WO2007053114A1 - Method of predicting a response to hdac inhibitors - Google Patents

Abstract

The present invention provides a method for predicting a response of a cell or a condition to exposure to a HDAC inhibitor, the method comprising the steps: providing a sample of a histone from the cell; and detecting an amount of a methylated lysine in the histone. In certain embodiments of the invention, the step of detecting comprises determining the amount of the methylated lysine. In certain embodiments of the invention substantial absence of the methylated lysine is indicative of a cell being responsive to the HDAC inhibitor and presence of the methylated lysine is indicative of a cell being non-responsive to the HDAC inhibitor. The present invention further provides kits for detecting a methylated lysine in histone.

Description

METHOD OF PREDICTING A RESPONSE TO HDAC INHIBITORS

FIELD OF THE INVENTION

The present invention relates to predicting a cell's response to a histone deacetylase (HDAC) inhibitor. Methods of the invention may find application in improving the efficiency and/or efficacy of treatments involving administration of a HDAC inhibitor.

BACKGROUND TO THE INVENTION

An important building block of chromatin, the nucleosome comprising two copies of each core histones H2A, H2B, H3 and H4, is subject to a variety of posttranslational modifications. These covalent changes of histone polypeptides form the so-called epigenetic code, which regulates gene expression in a temporal and spatial manner (Jenuwein and AlMs, 2001 ; Cosgrove and Wolberger, 2005). Amongst the known covalent histone modifications are acetylation and phosphorylation (Peterson and Laniel, 2004). In recent years additional histone tail modifications such as methylation and ubiquitination have emerged, whose functions have just begun to be revealed (Cheung and Lau, 2005). A wealth of information is available regarding the reversible pathways of histone acetylation in transcriptional control and many .enzymes involved in its regulation have been identified and well characterized (Verdone et al, 2005; Khan and Krishnamurthy, 2005). HDACs are a family of 18 different enzymes from three different classes that are involved in gene expression. In line with the current view, deacetylation of histones is generally linked to transcriptional silencing by . virtue of compacting the chromatin structure surrounding promoters and catalyzed by HDACs, which are co- recruited to target-promoters by DNA binding proteins. Because of the involvement of HDACs in gene expression, it is believed that they are "master regulators" of many diseases.

The field of histone deacetylation continues to receive considerable interest due to the ability of HDAC inhibitors to modulate transcriptional activity. Molecules from this therapeutic class can induce cell cycle arrest, differentiation and apoptosis and therefore have the potential to occupy an indomitable position in the fast-moving cancer therapeutic market.

HDAC inhibitors are emerging as an exciting new class of potential anti-cancer agents for the treatment of solid and haematological malignancies. In recent years, an increasing number of structurally diverse HDAC inhibitors have been identified that inhibit proliferation and induce differentiation and/or apoptosis of tumour cells in culture and in animal models. HDAC inhibition causes acetylated nuclear histones to accumulate in both tumour and normal tissues, providing a surrogate marker for the biological activity of HDAC inhibitors in vivo. The effects of HDAC inhibitors on gene expression are highly selective, leading to transcriptional activation of certain genes such as the cyclin- dependent kinase inhibitor p21^WAF1/clp1, but repression of others. HDAC inhibition not only results in acetylation of histones but also transcription factors such as p53, GATA-1 and estrogen receptor-α. The functional significance of acetylation of non-histone proteins and the precise mechanisms whereby HDAC inhibitors induce tumour cell growth arrest, differentiation and/or apoptosis are currently the focus of intensive research. Several HDAC inhibitors have shown evidence of anti-tumour activity in vivo in preclinical studies and are currently in clinical trials. The table below illustrates the importance various companies have placed on the potential utility of different HDAC inhibitors.

Table 1 : Examples of HDAC inhibitors in clinical trials

Compound Company Stage

SAHA , Merck & Co (New Jersey, US) Phase 2 ,

Deosin_pntiri_P Astellas (Japan)/Gloucester Phase 2

Depsⁱpeptⁱde _Pharmaceuticals (Cambridge, US) ^{Phase l}

Phenylbutyrate Elan Pharmaceuticals (Dublin) Phase 2

LBH859 Novartis (Basel) Phase 1

PXD101 TopoTarget (Copenhagen) Phase 1 I

MS-275 Schering AG (Berlin) Phase 1

Pyroxamide Merck & Co (New Jersey, US) Phase 1

MGCD0103 MethylGene (Montreal, Canada) Phase 1

PCI-24781 Pharmacyclics (Sunnyvale, US) Phase 1 Entering

SAVICOL TopoTarget (Copenhagen) Phase 2 Entering

BAECA TopoTarget (Copenhagen) Phase 2

In addition to the oncological applications for HDAC inhibitors, there are also potential non-oncology applications for HDAC inhibitors, including neurodegenerative diseases, metabolic diseases, inflammatory and autoimmune disorders, infectious diseases, and cardiovascular disease indications. All of these conditions may involve aberrant gene expression, which may be modulated by HDAC inhibitors.

While there are particular advantages to treating a cell with HDAC inhibitors to arrest growth and/or induce apoptosis, certain cells have been found to be insensitive to HDAC inhibitor treatment. Little is known about the molecular mechanisms of resistance towards HDAC inhibitors, but non-sensitive cell lines have been described in the literature. However, the molecular mechanisms and pathways involved in mediating resistance to HDAC inhibition remain poorly understood and there is no method or biomarker described yet, which would help to predict the cellular response to HDAC inhibitor treatment.

The inability to predict the response of a cell to exposure to a HDAC inhibitor causes significant problems in the treatment of patients. One class of conditions for which HDAC inhibitors have been used as a treatment is cancers. As would be appreciated by the skilled addressee, chemotherapy using HDAC inhibitors is generally carried out on patients with advanced stages of disease due to the generally late diagnosis of the condition. If the cancer is one that is not responsive to exposure to HDAC inhibitors then the use of a compound of this type can significantly compromise the prospects for successful treatment of the patient.

The ability to accurately predict the response of a cell to exposure to a HDAC inhibitor would allow the clinician to know whether such a treatment is likely to be successful and can thus choose an alternative treatment if the results indicate that the cell will not respond to exposure to a HDAC inhibitor. The present invention seeks to address this lack of predictability of cellular response to HDAC inhibitor treatment by providing methods and kits, which may be used to identify responsive cells. This is then expected to improve the success rate of the use of HDAC inhibitors.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

The present invention is based on the finding that methylation of histones is a predictor of a cell's response to HDAC inhibitors. This finding may also be used to predict the response of a medical condition to treatment with HDAC inhibitors.

In a first aspect the present invention provides a method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a sample of a histone from the patient; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence or substantial absence of methylation of the lysine with a predicted response of the conditio'n. In some forms of the invention, a substantial absence of methylation includes an absence of methylation. In some forms of the invention, a substantial absence of methylation includes a total absence of methylation. In some forms of the invention, the substantial absence or absence, of methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor. In some forms of the invention, the presence of methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor. In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention the histone is histone H3. In further forms of the invention, the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

In some forms of the invention, the sample of histone is obtained from a cancer cell, a peripheral blood lymphocyte or plasma of the patient. In some forms of the invention, the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or autoimmune disorder, a cardiovascular disease and an infectious disease.

In some forms of the invention, detecting the presence or substantial absence of methylation of a lysine is carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

In a second aspect the present invention provides a method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a histone-containing cell from the patient; exposing the his,tone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change in methylation of the lysine with a predicted response of the condition.

In some forms of the invention, an increase in methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor. In some forms of the invention, a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor. In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention the histone is histone H3. In further forms of the invention, the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

In some forms of the invention, the histone-containing cell is a cancer cell or a peripheral blood lymphocyte of the patient. In some forms of the invention, the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or autoimmune disorder, a cardiovascular disease and an infectious disease.

In some forms of the invention, detecting a change in methylation of a lysine is carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

In a third aspect the present invention provides a method for monitoring a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a first histone from a cell from the patient prior to treatment with the HDAC inhibitor; providing a second histone from a cell from the patient following treatment with the HDAC inhibitor; detecting a difference in methylation of a lysine between the first histone and the second histone; and correlating the difference in methylation of the lysine with a response of the condition. l'

In some forms of the invention, increased methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor. In some forms of the invention, a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor. In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention the histone is histone H3. In further forms of the invention, the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79. In some forms of the invention, the histone is obtained from a cancer cell or a peripheral blood lymphocyte of the patient. In some forms of the invention, the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or autoimmune disorder, a cardiovascular disease and an infectious disease.

In some forms of the invention, detecting a difference in methylation of a lysine is carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

In a fourth aspect the present invention provides a method for predicting a response of a cell to exposure to a HDAC inhibitor, the method comprising: providing a sample of a histone from the cell; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence or substantial absence of methylation of the lysine with a predicted response of the cell. In some forms of the invention, a substantial absence of methylation includes an absence of methylation. In some forms of the invention, a substantial absence of methylation includes a total absence of methylation.

In some forms of the invention, the substantial absence or absence of methylation of the lysine is indicative of the cell being substantially responsive to the HDAC inhibitor. In .some forms of the invention, the presence of _t methylation of the lysine is indicative of the cell being substantially non-responsive to the HDAC inhibitor. In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention' the histone is histone H3. In further forms of the invention, the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79. In some forms of the invention, the histone is obtained from a cancer cell. In further forms of the invention, the cancer cell is obtained from a patient with a cancer. In other forms of the invention, the histone is obtained from a peripheral blood lymphocyte obtained from a patient.

