 and 5,680,331 which are directed to more general computational approaches to protein modelling and structural mimicry of protein activity. Generally, other applicable methods include any of a variety of biophysical techniques which identify molecular interactions. Methods applicable to potentially useful techniques such as competitive radioligand binding assays, electrophysiology, analytical ultracentrifugation, microcalorimetry, surface plasmon resonance and optical biosensor-based methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.

A person skilled in the art will appreciate that cancer therapeutic agents may be in the form of a binding partner and as such, identified by interaction assays such as yeast two-hybrid approaches and the like, but without limitation thereto. Two-hybrid screening methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.

In a preferred embodiment, cancer therapeutic agents of the invention are small organic molecules which inhibit ELK4 or McI-I activity or expression.

By way of example only, the invention contemplates screening the National Cancer Institute (NCI) "Diversity Set", a collection of -2000 compounds selected to represent the greater chemical diversity of the NCI chemical repository (Rapisarda et al., 2002, Cancer Res. 62 4316), for small molecule inhibitors of ELK4 using glioma cell lines transfected with a destabilised (short half life) green fluorescent protein (GFP) reporter gene expressed under the control of the McI-I promoter and a destabilised red fluorescent protein (RFP) reporter gene expressed under the control of the ubiquitin promoter. Cells will be cultured in 96-well plates (typically at 10⁴ per well) containing candidate agents at the appropriate concentrations and both GFP and RFP intensity will be measured by plate reader after 24 hours' exposure. GFP fluorescence may be recorded relative to RFP fluorescence to control for cell number, viability and nonspecific effects on cell metabolism. It is anticipated that this should identify small molecules that specifically inhibit the McI-I promoter, independent of their mechanism or site of action, as opposed to candidate agents that non-specifically affect transcription, translation or general cell "health". If an agent simply reduces McI-I promoter activity, and thus GFP fluorescence, by affecting cell health and viability, it will also affect the ubiquitin promoter and reduce RFP fluorescence as well.

To identify small organic molecules specifically targeting ELK4, experiments may be performed in parallel with cancer (e.g. glioma) cells transfected with the G>A SNP in the ETS site in the McI-I promoter in place of the wild type McI-I promoter- GFP reporter together with the ubiquitin-RFP reporter.

This should discriminate small molecules that specifically inhibit the activity of ELK4 at the McI-I promoter e.g. binding to DNA, ternary complex formation with serum response factor, interaction with components of the general transcription machinery, signalling to ELK4 (e.g. binding by MAPK and phosphorylation of Ser381 and Ser387). This may facilitate identifying one or more candidate agents that specifically reduces McI-I promoter activity, dependent on the presence of the ELK4 binding site.

In another non-limiting example, cancer therapeutic agents may be screened for an ability to inhibit nucleoprotein complex formation between ELK4 and a wild-type

McI-I promoter or promoter fragment. Such an agent, preferably in the form of a small organic molecule, could be useful in down-regulating ELK4-driven expression of McI-I

RNA transcription, thereby reducing expression of McI-I protein.

In still further aspects, the invention relates to compositions and/or methods of treating cancers, including but not limited to glioma.

In certain embodiments, pharmaceutical compositions and treatment methods may utilize cancer therapeutic agents produced according to methods as hereinbefore described.

In another embodiment, pharmaceutical compositions and treatment methods may utilize a cancer therapeutic agent in the form of a synthetic peptide (T AT-DEF-

ELK4). This is designed to mimic one of the two MAPK docking domains of ELK4

(the DEF domain) and thereby block phosphorylation of ELK4 by ERK, ELK4 nuclear translocation and ELK4-dependent gene regulation in a manner similar to that demonstrated for the related ternary complex ETS factor Elkl (Lavour et ah, 2007, J. Neurosci. 27 14448). In yet another embodiment, pharmaceutical compositions and treatment methods may utilize a peptide which inhibits ELK4 activation of McI-I gene transcription. A non-limiting example of an inhibitory peptide could be based on a HLH domain of an Id protein which inhibits DNA binding by basic HLH proteins (e.g.. ELK4) in trans (Stinson et al., 2003, NAR, 31 16). Alternatively or additionally, an inhibitory peptide could be based on an NID (Net Interacting Domain) of ELK4, which inhibits DNA binding in cis. This inhibitory activity is overcome by phosphorylation. A peptide or small organic molecule that would inhibit ELK4 binding to the ETS site in the McI-I promoter in glioma or other McI-I expressing cancers, may thereby reduce McI-I expression in these tumours, sensitizing them to pro-apoptotic agents (e.g. induced by ABT-737 chemotherapy or radiotherapy).

In general, cancer therapeutic peptides and proteins may be conjugated or complexed with cell-permeable agents such as a HIV-TAT, Penetratin (Antp), PoIy- arginine,VP22, Transportan, MAP, MTS or PEP-I membrane permeability or protein transduction domain, or lipids such as cholesterol, and modified to improve structural stability or provide resistance to degradation by serum and cellular proteases by modifications such as hydrocarbon-stapling or use of D-amino acids in reverse sequence (i.e. retro-inversion) (Prive & Melnick, 2006, Curr Opin Genet Dev 16:71-77).

In other embodiments, pharmaceutical compositions and treatment methods may utilize nucleic acid constructs (including but not limited to inhibitory RNA constructs) for treatment of cancers ^cancer therapeutic nucleic acids").

Generally, a cancer therapeutic nucleic acid construct may be any recombinant nucleic acid that facilitates delivery, expression, propagation or manipulation of a desired nucleic acid component of the construct. By way of example only, a construct may be a plasmid, a cosmid, a modified virus or containing virus-derived elements, an artificial chromosome, a phagemid, an anti-sense oligonucleotide, RNA (e.g. siRNA or shRNA) or the like.

Particular virus-derived expression constructs suitable for human delivery include constructs comprising adenovirus-, adeno-associated virus-, lentivirus-, flavivirus- and/or vaccinia virus-derived elements. One particular example of a nucleic acid construct suitable for cancer therapy is an inhibitory RNA construct, such as but not limited to an inhibitory RNA that is double-stranded or otherwise comprises internal base pairing.

In particular embodiments, inhibitory RNA constructs includesiRNA or shRNA construct that down-regulate expression of an ELK4 protein.

Particular non-limiting examples of siRNA and shRNA constructs are described hereinafter.

It will also be appreciated that the invention provides a DNA construct that encodes an inhibitory RNA construct. Suitably, pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, diluent or excipient.

By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991) which is incorporated herein by reference.

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular and transdermal administration may be employed.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like.

These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be affected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be affected by using other polymer matrices, liposomes and/or microspheres.

Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. While methods and compositions of the invention are preferably directed to human therapy, the invention also contemplates extension to veterinary treatments, such as for livestock, domestic pets and performance animals, although without limitation thereto.

In further aspects, the invention relates to diagnosis of cancers and/or to determining or predicting the sensitivity or responsiveness of cancers to pro-apoptotic agents. In particular embodiments, diagnostic methods described herein may be used in conjunction with treatment methods described herein to determine the suitability of a patient for a particular drug therapy, for example a combination therapy.

Diagnostic methods of the invention are at least partly predicated on the discovery of a SNP in the myeloid cell leukemia- 1 (McI-I) promoter which is associated with a susceptibility to cancer.

In one aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence of at least a fragment of a myeloid cell leukemia- 1 (McI-I) promoter, which nucleotide sequence comprises SNP in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor. In this context, by "fragment" is meant a region, portion, sub-sequence or segment of an McI-I promoter which comprises a SNP in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor.

In particular non-limiting embodiments, the fragment may comprise at least 10, 20, 50, 100, 150, 200, 250, 300, 400 or more nucleotides of an McI-I promoter. In one particular embodiment, the SNP is a G>A SNP in an ELK4 binding site at position -126 relative to the major transcription start site in a human McI-I promoter.

In particular embodiments, said fragment comprises or consists of a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

The invention also includes an isolated nucleoprotein complex comprising at least a fragment of a myeloid cell leukemia- 1 (McI-I) promoter, as hereinbefore defined, which nucleotide sequence comprises an ETS transcription factor binding site; and an ETS transcription factor.

Preferably, the ETS transcription factor is ELK.4.

The invention also provides a chimeric gene comprising a nucleotide sequence of at least a fragment of a myeloid cell leukemia- 1 (McI-I) promoter, which nucleotide sequence comprises a polymorphism in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor operably linked or connected to an expressible nucleotide sequence.

By "operably linked or connected" is meant that said promoter sequence is/are positioned relative to said expressible nucleotide sequence to initiate, regulate or otherwise control transcription. A non-limiting example of an expressible nucleotide sequence is a "reporter gene" such as luciferase, β-galactosidase or green fluorescence protein (GFP) to facilitate measurement of promoter activity.

