WO2009144460A1

WO2009144460A1 - Markers for prostate cancer

Info

Publication number: WO2009144460A1
Application number: PCT/GB2009/001334
Authority: WO
Inventors: Colin Cooper; Jeremy Clark; Gerhardt Attard; Johann De Bono; Jack Cuzick
Original assignee: The Institute Of Cancer Research: Royal Cancer Hospital
Priority date: 2008-05-28
Filing date: 2009-05-28
Publication date: 2009-12-03
Also published as: GB0809689D0

Abstract

The present invention relates to markers for prostate cancer. In particular, the invention relates to the identification of novel fusions between ETS family genes, particularly ETVl, and the ACSL3 gene. Such fusion genes and their expression products can be used as diagnostic and prognostic markers for prostate cancer and as clinical targets for the treatment of prostate cancer.

Description

Markers for prostate cancer

Field of the invention

The present invention relates to markers for prostate cancer. In particular, the invention relates to gene fusions as diagnostic and prognostic markers for prostate cancer and as clinical targets for the treatment of prostate cancer.

Background of the invention Fusion of the prostate specific androgen-regulated gene

TMPRSS2 gene to the ETS transcription factor gene ERG has been reported to be a common event in prostate cancer (Tomlins et al. , 2005; Tomlins et al., 2006; Clark et al., 2006; Hermans et al., 2006; Iljin et al., 2006; Perner et al., 2006; Soller et al., 2006; Wang J et al., 2006; Yoshimoto et al., 2006) . Less frequently, TMPRSS2 becomes fused to ETVl or ETV4 (Tomlins et al., 2005; Tomlins et al., 2006; Hermans et al., 2006) , or to ETV5 (Helgeson et al., 2008) . In all cases, a TMPRSS2-ETS chimaeric gene is generated, resulting in high level expression of the fused 3' -ETS gene sequences. The reported incidence of TMPRSS2 : ETVl fusion in these studies (1- 2%) is, however, considerably lower than the observed incidence of ETVl gene overexpression (-10%) in prostate cancer (Tomlins et al., 2005), suggesting that there are alterative mechanisms of ETVl overexpression. Further studies have led to the identification of other 5' -fusion ETVl partners, including the prostate specific androgen- induced gene SLC45A3/ Prostein, an endogenous retroviral element HERV- K, a prostate-specific androgen- repressed gene C15orf21 , and a strongly expressed housekeeping gene HNRPA2B1 (Tomlins et al., 2007) . SLC45A3/Prostein has also been identified as a 5'- fusion partner for ETV5 (Helgeson et al., 2008) .

In addition, it has been found that in the two prostate cancer cell lines, LNCaP and MDA-PCa2B, outlier expression of ETVl was caused through the entire ETVl locus at 7p21 becoming juxtaposed to sequences 14ql3.3-14q21.1. By characterizing the expression in LNCaP cells of four contiguous genes within the region 14ql3.3-14q21.1 (SLC25A21, MIPOLl, FOXAl and TTC6) , as well as the expression of ETVl, it has been shown that this region (i.e. 14ql3.3-14q21.1) exhibits prostate-specific expression that is co-ordinately regulated by androgens (Tomlins et al. , 2007) . In this study, only single cases of the fusion were reported, with the exception of the juxtaposition of ETVl sequences to 14ql3.3-14q21.1 where two cases were observed. Therefore, it was not possible to assess the relative importance of the different fusion partners and of TMPRSS2.

Several studies have demonstrated a link between ERG gene re- arrangements and clinicopathological indicators (Demichelis F et al., 2007; Wang J et al., 2006; Nam RK et al., 2007; Perner et al., 2006) . In a watchful waiting cohort of 111 patients, a significant link has been reported between the presence of ERG alterations and prostate cancer specific death (Demichelis F et al., 2007) . In a series of 165 patients who underwent prostatectomy, it was found that the presence of a TMPRSS2:ERG fusion was associated with a greater probability of biochemical disease relapse (Nam RK et al., 2007) . Additionally, the present inventors have recently demonstrated that loss of 5' -ERG sequences coupled with duplication of

TMPRSS2 : ERG fusion sequences predicts extremely poor cancer specific survival independently of Gleason score and PSA level at diagnosis (Attard G et al., 2007) . In contrast, very little is known about the clinical significance of alteration at the ETVl gene locus.

Several groups have published androgen receptor (AR) ChIP-chip studies mapping AR binding sites within the human genome (Bolton et al., 2007; Massie et al., 2007; Wang et al., 2007; Takayama et al., 2007) . Wang et al. identified a functional AR binding site 13.5kb upstream of the TMPRSS2 gene (Wang et al., 2007) . Both Massie et al., and Wang et al. have proposed mechanisms for AR recruitment to subsets of target sequences through associations between the AR and other transcription factors for example, GATA-2, Octl, FoxAl and ETSl (Massie et al., 2007; Wang et al., 2007) .

Summary of the invention

At its most general, the invention relates to gene fusions, which can act as markers for the diagnosis and prognosis of prostate cancer and as clinical targets for the treatment of prostate cancer.

The present inventors have identified the androgen- regulated acyl-CoA synthetase long-chain family member (ACSL3) gene as a 5' -fusion partner for the ETS family member gene, ETVl, in patients with prostate cancer. Thus, the present invention relates to this gene fusion, methods for its detection and its use as a marker for the diagnosis and prognosis of prostate cancer and as a target for the treatment of prostate cancer. Kits for detecting this gene fusion and for diagnosing prostate cancer are also provided.

According to one aspect of the invention, there may be provided an isolated nucleic acid molecule comprising an upstream component fused to a downstream component at a fusion site, wherein the upstream component comprises a portion of an acyl-CoA synthetase long-chain family member (ACSL3) gene and the downstream component comprises a portion of an ETS family member gene . The upstream component preferably includes an upstream region of the ACSL3 gene. The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5, but is preferably ETVl. The invention also encompasses the reverse complements of the nucleic acid molecules described herein. The isolated nucleic acid molecule of the invention may be genomic DNA, cDNA or mRNA.

At the fusion site of the isolated nucleic acid molecule, a portion of the ACSL3 gene is contiguous with a portion of the ETS family member gene. Fusion between the ACSL3 gene and the ETS family member gene may occur in any region of the ACSL3 gene sequence and in any region of the ETS family member gene sequence. The portion of the ACSL3 gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the portion of the ETS family member gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the portion of the ACSL3 gene may be at least 20 nucleotides in length and the portion of the ETS family member gene may be at least 20 nucleotides in length. In one embodiment, the upstream component includes exons 1 and 2 and at least a portion of exon 3 of the ACSL3 gene and the downstream component includes exons 6 to 12 of the ETVl gene.

The invention also provides an expression vector comprising the isolated nucleic acid molecule described above and a host cell comprising this expression vector.

According to a further aspect of the invention, there may be provided a transgenic animal having a transgene comprising an upstream component fused to a downstream component at a fusion site, wherein the upstream component is a portion of an ACSL3 gene and the downstream component is a portion of an ETS family member gene. The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5 , but is preferably ETVl.

At the fusion site of the transgene, a portion of the ACSL3 gene is contiguous with a portion of the ETS family member gene. Fusion between the ACSL3 gene and the ETS family member gene may occur in any region of the ACSL3 gene sequence and in any region of the ETS family member gene sequence. The portion of the ACSL3 gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the portion of the ETS family member gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the portion of the ACSL3 gene may be at least 20 nucleotides in length and the portion of the ETS family member gene may be at least 20 nucleotides in length. In one embodiment, the upstream component includes exons 1 and 2 and at least a portion of exon 3 of the ACSL3 gene and the downstream component includes exons 6 to 12 of the ETVl gene.

In a further aspect of the invention, there may be provided a targeting oligonucleotide comprising a first nucleic acid sequence capable of hybridising to the sense or antisense strand of an ACSL3 gene, or to an mRNA sequence encoded thereby, and a second nucleic acid sequence capable of hybridising to the sense or antisense strand of an ETS family member gene, or to an mRNA sequence encoded thereby. Such targeting oligonucleotides inhibit, reduce or block expression of the fusion genes described herein (i.e. the nucleic acid molecules of the invention) by inhibiting, reducing or blocking their transcription or translation.

Preferably, the first nucleic acid sequence is complementary to a portion of the sense or antisense strand of an ACSL3 gene, or to the mRNA sequence transcribed therefrom, and the second nucleic acid sequence is complementary to a portion of the sense or antisense strand of an ETS family member gene, or to the mRNA transcribed therefrom. The first nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid sequence may be at least 20 nucleotides in length and the second nucleic acid sequence may be at least 20 nucleotides in length.

The first nucleic acid sequence may or may not be contiguous with the second nucleic acid sequence, i.e. there may be a spacer region between the first and second nucleic acid sequences .

The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5, but is preferably ETVl.

The targeting oligonucleotide may comprise any type of nucleic acid, such as genomic DNA, cDNA or RNA, but it is preferably RNA. The targeting oligonucleotide may also comprise modified nucleotides .

The targeting oligonucleotide may be an antisense oligonucleotide comprising a first nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ACSL3 gene, and a second nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ETS family member gene .

The invention also provides an siRNA molecule, an miRNA molecule or a triple helix inhibitior comprising the targeting oligonucleotide described above.

According to a further aspect of the invention, there may be provided a nucleic acid probe comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is capable of hybridising to the sense or antisense strand of an ACSL3 gene, or to mRNA encoded thereby, and the second nucleic acid sequence is capable of hybridising to the sense or antisense strand of an ETS family- member gene, or to mRNA encoded thereby. Accordingly, the nucleic acid probe is able to hybridise across the fusion site of the nucleic acid molecule of the invention. The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5 , but is preferably ETVl. The nucleic acid probe may comprise DNA, cDNA or RNA and may bind to DNA, cDNA or mRNA.

The first nucleic acid sequence may be complementary to a portion of the sense or antisense strand of an ACSL3 gene and the second nucleic acid sequence may be complementary to a portion of the sense of antisense strand of an ETS family member gene. The first nucleic sequence may also be complementary to mRNA encoded by the ACSL3 gene and the second nucleic acid may be complementary to the mRNA encoded by the ETS family member gene.

The first nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid sequence may be at least 20 nucleotides in length and the second nucleic acid sequence may be at least 20 nucleotides in length.

The first nucleic acid sequence may or may not be contiguous with the second nucleic acid sequence, i.e. there may be a spacer region between the first and second nucleic acid sequences. The nucleic acid probe may be labelled, e.g. fluorescently or radioactively. As such, the nucleic probe may be used for fluorescent in situ hybridisation (FISH) , or for Southern blotting (e.g. following pulsed field gel electrophoresis) . The nucleic acid probe may also be labelled with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5.

Alternatively, the nucleic acid probe may be a primer for use in PCR.

According to a further aspect of the invention, there may be provided a fusion protein comprising an N-terminal component and a C- terminal component, wherein the N-terminal component comprises a portion of an ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein. Preferably, the N-terminal component includes an N-terminal portion of the ACSL3 protein. The portion of the ACSL3 protein is preferably at least 5 amino acids, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, or at least 50 amino acids in length. The portion of the ETS family member protein is preferably at least 5 amino acids, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, or at least 50 amino acids in length. Any combination of these lengths is possible. For example, the portion of the ACSL3 protein may be at least 10 amino acids in length and the portion of the ETS family member protein may be at least 10 amino acids in length.

The ETS family member protein may be any ETS family member protein, such as ETVl, ETV4 , ERG or ETV5 , but is preferably ETVl. According to a further aspect of the invention, there may be provided a method of detecting a nucleic acid molecule in a sample, the nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene at a fusion site. The nucleic acid molecule to be detected may be genomic DNA, mRNA or cDNA. Preferably, the portion of the ACSL3 gene is an upstream portion of the ACSL3 gene. The ETS family member gene may be any ETS family member gene, such as ETVl, ETVi, ERG or ETV5 , but is preferably ETVl.