In some forms of the invention, detecting the presence or absence of methylation of a lysine is carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

In a fifth aspect the present invention provides a method for predicting a response of a histone-containing cell to treatment with a HDAC inhibitor, the method comprising: providing a sample of the histone-containing cell; exposing the sample of the histone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change in methylation with a predicted response of the cell.

In some forms of the invention, an increase in methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor. In some forms of the invention, a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor. In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention the histone/ is histone H3. In further forms of the invention, the, lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

In some forms of the invention, the histone is obtained from a cancer cell. In further forms of the invention, the cancer cell is obtained from a patient with a cancer. In other forms of the invention, the histone is obtained from a peripheral blood lymphocyte obtained from a patient. In some forms of the invention, detecting a change in methylation of a lysine is carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELlSA.

In a sixth aspect the present invention provides a kit for performing the methods of the present invention, comprising an antibody or antibody fragment which binds to a methylated histone, and at least one reagent for detecting binding of the antibody or antibody fragment to the methylated histone.

In certain forms of the invention, the methylated histone is a methylated histone selected from the group consisting of: methylated histone H2A, methylated histone H2B, methylated histone H3 and methylated histone H4. In some forms of the invention, the methylated histone is methylated histone H3. In further forms of the invention, the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

In some forms of the invention, binding of the antibody or antibody fragment to the methylated histone carried out by an immunological method. In certain forms of the invention, the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis, ELISA, RIA, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

In a seventh aspect the present invention provides a method of detecting a histone with a methylated lysine within a sample comprising: (i) contacting the sample with a first binding agent to form a secondary sample, wherein the first binding agent is capable of specifically binding the histone with the methylated lysine; (ii) contacting the secondary sample with a isecond binding agent to form a tertiary sample, wherein the second binding agent is either: (A) an agent capable of specifically binding the first binding agent; or (B) an agent capable of specifically binding the histone; (iii) contacting the tertiary sample with a third binding agent to form a quaternary sample, wherein if the second binding agent is (A), then the third binding agent is (B)₁ or if the second binding agent is (B) then the third binding agent is (A); and (iv) detecting the presence or absence of the agent capable of specifically binding the histone (B), wherein detecting the presence of the agent capable of specifically binding the histone (B) is indicative of the histone with the methylated lysine in the sample.

In certain forms of the invention the histone is selected from the group consisting of: H2A, H2B, H3 and H4. In particular forms of the invention the histone is histone H3. In further forms of the invention, the lysine is lysine 79 of histone H3.

In certain forms of the invention the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3. In further forms of the invention the agent capable of specifically binding the first binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to methylated lysine. In further forms of the invention the agent capable of specifically binding the histone is an antibody or fragment thereof which is capable of specifically binding the histone H3. In particular forms of the invention the agent capable of specifically binding the histone is detectable.

In an eighth aspect the present invention provides a kit when used to perform an ELISA to detect a methylated lysine in a histone, the kit comprising: a first binding agent, wherein the first binding agent specifically binds the methylated lysine in the histone; a second binding agent, wherein the second binding agent specifically binds the first binding agent and is substantially immobilised on a surface; a third binding agent, wherein the third binding agent specifically binds the histone.

In certain forms of the invention the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4. In certain forms of the invention the histone is histone H3. In further forms of the invention, the lysine of histone H3 is lysine 79. In certain forms of the invention the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3. In further forms of the invention the second binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to methylated lysine. In further forms of the invention the third binding agent is an antibody or fragment thereof which is capable of specifically binding the histone H3. In particular forms of the invention the third binding agent is detectable.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A shows the establishment of sensitivity profiles of selected cell lines towards Cpd A, a HDAC inhibitor. Figure 1B shows the apoptotic response to SAHA, LBH589 and Cpd B, a HDAC inhibitor (in sensitive and insensitive cell lines).

Figures 2 illustrates histone H3 methylation at residue lysine 79 following treatment with HDAC inhibitors in cell lines with different apoptotic response to HDAC inhibitors. Two colon cancer cell lines with high apoptotic response (Figure 2A) are compared with additional sensitive and insensitive cell lines (Figure 2C). Increased endogenous histone H3 methylation at residue lysine 79 and lack of methylation response in cell lines insensitive to HDAC inhibitors was observed. The corresponding densitometric analyses of the methylation level of histone H3 Lysine 79 of the sensitive and insensitive cell lines as shown in Figures 2A and C are represented in Figures 2B and D, respectively.

Figure 3A shows the low apoptotic response of the insensitive Non Small Cellular Lung Cancer (NSCLC) cell lines to various HDAC inhibitors as compared to the sensitive colorectal cancer HCT116 cells, and Figure 3B shows the lack of response in lysine 79 methylation of histone H3 in the insensitive cell lines. Figure 4 illustrates a lack of histone H3 (Lys 79) methylation response in non- transformed fibroblast cell lines insensitive to HDAC inhibitors.

Figure 5 illustrates the in vitro characterization of an additional insensitive tumor cell line (A2780 cell line), as well as the validation of histone (lys 79) H3 expression in three cancer xenograft models. Figure 5A illustrates the slow apoptotic response of A2780 ovarian cancer cells in vitro as measured by the Annexin V assay after 24h. Numbers within the quadrants reflect the percentage of cells found viable (lower left), early and late apoptotic (lower right and upper right, respectively) as well as necrotic (upper left).

Figure 5B shows the effect of treatment with HDAC inhibitor Cpd C on histone (lys 79) H3 expression in mouse xenograft models derived from HCT116 colorectal cancer, H460 lung cancer and A2780 ovarian cancer cell lines. The histone methylation response data were quantified by densitometric analysis of western blots derived from two mice per treatment group (control and 3h post treatment with 100 mg/kg of Cpd C), whereby the specific histone (Iys79) H3 signal was normalized to the corresponding actin expression.

Figure 6 illustrates that histone H3 (Lys 79) methylation may be a specific biomarker for HDAC inhibitory efficacy. As depicted in Figure 6A, the colorectal cancer cell line Colo205 was treated with various compounds at the concentration indicated in the figure and cell death was determined 24h post treatment. Figure 6B shows the selective increase in histone H3 (lys 79) methylation in the HDAC inhibitor treated samples as measured by specific antibodies in standard western blot procedures.

Figure 7 shows the effect of treatment with HDAC inhibitors on the B cell lymphoma cell line Ramos and the measurement of levels of histone H3 methylation at lysine 79. The left panel demonstrates the treatment dependent increase in histone H3 (lys 79) methylation, which corresponds with increased number of non-viable cells as measured by the standard Annexin V assay (right panel). Figure 8 illustrates the quantitative detection of histone H3 (Lys 79) methylation using a specific ELISA. Figure 8A shows the results obtained with the HDAC inhibitor sensitive Colo205 cell line treated with increasing amounts of the HDAC inhibitor PXD101 as indicated. Data obtained with the insensitive cell line CAKI-1 under the same conditions are depicted in Figure 8B. In both cases, the ELISA data are shown together with the viability readout.

DETAILED DESCRIPTION OF THE INVENTION

In contrast to histone acetylation, histone methylation has been known to have differing effects on gene activity, depending on the residue methylated. Methylation of certain lysine residues generally results in gene silencing, while methylation of arginines and certain lysines results in gene activation. In recent years, a histone methyltransferase named Dot1L was identified first in yeast and subsequently in mammals, which has been the first HMT not possessing a SET domain (Feng et al., 2002). The enzymatic methylase activity appeared to be cell cycle regulated, specific for lysine 79 of histone H3 and the biological function likely to be transcriptional silencing of telomere associated genes. More recently, Dot1L has been linked to leukemogenesis by aberrantly sequestering MML fusion proteins (Okada et al, 2005) and to be involved in the recognition of DNA-double strand breaks (Huyen et al., 2004).

The amino acid lysine may be referred to herein according to any one or more of: lysine; its three-letter code, Lys; and its single letter code, K.

Numerous histone methyltransferases are specific for K9 of histone H3 and involved in gene silencing and imprinting pathways associated with heterochromatin, some of them such as EZH2 are linked to cancer (Kleer et al., 2004), whereas K4 specific histone methylases are thought to activate gene expression in euchromatin (for a review, see Bannister and Kouzarides, 2005).

Two major observations were made in experiments described in the examples presented in the present application. The endogenous level of histone methylation at lysine residues such as lysine 4, 9 and 79 is low or not detectable in cell lines sensitive to HDAC inhibitor treatment, whereas an increased level of histone methylation was observed for the cell lines insensitive to the treatment. This suggests that histone marks other than acetylation such as methylation can also serve as predictors for the cellular response to HDAC inhibitors. In addition, in cell lines sensitive to HDAC inhibitors, a response in histone methylation was observed, which paralleled the increase in histone acetylation, which by itself serves as a common marker for HDAC inhibition- mechanism of action. The response in histone methylation is substantially absent in insensitive cell lines, despite the increase in histone acetylation observed in these cell types. This emphasizes the fact, that acetylation of core histones may not be suitable to serve as a sole correlative biomarker for efficacy of HDAC inhibitor treatment.

