WO2005107782A2

WO2005107782A2 - Use of a modulator of gene expression in the treatment of cancer

Info

Publication number: WO2005107782A2
Application number: PCT/GB2005/001765
Authority: WO
Inventors: Helen Louise Rogers; Anne Willis; Martin Bushell
Original assignee: University of Nottingham
Current assignee: University of Nottingham
Priority date: 2004-05-11
Filing date: 2005-05-10
Publication date: 2005-11-17
Anticipated expiration: 2006-11-11
Also published as: GB0410379D0; WO2005107782A3

Abstract

The use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, in the treatment of cancer.

Description

USE OF A MODULATOR OF GENE EXPRESSION IN THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates to the use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, in the treatment of cancer. The invention also relates to a medicament comprising a modulator and methods for treatment of cancer using a modulator. In certain aspects, the invention further relates to screening methods for identifying potential targets for the treatment of cancer and other disorders, to methods for the diagnosis and prognosis of cancer, including kits to achieve this, and to methods for identifying compounds potentially useful in such treatments.

BACKGROUND TO THE INVENTION

Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world with about 2500 new cases diagnosed each year in the UK. The disease remains incurable with conventional chemotherapy, and the median survival is ten years. There appears to be an inherited predisposition to CLL (Rawstron et al 2002) and there are more males than females with the disease. Disease progression is very variable with some patients surviving for many years with little or no therapy whilst others die rapidly with aggressive forms of the disease (Rozman 1995).

In CLL there are elevated levels of clonal leukaemic B-cells that typically express CD19, CD23 and CD5 and have low levels of CD20 (Kipps 2003). These leukaemic B- CLL cells can be segregated further based on the mutational status of the immunoglobulin genes and this has consequences for patient survival.

Studies involving cDNA microarrays to detect transcriptional changes have found that CLL B-cells from different patients share common expression profiles (Rosenwald et al 2001; Klein et al 2001; Aalto et al 2001; Straowa et al 2001;Jenlink et al 2003). This would suggest that CLL B-cells share a common mechanism of transformation and probably cell origin (Rosenwald et al 2001; Klein et al 2001).

However, whilst these transcriptional studies are useful aids to disease diagnosis they have not given much insight into the biology of CLL. In particular, these markers do not provide a rationale as to why these cells are resistant to apoptosis in vivo (Osorio et al 1998) yet rapidly under go apoptosis in vitro (King et al 1998, Almond et al 2001; MacFarlane et al 2002).

In the present invention a number of genes have been identified which are translationally up or down-regulated in disease state cells, such as CLL cells, compared with normal cells. The genes have been identified by examining the mRNAs that are associated with the polysomes i.e. the pool of mRNAs associated with actively translating ribosomes (Carter et al 2000) in the cells. The invention also relates to the use of modulators to modulate the effect of these translational changes, allowing the identification of new uses for modulators and new treatments for diseases such as cancer.

SUMMARY OF THE INVENTION

According to a first aspect the invention provides the use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, in the treatment of cancer.

According to a further aspect the invention provides the use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, in the preparation of a medicament for the treatment of cancer. Preferably in the use of the present invention the one or more genes are selected from the group comprising the genes listed in Figures 2 A to 11B. More preferably the genes listed in Figures 4 A to 1 IB.

More preferably the one or more genes are selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTG1 gene; the IGF2R gene; the DAB2 gene; the BECN1 gene; the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAF1 gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

The TNFα signalling pathway genes, the CLU gene and the GZMA gene have all been shown to be downregulated in CLL cells. The IGF1 gene, the IGF2 gene, the chemokine 7 gene, the IL17 gene, the BTG1 gene, the IGF2R gene, the DAB2 gene, the BECN1 gene and the AGIIR gene have all shown to be upregulated in CLL cells.

The modulator may increase or decrease the activity of the one or more genes or increase or decrease the activity of the expression product of the one or more genes.

The modulator may be an antagonist of angiotensin II .

The cancer may be leukaemia. The cancer may be chronic lymphocytic leukaemia (CLL).

According to another aspect the invention provides a medicament comprising a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, in a pharmaceutically acceptable form, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression.

The medicament may also comprise a pharmaceutical carrier, diluent or excipient. Preferably the modulator in the medicament is at a concentration which upon administration to a patient is effective for the treatment of cancer. Effective treatment of cancer may be determined based on the extent of cell death amongst the cancer cells or survival of the treated cancer patients. Preferably effective treatment will result in the death of at least 25% of the cancer cells present at the time of administration. More preferably at least 30%, more preferably at least 50% of the cancer cells are killed by the treatment. Alternatively, the effectiveness may be measured on the basis that survival rates are improved by 25%, more preferably by 30% and more preferably still by 50%.