In one embodiment, the method may include the step of contacting the sample with a probe which binds to the ACSL3 gene and to the ETS family member gene, wherein binding of the probe to nucleic acid in the sample indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene. As such, the probe binds across the fusion site of the nucleic acid molecule. The probe may be the nucleic acid probe of the invention described above.

In another embodiment, the detection method may include the step of contacting the sample with a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to nucleic acid downstream of the ETS family member gene, such that co- localisation or juxtaposition of the first probe and the second probe indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene. Binding of the first and second probes to sequences on the same physical piece of nucleic acid (e.g. on the same chromosome or on the same piece of mRNA or cDNA) within 10 Mb, within 5 Mb, within 4 Mb, within 3 Mb, within 2 Mb, within 1 Mb, within 500 kb, within 400 kb, within 300 kb, within 200 kb or within 100 kb of each other indicates co-localisation or juxtaposition of the first and second probes, and therefore, the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene.

The nucleic acid (e.g. chromosomal DNA) upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene. The nucleic acid (e.g. chromosomal DNA) downstream of the ETS family member gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ETS family member gene. For example, the nucleic acid (e.g. chromosomal DNA) upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid (e.g. chromosomal DNA) downstream of the ETS family member gene may extend up to 2 Mb downstream of the ETS family member gene.

The first and second probes may be nucleic acid probes. The first nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. Similarly, the second nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 nucleotides in length.

Any combination of these lengths is possible. For example, the first nucleic acid probe may be at least 20 nucleotides in length and the second nucleic acid probe may be at least 20 nucleotides in length.

In one embodiment, the first probe is a nucleic acid probe having the same nucleic acid sequence as RP11-157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-27B1, RP11-138H16 or CTD-2008I15 shown in Table 4. The nucleic acid probes may be labelled, e.g. fluorescently or radioactively labelled. The nucleic acid probes may also be labelled with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5. Suitable detection methods include in situ hybridisation (ISH) (e.g. fluorescent in situ hybridisation (FISH)) and Southern blotting (e.g. following pulsed field gel electrophoresis) .

In yet another embodiment, the detection method may include the steps of: i) denaturing nucleic acid in the sample; (ii) annealing the first and second nucleic acid probes to a target nucleic acid sequence in the sample; and (iii) amplifying the target nucleic acid sequence; wherein amplification of the target nucleic sequence indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene. Such a detection method may be carried out using polymerase chain reaction (PCR) .

According to a further aspect of the invention, there may be provided a kit for detecting a nucleic acid molecule in a sample, the nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene at a fusion site. The nucleic acid molecule to be detected may be genomic DNA, mRNA or cDNA. Preferably, the portion of the

ACSL3 gene includes an upstream portion of the ACSL3 gene . The ETS family member gene may be any ETS family member gene, such as ETVl₁ ETV4, ERG or ETV5 , but is preferably ETVl. Therefore, the kit may be used in the detection method described above.

The kit may comprise the nucleic acid probe of the invention described above, said nucleic acid probe comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is capable of hybridising to the ACSL3 gene, or mRNA encoded thereby, and the second nucleic acid sequence is capable of hybridising to the ETS family member gene, or mRNA encoded thereby. Accordingly, the nucleic acid probe is able to hybridise across the fusion site of the nucleic acid molecule to be detected, which may be genomic DNA, cDNA or mRNA.

The first nucleic acid sequence is at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid sequence may be at least 20 nucleotides in length and the second nucleic acid sequence may be at least 20 nucleotides in length.

Alternatively, the kit may comprise a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to nucleic acid downstream of the ETS family member gene .

The nucleic acid further upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene. The nucleic acid downstream of the ETS family member gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ETS family member gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ETS family member gene may extend up to 2 Mb downstream of the ETS family member gene. The first and second probes may be nucleic acid probes. The first nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid probe may be at least 20 nucleotides in length and the second nucleic acid probe may be at least 20 nucleotides in length.

In one embodiment, the first probe is a nucleic acid probe having the same nucleic acid sequence as RP11-157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-27B1, RP11-138H16 or CTD-2008I15 shown in Table 4. The nucleic acid probes may be labelled, e.g. f luorescently or radioactively labelled. The nucleic acid probes may also be labelled with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5. Alternatively, the first and second nucleic acid probes may be suitable for use as PCR primers .

Accordingly, the kit may be used in a variety of detection methods, including in situ hybridisation (ISH) (e.g. fluorescent in situ hybridisation (FISH) ) , Southern blotting (e.g. following pulsed field gel electrophoresis), northern blotting and PCR.

According to a further aspect of the invention, there may be provided a method for detecting break-apart of an ACSL3 gene in a sample, wherein the method includes the step of contacting the sample with a first probe which binds to an upstream portion of the ACSL3 gene and/or to chromosomal DNA upstream of the ACSL3 gene and a second probe which binds to a downstream portion of the ACSL3 gene and/or to chromosomal DNA downstream of the ACSL3 gene, wherein binding of the first and second probes to physically separate, non-adjacent pieces of DNA (e.g. to different chromosomes) indicates break-apart of the ACSL3 gene.

The chromosomal DNA upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene.

The chromosomal DNA downstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ACSL3 gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ACSL3 gene may extend up to 2 Mb downstream of the ACSL3 gene.

The first and second probes may be nucleic acid probes. The first nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length.

In one embodiment, the first probe is a nucleic acid probe having the same nucleic acid sequence as RPIl- 157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-136M23 or RP11-749C15 shown in Table 4. The nucleic acid probe may be labelled, e.g. fluorescently or radioactively or with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5 and suitable detection methods include in situ hybridisation (ISH) (e.g. fluorescent in situ hybridisation (FISH) ) .

According to a further aspect of the invention, there may be provided a kit for detecting break-apart of the ACSL3 gene, the kit comprising a first probe which binds to an upstream portion of the ACSL3 gene and/or to chromosomal DNA upstream of the ACSL3 gene and a second probe which binds to a downstream portion of the ACSL3 gene and/or to chromosomal DNA downstream of the ACSL3 gene.

The first probe may bind to chromosomal DNA which extends up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene. The second probe may bind to chromosomal DNA which extends up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ACSL3 gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ACSL3 gene may extend up to 2 Mb downstream of the ACSL3 gene .

The first and second probes may be nucleic acid probes. The first nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid probe is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid probe may be at least 20 nucleotides in length and the second nucleic acid probe may be at least 20 nucleotides in length.

In one embodiment, the first probe may be a nucleic acid probe having the same nucleic acid sequence as RP11-157M20 and the second probe may be a nucleic acid probe having the same nucleic acid sequence as RP11-136M23 or RP11-749C15 shown in Table 4. The nucleic acid probes may be labelled, e.g. fluorescently or radioactively labelled, or labelled with digoxygenin (DIG) or biotin to allow detection using an anti- DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5 and suitable detection methods include in situ hybridisation (ISH) (e.g. fluorescent in situ hybridisation (FISH)) .

According to a further aspect of the invention, there may be provided a method of detecting the fusion protein of the invention in a sample, wherein the method includes the step of contacting the sample with an agent which binds specifically to the fusion protein. Preferably, the agent is an antibody which binds to an ACSL3 protein and to an ETS family member protein, such as ETVl, ETV4 , ERG or ETV5. There may also be provided a kit comprising this agent.

According to a further aspect of the invention, there may be provided a method of diagnosing prostate cancer in a patient.

In one embodiment, the method of diagnosing prostate cancer in a patient includes the steps of: (i) providing a sample from a patient; and (ii) detecting a nucleic acid molecule in the sample using the detection methods of the invention described above, said nucleic acid molecule comprising a portion, preferably an upstream portion, of an ACSL3 gene fused to a portion of an ETS family member gene; wherein the presence of said nucleic acid molecule indicates that the patient has prostate cancer, that the patient has an aggressive form of prostate cancer, or is likely to develop prostate cancer.

Suitable samples contain nucleic acid from the patient and include blood, semen and urine samples and biopsies from prostate tumours or other prostate tissue.

The nucleic acid molecule to be detected in the above method may be genomic DNA, mRNA or cDNA. The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5, but is preferably ETVl.

In another embodiment, the method of diagnosing prostate cancer in a patient includes the steps of: (i) providing a nucleic acid sample from the patient; and (ii) detecting break-apart of an ACSL3 gene in the sample using the method described above; wherein break-apart of the ACSL3 gene indicates that the patient has prostate cancer, an aggressive form of prostate cancer, or is likely to develop prostate cancer.

In yet another embodiment, the method of diagnosing prostate cancer in a patient includes the steps of: (i) providing a sample from the patient from the patient; and (ii) detecting the fusion protein of the invention in the sample using the method described above, wherein the fusion protein comprises an N-terminal component fused to a C-terminal component, wherein the N-terminal component is a portion of an ACSL3 protein and the C-terminal component is a portion of an ETS family member protein, wherein the presence of the fusion protein indicates that the patient has prostate cancer, an aggressive form of prostate cancer, or is likely to develop prostate cancer.

Any of the kits of the invention may be used for the diagnosis of prostate cancer.

According to a further aspect of the invention, there may be provided a method of determining whether a tumour from a patient with prostate cancer is suitable for anti -androgen therapy .

In one embodiment, the method includes the steps of: (i) providing a sample from a patient; and (ii) detecting a nucleic acid molecule in the sample using the detection methods of the invention, said nucleic acid molecule comprising a portion, preferably an upstream portion, of an ACSL3 gene fused to a portion of an ETS family member gene; wherein the presence of said nucleic acid molecule indicates that the tumour is suitable for anti -androgen therapy.

The nucleic acid molecule to be detected in the above method may be genomic DNA, mRNA or cDNA. The ETS family member gene may be any ETS family member gene, such as ETVl, ETV^'4 , ERG or ETV5, but is preferably ETVl.

In another embodiment, the method of determining whether a tumour from a patient with prostate cancer is suitable for anti-androgen therapy includes the steps of: (i) providing a nucleic acid sample from the patient; and (ii) detecting break-apart of an ACSL3 gene in the sample using the method described above; wherein break-apart of the ACSL3 gene indicates that the tumour is suitable for anti-androgen therapy .

In yet another embodiment, the method of determining whether a tumour from a patient with prostate cancer is suitable for anti-androgen therapy includes the steps of: (i) providing a sample from the patient from the patient; and (ii) detecting the fusion protein of the invention using the method described above, wherein the fusion protein comprises an N- terminal component and a C- terminal component, wherein the N- terminal component is a portion of an ACSL3 protein and the C-terminal component is a portion of an ETS family member protein, wherein the presence of the fusion protein indicates that the tumour is suitable for anti-androgen therapy.

Any of the kits of the invention may be used to determine whether a tumour from a patient with prostate cancer is suitable for anti-androgen therapy.

According to a further aspect of the invention, there may be provided a method of treating a patient with prostate cancer, the method including the step of administering to the patient an agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to a portion of an ETS family member gene, or (ii) activity of a fusion protein comprising an N- terminal component fused to a C-terminal component, wherein the N-terminal component comprises a portion, preferably an N-terminal portion, of the ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein.

The nucleic acid molecule to be targeted by the above method may be genomic DNA, cDNA or mRNA.

The ETS family member gene may be any ETS family member gene, such as ETVl₁ ETV4 , ERG or ETV5 , but is preferably ETVl. Similarly, the ETS family member protein may be any ETS family member protein, such as ETVl, ETV4 , ERG or ETV5 , but is preferably ETVl. The invention also provides an agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to an ETS family member gene, or (ii) activity of a fusion protein comprising a portion of a ACSL3 protein fused to a portion of an ETS family member protein, for use in a method for treatment of disease and for use in a method for treatment of prostate cancer.

The ETS family member gene may be any ETS family member gene, such as ETVl, ETV4 , ERG or ETV5, but is preferably ETVl . Similarly, the ETS family member protein may be any ETS family member protein, such as ETVl, ETV4 , ERG or ETV5, but is preferably ETVl .

The invention also provides the use of the above agent for the manufacture of a medicament for the treatment of prostate cancer.