It would be desirable to be able to predict a cell's response to exposure to an HDAC inhibitor. As discussed herein, HDAC inhibitors may find application in the treatment of many conditions. Accordingly, it would be beneficial if, before treatment commences, or shortly after treatment commences, a simple assay could be used to determine whether or not a cell will respond, or is responding, to the treatment.

A response may be any one or more of: cell death; an arrest of cell growth; an arrest of cell proliferation or an alteration in one or more cell functions. The person skilled in the art would readily be able to identify and employ suitable ^ests to monitor any of these possible responses. In the case of a cancer cell it would be desirable for the response to be cell death. As described in the Examples, an apoptosis assay is a convenient measure of cell death since it measures activation of cellular processes which lead to cell death.

The present invention is based on the finding of a correlation between methylation of histone lysine residues and sensitivity to HDAC inhibitors.

Endogenous level of histone methylation The following aspects of the present invention are based on the endogenous level of histone methylation and its correlation to cellular response to exposure to an HDAC inhibitor.

In one aspect the present invention provides a method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a sample of a histone from the patient; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence or substantial absence of methylation of the lysine with a predicted response of the condition. HDAC inhibitors may be used in the treatment of a condition involving, relating to or, associated with dysregulation of HDAC.

In another aspect the present invention provides a method for predicting a response of a cell to exposure to a HDAC inhibitor, the method comprising: providing a sample of a histone from the cell; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence or substantial absence of methylation of the lysine with a predicted response of the cell.

As noted above the endogenous level of histone methylation at lysine residues is low or not detectable in cells sensitive to HDAC inhibitor treatment, therefore detecting methylation of a lysine may comprise determining an amount of methylation of the lysine. Quantitative methods for performing such determinations may include, but_t are not limited to, densitometry of Western blots used to identify the methylated lysine. An example of such an analysis is shown in the Examples. Alternative quantitative methods such as, but not limited to, ELISA may also be used.

In these aspects of the invention a substantial absence of methylation of the lysine is indicative of the condition or cell being substantially responsive to the HDAC inhibitor. When a condition or cell is said to be responsive, this means that the condition is ameliorated to some degree or cell growth is arrested, proliferation is inhibited or apoptosis is induced. In addition, the presence of methylation of the lysine is indicative of the condition or cell being substantially non-responsive to the HDAC inhibitor.

The Examples and Figures provide illustration of certain embodiments of the present invention, included in those embodiments are cell lines which are considered to be substantially responsive to HDAC inhibitors and other cells lines which are considered to be substantially non-responsive to HDAC inhibitors. Some of the substantially responsive cell lines show a level of endogenous lysine methylation which indicates the substantial absence of methylation of lysine.

As used herein the term "substantial absence" is intended to encompass situations in which there is absence or total absence of methylated lysine. It is also intended to encompass situations where the level of methylated lysine detected is less than 20% of an internal control.

In order to detect the substantial absence of methylated lysine, the level of endogenous methylated lysine may be compared to an internal control. In the accompanying Examples and Figures, actin is used as the internal control. Other possible internal control proteins may also be used. In order to identify whether there is a substantial absence of lysine methylation, the skilled addressee may choose to consider control samples for comparison with the test sample. As illustrated in the accompanying Examples and Figures, HCT116 or Colo205 colorectal cancer cells may be used as examples of substantial absence or absence of endogenous lysine methylation, while MCF7 cells may be used as an example of the presence of endogenous lysine methylation. It is not intended that the invention be limited to any particular cell lines or types, these are merely illustrated examples. >

In certain forms of the invention the methods may be used to analyse the methylation of a lysine within a histone selected from the group consisting of: H2A, H2B, H3 and H4. In particular embodiments of the present invention the methods may be used to analyse the methylation of a lysine within the histone H3. In particular embodiments of the invention, the lysine of histone H3 for analysis is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

There are a number of conditions that have been implicated in or known to be mediated at least in part by HDAC activity, where HDAC activity is known to play a role in triggering disease onset, or whose symptoms are known or have been shown to be alleviated by HDAC inhibitors. Such conditions may respond to treatment with a HDAC inhibitor. Conditions of this type that would be expected to be amenable to treatment with a HDAC inhibitor include, but not limited to, the following: proliferative disorders (e.g. cancer); neurodegenerative diseases including Huntington's disease, polyglutamine diseases, Parkinson's disease, Alzheimer's disease, seizures, striatonigral degeneration, progressive supranuclear palsy, torsion dystonia, spasmodic torticollis and dyskinesis, familial tremor, Gilles de Ia Tourette syndrome, diffuse Lewy body disease, Pick's disease, intracerebral haemorrhage, primary lateral sclerosis, spinal muscular atrophy, amyotrophic lateral sclerosis, hypertrophic interstitial polyneuropathy, retinitis pigmentosa, hereditary optic atrophy, hereditary spastic paraplegia, progressive ataxia and Shy-Drager syndrome; metabolic diseases including type 2 diabetes; degenerative diseases of the eye including glaucoma, age-related macular degeneration, macular myopic degeneration, rubeotic glaucoma, interstitial keratitis, diabetic retinopathy, Peter's anomaly, retinal degeneration, cellophane retinopathy; Cogan's dystrophy; corneal dystrophy; iris neovascularization (rubeosis); neovascularization of the cornea; retinopathy of prematurity; macular edema; macular hole; macular pucker; marginal blepharitis, myopia, nonmalignant growth of the conjunctiva; inflammatory diseases and/or immune system disorders including rheumatoid arthritis (RA), osteoarthritis, juvenile chronic arthritis, graft versus¹ host disease, psoriasis, asthma, spondyloarthropathy, Crohn's disease, inflammatory bowel disease, colitis ulcerosa, alcoholic hepatitis, diabetes, Sjoegrens's syndrome, multiple sclerosis, ankylosing spondylitis, membranous glomerulopathy, discogenic pain, systemic lupus erythematosus, allergic contact dermatitis; disease involving angiogenesis including cancer, psoriasis, rheumatoid arthritis; psychological disorders including bipolar disease, schizophrenia, depression and dementia; cardiovascular diseases including heart failure, restenosis, cardiac hypertrophy and arteriosclerosis; fibrotic diseases including liver fibrosis, lung fibrosis, cystic fibrosis and angiofibroma; Infectious diseases including Fungal infections, such as Candida albicans, bacterial infections, viral infections, such as herpes simplex, protozoal infections, such as malaria, Leishmania infection, Trypanosoma brucei infection, toxoplasmosis and coccidiosis, and haematopoietic disorders including thalassemia, anemia and sickle cell anemia.

One class of conditions for which treatment with HDAC inhibitors have shown promise is cancer. Accordingly, the present invention may find particular use for predicting a cancer patient's response to HDAC inhibitor treatment and also for monitoring the treatment of the cancer, as discussed in detail below. While the invention is described with particular focus on cancer, it is to be understood that the invention may be applied in many more situations, such as in conditions described above.

As used herein the term 'cancer' is a general term intended to encompass the vast number of conditions that are characterised by uncontrolled growth of cells. It is anticipated that the various cancers may be treated using HDAC inhibitors including but not limited to: bone cancers including Ewing's sarcoma, osteosarcoma, chondrosarcoma and the like, brain and CNS tumours including acoustic neuroma, neuroblastomas, glioma and other brain tumours, spinal cord tumours, breast cancers including ductal adenocarcinoma, metastatic ductal breast carcinoma, colorectal cancers, advanced colorectal adenocarcinomas, colon cancers, endocrine cancers including adenocortical carcinoma, pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid cancer, thymus cancer, multiple endocrine neoplasma, gastrointestinal cancers including stomach cancer, esophageal cancer, small intestine cancer, liver cancer, extra hepatic bile duct cancer, gastrointestinal carcinoid tumour, gall bladder cancer, genitourinary cancers including testicular cancer, penile cancer, prostate cancer, gynaecological cancers including cervical cancer, ovarian cancer, vaginal cancer, uterus/endometrium cancer, vulva cancer, gestational trophoblastic cancer, fallopian tube cancer, uterine sarcoma, head and neck cancers including oral cavity cancer, Up cancer, salivary gland cancer, larynx cancer, hypopharynx cancer, orthopharynx cancer, nasal cancer, paranasal cancer, nasopharynx cancer, leukemias including childhood leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia, erythroleukemia, myelomas, haematological disorders including myelodysplastic syndromes, myeloproliferative disorders, aplastic anemia, Fanconi anemia, Waldenstroms macroglobulinemia, lung cancers including small cell lung cancer, non-small cell lung cancer, mesothelioma, lymphomas including Hodgkin's disease, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, AIDS related Lymphoma, B-cell lymphoma, Burkitt's lymphoma, eye cancers including retinoblastoma, intraocular melanoma, skin cancers including melanoma, non-melanoma skin cancer, squamous cell carcinoma, merkel cell cancer, soft tissue sarcomas such as childhood soft tissue sarcoma, adult soft tissue sarcoma, Kaposi's sarcoma, urinary system cancers including kidney cancer, Wilms tumour, bladder cancer, urethral cancer, and transitional cell cancer.