The chimeric gene may be present in a recombinant vector to facilitate recombinant manipulation, propagation in host cells (e.g. bacteria such as E. coli) and the like. Accordingly, the vector may comprise one or more additional nucleotide sequences such as an origin of replication, a selection marker gene, an epitope tag- encoding sequence and/or other vector sequences as are well known in the art.

Particular aspects the invention relate to methods of diagnosis of cancer. Such methods may assist in determining whether a mammal is susceptible to cancer.

Accordingly, methods of the invention may be useful in determining whether or not a mammal suffers from cancer and/or is genetically predisposed to cancer.

By "predisposed" is meant having a higher probability, risk or susceptibility than normal for contracting or suffering from a cancer. Normal probability or risk may be assessed with reference to non-affected individuals, cohorts or populations of individuals as is well understood in the art.

Other particular aspects of the invention provide methods for determining the responsiveness of a mammal to cancer therapy.

In particular embodiments, the invention provides methods to determine or assess whether a mammal is more or less responsive to one or more anti-cancer agents.

Examples of anti-cancer agents are pro-apoptotic drugs that target members of the Bcl-2 protein family.

A non-limiting example of a pro-apoptotic cancer agent is ABT-377.

A summary of the relationship between McI-I promoter alleles, McI-I and Elk4 protein and/or nucleic acid expression levels, cancer predisposition and responsiveness to cancer therapy is provided in Tables 1 and 2.

In one embodiment, a -126 A SNP in an ELK4 binding site in a myeloid cell leukemia- 1 (McI-I) gene promoter is associated with a relatively reduced or lower level of expression of McI-I and a non-cancerous state and/or a relatively reduced susceptibility to cancer. Typically, a -126 A SNP in an ELK4 binding site in a myeloid cell leukemia- 1 (McI-I) gene promoter is associated with a relatively increased or greater sensitivity or responsiveness to cancer therapy.

In another embodiment, a -126G SNP in an ELK4 binding site in a myeloid cell leukemia- 1 (McI-I) gene promoter is associated with a relatively increased or higher level of expression of McI-I and cancer and/or increased susceptibility to cancer. Typically, a -126 G SNP in an ELK4 binding site in a myeloid cell leukemia- 1 (McI-I) gene promoter is associated with a relatively reduced or lower sensitivity or responsiveness to cancer therapy.

In yet another embodiment, a relatively increased or higher level of expression of ELK4 and/or McI-I is associated with cancer and/or an increased or greater susceptibility to cancer; a decreased or lower level of expression of ELK4 and/or McI-I is associated with a non-cancerous state and/or a reduced or lower susceptibility to cancer.

Typically, a relatively increased or higher level of expression of ELK4 and/or McI-I is associated with a relative resistance or lower sensitivity to cancer therapy; a relatively decreased or lower level of expression of ELK4 and/or McI-I is associated with a relatively increased or higher sensitivity to cancer therapy.

Accordingly, the invention provides diagnostic methods that may identify one or more of the following: (i) a relative level of expression of McI- 1 protein or encoding nucleic acid;

(ii) a relative level of expression of ELK-4 protein or encoding nucleic acid; and (iii) determination of a G or A residue at position -126 in an ELK4 binding site in an McI- 1 gene promoter. Preferably, diagnostic methods are performed using a biological sample obtained, or obtainable from, a mammal. Suitably, said biological sample includes cells, tissues, organs or organ biopsies, proteins, nucleic acids or other isolated biological material as appropriate for the particular diagnostic method.

Accordingly, diagnostic methods may be protein-based or nucleic acid-based. Nucleic acid-based detection is well known in the art and may utilize one or more techniques including nucleic acid sequence amplification, probe hybridization, mass spectrometry, nucleic acid arrays and nucleotide sequencing, although without limitation thereto.

In one embodiment, the invention contemplates nucleic acid sequence amplification and subsequent detection of one or more amplification products. Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999); strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al., 1996, J. Am. Chem. Soc.

118 1587 and International application WO 92/01813 and by Lizardi et al., in

International Application WO 97/19193; nucleic acid sequence-based amplification

(NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17 1077;

Q-β replicase amplification as for example described by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93 5395 and helicase-dependent amplification as described in

International Publication WO2004/02025.

Non-limiting examples of primers that may be used according to such methods are provided in the Examples and in Table 4.

The abovementioned are examples of nucleic acid sequence amplification techniques but are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.

For example, the invention contemplates use of particular techniques that facilitate quantification of nucleic acid sequence amplification products (e.g. referred as "qPCR") such as by "competitive PCR", or techniques such as "Real-Time" PCR amplification.

Non-limiting examples of ETS transcription factor (e.g. ELK4) and McI-I primers that may be used according to qPCR methods are provided in the Examples and in Table 4. As used herein, an "amplification product" is a nucleic acid generated by a nucleic acid sequence amplification technique as hereinbefore described.

Detection of amplification products may be achieved by detection of a probe hybridized to an amplification product, by direct visualization of amplification products by way of agarose gel electrophoresis, nucleotide sequencing of amplification products or by detection of fluorescently-labeled amplification products.

As used herein, a "probe"' is a single- or double-stranded oligonucleotide or polynucleotide, one and/or the other strand of which is capable of hybridizing to another nucleic acid, to thereby form a "hybrid" nucleic acid. A non-limiting example of a probe comprises nucleotides -134 to -115 of the

McI-I promoter with either an A or G at position -126, or a nucleotide sequence complementary thereto.

Probes and/or primers of the invention may be labeled, for example, with biotin or digoxigenin, with fluorochromes or donor fluorophores such as FITC, TRITC, Texas Red, TET, FAM6, HEX, ROX or Oregon Green, acceptor fluorophores such as LC- Red640, enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with radionuclides such as ¹²⁵I, ³²P, ³³P or ³⁵S to assist detection of amplification products by techniques as are well known in the art.

As used herein, "hybridization", "hybridize" and "hybridizing" refers to formation of a hybrid nucleic acid through base-pairing between complementary or at least partially complementary nucleotide sequences under defined conditions, as is well known in the art. Normal base-pairing occurs through formation of hydrogen bonds between complementary A and T or U bases, and between G and C bases. It will also be appreciated that base-pairing may occur between various derivatives of purines (G and A) and pyrimidines (C, T and U). Purine derivatives include inosine, methylinosine and methyladenosines. Pyrimidine derivatives include sulfur-containing pyrimidines such as thiouridine and methylated pyrimidines such as methylcytosine. For a detailed discussion of the factors that generally affect nucleic acid hybridization, the skilled addressee is directed to Chapter 2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra. More specifically, the terms "anneal" and "annealing" are used in the context of primer hybridization to a nucleic acid template for a subsequent primer extension reaction, such as occurs during nucleic acid sequence amplification or nucleotide sequencing, as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. (John Wiley & Sons NY 1995- 1999).

In another embodiment, detection may be performed by melting curve analysis using probes incorporating fluorescent labels that hybridize to amplification products in a sequence amplification reaction. A particular example is the use of Fluorescent

Resonance Energy Transfer (FRET) probes to hybridize with amplification products in "real time" as amplification products are produced with each cycle of amplification.

In yet another embodiment, the invention contemplates use of melting curve analysis whereby nucleic acid-intercalating dyes such as ethidium bromide (EtBr) or SYBR Green I bind amplification products and fluorescence emission by the intercalated complexes is detected. More specifically, detection of a G>A SNP in an ELK4 binding site in an McI-I gene promoter may be achieved by any method applicable to mutation detection.

In one particular embodiment, said promoter polymorphism may be identified by mass spectrometry analysis of primer extension products.

Mass spectrometry is preferably performed using a MALDI-TOF mass spectrometer. More preferably, mass spectrometry utilizes a Sequenom MassARRAY™ genomics platform.

It will also be appreciated that methods of the invention may employ other well known techniques and/or modifications thereof.

In one example, PCR-based restriction fragment length polymorphism analysis may be used.

In another example a PCR method that may also be useful is Bi-PASA (Bidirectional PCR Amplification of Specific Alleles), as for example described in Liu et al. 1997, Genome Res. 7 389-399.

Another potentially useful PCR method uses allele-specification oligonucleotide hybridization to PCR products, as for example described in Aitken et al., 1999, J Natl Cancer Inst 91 446-452. It will also be well understood by the skilled person that identification of McI-I promoter polymorphisms may be performed using any of a variety of other techniques such as fluorescence-based melt curve analysis, SSCP analysis, denaturing gradient gel electrophoresis (DGGE) or direct sequencing of amplification products. Particularly for the purpose of clinical diagnosis, although without limitation thereto, the invention provides a kit comprising one or more probes and/or primers (such as hereinbefore described) that facilitate detection of McI-I and/or ELK-4 nucleic acid and/or a presence of a G or A SNP in an ELK.4 binding site in an McI-I promoter. Said kit may further comprise other reagents such as a thermostable DNA polymerase, positive and/or negative nucleic acid control samples, molecular weight markers, detection reagents such as for colorimetric detection or fluorescence detection of amplification products and/or reaction vessels such as microtitre plates.