A medicament according to the invention may be used in the treatment of cancer. The cancer may be chronic lymphocytic leukaemia.

The modulator in the medicament may modulate the activity of one or more genes, or the expression product of one or more genes, selected from the group comprising the genes listed in Figures 2 A to 1 IB. More preferably from the genes as listed in Figures 4A and 11B. More preferably the one or more genes are selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTG1 gene; the IGF2R gene; the DAB2 gene; the BECN1 gene; the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAF1 gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

The modulator may be an antagonist of angiotensin II.

According to a further aspect the invention provides a method of treatment of cancer comprising modulating the activity of one or more genes, or modulating the activity of the expression product of one or more genes, wherein the one or more gene are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression. The data provided shows that genes falling into these groups are translationally up- or down-regulated in CLL cells compared with normal cells.

Preferably the method of treatment is applied to a human or non-human animal. The method of treatment may be applied to mammals, conveniently primates, and preferably humans.

The method may be used to treat leukaemia. The leukaemia may be chronic lymphocytic leukaemia (CLL).

The one or more genes may be selected from the group comprising the genes listed in Figures 2 A to 11B. More preferably the genes as listed in Figures 4 A to 11B. The genes are identified by standard abbreviated names, and the skilled person will be aware of the identity and nature of each gene. Preferably the one or more genes are selected from group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTG1 gene; the IGF2R gene; the DAB2 gene; the BECN1 gene; the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAF1 gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

In certain aspects of the invention, related genes to each of these may be modulated; for example, related genes being of the same family, or having a similar activity.

The activity of the expression product of one or more genes may be modulated by an antagonist of angiotensin II.

The expression product of the one or more genes may be the angiotensin II receptor (AGIIR).

In the method of treatment of the invention the step of modulating the gene activity, or expression product activity, may comprise either increasing or decreasing the activity. Wliere the gene has been identified as being upregulated in cancer cells, modulating may comprise decreasing activity, and conversely increasing activity may be used where the gene has been identified as being downregulated.

In all aspects of the invention the gene activity may be modulated by any suitable means available to the skilled man, for example, the means may comprise altering the transcription or translation of the gene, using a selective chemical inhibitor, adding an antisense nucleic acid, using RNAi, adding a repressor, adding an antibody or the like, introducing a vector for expression of the gene in a patient, introducing additional copies of the gene product into a cell, and the like.

In all aspects of the invention the modulation of an expression product of a gene may comprise increasing or decreasing the activity of the expression product; where the gene has been identified as being upregulated in cancer cells, modulating may comprise decreasing activity, and conversely increasing activity may be used where the gene has been identified as being downregulated. Modulation of expression product activity may be achieved by any suitable means available to the skilled person; for example, the modulator may be an agonist, antagonist, activator, stimulator, suppressor or inhibitor, such as a selective chemical inhibitor, of the expression product.

The method of treatment of the invention may be performed on/in tumour cells in a patient. Preferably the method of treatment is performed substantially only on/in tumour cells.

According to a yet further aspect the invention provides a method of treatment of cancer, comprising modulating the activity of an angiotensin II receptor in a patient.

The method may also comprise the step of administering an antagonist of angiotensin II to the patient.

The method may also comprise administering an antagonist of the angiotensin- converting enzyme to the patient.

Examples of suitable antagonists are known to the skilled man. The method may be performed where the cancer is leukaemia, preferably chronic lymphocytic leukaemia.

According to a further aspect the invention provides a method of screening a sample to identify potential targets for treatment of a disorder characterised by altered gene activity, the method comprising comparing the level of mRNA translation of one or more genes in a cell suffering from the disorder with that of a normal cell.

The disorder may be cancer. The cancer may be leukaemia.

The cell is preferably taken from a patient suffering from the disorder, this allows for treatment to be targeted to that particular patient, since it is possible that patterns of altered gene translation may differ among patients.

Preferably the comparison step comprises identifying one or more mRNA molecules present in polysomes of each cell, and comparing the identified mRNAs. The quantity of mRNA of each type present in each cell is preferably also determined and compared. Conveniently the comparison step may make use of one or more nucleic acid arrays containing each type of mRNA to be detected.

According to another aspect the invention provides a method of identifying potential treatments for cancer, the method comprising the steps of screening a sample to identify potential targets for treatment by comparing the levels of mRNA translation of one or more genes in a cancer cell with that of a normal cell, identifying one or more target genes or gene expression products where the level of mRNA translation is different in the cancer cell compared to the normal cell, treating a cancer cell with a candidate treatment, and determining whether the activity of an identified target is modulated by the candidate treatment and whether there is any cell death.