Exemplary cancers that may be treated by HDAC inhibitors include, but are not limited to: breast cancer, lung cancer, ovarian cancer, prostate cancer, head and neck cancer, renal cancer (e.g. renal cell carcinoma), gastric cancer, colon cancer, colorectal cancer and brain cancer. l" L

Further exemplary cancers that may be treated by HDAC inhibitors include but are not limited to leukemias such as erythroleukemia, acute promyelocytic leukemia, acute myeloid leukemia, acute lymophocytic leukemia, acute T-cell leukemia and lymphoma such as B-cell lymphoma (e.g. Burkitt's lymphoma), cutaneous T-cell lymphoma (CTCL), and peripheral T-cell lymphoma.

Yet further exemplary cancers that may be treated by HDAC inhibitors include solid tumors and hematologic malignancies. Accordingly, HDAC inhibitors may be used to treat any one or more of the following cancers: colon cancer, prostate cancer, hepatoma, ovarian cancer, non small cell lung cancer, small cell lung cancer, mesothelioma, clear cell carcinoma/mesonephroma, intestinal cancer and pancreatic cancer.

HDAC inhibitors may also be used in the treatment of a disorder involving, relating to or, associated with dysregulation of histone deacetylase (HDAC).

HDAC inhibitors may be useful for treating a proliferative disease that is refractory to the treatment with other chemotherapeutics; and for treating hyperproliferative condition such as leukemias, psoriasis and restenosis.

HDAC inhibitors may be used to treat pre-cancer conditions or hyperplasia including familial adenomatous polyposis, colonic adenomatous polyps, myeloid dysplasia, endometrial dysplasia, endometrial hyperplasia with atypia, cervical dysplasia, vaginal intraepithelial neoplasia, benign prostatic hyperplasia, papillomas of the larynx, actinic and solar keratosis, seborrheic keratosis and keratoacanthoma.

It should be noted that any of the above mentioned conditions and cancers may be cause for treatment with an HDAC inhibitor and therefore may signal the requirement for using the methods of the present invention to determine the suitability of HDAC inhibitor treatment.

Patients' histones may be tested for a response to HDAC inhibitor to determine the suitability or effectiveness of treatment with HDAC inhibitors. Patients to be tested may have any one of a number of conditions, these conditions include, but are not limited to: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer, haematological cancer, gastric cancer, colorectal cancer, brain cancer, B-cell lymphoma (e.g. Burkitt's lymphoma), leukemias (e.g. Acute promyelocytic leukemia), cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma. Cancers are intended to encompass solid tumours and haematological malignancies.

In addition to cancers, it is envisaged that the methods of the present invention will find application in the case where a patient has a condition selected from the group consisting of: a neurodegenerative disease, a metabolic disease, an inflammatory or auto-immune disorder, a cardiovascular disease and an infectious disease.

In certain embodiments of the present invention, a sample of a histone is taken from a patient. In these embodiments, the sample of histone may be obtained from a cancer cell, a peripheral blood lymphocyte or plasma of the patient. In other embodiments of the present invention, a sample of histone is obtained from a cell. In certain embodiments, the cell may be obtained from a patient. In these embodiments, the sample of histone may be obtained from a cancer cell or a peripheral blood lymphocyte.

In the case of a cancer cell, a biopsy of a cancer may be used to provide a cell from which the histone sample may be obtained. Peripheral blood lymphocytes may be obtained by procedures, including, but not limited to, Ficoll gradient separation of whole blood. Cancer cells and peripheral blood lymphocytes require lysing in order to obtain the sample of histone. Since histones are intranuclear proteins, histones present in plasma have come from lysed cells and thus the plasma does not require lysis in order to provide the sample of histone. The choice of source of histone will depend on the condition being examined, for example if a cancer is being examined, a cancer cell should be the source of histone.

In discussions regarding patients with conditions that may be responsive to i treatment with HDAC inhibitors, "treatment" is intended to encompass administration of a HDAC inhibitor by any of the accepted modes for enteral administration such as oral or rectal, or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Injection can be bolus or via constant or intermittent infusion. The HDAC inhibitor is typically included in a pharmaceutically acceptable carrier or diluent and in an amount sufficient to deliver to the patient a therapeutically effective dose. In various embodiments the inhibitor compound may be selectively toxic or more toxic to rapidly proliferating cells, e.g. cancerous tumors, than to normal cells.

The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. A therapeutically effective amount can be readily determined by a skilled practitioner by the use of conventional techniques and by observing results obtained in analogous circumstances. In determining the effective amount a number of factors are considered including the species of the patient, its size, age, general health, the specific disease involved, the degree or severity of the disease, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability of the compound, the dose regimen selected, the use of other medication and other relevant circumstances.

Pharmaceutical compositions for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Solid dosage forms for oral administration include capsules, dragees, tablets, pills, powders, and granules. In such solid dosage forms, the HDAC inhibitor is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the HDAC inhibitor, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the HDAC inhibitor with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Dosage forms for topical administration of a HDAC inhibitor include powders, patches, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.

In certain embodiments, any agent that modulates or leads to the modification of histone methylation levels such as, but not limited to: lysine residues 4, 9 and/or 79 of histone H3 may be suitable as a potential therapy in combination with HDAC inhibitors to enhance the response mediated by HDAC inhibitors. In certain embodiments, the combination will enhance the apoptotic cellular response. Examples of potential agents that can be combined with HDAC inhibitors include histone methyltransferase inhibitors (such as DOT1L, EZH2, SET7/9) or using oligonucleotide approaches such as antisense and/or RNA interference.

Response in histone methylation

The following aspects of the present invention are based on the response in histone methylation following exposure to an HDAC inhibitor.

In one aspect the present invention provides a method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method , comprising: providing a histone-containing cell from the patient; exposing the histone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change in rηethylation of the lysine with a predicted response of the condition.

In another aspect the present invention provides a method for predicting a response of a histone-cbntaining cell to treatment with a HDAC inhibitor, , the method comprising: providing a sample of the histone-containing cell; exposing the sample of the histone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change in methylation of the lysine with a predicted response of the cell.

In yet another aspect the present invention provides a method for monitoring a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a first histone from a cell from the patient prior to treatment with the HDAC inhibitor; providing a second histone from a cell from the patient following treatment with the HDAC inhibitor; detecting a difference in methylation of a lysine between the first histone and the second histone; and correlating the difference in methylation of the lysine with a response of the condition.

In these aspects of the invention an increase in methylation of the lysine is indicative of the condition or cell being substantially responsive to the HDAC inhibitor. In certain embodiments the increase is at least a two fold increase. In other embodiments the increase is at least a 5 fold increase. In other embodiments the increase is at least a 10 fold increase. In other embodiments the increase is at least a 20 fold increase. In other embodiments the increase is at least a 100 fold increase. The level of increase is typically dependent on the identity of the cell and the identity of the HDAC inhibitor. When a condition or cell is said to be responsive, this means that the condition, is ameliorated to some degree, or cell growth is arrested, proliferation is inhibited or apoptosis is induced. In addition, a lack of increase in methylation of the lysine is indicative of the condition or cell being substantially non-responsive to the HDAC inhibitor. _t In certain embodiments of the present invention, a "lack of increase" may include a decrease. For example, it is possible that following treatment with an HDAC inhibitor, the amount of histone lysine methylation may decrease. This is also indicative of the condition or cell being substantially non-responsive to the HDAG inhibitor. In certain embodiments the decrease is at least a 5% decrease. In certain embodiments the decrease is at least a 10% decrease. In certain embodiments the decrease is at least a 20% decrease. In certain embodiments the decrease is at least a 50% decrease. In certain embodiments the decrease is at least a 80% decrease. In certain embodiments of the present invention, an internal control is used so that changes in methylation levels may be readily detected. For example, in the accompanying Examples and Figures, actin is used as an internal control. Other possible internal control proteins may also be used.

In certain embodiments of the present invention the methods may be used to analyse the methylation of a lysine within the histone H3. In particular embodiments of the invention, the lysine for analysis is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

In the methods described herein, a histone-containing cell is typically provided. In certain embodiments of the invention, the histone-containing cell may be representative of a condition being examined. For example, if the condition being examined is a colon cancer, a colon cancer cell would most likely represent the condition and therefore be the histone-containing cell for the methods of that embodiment of the invention.

As noted above, these aspects of the invention are based on the change in histone lysine methylation being indicative of a response to exposure to the HDAC inhibitor. Therefore, these methods typically involve a comparison of methylation prior to and following HDAC exposure of the histone-containing cells. The histone-containing cells may be obtained in any manner described above. The histone-containing cells are lysed and then the histone is examined for methylation on lysine residues.

As discussed above, there are a large number of conditions that may be treated using HDAC inhibitors. A cell which is representative of any of these conditions may be obtained according to well Known methodologies, including, but not limited to biopsies and isolation from blood. In certain embodiments of the present invention, the methods may be used to predict whether or not a condition or, more generally, a cell is likely to respond to treatment with or exposure to a HDAC inhibitor. For these predictive methods, it is envisaged that there are two samples of the histone-containing cell: one is kept as a control and the other is exposed to a HDAC inhibitor. Following treatment, or lack of treatment, both samples are treated in parallel to analyse the methylation of specific lysine residues in the histones. Detection of methylated lysine is discussed below. As noted above, a change in the methylation of the histone lysine may be predictive of a response to a HDAC inhibitor.