A non-limiting example of a probe comprises nucleotides -134 to -115 of the McI-I promoter with either an A or G at position -126, or a nucleotide sequence complementary thereto.

It will also be appreciated that the method of the invention may be used alone or combined with other forms of molecular and/or clinical diagnosis to improve the accuracy of diagnosis.

In this regard, the invention contemplates nucleic acid array detection wherein one or more other nucleic acid markers associated with other cancers, or other diseases or conditions, may be provided on the array.

Nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2001).

The invention also contemplates protein based methods, although these are primarily directed to measurement of relative levels of McI-I and/or ELK4 protein.

Protein-based techniques applicable to the invention are well known in the art and include western blotting, ELISA, two dimensional protein profiling, protein arrays, immunoprecipitation, radioimmunoassays and radioligand binding, although without limitation thereto. In this regard, antibodies may be particularly useful in immunoassays such as ELISA, which are capable of high throughput analysis of multiple protein samples.

Alternatively, antibodies may be used in a protein array format, which is particularly suited to larger scale expression analysis. So that the present invention can be readily understood and put into practical effect, reference is made to the following non-limiting examples.

EXAMPLES

INTRODUCTION

Glioblastoma multiforme (GBM) (World Health Organization grade 4 astrocytoma) is the most common malignant primary brain tumour. Its mean age of onset is 53 years. Treatment involves surgical resection (where possible), followed by radiation and chemotherapy (Behin et al., 2003). Therapy is almost never curative due in part to the widely infiltrative nature of these tumours and the intrinsic resistance of high grade glioma to radiation and cytotoxic chemotherapy. Even with optimal treatment, including post-operative radiation and concurrent and adjuvant temozolomide chemotherapy, the median survival is less than 15 months and only about 10% of patients survive two years without disease recurrence (Stupp et al., 2005). This dismal situation highlights a pressing need to identify new therapeutic targets if the outlook for glioma is to be improved.

One potentially important target for molecular therapy is the cell death 'apoptotic' machinery of the cell. The p53 tumour suppressor, which is often mutated in human cancers, normally controls cell proliferation by Gl cell cycle arrest or by inducing apoptosis following DNA damage (Vogelstein et al., 2000). The p53 gene is often mutated in secondary GBM (67%) and primary GBM (11%) and ablates induction of the BH3-only proteins Puma and Noxa, preventing apoptosis (Watanabe et al., 1996; Jeffers et al., 2003; Shibue et al., 2003). Therefore anti-cancer agents that mimic BH3 proteins might prove efficacious even in tumours harbouring this mutation. One such agent, ABT-737, which has been proven to induce cell death in tumour cell lines including lymphoma and small-cell lung cancer, is a potent inhibitor of the anti- apoptotic cell death regulatory proteins B-cell leukemia-2 (Bcl-2) and B-cell leukemia-x long isoform (Bcl-xL). The Bcl-2 family of proteins contains both anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, McI-I and Bfl/Al) and pro-apoptotic (Bax, Bak, Bim, tBid, Bad, Bik, Bmf, Hrk, Noxa, Puma) members and imbalances between these underlie a number of neoplastic malignancies (Cory et al., 2003). ABT-737 binds Bcl-2, Bcl-xL and Bcl-w with high affinity (K,< InM), but has far lower affinity for myeloid cell leukemia- 1 (McI-I) (K,>lμM) (Oltersdorf et al., 2005). In keeping with this, overexpression of McI-I has been shown to attenuate ABT-737 sensitivity both in a mouse lymphoma model and in acute myeloid leukemia (van Delft et al., 2006; Konopleva et al., 2006) while ABT-737 has been shown to induce apoptosis in human carcinoma cell lines via Bak/Bax when McI-I was neutralised.

In this report we show that McI-I is the most highly expressed anti-apoptotic Bcl-2 family member in high grade glioma. In addition we report that in analysing the McI-I promoter region we identified a novel, functional G>A SNP in a consensus ETS transcription factor binding site. We show that while the WT (G) form of the SNP actively binds a nuclear protein from GBM cells, the SNP (A) form does not, and that this correlates with significantly decreased promoter activity and lower levels of McI-I mRNA. GBM cell lines harbouring the SNP showed increased sensitivity to inhibition of Bcl-2 and Bcl-xL following ABT-737 treatment and greater susceptibility to cisplatin treatment. Furthermore, apoptosis was observed in GBM cells lines and brain tumour stem cell 'neurosphere' lines free of the ETS SNP when McI-I was neutralised. We show that ELK4 is the dominant ETS domain transcription factor in GBM and binds to the identified McI-I promoter ETS site. Furthermore, a highly significant correlation (r=0.96) between ELK4 and McI-I mRNA expression in high grade glioma (n=38) was found. Down regulation of ELK4 by siRNA resulted in loss of McI-I expression and increased sensitivity to ABT-737 and cisplatin. Conversely, ELK4 overexpressing GBM cells showed increased levels of McI-I and this was shown to be protective against higher concentrations of cisplatin. These findings demonstrate ELK4 to be a critical regulator of McI-I and highlight both ELK4 and McI-I as therapeutic targets in high grade glioma. EXPERIMENTAL PROCEDURES Cell culture

Primary glioma resected tissue was obtained from the Department of Neurosurgery, Royal Brisbane Hospital. GBM cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were cultured in RPMI- 1640 medium with 10% fetal bovine serum (JRH Biosciences, Lenexa, KAN) at 37°C in humidified air/5% CO₂. 10⁷ cells were used for preparation of genomic DNA, RNA and protein lysates. Tissue and cells were snap frozen in dry ice. Neurosphere culture Neurospheres were generated from a primary resected GBM sample (LIB) and the GBM cell line U87 (U87 NS). Culture and media conditions were as previously described (Rietze and Reynolds, 2006). DNA extraction and sequence analysis

Genomic DNA was extracted using a salt precipitation approach described by Miller et al, 1988. The McI-I promoter from nucleotide -291 to the ATG translation start site was amplified from genomic DNA by polymerase chain reaction using AmpliTaq^® Gold (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Primers sequences (5' to 3') TGAGCTTGCTCACCTTTCCT (sense; SEQ ID NO:3) and ATTATAAGCTTTTGCCAGTCGC (anti-sense; SEQ ID NO:4). RNA analysis

RNA was extracted from tissue samples using TRIzol^® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA concentrations were determined using a spectrophotometer and stored at -70⁰C. Prior to qPCR, 2 μg of sample RNA (1 μg/μl) was DNase-treated with 2 μl of RQl DNase I enzyme (Promega, Madison, WI). First strand cDNA was primed with random hexamers and synthesised by reverse transcription (RT) using Superscript™ III (Invitrogen, Carlsbad, CA). cDNA was synthesised in duplicate, pooled and diluted in 10 mM Tris pH 8.5 to a final concentration of 50 ng/μl for qPCR. Relative quantitation by real-time PCR Real-Time PCR was carried out using SYBR^® Green PCR Master Mix (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. Briefly, 250 ng of cDNA was added to SYBR^® Green PCR Master Mix. Forward and reverse primers were added to a final concentration of 0.5 μM. Primer sequences (5' to 3') were McI-I (GenBank Ace. No.: NM_021960) TAACAAACTGGGGCAGGATT (sense; SEQ ID NO:5) and TCCCGTTTTGTCCTTACGAG (anti-sense; SEQ ID NO:6), ELK4 (GenBank Ace. No.: NM 021795) CTGGTGCCAAGACCTCTAGC (sense; SEQ ID NO:7) and TCGGCTGGATTCTCAGTCTT (anti-sense; SEQ ID NO:8) and 18S rRNA (GenBank Ace. No.: K03432) GACTC AAC ACGGGAAACCTC (sense; SEQ ID NO:9) and AGCATGCC AGAGTCTCGTTC (anti-sense; SEQ ID NO: 10). A standard curve using cDNA serially diluted further to 10^"2, 10^"4 and 10^"6 and 18S ribosomal RNA primers was created. For ChIP analysis forward and reverse primers were added to a final concentration of 4 μM. Primer sequences for McI-I (GenBank Ace. No.: NM_021960) were (5' to 3') ACTCAGAGCCTCCGAAGACC (sense; SEQ ID NO:11) and ATTATAAGCTTTTGCCAGTCGCCGCCGCC (anti-sense; SEQ ID NO: 12). All reactions were performed in duplicate. Real-time PCR was carried out in a Corbett Research Rotor-Gene 3000™ (Corbett Research, Australia). The PCR cycling conditions included activation for 15 minutes at 95°C and 30 cycles of 30 seconds at 95⁰C, 30 seconds at 55⁰C, and 30 seconds at 72⁰C. Fluorescence data was recorded at the end of each 72 ⁰C step. A DNA melt profile was run subsequently from 72°C to 95°C with a ramp of l°C/5 seconds. Fluorescence data was recorded continuously during the melt profile, data was analysed using Rotorgene 6, V6.1 software, (Corbett Research, Australia). A complete list of all qPCR primers is provided in Table 4. Reporter vector constructs The pGL2 luciferase reporter vector (Promega, Madison, WI) was used to determine the relative activity of the McI-I promoter region extending from nucleotide - 291 to the ATG translation start site of the wild type (WT) promoter versus comparable regions containing the 6 and 18 bp McI- 1 promoter insertions. These were amplified by PCR from genomic DNA as described above. The PCR products were gel purified using a QIAquick Gel Extraction Kit (QIAGEN, Germantown, MD), digested with the restriction enzymes Xhol and HindIII (New England Biolabs, Beverly MA), and ligated into Xhol- and Hindlll-digested pGL-2 reporter vector using T4 DNA ligase (New England Biolabs, Beverly MA). ETS SNP constructs were generated in the WT McI-I promoter construct using the QuikChange^® site directed mutagenesis kit (Stratagene La Jolla, CA) as per the manufacturer's instructions. Luciferase reporter assay

