WO2007053114A1 - Méthode permettant de prévoir une réponse aux inhibiteurs hdac - Google Patents

Méthode permettant de prévoir une réponse aux inhibiteurs hdac Download PDF

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Publication number
WO2007053114A1
WO2007053114A1 PCT/SG2006/000317 SG2006000317W WO2007053114A1 WO 2007053114 A1 WO2007053114 A1 WO 2007053114A1 SG 2006000317 W SG2006000317 W SG 2006000317W WO 2007053114 A1 WO2007053114 A1 WO 2007053114A1
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Prior art keywords
histone
lysine
cancer
binding agent
methylation
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PCT/SG2006/000317
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English (en)
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Walter STÜNKEL
Vasantha Malar Sabanayagam
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S*Bio Pte Ltd
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Publication of WO2007053114A1 publication Critical patent/WO2007053114A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds

Definitions

  • the present invention relates to predicting a cell's response to a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • 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.
  • HDAC inhibitors are emerging as an exciting new class of potential anti-cancer agents for the treatment of solid and haematological malignancies.
  • 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- ⁇ .
  • 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.
  • 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.
  • HDAC inhibitors 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.
  • 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.
  • HDAC inhibitors have been used as a treatment.
  • 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.
  • 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.
  • 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.
  • a substantial absence of methylation includes an absence of methylation.
  • a substantial absence of methylation includes a total absence of methylation.
  • the substantial absence or absence, of methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor.
  • the presence of methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor.
  • the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone is histone H3.
  • the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • the sample of histone is obtained from a cancer cell, a peripheral blood lymphocyte or plasma of the patient.
  • 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.
  • detecting the presence or substantial absence of methylation of a lysine is carried out by an immunological method.
  • the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.
  • 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.
  • an increase in methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor.
  • a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor.
  • the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone is histone H3.
  • the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • the histone-containing cell is a cancer cell or a peripheral blood lymphocyte of the patient.
  • 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.
  • detecting a change in methylation of a lysine is carried out by an immunological method.
  • the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.
  • 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.
  • 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.
  • detecting a difference in methylation of a lysine is carried out by an immunological method.
  • the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.
  • 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.
  • a substantial absence of methylation includes an absence of methylation.
  • a substantial absence of methylation includes a total absence of methylation.
  • the substantial absence or absence of methylation of the lysine is indicative of the cell being substantially responsive to the HDAC inhibitor.
  • the presence of t methylation of the lysine is indicative of the cell being substantially non-responsive to the HDAC inhibitor.
  • the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone is histone H3.
  • the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • the histone is obtained from a cancer cell.
  • the cancer cell is obtained from a patient with a cancer.
  • the histone is obtained from a peripheral blood lymphocyte obtained from a patient.
  • detecting the presence or absence of methylation of a lysine is carried out by an immunological method.
  • the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELISA.
  • 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.
  • an increase in methylation of the lysine is indicative of the condition being substantially responsive to the HDAC inhibitor.
  • a lack of increase in methylation of the lysine is indicative of the condition being substantially non-responsive to the HDAC inhibitor.
  • the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone/ is histone H3.
  • the, lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • the histone is obtained from a cancer cell.
  • the cancer cell is obtained from a patient with a cancer.
  • the histone is obtained from a peripheral blood lymphocyte obtained from a patient.
  • detecting a change in methylation of a lysine is carried out by an immunological method.
  • the immunological method is selected from the group consisting of: in situ immunohistochemistry, Western analysis and ELlSA.
  • 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.
  • 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.
  • the methylated histone is methylated histone H3.
  • the lysine of histone H3 is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • binding of the antibody or antibody fragment to the methylated histone carried out by an immunological method.
  • 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.
  • 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 agent capable of specifically
  • the histone is selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone is histone H3.
  • the lysine is lysine 79 of histone H3.
  • 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.
  • the agent capable of specifically binding the histone is an antibody or fragment thereof which is capable of specifically binding the histone H3.
  • the agent capable of specifically binding the histone is detectable.
  • 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.
  • the histone is a histone selected from the group consisting of: H2A, H2B, H3 and H4.
  • the histone is histone H3.
  • the lysine of histone H3 is lysine 79.
  • the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3.
  • 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.
  • the third binding agent is an antibody or fragment thereof which is capable of specifically binding the histone H3.
  • the third binding agent is detectable.
  • 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).
  • Figure 2A Two colon cancer cell lines with high apoptotic response
  • Figure 2C additional sensitive and insensitive cell lines
  • 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
  • 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.
  • 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.
  • histone methylation 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • the response In the case of a cancer cell it would be desirable for the response to be cell death.
  • 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.
  • 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.
  • 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.
  • 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.
  • a substantial absence of methylation of the lysine is indicative of the condition or cell being substantially responsive to the HDAC inhibitor.
  • 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.
  • the presence of methylation of the lysine is indicative of the condition or cell being substantially non-responsive to the HDAC inhibitor.
  • 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.
  • substantially 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.
  • the level of endogenous methylated lysine may be compared to an internal control.
  • actin is used as the internal control.
  • Other possible internal control proteins may also be used.
  • the skilled addressee may choose to consider control samples for comparison with the test sample.
  • HCT116 or Colo205 colorectal cancer cells may be used as examples of substantial absence or absence of endogenous lysine methylation
  • 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. >
  • 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.
  • the methods may be used to analyse the methylation of a lysine within the histone H3.
  • the lysine of histone H3 for analysis is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • 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.
  • 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
  • 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.
  • 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 hepati