The modulation of activity may be determined by assessing the candidate treatment effect on apoptosis of the cancer cell compared with a control non-cancer cell. According to a further aspect the invention provides a method of diagnosing a cancer in a sample from a human or non-human animal comprising determining the expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, wherein an increase or decrease in expression compared to a normal sample is indicative of cancer.

The one or more genes may be selected from the group comprising the genes listed in Figures 2 A to 1 IB. More preferably the genes listed in Figures 4 A to 1 IB. Preferably the one or more genes are selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTG1 gene; the IGF2R gene; the DAB2 gene; the BECN1 gene; the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAF1 gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene, is determined.

The cancer may be leukaemia, preferably chronic lymphocytic leukaemia.

The gene expression may be determined at the level of mRNA translation. The level of mRNA translation may be determined by isolating and analysing polysomes.

According to a yet further aspect the invention provides a method of determining the prognosis for an individual with cancer comprising determining the level of expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, administering a modulator intended to modulate the activity of one or more of the genes, or the activity of one or more of the expression products of the one or more genes, and assessing the effect of the modulator on the cancer progression. Preferably the modulator increases or decreases the activity of a gene or a gene expression product whose activity is increased or decreased in the individual with cancer when compared to an individual without cancer.

Preferably the modulator increases activity if activity was decreased in the individual with cancer, and the modulator decreases activity if activity was increased in the individual with cancer.

According to a further aspect the invention provides a kit for diagnosing a cancer, or providing a prognosis following treatment, in a sample from a human or non-human animal comprising means to determine the expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, and instructions to compare the results with expression levels in a normal sample from a human or non-human animal without the cancer of interest.

Preferably a kit according to the invention includes instructions that an increase or decrease in expression compared to a noraial, non-cancerous, sample is indicative of cancer.

Preferably a kit according to the invention includes means to determine the expression of one or more genes selected from the group comprising the genes listed in Figures 2 A to 1 IB. More preferably the genes listed in Figures 4 A to 1 IB. The kit may include means to determine the expression of one or more genes selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTG1 gene; the IGF2R gene; the DAB2 gene; the BECN1 gene; the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAF1 gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

The kit may be used where the cancer is leukaemia, preferably where the cancer is chronic lymphocytic leukaemia. The kit may include a microarray designed to determine the level of expression of the one or more genes.

The kit may be designed for use with polysomes.

Altematively, the kit may be ELISA based, and may include one or more antibodies directed to the expression product of the one or more genes.

According to another aspect the invention provides a method for identifying potential treatments for cancer, the method comprising the steps of identifying a gene whose level of translation is altered in a cancer cell compared with a normal cell; and contacting a cell suffering from the cancer with a candidate treatment, and determining whether the activity of the identified gene is modulated by the candidate treatment.

According to a further aspect the invention provides a method of treatment of cancer comprising modulating the activity of a gene identified by comparing the level of mRNA translation of one or more genes in a cell suffering from cancer with that of a noraial cell, the method comprising administering a modulator of the activity of the identified gene.

The identification of the gene may be carried out on a cancer cell of a patient to be treated by the method.

According to a still further aspect the invention provides the use of a gene selected from the group comprising cell surface receptors, genes associated with apoptosis, genes associated with cell proliferation, and genes associated with cell cycle progression in the preparation of a medicament for the treatment of cancer. Preferred genes are selected from the TNFα signalling pathway, CLU, GZMA (all genes which have been found to be downregulated), IGF1, IGF2, chemokine 7, IL17, BTG1, IGF2R, DAB2, BECN1, AGIIR (all genes which have been found to be upregulated). A particularly preferred embodiment of the invention provides the use of the angiotensin II receptor (AGIIR) in the preparation of a medicament for the treatment of cancer. The identification of the gene may be carried out on a cancer cell of a patient to be treated by the method.

In a yet further aspect the invention provides a method of treatment of cancer comprising modulating the activity of a gene identified as having an increased level of translation in cancer cells.

The skilled man will appreciate that preferred features of the invention discussed with reference to only some aspects of the invention can be applied to other aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example only, with reference to the accompanying drawings, in which

Figure 1 illustrates the method employed for microarray analysis and identification of translationally-altered genes;

Figures 2A and 2B shows a cluster analysis of the top 50 translationally up-regulated mRNAs in CLL cells. Figure 2A is a numerical representation of the data and Figure 2B is a pictorial representation of the data;

Figures 3A and 3B shows a cluster analysis of the top 50 translationally down-regulated mRNAs in CLL cells. Figure 3 A is a numerical representation of the data and Figure 3B is a pictorial representation of the data;