In certain embodiments of the present invention, the methods may be used to monitor a response of a condition in a patient to treatment with a HDAC inhibitor. Similar to the method described immediately above, this method may use a control histone-containing cell whose lysine methylation is compared to that of a cell which has been treated with HDAC inhibitor. In this case the treatment may have been in vivo and the present method is used to determine whether or not there is likely to have been the desired response to the treatment. The level of lysine methylation may be used as an indicator of an apoptotic response to HDAC inhibitor treatment. In a medical setting such an indication may be invaluable in the absence of other indications of cellular response to treatment.

In particular embodiments of the present invention, the histone-containing cell used in these methods is obtained from a patient. In certain embodiments the histone-containing cell is a cancer cell. In certain embodiments of the invention the cell is a colon cancer cell or an ovarian cancer cell. In other embodiments, the cell can be a breast, lung, prostate, liver, pancreatic, head and neck, renal or hematologic cell. In particular embodiments, the hematologic cell is a B-cell lymphoma cell.

The methods of the present invention typically involve examination of methylation of lysines in histones. It is therefore desirable to provide a sample of a histone from the type of cell which may be exposed to the HDAC inhibitor. It should be understood that providing a sample of a histone from the cell is intended to direct the skilled addressee to obtain the sample from a cell, which is representative of the cell which is to be exposed to the HDAC inhibitor. For example, if a tumour is to be exposed to the HDAC inhibitor, a small portion of the tumour may be taken for examination of histone methylation, the remaining part being the part which is be exposed to the HDAC inhibitor.

A sample of histone may be conveniently provided as a cell lysate of the cell of interest. In particular embodiments of the invention it is envisaged that the histone sample may be obtained from a tissue sample from a patient before and/or after patient treatment. In certain embodiments of the invention the tissue sample is a cancer tissue sample. In certain embodiments of the invention employing the use of patient tissue samples, a small amount of the sample would be lysed, according to standard protocols, in order to provide the sample of histone. Alternatively, peripheral blood lymphocytes or plasma may be prepared from patient whole blood samples; the histone proteins may be extracted therefrom and subjected to analyses.

Detecting methylation of lysine is intended to include determining an amount of the methylated lysine. That is to say, not only may the method detect zero amount of methylated lysine or some amount of methylated lysine, the method may be used to at least partially quantify the amount of methylated lysine. Such quantitation may be performed relative to the level of an internal control protein such' as actin.

The method of detection of methylated lysine may be carried out using any technique well known in the art. For example, it may conveniently be performed by an immunological method using an antibody, antibody fragment or the like, which binds the methylated lysine of interest. As used herein, the term "antibody or antibody fragment" is used in the broadest sense and includes molecules including, but not limited to: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, chimeric antibodies, antibody fragments such as Fv, Fab or F(ab')2 fragments and single chain antibodies, so long as they exhibit the desired biological activity. Further encompassed are molecules such as phage display molecules or antibodies. Exemplary, commercially available antibodies suitable for use in the methods of the present invention include, but are not limited to: UPSTATE antibodies anti-dimethyl-Histone H3 (Lys79); # 07-366; anti-dimethyl-Histone H3 (Lys4); # 07-030; and anti-dimethyl-Histone H3 (Lys9); # 07-521.

Various immunological methods for detection of the methylated lysine are known to the person skilled in the art, these include, but are not limited to: ELISA, immunohistochemistry, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

In certain embodiments an ELISA may be used to detect and/or determine the amount of methylated lysine. An exemplary ELISA may be carried out as follows; a histone-containing sample is incubated on a solid support, (e.g., a polystyrene dish) that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the methylated lysine-specific anti-histone antibody (primary antibody) is incubated in the dish, during which time the antibodies attach to the specific methylated lysine attached to the polystyrene dish. Unbound primary antibody is washed out with buffer. The reporter antibody linked to, for example, horseradish peroxidase is placed in the dish, resulting in binding of the reporter antibody to the primary antibody bound to histone with methylated lysine. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a colorimetric substrate, are then added to the dish. Immobilized peroxidase, linked to histone with methylated lysine through the primary and reporter antibodies, produces a coloured reaction product. The amount of colour developed in a given time period indicates the amount of histone with methylated lysine present in the sample. Quantitative results typically are obtained by reference to a standard curve.

A further aspect of the present invention provides a kit for performing the methods of the present invention, the kit comprising an antibody or antibody fragment which binds to a methylated histone and at least one reagent for detecting binding of the antibody or antibody fragment to the methylated histone. In certain embodiments of the invention the methylated histone is methylated histone H3. In particular embodiments, the methylated histone H3 is methylated at any one or more of lysine 4, lysine 9 and lysine 79, H3.

In order to detect binding of the antibody or antibody fragment to the methylated histone H3 an assay may be employed wherein the assay is selected from the group consisting of: immunohistochemistry, Western analysis, ELISA, RIA, FACS analysis, an immunofluorescence assay and a light emission immunoassay.

In certain embodiments of the invention, the antibody or antibody fragment is coupled to an enzyme. In particular embodiments the kit comprises an enzyme- linked immunosorbent assay (ELISA) kit.

In other embodiments of the present invention the antibody or antibody fragment may be coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

In a yet further aspect the present invention provides a method of detecting a histone with a methylated lysine within a sample comprising: (i) contacting the sample with a first binding agent to form a secondary sample, wherein the first binding agent is capable of specifically binding the histone with the methylated lysine; (ii) contacting the secondary sample with a second binding agent to form a tertiary sample, wherein the second binding agent is either: (A) an agent capable of specifically binding the first binding agent; or (B) an agent capable of specifically binding the histone; (iii) contacting the tertiary sample with a third binding agent to form a quaternary sample, wherein if the second binding agent is (A), then the third binding agent is (B), or if the second binding agent is (B) then the third binding agent is (A); and (iv) detecting the presence or absence of the agent capable of specifically binding the histone (B), wherein detecting the presence of the agent capable of specifically binding the histone (B) is indicative of the histone with the methylated lysine in the sample. In certain embodiments of the invention the histone is histone H3. In further embodiments of the invention, the lysine of histone H3 is lysine 79.

In particular embodiments of the invention the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3; the agent capable of specifically binding the first binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to methylated lysine; and the agent capable of specifically binding the histone is an antibody or fragment thereof which is capable of specifically binding the histone H3.

In particular embodiments of the invention the first binding agent is selected from the group consisting of: an anti-dimethyl-Histone H3 (Lys79) antibody; Upstate #07-366; an anti-dimethyl-Histone H3 (Lys4) antibody; Upstate #07-030; an anti-dimethyl-Histone H3 (Lys9) antibody; Upstate #07-521 ; and an anti-dimethyl Histone H3 (Lys79) polyclonal antibody; Upstate #05-835. In particular embodiments of the invention the agent capable of specifically binding the first binding agent is an anti-rabbit IgG antibody or anti-mouse IgG antibody. In particular embodiments of the invention the agent capable of specifically .binding the histone is an anti-Histone H3 antibody .(Capralogics P00006 sheep anti-histone H3). In particular embodiments including a fourth binding agent, the fourth binding agent is an anti-sheep IgG antibody.

In specific embodiments of this aspect of the invention, an antibody-histone complex is formed wherein the antibody is bound to a histone having a methylated lysine. Unbound antibody is then removed and the antibody-histone complex is bound to a second antibody which specifically binds the antibody which is bound to the histone having a methylated lysine. The second antibody may be immobilised on a surface such as in a well of an ELISA plate. Unbound material is then washed away and any part of the immobilizing surface which is still exposed, is blocked with a blocking agent. Suitable blocking agents include, but are not limited to: BSA or skim milk. The bound antibody-histone complex is then contacted with a third antibody which specifically binds to the histone. The third antibody should not disrupt the antibody-histone complex. Unbound third antibody is then washed away. (In certain other embodiments of the invention, the antibody-histone complex may be contacted with the third antibody prior to being immobilised via the second antibody.) Binding of the third antibody to the bound antibody-histone complex may be detected by any one of a number of methods. For example, if the third antibody is conjugated with a reporter enzyme (for example, horse radish peroxidase or alkaline phosphatase) or other detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light- emitting moiety. Detection of the third antibody may be carried out using standard methodologies for example in the case of the antibody being conjugated to an enzyme, that enzyme may be used in a colorimetric assay. The amount of colour developed in a given time period indicates the amount of histone with methylated lysine present in the sample. Quantitative results typically are obtained by reference to a standard curve. Alternatively, a fourth antibody may be used as a reporter to specifically bind the third antibody. Unbound fourth antibody is then washed away. The fourth antibody may typically include a reporter, such as an enzyme (for example, horse radish peroxidase or alkaline phosphatase) or other detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety. Detection of the fourth antibody may be carried out using standard methodologies for example in the case of the antibody being conjugated to an enzyme, that enzyme may be used in a colorimetric assay. The amount of colour developed in a given time period indicates the amount of histone with methylated lysine present in the sample. Quantitative results typically are obtained by reference to a standard curve.

In a still further aspect the present invention provides a kit when used to perform an ELISA to detect a methylated lysine in histone H3, the kit comprising: a first binding agent, wherein the first binding agent is capable of specifically binding a histone H3 which has the methylated lysine; a second binding agent, wherein the second binding agent which is capable of specifically binding the first binding agent and is substantially immobilised on a surface; a third binding agent, wherein the third binding agent which is capable of specifically binding histone H3.

In certain forms of the invention the histone is histone H3. In further forms of the invention, the lysine of histone H3 is lysine 79.