  • 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
  • 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.
  • leukemias such as erythroleukemia, acute promyelocytic leukemia, acute myeloid leukemia, acute lymophocytic leukemia, acute T-cell leukemia and lymphoma
  • B-cell lymphoma e.g. Burkitt's lymphoma
  • CCL cutaneous T-cell lymphoma
  • peripheral T-cell lymphoma peripheral T-cell lymphoma
  • 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 histone deacetylase
  • 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.
  • 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
  • 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.
  • 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.
  • a sample of a histone is taken from a patient.
  • the sample of histone may be obtained from a cancer cell, a peripheral blood lymphocyte or plasma of the patient.
  • a sample of histone is obtained from a cell.
  • the cell may be obtained from a patient.
  • the sample of histone may be obtained from a cancer cell or a peripheral blood lymphocyte.
  • 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.
  • 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.
  • the inhibitor compound may be selectively toxic or more toxic to rapidly proliferating cells, e.g. cancerous tumors, than to normal cells.
  • terapéuticaally 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.
  • 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.
  • 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.
  • Solid dosage forms for oral administration include capsules, dragees, tablets, pills, powders, and granules.
  • 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
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • 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.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • 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.
  • 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.
  • 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.
  • the combination will enhance the apoptotic cellular response.
  • 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.
  • the following aspects of the present invention are based on the response in histone methylation following exposure to an HDAC inhibitor.
  • 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 r ⁇ ethylation of the lysine with a predicted response of the condition.
  • 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.
  • 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.
  • an increase in methylation of the lysine is indicative of the condition or cell being substantially responsive to the HDAC inhibitor.
  • 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.
  • a lack of increase in methylation of the lysine is indicative of the condition or cell being substantially non-responsive to the HDAC inhibitor.
  • a "lack of increase" may include a decrease.
  • the decrease is at least a 5% decrease.
  • the decrease is at least a 10% decrease.
  • the decrease is at least a 20% decrease.
  • the decrease is at least a 50% decrease.
  • the decrease is at least a 80% decrease.
  • an internal control is used so that changes in methylation levels may be readily detected.
  • actin is used as an internal control.
  • Other possible internal control proteins may also be used.
  • the methods may be used to analyse the methylation of a lysine within the histone H3.
  • the lysine for analysis is selected from the group consisting of: lysine 4, lysine 9 and lysine 79.
  • a histone-containing cell is typically provided.
  • 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.
  • 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.
  • HDAC inhibitors 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.
  • 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.
  • a condition or, more generally, a cell is likely to respond to treatment with or exposure to a HDAC inhibitor.
  • 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.
  • a change in the methylation of the histone lysine may be predictive of a response to a HDAC inhibitor.
  • 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.
  • the histone-containing cell used in these methods is obtained from a patient.
  • the histone-containing cell is a cancer cell.
  • the cell is a colon cancer cell or an ovarian cancer cell.
  • the cell can be a breast, lung, prostate, liver, pancreatic, head and neck, renal or hematologic cell.
  • 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.
  • the histone sample may be obtained from a tissue sample from a patient before and/or after patient treatment.
  • the tissue sample is a cancer tissue sample.
  • a small amount of the sample would be lysed, according to standard protocols, in order to provide the sample of histone.
  • 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.
  • 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.
  • immunological methods for detection of the methylated lysine include, but are not limited to: ELISA, immunohistochemistry, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.
  • 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.
  • 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 album
  • 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.
  • the methylated histone is methylated histone H3.
  • the methylated histone H3 is methylated at any one or more of lysine 4, lysine 9 and lysine 79, 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.
  • the antibody or antibody fragment is coupled to an enzyme.
  • the kit comprises an enzyme- linked immunosorbent assay (ELISA) kit.
  • 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.
  • 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
  • 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;
  • the agent capable of specifically binding the histone is an antibody or fragment thereof which is capable of specifically binding the histone H3.
  • 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.
  • the agent capable of specifically binding the first binding agent is an anti-rabbit IgG antibody or anti-mouse IgG antibody.
  • the agent capable of specifically .binding the histone is an anti-Histone H3 antibody .(Capralogics P00006 sheep anti-histone H3).
  • the fourth binding agent is an anti-sheep IgG antibody.
  • 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.
  • a reporter enzyme for example, horse radish peroxidase or alkaline phosphatase
  • 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.
  • 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.
  • 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.
  • the histone is histone H3.
  • the lysine of histone H3 is lysine 79.
  • the first binding agent is an antibody or fragment thereof which is capable of specifically binding methylated lysine 79 of histone H3.
  • 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.
  • the third binding agent is an antibody or fragment thereof which is capable of specifically binding the histone H3.
  • 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.
  • 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.
  • the second binding agent is an anti-rabbit IgG antibody.
  • the third binding agent is an anti-Histone H3 antibody (Capralogics P00006 sheep anti- histone H3).
  • 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 MgCI 2 , 1 mg/ml BSA, tested compounds, 600 nM HDAC1 enzyme, 500 ⁇ M 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.
  • 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 IC 50 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.
  • 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 ⁇ M for 24 hours and subjected to either Annexin i V apoptosis assays or immunochemistry western blot assay for detection of histone modifications.
  • 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.
  • 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).
  • 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 21-22 0 C 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.
  • mice Female athymic nude mice, 8-10 weeks of age, were implanted subcutaneously in, the flank with 5 *10 6 cells of or A2780, HCT116, or H460 cells.
  • mice were pair-matched prior to treatment when the tumour reached a size around 100 mm 3 .
  • Tumour S 1 JZe was measured twice a week and the tumour volume calculated as follows:
  • 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.
  • 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.
  • ACHN renal adenocarcinoma
  • CAKI-1 renal adenocarcinoma
  • RAMOS B-cell lymphoma
  • 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.
  • 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.
  • 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.
  • 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).
  • chemotherapeutic drugs such as the general kinase inhibitor staurosporine and the DNA damaging agent etoposide
  • 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).
  • 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.
  • H3-lysine 79 is mediated by a new family of HMTases without a SET domain.
  • Chinnaiyan AM is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl

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Abstract

La présente invention concerne une méthode pour prévoir une réponse d'une cellule ou une condition suite à une exposition à un inhibiteur HDAC. Ladite méthode consistant à : obtenir un échantillon d'histone de la cellule ; et détecter une quantité d'une lysine méthylée dans l'histone. Dans certains modes de réalisation de l'invention, l'étape de détection consiste à déterminer la quantité de lysine méthylée. Dans certains modes de réalisation de l'invention l'absence substantielle de lysine méthylée indique qu’une cellule est sensible à l'inhibiteur HDAC et la présence de lysine méthylée indique qu’une cellule n’est pas sensible à l'inhibiteur HDAC. La présente invention permet en outre d’obtenir des trousses pour détecter une lysine méthylée dans de l’histone.
PCT/SG2006/000317 2005-10-31 2006-10-30 Méthode permettant de prévoir une réponse aux inhibiteurs hdac WO2007053114A1 (fr)

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WO2013067302A1 (fr) * 2011-11-04 2013-05-10 Glaxosmithkline Intellectual Property (No. 2) Limited Méthode de traitement
WO2013067300A1 (fr) * 2011-11-04 2013-05-10 Glaxosmithkline Intellectual Property (No. 2) Limited Méthode de traitement
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013067296A1 (fr) * 2011-11-04 2013-05-10 GLAXOSMITHKLINE INTELLECTUAL PROPERTY (No 2) LIMITED Méthode de traitement
WO2013067302A1 (fr) * 2011-11-04 2013-05-10 Glaxosmithkline Intellectual Property (No. 2) Limited Méthode de traitement
WO2013067300A1 (fr) * 2011-11-04 2013-05-10 Glaxosmithkline Intellectual Property (No. 2) Limited Méthode de traitement
CN104039956A (zh) * 2011-11-04 2014-09-10 葛兰素史密斯克莱知识产权（第2号）有限公司 治疗方法
US9242962B2 (en) 2011-11-04 2016-01-26 Glaxosmithkline Intellectual Property (No. 2) Limited Method of treatment
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RU2621148C2 (ru) * 2011-11-04 2017-05-31 Глэксосмитклайн Интеллекчуал Проперти (Но.2) Лимитед Способ лечения
US10246498B2 (en) 2014-05-30 2019-04-02 The Johns Hopkins University Genetically encoded histone reporter allele constructs

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