Figures 4A and 4B shows a cluster analysis of the up-regulated apoptosis related mRNAs in CLL cells. Figure 4A is a numerical representation of the data and Figure 4B is a pictorial representation of the data; Figures 5A and 5B shows a cluster analysis of the down-regulated apoptosis related mRNAs in CLL cells. Figure 5A is a numerical representation of the data and Figure 5B is a pictorial representation of the data;

Figures 6A and 6B shows a cluster analysis of the up-regulated cell cycle related mRNAs in CLL cells. Figure 6A is a numerical representation of the data and Figure 6B is a pictorial representation of the data;

Figures 7A and 7B shows a cluster analysis of the down-regulated cell cycle related mRNAs in CLL cells. Figure 7A is a numerical representation of the data and Figure 7B is a pictorial representation of the data;

Figures 8A and 8B shows a cluster analysis of the up-regulated proliferation related mRNAs in CLL cells. Figure 8 A is a numerical representation of the data and Figure 8B is a pictorial representation of the data;

Figures 9 A and 9B shows a cluster analysis of the down-regulated proliferation related mRNAs in CLL cells. Figure 9A is a numerical representation of the data and Figure 9B is a pictorial representation of the data;

Figures 10 A and 10B shows a cluster analysis of the up-regulated receptor related mRNAs in CLL cells. Figure 10A is a numerical representation of the data and Figure 1 OB is a pictorial representation of the data;

Figures 11 A and 11B shows a cluster analysis of the down-regulated receptor related mRNAs in CLL cells. Figure 11A is a numerical representation of the data and Figure 1 IB is a pictorial representation of the data;

Figure 12 shows the effect of incubating CLL cells with angiotensin II and angiotensin antagonists cDNA MICROARRAY ANALYSIS

cDNA microarray analysis on polysomally associated mRNAs allowed those genes that are up-regulated or down regulated at the level of translation in chronic lymphocytic leukaemia (CLL), when compared to control CD 19 positive B-cells isolated from tonsils of healthy adults, to be identified. By performing this type of analysis, in comparison to transcriptional cDNA microarrays, the changes identified are more closely related to changes in the protein expression profile in CLL B-cells. The data from the mircoarray studies suggest that a number of separate pathways are required for survival of CLL B-cells which include extrinsic factors such as cytokines, cell surface receptors expressed on CLL B-cells that are involved in either survival factor binding or binding to extra-cellular matrix and de-regulated expression of proteins that form part of the apoptotic machinery.

Methodology

The following methodology for cDNA microarray analysis was followed to identify up and down-regulated genes.

Sample preparation - CD 19 positive B cells were purified from CLL patients (11 patients) and from tonsils from healthy individuals (10 people). Contaminating T-cells were removed by incubation with anti-CD3 magnetic beads. The B cells were lysed and applied to 10-55% sucrose gradients that were centrifuged and then fractionated with continuous monitoring at A254. Pools of actively translating ribosomes were distinguished from the monosomes that are not translating. Interestingly, 2.5 times more CLL B-cells than tonsil B-cells were lysed yet this gave an equivalent number of polysomes when compared to the control cells. This suggests that the CLL B-cells are translationally repressed, consistent with studies that have shown that these cells are quiescent (Osario et al 1998). Polysome and monosomal fractions were identified from the sucrose gradients and RNA isolated.

Microarray - Minimum Information About a Microarray Experiment" - MIAME recommendations were used in the experimental design. The microarrays slides used in this study were generated by the laboratory of Dr T Gant (MRC centre for Mechanisms in Human Toxicity, Leicester). The arrays contain 10,000 named genes represented by cDNA clones including most known oncogenes, tumour suppressor and cell cycle related genes. The genes that had been identified in other microarray studies of CLL (Jelinek et al 2003; Klien et al 2001; Rosenwald et al 2001) were present on these array chips. The arrays were prepared using a Stanford type arrayer (see http://www.le.ac.uk/cmht/twgl/array-fp.html) in batches of 137. Clone databases are maintained and updated with all of the latest clone information from GenBank to ensure that the highest accuracy is maintained in the clone identification and Unigene cluster membership. Initially the polysomal and monosomal RNAs were labelled as shown and samples were then hybridised to array slides at 42°C for 18 hours in a 60μl volume containing 20μg COT 1 DNA, 20μg Poly A RNA and 20 μg tRNA in 1.3 x SSC. After washing the microarray slides were scanned with an Axon scanner. All micro arrays were performed in triplicate with reverse dye labelling.