Prior to transfection, 1.5x10⁵ cells per well were plated in triplicate in 6-well tissue culture plates and cultured overnight in 2 ml of RPMI containing 10% FCS. Cells were transfected with 1 μg of pGL2 reporter plasmid (McI-I WT promoter, 6 bp insertion or 18 bp insertion) together with 100 ng of the Renilla plasmid pRL-TK (Promega), to control for transfection efficiency. In addition pGL2-Basic (Promega) plasmid was included as a negative control and pGL2-Promoter (Promega) plasmid as positive control in all experiments. Transfections were conducted using FuGene^® 6 transfection reagent (Roche, Indianapolis, IN) as per the manufacturer's instructions. Reporter assays were conducted 48 hours post transfection using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI) and a TD-20/20 Luminometer (Turner Designs CA, USA). All experiments were performed in triplicate. The pGL2-Promoter plasmid was used to normalise and combine data from a minimum of two independent experiments carried out in triplicate. Electrophoretic mobility shift assay (EMSA) EMSAs were performed with double stranded (ds) oligonucleotides, radiolabeled using T4 polynucleotide kinase and [γ-³²P]ATP, and nuclear extracts prepared essentially according to Dignam et al. (1983). Binding reactions contained 20 mM HEPES pH 7.9, 100 mM KCl, 15 mM MgCl₂, 1 mM DTT, 0.1% Tween20, 1 μg poly[d(I-C)], 5 μg of nuclear extracts and 4 fmol of radiolabeled ds oligonucleotide (added last), in a total volume of 20 μl. For cold competition EMSAs, unlabelled ds oligonucleotide at 10- or 100-fold molar excess was mixed with radiolabeled ds oligonucleotide prior to addition to the binding reaction. Supershift EMSAs included 4 μg of ELK4 antibody (Santa Cruz, sc-1426X). Binding reactions were incubated at room temperature for 20 minutes before electrophoresis on 5% polyacrylamide gels (29:1 acrylamide:bisacrylamide) in 1 x TBE buffer at room temperature. Gels were dried under vacuum at 80⁰C and exposed to photographic film at -8O⁰C between intensifying screens.

Chromatin immunoprecipitation assay (ChIP)

ChIP Assays were performed as described: http://genomics.ucdavis.edu/farnham/protocols/chips.html. Nucleic DNA was sonicated to an average length of approximately 600bp using four 20 second pulses at 1.5 power constant setting on a Branson Sonifier^® 250 (Branson, Danbury, CT). Chromatin was pre-cleared using 'blocked' protein A sepharose (Pharmacia Biotech, Sweden) containing 10% salmon sperm DNA (10 mg/ml) and 10% BSA (10 mg/ml). Two ELK4 specific antibodies were used (C-20 and H- 167, Santa Cruz Biotechnology, Santa Cruz, USA). PCR fragments were amplified using primers specific for McI-I (GenBank Ace. No.: NM_021960) (5' to 3') ACTCAGAGCCTCCGAAGACC (sense; SEQ ID NO: 13) and ATTATAAGCTTTTGCCAGTCGCCGCCGCC (anti-sense; SEQ ID NO: 14) primers. Each 50 μl PCR consisted of 1 x AmpliTaq^® Gold PCR II buffer, 2mM MgCl₂, IM Betaine (Sigma), 0.4mM dNTPs, 0.4 μM primer pair, 0.2 μl AmpliTaq^® Gold, and 200ng ChIP DNA. PCR cycling conditions included activation for 10 minutes at 95°C and 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 1 minute at 72°C. Enrichment of ELK4-bound McI-I promoter fragments was confirmed by q-PCR as analysed previously. siRNA and shRNA mediated gene knockdown

Target proteins (McI-I, ELK4) were down regulated using the shRNA vector, pSuperior.neo+gfp (Oligoengine, Seattle W.A). Gene specific shRNA sequences were: McI-I 5'-GAT CCC CCG GGA CTG GCT AGT TAA ACT TCA AGA GAG TTT AAC TAG CCA GTC CCG TT TTTA-3' (Taniai et al., 2004; SEQ ID NO: 15) and ELK4 siRNA Validated Stealth™ DuoPak (cat # 1293609) (Invitrogen, Carlsbad, CA). Sequences were used for both siRNA and shRNA mediated knockdown. shRNA sequences were: ELK4 sequence #1 5'-GAT CCC CGC AAT GAC TAC ATA CAC TCT GGC TTT TCA AGA GAA AGC CAG AGT GTA TGT AGT CAT TGC TTT TTA-3' and ELK4 sequence #2 5'-GAT CCC CGG ATT CGC AAG AAC AAG CCT AAC ATT TCA AGA GAA TGT TAG GCT TGT TCT TGC GAA TCC TTT TTA-3' (SEQ ID NO: 16) with luciferase control 5'- GAT CCC CCG TAC GCG GAA TAC TTC GAT TCA AGA GAT CGA AGT ATT CCG CGT ACG TTT TTA-3' (SEQ ID NO: 17). Complementary pairs of oligonucleotides were annealed and ligated to the linearised pSuperior.neo+gfp vector according to the manufacturer's instructions. Insertion of oligonucleotides was confirmed by DNA sequencing. Transfections were conducted using FuGene^® 6.

ELK4 cDNA expression construct

ELK4 cDNA was PCR-amplified and cloned into the pEF-IRES-puro6 mammalian expression vector (Hobbs et al., 1998). pEF-IRES-puro6-ELK4 cDNA transfected cells were compared to vector only control transfected cells. For western blotting 1 ^χ 10⁶ cells were pelleted following two weeks' selection with lμg/ml puromycin and snap frozen in dry ice. NOXA peptide treatment

McI-I was neutralised using a NOXA peptide approach previously described in (Chen et al., 2005). Briefly NOXA sequence (10 μM): AELEVECATQLRRFGDKLNFRQKLLRRRRR (3758.396 D; SEQ ID NO: 18) and control sequence (10 μM) LPRFDTQGRVRANEAQLKELEKLLFRRRRR (3752.370 D; SEQ ID NO: 19) were applied to U87 and LlB-NS cultures alone and in combination with cisplatin (1 μM) or ABT-737 (1 μM). Protein analysis Anti-apoptotic protein level was visualised by Western blot analysis. Briefly, 1 x 10⁶ cells were pelleted and lysed with 50 μl of lysis buffer (150 mM NaCl, 1% Triton XlOO, 5 mM EDTA pH 8, 10 mM Tris-Cl pH 7.4 and 0.1 mM PMSF). Protein concentration was determined by Bradford protein assay. Protein lysate (200 μg/well) was mixed with an equal volume of 2 x sample buffer (130 mM Tris-Cl pH 8.0, 20% (v/v) glycerol, 4.6% (w/v) SDS, 0.02% bromophenol blue, 2% DTT and 5% β- mercaptoethanol) and boiled for 5 minutes. Samples were run on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels at 130 V for one hour and transferred overnight at 30 V onto Immuno-Blot™ PVDF Membrane (BIORAD, Hercules, CA). The membrane was blocked with 5% milk powder in TBST (137 mM NaCl, 10 mM Tris, pH 7.4, 0.02% Tween 20) for one hour. Primary antibodies, anti-Mcl-1 (S22) mouse monoclonal (Santa Cruz Biotechnology, Santa Cruz USA) (1: 100), anti-Bcl-2 (clone 124) mouse monoclonal (Dako Cytomation, CA USA) (1 :200), anti-Bcl-xL (S 18) rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz USA) (1:200) and anti-ELK4 (H- 167) rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, USA) (1: 1000), were diluted in 5% milk powder in TBST. Secondary detection antibodies used were ECL mouse IgG conjugated to horse radish peroxidase (HRP) (Amersham Biosciences, Piscataway, USA) (1 :1000) and rabbit anti-human IgG-HRP (Dako Cytomation, CA USA) (1:1000). The housekeeping protein β-actin was used as a loading control and detected with mouse anti-human β-actin monoclonal antibody (Sigma, St Louis MS) (1 :2000). Protein was detected using EC™ Western Blotting Detection Reagents as per manufacturer's instruction (Amersham Biosciences). Immunohistochemistry