The agent may be a targeting oligonucleotide, an antisense oligonucleotide, a ribozyme, an siRNA or miRNA molecule, or a triple helix inhibitor, as described above. Preferably, the agent is a targeting oligonucleotide according to the invention. The agent may also be an antibody which binds to a fusion protein in which a portion of the ACSL3 protein is fused to a portion of an ETS family member protein.

In a further aspect of the invention, there may be provided a pharmaceutical composition comprising the agent described above in admixture with a pharmaceutically excipient carrier.

In a further aspect of the invention, there is provided a method of determining a prognosis for prostate cancer. In one embodiment, the method includes the steps of: (i) providing a sample from a patient with prostate cancer; and (ii) detecting a nucleic acid molecule in the sample using the detection methods described above, said nucleic acid molecule comprising an upstream component fused to a portion of an ETVl gene,- wherein the detection of said nucleic acid molecule indicates a higher Gleason score and/or a later clinical stage of disease than if said nucleic acid molecule is not detected. Typically, a high Gleason score and/or a late clinical stage are associated with a poor prognosis.

The upstream component preferably comprises a portion, more preferably an upstream portion, of a gene selected from the group: TMPRSS2, SLC45A3/Prostein, HERV-K, C15orf21, HNRPA2B1, SLC25A21, MIPOLl, FOXAl, TTC6, or ACSL3.

The nucleic acid molecule to be detected by the above method may be genomic DNA, cDNA or mRNA.

In another embodiment, the method includes the steps of: (i) providing a sample from a patient with prostate cancer; and (ii) detecting a fusion protein in the sample using the detection methods described above, said fusion protein comprising an N- terminal component fused to a C-terminal component, wherein the C- terminal component comprises a portion of an ETVl protein; wherein detection of said fusion protein indicates a higher Gleason score and/or a later clinical stage of disease than if said fusion protein is not detected. The N-terminal component preferably comprises a portion, more preferably an N-terminal portion, of a protein selected from the group: TMPRSS2 , SLC45A3/Prostein, HERV-K, C15orf21, HNRPA2B1, SLC25A21, MIPOLl, FOXAl, TTC6 , or ACSL3.

The invention will now be described in detail, by way of example, with reference to the accompanying figures. Description of the figures

Figure 1 shows the cDNA sequence of the human ACSL3 gene . The putative open reading frame (ORF) is in capital letters. The putative start and stop codons are underlined.

Figure 2 shows the cDNA sequence of the human ETVl gene . The putative open reading frame (ORF) is in capital letters. The putative start and stop codons are underlined.

Figure 3 shows the cDNA sequence of an ACSL3.-ETV1 gene fusion. The ACSL3 sequence (exons 1-3) is underlined and the putative open reading frame is in capital letters. The putative start and stop codons are underlined twice and highlighted.

Figure 4 shows the cDNA sequence of the human ERG gene . The putative open reading frame (ORF) is in capital letters. The putative start and stop codons are underlined.

Figure 5 shows the cDNA sequence of the human ETV4 gene . The putative open reading frame (ORF) is in capital letters.

Figure 6 shows the cDNA sequence of the human ETV5 gene .

Figure 7 shows FISH detection of ETVl gene re -arrangements . Top: Interphase nuclei are hybridised to probes that detect sequences immediately 5' to the ETVl gene (Probe I) and immediately 3' to the ETVl gene (Probe II) . The signals from each probe are separated when an ETVl gene rearrangement occurs. A) Signals from normal un-rearranged ETVl loci (Class N) . B) Rearranged ETVl gene with separate 3' and 5' probes (Class ETVl Esplit) . Bottom: Map of the ETVl gene showing the position of the BACs used as probes in FISH assays. Probe I: El (RP11-27B1) , E2 (RP11-138H16) , E3 (CTD-2008I15) labelled with biotin and visualised with streptavidin bound Cy3 fluorophore. Probe II E4 (RP11-905H4) , E5 (RP11-621E24) , E6 (RP11-115D14) labelled with DIG and visualised with anti-DIG antibody bound to FITC. The direction of transcription of genes at this locus are indicated by arrows.

Figure 8 shows Kaplan-Meier analyses comparing prostate cancer outcomes for different categories of ETS alteration. The graphs compare cancer harbouring ETVl gene alteration (class ETVl Esplit) with cancers lacking both ETVl and ERG gene alterations (class N) . (A) Cause-specific survival. (B) Overall survival.

Figure 9 shows an assay to detect translocation of ETVl to chromosome 14q.

Figure 10 shows an ACSL3-ETV1 fusion. A) ACSL3 and ETVl transcripts with open reading frames (ORFs) in dark colour. Exons are numbered. A fusion transcript of ACSL3 exon 3 fused to ETVl exon 6 was detected by 5'-RACE from exon 6 ETVl sequences in prostate cancer sample 23. The ORF shown was predicted using software at www . dnalc . org . B) Sequence across the fusion gene boundary is shown. Underlined regions indicate the position of primers. The predicted initiation codon is indicated. ACSL3 sequence is in lower case and ETVl in upper case. C) RT-PCR detection of an ACSL3:ETV1 fusion transcript in RNA extracted from formalin- fixed paraffin-embedded prostate cancer samples: lanes 1-12 are ETVl- rearranged tumour samples, lane 12: tumour sample 23, lane 13 negative control. D) FISH assays to confirm fusion of ACSL3 with ETVl . Panel i: The ETVl break-apart assay utilises probes corresponding to 3'-ETVl sequences and 5' -ETVl sequences (see also Figure 1) . A nucleus with separated 3' and 5' probes confirming rearrangement of ETVl is shown. Panel ii: The ACSL3 break-apart assay- hybridised the same TMA slice used in the ETVl break-apart assay to 3' -ACSL3 sequences and 5' -ACSL3 sequences. These signals are coincident in the wild type, but are split on translocation of ACSL3 . Comparison of the images in panels i and ii indicates co-localisation of 3'-ETVl with 5' -ACSL3 and co-localisation of 5' -ETVl and 3' -ACSL3. This is confirmed by ETV1-ACSL3 co-localisation assays (panel iii) demonstrating co-localisation of 3' -ETVl sequences and 5' -ACSL3 sequences and (panel iv) demonstrating co-localisation of 3' -ACSL3 sequences and 5' -ETVl sequences in the same cell. Superimposition of the images in panels iii and iv confirms co-localisation of wild-type 3' -ETVl (panel iii) with 5' -ETVl (panel iv) and of wild-type 3' -ACSL3 (panel iv) with 5' -ACSL3 (panel iii) . The genes and their direction of transcription are indicated by the arrowheads. (E) Map of the ACSL3 gene showing the position of the BACs used as probes in FISH assays. Probe XV: Al (RP11-157M20) labelled with DIG and visualised with anti -DIG antibody bound to FITC. Probe XIV: A2 (RP11-136M23) and A3 (RP11-749C15) labelled with biotin and visualised with streptavidin bound Cy3 fluorophore. Probes XV and Probes XIV correspond, respectively, to sequences immediately 5' and 3' to the ACSL3 gene. Direction of gene transcription indicated by arrowheads.

Figure 11 shows the cDNA sequences of known 5¹ fusion partners of ETVl. (a) The cDNA sequence of the SLC45A3 gene, (b) The cDNA sequence of the HerV-K gene, (c) The cDNA sequence of the C150RF21 gene, (d) The cDNA sequence of the HNRNPA2B1 gene, (e) The cDNA sequence of the TMPRSS2 gene.

Detailed description of the invention

Nucleic acid molecules An isolated nucleic acid molecule is one which has been separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Therefore, it is present in a different form or setting from that in which it is found in nature. However, isolated nucleic acid molecules can be inserted into expression vectors for expression in host cells. Similarly, an isolated nucleic acid molecule can be incorporated as a transgene into the genome of an animal to generate a transgenic animal .

Nucleic acid molecules can be any nucleic acid- containing molecule, including, but not limited to genomic DNA, RNA (e.g. mRNA) and cDNA. As such, the term "nucleic acid sequence", as used herein, encompasses genomic DNA, mRNA and cDNA sequences. If a nucleic acid sequence includes the base thymine (T) , it should be taken to encompass an mRNA sequence having the base (U) . The nucleic acid molecules may also comprise modified nucleotides .

The nucleic acid molecules described herein comprise an upstream component fused to a downstream component at a fusion site. As such, the upstream and downstream components are adjacent to each other on the same nucleic acid molecule with no intervening sequence (s) , i.e. the upstream and downstream components are contiguous with each other and are joined at the fusion site.

Typically, the upstream component includes one or more of the following nucleic acid sequences: a transcriptional regulatory region (or regions) e.g. a promoter and/or an enhancer, a transcription initiation site, at least a portion of the 5' untranslated region (5¹UTR), a start codon, one or more complete exon sequences and at least part of the open reading frame (ORF) , or the reverse complement thereof. The downstream component typically includes one or more of the following nucleic acid sequences: a transcriptional termination site, at least a portion of the 3¹ untranslated region (3' UTR) , a stop codon, one or more complete exon sequences and at least part of the ORF, or the reverse complement thereof.

The use of the terms "upstream" and "downstream" is intended to convey that the upstream component is 5' of the downstream component on the sense strand of DNA. However, the orientation of these two components on a single strand of nucleic acid is not important, as on the corresponding anti- sense strand of DNA, the "upstream component" will be downstream (i.e. 3') of the "downstream component" . Accordingly, all references to nucleic acid molecules throughout this specification should be construed as including the reverse complements of these nucleic acid molecules.

In the nucleic acid molecules of the present invention, the upstream component comprises a portion of an ACSL3 gene and the downstream component comprises a portion of an ETS family member gene. The portion of the ACSL3 gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the portion of the ETS family member gene is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the portion of the ACSL3 gene may be at least 20 nucleotides in length and the portion of the ETS family member gene may be at least 20 nucleotides in length.

The ETS family member gene may be any ETS family member gene, such as ETVl, ETVA, ERG or ETV5 , but is preferably ETVl.

A portion includes any part of the specified gene, but not normally the whole gene sequence. Preferably, the upstream component comprises an upstream portion of an ACSL3 gene. Typically, an upstream portion of a gene includes one or more of the following nucleic acid sequences: a transcriptional regulatory region (or regions) , a promoter region, a transcription initiation site, at least a portion of the 5' untranslated region (5¹UTR) , a start codon, one or more complete exon sequences and at least part of the open reading frame (ORF) . As such, the upstream portion of the ACSL3 gene preferably comprises one or more transcriptional regulatory sequences, such as an androgen response element (ARE) . The upstream portion of the ACSL3 gene may also comprise a promoter region, a transcription initiation site, the 5¹UTR and/or one or more exon sequences, such as exons 1 and 2 and a portion of exon 3. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in Figure 3.

Fusion between the ACSL3 gene and the ETS family member gene can occur anywhere in the ACSL3 gene and anywhere in the ETS family member gene. However, as mentioned above, the upstream component preferably comprises an upstream portion of the ACSL3 gene. In a preferred embodiment, the downstream component comprises a portion of an ETS family member gene which includes a start (i.e. initiation) codon, which is downstream from its native start codon, so that when the nucleic acid molecule is translated, a truncated version of the ETVl protein is produced. Expression of this truncated protein may be partially or completely under the control of the upstream component. In another embodiment, the portion of the ACSL3 gene forming the upstream component may include a start codon and at least part of the ORF, such that a chimeric protein containing a portion of the ACSL3 protein fused to a portion of the ETVl protein is produced.

The nucleic acid molecules of the invention may be genomic DNA, RNA (e.g. mRNA) or cDNA. If the nucleic acid molecules are mRNA or cDNA molecules, the upstream component will not include any transcriptional regulatory sequences or a promoter region, but may include at least part of the 5' UTR and optionally, an initiation codon, one or more complete exon sequences and/or at least part of the ORF.