Results

The translational microarray study described above was used to assess the differences at a translational level in CLL B-cells derived from CLL patients and B-cells from healthy adults (Figure 1). The data, when analysed, identified 500 significant changes in the mRNAs that are present on the polysomes between the CLL B-cells and the control B- cells from tonsils, and 50 of the most and least changed mRNAs are shown (Figures 2 A to 3B). Interestingly, using this method, a very different sub-set of mRNAs were obtained as translationally up-regulated when compared to the data that has been obtained from the transcriptional studies (Jelinek et al 2003; Table 1; Figure 2A to 3B). Analysis of the total pool of mRNAs demonstrated significant changes in the polysomal association of mRNAs whose protein products are receptors, associated with processes of apoptosis, required for cell proliferation or necessary for cell cycle progression (Figures 4A and 11 B).

The examination of mRNAs that show a significant decrease in CLL B-cells, as illustrated in Figures 4 A and 11B, interestingly demonstrate that TNF family members are down-regulated. This is family of related ligands and receptor proteins the pleiotropic actions of which include apoptotic and necrotic cell death mechanisms (Dempsey et al 2003). The proteins and receptors that show a decreased association with the polysomes in CLL B-cells include FADD, TRAF1, TRAF3, TRAF4, NGFR, RIPK1, RIPK2, TNF and TNFRSF10, 11 and 12. In addition, there is a reduction in polysomal association of other proteins associated with pro-apoptotic functions including, Clusterin (Santinlli et al 2003), PCDC5 and Siva (Prasad et al 1997). There are also distinct decreases in the polysomal association of mRNAs whose protein products regulate cell cycle progression including CDC34 (mitosis), CDC20 (mitosis), CCNB1 (cyclin Bl mitosis), CDKN1A (cyclin dependent kinase 1A, regulated progression tlirough Gl), suggesting a cell-cycle block in CLL B-cells.

Figures 4A to 11B illustrates an increased expression of several cytokines/chemokines and receptors thereof including, IL17 receptor, IL24, IL2 receptor, chemokine receptor 4 and 7 (Burger and Kipps 2002; Kipps 2003, Muller and Lipp 2003). There is also an increase in polysomal association of, and number of, mRNAs that would contribute to the quiescent nature of CLL B-cells e.g. B-cell translocation gene 1 (BTG1; Prevot et al 2003). Finally several mRNAs that have anti-apoptotic functions, including Bax inhibitor 1 (TEGT;Jean et al 1999) and serine protease inhibitor 1 (serpinB2), also display an increase in polysomal association. There was a large difference in BECN1 (Beclin) mRNA association with the polysomes.

The data from the microarray studies correlate with the biology of the disease and in part provide a rationale as to why these CLL B-cells do not undergo apoptosis in vivo. Thus the data for the mRNAs that are down-regulated clearly show that there is a defect in the tumour necrosis factor alpha (TNF alpha) family signalling pathway (Figures 5A, 5B, 7 A, 7B, 9A, 9B 11 A and 1 IB). Moreover the data also suggest that there is a cell cycle block in CLL B-cells with reduced polysomal association of mRNAs that are required for progression through mitosis (Figures 5 A, 5B, 7A, 7B, 9A, 9B 11A and 1 IB). In CLL B-cells there is an increased association of a number of mRNAs encoding cytokines and their ligands with the polysomes and this could contribute to the survival of these cells in vivo (Figures 4A, 4B, 6 A, 6B, 8 A, 8B 10A and 10B). Analysis of the translational data suggests that the CLL B-cells share a common mechanism of transformation since many of the same mRNAs are translationally upregulated in all cases. It is clear however, that some of the patients fall into different sub-groups. For example, patients 6-8 have more related translation profiles, particularly in regard to decreased polysomal association, than the others (Figures 2a to 3B).

Northern analysis (to compare mRNA levels in the polysomes/ subpolysomes) and/or Western analysis (where antibodies are available) may be used to validate the results of the mRNA polysome analysis to confirm up and down regulation in translation.

(i) Testing the effects of addition of exogenous ligands on cell survival

CLL B-cells rapidly undergo apoptosis in vitro (6-24 hours; MacFarlane et al 2002) and therefore can be used to test whether the addition of either the ligands for receptors identified in the cDNA microarray screen and/or the ligands that show increased translation in CLL B-cells, promote cell survival in vitro.

CD 19 positive CLL and control B-cells can be purified as described (Figure 1; MacFarlane et al 2002) and the cells re-suspended in RPMI media and then incubated for up to 24 hours in the presence/absence of the ligands either singly or in combination. The degree of apoptosis in these cells can be assessed by measuring the increase in externalised phosphatidylserine by Annexin V labelling (King et al 1998). In addition, changes in the apoptotic machinery can be determined by Western analysis to examine caspase processing and any changes in the levels of anti-apoptotic molecules such as c- FLIP, XIAP and Bel family members (MacFarlane et al 2002).