In certain forms of the invention the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3. In further forms of the invention the second binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to methylated lysine. In further forms of the invention the third binding agent is an antibody or fragment thereof which is capable of specifically binding the histone H3. In particular forms of the invention the third binding agent is detectable. Detection of binding of the third binding agent to the histone H3 may be carried out according to the methodologies described above.

In particular embodiments of the invention the first binding agent is selected from the group consisting of: an anti-dimethyl-Histone H3 (Lys79) antibody; Upstate #07,-366; an anti-dimethyl-Histone H3 (Lys4) antibody; Upstate #07-030; an anti-dimethyl-Histone H3 (Lys9) antibody; Upstate #07-521 ; and an anti-dimethyl Histone H3 (Lys79) polyclonal' antibody; Upstate #05-835. In particular embodiments of the invention the second binding agent is an anti-rabbit IgG antibody. In particular embodiments of the invention the third binding agent is an anti-Histone H3 antibody (Capralogics P00006 sheep anti- histone H3).

Specific Embodiments and applications of the present invention will now be discussed in detail by reference to the accompanying examples. This discussion is in no way intended to limit the scope of the invention.

EXAMPLES 1. In vitro HDAC assay for determination of IC₅₀ values

In order to demonstrate HDAC inhibitor activity of compounds used in these studies, each compound was tested against HDAC. The assay has been carried out in 96 well format and the BIOMOL fluorescent-based HDAC activity assay has been applied. The reaction composed of assay buffer, containing 25 mM Tris pH 7.5, 137 mM NaCI, 2.7 mM KCI, 1 mM MgCI₂, 1 mg/ml BSA, tested compounds, 600 nM HDAC1 enzyme, 500 μM Fluor de Lys generic substrate for HDAC1 enzyme and subsequently was incubated at room temperature for 2 h. Fluor de Lys Developer was added and the reaction was incubated for 10 min. Briefly, deacetylation of the substrate sensitizes it to the developer, which then generates a fluorophore. The fluorophore is excited with 360 nm light and the emitted light (460 nm) is detected on a fluorometric plate reader (Tecan Ultra Microplate detection system, Tecan Group Ltd.). The analytical software, Prism 3.0 has been used to generate IC₅₀ from a series of data. The HDAC enzyme inhibition results of compounds mentioned in this application are shown in Table 2 indicating that they are inhibitors of HDAC activity. IC50 is defined as the concentration of compound required for 50% inhibition of HDAC enzyme activity.

Table 2: HDAC inhibitor activity

2. Identifying cell lines responsive to treatment with HDAC inhibitor

Cell culture conditions

The breast cancer line MCF7 was propagated in EMEM containing 10% FBS and 2 mM L-Glutamine, 0.1 mM non-essential amino acids, 1mM sodium pyruvate, 0.01 mg/ml Insulin. Fibroblast cell lines were propagated in EMEM containing 2 mM L-glutamine, 10% FBS, 1% non-essential amino acids, and 1 mM sodium pyruvate. All other cell lines were propagated in RPMI 1640 medium containing 5% L-glutamine and 10% FBS. All cell lines were treated with proprietary and other known HDAC inhibitors (e.g. SAHA, PXD101 and

LBH589) at concentrations of 2 μM for 24 hours and subjected to either Annexin i V apoptosis assays or immunochemistry western blot assay for detection of histone modifications.

Apoptosis assays

To measure induced cell death, the Annexin V staining method was applied (Becton Dickinson, BD). All incubation steps were carried out according to the manufacturer's instructions using a FACS Calibur machine. The percentage of non-viable cells was determined by adding the amount of cells in early and late apoptosis, as well as necrosis. Values were normalized by subtracting the background of non-viable cells from non-treated control cells.

Immunobiochemistrv analyses of treated samples.

For the detection of histone H3 modifications such as acetylation and methylation at residues K4, K9 and K79, specific antibodies were obtained from UPSTATE (Anti-dimethyl-Histone H3 (Lys79); # 07-366; lot-Nr.: 26207; Anti- dimethyl-Histone H3 (Lys4); # 07-030; lot-Nr.: 24747; Anti-dimethyl-Histone H3 (Lys9); # 07-521; lot-nr.: 30850). Histone modifications were detected according to standard western blot based procedures. The best detection of Lys79 methylated histone H3 was obtained, when cells were lysed in a buffer containing Tris-HCI 5OmM pH 7.4, NaCI 1OmM, 5mM EDTA, NP-40 0.5%, SDS 0.1 %, and a protease Inhibitor Cocktail (P8340, SIGMA).

In vivo xenograft models

BALB/c athymic (nude) female mice approximately 6-8 weeks of age were purchased from Animal Resources Centre, Western Australia. Mice were housed in static micro-isolators on 12-hour light cycle at 21-22⁰C and 40-60% humidity. Animals were provided with food and water ad libitum. All the animal procedures comply with the recommendations of the Singapore Guidelines on the Care and Use of Animals for Scientific Purposes with .respect to restraint, husbandry, feed and fluid regulation, and veterinary care.

For the in vivo efficacy and biomarker studies, female athymic nude mice, 8-10 weeks of age, were implanted subcutaneously in, the flank with 5 *10⁶ cells of or A2780, HCT116, or H460 cells.

For in vivo efficacy studies, the mice were pair-matched prior to treatment when the tumour reached a size around 100 mm³. Tumour S¹JZe was measured twice a week and the tumour volume calculated as follows:

Tumour volume (mm³) = (w² x l)/2, where w = width and I = length in mm. Cpd C was used as an example of a HDAC inhibitor. Cpd C was prepared in 0.5% methylcellulose/0.1 % Tween 80 for oral administration. Cpd C was orally administered everyday using a gavage for up to a period of 14 days at a dose of 50 mg/kg once daily.

Tumour growth inhibition (%TGI) was calculated according to: %TGI = [(Ct-TtV(Ct-CtI )* 100] Where: Ct = the median tumour size of the vehicle control group at time t, Tt = the median tumour size of the treatment group at the time t,

CtI = the median tumour size of the vehicle control group on the first day of treatment.

Animal body weights were determined every day during the treatment period and then twice a week until the end of the study. Acceptable toxicity for cancer drugs in mice is defined by the NCI as mean group loss less than 20% during the test, and not more than one toxic death among ten treated animals. The treatment was stopped on the group when more than 1 animal treatment-related death was found in the group.

For the biomarker studies, a separate experiment was conducted. When the tumour size reached 200-500 mm³, a single dose of Cpd C was orally administered at 100 mg/kg to the mice implanted with either A2780 cancer cells, HCT116 cancer cells or H460 cancer cells. 3h after dosing, the mice were sacrificed. Samples from the 3h group as well as the Oh control group were processed for determination of histone methylation response using Western blot analysis.

Development of a K79 specific Enzyme Linked Immunosorbent Assay (ELISA) First, 100 μl of anti-Rabbit IgG antibody (2 μg/ml) (R2004 - Sigma) was added to each well (except the blank wells) in a high binding clear 96-well ELISA plate overnight. Samples (10 μg protein concentration/100 μl buffer) and a standard peptide ('N'ART/KQT/ARK/STG/GKA/PRK/QLC/GGG/GGG/GGG/GDF/K

(dimethyl) TD/L— 'C) were incubated overnight with an anti-dimethyl Histone H3 (Lys79) polyclonal antibody (Upstate #05-835). Pre-coated (anti-rabbit IgG) ELISA plates were washed with PBS+0.05% Tween20 and 100 μl of SuperBIock Blocking Buffer in PBS (Pierce #37515) was added to each well and incubated for 1 hour. After repeating this wash step, the samples and standard peptides previously incubated with the anti-dimethyl histone H3 antibody were added and further incubation allowed to proceed for another 2 hours. The plates were subsequently washed before adding 100 μl of anti- Histone H3 antibody (05-499 Upstate) and incubated for another hour.

After another washing step, 100 μl of anti-mouse IgG HRP conjugated antibody was added to each well and incubated for 1 hour. The ELISA plate was washed as described before adding 100μl substrate (1-Step™ Turbo TMB-ELISA (Pierce #34022)) to all the wells. Upon significant colour development, 100 μl of 1 M sulphuric acid was added to each well to stop the reaction and absorbance was measured at OD450nm.

Results

Cell lines exerting a decreased sensitivity to HDAC inhibitors as measured in Annexin V-apoptosis ' assays in direct comparison with known sensitive cells with high apoptotic response were identified. The results are shown in Figure 1.

8 tumour derived cell lines and 2 non-transformed fibroblast cell lines were identified which exhibited a low apoptotic response rate towards the HDAC inhibitors used for the treatment. These cell lines are:

• MCF7 : breast cancer

• SKOV3: ovarian cancer

• A2780: ovarian cancer

• ACHN: renal adenocarcinoma

• CAKI-1 : renal adenocarcinoma

• 786-0: renal adenocarcinoma

• H322M: Non Small Lung Cancer • H460: Non Small Lung Cancer

• WI38: non-transformed fibroblast

• MRC9: non-transformed fibroblast

There were 4 cell lines with high apoptotic response to HDAC inhibition. These sensitive cell lines are:

• HCT116: colon cancer

• Colo205: colon cancer

• Lovo: colon cancer

• RAMOS: B-cell lymphoma

3. Characterization of histone methylation pattern in response to treatment with HDAC inhibitors

This study sought to shed light on the methylation of histone H3 at specific lysine residues in response to HDAC inhibitor treatment. The residues of interest in this study were Lys 4, Lys 9 and Lys 79 at the N-terminal tail of core histone H3.