The nucleotide sequence of the human ACSL3 gene is shown in Figure 1. This sequence includes 17 exons, as shown in Figure 4A, and is located on chromosome 2. The protein encoded by the ACSL3 gene is an isozyme of the long-chain fatty-acid-coenzyme A ligase family that converts free long-chain fatty acids into fatty acyl-CoA esters, and thereby plays a key role in lipid biosynthesis and fatty acid degradation. Insights into the regulation of ACSL3 expression arise from examining expression array data in which the LNCaP cell -line was treated with the synthetic androgen R1881. In two independent expression array data sets, ACSL3 was up-regulated by androgen treatment (Hendriksen et al., 2006; Wang et al., 2006) . One study showed ACSL3 up-regulation at time intervals of 2 , 4, 6 and 8 hours following androgen treatment (Hendriksen et al., 2006) and another study showed ACSL3 up-regulation after 16 hours (Wang et al., 2006) . Expression of ACSL3 was also elevated in a panel of 'androgen- sensitive' (LAPC-4, LNCaP, MDA PCa2a, MDA PCa2b and 22RvI) versus 'androgen- insensitive' (PPCl, PC3 and DU145) lines (Zhao et al., 2005; Tomlins et al., 2007) . Expression of TMPRSS2 and SLC45A3 follows the same pattern within these datasets (Zhao et al., 2005) .

Members of the ETS family of genes are transcription factors and are involved in regulating gene expression by activating or repressing the expression of specific target genes. In particular, they control the spatial and temporal expression of many growth factor receptor genes. Examples of ETS family member genes include human ERG, which is located on chromosome 21 (GenBank accession no. NM_004449; GenBank accession no. M17254 for the cDNA sequence; GenBank accession no. NP_04440 and Swiss Protein ace. no. P11308 for the protein sequence), human ETVl, which is located on chromosome 7 (GenBank accession nos. NC_000007.11, NC_086703.11 and NT_007819.15 ; NM_004956 for the cDNA sequence; NP_004947 and Swiss Protein ace. no. P50549 for the protein sequence) , human ETV4 located on chromosome 17 (GenBank accession nos. NC_000017.9 and NT_010783.14 ; GenBank accession no. NM_001986 for the cDNA sequence and GenBank accession no. NP_01977 and Swiss protein ace. no. P43268 for the protein sequence) and human ETV5 located on chromosome 3 (GenBank accession no. NM_004454.1) . The nucleotide sequence of the human ETVl gene is shown in Figure 2 and includes 12 exons, as shown in Figure 1OA. The nucleotide sequences of the human ERG, ETV4 and ETV5 genes are shown in Figures 4 to 6, respectively.

Expression vectors

Expression vectors are known in the art and are used to allow delivery of nucleic acid to target host cells and the expression of nucleic acid contained within the vector in these host cells. Expression vectors may be introduced into host cells by techniques known in the art, such as transfection. Suitable expression vectors include those which allow expression of nucleic acid in mammalian cell lines.

The isolated nucleic acid molecules of the invention may be ligated into an expression vector so that they can be introduced into host cells, where they can then be expressed to produce mRNA and/or protein. Suitable host cells include bacterial cells, e.g. E.coli, yeast cells and mammalian cells.

Transgenic animals

Transgenic animals are non-human animals which contain non- native nucleic acid sequences i.e. sequences which are not normally present in the genome of those animals. These sequences are known as transgenes. Suitable animals for use in transgenic studies include rodents and non-human primates. Transgenic animals expressing the nucleic acid molecules according to the invention can be used to study the effects of over-expression of these nucleic acid molecules and to screen for drugs which inhibit the activity of these nucleic acid molecules.

Targeting oligonucleotides

Targeting oligonucleotides are short nucleic acid sequences, usually less than 200 or less than 100 nucleotides in length, which are able to hybridise to a target nucleic acid sequence and inhibit its expression (i.e. its transcription or translation) . Such targeting oligonucleotides may hybridise to the sense or antisense strand of a DNA molecule (i.e. to the strand which codes for protein), or to an mRNA molecule. The nucleotide sequence of a targeting oligonucleotide will usually be complementary to that of its target sequence.

Targeting oligonucletides may be less than 100, less than 50, less than 40, less than 30, less than 25 or less than 20 nucleotides in length. Preferably, they are between 9-25 nucleotides in length and are at least 9, at least 10, at least 15 or at least 20 nucleotides in length.

Suitable hybridisation conditions include medium stringency conditions (e.g. at about 10-20°C below the melting temperature (T₀₁) of the probe; IxSSC) and high stringency conditions (e.g. at about 5-10⁰C below the melting temperature (TJ of the probe; 0.5xSSC) .

Targeting oligonucleotides according to the invention comprise a first nucleic acid sequence capable of hybridising to the sense or antisense strand of an ACSL3 gene, or to an mRNA sequence encoded thereby, and a second nucleic acid capable of hybridising to the sense strand or antisense strand of an ETS family member gene, or to the mRNA encoded thereby. The first nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. There may also be intervening nucleotides (i.e. a spacer region) between the first and second nucleic acid sequences.

The targeting oligonucleotides of the invention can hybridise to the nucleic acid molecules of the invention to inhibit their expression. If these target nucleic acid molecules are DNA molecules, the targeting oligonucleotide will bind to the sense or antisense strand of the DNA molecule. Alternatively, if the target nucleic acid molecules are mRNA molecules, the targeting oligonucleotides will bind to the mRNA. Examples of such targeting oligonucleotides (i.e. which bind to mRNA) include antisense oligonucleotides comprising a first nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ACSL3 gene, and a second nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ETS family member gene.

RNA interference

Similarly, other small RNA molecules may be employed to regulate gene expression. This includes targeted degradation of mRNAs by small interfering RNAs (siRNAs) , post transcriptional gene silencing (PTGs) , developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.

A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double- stranded RNA (dsRNA) -dependent post transcriptional silencing, also known as RNA interference (RNAi) , is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20- nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.

In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending in their origin. Both types of sequence may be used to down- regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro- interfering RNAs (miRNA) are endogenousIy encoded small non- coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially- complementary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

Accordingly, the present invention provides the use of these sequences for downregulating the expression of the nucleic acid molecules of the invention.

The siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down- regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to produce a host response.

miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from

DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single -stranded RNA molecule, the miRNA sequence and its reverse -complement base pair to form a partially double stranded RNA segment. The design of miRNA sequences is discussed on John et al, PLoS Biology, 11(2) , 1862-1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof) , more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double -stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design of suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http: //www. ambion. com/techlib/misc/siRNA_finder.html . siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) . In a preferred embodiment the siRNA is synthesized synthetically.

Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21: 324-328) . The longer dsRNA molecule may have symmetric 3' or 5 ' overhangs, e.g. of one or two

(ribo) nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs of 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev. , 17, 1340-5, 2003) .

Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complementary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene raRNA and suppresses expression. In a preferred embodiment, the shRNA is produced endogenousIy (within a cell) by- transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human Hl or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector.

The siRNA, longer dsRNA or miRNA may be produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue-specific promoter. Alternatively, the siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.

The vector may comprise a nucleic acid sequence in both the sense and antisense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA.

Alternatively, siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(O)S, (thioate) ; P(S)S, (dithioate) ; P (O)NR '2; P(O)R¹; P(0)0R6; CO; or CONR ' 2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-0-or-S- .

Modified nucleotide bases can be used in addition to the naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.

For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing. The provision of modified bases may also provide siRNA molecules which are more, or less, stable than unmodified siRNA.

The term 'modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 'position and other than a phosphate group at the 5 'position. Thus modified nucleotides may also include 2 ' substituted sugars such as 2'-0-methyl- ; 2-0-alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2 ' -halo or 2; azido-ribose, carbocyclic sugar analogues α- anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose .

Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles . These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N6- methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6- isopentyl -adenine, 1- methyladenine, 1-methylpseudouracil , 1- methylguanine , 2 , 2-dimethylguanine, 2methyladenine, 2- methylguanine , 3-methylcytosine, 5-methylcytosine, N6- methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5- methoxy amino methyl -2 - thiouracil , -D-mannosylqueosine, 5- methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-N6- isopentenyladenine, uracil- 5 -oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2- thiouracil, 4 -thiouracil, 5methyluracil, N-uracil- 5 -oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2- thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil , 5- pentylcytosine, and 2 , 6, diaminopurine, methylpsuedouracil, 1- methylguanine, 1-methylcytosine .

Methods relating to the use of RNAi to silence genes in C. elegans , Drosophila , plants, and mammals are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999) ; Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001) ; Tuschl , T. Chem. Biochem. 2, 239-245 (2001) ; Hamilton, A. et al., Science 286, 950-952 (1999) ; Hammond, S. M., et al., Nature 404, 293-296 (2000) ; Zamore, P. D., et al., Cell 101, 25-33 (2000) ; Bernstein, E., et al., Nature 409, 363-366 (2001) ; Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001) ; WO0129058; WO9932619, and Elbashir S M, et al., 2001 Nature 411:494-498) .

Nucleic acid probes Nucleic acid probes are able to detect specific nucleic acid sequences, usually by hybridising to such sequences. As such, nucleic acid probes typically have a complementary sequence to that of their target molecule. Nucleic acid probes can be used to detect DNA or RNA sequences and may be single -stranded or double -stranded. Accordingly, the nucleic acid probe may hybridise to the sense strand or the antisense strand of genomic DNA, or to both, to cDNA, or to mRNA. The nucleic acid probes are preferably less than 500kb, less than 400kb, less than 300kb, less than 200kb, less than 100kb, less than 50kb, less than 40kb, less than 30 kb, less than 20kb or less than 10kb length. Specifically, the nucleic acid probes may be bacterial artificial chromosomes (BACs) , which are preferably 100-200kb in length, fosmid probes, which are preferably 20- 50kb in length, cDNA sequences, which are preferably 0.5 to 20kb in length, or PCR primers, which are preferably 10-lOObp in length.

The nucleic acid probes of the invention comprise a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is capable of hybridising to an ACSL3 gene and the second nucleic acid sequence is capable of hybridising to an ETS family member gene. Accordingly, the nucleic acid probe is able to hybridise across the fusion site of the nucleic acid molecule of the invention. The first nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Similarly, the second nucleic acid sequence is preferably at least 5, at least 10, at least 20, at least 30, at least 40 or at least 50 nucleotides in length. Any combination of these lengths is possible. For example, the first nucleic acid sequence may be at least 20 nucleotides in length and the second nucleic acid sequence may be at least 20 nucleotides in length. There may also be intervening nucleotides (i.e. a spacer region) between the first and second nucleic acid sequences. Therefore, the first nucleic acid sequence is not necessarily contiguous with the second nucleic acid sequence.

If the target nucleic acid molecule is DNA, the nucleic acid probe may hybridise to the sense strand or to the anti-sense strand of the target DNA molecule. Alternatively, if the target nucleic acid molecule is an mRNA molecule, the nucleic acid probe will hybridise to the mRNA itself.

As set out above, suitable hybridisation conditions include medium stringency conditions (e.g. at about 10-20⁰C below the melting temperature (Tj of the probe; IxSSC) and high stringency conditions (e.g. at about 5-10°C below the melting temperature (T_m) of the probe; 0.5xSSC) .

Nucleic acid probes may be labelled so that they are detectable in a detection system, such as an enzyme -linked immunosorbent assay (ELISA) , enzyme-based histochemical assays, and fluorescent, luminescent or radioactive systems. Suitable detection systems are standard in the art and include Southern blotting (e.g. following pulsed field gel electrophoresis) and in situ hybridisation (ISH) , e.g. fluorescent in situ hybridisation (FISH) . As such, nucleic acid probes may be labelled radioactively or with fluorescent reporter molecules. The nucleic acid probes may also be labelled with digoxygenin (DIG) or biotin to allow detection using an ant i-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5. Nucleic acid probes may also be used as primers for nucleic acid amplification and therefore, the binding of nucleic acid probes to a target sequence can also be detected using the polymerase chain reaction (PCR) .

The BAC accession numbers of nucleic acid probes which bind to various nucleic sequences, including cDNA sequences from the human ACSL3 and ETVl genes, are shown in Table 4.