Figure 12 shows that in the presence of an angiotensin II antagonist the CLL cells rapidly undergo apoptosis, and this apoptosis is prevented by the addition of angiotensin II to the media. This clearly shows that CLL cells are responding to angiotensin II and suggests that this compound may be a survival factor for CLL. There are a number of angiotensin II antagonists that are used clinically, and these compounds would reduce tumour load in patients and prolong survival. To obtain the data illustrated in Figure 12 B-cells were purified and were incubated in RPMI with 5% FCS and the concentration of angiotensin and inhibitor shown. After 16 hours cells were washed in PBS and resuspending in buffer containing annexin V- and were separated by FACS.

(ii) Testing the effect of increased expression of proteins that are translationally upregulated in CLL

To investigate how modulating the levels of mRNAs that show altered translational expression in CLL affects control B-cell growth and survival, cDNAs which showed an increased polysomal association in patient samples, such as B-cell translocation gene 1 (BTG1), insulin like growth factor receptor 2 (IGF2R), disabled homologue 2 (DAB2) and Beclin (BECN1) can be subcloned into expression vectors and the AMAXA system be used to transfect B-cells purified from adult tonsils. Western analysis can be performed to determine the degree of expression of these proteins in B-cells. To test whether these cells become resistant to apoptosis as a result of over expression of these proteins (in the case of IGFR2 in the presence of the appropriate ligand), cells can be treated with apoptotic inducing agents including death receptor ligands, etoposide and UV light and the degree of apoptosis will be assessed measuring Annexin V labelling. The effects that the expression of these proteins has on cell growth will be determined by measuring 35S methionine and 3H thymidine incorporation.

(iii) Testing the effect of decreased expression of proteins that are translationally downregulated in CLL

To determine the effects of decreased expression of proteins that show a decrease in polysomal association on cell growth and survival, RNA interference technology (RNAi) can be used. The vector pSuper can be used and the relevant section of cDNAs that show decreased expression and are likely to affect survival of CLL B-cells in vivo including members of the TNF pathway (described with reference to Figure 4A to 1 IB), clusterin (CLU), and granzyme A (GZMA) can be subcloned into this vector. The efficacy of these vectors can be tested in a control B-CLL line (GM1 53), to ensure that they are capable of reducing expression of the corresponding proteins. These vectors can then be transfected into control primaiy B-cells using the Amaxa system and the effect that this has on survival of these cells following exposure to apoptotic stimuli will be determined.

Mechanisms of aberrant translational regulation of mRNAs in CLL B-cells.

The data shows that B-CLL cells are in a translationally repressed state: there are approximately 50% fewer polysomes on CLL B-cells than on control B-cells (Figure 1); the microarray data shows that the majority of TOP messages are not present on the polysomes (Meyhaus and Hornstein 2000; data not shown); there are alterations in the phosphorylation states of many eukaryotic initiation factors (elFs), which would reduce the ability of these cells to carry out translation, including eIF4E, eIF2a, 4EBP1 and 2 (data not shown). Therefore it is important to determine whether there are any unusual features in these mRNAs that allows their translation. There are two mechanisms of translation initiation that are used by mammalian cells, cap-dependent scanning (Gray and Wickens 1998) and internal ribosome entry (Hellen and Sarnow 2001). The latter mechanism requires the formation of a complex RNA structural element and this, in the presence of trans-acting factors, recruits the ribosome. This mechanism accounts for approximately 10% of all translation in a cell and is used by mRNAs whose protein products are involved in control of cell growth and cell death including Apaf-1, XI AP, Bag-1 and the Myc family of genes (Hellen and Sarnow 2001).

The sequences of the genes that have been identified by the microarray screen (translationally up or down regulated) are available from data bases and can be examined to determine whether they contain any unusual features e.g. long GC rich 5' UTRs or long 3' UTRs. The cDNAs that correspond to these sequences will be obtained either from gene banks, by RT-PCR or by RACE.

To determine the mechanism of translational regulation of the mRNAs that showed altered expression in CLL B-cells the regions of 5' and 3' UTRs of cDNAs under test can be inserted 'in frame' into a range of constructs. These are based on the firefly luciferase reporter vector pGL3 and include the dicistronic construct pRF (to test for the presence of an IRES; this vector contains Renilla and Firefly luciferase genes upstream and downstream respectively; Stoneley et al 2000) and derivatives of these vectors that contain stable hairpins phpRF, phpL, (Stoneley, et al. 2000). The resulting plasmids can be transfected into control B-cell lines (and for comparison other cell lines) and luciferase activities assayed to determine the effects that these sequences have on the expression of the reporter gene(s). Initial studies show that the 5' UTR of BTG1 contains an IRES (Rogers et al in preparation).