Compound can be sometimes abbreviated as Cpd or cpd. Control group in the various studies can be sometimes abbreviated as C or DMSO depending on the experiments conducted.

As illustrated in Figures 2 and 3, treatment with HDAC inhibitors was found to increase acetylation of histone H3 for all cell lines studied as a control, regardless of whether or not an apoptotic response was observed. In contrast, there was a strong increase in the amount of histone H3 lysine-methylation at residue 79 for all sensitive cell lines studied. This HDAC inhibitor induced histone methylation response was absent in all non-sensitive cell lines, whereby a strong background signal was noted in non-treated control cells (Figure 2C and Fig 3B). Increased levels of endogenous expression of histone populations carrying lysine methylation at residues 79, 4 and 9 was observed in this study as a characteristic feature of cell lines with lower apoptotic response. The degree of such modifications was either very low or not detectable in sensitive cancer cells undergoing apoptosis as a result of treatment with HDAC inhibitors.

Interestingly, as shown in Figure 4, non-transformed fibroblast cell lines tested in the study, are insensitive to HDAC inhibitor treatment and do not exert the methylation response on lysine 79 as compared to sensitive cancer cell lines.

Following the in vitro evaluation in various cell lines, it is of primary importance to validate the K79 methylation response using in vivo xenograft models. Mice with xenografts of HCT116 colorectal cancer cell line, NCI-H460 lung cancer cell line and A2780 ovarian cancer cell line have been dosed with 100 mg/kg of Cpd C and the response in histone H3(Lys 79) were determined at Oh and 3h after dosing.

In vivo xenograft studies with Cpd C, a HDAC inhibitor, revealed that HCT116 tumour growth was significantly inhibited with TGI >50%, whereas the growth of NSCLC and ovarian tumours was less sensitive to treatment (TGI < 50 % for both H460 and A2780 xenograft models). This confirms the in vitro observations shown in Figure 3, where all lung cancer cell lines tested in this study were identified as low apoptotic responders to treatment with HDAC inhibitors. In vitro apoptosis assays carried out under the conditions described for the data shown in Figure 1 confirmed low apoptotic response of the ovarian cancer cell line A2780 (Figure 5A).

In line with the in vitro ob/servations, response in histone H3 (Lys 79) expression was maximal after 3 hours of treatment in HCT116, whereas no increase could be detected in the insensitive H460 and A2780 models (Figure 5B). In addition, high endogenous levels of the control mice were found in the insensitive models, but not in the sensitive (HCT116) xenograft. The data shown were derived form densitometric quantification of two individual mice per treatment group, whereby the histone H3 (lys 79) signal was normalized against the respective actin signal and relative intensity of the bands expressed as arbitrary units. Control mice could be either vehicle treated or naive mice. Remarkably, histone H3 methylation at lysine 79 in HDAC inhibitor sensitive Colo 205 cells was not observed following induction of apoptosis upon treatment with chemotherapeutic drugs such as the general kinase inhibitor staurosporine and the DNA damaging agent etoposide, which indicates, that this histone modification may serve as a biomarker specific for apoptosis initiated by HDAC inhibition (Figure 6).

It is of importance to validate the K79 methylation response in cell lines from various tissues of origin, for instance from cancer cells stemming from solid tumours as the examples shown above or from haematological cancers. For this purpose, the B cell lymphoma cell line Ramos, which is an example of a haematological cancer cell line, was treated with HDAC inhibitors and the level of histone H3 methylation at lysine 79 was measured. As presented in Figure 7, all HDAC inhibitors tested provoked a methylation response, albeit to different extents depending on the degree of induced apoptosis based on the differences in potency of the compounds used. This K79 methylation response is therefore seen in both solid tumours and haematological cancer cell lines following treatment with HDAC inhibitors thus confirming its application for both solid tumours and haematogical cancers.

In order to quantify the degrees of the histone methylation response to HDAC inhibitors as described above, we developed a Lys79-specific ELISA. Figure 8A shows the detection of increasing amounts of Lys79-methylated histone H3, which; parallels the increase in non-viable Colo205 colorectal cells. In contrast, no increase in methylated histones and no induction of apoptosis could be detected in the renal cell carcinoma cell line CAKI-1 (Figure 8B).

4. Envisaged use of histone methylation pattern to predict and monitor a response to treatment with a HDAC inhibitor i) A patient having an early stage cancer presents at a clinic. The cancer is of a type which would normally be treated with a HDAC inhibitor as a first course of action. A doctor at the clinic decides to determine whether or not a HDAC inhibitor should be used. A small biopsy of the cancer is taken. A sample of the biopsied cancer is then lysed and the proteins separated by SDS-PAGE, the proteins are then transferred to a membrane by Western blotting. The Western blot is then probed with an anti-histone H3 (Lys79) antibody. Another Western blot is probed with an anti-actin antibody. The Western analysis shows only a faint band indicting methylated histone H3 (Lys79), but only after a long exposure/development time. This is considered a low level of methylation and the doctor commences treatment with an HDAC inhibitor.

ii) After several weeks of treatment the cancer has not reduced in size. The patient's cancer is biopsied again to determine whether there has been any response to the HDAC inhibitor. A sample from the original biopsy and the post treatment biopsy are subjected to the same Western analysis as described above. The results show virtually undetectable levels of methylated histone H3 (Lys 79) in the untreated lane, while there is considerable levels of methylated histone H3 (Lys 79) in the treated lane. Since such a response to HDAC inhibitor is indicative of a desirable response to the treatment, the doctor continues treating the patient with the HDAC inhibitor.

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Claims

Claims

1. A method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a sample of a histone from the patient; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence or substantial absence of methylation of the lysine with a predicted response of the condition.

2. A method according to claim 1, wherein detecting comprises determining an amount of methylation of the lysine.

3. A method according to claim 1 or 2, wherein substantial absence of methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor.

4. A method according to claim 1 or 2, wherein the presence of methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor.

5. A method according to any one of claims 1 to 4, wherein the histone is a histone H3.

6. A method according to claim 5, wherein the, lysine is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

7. A method according to claim 5 or 6, wherein the lysine is lysine 79.

8. A method according to any one of claims 1 to 7, wherein the sample of histone is obtained from a cancer cell, a peripheral blood lymphocyte or plasma of the patient.

9. A method according to any one of claims 1 to 8, wherein the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or auto-immune disorder, a cardiovascular disease and an infectious disease.

10. A method according to claim 9, wherein the cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer and haematological cancer.

11. A method according to any one of claims 1 to 10, wherein detecting the presence or substantial absence of methylation of a lysine is carried out by an immunological method.

12. A method according to claim 11 , wherein the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

13. A method for predicting a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a histone-containing cell from the patient; exposing the histone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change ₍ in methylation of the lysine with a predicted response of the condition.

14. A method according to claim 13, wherein the histone-containing cell is representative of the condition.

15. A method according to claim 13 or 14, wherein an increase in methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor.

16. A method according to claim 13 or 14, wherein a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor.

17. A method according to any one of claims 13 to 16, wherein the histone is a histone H3.

18. A method according to claim 17, wherein the lysine is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

19. A method according to claim 17 or 18, wherein the lysine is lysine 79.

20. A method according to any one of claims 13 to 19, wherein the histone- containing cell is a cancer cell or a peripheral blood lymphocyte of a patient.

21. A method according to any one of claims 13 to 20, wherein the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or auto-immune disorder, a cardiovascular disease and an infectious disease.

22. A method according to claim 21 , wherein the cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer and haematological cancer.

23. A method according to any one of claims 13 to 22, wherein detecting a change in møthylation of a lysine is carried out by an immunological method.

24. A method according to claim 23, wherein the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

25. A method for monitoring a response of a condition in a patient to treatment with a HDAC inhibitor, the method comprising: providing a first histone from a cell from the patient prior to treatment with the HDAC inhibitor; providing a second histone from a cell from the patient following treatment with the HDAC inhibitor; detecting a difference in methylation of a lysine between the first histone and the second histone; and correlating the difference in methylation of the lysine with a response of the condition.

26. A method according to claim 25, wherein the histone-containing cell is representative of the condition.

27. A method according to claim 25, wherein increased methylation of the lysine of the second histone when compared to methylation of the lysine of the first histone is indicative of the condition responding to the HDAC inhibitor.

28. A method according to claim 25, wherein a lack of increase in methylation of the lysine of the seco₍nd histone when compared to methylation of the lysine of the first histone is indicative of the condition not responding to the HDAC inhibitor.

29. A method according to any one of claims 25 to 28, wherein the histone is a histone H3.

30. A method according to claim 29, wherein the lysine is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

31. A method according to claim 29 or 30, wherein the lysine is lysine 79.

32. A method according to any one of claims 25 to 31 , wherein the cell is a cancer cell or a peripheral blood lymphocyte of the patient.

33. A method according to any one of claims 25 to 32, wherein the condition is selected from the group consisting of: a cancer, a neurodegenerative disease, a metabolic disease, an inflammatory or auto-immune disorder, a cardiovascular disease and an infectious disease.