Fusion proteins

Fusion proteins comprise an N- terminal component fused in frame to a C- terminal component to form a chimeric protein. In the fusion protein of the invention, the N-terminal component comprises a portion of an ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein. Preferably, the portion of the ACSL3 protein is an N-terminal portion comprising a methionine residue encoded by the native start codon.

Detection methods

Methods for detecting fusion of specific nucleic acid sequences may be carried out in vitro, ex vivo or in vivo. Probes used to detect specific nucleic acid sequences include, but are not limited to, the nucleic acid probes described herein.

The detection methods of the invention are used to detect a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene.

The detection method may include the step of contacting the sample with a probe which binds to the ACSL3 gene and to the ETS family member gene, wherein binding of the probe to nucleic acid in the sample indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene at a fusion site. As such, the probe binds across the fusion site of the nucleic acid molecule. The probe may be the nucleic acid probe described above and may hybridise to the upstream component and to the downstream component of the nucleic acid molecule to be detected.

As set out above, suitable hybridisation conditions include medium stringency conditions (e.g. at about 10-20⁰C below the melting temperature (T₁J of the probe; IxSSC) and high stringency conditions (e.g. at about 5-10°C below the melting temperature (TJ of the probe,- 0.5xSSC) . The nucleic acid probe may be labelled, e.g. f luorescently or radioactively, or with digoxygenin (DIG) or biotin to allow detection using an anti- DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5. The target nucleic acid sequence (s) can be detected by any suitable detection method, such as ISH, FISH or Southern blotting. Specifically, the nucleic acid probe may be a BAC probe, a fosmid probe or a cDNA probe for use in ISH. Alternatively, the nucleic acid probe may be a primer for use in nucleic acid amplification, so that the target nucleic acid sequence can be detected by PCR .

Alternatively, the detection method may include the step of contacting the sample with a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to nucleic acid downstream of the ETS family member gene, such that co- localisation or juxtaposition of the first probe and the second probe indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene.

Binding of the first and second probes to sequences on the same physical piece of nucleic acid (e.g. on the same chromosome or on the same piece of cDNA or mRNA) within 10 Mb, within 5 Mb, within 4 Mb, within 3 Mb, within 2 Mb, within 1

Mb, within 500 kb, within 400 kb, within 300 kb, within 200 kb or within 100 kb of each other indicates co-localisation or juxtaposition of the first and second probes, and therefore, the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene .

The nucleic acid (e.g. chromosomal DNA) upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene. The nucleic acid (e.g. chromosomal DNA) downstream of the ETS family member gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ETS family member gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ETS family member gene may extend up to 2 Mb downstream of the ETS family member gene. Preferably, the nucleic acid upstream of the ACSL3 gene is upstream of the ACSL3 gene in its native chromosomal state. Similarly, the nucleic acid downstream of the ETS family member gene is preferably downstream of the ETS family member gene in its native chromosomal state.

The first and second probes may be labelled, e.g. fluorescently or radioactively) or with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC,

Sprectrum Aqua or Cy5. Co-localisation of the probes (which are preferably nucleic acid probes) may be detected by any suitable detection method, such as ISH, FISH or Southern blotting. In FISH, the first and second probes nucleic probes (e.g. BAC, fosmid or cDNA probes) and are fluorescently labelled, either directly or indirectly, so that they can be visualised using fluorescence microscopy. In a sample containing the nucleic acid molecule to be detected (i.e. a portion of an ACSL3 gene is fused to a portion of an ETS family member gene) , the fluorescently labelled nucleic acid probes will be visible by fluorescence microscopy as adjacent, co-localised signals. However, if the sample does not contain the nucleic acid molecule to be detected (i.e. a portion of an ACSL3 gene is not fused to a portion of an ETS family member gene) , the fluorescently labelled nucleic acid probes will be visible as separate, non-adjacent signals. The first and second probes may also be primers for nucleic acid amplification, e.g. for PCR. In this process, the first and second probes are added to a sample containing nucleic acid under annealing conditions. If the first and second markers bind to the same physical piece of nucleic acid (e.g. to the same chromosome) within 200kb, within 100kb, within 50 kb, within 20kb, within 10 kb, within 5 kb or within lkb of each other (i.e. they are co-localised) , the intervening nucleic acid sequence will be amplified, indicating co- localisation of the first and second probes (and therefore, the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene) . Fusion of a portion of an ACSL3 gene to a portion of an ETS family member gene can be confirmed by sequencing.

The detection methods of the invention may be used to detect any type of nucleic acid molecule, including genomic DNA, cDNA or mRNA.

Methods of detecting the fusion protein of the invention are also provided. Such a method may include the step of contacting the sample with an agent which binds specifically to the fusion protein, such as an antibody which binds to an ACSL3 protein and an ETS family member protein. Binding of an antibody to the fusion protein can be detected by any suitable method know in the art, such as immunocytochemistry, immunohistochemistry or western blotting.

Break-apart assay

The invention provides a method for detecting "break-apart" of an ACSL3 gene. "Break-apart" of a gene means that portions of that gene, which were originally adjacent to each other in native chromosomal DNA have become separated from each other so that they are no longer adjacent to each other or juxtaposed. Typically, a portion of the gene becomes localised on a different chromosome, e.g. adjacent to an ETS family member gene, while another portion remains localised in its native chromosomal position.

The method for detecting break apart of an ACSL3 gene includes the step of contacting the sample with a first probe which binds to an upstream portion of the ACSL3 gene and/or to chromosomal DNA upstream of the ACSL3 gene and a second probe which binds to a downstream portion of the ACSL3 gene and/or to chromosomal DNA downstream of the ACSL3 gene, wherein binding of the first and second probes to physically separate, non-adjacent pieces of DNA (e.g. to different chromosomes) indicates break-apart of the ACSL3 gene.

Chromosomal DNA upstream or downstream of the native ACSL3 gene can also become localised on a different chromosome, along with the portion of the ACSL3 gene which is re- localised. The chromosomal DNA upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the native ACSL3 gene . The chromosomal DNA downstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the native ACSL3 gene.

The first and second probes are preferably nucleic acid probes and may be labelled, e.g. fluorescently . Break-apart of the ACSL3 gene may be detected by any suitable detection method, such as FISH. In FISH, the first and second probes are nucleic acid probes (e.g. BAC, fosmid or cDNA probes) and are fluorescently labelled, either directly or indirectly, so that they can be visualised using fluorescence microscopy. In a sample containing the ACSL3 gene in its native chromosomal location (i.e. in which the ACSL3 gene is intact) , the f luorescently labelled nucleic acid probes will be visible by- fluorescence microscopy as adjacent, co- localised signals. However, if the ACSL3 gene has been broken apart (i.e. it is no longer intact) , the fluorescently labelled nucleic acid probes will be visible as separate, non-adjacent signals.

Kits

Kits for detecting a nucleic acid molecule in a sample, wherein the nucleic acid molecule comprises a portion of an ACSL3 gene fused to a portion of an ETS family member gene, typically contain: (i) a probe comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is capable of binding to the ACSL3 gene and the second nucleic acid sequence is capable of binding to the ETS family member gene; or (ii) a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to chromosomal DNA downstream of the ETS family- member gene . The probes are preferably nucleic acid probes and may labelled, e.g. fluorescently or radioactively labelled. The probes may also be labelled with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5.

The nucleic acid upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the ACSL3 gene. The nucleic acid downstream of the ETS family member gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the ETS family member gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ETS family member gene may extend up to 2 Mb downstream of the ETS family member gene .

The kits of the invention can be used in any of the methods for detecting a nucleic acid molecule described above.

The invention further provides a kit for detecting break-apart of the ACSL3 gene, using the break-apart assay described above. Such a kit comprises a first probe which binds to an upstream portion of the ACSL3 gene and/or to chromosomal DNA upstream of the ACSL3 gene and a second probe which binds to a downstream portion of the ACSL3 gene and/or to chromosomal DNA downstream of the ACSL3 gene .

The chromosomal DNA upstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb upstream of the native ACSL3 gene . The chromosomal DNA downstream of the ACSL3 gene may extend up to 10 Mb, up to 5 Mb, up to 4 Mb, up to 3 Mb, up to 2 Mb, up to 1 Mb, up to 500 kb, up to 400 kb, up to 300 kb, up to 200 kb, up to 100 kb or up to 50 kb downstream of the native ACSL3 gene. For example, the nucleic acid upstream of the ACSL3 gene may extend up to 2 Mb upstream of the ACSL3 gene and the nucleic acid downstream of the ETS family member gene may extend up to 2 Mb downstream of the ETS family member gene .

The first and second probes are preferably nucleic acid probes and may be labelled, e.g. fluorescently. The probes may also be labelled with digoxygenin (DIG) or biotin to allow detection using an anti-DIG antibody bound to a fluorophore or a streptavidin bound fluorophore respectively. Suitable fluourophores include Cy3 , FITC, Sprectrum Aqua or Cy5. The invention also provides a kit for detecting the fusion protein of the invention, wherein the kit comprises an agent that binds specifically to the fusion protein. Preferably, the agent is an antibody which binds to an ACSL3 protein and to an ETS family member protein.

All the kits described herein may also contain instructions for carrying out the detection methods of the invention and/or a positive control sample, such as an expression vector according to the invention.

The kits of the invention may also be used for diagnostic purposes, e.g. for methods of diagnosing prostate cancer according to the invention.

Methods of diagnosis

Methods of diagnosing prostate cancer as described herein may include the step of obtaining a sample from a patient or may be performed on a sample which has already been obtained from a patient. Suitable samples contain nucleic acid from the patient and include blood, semen and urine samples and biopsies from prostate tumours or other prostate tissue. The methods of diagnosis according to the invention include the step of detecting a nucleic acid molecule in the sample using any of the detection methods of the invention described above, said nucleic acid molecule comprising a portion, preferably an upstream portion, of an ACSL3 gene fused to a portion of an ETS family member gene; wherein the presence of said nucleic acid molecule indicates that the patient has prostate cancer, is likely to develop prostate cancer, or has a more aggressive form of prostate cancer than if said nucleic acid molecule is not detected.

An ti -androgen therapy The prostate glands require androgens (i.e. male hormones, such as testosterone) to function properly. Anti -androgen therapy is known as a treatment for prostate cancer and blocks the activity of dihydrotestosterone (DHT) , which is produced in the prostate and required for the growth and spread of most prostate cancer cells.

Expression of the ACSL3 gene is regulated by androgens and the ACSL3 gene includes an upstream androgen response element (ARE) . Therefore, fusion of a portion of an ACSL3 gene containing an ARE to a downstream component, such as a portion of an ETS family member gene, brings expression of the downstream component (e.g. a portion of ETVl) under androgen- regulated control. Therefore, the downstream component (e.g. a portion of ETVl) will be overexpressed in the prostate, which produces high levels of androgens. As ETVl has been shown to be overexpressed in human prostate tumours and to confer a neoplastic phenotype in transgenic mouse models (Tomlins et al., 2007) , anti-androgen therapy may be suitable for patients in which a portion of an ACSL3 gene (which includes an ARE) has been fused to an ETS family member gene, such as ETVl. Such fusions can be detected using the detection methods of the invention.

Methods of treatment

The methods of treatment according to the invention include the step of administering to a patient an agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to a portion of an ETS family member gene, e.g. ETV 1, at a fusion site, or (ii) activity of a fusion protein comprising an N-terminal component fused to a C-terminal component, wherein the N-terminal component comprises a portion, preferably an N-terminal portion, of the ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein, e.g. ETVl. Therefore, the agent may prevent or inhibit transcription of the nucleic acid molecule to produce mRNA or it may prevent or inhibit translation of the mRNA into protein. For example, the agent may do this by preventing or inhibiting the access of transcription factors to DNA or of the translation machinery to mRNA. Alternatively, the agent may promote the degradation of its target DNA or mRNA.

The agent may also inhibit the activity of the fusion protein. Such activity includes any biological activity, such as its interaction with and signalling through other molecules. In particular, the agent may prevent the interaction between the truncated ETS family member protein and other transcription factors, or it may inhibit the binding of the truncated ETS family member protein to DNA.