Immunohistochemistry was used to determine the level of McI-I in the GBM cell lines U87, Ul 18, D645 and U373. Neurosphere lines LIB and U87 NS were stained for glial and neuronal lineage markers following 7 days differentiation in media containing serum (2%). 2 * 10⁴ cells were allowed to grow on glass cover slips in 24- well plates prior to staining. Briefly, cells were washed twice with 5% FCS in PBS and fixed using 4% PFA. Primary detection antibodies anti-Mcl-1 (S22) mouse mAb (Santa Cruz Biotechnology, Santa Cruz USA) (1 :100), anti-βlll tubulin (5G8) mouse mAb (Promega, Madison, WI) (1:2000), anti-GFAP rabbit polyclonal (20334) (Dako Cytomation, CA USA) (1 :500), anti-MBP (AB980) (Chemicon, Aus) (1 :200), were incubated for 20 mins at room temperature. Secondary detection antibodies for McI-I staining were anti-mouse Ig-FITC conjugated (Chemicon, Aus) (1:250), for GFAP staining, alexa goat anti-rabbit 488 (AI lOOl) (Invitrogen, Carlsbad, CA) (1:1000), and for βlll tubulin and MBP, alexa goat anti-mouse 568 (Al 1011) (Invitrogen, Carlsbad, CA) (1:1000). Secondary antibodies were incubated for 15 mins in the dark at room temperature. Cover slips were mounted on glass slides for McI-I staining using fluorescent mounting medium (Dako Cytomation, CA USA) and glial and neuronal markers using ProlongGold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA), and visualised using fluorescent microscopy. Neurosphere assay Neurosphere assays were performed as described (Reynolds and Rietze, 2005).

LIB neurospheres were plated in triplicate at a density of 5 x 10⁴ cells per ml. ABT-737

(0.5 μM) in DMSO and cisplatin (0.5 μM) in PBS were applied after 24 hours and cells were trypsinised after 7 days and cell counts performed. 5 x 10⁴ cells per ml were replated and the procedure repeated for 8 weeks or until no cells remained. DMSO only, at matching concentrations, was used in control wells.

Sphere forming assay

Sphere forming and diameter assays were performed in 96 well plates. LlB-NS cultures were plated in triplicate at a density of 3 x 10³ cells per well. Cultures were treated with ABT-737 (0.5μM), cisplatin (0.5μM) or ABT-737/cisplatin (0.5μM) combined. Neurospheres were cultured for 7 days and image capture was conducted and sphere formation and diameter (>50μM) quantitated.

Annexin V staining

Apoptosis was determined by Annexin V cell staining. Briefly, samples were washed in PBS, trypsinised (2% trypsin in PBS), and resuspended in 2 ml of 1 x

Annexin V binding buffer (1O mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂).

Annexin V-PE (5 μl) (BD Pharmingen, San Jose, CA) was added, mixed and incubated at room temperature for 15 mins. Samples were sorted on a FACSCalibur (BD

Biosciences, San Jose, CA), and data analysed using Summit® V 3.1 software, (Cytomation Inc, USA).

Statistical analysis

A two-tailed Student's t-test determined the probability of difference and a p- value <0.05 was considered significant. A Chi-square test was used to evaluate significance of McI-I promoter inserts in GBM versus normal control samples. All statistical tests were two-sided. Correlation was determined using a parametric method and correlation coefficient determined.

RESULTS McI-I is the dominant anti-apoptotic Bcl-2-related protein in high grade glioma Expression of anti-apoptotic Bcl-2 family members was assessed in a series of resected gliomas and glioma cell lines. Expression of Bcl-2, Bcl-xL and McI-I mRNA was determined by quantitative PCR (qPCR) in 37 resected brain tumours as well as normal brain (Figure IA). The expression profile was characterised by substantial levels of mRNA encoding McI-I and to a lesser degree Bcl-xL in glioma tissues; 46% (17/37) of samples expressed > 1.5* 10⁴ transcript copies of McI-I per 10⁸ copies of 18S. Significantly lower levels of Bcl-2 were noted in all clinical samples. In fact, McI-I and Bcl-xL expression was in general three orders of magnitude higher than Bcl-2, which was not expressed at significant levels in any of these cases. Interestingly, high correlation (r=0.90) between Bcl-xL and McI-I was noted in glioma tissues while no significant correlation with Bcl-2 was identified. Anti-apoptotic protein levels also were evaluated in four common GBM lines.

Notably three (U87, Ul 18 and D645) of the four lines tested by western blot (Figure IB) expressed McI-I, the exception being U373. All four lines expressed Bcl-xL, with U87 and D645 expressing high levels, while Bcl-2 was detected in Ul 18 only. Immunocytochemical analysis of McI-I in these lines highlighted the same expression pattern (Figure 1C). Taken together, this expression profile suggested a significant role of McI-I, and to a lesser extent Bcl-xL rather than Bcl-2, in brain tumour cell survival. Identification of a novel SNP in an ETS site in the Md-I promoter

Sequence analysis of GBM tissue samples and normal control DNA revealed the presence of a previously unidentified single nucleotide polymorphism (SNP) in the McI-I promoter in high grade glioma. The G>A SNP was located in a consensus ETS transcription factor binding site located 126 bp upstream of the major transcription start site (Figure 2A). The -126G>A SNP was identified in the GBM cell lines U251 and U373 and in 2/75 normal control PBMC samples tested. The SNP was not identified in any of 45 GBM/astrocytoma tumour samples, consistent with both the low frequency in normal samples and an anti-apoptotic/ tumour promoting function of McI-I .

Notably, the cells lines U251 and U373, which harboured the ETS SNP, had the lowest McI-I mRNA expression of 12 GBM cell lines tested (Figure 2B and Figure IB & C for U373 protein expression). This data provided an insight that the SNP may affect gene transcription. To determine if indeed the SNP was functional, the effect of the -126G>A substitution on McI-I promoter activity was investigated by reporter gene assay in four GBM cell lines (Figure 2C). The McI-I promoter -126G>A SNP resulted in statistically significant reductions (p<0.0l) in promoter activity (U87 36%, Ul 18 50%, U251 42% and D645 53%) when compared to WT promoter activity.

To determine if the -126G>A SNP reduced or ablated DNA binding by nuclear protein(s), electrophoretic mobility shift assays (EMSA) were performed. Nuclear extracts were prepared from four GBM cell lines and the ability to bind nuclear extracts of a probe representing nucleotides -134 to -1 15 of the WT McI-I promoter and an otherwise identical probe containing the -126G>A substitution, was compared (Figure 2D). EMSA results revealed two complexes formed between WT probe and nuclear extracts from each of the four GBM cell lines (arrowed in Figure 2D) which were not detected or only weakly detected (Ul 18) with the -126G>A SNP probe. Cold competition EMSA with Ul 18 nuclear extracts demonstrated these complexes were specific as they were competed out by the WT probe but not the -126G>A SNP probe, nor by an unrelated double stranded oligonucleotide (Figure 2E). Taken together, this data demonstrates that the -126G>A SNP specifically ablates DNA binding by nuclear protein(s) to this site.

ELK4 is the dominant ETS family member in high grade glioma

Given the identification of a functional consensus ETS-binding site within the McI-I promoter we sought to confirm that this site bound an ETS family transcription factor in GBM. As a first step we surveyed ETS family member expression in GBM. Five primary GBM tumour samples and five GBM cell lines were measured for ETS family expression by qPCR. Twenty seven ETS family members were investigated, of which 12 showed some degree of expression (Figure 3A,B)- Notably, both four of the five GBM tumour samples and four of the five GBM cell lines expressed substantial levels of ELK4 compared to other ETS family members. ETS-I and ETS-2 also were detected but at generally lower levels in selected samples. Given this expression data, ELK4, and to a lesser extent ETS- 1 or ETS-2, were the strongest candidates for binding to the putative ETS transcription factor binding site in the McI-I promoter.