34. A method according to claim 33, wherein the cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer and haematological cancer.

35. A method according to any one of claims 25 to 34, wherein the step of detecting is carried out by an immunological method.

36. A method according to claim 35, wherein the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.

37. A method for predicting a response of a cell to exposure to a HDAC inhibitor, the method comprising: providing a sample of a histone from the cell; detecting the presence or substantial absence of methylation of a lysine in the histone; and correlating the presence ør substantial absence of methylation of the lysine with a predicted response of the cell.

38. A method according to claim 37, wherein detecting comprises determining an amount of methylation of the lysine.

39. A method according to claim 37 or 38, wherein substantial absence of methylation of the lysine is indicative of the cell being substantially responsive to the HDAC inhibitor.

40. A method according to claim 37 or 38, wherein the presence of methylation of the lysine is indicative of the cell being substantially non-responsive to the HDAC inhibitor.

41. A method according to any one of claims 37 to 40, wherein the histone is a histone H3.

42. A method according to claim 41 , wherein the lysine is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

43. A method according to claim 41 or 42, wherein the lysine is lysine 79.

44. A method according to any one of claims 37 to 43, wherein the histone is obtained from a cancer cell.

45. A method according to claim 44, wherein the cancer cell is obtained from a patient with a cancer.

46. A method according to claim 45, wherein the cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer and haematological cancer.

47. A method according to any one of claims 37 to 45, wherein the histone is obtained from a peripheral blood lymphocyte obtained from a patient.

48. A method according to any one of claims 37 to 47, wherein detecting the presence or substantial absence of methylation is carried out by an immunological method.

49. A method according to claim 48, wherein the immunological method is selected from the group consisting of: in situ immunohistochemistry and ELISA.

50. A method for predicting a response of a histone-containing cell to treatment with a HDAC inhibitor, the method comprising: providing a sample of the histone-containing cell; exposing the sample of the histone-containing cell to the HDAC inhibitor; detecting a change in methylation of a lysine of a histone following exposure to the HDAC inhibitor; and correlating the change in methylation of the lysine with a predicted response of the cell.

51. A method according to claim 50, wherein an increase in methylation of the lysine is indicative of the cell being substantially responsive to the HDAC inhibitor.

52. A method according to claim 50, wherein a lack of increase in methylation of the lysine is indicative of the cell being substantially non-responsive to the HDAC inhibitor.

53. A method according to any one of claims 50 to 52, wherein the histone is a histone H3.

54. A method according to claim 53, wherein the lysine is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.

55. A method according to claim 53 or 54, wherein the lysine is lysine 79.

56. A method according to any one of claims 50 to 55, wherein the cell is a cancer cell.

57. A method according to claim 56, wherein the cancer cell is obtained from a patient with a cancer.

58. A method according to claim 57 wherein the cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, head and neck cancer, renal cancer and haematological cancer.

59. A method according to any one of claims 50 to 55, wherein the cell is a peripheral blood lymphocyte obtained from a patient.

60. A method according to any one of claims 50 to 59, wherein the step of detecting is carried out by an immunological method.

61. A method according to claim 60, wherein the immunological method is selected from the group consisting of: in situ immunohistochemistry and ELISA.

62. A kit for performing the method of any of the preceding claims comprising an antibody or antibody fragment which binds to a methylated histone, and at least one/reagent for detecting binding of the antibody or antibody fragment to the methylated histone.

63. Kit according to claim 62, wherein the methylated histone is methylated histone H3. ,

64. Kit according to claim 63, wherein the methylated histone H3 is methylated at any one or more of lysine 4, lysine 9 and lysine 79.

65. Kit according to claim 63 or 64, wherein detecting binding of the antibody or antibody fragment to the methylated histone H3 is effected by an assay selected from the group consisting of: in situ immunohistochemistry, Western analysis, ELISA, RIA, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

66. Kit according to any one of claims 62 to 64, wherein the kit comprises an enzyme-linked immunosorbent assay (ELISA) kit.

67. Kit according to any one of claims 62 to 66, wherein the antibody or antibody fragment is coupled to an enzyme.

68. Kit according to any one of claims 62 to 65, wherein the antibody or antibody fragment is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

69. A method of detecting a histone with a methylated lysine within a sample comprising:

(i) contacting the sample with a first binding agent to form a secondary sample, wherein the first binding agent is capable of specifically binding the histone with the methylated lysine;

(ii) contacting the secondary sample with a second binding agent to form a tertiary sample, wherein the second binding agent is either:

(A) an agent capable of specifically binding the first binding agent; or

(B) an agent capable of specifically binding the histone;

(iii) contacting the tertiary sample with a third binding agent to form a quaternary sample, wherein if the second bindjng agent is (A), then the third binding agent is (B), or if the second binding agent is (B) then the third binding agent is (A); and

(iv) detecting the presence or absence of the agent capable of specifically binding the histone (B), wherein detecting the presence of the agent capable of specifically binding the histone (B) is indicative of the histone with the methylated lysine in the sample.

70. A method according to claim 69 comprising:

(i) contacting the sample with a first binding agent to form a secondary sample, wherein the first binding agent is capable of specifically binding the histone with the methylated lysine;

(ii) contacting the secondary sample with a second binding agent to form a tertiary sample, wherein the second binding agent is an agent capable of specifically binding the first binding agent;

(iii) contacting the tertiary sample with a third binding agent to form a quaternary sample, wherein the third binding agent is an agent capable of specifically binding the histone; and

(iv) detecting the presence or absence of the third binding agent, wherein detecting the presence of the third binding agent is indicative of the histone with the methylated lysine in the sample.

71. A method according to claim 69 comprising:

(i) contacting the sample with a first binding agent to form a secondary sample, wherein the first binding agent is capable of specifically binding the histone with the methylated lysine;

(ii) contacting the secondary sample with a second binding agent to form a tertiary sample, wherein the second binding agent is an agent capable of specifically binding the histone;

(iii) contacting the tertiary sample with a third binding agent to form a quaternary sample, wherein the third binding agent is an agent capable of specifically binding the first binding agent; and

(iv) detecting the presence or absence of the second binding agent, wherein detecting the presence of the second binding agent is indicative of the histone with the methylated lysine in the sample.

72. A method according to any one of claims 69 to 71 , wherein the histone is a core histone selected from the group consisting of histones: H2A, H2B, H3 and H4.

73. A method according to any one of claims 69 to 72, wherein the histone is histone H3.

74. A method according to claim 72, wherein the lysine is lysine 79.

75. A method according to claim 74, wherein the first binding agent is an antibody or fragment thereof which is capable of specifically binding a histone H3 which is methylated on lysine 79.

76. A method according to any one of claims 69 to 75, wherein the agent capable of specifically binding the first binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to the histone with the methylated lysine.

77. A method according to any one of claims 69 to 76, wherein the agent capable of specifically binding the first binding agent is immobilised in a well of an ELISA plate.

78. A method according to any one of claims 72 to 77, wherein the agent capable of specifically binding the histone is an antibody or fragment thereof whiph is capable of specifically binding the histone H3.

79. A method according to any one of claims 69 to 78, wherein the agent capable of specifically binding the histone is coupled to an enzyme.

80. A method according to any one of claims 69 to 78, wherein the agent capable of specifically binding the histone is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

81. A method according to any one of claims 69 to 78 further comprising contacting the agent capable of specifically binding the histone with a fourth binding agent, wherein the fourth binding agent specifically binds the agent capable of specifically binding the histone.

82. A method according to claim 81 , wherein the fourth binding agent is coupled to an enzyme.

83. A method according to claim 81, wherein the fourth binding agent is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

84. A method according to claim 79 or 82, wherein the enzyme is selected from the group consisting of: horse radish peroxidase and alkaline peroxidase.

85. A kit when used to perform an ELISA to detect a methylated lysine in a histone, the kit comprising: a first binding agent, wherein the first binding agent specifically binds the methylated lysine in the histone; a second binding agent, wherein the second binding agent specifically binds the first binding agent and is substantially immobilised on a surface; a third binding agent, wherein the third binding agent specifically binds the histone.

86. . A kit according to claim 85 wherein the histone is $ histone selected from the group consisting of: H2A, H2B, H3 and H4.

87. A kit according to claim 86 wherein the histone is histone H3.

88. A kit according to claim 86 or 87, wherein the lysine is lysine 79.

89. A kit according to claim 86, wherein the first binding agent is an antibody or fragment thereof which is capable of specifically binding a histone H3 which is methylated on lysine 79.

90. A kit according to claim 87, wherein the second binding agent is an antibody or fragment thereof which is capable of binding the first binding agent when the first binding agent is bound to methylated lysine.

91. A kit according to claim 88, wherein the second binding agent is immobilised in a well of an ELISA plate.

92. A kit according to any one of claims 85 to 89, wherein the third binding agent is an antibody or fragment thereof which is capable of specifically binding the histone H3.

93. A kit according to any one of claims 85 to 90, wherein the third binding agent is coupled to an enzyme.

94. A kit according to any one of claims 85 to 90, wherein the third binding agent is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

95. A kit according to any one of claims 85 to 90 further comprising a fourth binding agent, wherein the fourth binding agent specifically binds the third binding agent.

96. A kit according to claim 93, wherein the fourth binding agent is coupled to an enzyme. A kit according to claim 93, wherein the fourth binding agent is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.