Summary

The B-cells associated with CLL are essentially quiescent and exhibit a very reduced rate of translation. The data provided shows that by examining the translationally active mRNAs it is possible to predict more accurately the protein expression profile in the diseased cells, to develop of diagnostics, for example based on ELISAs, for the proteins whose expression has been altered and to identify new therapeutic targets.

The use of translational profiling directly on patient samples is unique.

In CLL the leukaemic B-cells survive in the patients and this leads to a high tumour load, but once the cells are isolated in vitro they rapidly undergo apoptosis. Two possibilities as to how this is occurring include: A) The CLL B-cells express a receptor that allows them to respond to a survival cytokine/hormone in vivo and this prevents apoptosis in vivo. B) They express cell surface proteins e.g. integrins that allows them to interact with another cell type that again prevents apoptosis in vivo.

In each case removing these cells from the patients would cause apoptosis in vitro.

The data shows a number of receptors that have not previously been shown to be associated with CLL, these include the angiotensin II type I receptor, the interferon receptor, interleukin 17, amongst others. References

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Claims

1. Use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, in the treatment of cancer.

2. Use of a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, in the preparation of a medicament for the treatment of cancer.

3. The use according to claim 1 or claim 2 wherein the one or more genes are selected from the group comprising the genes listed in Figures 2 A, to 1 IB.

4. The use according to claim 1 or claim 2 wherein the gene is selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTGl gene; the IGF2R gene; the DAB2 gene; the BECN1 gene and the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAFl gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

5. The use according to any preceding claim wherein the modulator is an antagonist of angiotensin II .

6. The use according to any of claims 1 to 4 wherein the modulator increases the activity of the one or more genes or increases the activity of the expression product of the one or more genes.

7. The use according to any of claims 1 to 4 wherein the modulator decreases the activity of the one or more genes or decreases the^' activity of the expression product of the one or more genes.

8. The use according to any preceding claim wherein the cancer is leukaemia.

9. The use according to claim 8 wherein the leukaemia is chronic lymphocytic leukaemia (CLL).

10. A medicament comprising a modulator of the activity of one or more genes, or a modulator of the activity of the expression product of one or more genes, in a pharmaceutically acceptable form, wherein the one or more genes are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression.

11. A medicament according to claim 10 comprising a pharmaceutical carrier, diluent or excipient.

12. A medicament according to claim 10 or claim 11 wherein the modulator is at a concentration which upon administration is effective for the treatment of cancer.

13. A medicament according to any of claims 10 to 12 for use in the treatment of cancer.

14. A medicament according to claim 13 wherein the cancer is chronic lymphocytic leukaemia.

15. A medicament according to any of claims 10 to 14 wherein the one or more genes are selected from the group comprising the genes listed in Figures 2A to 11 B.

16. A medicament according to any of claims 10 to 14 wherein the gene is selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTGl gene; the IGF2R gene; the DAB2 gene; the BECN1 gene and the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAFl gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene.

17. A method of treatment of cancer comprising modulating the activity of one or more genes, or modulating the activity of the expression product of one or more genes, wherein the one or more gene are selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression.

18. The method according to claim 17 applied to a human or non-human animal.

19. The method according to claim 17 or 18 wherein the cancer is leukaemia.

20. The method according to claim 19 wherein the leukaemia is chronic lymphocytic leukaemia (CLL).

21. The method according to any of claims 17 to 20 wherein the one or more genes are selected from the group comprising the genes listed in Figures 2 A to 1 IB.

22. The method according to any of claims 17 to 21 wherein the one or more genes are selected from group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTGl gene; the IGF2R gene; the DAB2 gene; the BECN1 gene and the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAFl gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene;.

23. The method according to any of claims 17 or 22 wherein the activity of the expression product of one or more genes is modulated by an antagonist of angiotensin II.

24. The method according to any of claims 17 to 23 wherein the expression product of the one or more genes is the angiotensin II receptor (AGIIR).

25. The method according to any of claims 10 to 15 wherein the step of modulating the gene activity, or expression product activity, comprises either increasing or decreasing the activity.

26. The method according to claim 28 wherein the gene activity or expression product activity is modulated by any one of the means selected from the group comprising altering the transcription or translation of the gene, adding an antisense nucleic acid, using RNAi, adding a repressor, adding an antibody, introducing an agonist or an antagonist to the relevant expression product/protein, introducing a vector to introduce an additional copy of the gene product into a cell.

27. The method according to any of claims 17 to 26 wherein the method is performed on tumour cells in a patient.

28. A method of treatment of cancer, comprising modulating the activity of an angiotensin II receptor in a patient.

29. The method according to claim 28 comprising the step of administering an antagonist of angiotensin II to the patient.