Agents according to the invention may be used in a method for the treatment of disease, or specifically in a method for the treatment of prostate cancer. Accordingly, agents of the invention can be used in the preparation of a medicament for the treatment of prostate cancer.

Suitable agents for use in the methods of treatment according to the invention include antisense oligonucleotides, siRNA molecules, miRNA molecules (e.g. the antisense oligonucleotides, siRNA and miRNA molecules of the invention) , ribozymes, triple helix inhibitors and antibodies.

The agents of the present invention may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Preferably, the composition, pharmaceutical, medicament or drug comprises an agent of the invention in combination with a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. These compositions, pharmaceuticals, medicaments or drugs may be administered to individuals and the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or sub- cutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection.

Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Methods of determining prognosis Methods of determining prognosis give a prediction of the likely outcome and/or progression of a disease and its likely- response to treatment. The most widely used prognostic indicators for prostate cancer are clinical stage of disease, pre-therapy prostate serum antigen (PSA) level and Gleason score, which is a score given to prostate cancer on the basis of its microscopic appearance (Johansson JE et al., 2004; Albertsen et al . , 2005) . Typically, several parameters (which usually include the Gleason score) are used for "prostate cancer staging" , which can be used to predict prognosis and to guide therapy.

Specifically, a high Gleason score indicates that a prostate cancer is aggressive and is associated with a poor prognosis. In particular, a high Gleason score has been shown to be an important determinant of cancer- specific mortality, although baseline PSA and, to a lesser extent, stage of disease add further predictive value.

The inventors have shown a significant association between the presence of ETVl gene rearrangements and Gleason score, baseline PSA levels and clinical stage of disease (see Example 1 below and Figure 2) . Therefore, ETVl gene rearrangements can be used as markers (either alone or in combination with other prognostic indicators, such as Gleason score) to determine a prognosis for prostate cancer, such that an ETVl rearrangement is associated with a higher Gleason score and/or a later clinical stage of disease for a patient with prostate cancer than if an ETVl rearrangement is not detected. Therefore, the presence of an ETVl rearrangement may be indicative of a poor prognosis. Such ETVl rearrangements include the fusion of a first nucleic acid sequence, such as an upstream portion of a TMPRSS2 (accession number NM_005656.2) , SLC45A3/Prostein (accession number NM_033102.2) , HERV-K (sequence BC020811 from IMAGE clone ID:4294287), C15orf21 (accession no. NM_001005266.1) , HNRPA2B1 (NM_002137.3 ) , SLC25A21, MIPOLl, FOXAl and TTC6 from the chromosomal region 14ql3.3 - 14q21.1, or an ACSL3 gene (accession number NM_004457.3 ) , to a portion of the ETVl gene resulting in the production of a truncated ETVl gene. These ETVl gene rearrangements can be detected using the detection methods of the invention, or by using the ETVl break-apart assay described in Figure 7. Examples

Example 1 - Identification of biomarkers for prostate cancer To identify biomarkers that may be of use in the management of men with prostate cancer, a retrospective cohort of men whose cancers were conservatively managed was established (Cuzick J et al., 2006) . The analyses included centrally assigned Gleason scores determined by modern grading criteria, and allowed comparisons with several additional clinical parameters. In agreement with previous studies (Johansson JE et al., 2004; Albertsen et al., 2005), Gleason score was found to be an important determinant of cancer- specific mortality, although baseline PSA and, to a lesser extent, stage of disease added further predictive value.

A fluorescence in situ hybridization (FISH) ETVl gene 'break- apart' assay was used to screen for ETVl rearrangements in a set of 945 Tissue Micorarray (TMA) trans -urethral resection of the prostate (TURP) cancer cores from 429 patients. The ETVl break-apart assay hybridized to 3' -ETVl sequences and to 5'- ETVl sequences (see panel i of Figure 10D) . A nucleus with a pair of split probes confirming rearrangement of ETVl is shown in panel i of Figure 10D. Three overlapping BAC probes at the centromeric 3 '-end and three BAC probes at the telomeric 5'- end were used (see Figure 7) . Normal ETVl loci were visualized in interphase nuclei as immediately adjacent signals (see Figure 7, Class N) . When rearrangements involving the ETVl gene were present, the 3 ' -centromeric and 5' -telomeric ends of ETVl were present as separated signals (see Figure 7, Class ETVl Esplit) . These analyses identified ETVl gene rearrangements in cancer from 23 patients (5.4% of all cancers) .

An ERG gene break-apart assay, completed as previously described (Attard G et al., 2007) , demonstrated that an additional 155 cancers (36%) in this series contained ERG gene rearrangements, including one patient who had both ERG and ETVl rearrangements in distinct foci of cancer in the same prostate, as reported previously (Clark J et al. , 2007) . However, rearrangements of ERG and ETVl were invariably mutually exclusive in the same cancer core. Cancers with ERG alterations were excluded from further analysis comparing cancers that contained solely an ETVl gene re - arrangement (22 tumours) with cancers that lacked rearrangements of both ERG and ETVl (class N) (252 tumours) .

Tumour demographics and characteristics for the patients that lacked ERG gene rearrangements and correlations to clinicopathological parameters are shown in Table 1. The precise number of ETVl rearrangements within each Gleason category is listed in Table 3. Tissue microarrays (TMAs) for these studies were constructed from 429 unselected transurethral resection of the prostate specimens taken from patients managed with no initial treatment (385) or who were initially treated with hormone therapy (44) in a cohort of men with prostate cancer who did not receive up- front radical treatment for their cancer (Cuzick J et al., 2006) . The median age of diagnosis was 71 years (54-76 years) and the median follow-up was 90 months (3-169 months) . National approval for the collection of the cohort was obtained from the Northern Multi -Research Ethics Committee followed by local ethics committee approval at each of the collaborating hospital trusts. This work was approved by the Clinical Research and Ethics Committee at the Royal Marsden NHS Foundation Trust and Institute of Cancer Research. TMAs were constructed in 35*2*7mm blocks of Lamb paraffin wax using a manual tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA) . Up to four cores of 600 mm diameter were taken from each tumour. Reassignment of areas of 'cancer' or 'normal' in each core was carried out on the basis of histopathological examination of haematoxylin and eosin and p63 /AMACR- stained sections that flanked the TMA slice used for FISH studies. The morphological criteria for selection of 'normal' and 'malignant' prostatic epithelium conformed to previously published definitions (Foster et al., 2000; Foster, 2000; Foster et al., 2004) . 'Hyperplasia' , 'dysplasia' and 'PIN' were not scored in this study. The FISH method used for detecting ETVl gene rearrangements is described in Figure 7. All procedures used including the preparation of FISH probes, hybridization of FISH probes to TMA slices (4 μm) cut onto SuperFrostPlus glass slides (VWR International, Poole, UK) , washing slices and scanning and storage of images were carried out exactly as described previously (Clark J et al., 2007; Attard G et al., 2007) . There were significant associations between the presence of ETVl gene re -arrangement and Gleason score (p=0.001) , baseline PSA (P=<0.0001) , clinical stage (p=0.017) and age (0.04) (see Table 1) . However, despite these links to indicators of more aggressive disease, there was no evidence for a difference in overall and cancer-specific survival between those cancers harbouring ETVl gene alterations (class ETVl Esplit) and those cancers retaining normal ERG and ETVl loci (class N) (HR = 1.61, CI = 0.69 - 3.76, P=O.27 and HR = 1.49, CI = 0.86 - 2.59, P = 0.16 respectively) (see Figure 8) . The Kaplan-Meier curve for all ETVl fusions does not show a difference in survival but a difference may be observed when individual fusion gene partners are examined separately.

A single TMA block containing cores from all of the cancers harbouring ETVl re -arrangements (23 tumours) and 6 randomly selected cancers with an ERG gene rearrangement was constructed. Slices of this TMA were used to carry out break- apart assays for previously identified 5 '-partners (Tomlins et al., 2005; Tomlins et al., 2006; Tomlins et al., 2007) : namely TMPRSS2, SLC45A3, HERV-K, C15orf21 and HNRPA2B1. FISH assays were used to confirm co-localization of 3'-ETVl with 5' sequences from each of the above partners as previously described (Tomlins et al., 2007) . To identify tumours with translocation of ETVl to the androgen- regulated prostate specific region at 14ql3.3-14q21.1, a TMA slice with a 3' -ETVl FISH probe and a FISH probe consisting of 6 BACs spanning the entire region of 14ql3.3 - 14q21.1 were co-hybridised. Co- localisation or immediate juxtaposition of the two probes were taken as evidence of translocation of ETVl to this region (Figure 9) .

The results demonstrated that all of the ERG rearranged cancers had a TMPRSS2:ERG fusion, but none of the cancers contained a fusion involving TMPRSS2 or HERV-K retroviral sequence. In four cancers, 3' -ETVl exhibited fusion to C15orf21 , two cancers contained translocation to 14ql3.3- 14q21.1, one cancer contained fusion to HNRPA2B1 and one cancer contained fusion to SLC45A5/Prostein (Table 2) . Thus, only 8 of the cancers with re-arranged ETVl genes had identified fusion partners. Co-localization of a FISH probe to 3'-ETVl sequences with a probe to C150RF21 in the previously reported case with ERG and ETVl rearrangements in distinct foci of the same prostate was also demonstrated. The recurrent fusions of the prostate-specific androgen-repressed gene C15orf21 to 3' -ETVl sequences is of particular interest because it implies a small proportion of prostate cancers are not driven by exposure to androgens, and hence may exhibit growth resistance to androgen deprivation therapies. Joining of ETVl to individual partners was too uncommon to allow survival analysis for specific gene fusions. Of the 4 cases with a C15ORF21.-ETV1 fusion, 3 are still alive and one has died of unrelated causes. Interestingly, there was no significant difference in the expression of nuclear-localized androgen receptor (AR) using immuno-histochemical staining between C15ORF21-ETV1 tumours and tumours with ETVl or ERG fused to androgen-regulated partners implying activated AR signalling in these tumours.

As stated above, the objective of the current study was initially to use this cohort of conservatively managed prostate cancer cases to assess the potential clinical significance of ETVl gene alterations and in parallel to assess the relative frequency of the each of the known ETVl fusion partners. However, since these only accounted for about 34% of all ETVl re -arrangements, rapid amplification of 5' cDNA ends (5'-RACE) studies was carried out to identify novel ETVl fusion partners .

Example 2 - Identification of a novel fusion partner for ETVl 5'-RACE was performed to identify novel partners that are fused to 3'-ETVl sequences. Such studies were limited by the small amounts and poor quality RNA that could be prepared from the formalin fixed cancers containing ETVl re -arrangements in this series. However, 5'-RACE was successfully performed on a single RNA sample leading to the identification of ACSL3 as a novel ETVl fusion partner. The 5'-RACE product contained exon 3 of ACSL3 fused to exon 6 of the ETVl gene . The structure of this fusion which is predicted to encode a truncated ETVl protein is shown in Figure 1OA. This fusion transcript of ACSL3 exon 3 sequences fused to ETVl exon 6 sequences was found in prostate cancer sample 23. The ORF shown in Figure 1OA was predicted using software at www.dnalc.org.