To determine if ELK4 bound the McI-I promoter ETS site, EMSA supershift and chromatin immunoprecipitation (ChIP) assays were conducted. Nuclear extracts from four GBM cell lines were tested in the supershift assay using an ELK.4 specific antibody (Figure 3C). In the presence of the antibody two super-shifted complexes (arrowed in Figure 3C) were detected in all four lines. The appearances of these bands correlated with a decrease in intensity of the primary bands in all samples. The demonstration of an ELK4/Mcl-1 promoter probe/antibody complex prompted us to use a ChIP assay to determine whether ELK4 bound the endogenous McI-I promoter in GBM cell lines (Figure 3D). ELK4 binding to the McI-I promoter was detected using two ELK4 antibodies. Greater immunoprecipitation was noted with the H- 167 antibody which binds ELK4 furthest from its ETS DNA binding domain. The H- 167 antibody was selected for testing by qPCR in the Ul 18 line (Figure 3E), and a four cycle threshold difference between the antibody and control was found. Together these results show that ELK4 binds to the McI-I promoter ETS site in GBM cells. ELK4 is a key regulator of McI-I expression in high grade glioma

These results suggested to us that ELK4 is likely a positive regulator of McI-I expression. To further establish a connection between ELK4 and McI-I expression in high grade glioma we investigated the correlation between ELK4 and McI-I mRNA expression using qPCR in 38 high grade glioma tissue samples (Figure 4A). Sequence analysis determined that none of the glioma tissue tested contained the -126G>A SNP. Expression results show a highly significant correlation (r=0.96) between ELK4 and McI-I mRNA levels in GBM tissue.

We also compared the level of ELK4 and McI-I mRNA in three GBM cell lines, two of which contained the -126G>A SNP (Figure 4B). Significant and comparable ELK4 expression was detected in all lines, however U87, which does not harbour the - 126G>A SNP, showed a significantly greater level of McI-I expression. Both U251 and U373 are heterozygous for the -126G>A (ETS) SNP. Although expressing equivalent or in the case of U373 elevated levels of ELK4 mRNA relative to U87, both showed significantly reduced McI-I mRNA, expression, far less than normally would be expected from the remaining WT McI-I allele. The reason for this is currently unclear. To determine if ELK.4 and not ETS-I or ETS-2 was the critical ETS family member regulating McI-I expression in GBM we used siRNA technology to down regulate ELK4 (Figure 4C). Two specific siRNAs were selected which mapped to different regions of ELK4. Results show substantial down regulation of ELK.4 following 24 hours treatment with both siRNA duplexes in the GBM cell line U87. Significantly, the reduction in ELK4 levels resulted in loss of McI-I expression. Although greater knockdown was achieved with siRNA#2, the small amounts of ELK4 detected using siRNA#l still resulted in loss of the McI-I signal. To determine if increased levels of ELK4 would have the reverse effect on McI-I levels, ELK4 cDNA was stably overexpressed in the Ul 18 GBM cell line (Figure 4D). Results show a four-fold increase in McI-I expression accompanying the increase in ELK4. Together these results establish ELK4 as a key regulator of McI-I expression in glioma cells. Md-I is a critical survival determinant in GBM

To confirm McI-I was conferring a survival advantage in cell lines free of the ETS SNP we utilised shRNA to down regulate McI-I in U87, D645 and U373 cells. The McI-I shRNA sequence was shown to effectively down regulate McI-I (Figure 5A). McI-I down regulation induced apoptosis in both U87 (64%) and D645 (33%) while the already low McI-I U373 ETS SNP line was only mildly affected (Figure 5B). Partial apoptotic responses when McI-I was down regulated could in part be explained by the relatively high levels of Bcl-xL which maintained survival in a subset of the cell population. To explore this, the BH3 mimetic ABT-737 (Abbott Pharmaceuticals) was employed. ABT-737 binds and inhibits Bcl-2 and Bcl-xL with high affinity but has no effect on McI-I at concentrations of < lμM (Oltersdorf et al., 2005). ABT-737 was applied to U87 and D645 GBM cells when McI-I was down regulated using shRNA. Glioma cells positively expressing McI-I shRNA and control shRNA were subjected to ABT-737 (lμM) 24 hours post shRNA treatment (Figure 5C). Results show increases in cells undergoing apoptosis when McI-I was down regulated in conjunction with ABT-737 treatment, U87 (94%) and D645 (77%). This data suggests either Bcl-xL or Bcl-2 could promote survival in a sub-population of these lines. Given the elevated expression of Bcl-xL over Bcl-2 it could be expected that Bcl-xL may be causing the effects seen in these two lines.

To further substantiate this data we also inhibited McI-I function via an alternative method. A NOXA peptide previously shown to neutralise McI-I (Chen et al., 2005), was utilised. U87 was treated with NOXA and control peptides alone and in combination with ABT-737 (l μM) or cisplatin (lμM) and assayed for apoptosis using Annexin V staining (Figure 5D). Results show increased apoptosis (>20%) (p<.05) compared to the control peptide. Furthermore increased apoptosis was achieved by the addition of ABT-737 or cisplatin treatment.

Targeted reduction of ELK4 increases GBM cell line sensitivity to ABT-737 and cisplatin treatment To determine the sensitivity of glioblastoma to chemotherapy treatment and to inhibition of both Bcl-xL and Bcl-2, five high grade glioma cell lines were treated with either cisplatin (cis-diamminedichloroplatinum(II)/cisplatinum) (lμM), ABT-737 (lμM), or both agents combined. Forty-eight hours post treatment cells were assayed for apoptosis by Annexin V staining (Figure 6A). U87, Ul 18 and D645 lines were largely resistant to these treatments, however the low McI-I -126G>A ETS SNP lines U251 and U373 showed greater sensitivity (p<.05) to both ABT-737 and cisplatin as well as combined ABT-737/cisplatin treatment. To determine if stable reduction of ELK4 would increase GBM sensitivity to ABT-737 or cisplatin treatment, the more effective ELK4 siRNA sequence (#2, Figure 4C) was cloned into the pSuperior shRNA vector. The U87 GBM cell line was stably transfected with ELK4 shRNA and a control shRNA sequence. Following stable knockdown of ELK4, U87 cells were treated with ABT-737 (lμM), cisplatin (lμM) or ABT-737/cisplatin (lμM) (Figure 5B). As expected, stable down regulation of ELK4, resulting in lower McI-I, sensitised these cells to both agents resulting in increased apoptosis compared to control treated cells. Combined treatment with ABT-737 and cisplatin (lμM) resulted in >20% (p<.05) of cells undergoing apoptosis compared with <10% apoptosis for control treated cells.

Given that ELK4 knockdown sensitised glioma cells to chemotherapy-induced apoptosis, overexpression of ELK4, resulting in increased levels of McI-I protein, was anticipated to confer increased survival in GBM cells. To determine this ELK.4 was overexpressed in the Ul 18 GBM cell line leading to increased McI-I (Figure 4D). Ul 18 control cells and ELK4 overexpressing cells were treated with the common chemotherapy agent cisplatin (lOμM). Induction of apoptosis was determined using Annexin V staining 72 hours post cisplatin treatment (Figure 6C). As expected, results show increased protection from apoptosis when ELK4 was overexpressed (17% apoptosis) (p<.05) relative to the control cells (25% apoptosis), highlighting the protective effect of McI-I against elevated concentrations of cisplatin treatment. Expression of Bcl-2-related proteins in GBM neurosphere cultures

Neurosphere cultures represent a serum-free selective culture for the expansion of adult neural stem cells in the presence of mitogens EGF and bFGF (Reynolds et al., 1992; Rietze and Reynolds, 2006). Growth under neurosphere conditions is a selective culture in which differentiated cells die whereas stem and progenitor cells respond to growth factors and divide to form 'neurospheres'. Recently identified 'brain tumour stem cells' are yet to be fully characterised, but also grow as neurospheres and exhibit self renewal and maintenance, generate large numbers of progeny and retain glial and neuronal multilineage potential (Vescovi et al, 2006). Interestingly, GBM cells grown as neurospheres more closely mirror the phenotype and genotype of primary tumours than do serum grown lines and therefore may represent a better approach to analyse GBM in-vitro (Lee et al., 2006).