30. The method according to claim 28 comprising administering an antagonist of the angiotensin-converting enzyme.

31. The method according to any one of claims 28 to 30 wherein the cancer is leukaemia.

32. The method according to claim 31 wherein the leukaemia is chronic lymphocytic leukaemia.

33. A method of screening a sample to identify potential targets for treatment of a disorder characterised by altered gene activity, the method comprising comparing the level of mRNA translation of one or more genes in a cell suffering from the disorder with that of a normal cell.

34. The method according to claim 33 wherein the disorder is cancer.

35. The method according to claim 34 wherein the cancer is leukaemia.

36. The method according to any of claims 33 to 35 wherein the comparison step comprises identifying one or more mRNA molecules present in polysomes of each cell, and comparing the identified mRNAs.

37. A method of identifying potential treatments for cancer, the method comprising the steps of screening a sample to identify potential targets for treatment by comparing the levels of mRNA translation of one or more genes in a cancer cell with that of a normal cell, identifying one or more target genes or gene expression products where the level of mRNA translation is different in the cancer cell compared to the normal cell, treating a cancer cell with a candidate treatment, and determining whether the activity of an identified target is modulated by the candidate treatment and whether there is any cell death.

38. The method according to claim 37 wherein the modulation of activity is determined by assessing the candidate treatment effect on apoptosis of the cancer cell compared with a control non-cancer cell.

39. A method of diagnosing a cancer in a sample from a human or non-human animal comprising determining the expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, wherein an increase or decrease in expression compared to a normal sample is indicative of cancer.

40. The method of claim 39 wherein the expression of one or more genes selected from the group comprising the genes listed in Figures 2 A to 1 IB is determined.

41. The method according to claim 39 or claim 40 wherein the expression of one or more genes selected from the group comprising the CLU gene; the GZMA gene; the IGF1 gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTGl gene; the IGF2R gene; the DAB2 gene; the BECNl gene and the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAFl gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS12 gene, is determined.

42. The method according to any of claims 39 to 41 wherein the cancer is leukaemia.

43. The method according to claim 42 wherein the cancer is chronic lympocytic leukaemia.

44. The method according to any of claims 39 to 43 wherein the gene expression is determined at the level of mRNA translation.

45. The method of claim 44 wherein mRNA translation is determined by isolating and analysing polysomes.

46. A method of determining the prognosis for an individual with cancer comprising determining the level of expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, administering a modulator intended to modulate the activity of one or more of the genes, or the activity of one or more the expression products of the genes, and assessing the effect of the modulator on the cancer progression.

47. A method according to claim 46 wherein the modulator increases or decreases the activity of a gene or a gene expression product whose activity is increased or decreased in the individual with cancer when compared to an individual without cancer.

48. A method according to claim 46 or claim 47 wherein the modulator increases activity if activity was decreased in the individual with cancer, or wherein the modulator decreases activity if activity was increase in the individual with cancer.

49. A kit for diagnosing a cancer, or determining the prognosis of an individual treated for cancer, in a sample from a human or non-human animal comprising means to determine the expression of one or more genes selected from the group comprising cell surface receptor genes, genes associated with apoptosis, genes associated with cell proliferation and genes associated with cell cycle progression, and instructions to compare the results with expression levels in a normal sample from a human or non- human animal without the cancer of interest.

50. The kit according to claim 49 including instructions that an increase or decrease in expression compared to a normal sample is indicative of cancer.

51. The kit according to claim 49 or claim 50 including means to determine the expression of one or more genes selected from the group comprising the genes listed in Figures 2 A to 11B.

52. The kit according to any of claims 49 to 51 including means to deteπnine the expression of one or more genes selected from the group comprising the CLU gene; the GZMA gene; the IGFl gene; the IGF2 gene; the chemokine 7 gene; the IL17 gene; the BTGl gene; the IGF2R gene; the DAB2 gene; the BECNl gene and the AGIIR gene; and one or more genes in the TNFα signalling pathway which includes the FADD gene, the TRAFl gene, the TRAF3 gene, the NGFR gene, the RIPK1 gene, the RIPK2 gene, the TNF gene, the TNFRS10 gene, the TNFRS11 gene and the TNFRS 12 gene.

53. The kit according to any of claims 49 to 52 wherein the cancer is leukaemia.

54. The kit according to claim 53 wherein the cancer is chronic lymphocytic leukaemia.

55. The kit according to any of claims 49 to 54 comprising a microarray designed to determine the level of expression of one or more genes.

56. The kit according to any of claims 49 to 55 for use with polysomes.

• 57. The kit according to any of claims 49 to 54 comprising one or more antibodies directed to the expression product of the one or more genes.

58. The kit according to claim 57 including ELISA technology.