The presence of the ACSL3-ETV1 fusion was confirmed in this specimen by RT-PCR using 5' -ACSL3 and 3'-ETVl primers (Figure 10C) and by co-localization on FISH hybridization of BAC probes corresponding to 5' -ACSL3 sequences and 3'-ETVl sequences (Figure 1OD panel iii) . Using this and an ACSL3 break-apart assay to screen the entire TMA containing the 23 cancers with rearrangement of the ETVl gene failed to identify additional cancers of this particular fusion. Therefore, like fusion to TMPRSS2, HNRPA2B1 , HERV-K or SLC45A5/Prostein, the fusion of 3' -ETVl sequences to 5' -ACSL3 sequences does not appear to be a particularly common event. Fusion of 5' -ETVl sequences with 3' -ACSL3 sequences indicating the mechanism underlying formation of this fusion gene was shown to be a balanced translocation (Figure 1OD panel iv) . The ACSL3 break-apart assay hybridized the same Tissue Microarray (TMA) slice used in the ETVl break-apart assay with 3' -ACLS3 sequences with 5' -ACLS3 sequences. These signals are coincident in the wild type, but are split when ACLS3 is involved in a translocation. Super-imposition of the images in panels (i) and (ii) of Figure 1OD confirms co- localization of 3'-ETVl with 5' -ACSL3 and co-localiztion of 5' -ETVl and 3'- ACSL3. This is confirmed by a ETV1-ACSL3 co-localization assay (panel iii of Figure 10D) demonstrating co-localization of 3'- ETVl sequences and 5' -ACSL3 sequences and (panel iv) co- localization of 3' -ACSL3 sequences and 5' -ETVl sequences. Superimposition of the images in panels iii and iv of Figure 1OD confirms co-localization of wild-type 3'-ETVl (panel iii) with 5' -ETVl (panel iv) and of wild-type 3 ' -ACSL3 (panel iv) with 5' -ACSL3 (panel iii) . Direction of transcription of genes is indicated by the arrowheads. All procedures used including preparation of FISH probes, hybridization of probes to cancer slices, washing slices and scanning and storage of images were carried out exactly as described previously (Clark J et al., 2007; Attard G et al., 2007) .

The protein encoded by the ACSL3 gene is an isozyme of the long-chain fatty-acid-coenzyme A ligase family that converts free long-chain fatty acids into fatty acyl-CoA esters, and thereby plays a key role in lipid biosynthesis and fatty acid degradation. Insights into the regulation of ACSL3 expression arise from examining expression array data in which the LNCaP cell-line was treated with the synthetic androgen R1881. In two independent expression array data sets, ACSL3 was up- regulated by androgen treatment (Hendriksen et al., 2006; Wang et al. , 2006) . One study showed ACSL3 up-regulation at time intervals of 2, 4, 6 and 8 hours following androgen treatment (Hendriksen et al., 2006) and another study showed ACSL3 up- regulation after 16 hours (Wang et al., 2006) . Expression of ACSL3 was also elevated in a panel of 'androgen- sensitive' (LAPC-4, LNCaP, MDA PCa2a, MDA PCa2b and 22RvI) versus 'androgen- insensitive' (PPCl, PC3 and DU145) lines (Zhao et al., 2005; Tomlins et al., 2007) . Expression of TMPRSS2 and SLC45A3 follows the same pattern within these datasets (Zhao et al. , 2005) .

Example 3 - identification of androgen-response elements upstream of ETVl 5' fusion partners

Using sequences searches, an ARE (androgen response element) has been identified about 4kb upstream of the transcription start site for ACSL3. This ARE lies within a cluster of motifs for AR pioneer or collaborating transcription factors (OCTl, GATA and ETS factors) . This makes the region spanning 3.5kb- 4.2kb upstream of ACSL3 a candidate control region for the androgen regulation of ACSL3. The predicted ARE sites in other ETS fusion partner genes are shown in Table 6.

Summary

In summary, a FISH assay was used to screen for ETVl gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans -urethral resection of the prostate. The presence of ETVl gene alterations (found in

23 cases, 5.4%) was correlated with higher Gleason Score

(p=0.001) , PSA level at diagnosis (p=<0.0001) and clinical stage (p=0.017) , but was not to linked poorer survival. It was shown that the six previously characterized 5 '-fusion partners of ETVl only accounted for 34% of ETVl re -arrangements (8/23) in this series, with joining to the androgen-repressed gene C15orf21 representing the commonest fusion event (4/23) . In 5'-RACE experiments, the androgen-regulated gene ACSL3 was identified as a new 5 '-partner of ETVl. These studies report a novel fusion partner for ETVl and highlight the considerable heterogeneity of ETVl gene rearrangements in human prostate cancer. With the exception of Tomlins et al. (2007) , who reported 4 cases, previous studies have each reported only one cancer with an ETVl rearrangement (Tomlins et al., 2005; Hermans et al., 2006; Tu et al., 2007) . The present study therefore represents the largest single series of prostate cases with an ETVl rearrangement. This study confirms previous observations that ETVl may form a fusion gene with a variety of partners but shows that each individual fusion is relatively rare. Importantly, it is shown that the known fusion partners, including the novel ACSL3 :ETV1 fusion gene, only account for 39% of cancers with an ETVl rearrangement and it is therefore likely that many new partner genes remain to be identified.

Table 1 Relationship of ETVl gene status with demographics and tumour characteristics.

ETV1 gene status

Variable p-value

Class N Class Esplit

Mean age ± SD (years) 70 ± 5 67 + 6 ' 0.04

Early hormone management 0.37

Yes 39(15%) 5 (23%)

No 213(85%) 17 (77%)

Gleason score* 0.001

<7 143(57%) 3 (14%)

=7 59 (24%) 11 (50%)

>7 49(19%) 8 (36%)

Clinical stage** 0.017

T1 76 (53%) 4 (27%)

T2 50 (35%) 6 (40%)

T3 18(12%) 5 (33%)

Baseline PSA < 0.0001

<4 102(41%) 4(18%)

>4- 10 59 (23%) 4(18%)

> 10-25 48 (19%) 3 (14%)

> 25-50 27(11%) 5 (23%)

> 50 -100 16(6%) 6 (27%)

* restricted to patients for which Gleason score is available ** restricted to patients for which clinical stage is available Abbreviation: PSA₁ Prostate Specific Antigen.

Table 1 shows the relationship of ETVl gene status with demographics and tumour characteristics. Table 2 Frequency of fusion of ETVl with previously described 5 ' partners . Class of ETV1 rearrangement Number of cases

(% of total number of ETW-rearranged cancers)

1. Fusion with C15ORF21 4 (18%)

2. Rearrangement to 14q13.3-14q21.1 2 (9%)

3. Fusion with HNRPA2B1 1 (4.5%)

4. Fusion with SLC45A5/Prostein 1 (4.5%)

5. Fusion with HERV-K 0

6. Fusion with TMPRSS2 0

Table 2 shows the frequency of fusion of ETVl with previously described 5¹ partners. The number of cases of ETVl rearrangement involving each of the 6 previously described classes, namely fusion with C150RF21 , rearrangement to 14ql3.3-14q21.1, fusion with HNRPA2B1, fusion with SLC45A5/Prostein, fusion with HERV-K and fusion with TMPRSS2, in the series of 23 ETVl -rearranged cancers is given.

Table 3 ETVl classi fication and revised Gleason score . Gleason Class N Class ETV1 Esplit Total score

4 2 δ 2

5 11 1 12

6 130 2 132

7 59 11 70

8 27 3 30

9 20 5 25

10 2 0 2 unknown 1 0 1

Total 252 22 274

Table 3 shows ETVl classification and revised Gleason score. Revised Gleason score for cancers lacking ERG and ETVl rearrangements (class N) is compared to cancers with rearrangement of ETVl (class ETVl Esplit) .

Table 4 Fluorescence in situ hybridization probes.

Probe # Gene/region Localization Probe (BAC accession number)

I ETV1 3' RP11-27B1, RP11-138H16, CTD-2008115

Il ETV1 5' RP11-905H4, RP11-621 E24, RP11-115D14

III C15ORF21 3' RP11-626F7

IV C15ORF21 5' RP11-112513

V Chromosome 14q 13.3 Chr 14q RP11-945C4, RP11-381L10, RP11-666J24,

- 21.1 13.3 - 21.1 RP11-796F21, RP11-588D7, RP11-107E23

Vl SLC45A5/Prostein 3' RP11-379J10, RP11-1143H2

VII SLC45A5/Prostein 5¹ RP11-219P13

VIII HNRPA2B1 3" RP11-91F20

IX HNRPA2B1 5' RP11-1006O2, RP11-379M24

X HERV-K _22q11.23 3¹ RP11-71G19

Xl HERV-K _22q 11.23 5¹ RP11-947A12

XII TMPRSS2 3¹ RP11-114H1, 11:RP11-662D5

XIII TMPRSS2 5' three pooled fosmids (G248P89444D12,

G248P800876A1, G248P8239C5), RP11-

35C4. RP11-282120

XIV ACSL3 3' RP11-136M23,

RP11-749C15

XV ACSL3 5¹ RP11-157M20

Table 4 lists the probes used in FISH assays. The probe number, gene/region, localization (5'/3') to the transcript, and BAC accession number for all the probes utilized are given.

Table 5 Fluorescent in situ hybridization assays.

FISH assay Genes/region tested Probe #

1. ETV1 break-apart 3'-ETV1 + 5'-ETV1 I + Il

2. C15ORF21 break-apart 3'-C15ORF21 + 5'-C15ORF21 III + IV

3. ETV1 to C15ORF21 co-localization 3'-ETV1 + 5'-C15ORF21 I + IV

4. ETV1 to chromosome 14q13.3-21.1 3'-ETV1 + chr 14q13.3 - 21.1 I +V

5. SLC45A5/Prostein break-apart 3'-SLC45A5 + 5'-SLC45A5 Vl + VII

6. ETV1 to SLC45A5 co-localization 3'-ETV1 + 5'-SLC45A5 I + Vl

7. HNRPA2B1 break-apart 3'-HNRPA2B1 + 5'-HNRPA2B1 VIII + IX

8. ETV1 to HNRPA2B1 co-localization 3'-ETV1 + 5'-HNRPA2B1 I + VIII

9. HERV-K _22q11.23 break-apart 3'-HERV-K + 5'-HERV-K X + XI

10. ETV1 to HERV-K _22q11.23 co-localization 3'-ETV1 + 5'-HERV-K I + X

11. TMPRSS2 break-apart 3'-TMPRSS2 + 5'-TMPRSS2 XII + XIII

12. ACSL3 break-apart 3'-ACSL3 + 5'-ACSL3 XIV + XV

13. ETV1 to ACSL3 co-localization 3'-ETV1 + 5'-ACSL3 I + XIV

14. 5'-ETV1 to 3'-ACSL3 co-localization 5'-ETV1 + 3'-ACSL3 Il + XV

Table 5 shows the gene/region studied and the probes used for the FISH assays utilized in this study.

Table 6 Androgen receptor (AR) binding sites (AREs) in the genomic regions of ETS fusion partner genes .

Table 6 shows androgen receptor (AR) binding sites (AREs) in the genomic regions of ETS fusion partner genes. Mapped AR binding sites were identified from four published AR chromatin immunoprecipitation (ChIP) -on-chip studies (Bolton et al., 2007; Massie et al., 2007; Wang et al., 2007; Takayama et al., 2007) . AR ChlP-chip data were retrieved, converted to GFF format and uploaded to the UCSC Genome Browser. Genomic loci around the ETS fusion partner genes ('Targets') were examined for the nearest AR binding sites found in any of these studies. In a parallel approach we searched for predicted AR binding sites in the 5kb promoter sequences of the ETS fusion partner genes ('Targets') . Sequences corresponding to the 5kb regions immediately upstream of the transcriptional start site for each gene were retrieved from the UCSC Genome Browser and these sequences were analysed for the occurrence of the consensus ARE sequence using JASPAR. References

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Claims

1. An isolated nucleic acid molecule comprising an upstream component fused to a downstream component at a fusion site, wherein the upstream component comprises a portion of an acyl- CoA synthetase long- chain family member {ACSL3) gene and the downstream component comprises a portion of an ETS family- member gene , or the reverse complement thereof .

2. An isolated nucleic acid molecule according to claim 1 wherein the upstream component includes an upstream region of the ACSL3 gene.

3. An isolated nucleic acid molecule according to claim 1 or claim 2 wherein the ETS family member gene is ETVl.

4. An isolated nucleic acid molecule according to any one of claims 1 to 3 wherein, at the fusion site, a portion of the ACSL3 gene is contiguous with a portion of the ETS family member gene .