Neurosphere lines were generated from a primary resected GBM tissue sample (LIB) and the GBM cell line U87 (U87-NS). Both neurosphere cultures showed self renewal capability, with U87-NS and LIB persisting over 50 cell passages with no apparent morphological or growth characteristic changes. The ability of long term serum cultured GBM cell lines to be grown as neurospheres in the presence of mitogens without serum suggests a stem-like cell is maintained within this population. After removal of mitogens (bFGF and EGF) and the addition of serum (2%), neurosphere cultures showed expression increases in mRNA levels and stained positive for neuronal (Dlll-tubulin) and glial (glial fibrillary acid protein, myelin basic protein) lineage markers (Figure 7A,B). Consistent with previous reports (Hemmati et al, 2003; Galli et al., 2004), differentiated cultures co-stained for multilineage markers highlighting the incomplete differentiation status of these cultures. Expression of anti-apoptotic Bcl-2 family members was assessed in neurosphere cultures using qPCR. Bcl-2, Bcl-xL and McI-I expression was two to three orders of magnitude lower in neurospheres compared to when differentiated (2% serum). Interestingly, the expression pattern was consistent between the primary tumour isolated neurosphere line and the GBM cell line derived neurosphere line (Figure 7C). Effects of ABT-737 and cisplatin on brain tumour stem/progenitor cells Given the lower expression levels of Bcl-2 family members, particularly McI-I and Bcl-xL, in the neurosphere cultures we sought to determine whether they display greater sensitivity to ABT-737 and cisplatin treatment. The short-term effects on neurosphere cultures were compared with neurosphere cells differentiated in serum. Analysis of apoptosis was conducted 48-72 hours post agent application (Figure 8A). Results for the LIB neurosphere line are shown, demonstrating partial sensitivity to ABT-737 (0.5μM) and cisplatin (0.5μM) with a 24% and 15% increase respectively in apoptotic cells. A greater than additive effect was observed when ABT-737 and cisplatin were combined, resulting in 48% of cells Annexin V positive as opposed to only 6% when the cells were differentiated. The differentiated cells remained largely resistant to both these agents applied alone and in combination. We also sought to determine the sensitivity of neurospheres to McI- 1 inhibition. To assess this we treated the LIB neurospheres with NOXA and control peptides alone and in combination with ABT-737 (lμM) or cisplatin (lμM) and assayed for apoptosis using Annexin V staining (Figure 8B). Results show increased apoptosis (>50%) (p<.05) compared to the control peptide. Furthermore increased apoptosis was achieved by the addition of ABT-737 or cisplatin treatment.

The ability of these lines to form neurospheres in the presence of cisplatin and ABT-737 treatment was determined (Figure 8C). LIB neurospheres show a 15% reduction (p=.O2) in total sphere number after single agent treatment, and a 93% reduction (p=.0003) in total spheres when these agents were combined. The effects of these treatments on sphere diameter were also determined (Figure 8D). Results show that cisplatin increased sphere diameter, however this was not found to be significant. ABT-737 treatment alone resulted in a 16% reduction (p=.O5) and a 35% reduction (p=.0l) in sphere diameter (>50μM) when these agents were combined.

A recent report by Louis et al., 2008, has highlighted that secondary or tertiary spheres contain progenitor cells only and exhibit a limited proliferative potential, resulting in smaller sphere size, while spheres that show a high proliferative potential with the ability to form large spheres in semi-solid medium (<3%) exhibited the cardinal in-vitro properties of a stem cell. Based on these findings agents alone or in combination that result in a decrease in sphere size may be affecting the underlying stem cell population.

To determine the long term effects of these agents on the tumour initiating cells within this population the neurosphere assay was used (Reynolds and Rietze, 2005). LIB neurospheres were serially passaged seven times (over seven weeks) and the total theoretical cell number calculated. All cultures were grown in triplicate and agents reapplied after each passage (Figure 8E). LIB control cultures generated IxIO⁹ cells over the course of the experiment; this was reduced to 1.2* 10⁸ cells following ABT-737 (0.5 μM) treatment. Cisplatin had an even greater effect. Following seven weeks of cisplatin treatment no cells remained within this population. Notably when these agents were combined significant cell death was observed, and after two weeks of treatment no cells remained. ABT-737 and cisplatin combined resulted in complete cell death five weeks earlier than cisplatin treatment alone. These findings were consistent with the combined agent result from the sphere formation and diameter assays. Importantly cisplatin and ABT-737/cisplatin combined were able to prevent extensive self renewal and generation of a large number of progeny in these cultures.

ELK4/Mcl-1 correlation in clinical tumour specimens

ELK4 and McI-I mRNA expression was determined by qPCR in kidney, lung, thyroid and colon tumour specimens. Eight clinical tumour specimens were tested for kidney (renal cell carcinoma), lung and thyroid cancer using an OriGene TissueScan

Oncology Survey Tissue qPCR Array I (Cat. no. CSRT501, Lot no. 0508). Colon expression data was obtained from seven colon cancer cell lines. Correlation was determined using a parametric method and the correlation coefficient determined, β- actin was used in all cases as the reference gene. The results, shown in Table 3, indicate a very high statistical correlation also exists between ELK4 and McI-I mRNA expression in kidney, lung, thyroid and colon cancer in addition to glioblastoma.

DISCUSSION For anyone newly diagnosed with GBM the prognosis is dismal. Current treatment, involving surgical debulking of the tumour, where possible, followed by radiotherapy and chemotherapy (with cisplatin or temozolomide), is almost never curative. There is a pressing need to identify new therapeutic targets if the outlook is to be improved.

One potential new small molecule inhibitor with promise in this regard is the BH3 mimetic ABT-737. ABT-737 targets prosurvival members of the Bcl-2 family of proteins which are key regulators of apoptosis. Impaired apoptosis is a feature of many cancers that contributes to the resistance of malignant cells to conventional cytotoxic therapy. Frequently the capacity of the Bcl-2 family proteins to induce apoptosis in malignant cells is subverted either because a prosurvival/anti-apoptotic member of the family is overexpressed or because mutations in the p53 pathway ablate induction by p53 of the BH3-only proteins Puma and Noxa, which otherwise trigger apoptosis (van delft et al., 2006). ABT-737 targets prosurvival/anti-apoptotic Bcl-2 proteins Bcl-2, BcI- xL and Bcl-w, but not McI-I which confers resistance to this novel agent. McI-I thus represents a critical determinant of ABT-737 sensitivity and resistance, such that McI-I down regulation by various pharmacologic agents or genetic approaches dramatically increases ABT-737 lethality in diverse malignant cell types (Dai and Grant, 2007).

In this report we investigated expression of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL and McI-I in both human primary brain tumours and GBM cell lines and found McI-I to be the most highly expressed. Down regulation of McI-I by shRNA-mediated RNA interference sensitised GBM cell lines to ABT-737-induced apoptosis as did treatment with a Noxa peptide. This prompted us to investigate the molecular basis for elevated McI-I expression in GBM with the aim of identifying potentially novel therapeutic targets.

Genomic analysis of the McI-I promoter revealed that insertions of 6bp and 18bp, reported previously in chronic lymphocytic leukaemia (CLL), were common to both GBM tissue samples and normal control samples. Whilst a previous study (Moshynska et al., 2004) suggested that these insertions resulted in increased promoter activity in leukaemic cells, we found that only small and statistically insignificant increases in promoter activity were observed in GBM cells. Furthermore, no correlation was found between McI-I mRNA expression and the presence of insertions in GBM. This data, taken together, suggests minimal effects of these insertions on McI-I promoter activity in both GBM and CLL and, given the high frequency in normal subjects, suggests that they are unlikely to be a significant determinant of disease progression.

More significantly though, we identified a novel, functional SNP (-126G>A) in the McI-I promoter in GBM, the presence of which resulted in a marked decrease in promoter activity. Two GBM cell lines, identified as being heterozygous for the - 126G>A SNP, also exhibited significantly reduced McI-I levels. Interestingly, the extent of McI-I suppression was far greater than expected in these lines given that only one allele harboured the SNP. The potential therapeutic significance of this is clear and the molecular basis responsible for it warrants further investigation.

Given that the SNP was contained in a potential ETS transcription factor binding site, and appeared to disrupt the consensus binding sequence, we sought evidence of binding by a cognate ETS family transcription factor. We demonstrate this to be ELK4 in GBM cells and furthermore provide compelling evidence, for the first time, that ELK4 is both a key regulator of McI-I promoter activity and McI-I expression in GBM and a potential target for downregulating McI-I and enhancing sensitivity to both ABT- 737 and cisplatin treatment in high grade glioma.

The elevated expression levels of both ELK4 and McI-I in a significant proportion of brain tumours suggests ELK4 may play a critical role in mediating McI-I expression in the oncogenic pathway leading to GBM.

Both ELK4 and McI-I are highlighted by this study as potential targets for therapeutic intervention in high grade gliomas. Consideration of the respective phenotypes of McI-I and ELK4 knockout mice may be instructive in this regard. Previous reports indicate McI-I knockout mice die at early embryonic stages (Rinkenberger et al., 2000), with McI-I critical for lymphocyte and haematopoietic stem cell survival (Opferman et al., 2003; Opferman et al., 2005). Conditional deletion of McI-I in mice shows it to be essential for neutrophil survival (Dzhagalov., et al 2007), while inhibition of McI-I expression by antisense .oligonucleotides also results in human macrophage apoptosis (Liu., et al 2001). In contrast, ELK4-deficient mice reportedly are viable and fertile with no gross physical abnormalities although some immune suppression was noted with a reduction in single-positive thymocytes and peripheral T cell numbers (Costello et al 2004). Given the much milder phenotype of ELK.4 knockout mice, targeting ELK.4 may result in fewer unwanted side effects and thus provide a more attractive target for down regulating McI-I and sensitising to apoptosis in GBM. The data in Table 3 extend these GBM observations to other cancers such as lung, colon, thyroid and kidney, which indicates that McI-I and/or Elk4 may also be useful in the diagnosis of these cancers, determining the sensitivity of patients to cancer treatment and also for the development of anti-cancer agents for these particular cancers. Therefore the novel, functional McI-I promoter ETS SNP, although identified at low frequencies, clearly reduces McI-I expression and may prove a useful prognostic marker for cancer survival outcome or a marker which determines treatment strategies.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Table 1

Table 2

Table 3

(ETS family transcription factor Q-PCR primers)

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REFERENCES

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Claims

1. A method of designing, engineering, screening or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly reduces or inhibits the activity and/or expression of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter.