5. An isolated nucleic acid molecule according to claim 3 wherein the upstream component includes exons 1 and 2 and at least a portion of exon 3 of the ACSL3 gene and the downstream component includes exons 6 to 12 of the ETVl gene.

6. An expression vector comprising an isolated nucleic acid according to any one of claims 1 to 5.

7. A host cell comprising an expression vector according to claim 6.

8. A non-human transgenic animal having a transgene comprising a nucleic acid molecule as described in any one of claims 1 to 5.

9. A targeting oligonucleotide comprising a first nucleic acid sequence capable of hybridising to the sense or antisense strand of an ACSL3 gene , or to an mRNA sequence encoded thereby, and a second nucleic acid sequence capable of hybridising to the sense or antisense strand of an ETS family member gene, or to an mRNA sequence encoded thereby, wherein said targeting oligonucleotide is capable of inhibiting, reducing or blocking expression of a fusion between said ACSL3 gene and said ETS family member gene.

10. A nucleic acid probe comprising a first nucleic acid sequence capable of hybridising to the sense or antisense strand of an ACSL3 gene, or to an mRNA sequence encoded thereby, and a second nucleic acid sequence capable of hybridising to the sense or antisense strand of an ETS family member gene, or to an mRNA sequence encoded thereby.

11. A targeting oligonucleotide or nucleic acid probe according to claim 9 or claim 10 wherein the first nucleic acid sequence is complementary to a portion of the sense or antisense strand of an ACSL3 gene, or to the mRNA sequence transcribed therefrom, and the second nucleic acid sequence is complementary to a portion of the sense or antisense strand of an ETS family member gene, or to the mRNA transcribed therefrom.

12. A targeting oligonucleotide or nucleic acid probe according to any one of claims 9 to 11 wherein the first nucleic acid sequence is at least 20 nucleotides in length and the second nucleic acid sequence is at least 20 nucleotides in length.

13. A targeting oligonucleotide or nucleic acid probe according to any one of claims 9 to 12 wherein the first nucleic acid sequence is contiguous with the second nucleic acid sequence.

14. A targeting oligonucleotide or nucleic acid probe according to any one of claims 9 to 12 wherein a spacer region is present between the first and second nucleic acid sequences .

15. A targeting oligonucleotide or nucleic acid probe according to any one of claims 9 to 14 wherein the ETS family member gene is ETVl.

16. A targeting oligonucleotide or nucleic acid probe according to any one of claims 9 to 15 wherein the targeting oligonucleotide is an antisense oligonucleotide comprising a first nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ACSL3 gene, and a second nucleic acid sequence capable of hybridising to an mRNA sequence encoded by an ETS family member gene.

17. A siRNA molecule, miRNA molecule or triple helix inhibitor comprising a targeting oligonucleotide according to any one of claims 9 or 11 to 16.

18. A nucleic acid probe according to any one of claims 10 to 16 wherein the probe is labelled or is a primer for use in PCR.

19. A fusion protein comprising an N-terminal component and a C- terminal component, wherein the N-terminal component comprises a portion of an ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein.

20. A fusion protein according to claim 19 wherein the N- terminal component includes an N-terminal portion of the ACSL3 protein.

21. A fusion protein according to claim 18 or claim 19 wherein the ETS family member protein is ETVl.

22. A fusion protein according to any one of claims 19 to 21 wherein the fusion protein is encoded by a nucleic acid as described in any one of claims 1 to 5.

23. A method of detecting a nucleic acid molecule in a sample, the nucleic acid molecule comprising an upstream component fused to a downstream component at a fusion site, wherein the upstream component comprises a portion of an acyl- CoA synthetase long- chain family member {ACSL3} gene and the downstream component comprises a portion of an ETS family- member gene , or the reverse complement thereof .

24. A method according to claim 23 wherein the nucleic acid molecule to be detected is genomic DNA, mRNA or cDNA.

25. A method according to claim 23 or claim 24 comprising the step of contacting the sample with a probe which binds to the

ACSL3 gene and to the ETS family member gene, wherein binding of the probe to nucleic acid in the sample indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene.

26. A method according to claim 25 wherein the probe is a nucleic acid probe according to any one of claims 10 to 16.

27. A method according to any one of claims 23 to 26 comprising in situ hybridisation (e.g. fluorescent in situ hybridisation) , Southern blotting (e.g. following pulsed field gel electrophoresis) , northern blotting or PCR.

28. A method according to claim 23 or claim 24 comprising the step of contacting the sample with a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to nucleic acid downstream of the ETS family member gene .

29. A method according to claim 28 wherein binding of the first and second probes to sequences on the same piece of nucleic acid (e.g. on the same chromosome or on the same piece of mRNA or cDNA) indicates co-localisation or juxtaposition of the first and second probes.

30. A method according to claim 28 or claim 29 wherein the first probe is a nucleic acid probe having the same nucleic acid sequence as RP11-157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-27B1, RP11-138H16 or CTD-2008I15 shown in Table 4.

31. A method according to any one of claims 28 to 30 comprising the steps of: i) denaturing nucleic acid in the sample; (ii) annealing the first and second nucleic acid probes to a target nucleic acid sequence in the sample; and (iii) amplifying the target nucleic acid sequence; wherein amplification of the target nucleic sequence indicates the presence of a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene.

32. A method according to claim 31 which is carried out using polymerase chain reaction (PCR) .

33. A kit for detecting a nucleic acid molecule in a sample, the nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene at a fusion site, comprising

(i) a nucleic acid probe according to any one of claims 10 to 16; or (ii) a first probe which binds to the ACSL3 gene and/or to nucleic acid upstream of the ACSL3 gene, and a second probe which binds to the ETS family member gene and/or to nucleic acid downstream of the ETS family member gene.

34. A kit according to claim 33 wherein the first probe is a nucleic acid probe having the same nucleic acid sequence as RPIl- 157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-27B1, RPIl- 138H16 or CTD-2008I15 shown in Table 4.

35. A method for detecting break-apart of an ACSL3 gene in a sample, wherein the method includes the step of contacting the sample with a first probe which binds to an upstream portion of the ACSL3 gene and/or to chromosomal DNA upstream of the ACSL3 gene and a second probe which binds to a downstream portion of the ACSL3 gene and/or to chromosomal DNA downstream of the ACSL3 gene, wherein binding of the first and second probes to physically separate pieces of DNA (e.g. to different chromosomes) indicates break-apart of the ACSL3 gene.

36. A method according to claim 35 wherein the first probe is a nucleic acid probe having the same nucleic acid sequence as RP11-157M20 and/or the second probe is a nucleic acid probe having the same nucleic acid sequence as RP11-136M23 or RPIl- 749C15 shown in Table 4.

37. A kit for detecting break-apart of the ACSL3 gene, the kit comprising first and second probes as described in claim 35 or claim 36.

38. A method of detecting a fusion protein as described in any one of claims 19 to 22 in a sample, wherein the method includes the step of contacting the sample with an agent which binds specifically to the fusion protein.

39. A method according to claim 38 wherein the agent is an antibody which binds to an ACSL3 protein and to an ETS family member protein, such as ETVl, ETV4 , ERG or ETV5.

40. A method of diagnosing prostate cancer in a patient comprising the steps of:

(i) providing a sample from a patient; and (ii)

(a) detecting a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene by a method according to any one of claims 23 to 32; (b) detecting break-apart of an ACSL3 gene in the sample by a method according to claim 35 or claim 36; or (c) detecting a fusion protein in the sample by a method according to claim 38 or claim 39, wherein the fusion protein comprises an N-terminal component fused to a C-terminal component, wherein the N-terminal component is a portion of an ACSL3 protein and the C-terminal component is a portion of an ETS family member protein, ; wherein the respective presence of said nucleic acid molecule, break-apart of said ACSL gene, or presence of said fusion protein indicates that the patient has prostate cancer, that the patient has an aggressive form of prostate cancer, or is likely to develop prostate cancer.

41. A method according to claim 40 wherein the sample from the patient is a blood, semen or urine sample, or a biopsy from a prostate tumour or prostate tissue.

42. An agent capable of binding specifically to a fusion protein as described in any one of claims 19 to 22, a nucleic acid probe according to any one of claims 10 to 16, or a kit according to any one of claims 33, 34 or 36 for use in the diagnosis of prostate cancer.

43. A method of determining whether a tumour from a patient with prostate cancer is suitable for anti -androgen therapy comprising the steps of:

(i) providing a sample from a patient; and (ii)

(a) detecting a nucleic acid molecule comprising a portion of an ACSL3 gene fused to a portion of an ETS family member gene by a method according to any one of claims 23 to 32; (b) detecting break-apart of an ACSL3 gene in the sample by a method according to claim 35 or claim 36; or

(c) detecting a fusion protein in the sample by a method according to claim 38 or claim 39, wherein the fusion protein comprises an N-terminal component fused to a C-terminal component, wherein the N-terminal component is a portion of an ACSL3 protein and the C-terminal component is a portion of an ETS family member protein, ; wherein the respective presence of said nucleic acid molecule, break-apart of said ACSL gene, or presence of said fusion protein indicates that the tumour is suitable for anti- androgen therapy.

44. An agent capable of binding specifically to a fusion protein as described in any one of claims 19 to 22, a nucleic acid probe according to any one of claims 10 to 16, or a kit according to any one of claims 33, 34 or 36 for use in determining whether a tumour from a patient with prostate cancer is suitable for anti -androgen therapy.

45. A method of treating a patient with prostate cancer, the method including the step of administering to the patient an agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to a portion of an ETS family member gene, or (ii) activity of a fusion protein comprising an N-terminal component fused to a C-terminal component, wherein the N-terminal component comprises a portion, preferably an N-terminal portion, of the ACSL3 protein and the C-terminal component comprises a portion of an ETS family member protein.

46. A method according to claim 45 wherein the ETS family- member gene or protein is ETVl.

47. An agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to an ETS family member gene, or (ii) activity of a fusion protein comprising a portion of a ACSL3 protein fused to a portion of an ETS family member protein, for use in a method for treatment of prostate cancer .

48. Use of an agent which inhibits (i) transcription or translation of a nucleic acid molecule in which a portion, preferably an upstream portion, of an ACSL3 gene is fused to an ETS family member gene, or (ii) activity of a fusion protein comprising a portion of a ACSL3 protein fused to a portion of an ETS family member protein, in the manufacture of a medicament for the treatment of prostate cancer.

49. Use, or an agent for use in a method of treatment, according to claim 47 or 48 wherein the ETS family member is ETVl.

50. Use, or agent for use in a method of treatment, according to any one of claims 47 to 49 wherein the agent is a targeting oligonucleotide, an antisense oligonucleotide, a ribozyme, an siRNA or miRNA molecule, a triple helix inhibitor, or an antibody which binds to a fusion protein in which a portion of the ACSL3 protein is fused to a portion of an ETS family member protein.

51. A pharmaceutical composition comprising a targeting oligonucleotide, an antisense oligonucleotide, a ribozyme, an siRNA or miRNA molecule, a triple helix inhibitor, or an antibody which binds to a fusion protein in which a portion ^"of the ACSL3 protein is fused to a portion of an ETS family member protein, in admixture with a pharmaceutically acceptable carrier.

52. A method of determining a prognosis for prostate cancer comprising the steps of:

(i) providing a sample from a patient with prostate cancer; and

(ϋ)

(a) detecting a nucleic acid molecule comprising an upstream component fused to a portion of an ETVl gene; or

(b) detecting a fusion protein in the sample, said fusion protein comprising an N-terminal component fused to a C- terminal component, wherein the C- terminal component comprises a portion of an ETVl protein; wherein the detection of said nucleic acid molecule or fusion protein indicates a higher Gleason score and/or a later clinical stage of disease than if said nucleic acid molecule or fusion protein is not detected.

53. A method according to claim 51 wherein the upstream or N- terminal component comprises a portion, more preferably an upstream portion, of a gene or protein selected from the group: TMPRSS2 , SLC45A3/Prostein, HERV-K, C15orf21, HNRPA2B1, SLC25A21, MIPOLl, FOXAl, TTC6 , or ACSL3.