2. The method of Claim 1 wherein the cancer is selected from the group consisting of glioma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

3. A method of designing, engineering, screening or otherwise producing a cancer therapeutic agent, said method including the step of identifying a candidate agent which at least partly reduces inhibits the activity of a myeloid cell leukemia- 1 (McI-I) protein, or transcription of an mRNA encoding said protein, wherein the cancer is selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

4. The method of any one of Claims 1-3, for designing, engineering, screening or otherwise producing a cancer therapeutic agent that sensitizes, or increases the responsiveness of, a cancer cell to one or more pro-apoptotic agents.

5. The method of Claim 4, wherein the one or more pro-apoptotic agents target one or more members of the Bcl-2 protein family.

6. A cancer therapeutic agent designed, engineered, screened or otherwise produced according to the method of any one of Claims 1 -5.

7. A pharmaceutical composition comprising the cancer therapeutic agent of Claim 6 and a pharmaceutically acceptable carrier, diluent or excipient.

8. The pharmaceutical composition of Claim 7, further comprising one or more pro-apoptotic agents.

9. The pharmaceutical composition of Claim 8, wherein the one or more pro- apoptotic agents target one or more members of the Bcl-2 protein family.

10. The pharmaceutical composition of Claim 9, wherein the one or more pro- apoptotic agents include ABT-377.

11. A method of treating cancer in a mammal, said method including the step of reducing the activity and/or expression an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter in said mammal to thereby treat, sensitize or improve the responsiveness of said mammal to cancer treatment.

12. The method of Claim 11, wherein the cancer is selected from the group consisting of glioma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

13. A method of treating cancer in a mammal, said method including the step of reducing myeloid cell leukemia- 1 (McI-I) protein activity and/or expression, or transcription of an mRNA encoding said protein, in said mammal to thereby treat, sensitize or improve the responsiveness of said mammal to cancer treatment, wherein the cancer is selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

14. The method of any one of Claims 1 1-13, wherein the method includes administering the pharmaceutical composition of any one of Claims 7-10.

15. The method of any one of Claims 1 1-14 wherein the cancer is glioma.

16. The method of any one of Claims 11-15, wherein the mammal is a human.

17. An isolated nucleic acid comprising a nucleotide sequence of at least a fragment of a myeloid cell leukemia- 1 (McI-I) gene promoter, which nucleotide sequence comprises a polymorphism in an ETS transcription factor binding site associated with reduced binding and/or activity of an ETS transcription factor.

18. The isolated nucleic acid of Claim 17, wherein the polymorphism is a single nucleotide polymorphism (SNP).

19. The isolated nucleic acid of Claim 18, wherein the SNP is located at position - 126 relative to the major transcription start site of the myeloid cell leukemia- 1 (McI-I) promoter in an ETS transcription factor binding site.

20. The isolated nucleic acid of Claim 19, wherein the SNP is a G>A SNP.

21. The isolated nucleic acid of any preceding claim, wherein the ETS transcription factor is ELK4.

22. An isolated nucleoprotein complex comprising the isolated nucleic acid of any one of Claims 17-21; and said ETS transcription factor.

23. A chimeric gene comprising the isolated nucleic acid of any one of Claims 17- 22, operably linked or connected to an expressible nucleotide sequence.

24. A recombinant vector comprising the chimeric gene of Claim 23.

25. A method of determining the susceptibility of a mammal to cancer, said method including the step of determining whether a myeloid cell leukemia- 1 (McI-I) gene promoter of said mammal comprises a nucleotide sequence polymorphism in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor, wherein said polymorphism indicates the susceptibility of said mammal to cancer

26. The method of Claim 25, wherein the polymorphism is a single nucleotide polymorphism (SNP).

27. The method of Claim 26, wherein the SNP is located at position -126 relative to the major transcription start site of the myeloid cell leukemia- 1 (McI-I) promoter in an ETS transcription factor binding site.

28. The method of Claim 27, wherein: an A at position -126 indicates that said mammal has a relatively lower or reduced susceptibility to cancer; and a G at position -

126 indicates that said mammal has a relatively higher or increased susceptibility to cancer

29. A method of determining responsiveness of a mammal to cancer therapy, said method including the step of determining whether a myeloid cell leukemia- 1 (McI-I) gene promoter of said mammal comprises a nucleotide sequence polymorphism in an ETS transcription factor binding site that affects binding and/or activity of an ETS transcription factor, wherein said polymorphism indicates responsiveness of said mammal to cancer therapy.

30. The method of Claim 29 wherein the polymorphism is a single nucleotide polymorphism (SNP).

31. The method of Claim 30, wherein the SNP is located at position -126 relative to the major transcription start site of the myeloid cell leukemia- 1 (McI-I) promoter in an ETS transcription factor binding site.

32. The method of Claim 31, wherein: (a) an A at position -126 indicates relatively increased or greater responsiveness of said mammal to cancer therapy; and (b) a G at position -126 indicates relatively decreased or lower responsiveness of said mammal to cancer therapy.

33. A method of determining the susceptibility of a mammal to a cancer selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer, said method including the step of determining a level of expression of a myeloid cell leukemia- 1 (McI-I) protein, or nucleic acid encoding said myeloid cell leukemia- 1 (McI-I) protein, in said mammal to thereby determine the susceptibility of said mammal to said cancer.

34. The method of Claim 33, wherein (a) a relatively increased or higher level of expression of McI-I protein or encoding nucleic acid indicates a relatively higher or increased susceptibility to said cancer; and (b) a decreased or lower level of expression of McI-I protein or encoding nucleic acid indicates a relatively reduced or lower susceptibility to said cancer.

35. A method of determining responsiveness of a mammal to cancer therapy, said cancer selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer, said method including the step of determining a level of expression of a myeloid cell leukemia- 1 (McI-I) protein, or nucleic acid encoding said myeloid cell leukemia- 1 (McI-I) protein, in said mammal to thereby determine responsiveness of said mammal to cancer therapy.

36. The method of Claim 35, wherein (a) a relatively increased or higher level of expression of McI-I protein or encoding nucleic acid indicates a relatively decreased or lower responsiveness of said mammal to cancer therapy; and (b) a relatively decreased or lower level of expression of McI-I protein or encoding nucleic acid indicates a relatively increased or greater responsiveness of said mammal to cancer therapy.

37. The method of any one of Claims 25-36, wherein the cancer is selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

38. A method of determining the susceptibility of a mammal to cancer, said method including the step of determining a level of expression of an ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter, or a nucleic acid encoding said ETS transcription factor, in said mammal to thereby determine the susceptibility of said mammal to cancer.

39. The method of Claim 38, wherein (a) a relatively increased or higher level of expression of said ETS transcription factor indicates an increased susceptibility to cancer; and (b) a decreased or lower level of expression of said ETS transcription factor indicates a relatively reduced or lower susceptibility to cancer.

40. A method of determining the responsiveness of a mammal to cancer therapy, said method including the step of determining a level of expression of an ETS transcription factor, or a nucleic acid encoding said ETS transcription factor that binds a myeloid cell leukemia- 1 (McI-I) gene promoter, in said mammal to thereby determine the responsiveness of said mammal to cancer therapy.

41. The method of Claim 40, wherein (a) a relatively increased or higher level of expression of said ETS transcription factor indicates a relatively decreased or lower responsiveness of said mammal to cancer therapy; and (b) a relatively decreased or lower level of expression of said ETS transcription factor indicates a relatively increased or greater responsiveness of said mammal to cancer therapy.

42. The method of any one of Claims 1-5 and 1 1-41, wherein the ETS transcription factor is ELK4/Sapla.

43. The method of any one of Claims 25-37, wherein the cancer is selected from the group consisting of glioma, melanoma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

44. The method of any one of Claims 38-41, wherein the cancer is selected from the group consisting of glioma, kidney cancer, colon cancer, thyroid cancer and lung cancer.

45. The method of Claim 43 or Claim 44 wherein the cancer is glioma.

46. The method of any one of Claims 29-32, 35-37 and 40-45 wherein cancer therapy includes treatment of said mammal with one or more pro-apoptotic agents.